INCREASING THE BIODEGRADATION RATE OF POLY(LACTIC ACID) IN COMPOSTING CONDITIONS By Edgar Castro Aguirre A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Packaging Doctor of Philosophy 201 8 ABSTRACT INCREASING THE BIODEGRADATION RATE OF POLY(LACTIC ACID) IN COMPOSTING CONDITIONS By Edgar Castro Aguirre P oly(lactic acid) (PLA) , a well - known compostable and bio - based aliphatic polyester , has found applications in the medical, textile, plasticulture, and packaging industries . PLA has been blended with several polymers and compounded with different micro and nanoparticles t o fulfill desirable properties and to extend its range of applicati ons. The growing interest in PLA - based materials and other biodegradable polymers has required the development of methodologies to evaluate their biodegrada bility and understand the different factors affecting t heir biodegradation mechanisms and rate . One of the current limitations of biodegradable polymers, like PLA, is that they do not biodegrade as fast as other organic wastes during composting , affecting the ir general acceptance in industrial compos ting facilities. In this work, the results of two diffe rent approaches to accelerate the biodegradation rate of PLA are presented: 1) the addition of layered silicate nanoparticles to the PLA matrix, and 2) the addition of selective PLA - degrading microbial strain s to the media , i.e., bioaugmentation. For stru ctural changes, t hree different nanoclays were used as model systems due to their different surface characteristics but similar chemistry : organo - modified montmorillonite (OMMT), Halloysite nanotubes (HNT), and Laponite ® RD (LRD). Additionally, the organo - modifier of OMMT ( Cloisite ® 30B ) , methyl, tallow, bis - 2 - hydroxyethyl, quaternary ammonium (QAC) was used to investigate its effects on the biodegradation of the polymer . PLA and PLA bio - nanocomposite films (BNC s ) were produced and fully characte rized . Films were tested for biodegradation in simulated composting conditions by analysis of evolved CO 2 with an in - house built direct measurement respirometer. The molecular weight of the films was monitored during the biodegradation tests and correlated with the degradation kinetics. Additionally, a biofilm formation essay and scanning electron microscopy were used to evaluate microbial attachment on the surface of PLA and BNCs. The biodegradation test results showed a higher mineralization and microbial attachment of the films containing nanoclay in comparison to the pristine PLA . However, t he effect of the nanoclays on the initial molecular weight and thickness played a crucial role in the evolution of CO 2 . For bioaugmentation, m icroorganisms present in the compost and capable of degrading PLA were isolated through an enrichment technique with PLA as the sole carbon source at 58°C . The isolates were identified as Geobacillus using 16S rRNA gene sequencing and further used to study the effect of bioaugmen tation on the biodegradation rate of PLA and BNCs in solid environments. The results showed that b ioaugmentation with Geobacillus increased the evolution of CO 2 and accelerated the biodegradation phase of PLA and BNCs when tested in compost and vermiculite inoculated with a compost - derived mixed culture. This work provides the insights gained during the performance of different biodegradation tests and unique understanding about the biodegradation mechanism of PLA . Increasing the biodegradation rate of PLA - based materials will greatly benefit the ir general use and their acceptance in industrial composting facilities at their end of life . Copyright by EDGAR CASTRO AGUIRRE 201 8 v To my parents Roberto and Clementina, brother and sister, Roberto Carlos and Denise vi ACKNOWLEDGMENTS I would like to acknowledge those who without their support, contribution, encouragement and inspiration this dissertation would not have been possible. I would like to express my deep gratitude to Dr. Rafael Auras, my research supervisor, for his patient guidance, enthusiastic encouragement a nd useful critiques during the planning and development of this work. Dr. Auras encouraged me to always give the best of me and taught me that every single problem has a simple solution if we try harder and think differently . I would like to express my ver y great appreciation to Dr. Susan Selke . Her comments and criticisms taught me about critical thinking and reasoning. I would also like to thank Dr. Maria Rubino for her valuable and constructive suggestions. I would like to offer my special thanks to Dr. Terence Marsh for his guidance in microbiology. His willingness to give his time so generously has been very much appreciated. group (RAA group) who are not only partners but fr iends. Special thanks should be given to Fabiola Iniguez - Franco , Javiera Rubilar - Parra, Hayati Samsudin, Pooja Mayekar, Anibal Bher, Sadia Satti, and Wanwarang Limsukon. I would also like to extend my thanks to the undergraduate students: Connor Pettengill , Jason Shaffer, Julia Beaumier, Peter Kieffer, and Austin Barnaby for their invaluable help and time. I am very thankful to my friends who became my Lansing family: Fabiola Iniguez - Franco , Javiera Rubilar - Parra , Hayati Samsudin , Pooja Mayekar , Jiyon Lee , Lisette Delgado - Aquije , Woranit Muangmala , Jin Zhang , Ki kyung Kim, Trey Gase , Rijosh Cheruvathur , Anibal Bher , Ismael Povea - Garcerant , and Felipe Vogelsang for their support, encouragement and inspiration , and for making this journey better. vii I would like to acknowledge the Mexican National Council for Science and Technology (CONACYT), and the Mexican Secretariat of Public Education (SEP) for providing a scholarship . I would like to extend my thanks to the School of Packaging (SoP), and the Center for Packaging Innovation and Sustainability (CPIS) for research funding support , t o the Environmental Science and Policy Program (ESPP) for a summer research fellowship , and to the College of Agriculture and Natur a l Resources (CANR) for providing a dissertatio n completion fellowship. W ithout all th is financial support my Ph. D. studies would not have been completed. I would like to express my gratitude to the faculty from the School of Packaging, their teaching , guidance, and advices have not only helped me und erstand and improve my research, but also inspired me to continue working in the packaging field . I would also like to extend my gratitude to the staff from the School of Packaging for their support and constant willingness to help. Finally, I wish to tha nk my family, especially my parents, Roberto Castro Diaz and Clementina Aguirre Jaimes , and siblings, Roberto Carlos and Denise Castro Aguirre , for their love and continuous support and encouragement, and for believing in me. I have no words to express my gratitude to them. I would also like to extend my gratitude to those who unintentionally I did not mention. viii TABLE OF CONTENTS LIST OF TABL ES ................................ ................................ ................................ .......... x ii LIST OF FIGURES ................................ ................................ ................................ ........ x iv KEY TO SYMBOLS AND ABBREVIATIONS ................................ .............................. x x ii CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ............. 1 1.0 Background and motivation ................................ ................................ .......... 1 1.1 Overall goal and objectives ................................ ................................ .......... 4 1.2 Dissertation overview ................................ ................................ ................... 5 REFERENCES ................................ ................................ ................................ ................ 7 CHAPTER 2 POLY(LACTIC ACID) MASS PRODUCTION, PROCESSING, INDUSTRIAL APPLICATIONS, AND END OF LIFE ................................ ................................ .......... 1 3 2.0 Abstract ................................ ................................ ................................ ...... 1 4 2.1 Introduction ................................ ................................ ................................ . 1 4 2.2 PLA Resin Production ................................ ................................ ................ 1 6 2.3 PLA Processing ................................ ................................ .......................... 2 5 2.3.1 Extrusion ................................ ................................ .............................. 2 6 2.3.2 Injection molding ................................ ................................ .................. 3 0 2.3.3 Injection stretch blow molding ................................ .............................. 3 2 2.3.4 Cast film and sheet ................................ ................................ .............. 3 3 2.3.5 Thermoforming ................................ ................................ .................... 38 2.3.6 Other processes Foaming and fibers ................................ ................ 4 0 2.4 Tailoring PLA Properties ................................ ................................ ............. 4 5 2.5 PLA Industrial Applications ................................ ................................ ......... 5 0 2.5.1 Medical ................................ ................................ ................................ 5 1 2.5.2 Fibers and textiles ................................ ................................ ................ 5 3 2.5.3 Packaging and serviceware ................................ ................................ . 5 6 2.5.4 Plasticulture ................................ ................................ ......................... 6 3 2.5.5 Environmental remediation ................................ ................................ .. 6 6 2.5.6 Other applications ................................ ................................ ................ 68 2.6 PLA Degradation ................................ ................................ ........................ 69 2.6.1 Hydrolysis ................................ ................................ ............................ 7 0 2.6.2 Thermal degradation ................................ ................................ ............ 7 3 2.6.3 Photodegradation ................................ ................................ ................ 77 2.7 End - of - life Scenarios for PLA ................................ ................................ ..... 8 1 2.7.1 Source reduction (reuse) ................................ ................................ ..... 8 3 2.7.2 Recycling ................................ ................................ ............................. 8 4 2.7.3 Composting ................................ ................................ .......................... 8 6 2.7.4 Incineration with energy recovery ................................ ........................ 9 1 ix 2.7.5 Landfill ................................ ................................ ................................ . 9 2 2.8 Environmental Footprint of PLA ................................ ................................ .. 9 3 2.9 Final Remarks ................................ ................................ .......................... 10 3 REFERENCES ................................ ................................ ................................ ........... 1 07 CHAPTER 3 INSIGHTS ON THE AEROBIC BIODEGRADATION OF POLYMERS BY ANALYSIS OF EVOLVED CARBON DIOXIDE IN SIMULATED COMPOSTING CONDITION .......... 1 3 7 3.0 Abstract ................................ ................................ ................................ .... 1 3 8 3.1 Introduction ................................ ................................ ............................... 1 3 9 3.2 Materials and Methods ................................ ................................ ............. 1 5 1 3.2.1 Materials ................................ ................................ ............................ 1 5 1 3.2.1.1 Material processing and characterization ................................ .... 1 5 1 3.2.2 Biodegradation test ................................ ................................ ............ 1 5 2 3.2.2.1 Compost source ................................ ................................ .......... 1 5 3 3.2.2.2 Compost characterization ................................ ........................... 1 5 4 3.2.2.3 Preparation of inoculum solution ................................ ................. 1 5 4 3.2.2.4 Biodegradation in compost ................................ .......................... 1 5 5 3.2.2.5 Biodegradation in vermiculite ................................ ...................... 1 55 3.3 Results and Discussion ................................ ................................ ............ 1 5 6 3.3.1 Biodegradation: CO 2 evolution and mineralization ............................. 1 56 3.3.1.1 Biodegradation in compost ................................ .......................... 1 57 3.3.1.2 Biodegradation in vermiculite ................................ ...................... 1 65 3.3.2 Environment - related factors affecting biodegradation ........................ 1 68 3.3.2.1 Microorganisms ................................ ................................ ........... 1 68 3.3.2.2 Temperature ................................ ................................ ............... 1 70 3.3.2.3 Oxygen availability ................................ ................................ ...... 1 71 3.3.2.4 Water availability ................................ ................................ ......... 1 7 2 3.3.3 Inoculum - related factors affecting biodegradation ............................. 1 75 3.3.3.1 Dry solids and volatile solids ................................ ....................... 1 76 3.3.3.2 pH ................................ ................................ ............................... 1 77 3.3.3.3 C/N ................................ ................................ .............................. 1 77 3.3.3.4 Compost activity ................................ ................................ .......... 1 79 3.3.3.5 Other nutrients ................................ ................................ ............ 1 81 3.3.3.6 Priming effect ................................ ................................ .............. 1 83 3.3.4 Material - related factors affecting biodegradation ............................... 1 84 3.3.4.1 Chemical structure and properties ................................ .............. 1 84 3.3.4.2 Concentration ................................ ................................ .............. 1 88 3.3.4.3 Shape ................................ ................................ .......................... 189 3.3.4.4 Comparison among different biodegradation tests ...................... 190 3.3.5 Study Case: Biodegradation of Poly(lactic acid) ................................ 192 3.4 Final Remarks ................................ ................................ .......................... 197 APPENDICES ................................ ................................ ................................ ............. 2 00 APPENDIX 3A: Material processing ................................ ................................ . 2 01 APPENDIX 3B: Elemental analysis ................................ ................................ .. 2 02 APPENDIX 3C: Molecular weight determination ................................ .............. 2 03 APPENDIX 3D: Compost source ................................ ................................ ...... 2 04 x APPENDIX 3E: Respirometric system ................................ .............................. 2 05 APPENDIX 3F: Calculation method ................................ ................................ .. 2 07 APPENDIX 3G: Determination of the calibration factor, optimal flow and cycle time ................................ ................................ ................................ ................. 2 11 APPENDIX 3H: Compost physicochemical characteristics before and after the test ................................ ................................ ................................ ................. 2 16 APPENDIX 3I: Compost nitrate and ammonium concentration ........................ 2 17 APPENDIX 3J: Summary of the results obtained from the eight different biodegradation tests ................................ ................................ ......................... 2 18 REFERENCES ................................ ................................ ................................ ............ 2 20 CHAPTER 4 IMPACT OF NANOCLAYS ON THE BIODEGRADATION OF POLY(LACTIC ACID) NANOCOMPOSITES ................................ ................................ ................................ .. 2 31 4.0 Abstract ................................ ................................ ................................ .... 2 32 4.1 Introduction ................................ ................................ ............................... 2 33 4.2 Materials and Methods ................................ ................................ ............. 2 37 4.2.1 Materials ................................ ................................ ............................ 2 37 4.2.2 Processing of the PLA bio - nanocomposites ................................ ...... 2 38 4.2.3 Characterization of the PLA bio - nanocomposites .............................. 2 38 4.2.4 Biodegradation evaluation ................................ ................................ . 2 39 4.2.5 Size Exclusion Chromatography (SEC) ................................ ............. 2 41 4.2.6 Microbial attachment ................................ ................................ .......... 2 41 4.2.7 Statistical Analysis ................................ ................................ ............. 2 43 4.3 Results and Discussion ................................ ................................ ............ 2 43 4 .3.1 Characterization of the PLA bio - nanocomposites .............................. 2 43 4.3.2 Biodegradation evaluation ................................ ................................ . 2 46 4.3.3 Molecular Weight ................................ ................................ ............... 2 57 4.3.4 Microbial attachment ................................ ................................ .......... 2 62 4.4 Final Remarks ................................ ................................ .......................... 2 66 APPENDICES ................................ ................................ ................................ ............. 2 68 APPENDIX 4A: Material processing ................................ ................................ . 2 69 APPENDIX 4B: Material characterization ................................ ......................... 2 71 APPENDIX 4C: Physicochemical characteristics of the compost ..................... 2 76 APPENDIX 4D: Molecular weight determination ................................ .............. 2 77 APPENDIX 4E: Biofilm formation ................................ ................................ ..... 284 REFERENCES ................................ ................................ ................................ ............ 287 CHAPTER 5 ENHANCING THE BIODEGRADATION RATE OF POLY(LACTIC ACID) FILMS AND PLA BIO - NANOCOMPOSITES IN SIMULATED COMPOSTING THROUGH BIOAUGMENTATION ................................ ................................ ................................ . 295 5.0 Abstract ................................ ................................ ................................ .... 296 5.1 Introduction ................................ ................................ ............................... 297 5.2 Materials and Methods ................................ ................................ ............. 299 5.2.1 Materials ................................ ................................ ............................ 299 5.2.1.1 Material processing and characterization ................................ .... 299 xi 5.2.2 Isolation of PLA - degrading microbial strain ................................ ........ 3 00 5.2.3 Identification of PLA - degrading microbial strain ................................ . 3 01 5.2.4 Biodegradation evaluation ................................ ................................ . 3 02 5.2.4.1 Preparation of the compost and vermiculite ................................ 3 02 5.2.4.2 Bioreactor setup ................................ ................................ .......... 3 03 5.2.4.3 Bioaugmentation ................................ ................................ ......... 3 04 5.2.5 Biofilm formation ................................ ................................ ................ 3 04 5.2.6 Size Exclusion Chromatography (SEC) ................................ ............. 3 05 5.2.7 Statistical Analysis ................................ ................................ ............. 3 05 5.3 Results and Discussion ................................ ................................ ............ 3 05 5.3.1 Isolation and identification of PLA - degrading bacteria ....................... 3 05 5.3.2 Biodegradation Test ................................ ................................ ........... 3 07 5.3.3 Molecular Weight ................................ ................................ ............... 3 12 5.3.4 Biofilm test ................................ ................................ ......................... 3 15 5.4 Final Remarks ................................ ................................ .......................... 3 18 APPENDICES ................................ ................................ ................................ ............. 3 20 APPENDIX 5A: Compost and vermiculite nutrient analysis .............................. 3 21 APPENDIX 5B: Molecular weight ................................ ................................ ..... 3 22 APPENDIX 5C: Biofilm Test ................................ ................................ ............. 3 24 REFERENCES ................................ ................................ ................................ ............ 3 25 CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS ................................ ........................... 3 3 1 6.0 Conclusions ................................ ................................ .............................. 3 3 1 6.1 Recommendations ................................ ................................ .................... 3 3 4 xii LIST OF TABLES Table 2.1 Properties and processing temperatures of selected commercially available Ingeo TM PLA resins ................................ ................................ ................................ ....... 29 Table 2.2 Selected average optical, physical, mechanical, and barrier properties of PLA films reported from a number of studies using different grades of PLA, adapted and modified from Auras [49]. ................................ ................................ ............................. 3 6 Table 2.3 Selected examples of packaging containers produced fro m PLA ................. 6 0 Table 2.4 General requirements to test biodegradation under laboratory conditions and comparison between ASTM D5338 and ISO 14855 standards [334, 335], reproduced from Castro - Aguirre, E. [336]. ................................ ................................ ....................... 89 Table 2.5 Environmental footprint of 1 kg of selected commercial polymer resins as available in Ecoinvent 3.2 and reported using Simapro 8.0.5 with ReCiPe (E) Midpoint Indicator considering the World as the geographical region Table 3.1 Selected biode gradation tests in composting conditions reported in the literature and presented in reverse chronological order for 2015 through 1990, including information about the samples, compost and the main methods for assessing biodegradation. ................................ ................................ ................................ ........... 1 4 2 Table 3.2 Initial M n , M w , and PI of the PLA samples ................................ ................... 1 5 2 Table 3.3 Biodegradation test, materials, and media used for testing ........................ 1 5 3 Table 3.4 Detailed composition of 1 L of mineral solution ................................ ........... 1 5 5 Table 3.5 Characteristics of the compost samples for each test and require ments according to ISO 14855 standard ................................ ................................ ................ 17 6 Table 3.6 Physicochemical parameters and total nutrient analysis of different media used in the Nov15 test ................................ ................................ ................................ ......... 1 83 Table 3A.1 Temperature profile and screw speed used for the production of the PLA films ................................ ................................ ................................ ................................ .... 2 01 Table 3B.1 Carbon, hydrogen, and nitrogen content of the tested materials .............. 2 02 Table 3H.1 Physicochemical characteristics of the compost from the Feb13 and the Nov14 tests determined before and after the test ................................ ....................... 2 16 Table 3J.1 Summary of the results obtained from the eight different biodegradation tests ................................ ................................ ................................ ................................ .... 2 18 xiii Table 4.1 Key for biodegradation test and labels of the samples ................................ 2 40 Table 4A.1 Processing conditions of the sample materials ................................ ......... 2 70 Table 4B.1 Carbon, hydrogen, and nitrogen content of the tested materials .............. 2 71 Table 4B.2 Thermal properties of the PLA and BNCs ................................ ................ 2 73 Table 4B.3 Resisitivity of the PLA and BNCs ................................ ............................. 2 74 Table 4B.4 Contact angle of the PLA and BNCs measured with water at room temperature ................................ ................................ ................................ ................. 2 75 Table 4C.1 Physicochemical characteristics of the compost used in the different biodegradation tests ................................ ................................ ................................ .... 2 76 Table 4D.1 Initial M n , M w , and PDI of the PLA samples ................................ .............. 2 78 Table 4D.2 Initial molecular weight and reduction rate of PLA and BNCs as estimated by the first order reaction of the form M n = M n0 exp( - kt ) ................................ ................... 2 83 Table 4E.1 Absorbance (600 nm) of a) PA at 23°C first iteration ................................ 285 Table 4E.2 Absorbance (600 nm) of a) CE at 58°C first iteration ................................ 286 Table 4E.3 Absorbance (600 nm) of PA at 23°C during the biofilm test ..................... 286 Table 4E.4 Absorbance (600 nm) of CE at 58°C during the biofilm test ..................... 286 Table 5.1 Carbon, hydrogen, and nitrogen content of the tested materials ................. 3 00 Table 5.2 Initial physicochemical parameters of the compost and vermiculite used for biodegradation tests ................................ ................................ ................................ .... 3 03 Table 5.3 Identification of the microbial isolates using the MSU - RDP Sequence Match and the NCBI database. ................................ ................................ .............................. 3 07 Table 5.4 Absorbance (600 nm) of biofilm for mation samples with Pseudomonas aeruginosa (PA) at 23°C ................................ ................................ ............................. 3 16 Table 5.5 Absorbance (600 nm) of biofilm formation samples with Geobacillus at 58°C ................................ ................................ ................................ ................................ .... 3 17 Table 5A.1 Physicochemical parameters and total nutrient analysis of different media used in the biodegradation test ................................ ................................ ................... 3 21 Table 5B.1 Molecular weight reduction rate of PLA and PLA - OMMT5 in vermiculite with different levels of inoculation as estimated by the first ord er reaction of the form M n / M n0 = exp( - kt ). ................................ ................................ ................................ ....................... 3 22 xiv LIST OF FIGURES Figure 2.1 Number of research reports published since 1990 based on the Web of Science search using keywords "PLA", "PLLA", "PDLA", "polylactic acid", "polylactide", ................................ ................................ .............................. 1 5 Figure 2.2 Chemical structure of L(+) and D( - ) lactic acid. ................................ ........... 1 7 Figur e 2.3 The manufacturing processes to produce high molecular weight PLA, adapted from Hartmann [17]. ................................ ................................ ................................ ...... 19 Figure 2.4 NatureWorks LLC commercial process for producing high molecular weight PLA, adapted from Auras et al. [2] and Vink et al. [5]. ................................ ................... 2 1 Figure 2.5 D iastereomeric structures of lactide (3,6 - dimethyl - 1,4 - dioxane - 2,5 - dione). T m of L - lactide, D - lactide, meso - lactide, and rac - lactide are 96, 96 97, 53, and 125°C, respectively, adapted from Vert et al. [18]. ................................ ................................ .... 2 2 Figure 2.6 Major components of an injection mo lding machine showing the extruder et al ., Processing technologies for poly (lactic acid), 820 - 852, Copyright (2008), with [1]. ................................ ................................ ........................ 3 1 Figure 2.7 in Polymer Science, 33, Lim et al. , Processing technologies for poly (lactic acid), 820 - 852, ................................ .................... 3 3 Figure 2.8 Biaxial oriented Progress in Polymer Science, 33, Lim et al. , Processing technologies for poly (lactic acid), 820 - ................................ ..... 3 5 Figure 2.9 Production of a thermoformi Polymer Science, 33, Lim et al. , Processing technologies for poly (lactic acid), 820 - 852, ................................ .................... 39 Figure 2.10 Schematic of microcellular foaming process: a) ba tch process, b) continuous process; 1 to 6 are the main regions of the extruder; adapted from Matuana [64]. ....... 4 2 Figure 2.11 Schematic representation of melt spinning setup: (1) extruder drive, (2) single - extruder - 24 to 36:1 L/D ratio, (3) hopper, (4) scre w, (5) manifold, (6) static mixer, (7) metering pump, (8) metering pump drive, (9) spin pack, (10) mesh filters, (11) distributor, (12) spinneret, (13) cross - flow quench chamber, (14) freshly spun yarn, (15) godet, (16) idler roller, (17) friction - driven w inder, (18) yarn bobbin, adapted from Agrawal [67]. ................................ ................................ ................................ ............................... 4 5 xv Figure 2.12 Selected biodegradable and non - biodegradable blends of PLA polymers: PLA - LDPE [89], PLA - LLDPE [90], PLLA - LLDPE [90 - 92], PLLA - HDPE [91], PLA - PS [93], PLLA - PEVA [94], PLLA - EVOH [95], PLA - TPO [96], PLLA - ABS [97], PLLA - PIP [98], PLA - PVOH [99], PLA - PHB [100] PLLA - PBS [101, 102], PLA - PBSA [103], PLA - PBAT [104, 105], PLLA - PTAT [106], PLA - PAE [107], PLA - PU [108], PLA - PEG [109], PLA - SPI [110], PLA - SPC [110], PLA - SF [111], PLA - TKGM [112], PLA - Chitosan [83, 113], PDLLA - Chitosan [114], PLLA - Chitosan [114], PLLA - PBSL [115], PLLA - PEO [116], PLLA - PCL [117 - 119], PDLLA - PCL [117], PLA - PCL [120], PLA - Starch [121 - 124], PLA - PHBHxx [125], PLA - PPC [126], PLA - PP [127], PLA - PC [128], PLA - PGS [129], PLA - PTT [130], and PLA - EGMA [131]. ................................ ................................ ................................ .................. 4 7 Figure 2.13 Average scale dimensions of selected fillers in PLA composites: MMT (montmorillonite) [168], CNT (carbon nanotube) [169], Ag [170], sisal [171], wood [168, 172, 173], MCC (microcrystalline cellulose) [174], sepiolite [168], cotton [175, 176], ramie [177, 178], MWCNT (multiwall carbon nanotube), graphene, algal [179, 180], tunicin [181], halloysite [148], talc3 [182], CaCO 3 [183], talc2 [182], CaSO 4 [184], talc1 [182], glass fiber [185, 186], abaca fiber [187], jute fiber [187 ], cotton fiber [187], hemp fiber [187], and flax fiber [186, 188]. ................................ ................................ ...................... 5 0 Figure 2.14 (left) tomato plots covered with mulch films; (right) high tunnel or overwintering house. ................................ ................................ ................................ ..... 6 5 Figure 2.15 Cradle to gate, grave, and cradle life cycle flowchart of plastic mu lch films. After removal of conventional mulch films, they can be reused, recycled, incinerated, and/or landfilled. Biodegradable mulch provides the same end - of - life scenario routes and also can be composted. ................................ ................................ ................................ 6 6 Figure 2.16 Hydrolytic chain cleavage mechan isms of PLA in alkaline (a) and acidic (b) et al. , New insights into the hydrolytic degradation of poly(lactic acid): participation of the alcohol terminus, 2795 - 2802, Copyright (2001), with permission from Elsevier ................................ ................ 7 2 Figure 2.17 Ln ( M n ) as a function of time during hydrolysis of PLA films into water, 95% ethanol, or 50% ethanol at 40°C. ................................ ................................ .................. 7 3 Figure 2.18 Synthesis, structure, properties, processing, and applications, Nishida, Thermal degradation, 401 - 412, Copyright (2010), with permission from John Wiley & Sons, Inc." [286]. ................................ ................................ ................................ ............................. 7 5 Figur e 2.19 Photodegradation of PLA via Norish II mechanism, adapted from Tsuji et al. [302]. ................................ ................................ ................................ ............................. 78 Figure 2.20 Mechanisms of photodegradation of PLA, adapted from Janokar et al. [303]. ................................ ................................ ................................ ................................ ...... 79 Figure 2.21 Radicals generated during photodegradation of PLA from cleavage of C - C bonds, adapted from Copinet et al. [263]. ................................ ................................ ..... 8 0 xvi Figure 2.22 Radicals generated during photodegradation of PLA (a) from C - O and (b) ester bond cleavage, adapted from Copinet et al. [263]. ................................ ............... 8 0 Figure 2.23 Diagram of solid waste management, adapted from the U.S. Environmental Protection Agency in Advancing Sustainable Materials Management [314]. ................. 8 2 Figure 2.24 Large - International, 57, Kijchavengkul et al. , Compostability of polymers, 793 - 804, Copyright ................................ ................................ .... 87 Figure 2.25 Schematic of polymer biodegradation mechanism, adapted from Leejarkpai et al. [332]. ................................ ................................ ................................ .................... 88 Figure 2.26 a) Amount of CO 2 evolved from blank, cellulose, and PLA film; b) Percentage mineralizati on of cellulose and PLA film. ................................ ................................ ....... 9 0 Figure 2.27 Calorific values of selected materials, adapted from et al. [340]. ................................ ................................ ................................ ................................ ...... 9 2 Figure 2.28 Cradle - to - gate, cradle - to - grave, and cradle - to - cradle representations of production, consumption, and disposal of bio - based polymers from renewable resource et al. , Compostability of polymers, 793 - 804, Copyright (2008), with permission from Wiley" [329]. ................................ ................................ ................................ ............................. 9 4 Figure 2.29 Carbon cycle of fossil - based polymers and bio - based polymers. Renewable resource pathway (green arrows); fossil resource pathway (black arrows); and pathway for both re newable and fossil resources (gray arrow), adapted from Kijchavengkul et al. [329]. ................................ ................................ ................................ ............................. 96 Figure 2.30 GWP, primary energy of non - renewable resources expressed as higher heating values (HHV), and net water uptake for the production system of Ingeo TM resin, ada pted from Vink and Davies [5]. ................................ ................................ ................ 97 Figure 2.31 Climate change, non - renewable energy, water depletion for 1 kg of PLA and other commercial polymers as available in Ecoinvent 3.2 and reported using Simapro 8.0.5 with Recipe (E) Midpoint Indicator considering the World as the geographical region, and Ingeo TM adapted from Vink and Davies [5]. ................................ ................ 99 Figure 2.32 PLA life cycle and boundary of the studied system. Adapted from Rossi et al. [315]. ................................ ................................ ................................ ........................... 10 1 Figure 2.33 Comparison of dynamically - assessed global warming impacts over 100 years associated with the six end - of - life treatments for PLA. The bars on the left side present production impacts of the resin (cradle - to - gate) for comparison purposes, adapted from Rossi et al. [315]. ................................ ................................ .................. 10 2 xvii Figure 2.34 Comparison of end - of - life options for PLA for each midpoint category, adapted from Rossi et al. [315] 10 3 Figure 3.1 Example of biodegradation test parameters as a function of time. Fitted lines ( ) are included for visual guidance only. Air flow rate, temperature, moisture and pH monitored as a function of time in the May13 test during the first 60 days of testing . ................................ ................................ ................................ ................................ .... 1 5 7 Figure 3.2 Cumulative CO 2 evolution of blank bioreactors in the different biodegradation tests showing large variation of the CO 2 evolved although they were run under the same experimental conditions. ................................ ................................ .............................. 1 58 Figure 3.3 Cumulative CO 2 evolution (a) and mineralization (b) of cellulose bioreactors in the different biodegradation tests . While similar or different CO 2 values were observed, the % mineralization is highly driven by the evolved CO 2 values for the blank test. .... 1 59 Figure 3.4 Cumulative CO 2 evolution (a) and mineralization (b) of CS in the different biodegradation tests. Biode gradation tests show a fast increase in the mineralization during the first 10 days of testing. ................................ ................................ ............... 1 60 Figure 3.5 Cumulative CO 2 evolution and mineralization of LDPE (a & b) and PE (c & d) bioreactors in different biodegradation tests. Negative values of mineralization are observed in many tests. ................................ ................................ .............................. 1 63 Figure 3.6 Cumulative CO 2 evolution (a) and mineralization (b) of PLA bioreactors in the different biodegradation tests; solid, dashed and dotted lines represent PLA1 (93.5 kDa), PLA2 (82.9 kDa), and PLA3 (72.6 kDa), respectively. ................................ ................. 1 6 4 Figure 3.7 Cumulative CO 2 evolution and mineralization of CP and PLA tested in inoculated vermiculite i n the Jan14 test. Lower values of evolved CO 2 are seen when compared with compost tests, as expected. ................................ ................................ 1 66 Figure 3.8 Cumulative CO 2 (a) and mineralization (b) of CP, PLA1, PLA2, and PLA3 in the Nov15 test. Solid line, dashed line, and dotted line represent compost, inoculated vermiculite and uninoculated vermiculite, respectively. Large difference in CO 2 production can be observed between evolved CO 2 in inoculated and uninoculated vermiculite. .. 1 67 Figure 3.9 Cumulative CO 2 of blank and cellulose and % miner alization of cellulose of two different tests Sep12 test (a & b) and Nov15 test (c & d), respectively. The biodegradation process of cellulose was more homogeneous and more efficient in the test in which water was added twice a week seeing as a high % mine ralization in a short period of time. ................................ ................................ ................................ ............. 1 73 Figure 3.10 C/N of the compost as a function of time during the Nov14 test. The fitted line ( ) is included for visual guidance only. ................................ ...................... 1 79 xviii Figure 3.11 Microbial activity of the compost measured as the production of CO 2 per gram of VS. Variation between 30 and 80 mg of CO 2 per gram of VS is seeing at 10 d. ................................ ................................ ................................ ................................ .... 1 80 Figure 3.12 Cumulative CO 2 evolution (a) and mineralization (b) of CP, CS, and GC in the Jan14 test. Mineralization values larger than 100% are observed for GC. ............ 1 84 Figure 3.13 CO 2 evolution (a) and mineralization (b) of different materials in the Jun14 test. Large difference of % Mineralization is observed for the different materials. ....... 1 85 Figure 3. 14 Cumulative CO 2 (a) and mineralization (b) of CP, PLA2, and PLA4 in the Nov14 test. PLA 4032D shows faster and larger mineralization than PLA 2003D. ..... 1 87 Figure 3.15 Cumulative CO 2 and % mineralization of CP and PLA pellets with two different concentratio ns: 5% and 15%, in the Jan14 test. % Mineralization was not affected regardless of the initial amount of PLA. ................................ ......................... 189 F igure 3.16 Cumulative CO 2 evolution (a) and mineralization (b) of CP and of PLA provided in different forms: pellet and film, in the Jan14 test. % Mineralization was not extensively different regardless of the shape of the material. ................................ ...... 190 Figure 3.17 Comparison of the mineralization values obtained for PLA1 in the Jun14 and the Nov15 tests (a), and the mineralization values obtained for PLA2 in the Nov14 and the Nov15 tests (b). The mineralization ratio when adjusting the time span of the test seems t o be similar when comparing the same test material ................................ ...... 191 Figure 3.18 Biodegradation of CP (a) and PLA2 (b) during the Nov14 test. The black, red, blue, and green lines represent cumulative CO 2 , mineralization, evolved CO 2 per measurement, and M n reduction, r espectively. The dashed blue line represents the evolved CO 2 per measurement of the blank bioreactors. The green line indicates a fitting of an equation of the form M n = M n0 exp ( - kt), where M n0 is the initial M n , k is the rate constant and t is the time. T he black dash - dot lines are used as reference to indicate the beginning and end of the biodegradation phase, and the M n at which the biodegradation phase gets started. Different lag phases and biodegradation phases were observed for CP and PLA2. ................................ ................................ ................................ ............. 192 Figure 3.19 Cumulative CO 2 (a) and mineralization (b) of CP, PLA1, PLA2, and PLA3 in the Nov15 test. Solid lines and dotted lines represent inoculated vermiculite and uninoculated vermiculite, respectively. ................................ ................................ ........ 195 Figure 3.20 Molecular weight reduction as a f unction of time for PLA1, PLA2, and PLA3 in compost (solid line), inoculated vermiculite (dashed line), and uninoculated vermiculite (dotted line), in the Nov15 test. Lines indicate fitting of a first order reaction of the form M n = M n0 exp ( - kt ), where M n0 is the initial M n , k is the rate constant and t is the time .. 196 Figure 3E.1 Schematic diagram of a direct measurement respirometric system, reproduced from Castro - Aguirre, E. [40]. CO 2 from the incoming air is scrubbed by passing through a series of canisters containing soda lime. This CO 2 - free air enters a xix water tank, located inside the environmental chamber at 58 °C , to get humidified; then, CO 2 - free water - saturated air is provided to the bioreactors with an upward flow direction. The respired air st ream exits the bioreactors and the environmental chamber passing through a water trap, a mass flow controller (MFC) and a NDIR - CO2 sensor for CO 2 concentration measurement. Temperature, relative humidity (RH), air flow rate, time and CO 2 concentration are measured and recorded by a data acquisition system (DAS). 2 06 Figure 3F.1 Time vs. Concentration Plot , reproduced from Castro - Aguirre, E. [40] ... 2 09 Figure 3G.1 Calibration curve at 58 ± 2°C and 55 ± 5% RH. A linear relationship of the form [C]= c*k was fitted to the data, where [C] is the actual CO 2 concentration, c the response CO 2 concentration as measured by the NDIR analyzer, and k the calibration factor. ................................ ................................ ................................ .......................... 2 12 Figure 3G.2 Response CO 2 concentration obtained when using different injection volumes and air flow rates. The maximum concentration can be achieved in each case regardless the air flow rate used. Fitted lines of the form are included for visual guidance only, with = 0, and = 507, 2535, 5070, and 10140, corresponding to the differen t injection volumes used. ................................ ................................ ................. 2 13 Figure 3G.3 Time to reach the maximum CO 2 concentration when using different air flow rates. The longest time to reach the maximum concentration was observed when using the lowest air flow rate. ................................ ................................ ................................ 2 14 Figure 3G.4 Response concen tration and time required for a selected bioreactor to reach the peak concentration for different injection volumes and air flow rates. The longest time was observed with the highest CO 2 concentration and the lowest air flow rate. .......... 2 15 Figure 3I.1 Concentration of NO 3 - (left - black axis) and NH 4 + (right - red axis) as a function of time of the compost in blank bioreactors (a) and CP bioreactors (b) during the Nov14 test. ................................ ................................ ................................ ............................. 2 17 Figure 4.1 XRD spectra of the different nanoclays, PLA1, and (a) OMMT, (b) HN T, and (c) LRD bio - nanocomposite films. ................................ ................................ ............... 2 44 Figure 4.2 TEM micrographs of (a) PLA - OMMT5, (b) PLA - HNT5, and c) PLA - LRD5 bio - ........................ 2 44 Figure 4.3 CO 2 evolution of the three different nanoclays (Test I in compost) ............ 2 47 Figure 4.4 (a) CO 2 evolution and (b) % Mineralization of PLA and PLA - OMMT5 films (Test I in compost) ................................ ................................ ................................ ....... 2 49 Figure 4.5 (a) CO 2 evolution and (b) % Mineralization of PLA and PLA - OMMT films wit h three different levels of loading (1, 5, and 7.5%) (Test II in compost) ......................... 2 51 Figure 4.6 CO 2 evolution of OMMT nanoclay and QAC surfactant (Test II in compost) ................................ ................................ ................................ ................................ .... 2 52 xx Figure 4.7 CO 2 evolution and % Mineralization of PLA - OMMT films (a & b) and PLA - QAC films (c & d) (Test III in compost) ................................ ................................ ................. 2 53 Figure 4.8 (a) CO 2 evolution and (b) % Mineralization of PLA - HNT films (Test III in compost) ................................ ................................ ................................ ..................... 2 54 Figure 4.9 (a) CO 2 evolution and (b) % Mineralization of PLA - LRD films (Test III in compost) ................................ ................................ ................................ ..................... 2 55 Figure 4.10 (a) CO 2 evolution and (b) % Mineralization of PLA and PLA - OMMT5 films (Test IV in compost) ................................ ................................ ................................ .... 2 56 Figure 4.11 (a) CO 2 evolution and (b) % Mineralization of PLA, PLA - OMMT5, and PLA - QAC0.4 (Test IV in inoculated vermiculite (dashed lines) and uninoculated vermiculite (dotted lines)) ................................ ................................ ................................ .............. 2 57 Figure 4.12 Initial molecular weight of PLA and BNCs ................................ ............... 2 58 Figure 4.13 Change in molecular weight of PLA2 film (Test III in compost) ............... 2 59 Figure 4.14 Change in molecular weight of (a) PLA2, (b) PLA - OMMT1, (c) PLA - OMMT5, (d) PLA - QAC0.4, (e) PLA - QAC1.5, (f) PLA - HNT1, (g) PLA - HNT5, (h) PLA - LRD1, and (i) PLA - LRD5 films (Test III in compost). ................................ ................................ ......... 26 1 Figure 4.15 Absorbance (600 nm) of (a) PA at 23°C, and (b) CE at 58°C for second biofilm test. Columns with the same letter within a group ( i.e., wells , films, or total) are not ................................ ................................ 2 65 Figure 4.16 SEM micrographs of (a) PLA and (b) PLA - LRD at 1000x before incubation, (c) PLA and (d) PLA - LRD5 after incubation for 48 h at 58°C with compost extract in R2B. ................................ ................................ ................................ ................................ .... 2 66 Figure 4B.1 DSC of the PLA and BNCs films (1 st cycle) ................................ ............ 2 72 Figure 4B.2 TGA of the PLA and PLA - OMMT films ................................ .................... 2 72 Figure 4B.3 Moisture sorption isotherms of the nanoclays, PLA and BNCs films ...... 2 73 Figure 4D.1 Deconvolution of the PLA2 peaks at days (a) 7, (b) 14, (c) 21, and (d) 28 (Test III in compost) ................................ ................................ ................................ ..... 2 80 Figure 4D.2 (a) M n and (b) area fraction as function of time for PLA2 film (Test III in compost) ................................ ................................ ................................ ..................... 2 81 Figure 4D.3 Molecular weight reduction as function of time for PLA2 and (a) PLA - OMMT, (b) PLA - QAC, (c) PLA - HNT, and (d) PLA - LRD films (Test III in compost). Dashed lines indicate fitting of a first order reaction of the form M n / M n0 = exp ( - kt ), where M n0 is the initial M n , k is the rate constant and t is the time. ................................ ................................ .. 28 2 xxi Figure 4E.1 Absorbance (600 nm) of (a) PA at 23°C, and (b) CE at 58°C first iteration ................................ ................................ ................................ ................................ .... 284 Figure 5.1 (a) CO 2 evolution and (b) % Mineralization of cellulose, PLA, and PLA - OMMT5, and PLA - QAC0.4 in compost (solid lines), inoculated vermiculite with mixed culture (dashed lines), and uninoculated vermiculite (dotted lines); adapted from Castro - Aguirre et al. [3]. ................................ ................................ ................................ .......... 3 0 8 Figure 5.2 (a) CO 2 evolution and (b) % Mineralization of cellulose, PLA, and PLA - OMMT5 in compost without Geobacillus (solid lines) and with Geobacillus (dashed lines). ..... 3 09 Figure 5.3 % Mineralization of (a) Cellulose, (b) PLA, and (c) PLA - OMMT5 in vermi culite with different levels of inoculation. ................................ ................................ ............... 3 11 Figure 5.4 % Mineralization of cellulose, PLA, and PLA - OMMT5 in (a) compost (same as Figure 5.2b), (b) vermiculite inoculated with mixed culture, and (c) uninoculated vermiculite. Solid lines represent sampl es without Geobacillus while dashed lines represent samples inoculated with Geobacillus . ................................ ......................... 3 11 Figure 5.5 Molecular weight reduction as function of time for (a) PLA and (b) PLA - OMMT5 in vermiculite with different levels of inoculation. Lines represent the fi tting of the equation M n = M n0 exp ( - kt ), where M n0 is the initial M n , k is the rate constant and t is the time. 3 12 Figure 5.6 MWD of PLA samples in vermiculite with different levels of inoculation (a) uninoculated, (b) Geobacillus only, (c) mixed culture, (d) mixed culture and Geobacillus . ................................ ................................ ................................ ................................ .... 3 15 Figure 5B.1 MWD of PLA - OMMT5 samples in vermiculite with different levels of inoculation (a) uninoculated, (b) Geobacillus only, (c) mixed culture, (d) mixed culture and Geobacillus. ................................ ................................ ................................ ................ 3 23 Figure 5C.1 Absorbance (600 nm) of (a) PA at 23 ° C, and (b) Geobacillus at 58 ° C of biofilm formation samples. Bars with the same letter within a group ( i.e. , wells, films, or - Kramer test. .... 3 24 Figure 5C.2 Absorbance (600 nm) of (a) PA at 23 ° C, and (b) mixed culture at 58 ° C for biofilm test. Bars with the same letter within a group ( i.e. , wells, films, or total) are not - Kramer test). Adapted f rom Castro - Aguirre et al. [1]. ................................ ................................ ................................ ............................... 3 24 xxii KEY TO SYMBOLS AND ABBREVIATIONS A area under the curve in equation 3F.4 AATCC American Association of Textile Chemists and Colorists ABS acrylonitrile butadiene styrene AD anaerobic digestion A film surface area of the film ANOVA analysis of variance APR Association of Postconsumer Plastic Recyclers ASTM American Society for Testing and Materials ATBC acetyl - tri - n - butyl citrate Biopol poly(hydroxy butyrate)/poly(hydroxy valerate) blend BNC bio - nanocomposite C compost C concentration of CO 2 evolved during the measurement interval in equation 3F.3 c response concentration of CO 2 as measured by the NDIR analyzer in equation 3F.1 (CO 2 ) B average cumulative mass of CO 2 evolved from the blank in equation 3F.7 (CO 2 ) T average cumulative mass of CO 2 evolved from the sample in equation 3F.7 [C] actual concentration of CO 2 of each sample in equation 3F.1 [C] n concentration of CO 2 at time t n in equation 3F.4 [C] n - 1 con centration of CO 2 at time t n - 1 in equation 3F.4 C(CO 2 ) cumulative mass of CO 2 in equation 3F.5 xxiii C(CO 2 ) H cumulative mass of CO 2 at time t H in equation 3F.6 C(CO 2 ) L cumulative mass of CO 2 at time t L in equation 3F.6 C(CO 2 ) n - 1 cumulative mass of CO 2 until time t n - 1 in equation 3F.5 C/N carbon - nitrogen ratio CA cellulose acetate CAB cellulose acetate butyrate CCD charge - coupled device CD complete disintegration CE compost extract CFA chemical foaming agent CHN elemental analysis CMR cumulative measurement respirometry CNT carbon nanotube CP cellulose powder CS cassava starch C TOT proportion of total organic carbon in the total mass of test material in equation 3F.7 DAS data acquisition system DFS direct fuel substitution D - LA D - Lactic acid DMR direct measurement respirometry DNA deoxyribonucleic acid DS dry solids DSC differential scanning calorimetry E(CO 2 ) mass of evolved carbon dioxide in equation 3F.3 xxiv E(CO 2 ) n mass of CO 2 evolved from the sample at time t n in equat ion 3F.5 E act average energy of activation EFP environmental footprint EG ethylene glycol EGMA poly(ethylene - glycidyl methacrylate) EIS electrochemical impedance spectroscopy EPA Environmental Protection Agency EPS exopolymeric substances Eq. equation eq. equivalent ESIMS electrospray ionization - mass spectrometry ESR electron spin resonance EU European Union EVA e thylene vinyl acetate EVOH ethylene vinyl alcohol F air flow rate in equation 3F.3 FDA Food and Drug Administration GC gas chromatography GC glycerol GHG greenhouse gas GMR gravimetric measurement respirometry GPC gel permeation chromatography GRAS generally recognized as safe GWP global warming potential xxv HA hydroxyapatite HDPE high - density polyethylene HDT heat deflection temperature HHV higher heating values HNT halloysite nanotubes HPLC high - performance liquid chromatography HRC hydrogen release compound I(CO 2 ) interpolated cumulative mass of CO 2 at time t I in equation 3F.6 IC industrial composting IR infrared ISBM injection stretch blow molding ISO International Organization for Standardization IV inoculated vermiculite k rate constant k calibration factor in equation 3F.1 L/D ratio of flight length of the screw to its outer diameter LA lactic acid LCA life cycle assessment LDPE low - density polyethylene LF landfilling L - LA L - Lactic acid LLDPE linear low - density polyethylene LRD Laponite ® RD MA malonic acid xxvi MA maleic anhydride MCC microcrystalline cellulose MD machine direction MFC mass flow controller MFI melt flow index MFR melt flow rate MIC minimum inhibitory concentration % Mineralization percent carbon molecules converted to CO 2 in equation 3F.7 MMT montmorillonite M n number average molecular weight M n0 initial number average molecular weight MR mechanical recycling MSU Michigan State University MSW municipal solid waste MSWI municipal solid waste incineration M TOT mass of test material in equation 3F.7 M w weight average molecular weight MWCNT multi - wall carbon nanotube MWD molecular weight distribution NAPCOR National Association for PET Container Resources NCBI National Center for Biotechnology Information NCI non - inhibitory concentration NDIR non - dispersive infrared gas analyzer OM organic matter xxvii OMMT organo - modified montmorillonite PA polyamide PA Pseudomonas aeruginosa PAE polyamide elastomer PBAT poly(butylene adipate - co - terephthalate) PBS polybutylene succinate PBSA poly(butylene succinate - co - adipate) PBSL poly(butylene succinate - co - L - lactate) PC polycarbonate PCDI polycarbodiimide PCL poly( - caprolactone) PCR post - consumer recycled PDI polydispersity index PDLA poly(D - lactic acid) PDLLA poly(D,L - lactic acid) PE polyethylene PEG poly(ethylene glycol) PENNR primary energy from nonrenewable resources PEO poly(ethylene oxide) PET poly(ethylene terephthalate) PEVA poly(ethylene - co - vinyl acetate) PFA physical foaming agent PGA polyglycolic acid PGS poly(glycerol sebacate) pH potential of hydrogen xxviii PHAs poly( hydroxyalkanoates) PHB poly(hydroxybutyrate) PHBHxx poly[(3 - hydroxybutyrate) - co - (3 - hydroxyhexanoate)] PHBV poly(3 - hydroxybutyrate - co - 3 - hydroxyvalerate) PI polydispersity index PIP poly( cis - 1,4 - isoprene) PLA poly(lactic acid) PLLA poly(L - lactic acid) PP polypropylene PPC poly(propylene carbonate) ppm parts per million PS polystyrene PTAT poly(tetramethylene adipate - co - terephthalate) PTFE polytetrafluoroethylene PTT poly(trimentylene terephthalate) PU poly(ether)urethane PVC polyvinyl chloride PVOH polyvinyl alcohol QAC Tomamine TM (methyl, tallow, bis - 2 - hydroxyethyl, quaternary ammonium) R2B R2 broth RDP Ribosomal Database Project RH relative humidity RIC resin identification code ROP ring - opening polymerization xxix rRNA ribosomal ribonucleic acid RTSF Research Technology Support Facility SA succinic acid sccm standard cubic centimeters per minute SCE sterile compost extract SCORIM shear - controlled orientation in injection molding SEC size exclusion chromatography SEM scanning electron microscopy SF silk fibroin SIC solvent induced crystallization SoP School of Packaging SPC soy protein concentrate SPI soy protein isolate STP standard temperature and pressure t time T measurement interval in equation 3F.3 TCD thermal conductivity detector T d de composition temperature T d,0 initial decomposition temperature T d,1/2 half decomposition temperature TEM transmission electron microscopy T g glass transition temperature TGA thermal gravimetric analysis t H immediate higher value of the time interval in equation 3F.6 xxx THF tetrahydrofuran t i time at which the measurement was taken t I time interval in equation 3F.6 TKGM thermoplastic konjac glucomannan t L immediate lower value of the time interval in equation3F.6 T m melting temperature T mc melt crystallization temperature t n time at which each measurement was done in equation 3F.2 t n - 1 time in which the previous measurement was done in equation 3F.4 TNPP tris(nonylphenyl) phosphite TOC total organic carbon TPDAS thermoplastic dialdehyde starch TPO thermoplastic polyolefin elastomer TPS thermoplastic starch T - RFLP terminal restriction fragment length polymorphism t sn time stamp at time t n in equation 3F.2 t so time stamp at time to corresponding to the time at which the experiment started in equation 3F.2 t T total time of the test US United States USA United States of America UV ultraviolet light/radiation V volume xxxi V uninoculated vermiculite V c molar volume of semicrystalline polymer V g molar volume of glassy amorphous VS volatile solids V W Van der Waals volume wt. weight %X c percentage crystalinity XRD X - ray diffraction Z impedance m enthalpy p solubility parameter 1 CHAPTER 1 INTRODUCTION 1.0 Background and motivation Plastics represent 12.9 % of the 25 8 million tons of municipal solid waste (MSW) generated in the United States in 201 4 , from which only 9 .5 % was recovered, mostly polyethylene (PE) and polyethylene terephthalate (PET). Hence, most plastic waste ( 25 .1 million tons) ended up accumulating in landfills, creating a major environmental concern and representing a missing environmental opportunity to reduce greenhouse gases emissions [1] . Biodegradable polymers like poly(lactic acid) (PLA), poly(butylene adipate - co - terephthalate ) (PBAT), and thermoplastic starch (TPS) represent a promising way to divert plastic waste from l andfills, with composting as an alternative disposal route, and to replace conventional fossil - based plastics for some applications, especially in cases where the plastic waste is highly contami nated and /or difficult to recover through recycl ing [2] . Disposable products like packaging would greatly benefit from the biodegradable features of these materials , but such benefit is only realized if biodegradable products are disposed in an appr opriate waste management system. Ideally, biodegradable plastics could be treated together with other organic wastes in composting facilities and produce compost, a valuable soil conditioner and fertilizer [3] . In the last two decades , there has been extensive research focusing on ways to overcome some of the performance limitations of biodegradable and bio - based plastics , and to expand their applications . P olymer bio - nanocomposites (BNCs) h ave gained 2 great attention for developing new materials with improved and/or tailored performance properties. One particularly useful class of inorgan ic layered materials that has been used to produce bio - nanocomposites is inorganic layered silicate minera ls, or nanoclays, due to their availability, low cost, significant enhancements and r elative simple processability [4] . N atural nanoclays, such as montmorillonite (MMT), and syntheti c nanoclays, such as L aponite ® RD (L RD) and halloysite nanotubes (HNT), offer a unique route for enhancing the mechanical, physical and barrier properties of polymers like PLA at low levels of loading (<5% wt.), especially when the nanoclay particles are well dispersed in the polymer matrix [5,6] . For example, organically - modified montmorillonite (OMMT), has already been proven to be an effective nanofiller to improve properties of biodegradable materials [4,6,7] . Some researche rs reported that PLA - OMMT bio - nanocomposites have improved storage modulus, flexural, and tensile modulus, flexural strength, and elongation at break when compared to pristine PLA [8 10] . Similarly , PLA - HNT bio - nanocomposites have exhibited improvement in properties like tensile strength, Young and storage modulus, impact and flexural properties [11 14] . PLA - LRD bio - nanocomposites have also shown improvement in thermal stability, tensile strength and hydrophilicity [15 17] . Besides performance limitations, one of the drawbacks of some biodegradable polymers, like PLA, is t hat they do not biodegrade as fast as other organic wastes durin g composting , which in turn affect s the ir general acceptance in industrial composting facilities [18] . Therefore, increasing the ir biodegradation rate in the 3 compost ing environment should facilitate and encourage the ir disposal through these facilities by degrading in a time frame comparable with other organic materials. Several researchers studied the eff ect of O MMT on the biodegradation of biodegradable polymers like polycaprolactone (PCL) [19] , poly( 3 - hydroxybutyrate - co - 3 - hydroxyvalerate ) (PHBV) [20] , TPS [21] , and PLA [10,18,22 30] . Their results indicated that, in general, these BNCs biodegraded faster than their respective pristine polymer. Therefore, the incorporation of nanoclays into a bi odegradable polymer matrix represents a promising approach not only for enhancing the polymer performance but also for increasing its biodegradation rate in composting conditions. However, the effect of different nanocl ays and organo - modifiers, on the abio tic and biotic degradation of PLA is still unclear and needs further investigation. Even though it is well known that the biodegradation mechanism of PLA involves chemical hydrolysis, the role of microorganisms and how they are affected by the presence of nanoparticles is still not well understood [29] . B ioaugmentation is a nother promising technique that can be studied to accelerate the biodegra dation of compostable plastics. Some researchers have identified most of the microbial consortia present in the compost environment [31 33] , and some have report ed the isolation and identification of several species capable of biodegrading PLA [34 42] , and other polymers [43 51] by 16S ribosomal ribonucleic acid ( rRNA ) sequence analysis . These isolated microbial strains can be potentially used to investigate the effect of bioaugmentation in the biodegradation rate of PLA and PLA bio - nanocomposites. 4 Thus, t his study seeks to understand the bio degradation mechanisms of bio - nanocomposites made of PLA and the main factors contrib uting to th eir biodegradation rat e such as those related to the polymer structure and also those related to the soil/compost environment s or to the microbial populations that could be impacted by the presence of nanoparticles. 1.1 Overall goal and objectives The o verall goal of this research is to obtain fundamental knowledge and unique understanding about the biodegradation mechanisms of PLA, to evaluate the bio degradation rate of PLA in simulated composting conditions, and to propose and to test different mechanism s able to accelerate and/or to tailor this process , which could greatly benefit the general use of PLA and its acceptance in industrial composting facilities. As consequence, if more solid wastes can be disposed through composting, the amount of waste reaching landfills could be reduced along with the social and environmental impacts associated with landfilling, for example soil and water contamination and generation of greenhouse gas es like methane . To accomplish the overall goal, PLA and t hree different nanoclays (OMMT, LRD, and HNT) will be used in this study as model syst ems for testing biodegradation in simulated compost ing conditions . T he following specific objectives have been outlined : Objective 1: To evaluate the effect of nanoclays on the aerobic biodegradation and biodegradation rate of PLA in composting conditions and the ir impact on the microbial community of the compost. 5 Some researchers have reported an accelerated degradation of PLA after addition of nanoclays. Therefore, the biodegradation rate of pristine PLA and PLA bio - nanocomposites (PLA - OMMT, PLA - LRD, PLA - HNT) will be evaluated in composting conditions . T his objective should provide the necessary evidence to understand if the presence of nanoclay modifies the biodegrada ti on rate of polymers like PLA. Objective 2: To evaluate the effect of introducing microbial strains capable of degrading PLA during the composting process on the biodegradation rate of PLA and PLA bio - nanocomposites. Preliminary studies indicate that certa in microbial strains are capable of assimilating PLA. Therefore, the biodegradation rate of PLA and PLA - OMMT is evaluated in composting conditions with bioaugmentation, meaning that an isolated microbial strain capable of degrading PLA is introduced in the composting system. This objective provides the necessary evidence to understand if the addition of these microorganisms into the bioreactors helps increasing the biodegradation rate of PLA in solid environments like compost. This approach also allow s to f urther understand the abiotic and biotic contributions on the biodegradation process. 1.2 Dissertation overview To answer the objectives of this dissertation , t his document is organized as follows : Chapter 2 provides an extensive literature review on PLA, incl uding resin production, processing techniques , properties and applications . This chapter also covers the main degradation reactions, the different end - of - life scenarios , and the environmental footprint of PLA. 6 Chapter 3 provides a critical literature review about the biodegradation testing of polymers by analysis of evolved carbon dioxide in simul ated composting conditions. This chapter not only provides insights on the biodegradation testing but also experiment - relevant information about the b iodegrad ation mechanisms and the different abiotic and biotic factors controlling the biodegradation rate of PLA . Chapter 4 is a version of a published article that first provides a critical review on PLA bio - nanocomposites and then presents the results about the impact of nanoclays on the biodegradation of PLA and BNCs in simulated composting conditions . This chapter also presents the results of a biofilm formation essay and scanning electron microscopy that were used to evaluate the effect of nanoclays on the mi crobial attachment on the surface of PLA and BNCs . Chapter 5 investigates bioaugmentation , in which PLA - degrading bacteria were isolated from compost and identified as Geobacillus using 16S rRNA gene sequencing . These isolates were further used to study the effect of bioaugmentation on the biodegradation rate of PLA and BNCs in solid environments. This chapter also presents the results of a biofilm formation essay performed to assess the Geobacillus attachment on the surface of PLA and BNCs . Chapter 6 sum marizes all the work in this dissertation and concludes with future work recommendation s. 7 REFERENCES 8 R EFERENCES [1] EPA, Advancing Sustainable Materials Management: 2014 Tables and Figures, (2016) 1 65. [2] E. Castro - Aguirre, F. Iñiguez - Franco, H. Samsudin, X. Fang, R. Auras, Poly(lactic acid) Mass production, processing, industrial applications, and end of life, Adv. Drug Deliv. Rev. 107 (2016) 333 366. doi:10.1016/j.addr.2016.03.01 0. [3] T. Kijchavengkul, R. Auras, Compostability of polymers, Polym. Int. 57 (2008) 793 804. doi:10.1002/pi.2420. [4] H.M.C. De Azeredo, Nanocomposites for food packaging applications, Food Res. 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Stab. 129 (2016) 338 346. doi:10.1016/j.polymdegradstab.2016.05.018. 13 CHAPTER 2 POLY(LACTIC ACID) MASS PRODUCTION, PROCESSING, INDUSTRIAL APPLICATIONS, AND END OF LIFE A version of this chapter is published as: Castro - Aguirre, E., Iniguez - Franco, F., Samsudin, H., Fang, X., Auras, R. Poly(lactic acid) Mass production, processing, industrial applications, and end of life , Advanced Drug Delivery Reviews Journal , 107 ( 2016 ) 333 366 . 14 2.0 Abstract Global awareness of material sustainability h a s increased the demand for bio - based polymer s like p oly(lactic acid) (PLA) , which are seen as a desirable alternative to fossil - based polymers because they hav e less environmental impact . PLA is an aliphatic polyester , primarily p roduced by industrial polycondensation of lactic acid and/or ring - opening polymerization of lactide . Melt processing is the main technique used for mass production of PLA products for the medical, textile, plasticulture, and packaging industries . T o fulfill additional desirable product properties and extend product use, PLA has been blended with other resins or compounded with different fillers such as fibers, and micro and nanoparticles . This paper presents a review of the current status of PLA mas s production , processing techniques and current applications , and also covers the m ethods to tailor PLA properties , the main PLA degradation reactions, end - of - life scenarios and the environmental footprint of this unique polymer . 2.1 Introductio n Poly(lactic acid) (PLA) is a biodegradable and bio - based aliphatic polyester derived from renewable sources such as corn sugar, potato, and sugar cane . PLA has played a central role in replacing fossil - based polymers for certain applications [1, 2] . As a compostable polymer , PLA is considered a promising alternative to reduce the municipal solid waste (MSW) disposal problem by offering additional end - of - life scenarios [3] . High weight average molecular weight ( M w ) PLA is generally produced by polycondensation and/ or ring - opening polymerization (ROP) [4] . NatureWork s LLC is the major producer of PLA , with a capacity of 150,000 metric t on y ear in its U .S. manufacturing facility ( in Blair, N ebraska) [2, 5] . D ue to great market penetration , 15 worldwide attention , and the rise of PLA production [6] , the number of published research studies and reports about PLA have exponentially increased in the last 25 year s , as shown in Figure 2.1 . Figure 2 . 1 Number of research reports published since 1990 based on the Web of Science search using keywords "PLA", "PLLA", "PDLA", "polylactic acid", "polylactide", and poly(lactic acid) [7] . T he use of PLA was initially limited to medical applicatio ns due to its high cost and low availability, but high M w PLA now can be processed by i njection molding, sheet and film extrusion, blow molding, foaming, fiber spinning, and thermoforming. Also , PLA provides comparable optical, mechanical, thermal , and barrier properties when compared with commercially available commodity polymers such as polypropylene (PP), poly(ethylene terephthalate) (PET), and polystyrene (PS) , expandi ng its commercial range of applications [2, 5] . In the medical field, PLA is extensively used because of its biocompatibility with the h uman body, including for applications such as 16 medical implants, surgical sutures and medical devices [8 - 12] . In addition, PLA has be en used fo r applications such as fiber s , textile s , plasticulture, serviceware, packaging containers ( i.e. , food packaging for short - life products), and environmental remediation films [13] . PLA is cons idered as a Generally Recognized as Safe (GRAS) material by the U . S . Food and Drug Administration (FDA). However, PLA has also some limit ations ( e.g. , poor toughness) , so research efforts are centered on obtain ing PLA products with particular desired prope rties by blending PLA with other biodegradable and non - biodegradable resins, and/or by compounding PLA with fillers such as fibers or micro and nanoparticles. T his critical review focus es on the status of PLA polymer regarding its mass production, the main processing techniques, and methods that have been used to extend PLA applications on the basis of its intrinsic properties. Furthermore, th is review provides a panorama of the current main applications categorized according to PLA commercial usa ge , an d an overview of different environments to which PLA products can be exposed during their lifetime that lead to their degradation , including hydrolysis in non - medical applications . Finally, the end - of - life scenarios of PLA products as well as the cradle - to - grave and cradle - to - cradle environmental footprint (EFP) are discussed . 2.2 PLA Resin Production L actic acid (LA) , also named 2 - hydroxy propionic acid, is the basic monomer of PLA. The monomer exists as two stereo isomers, L - LA and D - LA . Figure 2.2 shows the different chemical structures of these two isomers [2] . 17 Figure 2 . 2 C hemical structure of L(+) a nd D( - ) lactic acid. The two main methods to produce LA are by bacterial fermentation of carbohydrates or by chemical synthesis [14] . B a cterial fermentation is the preferred industrial process used by NatureWorks LLC and Corbion ® , the two ma jor producers of PLA . C hemical synthesis has many limitations , including limited production capacity, inability to produce only the desired L - LA isom er, and high manufacturing cost s [15] . The bacterial ferme ntation processes to produce LA can be classified as homofermentative or heterofermentative method s , depending on the bacteria used . In the h eterofermentative method, 1 mole of hexose produce s less than 1.8 moles of LA , along with significant levels of oth er metabolites such as acetic acid, ethanol, glycerol, mannitol, and carbon dioxide. However, in the h omofermentative method, 1 mole of hexose can produce an average of 1.8 moles of LA , with minor levels of other metabolites , which means every 100 g of glu cose could yield more than 90 g of LA . T he homofermentative method is more frequently used by industry due to its greater production yield s and lower levels of by - products in comparison with the heterofermentative method [16] . In the homofermentative method, species of the Lactobacillus genus , such as L . delbrueckii , L. amylophilus , L. bulgaricus and L. leichmanii , are used under conditions of 18 a pH range from 5.4 to 6.4, a temperature range from 38 to 42 °C , and a low oxygen concentration. The nutrients used to feed the bacteria can be simple sugars , such as glucose and maltose from corn or potato or other sources such as vitamin - B , amino acids, and nucleotides prov ided by rich corn steep liquor. In general, batch production processes can yield 1 to 4.5 g . L - 1 . h - 1 of LA , whereas continuous processes can achieve 3 to 9.0 g . L - 1 . h - 1 of LA . On a larger scale, cell recycle reactors can produce up to 76 g . L - 1 . h .1 [16, 17] . After the initial production process, the LA must be purified by distillation if it will be used for pharmaceutical and food derivative purpose s . Natur eWorks LLC is currently us ing a lower pH process to produce LA, which reduces the amount of calcium hydroxide and sulfuric acid by - products, resulting in the lower production of calcium sulphate (gypsum) [5] . Corbion ® , through a proprietary technology , produces LA in a gypsum - free process, which uses second generation feedstocks ( i.e ., plant - based materials such as corn stover, bagasse , wheat straw , and wood chips) [18, 19 ] . LA can be used to produce PLA of variable molecular weight s ; however, usually only the high M w PLA has ma jor commercial value in the fiber, textile, plasticulture , and packaging industr ies . Figure 2.3 shows the three main methods available to produce high M w PLA from LA : (1) direct condensation polymerization; (2) direct polycondensation in an azeotropic solution; and (3) polymerization th rough lactide formation [17] . 19 Figure 2 . 3 The manufacturing process es to pr oduce high molecular weight PLA , a dapted from Hartmann [17] . The direct condensation polymerization process involves three main steps: 1) free water removal, 2) oligomer polycondensation, and 3) melt polycondensation of high M w PLA. A detail ed description of this process can be found in Hartmann [17] . Direct condensation polymerization is generally considered the least expensive process to produc e high M w PLA. However, the nec essity to use chain coupling agents and adjuv a nts to obtain a solvent - free PLA increases the costs of the products and the complexity of the process [14, 17, 20, 21] . 20 D irect polycondensation i n an azeotropic solution is the method applied by Mitsui Toatsu Chemicals, Inc. to produce high M w PLA [22] . In the process, no chain extenders or adjuv a nts are used . The PLA is produced by a direct condensation while the condensation water is continuously removed by the azeotropic distillation. T he process includes reduction of the distillation pressure of LA for 2 3 h at 130ºC , and t he majority of the condens ation water is removed. Catalyst i s added along with diphenyl ester. A tube packed with 3 - Å molecular sieves is attached to the reaction vessel, and the solvent is returned to the vessel via the molecular sieves for an additional 30 40 h at 130ºC. Finally, the polymer is isolated as is or it is dissolved and precipitated for further purification. The effect of difference catalysts on the azeotropic dehydration of LA in diphenyl ether a nd additional details of th e technique is reported elsewhere [17, 22] . NatureW orks LLC, the major produce r of high M w PLA based on the original Cargill - Dow patented process [23] , combine s a solvent - free process and a distillation process to produce PLA with controlled molecular weights in a multi - step p r ocess . T he LA is first condensed to form low M w prepolymer PLA . With controlled depolymeri z ation, the cyclic dimer, also referred as lactide, is produced from the low M w prepolymer PLA [24] . The lactide in the liquid form is purified by distillation. The PLA with controlled molecular weight is produced by the ring opening of lactide and then polymerization with catalyst [16, 17, 25, 26] . Figure 2.4 shows t he basic process design to produce high M w PLA . 21 Figure 2 . 4 NatureWorks LLC commercial process for producing high molecular weight PLA , a dapted from Auras et al. [2] and Vink et al. [5] . PLA produced from this process can be derived f rom different amounts of L - and D - lactide. The lactide reactor produces a combination of LA, LA oligomers, water, meso - lactide and impurities [18] . T he mixture must be purified , in this case by vacuum distillation through a series of columns. Due to the differen ce in boiling points of lactide and meso - lactide ( Figure 2.5 ) , the highest M w PLA is derived from L - lactide and a small amount of meso - lactide. The higher the stereochemical purity of the lactide mixture, the higher the stereochemical purity of the PLA . The NatureWorks LLC process results in a large amount of meso - lactide, so the properties of the PLA resin obtained through this process can vary according to the amount of meso - lactide in the mix . PLA with a large amount of 93% L - LA can crystallize. 22 Figure 2 . 5 Di a s tereomeric structures of lactide (3,6 - dimethyl - 1,4 - dioxane - 2,5 - dione). T m of L - lactide, D - lactide, meso - lactide , and rac - lactide are 96, 96 97, 53 , and 125°C, respectively , a dapted from Vert et al. [18] . Although a higher amount of meso - lactide in the monomer of PLA contribute s to some advantages , such as eas ier process ing and production of amorphous PLA, it s presence compromises the thermal stability of PLA ( i.e. , low heat deflection temperature (HDT)) for a wide range of applications [18] . T he presence of meso - lactide in poly(L - l actic acid) ( PLLA ) also may cause deteriorative changes of the crystallinity and biodegradation properties of the materials [27] . Therefore, it is generally desirable for PLA monomer to contain a low amount of meso - lactide. Although the production of meso - lactide is considered undesirable and often is associated with impurities, NatureWorks LLC has made it possible to refine this monomer into various functionalit ies . A pplications of the meso - lactide by - product include its use as chemical intermediates in v arious surfactants, coatings, and copolymers [28] . Meso - lactide can be separated easily from either ( S,S ) - lactide or ( R,R ) - lactide due to its volatile nature. 23 Commonly used methods to perform separation of me so - lactide from either ( S,S ) - lactide or ( R,R ) - lactide are fractional distillation, melt crystallization and solvent recrystallization [29] ; h owever, these methods pose some difficult ies in separating meso - lactide from other impurities. NatureWorks LLC has patented a process in which meso - lactide can be separated from crude lactide efficiently by means of an enriched stream of a minimum 0.8 - mole - fraction of meso - lactide and forming a purified ( S,S ) - and ( R,R) - lactide stream. Futerro S.A. , a joint venture company between Galactic and Total Petrochemicals , has also patented a method to produce meso - lactide, D - lactide , and L - lactide by back biting depolymerization of PLA . The process start s by employing a controlled temperature (200 290°C) and a reduced pressure in a presence of catalyst and co - catalyst to de polymerize PLA into its dimeric cyclic esters form. The resultant PLA components are depolymerized into a vaporized form in a reaction zone. This vaporized form is then condensed and the meso - lactide, D - lactide , and L - lactide produced are recovered separat ely or together. This invention , which is regarded as the second generation of PLA , can produce D - lactide and meso - lactide with high throughput for the production of poly(D - l actic acid) ( PDLA ) or co - polymers consist ing of L - and D - LA enantiomers without th e need to start off with LA [30] . Extensive research has also been conducted to produce lactide and PLA via low manufacturing and production cost s and with enhanced properties [31, 32] . Various catalysts , ranging from metal, cationic , and organic , have been used during polymerization of PLA to achieve high M w and high optical purity [33] . Metal complexes are reported to be one of the most efficient c atalysts for the production of stereoblock isotactic PLA via ROP of rac - lactide due to its ability to control parameters such as 24 molecular and chain microstructure [34] . Dusselier et al. [35] reported the production of lactide through a direct Br ø nsted acidic zeolite - based catalytic process , which obtains considerabl y larg er lactide yields tha n with the controlled ROP of low M w PLA . Yang et al. [34] investigated the production of PLA by rac - lactide using monoanionic aminophenolate ligands with metal complexes in the presence of solvent s such as tetrahydrofuran and 2 - propanol. Monomeric zinc silylamido complexes with acrylamine coordination ligands produced a low degree of heterotactic PLA , and similar complexes with alkylamine coordination ligands produced isotactic PLA. The stereoselectivity of the rac - lactide polymerization was affected by the patter n of the monoanionic aminophenolate ligands coordination. Moreover, complexes of tetrametallic lithium and sodium diamino - bis(phenolate) were investigated for their efficacy in the polymerization of rac - lactide , and these complexes were able to produce PLA with narrow M w dispersities [36] . A recent study reported the use of a biodiesel fuel by - product , glycerol , to produce rac - lactide, a monomer for producing stereoblock PLA [37] . The method employ ed a hydrothermal reaction in the presence of alkaline catalyst to produce racemic lactide . Th e lactide was further purified by acidizing sodium lactate with sulfuric acid , and the resultant lactide was extracted with ethyl acetate to obtain refined lactide . A mixture of lactide isomer s (crude lactide) containing both rac - lactide and meso - lactide were produced via dimerization of lactic acid ( i.e., reactive distillation at temperature of 210 230°C and a pressure of 5 10 mmHg). The levels of rac - lactide and meso - lactide in the crude lactide were reported to be 32.8 and 32.6%, respectively , with 34.6% impurities ( i.e., LA and oligomer). This crude lactide was then purified with ethyl acetate 25 in a N 2 atmospheric condition via three - time recrystallization to obtaine d a refined rac - lactide of 99.1% purity, with meso - lactide (0.2%) and other impurities ( 0.7% ) [37] . Zhu and Chen [38] also reported a new approach to convert meso - lactide to rac - lactide . On the basis of the frustrated Lewis pair concept, this approach utilized the epimerization of meso - lactide to rac - lactide using 1,4 - diazabicyclo [2.2.2] octane (DABCO)/ tris( pentafluorophenyl) borane 95% (B(C 6 F 5 ) 3 ) at 2M in toluene, which resulted in 95.4% conversion. This study investigated different types of Lewis acids with different molar concentrations and different polar and non - polar solvents to find optimized conversio n of meso - lactide to rac - lactide. The epimerization method used in this study is versatile since it was able to effectively conver t lactide stereoisomers regardless of ratio into rac - lactide [38] . 2.3 PLA Processing The methods for processing PLA are well - established polymer - manufacturing techniques used for ot her commercial polymers such as PS and PET [39] . Melt processing is the main technique for mass production of high M w PLA in which the PLA resin obtained ( as shown in Figure 2.4 ) is converted into end products such as consumer goods, packaging, and other applications. Melt processing is characterized by heating the material above its melting temperature, shaping the molten polymer into desired shapes, and finally cooling to stabili ze its final dimensions. Processing of PLA has been extensively reviewed [1, 40] . T he main objective s of this section are to summarize the key methods used to process PLA and then to provide a short update of new research since our last review of PLA processing [1] . Additionally, we direct the 26 reader to a number of contributions that explain each processing technique in further detail. The lim iting factors for processing PLA are similar to those for fossil - based polymers : degradation at the upper limits of temperature and shear, and poor homogeneity at the lower limits [39] . Ho wever, u crystallinity, and melt rheological behaviors is critical to optimize its processing and component qualities. Detail ed information about these properties is provided elsewhere [13] . PLA is a hygroscopic material and very sensitive to high relative humidity ( RH ) and temperature [39] . B efore PLA can be processed , it should be dried to a water content less than 100 ppm (0.01%, w/w) to avoid hydrolysis ( M w reduction) , as discussed in section 2. 6 .1 . During industrial production , PLA is mostly dried to values below 250 ppm water (0.025%, w/w). I f PLA is processed at temperatures higher than 240°C or with longer residence times, the PLA resin should be dried below 50 ppm water to avoid number average molecular weight ( M n ) reduction [1, 41] . To achieve effective drying, , with an airflow rate greater than 0.03 m 3 . h - 1 .kg - 1 of resin throughput. After the PLA resin is properly dried, melt extrusion is the most important technique for continuous melt processing of high M w PLA consumer goods. 2.3.1 Extrusion Extrusion of PLA in a heated screw is the first step before any further processing of PLA , such as injection, thermoforming or spinning , ta kes place . Commercial PLA resins can be processed by using conventional screws equipped with a general - purpose screw of L/D ratio (ratio of flight length of the screw to its outer diameter) of 24 to 30. If PLA is 27 processed in extruders designed for polyole fin s, and the extruder is working near to its maximum power, the extruder may not have enough torque to process PLA. So, it is recommended to process PLA in extruders regularly used for polyesters or PS , with similar performance profile . The recommended co mpression ratio (ratio of the flight depth in the feed section to the flight depth in the metering section) for PLA processing is in the range of 2 to 3. T he extruder provides the heat to melt the resins by heater bands wrapped around the barrel; however, the majority of heat input is provided by the friction of the resin between the screw and the barrel. Thus, to ensure that all the crystalline domains of the sem i cr y stalline PLA are melted , and to achieve an optimal melt viscosity for processing, the heate rs are usually set at 40 to 50°C higher than the melting temperature ( T m ). The m elt rheological properties of PLA play a n important role in how the polymer flows during extrusion. Melt viscosities of H igh M w PLA melt viscosities are in the order of 5 , 000 10,000 P (500 1000 Pa . s) at shear rates of 10 50 s - 1 ; t hese polymer grades are equivalent to M w of ~100,000 Da for injection molding to ~300,000 Da for cast film extrusion applications [1] . Extruding PLA at high temperature s can cause thermal degradation (as explained in section 2. 6 .2 ), so the temperature profile during extrusion of PLA should be tight ly controlled . The t hermal degradation of PLA can be attributed to several factors: ( a ) hydrolysis by trace amounts of water ; ( b ) zipper - like depolymerization ; ( c ) oxidative, random main - chain scission ; ( d ) intermolecular transesterification to monomer and oligomeric esters ; and ( e ) intramolecular transesterification resulting in formation of monomer and oligomer lactides of low M w [40] . Processing PLA above 200°C can degrade PLA through intra and intermolecular ester exchange, cis - elimination, and radical and concerted non - 28 radical reactions resulting in the produc tion of CO, CO 2 , acetaldehyde, and methyl ketone [42] . Depending on the rate of the degradation reaction, the end product can be lactide or acetaldehyde. Formation of lactide during extrusion can affect the optical purity of the final extruded PLA ; reduce the melt viscosity ; produce fuming of lactide ( i.e., lactide vapor produced during extrusion) ; and can condens e on equipment surfaces , such as chilled rollers and molds , which is known as plate out. To avoid lactide fuming and condensation, the temperature of the surfaces should be increased. Table 2.1 shows the recommended processing temperatures for a number of commercially available NatureWorks LLC PLA resins known as Ingeo TM PLA. 29 Table 2 . 1 P roperties and processing temperatures of selected commercially available Ingeo TM PLA resins 2500HP [43] 3001D [44] 4032D [45] 6060D [46] 7001D [47] 8052D [48] Application Extrusion - Crystalline sheets Injection m olding Biaxially oriented films Fiber melt spinning Injection stretch blow molding Foam Specific gravity, ASTM D792 1.24 1.24 1.24 * 1.24 1.24 1.24 MFR, g/10 min (210°C, 2.16 kg) ASTM D1238 8 22 N / A 8 - 10 6 14 Melt temperature, °C 210 200 210 N / A 200 - 220 200 Feed throat, °C 45 20 45 N / A 20 20 Feed temperature, °C 190 150/ 165 ** 180 N / A 180 165 Compression section, °C 200 195 190 N / A 210 195 Metering section, °C 210 205 200 N / A 210 - 220 205 Nozzle, N / A 205 200 N / A 210 - 220 205 Adapter, °C 210 N / A N / A N / A N / A N / A Die, °C 210 N / A N / A N / A N / A N / A Mold, °C N / A 25 200 N / A 21 - 38 25 Screw s peed, rpm 20 - 150 100 - 175 20 - 100 N / A N / A 100 - 175 Back pressure, MPa N / A 0.345 - 0.689 0.414 - 0.483 N / A 0.689 - 1.379 0.345 - 0.689 Note s : * ASTM 1505, ** 150 °C amorphous / 165 °C crystalline . N/A: Not available 30 2.3.2 Injection molding PLA is primarily injected on machines that have a reciprocating screw extruder , as shown in Figure 2.6 . In this case, the screw is designed to reciprocate wit hin the barrel to inject the molten polymer into the mold cavities. At the start , the molds close and the nozzle opens, and the screw moves forward , injecting the molten polymer into the mold cavity. Since the polymer shrinks during cooling, the screw is m aintained in the injection position by holding pressure steady . Then, the nozzle is closed and the screw starts retracting. During the cooling cycle of the molds , the screw rotates and convey s the melt polymer forward ; sufficient cooling time should be pro vided to produce stable parts. Cycle time of the injection part is extremely important to control shrinkage of the PLA injection - molded parts, which are generally brittle due to the accelerated physical aging of PLA , which is attributed to its low glass tr ansition temperature ( T g ) . PLA parts produced with low M n are subjected to faster aging. Likewise, PLA injection - molded parts could exhibit low crystallinity due to the slow crystallization rate of PLA. Furthermore, in order to avoid excessive shrinkage, processing parameters such as mold temperature, packing pressure, cooling rate, and post - mold cooling treatment should be properly controlled. A complete explanation of how to optimize the cycling time for PLA injection - mold ed parts and reduce shrinkage is provided elsewhere [1] . 31 Figure 2 . 6 Major components of an injecti on molding machine showing the extruder (reciprocal sc Reprinted from Progress in P olymer S cience , 33, Lim et al ., Processing technologies for poly (lactic acid) , 820 - 852, Copyright (2008), with [1] . Shear - controlled orientation in injection molding (SCORIM) is a technique that allows the enhancement of mechanical properties of semicrystall ine polymers , like PLA , by tailoring the morphology of the solidifying polymer melt using an in - mold shearing action that is externally controlled [39] . As in conventional injection moldin g, t he processing cycle begins with the filling of the cavity. T he SCORIM unit, which has two cylinders with their own melt flow path and three operation modes (A, B and C) , then can manipulate the melt . Mode - A consists of an out of phase reciprocation of the two pistons; Mode - B consists of an in - phase operation to pump more melt into the cavity; and Mode - C consists of applying hydrostatic pressure by two cylinders for offsetting volumetric shrinkage [3] . 32 2.3.3 Injection s tretch blow molding Injection stretch blow molding (ISBM) is primarily used t o produce bottles. Figure 2.7 shows a general ISBM process for making PLA bottles. ISBM requires the initial production of a parison or preform by injection molding. Then, the p reform is transferred to a blow molding machine where it is heated at around 90°C, and the preform is stretched in both the axial and hoop directions to achieve biaxial orientation , which improv es the physical a nd barrier properties of PLA bottles . Additives are added to the PLA resin s to optimize the absorption of energy by the preform from the infrared lamps, so that optimal stretching is achieved. PLA preform s tend to shrink after reheat in regions near the ne ck and the end cap ( i.e., regions where the residual injection stresses are largest.) Residual stresses can be minimized by properly designing the preform . ISBM can be conducted in one or two stages where the preform is produced during the same step as blo wing or it is just produced in two consecutive steps [1] . PLA resins show strain - hardening when stretched to a high strain ratio. Therefore , stretc hing of PLA should be programmed to obtain PLA bottles with optimal sidewall orientation and thickness. Under - stretched preform s result in bottles with large wall thickness variation and lower mechanical properties. Over - stretched preform s result in stress whitening due to the formation of micro - cracks on the bottle surface that diffract light. Preform axial stretch ratios of 2.8 3.2 and hoop stretch ratios of 2 3, with the desirable planar stretch ratio of 8 11, are recommended [1] . Introducing standard feature s in the bottle design , such as transition shape, step changes , and pinch points on the core and cavity , may help to improve PLA bottle perfor mance. Preform design s are also important for obtaining bottles with good clarity and physical properties, which usually 33 depend s on the bottle design and the blow mold equipment ; however , there is little information about th at in the literature due to the proprietary nature of this information . Figure 2 . 7 Injection stretch blow molding (ISBM) of PLA bottle . Progress in P olymer S cience , 33, Lim et al. , Processing technologies for poly (lactic acid), 820 - [1] . 2.3.4 Cast film and sheet Cast is the main method to produce 0.076 mm and sheets with 0.25 mm . During the production of cast films, molten PLA is extruded through a lip die and quenched on polished chrome rollers refrigerated with cooled water. C ast films usu ally have a low crystallinity and t ransparent appearance d ue to the rapid cooling provided by the chilled rolls. Cast film extrusion has the 34 advantages of providing good optical properties, high production rate, and good control of film thickness [39] . Deckle systems to control film and sheet edge trimming are generally avoided to reduce the effect that the degraded molten PLA introduce s in the edge instability. The gap of the die is set to around 10% higher than the thickne ss of the film and/or sheet to obtain the right film and sheet dimensions. Table 2.1 shows the recommended temperatures to extrude PLA films and sheets (PLA 4032D and 2500HP). Horizontal roll stacks are used to produce PLA films and sheets due to the low melt stren gth. Roller temperature between 25 50°C is recommended to avoid lactide condensation, and by using an exhaust system around the die, lactide buildup can be controlled. Additionally, good contact between the web and the rolls is recommended to minimize lact ide buildup. Slitting and web handling of PLA is similar t o that for PS. Rotary shear knives are recommended for trimming the edge of PLA web since razor knives could yield rough edges and break the web. Orientation between 2 and 10 times its original leng th will improve PLA thermal and impact sheet properties. PLA films produced from 98% L - lactide can be subjected to 2 to 3 times machine direction (MD) stretch ratios, and 2 to 4 times transverse stretch ratios. When a l arger amount of D - lactide is present in PLA, more amorphous sheet or film is produced, and larger stretch ratios can be obtained. Extruded PLA films and sheets have excellent toughness [40] . Table 2.2 shows the main optical, physical, thermal, mechanical , and barrier properties of PLA films . Figure 2.8 shows the production of biaxial ly oriented PLA extrusion cast film. 35 Production of PLA film by blown film technologies is rarely done since PLA has weaker melt strength, and so formation of a stable bubble during extrusion is challenging. Attempts to create PLA blown film have been conducted by using viscosity enhancers. Most of the additives used to increase the melt strength of the PLA are proprietary [40] . Beside s low melt strength, PLA is stiff, so when collapsing the bubble during blown film production, permanent wrinkles may be produced. The problem of dead - fold properties can be overcome b y also introducing additives. Figure 2 . 8 Biaxial oriented extrusion cast film machine, adapted from Progress in P olyme r S cience , 33, Lim et al. , Processing technologies for poly (lactic acid), 820 - [1] . 36 Table 2 . 2 Selected a verage optical, physical, mechanical , and barrier properties of PLA films reported from a number of studies using different grades of PLA , adapted and modified from Auras [49] . Optical Refractive index (a), [50] 1.35 - 1.45 Clarity Clear - yellow Thermo - Physical Density amorphous, [51] kg.m - 3 1250 Density 100 % crystalline, (b), [50] , kg.m - 3 1490 Van der Waals volume (V W ), (c), [50] , cm 3 .mol - 1 34.45 Molar volume of glassy amorphous (V g ), (c), [50] , cm 3 .mol - 1 55.12 Molar volume of semicrystalline polymer (V c ), (c), [50] , cm 3 .mol - 1 49.44 p ), 25°C, [2] , MPa 0.5 19 - 20.5 T g , [52] ,°C 50 - 80 T m , [52] ,°C 130 - 180 Initial decomposition temperature ( T d,0 ), [53] , °C 335 Half decomposition temperature ( T d,1/2 ), [53] , °C 395 Average energy of activation (E act ), [53] , kJ.mol - 1 205 - 297 Enthalpy ( H m ), [54] 100%, J.g - 1 93 Crystallinity, [53] , % 0 40 Heat deflection temperature, [55] , °C 55 65 Vicat penetration temperature, [55] , °C 59 Thermal conductivity x10 - 4 , [1] , cal.cm - 1 .s - 1 . °C - 1 2.9 Heat capacity, [1] , cal.g - 1 .°C - 1 0.39 Thermal expansion coefficient x10 - 6 , [1] , °C - 1 70 Surface tension, [2] , dyn.cm - 1 42.0 Friction coefficient, [2] 0.37 37 Table 2.2 Thermo - Physical Melt flow Index, d, [2] , g. min - 1 0.85 Rheological Mark - Houwink constants K , (e), [56] , mL.g - 1 0.0174 a (e), [56] 0.736 Mechanical Tensile strength @ yield, [2] , MPa 0.88 Elastic modulus, [2] , GPa 8.6 Elongation at break, [2] , % 3 - 30 Flexural strength, [55] , MPa 70 Flexural modulus, [55] ,GPa 3.8 Unnotech Izod Impact, [55] , J.m - 1 106 Notched Izod Impact, [55] , J. m - 1 26 Rockwell hardness, [55] 88 Impact strength Poor Barrier Oxygen permeability x10 - 18 , (f), [52, 54] , kg.m.m - 2 .s - 1 .Pa - 1 @25ºC 1.21 ± 0.07 Oxygen activation energy, (f), [5 2, 54] , kJ.mol - 1 [25 - 45ºC] 41.43 ± 3.5 Carbon dioxide permeability x10 - 17 , (g), [52] , kg.m.m - 2 .s - 1 .Pa - 1 @25ºC 2.77 ± 0.05 Ca rbon dioxide activation energy, (g), [52] , kJ. mol - 1 15.65 ± 0.63 Nitrogen permeability x10 - 19 , [57] , kg.m.m - 2 .s - 1 .Pa - 1 468 Nitrogen activation energy, [57] , kJ.mol - 1 11.2 Water permeability x10 - 14 , (h), [52, 54] , kg.m.m - 2 .s - 1 .Pa - 1 @25ºC 1.75 ± 0.05 Water activation energy, (h), [52 , 54] , kJ.mol - 1 - 9.73 ±0.27 d - limonene permeability x10 - 19 , (i), [58, 59] , kg.m.m - 2 .s - 1 .Pa - 1 <1.0 38 Table 2.2 Barrier Ethyl acetate permeability x10 - 19 , (i), [58, 59] , kg.m.m - 2 .s - 1 .Pa - 1 5.34* Methane permeability x10 - 18 , [57] , m 3 (STP).m.m - 2 .s - 1 .Pa - 1 7.50 Methane activation energy, [57] , kJ.mol - 1 13 Helium transmission rate x10 - 6 , (j), [60] , cm 3 .cm.m - 2 .s - 1 .kPa - 1 10.30 N/A: Not available Note: ( a) Refractive index values for PLA were calculated by Gladstone and Dale, Vogel, and Lloyenga meth ods ; ( b ) d ensity of 100% PLA was calculated according to the group contribution method ; ( c ) PLA value was calculated using the group contribution method; ( d ) PLA value measured at 200ºC and 5 kg according ASTM D1238; ( e ) PLA value was measured according to ASTM D445 and D446 (PLA values were determined in t etrahydrofuran at 30°C ); ( f ) o xygen ac tivation energy is reported for temperatures between 25 - 45 ° C; ( g ) carbon dioxide activation energy is reported for temperatures between 25 - 45 ° C; ( h ) water activation energy is reported for temperatures between 10 - 37.8 ° C; ( i ) e thyl acetate values of PLA at 3030 P a and 30°C and 9435 Pa and 30°C, respectively; d - limonene values of PLA at 245 Pa and 45°C a nd 45 Pa and 23°C, respectively ; ( j ) v alue of amorphous PLA at 23°C and 0% RH . 2.3.5 Thermoforming Thermoforming is a standard method to produce PLA containers , suc h as clamshell, cups, and food trays, extensively used for short shelf - life product packaging applications. Thermoforming is a process in which a pliable plastic is pressed into a final shape by vacuum or air pressure. Figure 2.9 shows the steps to produce a thermoformed PLA part. Generally, a PLA sheet (thickness >10 mil or 254 m) is extruded as previously described, heated, introduced to a mold where it is pre - stretched and then formed ( assisted or not by a plunger ) to obtain the final PLA container. Initial heating of the PLA film is by infrared (IR) lamps ; the IR wavelength should match the 39 maximum absorbance of the polymer being ther moformed. PLA sheets are thermoformed at temperature s around 80 110°C. Aluminum molds are recommended for thermoforming PLA . As in the case of ISBM, orientation improves toughness of PLA containers ; therefore, thermoformed parts are less brit tle than PLA sheet, especially in regions highly stretched during the forming operations rather than flanges and lips . Clamshell s produced from PLA sheets show better drop - impact properties at fr eezing temperature ( - 20°C) than PET and PS clamshell s [61] . PLA sheets produced with 100% recycled PLA flakes showed a reduction of M n of around 5% compar ed with the original PLA samples; however, this reduction did not affect the production of PLA containers with 100% post - consumer recycled (PCR) PLA content [62] . Figure 2 . 9 Production of a thermoforming part, reproduced Progress in P olymer S cience , 33, Lim et al. , Processing technologies for poly (lactic acid), 820 - 852, [1] . 40 2.3.6 Other process es Foaming and fibers PLA resins are used in foam and textile applications. Although methodologies for these applications are well established for other commercial polymers , until now the amount of commercial PLA ( by weight ) marketed in th e s e form s has been minor. Obtaining l ightweight materials with improv ed cushioning, insulati on , and structural performance is a ma jor reason to produce PLA foam parts. Initially, PLA foams were extensively used for medical applications ( suture s , implants, and screw s ) , but it is also a promising bioplastic for use in relatively short - lived applications like transport packaging ( loose - fill packaging, insulation , and cushioning ) or disposable cutlery. In this context, PLA would allow an alternative disposal route and replace fossil - based foams since the polymer is biodegradable and based on renewable resources [63] . Most commercial foaming of PLA is obtained by batch or continuous processes. In these processes, a physical or chemical foaming agent (PFA or CFA) is introduced in the PLA matrix. PFAs are dissolved in the molten PLA matrix and undergo a physi cal change , such as volatilization of a liquid or release of compressed gas , during foaming. Examples of PFAs are hydrocarbons and halogenated hydrocarbons and gases such as N 2 , CO 2 , and Ar. CFAs are chemical compounds which are stable at room temperature , such as sodium bicarbonate, azodicarbonamide, p , - o xybis(benzene) sulfonyl hydrazide, p - t oluene sulfonyl semicarbazide, and 5 - p henyltetrazole , but after a set change of temperature and pressure conditions the se compounds convert to gas by undergoing a chemical reaction that provid es gas to nucleate bubbles inside the PLA matrix and creat e the foam structure. CFAs can react endothermically ( i.e. , absorption of heat during decomposition), or exothermically ( i.e. , release of heat during 41 decomposition) [64] . Generally, CFAs are selected to be used at temperatures close to the pr ocessing temperature of the polymer. In the ca se of PLA, a number of PFA s and CFA s have been used such as CO 2 , N 2 , and BIH40 ( a CFA produced by Boehringer Ingelheim Chemicals ) [64] . During the batch process, a gas ( N 2 , CO 2 or a mix ture) is saturated into the PLA matrix at a pressure below 800 MPa at room temperature in a chamber . Then, the saturated PLA sample is removed from the chamber , and t he solubility of the blowing agents is suddenly reduced by increasing the temperature and/or reducing the pressure, so that bubbles can nucleate. Finally, the produced cells are vitrified by reducing the temper ature below the T g of the PLA matrix. Figure 2.10 a shows a representation of a batch process. The continuous microcellular foaming process was develop ed to overcome some of the drawbacks of the batch process , for example, the time required to saturate the samples. In a continuous process , a blowing agent, generally a gas, is introduced into the molten PLA matrix in a modified extruder ( Figure 2.10 b ). After that, the saturated gas PLA m atrix is solution mixed and transferred to a static mixer, which guarantee s the single - phase solution. Finally, the microcellular nucleation occurs in the nozzle of the extruder unit due to the rapid pressure drop. PLA foaming is affected by a number of parameters such as initial crystallinity, melt rheology, fillers, amount of C FAs, and processing conditions. A detailed review of PLA microcellular foams can be found elsewhere PLA foam samples ha ve been reported to increase Notched Izod impact strength by more than triple while reducing the specific density by almost half [64] . 42 PLA low melt strength is the main drawback of using it for foaming applications ; however, new modifiers are being investigated to induce crosslinking, chain extension , or grafting to increase the molecular weight and the melt properties such as shear and elongational viscosity. Gottermann et al . [63] reported that the use of modifiers , such as organic peroxi de, mul tifunctional epoxide, styrene ma leic anhydride, isocyanurate + diisocyanate, and bisoxazoline + diisocyanate , helps to increase the M w of commercially available PLA. In most cases the foam density decreased and cell size increased (except with multifunctional epoxide), and when modified with organic peroxide and multifunctional epoxide the elongational viscosity of PLA increased [63] . Figure 2 . 10 Schematic of microcellular foaming process : a) batch process, b) continuous process ; 1 to 6 are the main region s of the extruder ; adapt ed from Matuana [64] . Spinning of PLA fibers has been used to produc e PLA fibers for suture applications. PLA fibers are gaining importance since they have lower water barrier properties. Conventional processes and finishing technologies can be used for processing PLA fabrics; PLA shows similar properties to other synthetic fibers, but requires modified dyeing and finishing techniques due to its low affinity to conventional 43 water - soluble dyes [65] . PLA fibers can be used to produce breathable garments. Among the important criteria to produce fibers are: i ) to control moisture content to be less than 50 ppm to avoid any possible hydrolytic degradation , and ii ) to achieve an T g ) and drawing speed (200 9000 m . min - 1 ) to obtain appropriate crystallinity and strong PLA fibers [66] . During spinning of PLA fibers, the microstructure of the polymer chains is oriented in the axis direction of the fiber, so a fiber with very high aspect ratio (length to diameter) and orientation can be produced . Spinning of fibers produce s a controlled molecular orientation and spatial arrangement of the PLA structure. In modern spinning process es , a molten polymer or solution is extruded through a small orifice and is elongated by applying an external force. Then , the polymer filament is cooled and precipitated. F urther processing of the polymer filament may tak e place , such as drawing, unidirectional stretching, and texture control . PLLA with a M w around 0.5 to 3.5 x 10 5 Da is used for melt spinning through a two - stage process that includes melt spinning and hot drawing [67] . A standard melt spinning process is represented in Figure 2.11 . A typical melt temperature profile for PLA resin melt spinning is shown in Table 2.2 . A g eneral extrusion process , as described in section 2. 3.1 , is used to produce the fibers; however, the spin pack plays an extremely important role since it deliv ers the molten PLA previously filtered to remove impurities to the spinneret plate through the spinneret holes. The spinneret holes have a specific ratio of length to diameter to achieve the desirable shear flow mode. Spinneret plates can be monofilament o r multifilament. Deniers , the unit used to quantify filaments, is defined as weight in grams of a 9,000 - m long filament. For PLA, the recommended diameter of the spinneret holes range from 0.2 0.35 mm with a 44 typical ratio of 2 to 3. Larger hole diameters are necessary for filaments greater than 6 deniers. After the filament is produced, it is air cooled at temperatures about 15 30°C in an air quench zone or chamber, which cools the filament through its melt crystallization temperature ( T mc ) and t o its T g . When the spun filament temperature is below the T g , the spinning process is considered complete. Then, the spun filament needs to be finished and w ou nd up . A detailed description of the production of melt spinning PLA fibers can be found elsewher e [67] . S olution spinning of PLA can also be carried out to avoid the substa ntial hydrolytic degradation that happen s during melt spinning. During solution spinning , PLA is extruded as in melt spinning, but then the spinneret is submerged in a spinning bath, so that the PLA melting point is immediately depressed to below room temp erature. After that, the solvent is removed by solvent - assi s ted coagulation or evaporation. Two main methods are used for solution spinning : wet and dry spinning. During wet spinning, PLA is dissolved in a solvent such as tetrahydrofuran, chloroform and/or dichloromethane, and then it is extruded in a submerged bath with a mixture of a solvent and a non - solvent ( e.g. , toluene at 110°C) to indu ce coagulation. Generally, PLLA with M w <3x10 5 Da is not suitable for wet spinning. During dry spinning, after the PLLA dope solution ( e.g. , PLLA in chloroform) is extruded, and pump ed through a multi - hole spinneret, it is introduced in a chamber with circ ulating heate d air/gas, so that the solvent can evaporate. PLA fibers are used for textiles and medical applications ; e xamples of these applications are presented in section 2. 5 .2 . 45 Figure 2 . 11 Schematic representation of melt spinning setup: (1) extruder drive, (2) single - extruder - 24 to 36:1 L/D ratio, (3) hopper, (4) screw, (5) manifold, (6) static mixer, (7) metering pump, (8) metering pump drive, (9) spin pack, (10) mesh filters, (11) distributor, (1 2) spinneret, (13) cross - flow quench chamber, (14) freshly spun yarn, (15) godet, (16) idler roller, (17) friction - driven winder, (18) yarn bobbin , adapted from Agrawal [67] . 2.4 Tailoring PLA P roperties Al though PLA ha s many desirable properties for consumer good applications, there are limitation s for all - purpose use, as with any p olymer . R esearchers have been trying to ex pand PLA use and applications by blending PLA with a number of biodegradable and non - biodegradable resins, and/or by compounding PLA with a number of fillers such as fibers and micro and nanoparticles. Covering all of the blends and composites in a short 46 overview is a daunting task , and a number of review paper s ha ve been written to discuss the improvement s of PLA properties [68 - 73] . Therefore, this section provide s a summary of the main resin s used to blend and/or compound with PLA, and it will direct the reader to the or iginal work to obtain additional information. Blending of PLA with biodegradable and non - biodegradable polymers ha s been extensively reported [71 - 73] . Figure 2.12 shows the main biodegradable and non - biodegradable resins blended with PLA. PLA is considered a brittle polymer, so extensive research ha s been conducted to improve its toughness for differen t applications [74, 75] . In general, a rubbery polymer with low T g (generally below 20 ° C of the use temperature) is blended with PLA at a low ratio to create small rubber domains between 0.1 and 1.0 m with good interfacial adhesion to PLA, so that the rubber domains can dissipate the impact energy when PLA is failing through fracture [76] . Beside an improvement in toughness , different polymers have been blended to PLA to improve properties such as optical [75, 77] , barrier [78 - 83] , thermal [74] , and biodegradation [84 - 88] . 47 Figure 2 . 12 Select ed biodegradable and non - biodegradable blends of PLA polymers : PLA - LDPE [89] , PLA - L LDPE [90] , PLLA - L LDPE [90 - 92] , PLLA - HDPE [91] , PLA - PS [93] , PLLA - PEVA [94] , PLLA - EVOH [95] , PLA - TPO [96] , PLLA - ABS [97] , PLLA - PIP [98] , PLA - PVOH [99] , PLA - PHB [100] PLLA - PBS [101, 102] , PLA - PBSA [103] , PLA - PBAT [104, 105] , PLLA - PTAT [106] , PLA - PAE [107] , PLA - PU [108] , PLA - PEG [109] , PLA - SPI [110] , PLA - SPC [110] , PLA - SF [111] , PLA - TKGM [112] , PLA - Chitosan [83, 113] , PDLLA - Chitosan [114] , PLLA - Chitosan [114] , PLLA - PBSL [115] , PLLA - PEO [116] , PLLA - PCL [117 - 119] , PDLLA - PCL [117] , PLA - PCL [120] , PLA - Starch [121 - 124] , PLA - PHBHxx [125] , PLA - PPC [126] , PLA - PP [127] , PLA - PC [128] , PLA - PGS [129] , PLA - PTT [13 0] , and PLA - EGMA [131] . A polymer composite is defined as a material that has two or more distinct phases. One of the phases is a discontinuous phase considered as the reinforcement phase dispersed in a continuous or matrix phase. The reinforcement phase can be fibers and/or micro and nano particles. The main goal of adding a reinforcement phase to PLA is to ta ilor its properties , such as elongation at break [81, 132 - 141] , heat resistance [138, 142, 143] , dimensional stability [137, 144 - 147] , barrier [132, 137, 140, 48 148, 149] , and cost [150] , to overcome some of shortcoming properties compared with fossil commodity polymer s, as well as brittleness and low thermal stability [2] . Since the main engineering properties of a composite result from the discontinu ous phase, PLA has been reinforced with natural and synthetic fibers, micro and nano fillers [73, 151] . Fibers with a larger length to diameter ratio can be used to carry load in fiber - PLA composites, and increase their application s [73] . Natural and synthetic fibers have been used to reinforce PLA. Dispersion and orientation of the fibers play a crucial role in obtaining PLA composites with the desired properties. Adhesion between the PLA matrix and the fibers is a strong controlling parameter of the final composite properties since enhancement of the composite performance is strongly attributed to the adhesion between the continuous and disc ontinuous phases. Wood and non - wood natural fibers , such as cotton, jute, flax, kenaf, sisal, and hemp , are extremely attractive to be compounded with PLA since they are 100% renewable and so a fully bio - based composite is obtained . Synthetic fibers , such as glass and carbon - based , are also commonly used to reinforce PLA parts since they have extremely high tensile strength, which improv es the final mechanical properties of the composite [73] . Fillers in micro and nano sizes have played an increasing role in creating composites with lower cost and environmental footprint. Inorganic fillers , such as talc, mica, hydroxyapatite, carbon black, and gypsum , have been used for many decades to reinforce polymers since they can enhance PLA mechanical properties with a small amount of comp osite. Lately, the addition of nanoparticles has gained attention since adding nanoscale clay particles results in significant improvement of material 49 performance. Some of the specific nanocomposite properties that are enhanced through the exfoliation of these nanoparticles include mechanical [152 - 160] , barrier [132, 137, 140, 148, 149, 160, 161] , and thermal properties [156, 157, 160] . T he mechanical property of polymers with a nanoclay loading of 3 6% can achieve equivalent mechanical properties ( i.e., tensile strength, impact strength, flexural modulus) to a polymer with up to 30 wt% fillers at the microscale ( i.e ., glass and mineral fibers, etc.) [162, 163] . Since clay platelets are considered impermeable to small molecules ( e.g . , gasses, liquids) , and their presence in the polymer matrix extends the diffusion path of small molecules through a tortuous path, nanoclay s can improve the barrier of the nanocomposite [164] . Although a recent study showed that in the case of organic compounds, it is the sorption of the compound to the surfactant added to the nanoclay that modif ies the barrier property of PLA ( i.e ., by modifying the solubility parameters) [165] . The high thermal stability of clays allows their use in polymers for hea t - resistant and flame - retardant applications. Enhancement in polymer thermal stability is affected by the size of clay particles; nanoclays with an aspect ratio (lateral dimension vs thickness) greater than 100 are usually preferred [163] . Well - dispersed nanoclay particles in a polymer matrix can act as both a superior heat insulator [166] and mass transport barrier [167] . Figure 2.13 shows the diameter or thickness of selective micro and nanofiller s that have been added to PLA. 50 Figure 2 . 13 Average scale dimensions of selected fillers in PLA composites : MMT (montmorillonite) [168] , CNT (carbon nanotube) [169] , Ag [170] , sisal [171] , wood [168, 172, 173] , MCC (microcrystalline cellulose) [174] , sepiolite [168] , cotton [1 75, 176] , ramie [177, 178] , MWCNT (multiwall carbon nanotube), graphene, algal [179, 180] , tunicin [181] , halloysite [148] , talc3 [182] , CaCO 3 [183] , talc2 [182] , CaSO 4 [184] , talc1 [182] , glass fiber [185, 186] , abaca fiber [187] , jute fiber [187] , cotton fiber [187] , hemp fiber [187] , and flax fiber [186, 188] . 2.5 PLA I ndustrial A pplications Production of PLA for industrial applications has rise n s teeply due to its competitive cost and the positive public perception environmental footprint. Industrial applications for PLA can be categorized into two main groups: consumer durable goods and consumer non - durable goods. From an economic perspective, consumer durable goods are commodity products with a lifetime of more than three years such as 51 appliances, car s , and medical products . C onsumer non - durable goods are products having a lifetime up to three years such as packaging, short - term m edical items , and serviceware [189] . In some cases the se product categories may overlap , depending on the PLA design . T he next section lay s out the industrial application s for PLA according to commercial usage categories: medical, fibers and textile s , packaging and serviceware, environmental remediation , and others. 2.5.1 Medical S ince the early 1960s , PLA has been used for medical application s su ch as implants and medical devices. PLA found a favorable niche for medical implants since it degrades over time, therefore the removal step of an implant is not required. Also , LA is naturally produced by the body and has no known toxicity effect on human s . Various applications for PLA as medical implants include tissue growth, bone grafting, and fracture fixation devices. PLA is c ommonly used in combination with other polymers and/or protein s, such as polyglycolic acid (PGA), glass fiber, collagen, carbon fiber, and hydroxyapatite (HA) ceramic , to improve its functionality for stabilization of fractures, fixation of tendon s and ligament s , and improvement of mechanical propert ies . On the other hand, degradation of PLA has been reported to lower the pH of ce ll s /tissue s due to the accumulation of LA , leading to inflammation of the in - contact tissue [190] . Zhou and Li [191] reported that a composite of PLA - chitosan could alleviate this inflammation issue, as the presence of chitosan neutralizes the PLA - induced pH sites [10, 191] . In addition, PLA composite i mplants may help treat any organ loss or malfunction by stimulating the growth of the natural cells around the polymer part. The American Society of Plastic Surgeons has recently promoted dermal fillers made of PLA. Such a 52 filler works by stimulating the p roduction of collagen in the human body and is intended for facial improvement [192] . Although extensive documentation is available on the use of PLA c omposites as medical implants, report s on clinical practice using these implants are scarce , which could be due to possible compatibility issue s between the human body and PLA implants. Fast or slow degradation of PLA implants may cause some defense reacti on from the human host. Moreover, the toxicity effect may occur for long - term use [190] . T he use of PLA medica l devices to replace metallic medical devices has been researched for more than a decade. PLA has been sought as an alternative to solve issues associated with metal device implants such as possible corrosion and distortion of magnetic resonance image s [192] . For example, Zimmer Biomet ® , a musculoskeletal health solutions company , produced Bio - Statak ® , a tissue attachment device made of PLLA that is resorbable and was reported to have comparable pullout strength to metal device s [193] . Researchers from the Fraunhofer Institute in Germany in 2010 developed PLA composite screws that are claimed to closely mimic real bone strength as an alternative to titanium surgical im p lants [194] . O ther companies , such as Arthrex TM , Phusis, Gunze, Takiron, and Linvatec , ha ve commercialized PLA medical devices for use as interference screw s , miniplate s , rod s , and suture anchor s . Most of the aforementioned medical devices are made through a drawing process of PLLA with M w > 7 .0 x 10 4 Da [190] . This process helps to strengthen the property of the devices to be as close as possible to real bones , and is achieved as a result of the orientation and crystallinity of PLA . The d rawing process of PLLA also seems to affect the piezoelectricity property of the devices ; t his property is associated with stimulation of 53 bone growth [190] . PLA seems to be a better option than metal, but in the case of bone grafting, PLA has slower effect on bone resorption. Moreover, some mechanisms of PLA degradation in the human body are not fully understood. In 2005, Mitek Sports Medicine launched a bi ocomposite implant known as Biocryl ® Rapide ® , with the claim of superior function over PLA. By 2013, this biocomposite had reportedly been used for knee and shoulder implants in more than 250,000 patients [195] . Additional examples of PLA use in medical applications are provided in the other reviews of this series . A function as medical impl ants. 2.5.2 Fibers and t extile s PLA can be processed into fibers by spinning , as explained in section 2. 3 . 6 . PLA is suitable for fiber applications due to its ability to absorb organic compounds and its wicking propert ies . Since the polymer is fairly polar, it can absorb moisture, which makes PLA a suitable candidate for wipes. For example, Biovation ® developed a PLA single - use antimicrobial wipe. Fraunhofer UMSICHT and FKuR developed water filters based on PLA blend fibers (Bio - Flex ® S 9533) t his blend is repor ted to contain adsorbent carbon made of coconut shells [196] . Since PLA has excellent wicking propert ies , the polymer also can be used for disposable products. F or example, Biovation launched disposable antimicrobial blood pressure cuff shields called Bioarmour TM t his product is composed of 74 wt% PLA and is intended to protect a and provides comfort for patients due to its breathability [197] . Ahlstrom Corporation recently introduced a fine - 54 filament web filter for tea made from PLA fibers wicking ability allows the infusion of the tea flavor into hot water [198] . PLA fibers also are of interest to the automotive industry. Approximately 10% of a vehicle compartment is made of plastic. Various c ompanies , including Ford Motor Company , are looking into environmentally friendly polymer option s for car interior parts such as carpet s , floor mat s , and trim parts. Some companies have started producing parts with different bio - products such as PLA, flax, jute, and cotton . A conference on bio - based materials for automotive applications (bio!CAR) was held in Stuttgart, Germany , in September 2015. However, some obstacles must be solved , such as emission of undesirable odors when the polymer is at high temperature, time span of degradation process es , and moisture effect s towards material s, before PLA can be fully implemented for such applications [199] . Ford Motor Company performed a study comparing PET and PLA - based seat fabrics to investigate automotive requirement properties such as seam fatigue, flammability, resistance to abrasion and snagging. The study found that PLA met most of the requirements for automotive fabrics and had comparab le performance to PET, but failed in the flammability and abrasion tests [200] . Other biopolymers , such as polyurethane and soy - based polymer s, have bee n investigated for their use in the automotive industr y by Daimler AG, Fiat, and the Toyota Motor Group. T hese major car producers are primarily concerned with the durability of the biopolymers [201] . S ome improvements are needed before PLA can replace fossil - bas ed polymers in th e car industry. The use of PLA to replace major synthetic polymer s, such as nylon and PET , in the textile industry is increas ing . PLA textiles are being used by garment industries ( i.e., 55 apparel, homeware). Although PET - cotton blends are a common combination in apparel for established brands like Nike ® , Gap ® , and Under Armour ® , PLA itself is see n as a promising alternative due to its wicking propert ies and breathability, making PLA a comfortable material for apparel manufacturing. The Hohenstein Research Institute tested the use of PLA and PLA - cotton blends in garments , and found that PLA is suitable for sport s apparel due to its thermal insulation and buffering capacity to sweat , among other specifications [202] . PLA had high resiliency when used for making jackets. Also, t he ability of PLA to withstand laundry service with multiple washing was tested and was in accordance with the American Association of Textile Chemists and Colo rists (AATCC) standards. However, some issues are associated with PLA textile s, such as the pressing and ironing temperatures, which are limited to temperature s lower than those acceptable for PET and cotton [202] . The d y e ing and finishing process es for textile s often undergo conditions involving temp erature, pH , and time, thus imposing a challenge for PLA since the polymer is susceptible to degradation under the aforementioned conditions [202] . PLA has good retention and crimp properties, so it is suitable for knitted and embroider ed textile s . Another application for PLA textile is in homeware use such as curtain s , pillowcases, and rugs [203] . Early in 2015, Kansai University and Teijin TM developed a new wearable piezoelectric device to detect " directional changes and arbitrary displacement " t his device is made of laminated PLLA and PDLA [204] . In summary , there is significant potential for PLA to be used by the fiber an d textile industries, but its limitations remain an issue and more development and changes are needed for PLA to compete with existing fossil - based polymers. 56 2.5.3 Packaging and serviceware The use of PLA in packaging and serviceware has largely increased over t he last five years . R esearch is being performed by both academi a and industr y with collaborative works between the two to strengthen the green - packaging market to meet consumer demands for packaging derived from renewable resources. PLA has numerous challe nges for commercial packaging applications due to its limited mechanical and barrier performance. However, PLA package performance ha s been improved significantly by tailoring polymer processing, blending with other polymers, and adding compounds , such as nucleating agents, antioxidants, and plasticizers , to meet the end need s [205] . For example , oriented and non - oriented PLA can be produced by tailoring PLA processing. Oriented PLA has considerable thermal resistance with good clarity over non - oriented PLA. Although oriented PLA films pose desirable characteristics, their brittleness is still of concern due to the fragility and loud noise produced by the packages. Frito Lay introduced a compostable PLA bag for their Sunchips ® b rand in 2010 , but this bag under went major public scrutiny over the loud crinkling sounds during bag handling . The bags were later removed completely from the market [206, 207] . Oriented PLA films are also used for bakery packaging and gift cards [205] . Meanwhile, non - oriented PLA sheets are p referred for use as thermoformed clamshells to package fresh produc ts [208] and other products with short shelf life. These clamshells are still be i ng used to pack some Wal - Mart product s . Other c ompanies have claimed that the shelf life of the packaged fruits is 10 15% longer in PLA containers [209] . However, the low barrier properties of PLA towards moisture and gases may cause limitation s in other 57 applications. Table 2.3 shows examples of products that have been and/or continue to be packaged in PLA containers. Major European markets show ed early interest in the use of commercialized PLA. Danone ® , for exampl e, launched yogurt cups made of PLA for its Germany market, which accounted for 80% of the total volume of their Activia product line [210] . O ther thermoformed PLA products also are available by various companies ( Table 2.3 ). Packages produced from non - oriented PLA, however, are limited to non - heat applications. Some other commercial packaging applications for PLA include shrink films and shrink labels. For PLA to meet the requirement s for these types of applications, it needs to exhibit shrinkage, which is commonly observed for oriented PLA at temperature above 60 ° C with a reported shrinkage ratio of 70% [205] . ConAgra Foods uses recycled PLA shrink film ( produced and supplied by EarthFirst ® ) as tamper - evident seals for its three leading table spread brands : ® , Blue Bonnet ® , and Parkay ® [211] ( Table 2.3 ). T he use of oriented PLA as shrink labels does have a slight limitation since shrinkage ratio is low at around 70 ° C , which result s in whitening of the label due to the crystallization process. Thus, lamination with other polyester films or blending other polymer s with amorphous PLA are used to ameliorate PLA shrinkage properties [205] . Commercial us e of PLA shrink lab els was reported for soft drink products manufactured by S&B Foods, Nisshin Oilio, and Asahi [205] . PLA is used to produce bottles for water and juices ; h owever, this market is not extensive. Common production for PLA bottles is ISBM , as previously explained. A pplication for PL A bottles is limited only to non - carbonated beverage s due to the insufficient creep behavior of PLA and low barrier towards CO 2 (which results in product 58 with a lack of carbonation ) . Vitamore ® carbonated drink in PLA was reported to have shelf life of abou t 6 months with a moderate loss of CO 2 [212] . Further improvements are needed to tailor the b arrier properties of PLA for products with a longer shelf life to expand commercial applications. Some examples of PLA bottle d products for the beverage market are listed in Table 2.3 . Despite numerous efforts by manufacturers in introducing PLA - based bottles into the market, further development is needed to obtain PLA bottles with the required commercial properties to com pete with the established fossil - based polymers . Nevertheless, Coca Cola ® has shown interest in bio - based materials , such as high density poly(ethylene) (HDPE) made from sugarcane molasses , for their Odwalla juice beverage line [213] . Tetra Pak, one of the world's leading packaging companies , recently launched a new bio - based carton made of certified paperboard, and bio - based low - density poly(ethylene) (LDPE) films with bio - based HDPE caps named Tetra Rex ® . Valio, a dairy producer in Finland , is currently used these Tetra Pak ® cartons for Eila lactose - free semi skimmed milk drink for the Finnish market [214] . Production of PLA containers for serviceware applications , such as microwaveable container s and single - use disposable drinking cups , is challenging since PLA is susceptible to heat deformation. For such applications, a higher heat deflection temperature ( HDT ) is desirable as it allows the molten polymer to mold faster and to retain its dimensional shape once the formed polymer is removed from the mold. The HDT of PLA is repo rted to be between 55 and 65 ° C [1, 215] , which is too low for producing thermally stable PLA containers for a non - refrigerated supply chain. Therefore, nucleating agents , such as alkylene bisamide [205] , Ecopromote ® a 59 biodegradable nucleating agent by Nissan Chemical [216] , talc, and PDLA , are often i ncorporated into the PLA [205] . The presence of a nucleating agent helps to induce faster crystallization of PLA, so an increase in HDT can be achieved. For example , Corbion Purac ® produ ce s PURALACT ® lactide serviceware , which is microwaveable and has a comparable impact resistance to acrylonitrile butadiene styrene (ABS) ; production is acheived by manipulating the stereochemistry of PLA with D - lactide monomers , but no further process det ails have been disclosed by the company [217] . SelfEco, a company under VistaTek LLC , has produced party servi ceware items from PLA and its blends [218] . Teknor Apex has developed a PLA series (Terraloy BP - 34001) with improved impact strength and HDT of 135 J . m - 1 and 112 ° C , respectively, which are higher than those of standard PLA (impact strength = 33 J . m - 1 ; HDT = 55 65 ° C ). Teknor Apex claimed that this new compound contains 78 wt% PLA and has the ability for rapid processing with shorter cycle time s [219] . It is likely that thermal resistan t food serviceware made of PLA will become more available in the coming years. 60 Table 2 . 3 Select ed examples of packaging containers produced from PLA Trademark/ Commerciali zed Brand Year Active Improved Functions Applications Remarks Ref . Tenova, Sweden 2003 - curr ent None Shopping bags Bags composed of 45 wt% PLA and 55 wt% Ecoflex [220] Biota®, U.S . 2004 - 2006 None Bottled waters Advertised as biodegradable bottles No longer on market due to company bankruptcy [221, 222] Wal - Mart, U.S . 2005 - current None Strawberr ies , Brussel sprouts Advertised as biodegradable clamshells Among the first company to use commercialized PLA [208] Del - Monte, U.S . 2005 - current None Fresh - cut produce Advertised as biodegradable clamshells [223] SPAR, Austria 2005 - current None Organic pears, apples, tomatoes Advertised as biodegradable thermoformed with flexible PLA lid [209] Hypermarket chain Auchan, France 2005 - current* None Fresh salads Advertised as biodegradable containers [224] Own, U.S . 2005 - current* None Organic salads Advertised as biodegradable containers [225] Pacific Pre - Cut, U.S . 2005 - current* None Freshly prepared salads Advertised as biodegradable containers [226] Vitamore®, Ihr Platz (drugstore chain), Germany 2006 - current None Bottled beauty, energy and memory drinks Advertised as 100% bio - based bottles [212] 61 Table 2.3 Trademark/ Commerciali zed Brand Year Active Improved Functions Applications Remarks Ref. Huhtamaki, Finland 2006 - current None Dessert cups Advertised as biodegradable containers [227] Greenware® , Fabri - Kal, U.S. 2008 - current None Cold drink cups, lids and portion containers 100% biodegradable [228] Noble Juice, U.S. 2008 - current None Organic and non - organic citrus juice bottles 100% biodegradable [229] Apple Inc., U.S. 200 8 - current* None iTunes prepaid gift cards Current status in market unknown [230] Italy 2008 - current None Bottled water 100% biodegradable bottles with PE lids [231] ®, Blue Bonnet®, Parkay®; ConAgra Foods, U.S. 2009 - current Improved shrinkage performanc e Tamper evident seals for tablespreads Made of recycled PLA Claimed to reduce 20% of consumption [211] Reddi - Wip®, PAM®; ConAgra Foods, U.S. 2009 - current Improved shrinkage performanc e Shrink labels for cream whipped topping and cooking spray Made of recycled PLA Claimed to reduce 20% of consumption [211] Shiseido - Urara, China 2009 - current None Bottled shampoo Favorable reception in Chinese market as an environmentally friendly option Bottles are 50 wt % PLA and 50 wt% HDPE [232] 62 Table 2.3 Trademark/ Commerciali zed Brand Year Active Improved Functions Applications Remarks Ref. Wal - Mart; Sams - Club, Mexico 2010 - current* None Small white onion No longer on the market Advertised as biodegradable clamshells [233] Sunschips®, Frito Lay, U.S. 2010 - 2014 Thermal resistance Potato chips bags Bags withdrawn from the market within a year due to loud crinkling noise Original flavor was retained for a while after incident, but is no longer available Bags composed of 94 wt% PLA, 6 wt% adhesive and ink, 0.2 wt% aluminum liner [206, 207] Activia®, Danone, Germany 2010/2011 - current None Yogurt Improved carbon footprint by 25% 43% less fossil resource usage than original package [210] Stonyfield Farm®, U.S. 2010/2011 - current None Organic yogurt multipack cups Cups composed of 93 wt% PLA, 4 wt % titanium dioxide and 3 wt% compounded additives 48% reduction of greenhouse gas emissions [234] 63 Table 2.3 Trademark/ Commerciali zed Brand Year Active Improved Functions Applications Remarks Ref. Polenghi LAS, Italy 2010 - current None Bottled lemon juice Claimed to be first blown extrusion PLA bottle in EU market [235, 236] Ceramis®, Swiss 2011 - current High barrier towards O2, moisture, aroma compounds Snacks (pouches) Fruits and vegetables (thermoforme d) Breads Silicon oxide coating provides excellent barrier for PLA [237] Track & Field, Brazil 2011 - current* None Capsules for athletic apparel Current status in market is unknown [238] PURALACT®, Netherlands 2013 - current Thermoform ed containers able to tolerate boiling temperature Single - use hot bevera ge cups Conversion to PLA packaging line is feasible by using an existing PS line [239] PURALACT®, Netherlands 2013 - current Comparable impact resistance to ABS Serviceware Safe food contact application, and the containers are microwavable [217] * Current (2016) market availability could not be confirmed . 2.5.4 Plasticulture Plasticulture is the use of plastics for agricultural applications. Plastics are used for application s such as i) to protect soils from erosion and plants from weed, insects, and birds via mulch films, ii) to function as drip irrigation tubing, and iii) to cover tunnels of greenhouses ( Figure 2.14 and Figure 2.15 ) . The use of plastics for agricultural applications started in the 19 50s to improve and increase the growth and production of agricultural products [240] . Conventional non - renewable plas tics are the default choice 64 in the plasticulture industry , and poly (ethylene) ( PE ) is the main polymer in use . However, various issues regarding the use of non - renewable plastics are of increasing concern among agricultural personnel and consumers. Among the se issues are cost of waste management, end - of - life options , and consumer demands for more environmentally friendly option s . W aste management handling is expensive , due to the additional labor cost for the removal of conventional plastics after use and associated transportation costs. Also , the end - of - life option is not feasible since landfill soil may become contaminated with pesticide residues from the used plastics. Similarly, recycling is not an option and open burning is illegal in several states in the U.S . [241] . Therefore, biodegradable plastics , such as PLA, poly(hydroxyalka noates) (PHA s ), starch, and poly(butylene adipate - co - terephthalate) ( PBAT ) , are seen as attractive options to help solve these issues [242] . The implementation of biodegradable plastics in the plasticulture industry is still at the early stage and is mostly done at the research level due to the high per - pound cost of the polymer s . The most promising outcome to be expected from the use of biodegradable plastics for plasticulture is that they are able to biodegrade after use. 65 Figure 2 . 14 (left) tomato plots covered with mulch films; (right) high tunnel or overwintering house . Although , as discussed above, PLA has considerable potential for various industrial applications, the use of homopolymer PLA in the plasticulture industry has been limited due to its poor mechanical and thermal properties. M ulch films ( Figure 2.15 ) made wit h PLA alone are deemed insufficient to protect soils and plants due to brittleness. The relatively high T g of PLA and less available amorphous region limit the food sources for microorganisms to initiate the biodegradation process at low temperature s [240] . Consequently, PLA is blended with other biodegradable polyesters to produce commercialized PLA - based mulch fil ms [240] . Commercialized PLA - based mulch films are commonly made with plasticizer s, and those that incorporate LA der ivatives or oligomers demonstrate an accelerated biodegradation process [243, 244] . The accelerat ed biodegradation of plasticized PLA - based mulch films could be attributed to the introduction of free volu me in the PLA polymer matrix , allowing the diffusion of surrounding water into the polymer, thus promoting hydrolysis and, in turn , increasing the accessibility of microorganisms to their food sources. This described 66 phenomenon is called bulk erosion. A not her phenomenon involved in the biodegradation process is known as surface erosion [2] . Details on hydrolysis of PLA - based films in non - medical environments are provided in section 2. 6 . 1. Figure 2 . 15 Cradle to gate, grave , and cradle life cycle flowchart of plastic mulch films. After remov al of conventional mulch films, they can be reused, recycled, incinerated , and/or landfill ed . Biodegradable mulch provid es the same end - of - life scenario routes and also can be composted. 2.5.5 Environmental remediation Removal of contaminants from the environment is known as environmental remediation or bioremediation. Remediation is one waste management method available today to treat water and wastewater by employing mostly sorption and denitrification mechanisms. Theoretically, it is believed that the efficiency of these mechanisms relies 67 on Van de r Waals interaction and electronic affinity between contaminants and sorption/source media [245] . The sorption/source media may be adsorbents such as activated carbon, zeolite, and polymers. Biodegradable polymers , such as PCL, PBS, poly(3 - h ydroxybutyrate - co - 3 - hydroxyvalerate) (PHBV), and PLA , have the potential to be used for environmental remediation [246] . These polymers act either by absorbing the contaminants from any c ontaminated system (sorption mechanism) or by supplying carbon and energy to microorganisms to facilitate the denitrification mechanism. PLA , among other biodegradable polymers , is being investigated for possible use in environmental remediation due to its availability as a raw material and its relatively lower price. One example of commercialized PLA used for environmental remediation is Hydrogen Release Compound (HRC ® ), produced b y Regenesis Bioremediation Products (San Clemente, CA). This product is manufactured in a liquid and a gel - like form intended for controlled - release of LA for certain durations [247] . However, to a certain extent, PLA characterization and research for environm ental applications are limited. T he efficiency of biodegradable polymers for environmental application s relies on their T g . In the case of PLA, its ability to adsorb contaminants would be limited to condition s >60 ° C [246] . In addition, PLA has more resistance to microbial activity than that of other biodegradable polymers like PCL [248] . The resistan ce of PLA toward microbial activity is mainly due to the high molecular weight of PLA ( 2 . 0 x 10 5 Da). T hus, microorganisms need more time to use PLA as their food source ; hydrolysis should reduce the M w to be manageable for the microorganisms to use it. Consequently, lower molecular weight PLA is likely a better candidate for the denitrification mechanism. Studies on PLA with M w < 1 . 0 x 10 4 Da show ed a significant ly 68 greater removal rate of nitrogen than for PLAs of higher molecular weight [2 46] . Meanwhile, the focus for environmental remediation applications seems to lie with other aforementioned biodegradable polymers ( i.e., PCL, PBS, PHBV) due to their effectiveness in adsorb ing contaminants , such as chlorophenols , at room temperature [246] . 2.5.6 Other applications Some other commercialized or potential applications of PLA include paints, cigarette filters, 3D printing, and parts for space exploration [249] . A 3D portable on - board printer was developed by collaborative work of Altran Italia, Thales Alenia Space , and the Italian Institute of Technology for use in space ; the printer was produced with approximately 5.5 kg of PLA. PLA characteristics , such as gl oss iness and multicolor appearance , make PLA one of the main choices for 3D printing. A high accuracy for dimensional part s can be achieved with PLA because it poses less warp behavior than commonly used printing filament material s like ABS [249] . PLA ha s been used to develop tow fibers for cigarette filter s by D.M. Enterprises Pvt. Limited (Hong Kong) to replace cellulose acetate tow , an invention that may help to reduce cigarette litter. Although cellulose acetate is a natural product, its degradation i s relatively slow ; the addition of acid may improve the degradation rate. However, concentrated acid worked better to accelerate the degradation process of cellulose acetate, thus it is not a safe choice for such application s [250] . Therefore, the use of biodegradable polymer s like PLA may alleviate the current degradation issue associated with cigarette filters. Another newly developed application of PLA is as a water - based paint : Fujitsu Laboratories Ltd. developed this product to reduce the level of volatile organic 69 compounds commonly found in solvent - based paints. Isocy anate reactant was used to improve the stability of PLA emulsion and , in turn, the quality for its final application as water - based paint [251] . Additionally, Fujitsu in collaborat ion with Toray Industries, Inc. in 2005, developed a PLA alloy that ha d high heat resistance and flame retardant used to mass produce the Fujitsu's FMV - BIBLO notebook models [252] . Similarly, PEGA D&E of PEGATRON Corp . collaborated with the Plastic Industry Development Center (Taichung, Taiwan) to produce an alloy consisting of both PLA and medical recycled PC for use in consumer electronics [253] . M any newer a pplications of PLA surfaced with the help of existing and newly developed technologies. Most of these applications were achieved through a fundamental understanding of the physicochemical, mechanical, stereo - chemical, and morphological properties of PLA . 2.6 P LA D egradation PLA polymeric parts can be exposed to different environments during their lifetime, which may promote their degradation . Degradation leads to irreversible changes of the polymer until it gradually fails due to the loss of various properties . Such loss of properties can occur under different mechanism s , including chemical hydrolysis, microbial, photochemical, thermal , and enzymatic degradation , which mainly occur by main chain scission or side chain scission [2, 254, 255] . Depending on the application, degradation of PLA can be an advantage or a disadvantage. I n the case of mulch films or contaminated packages, degradation through different mechanism s is one of the advantage s of PLA. In this section, the main mechanism s of PLA degradation in commercial applications are discussed . 70 2.6.1 Hydrolysis Hydrolytic degradation takes place when PLA is exposed to moisture : the ester groups of the main chain of the polym er are cleav ed, resulting in a decrease of molecular weight and the release of soluble oligomers and monomers. The products of the hydrolysis self - catalyze the reaction [256 - 258] . Thus, h ydrolysis of PLA starts by the diffusion of water molecules into the amorphous regions , which in turn initiates the cleavage of the ester bonds. Then, degradation continues in the boundary layer of the crystalline domains [259, 260] . The following reaction shows the hydrolysis of the ester groups of aliphatic polyesters , as i n PLA , in the presence of water: Information regarding PLA hydrolysis in non - medical applications , such as in food packaging, plasticulture, and environmental remediation, is scarce . Only a few studies have been done in different environments and media (other than in medical settings ) in which PLA can be in contact with water during its use , leading to hydrolysis reactions. For example, PLA has been used in plasticulture as mulch films (as show n in section 2. 5 .4 ) where PLA can be affected by a number of abiotic factors , such as temperature, pH, soil moisture , and UV radiation , which all play relevant role s in the degradation . During abiotic degradation, the mulch films are fragmented, the tensile strength of the material weakens , and a slight reduction of M w occurs [240] , then PLA is converted into CO 2 , water , and inorganics . Hydrolysis is one of the mechanisms that help s degrad e PLA in this environment. Furthermore, in other agriculture applications , for example during controlled release of herbicides for stimulating the plant growth and 71 improving yield, hydrolysis of PLA takes place since the material is exposed to high RH or put in direct contact with water [8, 261, 262] . In packaging applications, when PLA is used for fresh produce or beverage containers , it is exposed to humid environments that can trigger hydrolysis reactions. Copinet et al . reported that at high RH the rate of hydrolysis increases due to absorption of water molecules into PLA. As a result , a decrease of the M w was observed, leading to a reduction in T g from 60 to 19.4°C when exposed to 100% RH, and a 50% reduction of of the initial percent e longation at break at 30°C in 15 and 10 weeks when PLA films were exposed to 50 and 100% RH, respectively [263] . PLA containers have been developed to be in contact with water, cold - chain dairy , and juice s, so the PLA is in contact with different environments at different pH and polarity. T he medium pH influences the rate of hydrolysis of PLA - based materials. In strong acidic and basic media, polymer chains are more easily degraded since the hydrolysis reactions are catalyzed by the presence of hydronium and hydroxide ions ( Figure 2.16 ) [264 - 267] . F or example , if PLA is used for a citrus juice bottle, the PLA will be exposed to an acidic medi um (pH <4) making the hydrol ysis mechanism proceed via chain - end scission [257, 268] . In PLA containers used for alcoholic products, ethanol will swell the PLA matrix, act as plasticizer , and increase the chain mobility ; as a result , the PLA will be subjected to solvent induced crystallization (SIC) [269 - 2 71] . T o certify polymers for food contact applications , c ommon food simulants are used for migr ation studies , including 95% ethanol, 50% ethanol , and water for fatty, alcoholic , and aqueous liquid products, respectively [272, 273] . PLA films exposed to alcohol solution s at 40°C under go 72 hydrolysis, in turn causing a large reduction of M n , especially when PLA films are exposed to 50% ethanol [ 274] ( Figure 2.17 ) . Figure 2 . 16 Hydrolytic chain cleavage mechanisms of PLA in alkaline (a) and acidic (b) media Polymer , 42, Jong et al. , New insights into the hydrolytic degradation of poly(lactic acid): participation of the alcohol terminus, 2795 - 2802, [257] . 73 Figure 2 . 17 Ln ( M n ) as a function of time during hydrolysis of PLA films into water, 95% ethanol , or 50% ethanol at 40°C. Temperature also plays a crucial role in the hydrolysis of PLA in non - medical applications. The rate of degradation of PLA increases with temperature , result ing in faster cleavage of the ester bonds [258, 263, 275, 276] . When PLA is immersed in water at 30, 40 , and 50°C, chain scission is accelerated as temperature increase s , and an increment of carbonyl index attributed to the formation of carboxyl groups during hydrolysis is also expected [277] . 2.6.2 Thermal d egradation PLA is susceptible to thermal degradation during processing , leading to a decrease in M w and the rheological and mechanical properties of processed PLA parts . Thermal degradation of PLA can be attributed to the hydrolysis initiated by residuals of water during processing, unzipping depolymerization reaction, random main - chain scission , 74 and intramolecular and intermolecular transesterification ( Figure 2.18 ) [278] . Therefore, drying PLA resins before processing is highly recommended . A number of studies have addressed the complex mechanism of the thermal degradation of PLA. Kopinke et al. [279, 280] proposed that the dominant pathway of the PLA thermal degradation above 200°C is intra - and intermolecular ester exchange, cis - elimination, radical and concerted nonradical reactions. McNeill and Leiper [281] stated that the mechanism is based upon a hydroxyl end - initiated ester interchan ge, non - radical process. Aoyagi et al . [282] and Abe e t al. [283] propose d that PLA not only follow s one mechanism during pyrolysis but this thermodegradation also involve s more than two pathways, such as random scission, unzipping depolymerization , and intermolecular transesterification. Furthermore, changes of activation energies of the thermal degradation process have been reported, wi th increasing weight loss using isothermal methods going from 103 to 72 kJ . mol - 1 , 80 to 160 kJ . mol - 1 and 170 to 190 kJ . mol - 1 involving complex kinetic mechanisms [282, 284, 285] . 75 Figure 2 . 18 Thermodegradation mechanisms of PLA : Poly(lactic acid). Synthesis, structure, prop erties, processing, and applications, Nishida, Thermal degradation, 401 - 412, Copyright (2010), with permission from John Wiley & Sons, Inc. " [286] . Therm al degradation of PLA is a complex phenomenon leading to the appearance of different compounds suc h as low molecular weight molecules and linear and cyclic oligomers with different M w and lactide. Other degradation products have been detected such as CO, CO 2 , acetaldehyde , and methyl ket o ne [280, 281, 285] . Kopinke et al . [280] found that temper ature s above 270°C lead to degradation of PLA and that the formation of acetaldehyde increase s with temperature. However, McNeill and Leiper [281] showed that during degradation temperatur es in the range of 230 440°C, acetaldehyde was formed in the highest concentration at 230°C and then a 76 decreasing effect was observed at 440°C. The decrease in proportion can be explained by the thermal degradation of acetaldehyde, involving chain reaction s to obtain the by - products CH 4 and CO. PLA thermal degradation is influenced by several factors such as initial M w , moisture , and residual polymerizing catalysts [287, 288] . Moisture in the resin, temperature , and residence time in the extruder during processing contribute to the decrease in M n and stress and strain at break due to the dependency of these parameters on M w [289, 290] . The presence of residual metals is a parameter that also causes drastic thermal degradation of PLA. Kopinke et al. [280] show ed that PLA in the presence of residual Sn from the polymerization process leads to a selective depolymerization step producing lactide. Cam and Marucci [291] observed that the presence of residual metals assists thermal degradation in PLA , affecting the onset temperature in the order of Fe > Al > Zn > Sn. Furthermore, the pr esence of stannous octoate catalyst (Sn(Oct) 2 ) in a proportion of 0.5, 1, and 5 wt%, accelerated the degradation of PLA . The presence of 5 wt% of Sn(Oct) 2 , even at the low temperature of 160°C , accelerates PLA degradation [278, 292] . Abe et al. [283] found that Zn catalyze s int ermolecular transesterification to produc e linear PLA oligomers , and selective unzipping depolymerization of cyclic PLA oligomers to produc e lactides. Improv ing the thermal stability of PLA to avoid the degradation of the polymer during processing, via end - protection or using chain extenders , has been studied . End - protection of the hydroxyl group has been assessed due to the mechanism of pyrolysis in PLA by a back - biting reaction, which causes an unzipping depolymerization start ing from the hydroxyl ends of the chains [280, 281, 284] . One method is by the acetylation 77 process, which not only achieves end protection, but also is capable of removing residual metals that accelerate degradation of PLA [283, 288, 291, 293] . Fan et al . [293] studied the relationship between the effects of the acetylation and metal content by acetic anhydride where the stabilization was due to the elimination of residual Sn. On the other hand, chain extenders , such as tr is (nonylphenyl) phosphite (TNPP), polycarbodiimide (PCDI) and Joncryl ® , ha ve been used where the onset temperature of degradation is increased due to the reduction of active sites on the chain end per mass by the production of longer polymer chains [273, 294, 295] . 2.6.3 Photod egradation PLA is exposed to sunlight during its lifetime for applications in plasticulture , packaging containers , or films , thereby inducing plastic degradation due to the low wavelength and high - energy UV radiation . Other applications involve the use of UV i rradiation for sterilization of biomedical and pharma ceutical products. The carbonyl group presen ce in the PLA chemical structure absorbs UV radiation at about 280 nm via n - transition , thus increasing the susceptibility of PLA to photodegradation [296] . Aliphatic polyesters ,including PLA , photodegrade under UV and sunlight exposure via the Norish II mechanism ( Figure 2.19 ) where by chain scission of the main chain occurs and the formation of C=C double bonds takes place along with carboxyl end groups and where the reaction is triggered by the electron transition at C=O [297 - 300] . The main - chain scission of PLA during p hoto degradration occurs randomly ; the photodegradability is higher in the amorphous regions than in crystalline regions, resulting in the reduction of M w w here anhydride groups are formed and decreasing the rate of crystallization [ 299, 301, 302] . When UV radiation penetrates the polyme r , the 78 degradation proceeds via bulk e rosion where the light penetrates the polymer without a significant reduction in its intensity regardless of the chemical structure and the crystallinity of the polymer [302] . Figure 2 . 19 Photodegradation of PLA via Norish II mechanism , a dapted from Tsuji et al. [302] . Other basic mechanisms have been proposed to predict the degradation of PLA products by UV irradiation. Janokar et al. [303] , stud ying the effect of wavelength on PLA photodegradation at a range of 232 500 nm , concluded that photodegradation mainly occurs between 200 300 nm and propos ed two mechanisms. One mechanism leads to breakage of the main chain C - O by a photolysis reaction ( Figure 2.20 a ), and the other leads to the formation of hydroperoxide derivatives and subsequent degradation compounds containing carboxylic acid and diketone end groups by photooxidation ( Figure 2.20 b ). 79 Figure 2 . 20 Mechanisms of photodegradation of P LA , a dapted from Janokar et al. [303] . UV irradiation can have diff erent effect s in PLA. UV irradiation can cause a decrease in M w of PLA ; UV also causes an increase i n M w distribution , which has an effect on mechanical properties , such as the decrease in stress and strain at break, where PLA becomes brittle over time [302 - 305] . F urthermore, a faster degradation takes places when exposure time to UV light increases [301] . The combination of different factor s can affect the degradation of PLA . Copinet et al . [263] studied the effect of temperature and humidity of PLA exposed to UV irradiat i on at 315 nm, where the UV light accelerated the reduction of molecular weight, T g , percentage of elongation at break , and crystallinity at different temperatures and RH. During this study, from the cleavage of C - C bonds of the main chain, two radicals were supposed to be produced ( Figure 2.21 ), and two other s from cleavage at C - O of the main chain ( Figure 2.22 a ), and from cleav age of ester bonds ( Figure 2.22 b ) . 80 Figure 2 . 21 Radicals generated during photodegradation of PLA from cleavage of C - C bonds , a dapted from Copinet et al. [263] . Figure 2 . 22 Radicals generated during photodegradation of PLA (a) from C - O and (b) ester bond cleavage , a dapted from Copinet et al. [263] . Besides UV light, PLA can be exposed to different kind s of radia - irradiation when the material undergoes a - - radiation has be en studied by Balbanalbi et al. [306] using electron paramagnetic resonance (ESR) spectroscopy, where the radicals formed during de gradation were the result from the scission of the ester bonds and hydrogen abstraction from the methane groups of PLA main chain. Birkinshaw et al. [307] examined the - radiation on the molded poly - D,L - lactide , where changes in mechanical properties and reduction in M w were observed , making the sample brittle due to random chain scission of the pol - radiation occurs mainly in the amorphous region of the polymer [308] . 81 2.7 End - of - life S cenarios for PLA According to the 2015 Global Sustainable Development Report, the increasing global awareness of sustainability is noticeably changing consumer preferences, and producers have to adapt to meet those preferences [309] , along with the need of tools that allow the assessment of environmental impacts of materials [310] . Consequently, there is increasing demand for bio - based polymers to replace traditional fossil - based polymers , as the latter are perceived to have higher environmenta l footprint s [3, 5, 311] . PLA is lik ely the most popular bio - based polymer. It is recyclable and biodegradable under industrial composting (IC) conditions through an initial hydrolysis process [2] , and it has been proposed to be used especially in cases where plastics become highly contaminated and are difficult to recover thro ugh recycling such as food packaging and agricultural mulch films. W hile PLA is derived from renewable resources and offer s an alternative disposal route ( i.e ., composting) , there are limitations to its implementation due to the lack of suitable infrastruc ture for sorting, recycling , and/or composting PLA products at their end of life [3] . The European Commission introduced a five - level waste hierarchy in the European Waste Directive 2008/98/EC , which includes: 1) p revention, 2) reuse , 3) recycling , 4) other recovery, and 5) disposal [312] . Likewise, the U.S. Environmental Protection Agency (EPA) has a four - level integrated waste management hierarchy , including: 1) s ource reduction (including reuse), 2) r ecycling (including composting), 3) c ombustion with energy recovery, and 4) d isposal through landfill [313, 314] . The four components are important within the in tegrated waste management system as shown in Figure 2.23 . 82 Figure 2 . 23 Diagram of solid waste management , a dapted from the U.S. Environmental Protection Agency in Advancing Sustainable Materials Management [314] . PLA is a specia l polymer since it can be treated in all levels of the hierarchy, including composting as an end - of - life scenario [315] . However, according to U.S. EPA Advancing Sustainab le Materials Management: Facts and Figures 2013 [314] , about 254,110 thousands of tons of MSW were generated in the U.S. in 2013, from which about 25.5% was recycled, 8.8% was composted, 12.9% was combusted with energy recovery , and 52.8% was discarded in landfills. Packaging and containers comprised the biggest portion (29.8%) of the MSW , from which only about 5.2% of plastic packaging was recovered, mostly PET (24.8%) and HDPE (16%). On the other hand, from the 50 thousand tons of PLA waste that was generated (mostly for plates and cups, packaging, and other non - durable goods), only a negligible amount (less than 5 thousand tons , 10% ) w as recovered through recycling and/ or composting [313, 314] . 83 2.7.1 Source reduction (reuse ) F or new applications of PLA to enter the market, it is important to analyze in more detail the preferred end - of - life scenarios [315] . W aste management hierarchy emphasizes source reduction by designing products, especially packaging, to achieve material reduction (ligh t wei g h t ing) , l onger product l ife, and reuse [313, 314, 316] . Lightweighting of PLA has been of great intere st not only for its economic value but also for waste reduction. Nevertheless, with this approach one must consider the possibility of reduced mechanical functionality of the PLA. E fforts made over the years to produce lightweight PLA materials with good p hysical and mechanical properties include produc ing reinforced PLA with fibers/fillers, PLA composites , and foamed PLA, to name a few. PLA sugar - beet composite materials produced by compression - heating technique s ha ve reduced density compared with PLA ; h ow ever, the mechanical properties of the composite materials were affected negatively with increasing amount s of sugar beet pulp [317] . Peinado et al. [318] fabricated PLA reinfor ced with functionalized s epiolite - aminosilane grafted filler and CFA to produce lightweight PLA with improved mechanical properties. Although the addition of CFA significantly reduced the material density, its mechanical properties were compromised in the absence of the functionalized s epiolite filler. A s ynergistic effect of the CFA and the functionalized s epiolite was reported , where the density of the material was significantly reduced with an improved modulus. Additionally, the application of lightweigh t PLA - cellulose fiber composite was extended to the automotive industry for use as floor - load materials [319] . The composite with 50 wt% fiber fraction demonstrated the highest tensile strength due to the ability of this fiber to form hydrogen bonding network s within PLA matrices. T he 84 composites with 50 wt% fiber fraction with fixed nominal dens ity of 0.2, 0.3 , and 0.4 g/cm 3 met the flexural stress and stiffness for the supportable load floor weight of vehicle specifications [319] . As the market for PLA parts increas e , we expect additional research of technologies to lightweight PLA. I n the case of retail packaging, a wide - scale reuse system for materials h as restricted potential due to the logistics and cost involved in returning empty containers to suppliers [320] . PLA packaging is not the exception. On a much smaller scale, PLA products and packaging could be reused (whether for its primary purpose or not) by households, assuming the PLA products and packaging maintain the desired properties, functionality, and safety. 2.7.2 Recycling Following the hierarchy, the next preferable disposal route of PLA would be recycl ing , which can be either chemical or mechanical [62, 321, 322] . PLA packag ing , such as water bottles or blisters , usually h as low contamination, making recycling a viable route to recover the material [62] . However, as previously mentioned , the lack of infrastructure to collect PLA and the logistics required to recover make it challenging to collect and recycle PLA . The economic cost involved in recycling at the post - consumer level does not usually favor the recovery and recycling of plastics other than HDPE and PET (mostly bottles); these t wo post - consumer resins have a big market demand since they can be used to form new bottles or other products like fibers, clothes, carpets, and textiles [62, 323] . D uring chemical recycling, PLA is hydrolyzed at a high temperature to yield LA, which can be readily polymerized to high M w PLA [62, 321, 322] . The disadvantage of 85 chemical recycling is that it is still complex and expensive [62] . NatureWorks LLC ha ve successfully recycled off - grade Ingeo TM by using chemical recycling [324] . Mechanical recycling (MR) would be the easie st and che apest way to recycle post - consumer PLA , and it involve s recovering, sorting, regrinding , and reprocessing ( i.e., melt processing) the PLA waste [62] . However, there is a debate on whether PLA can be successfully recycled in the current plastics recycling infrastructure due to the contamination of the recycling stream. According to Cornell [323] , for PLA to be mechanically recycled, it must be either completely fungible with existing recycled resins or be available in sufficient quantity to achieve the needed critical mass. On one hand, there are some initiatives to facilitate the recycling of PLA through the existing infrastructure for recovery and sorting. One initiative is to improve the material identification by establishing a new resin identification code (RIC) exclusive to PLA since it currently - 7611 Standard Practice for Coding Plastic Manufactured Articles for Resin Identification ; this category is shared with other uncoded materials such as polycarbonate, ethylene vinyl alcohol, to name a few . Other initiatives are focused on the use of technologies like near - infrared or black light illumination to facilitate the sorting of PLA from the waste stream [325] . Thus, if there are sufficient PLA containers entering the waste stream, recycling entrepreneurs may explore means of recovering and recycling PLA in a cost - effective fashion. Various o rganizations , such as the Bioplastics Recycling Con sortium and Greenplastics Inc. , were formed to develop solutions for post - consumer bioplastic materials [326, 327] . On the other hand, the National Association for PET Container Resources (NAPCOR) and the Association of Postconsumer Plastic Recyclers (APR) 86 have refuted the idea of mixing biopolymers like PLA into the ex isting stream of recycled containers , expressing concerns regarding the cost of separation and processing, increased contamination, and reduced quality of the recycled material [323, 328] . 2.7.3 Composting Biodegradation is consider ed to be nature s way of recycling [4, 329] . PLA is biodegradable under IC conditions starting with an hydrolysis process, in which ultimate PLA degradation results from the action of naturally occurring microorganisms at a high temperature (58 ° C) and 50% RH [329] . Figure 2.24 shows a typical large - scale composting process in which biodegradable materials decompose , resulting in compost, CO 2 , H 2 O , and minerals. There are three indispensable factors for polymer biodegradation to take place: substrate (chemical structure and conformation), environment (temperature, oxygen , and moistur e) and microorganisms (metabolic pathways and enzymes) [330, 331] . 87 Figure 2 . 24 Large - scale commercial composting process . International, 57, Kijchavengkul et al. , Compostability of polymers , 793 - 804, Copyright (2008 ), with permission from Wiley [329] . Biodegradation of polymers (including PLA) usually takes place in two main steps: primary degradation, in which fragmentation of the polymer chain occurs due to hydrolysis or another oxidative reaction, an d ultimate biodegradation, in which the microorganisms assimilate the low M w chains formed ( Figure 2.25 ) [330 - 332] . 88 Figure 2 . 25 Schematic of polymer biodegradation mechanism , a dapted from Leejarkpai et al. [332] . Biodegradation can be evaluated by different analytical t echniques, either in a direct or an indirect approach [333] , but respirometric methods are usually prefer red to evaluate biodegradation of polymers in laboratory settings [330] . R espirometric methods directly measure the consumption of oxygen or the evolution of CO 2 [333] . A number of standards have been developed to define the requirements and the methodologies to assess the biodegradability of plastic materials [4] . ASTM D5338 and ISO 14855 are the main standards describing the measurement o f an aerobic biodegradation of plastic materials under composting conditions by analysis of evolved CO 2 [334, 335] . Table 2.4 shows a basic comparison between these two standards. 89 Table 2 . 4 General requirements to test biodegradation under laboratory conditions an d comparison between ASTM D5338 and ISO 14855 standards [334, 335] , reproduced from Castro - Aguirre, E. [336] . Requirement ASTM D5338 ISO 14855 Apparatus Number of b ioreactors At least 12 At least 9 Volume of bioreactors 2 to 5 L (sufficient headspace) 2 L or higher (sufficient headspace) Aeration Water saturated CO 2 - free Accurate flow rate Dry or water saturated CO 2 - free At pre - set flow rate Sensor Specific sensors or appropriate gas chromatographs Infrared analyzer Gas chromatograph Compost Inoculum Age 2 - 4 months old 2 - 4 months old Homogeneity Sieved on a screen <10 mm Allows addition of structural material Sieved on a screen of about 0.5 to 1 cm Allows addition of structural material Dry solids Between 50 and 55% Between 50 and 55% Volatile solids Ash content < 70% No more than 15% of wet or 30% of dry solids pH Between 7 and 8.2 Between 7 and 9 Production of carbon dioxide Between 50 and 150 mg of CO 2 per gram of volatile solids over the first 10 days Between 50 and 150 mg of CO 2 per gram of volatile solids over the first 10 days C/N ratio Between 10 and 40 Between 10 and 40 Substrate Shape Granules, powder, film, simple shapes Granules, powder, film, simple shapes Surface a rea 2 × 2 cm max. 2 × 2 cm max. Positive c ontrol Cellulose (particle size Cellulose (particle size <20 Negative c ontrol Polyethylene Not required Other Temperature 58 ± 2°C 58 ± 2°C Water content About 50% About 50% Ratio of mixture 6:1 sample (dry s olids) 6:1 sample (dry solids) Frequency of measurement At least daily At least twice per day Test p eriod At least 45 days Not exceeding 6 months Incubation Dark or diffused light Dark or diffused light Oxygen concentration 6% or higher 6% or higher 90 T he aerobic biodegradation of PLA film ( ) in compost was evaluated by using an in - house - built direct measurement respirometer (DMR) following the methodology described by Selke et al. [337] , in which bioreactors containing PLA , blank (compost only) , and cellulose (positive reference) were tested. Figure 2.26 a show s that the PLA film produce d a significantly higher amount of CO 2 than the blank, meaning that microorganisms were able to use the carbon from the polymer for their metabolic processes. The amount of CO 2 produced by the PLA film is comparable with that for the positive reference in the same time period . Figure 2.26 b shows that the PLA film mineralized above 70% after two months of composting. PLA also presented a lag time during the first 3 weeks of the test , which is related to the primary degradation where the M w of the polymer should be reduced to around 9 .0 x 10 3 Da (data not shown) for the microorganisms to start the ultimate degradation or mineralization. Figure 2 . 26 ( a) Amount of CO 2 evolved from blank, cellulose , and PLA film ; ( b) Percentage mineraliz ation of cellulose and PLA film . C omposting would be the optimal end - of - life option for contaminated PLA . However, there are only few existing composting facilities that accept biodegradable 91 plastic materials since most are concerned that biodegradable plastics are not easily distinguishable from conventional pl astics and that quality control is difficult [315] . Simila r to recycling, there is a big challenge for collecting and sorting PLA waste from other MSW so that the PLA can be sent to the composting facilities. Hence, the benefit provided by PLA of offering an additional disposal route (biodegradation or composting) is only realized if PLA is disposed in an appropriate waste management system that uses their biodegradable fea tures [320] ; otherwise, the PLA would accumulate like other plastic materials in the landfill. 2.7.4 Incineration with energy recovery The incineration of waste is not only a volume - reduction practice , but it has evolved to waste - to - energy plants in which energy is recovered from waste materials to produce heat or electricity, followed by the disposal of the fly and bottom ashes . Incineration of waste with energy recovery also reduces the dependency of using fossil resources and other fuel sources. Even though air pollution is often the main concern about incinerat ion, the improvements in gas cleaning techno logy allow the reduction of pollutants released to the atmosphere [338, 339] . Thus, s ome of the energy content of plastics can be recovered by incineration, and reasonable energy efficiency can be achieved throug h various approaches such as co - fuelling of kilns [320] . D isposing PLA waste via incineration recover s the energy embedded in PLA , representing a CO 2 - neutral method of energy product ion , and it contributes to the conservation of fossil re sources [340] . However, energy recovery does not reduce the demand for raw material used in plastic production [320] , and it is also important to consider the composition of the emitted combustion gases [340] . 92 NatureWorks LLC reported that Ingeo TM resin heat content is about 19.5 MJ . kg - 1 [341] . This v alue et al. [340] ( Figure 2.27 ) , who carried out comparative experiments between biopolymers, fossil - based polymers , and fuels . They concluded that biopolymers , including PLA , are suitable for thermal energy recovery since they have calorific values comparable to cellulosic - based materials , and they do not produce additional toxicologically critical substances during combustion [340] . Figure 2 . 27 Calorific values of selected materials , a dapted from mann et al. [340] . 2.7.5 Landfill T he less preferable option to dispose PLA is landfilling (LF) . According to the U.S. EPA, although disposal of MSW to landfill decreased from 145.3 million tons in 1990 to 134.3 million tons in 2013, landfill remains the most economic and attractive method for handling MSW [313, 314] . LF has some environmental impacts primarily due to gas and leachate formation , including health hazards, fires and explosions, vegetation damage, unpleasant odors, landfill settlement, ground water pollution, air pollution , and global 93 warming [342] . T he drawback of disposing plastics in landfills lies in the fact that most plastic materials do not degrade in a practical period of time and end up ac cumulat ing [343] . Landfills usually do not provide the appropriate environment to promote degradation, and their conditions vary considerably by geography [344] . On the oth er hand, PLA biodegradation is highly dependent on temperature and moisture , since these two factors promote hydrolysis of the polymer chains , and in turn accelerate biodegradation. A t mesophilic temperatures little or no degradation of PLA is observed [344] . According to NatureWorks LLC , their resin Ingeo TM is stable in landfill conditions with no statistically significant quantity of methane released. S tudies performed under accelerated landfill conditions at different temperatures and moisture levels found that the amor phous PLA did generate a small amount of methane in the test at 35 ° C, but no methane was generated in the test at ambient temperature . S emicrystalline PLA did not generate a significant amount of methane in any of the tests. The company also pointed out th at it is likely that any degradation of PLA in a landfill would require a chemic al hydrolysis step prior to any biodegradation [344] . 2.8 Environmental F ootprint of PLA T he increasing global awareness of sustainability is changing the perception s and preferences of consumer s ; therefore, environmental assessment tools are being used to evaluate the EFP of systems and products [310] . A n EFP is a quantita tive measurement describing how human activities can inflict different impacts on global sustainability considering the environmental, social, and eco nomic indicators [345] . 94 Life cycle as sessment (LCA) can be used t o evaluate the EFP of PLA. LCA is a method to assess the environmental performance of products and /or potential impacts of a sy stem considering raw materials acquisition, production, use, and disposal [346, 347] . LCA is conventi onally thought of as a - to - grave ; however, in the - to - introduced [345] . Figure 2 . 28 Cradle - to - gate, cradle - to - grave , and cradle - to - cradle representations of production, consumption , and disposal of bio - based polymers from renewable resource via composting. et al. , Compostability of polymers, 793 - 804, Copyright (2008), with permission from Wiley " [329] . 95 In other words, LCA systematically evaluates each of the life stages of a product or product system, in which environmental inputs (resources) and environmental outputs (emission and waste) are produced and the impacts to huma n health and environment are calculated . LCA results are interpreted in relation to the objectives of the study [346, 347] . LCA studies are mostly conducted under the framework of the internationa l standard s ISO 14040 and 14044 [348, 349] , which provide requirements, recommendations, and guidelines about methods and techniques for quantifying inputs and outputs, and impact characterization [346, 347] . T he EFP of PLA resin s and/or PLA products can be evaluated using midpoint impact categories [350] . Additionally, measuring key indicators such as greenhouse gases (GHG) emission s and non - renewable energy use, and comparing the data between PLA and tradition al polymers ( e.g. , PET and PS) can give insights about PLA environmental performance. In 2003, NatureWorks LLC p ublished the first cradle - to - gate life cycle inventory data ( ec oprofile) for its PLA ( Ingeo TM ) based on the 140,000 - t/y pla nt design , in which they provided some information regarding the production technology [351] . I n 2007, the company provided an updated ecoprofile based on the actual data collected from its production facilities , and also provided a more accurate description of the manufacturing system and LCA calculation procedure [352] . I n 2010, NatureWorks LLC publish ed an updated ecoprofile based on the production technology improvements and also benchmarked the results for energy requirements and GHG emissio ns with data for a selection of foss il - based polymers [353] . Recently (2015), the company published an updated PLA ecoprofile providing a detailed description of the production of its resin 96 (now 150, 000 - t/y pla nt) and focused on the corn feedstock used to produce Ingeo TM and on the PLA intrinsic zero material carbon foo tprint , as explained below [5, 345] . O ne of the advantages of using bio - based biodegradable polymers like PLA is to he lp replenish the carbon cycle ( Figure 2.29 ) [329] . W hen using renewable carbon feedstock to manufacture plastic materials instead of fossil carbon fe edstock , there is an intrinsic zero material carbon footprint value proposition ; in other words, the carbon footprint reduction arises from the material itself and not necessarily from the process of converting the feedstock to produ cts (process carbon footprint) [354] . Figure 2 . 29 Carbon cycle of fossil - based polymers and bio - based polymers. R enewable resource pathway (green arrows); fossil resource pathway (black arrows); and pathway for both renewable and fossil resources (gray arrow) , a dapted from Kijchavengkul et al. [329] . Fossil resources could be consider ed renewable, but it takes more than a million years f or biomass to be converted into fossil fuels. Since the rate of consumption is much greater than the rate of r eplenishment, mass imbalance occurs in the carb on cycle. In contrast, biodegradab le polymers made from bio - based materials , s uch as corn and corn starc h , can be produced and converted into biomass in similar time 97 frames [329] . Figure 2.30 show s the global warming potential (GWP), primary energy from nonre newable resources (PENNR), such as oil, gas, coal, and ura nium, and water uptake for 1 kg of I ngeo TM PLA resin [5] . One of the main value proposition s for using PLA to replace other fossil - based polymers, is the lower GWP due to carbon sequestration during the corn - growing stage. Figure 2 . 30 GWP , primary energy of non - renewable resources expressed as higher heating values (HHV), and net water uptake for the production system of Ingeo TM resin , a dapted from Vink and Davies [5] . Several authors have done LCAs regarding the performance of PLA in comparison with other materials like PET and PS for different applications , in which PLA could be a good substitut e for clamshell con tainers, trays, and water bottles [311, 355 - 358] . Table 2.5 and Figure 2.31 show general information about the EFP of PLA in comparison with other polymers. 98 Table 2 . 5 Environmental footprint of 1 kg of selected commercial polymer resins as available in Ecoinvent 3. 2 and reported using Simapro 8.0.5 w ith ReCiP e (E) Midpoint Indicator considering the Wo rld as the geographical region. Impact category PLA Nylon 6 - 6 PET HDPE LLDPE LDPE PP PS Climate change, kg CO 2 eq . 2.7907 7.0460 2.4813 1.6815 1.6055 1.8154 1.7666 2.9690 Ozone depletion, kg CFC - 11 eq . 2.18E - 07 2.61E - 09 1.48E - 07 1.18E - 09 4.75E - 08 1.13E - 09 8.85E - 10 5.46E - 09 Terrestrial acidification, kg SO 2 eq . 0.0218 0.0295 0.0121 0.0064 0.0057 0.0078 0.0062 0.0112 Freshwater eutrophication, kg P eq . 0.0004 0.0003 0.0001 1.23E - 06 9.03E - 07 1.42E - 06 4.33E - 05 3.56E - 06 Marine eutrophication, kg N eq . 0.0065 0.0091 0.0002 0.0001 0.0001 0.0002 0.0002 0.0003 Human toxicity, kg 1,4 - DB eq . 9.4123 1.7760 4.6504 0.4946 0.2917 0.6967 0.4113 0.6617 Photochemical oxidant formation, kg NMVOC 0.0115 0.0205 0.0087 0.0086 0.0065 0.0093 0.0076 0.0096 Particulate matter formation, kg PM10 eq . 0.0063 0.0082 0.0040 0.0020 0.0021 0.0023 0.0019 0.0033 Terrestrial ecotoxicity, kg 1,4 - DB eq . 0.0089 0.0002 0.0018 1.51E - 05 1.13E - 05 2.04E - 05 1.21E - 05 0.0003 Freshwater ecotoxicity, kg 1,4 - DB eq . 0.0090 0.0037 0.0021 0.0004 0.0002 0.0005 0.0003 0.0009 Marine ecotoxicity, kg 1,4 - DB eq . 3.5355 2.5132 2.9761 0.2391 0.1561 0.3246 0.1915 0.9059 Ioni z ing radiation, kBq U235 eq . 0.1398 0.0006 0.0785 0.0002 0.0002 0.0003 0.0002 0.0004 Agricultural land occupation, m 2 a 1.1321 0.0009 0.1035 0.0004 0.0003 0.0003 0.0003 0.0006 Urban land occupation, m 2 a 0.0674 0.0006 0.0147 0.0002 0.0001 0.0002 0.0002 0.0004 Natural land transformation, m 2 0.0004 - 2.04E - 06 0.0004 - 3.77E - 07 - 4.39E - 08 - 7.27E - 07 - 3.98E - 07 - 1.25E - 06 Water depletion, m 3 0.2726 0.2262 0.0714 0.0136 0.0443 0.0176 0.0156 0.0524 Metal depletion, kg Fe eq . 0.1538 0.0046 0.1646 0.0015 0.0019 0.0029 0.0015 0.0108 Fossil depletion, kg oil eq . 0.8246 2.6814 1.5455 1.5908 1.5628 1.5684 1.5716 1.8711 Non - renewable energy, MJ primary 41.739 135.86 73.182 76.398 74.090 78.223 74.636 87.542 99 Figure 2 . 31 Climate change, non - renewable energy, water depletion for 1 kg of PLA and other commercial polymers as available in Ecoinvent 3.2 and reported using Simapro 8.0.5 with Recipe (E) Midpoint Indicator considering t he World as the geographical region , and Ingeo TM a dapted from Vink and Davies [5] . S uch a comparison is effective only if : a) polymer weights in the studied applicat ions are quite similar ; b) con tributions to impact categories are dominated by the polymer - pellet production ; c) energy requirements for converting the polymer into product are relati vely small or relatively similar ; d) use phase is similar ; e) t he same recycling or end - or - life routes are employed ; f) the same level of det ail in the life cycle inventory data - collection process was used ; g) the same LCA methodology was used ; h) the same database for upstream inventory data was used ; and i) the same life cycle impact assessment methodology, indicators, and cha racterization factors (+version) were used [5] . G iven the above , the climate change of Ingeo TM and PLA have large 100 differ ences since carbon sequestration has not been accounted for in PLA. If this factor is tak en in to consideration, a global warming potential of PLA would be 0.9 kg CO 2 eq per kg of resin vs 0.62 kg CO 2 eq per kg of resin for Ingeo TM . Thus a large benefit is obtained using the new reported data for Ingeo TM [5] . In the case of non - renewable energy , similar values are reported by Vink and Davies and the current data are available in Ecoinvent 3.2. I n the case of water depletion, the new values rep orted by Vink and Davies make sure to properly account for water uptake from river and ground for the Blair manufacturing plant , excluding the water for hydropower installations and rainwater. So, a much lower water EFP is reported for Ingeo TM , provid ed th at water consumption is similar to that for polyolefins. A large controversy exi s ts regarding the us e of the arable food land for plastic materials [359 - 361] . Vink and Davies reported , based on Carus [362] , that 0.00046% of the 5 billion hectares of agricul tural land available will be required to supply the corn needed for the 150,000 t /y Ingeo TM production in Blair, and if we imagine a scenario where the 300 million tons of plastics annually produced in the w orld were to be replaced by bio - based polymer s wi th the same land use per kg of PLA, 0.9% of the 5 billion hectares of agricultural land available will be required [5, 362, 363] . An LCA study ha s been done recently regarding the end - of - life options for PLA [315] . In their study, Ros si et al. performed an LCA of the end - of - life options for biodegradable packaging based on t he waste hierarchy mentioned in section 2. 7. Figure 2.32 shows the system boundary, which covers the primary material production and end - of - life trea tment processes such as M R , IC , anaerobic digestion (AD), direct fuel substitution in industrial fa cility (DFS), incineration with heat recovery in MSW 101 incinerator (MSWI) , and LF . Details of the life cycle inventory for each scenario can be found elsewhere [315] . Figure 2 . 32 PLA life cycle and boundary of the studied system . A dapted from Rossi et al. [315] . Rossi et al. [315] used IMPACT 2002+ LCIA method to evaluate the environmental impacts for the different end - of - life scenarios complemented by water withdrawal and turbined water indicators. Global warming impacts for PLA dynamic ally assessed over a 100 - year time horizon are presented in Figure 2.33 , in which IC has the highest net impact (measured in kg of CO 2 eq. per kilogram of dry PLA packaging without food contamination after deduction of the treatment credits ) and MR has the lowest net impact. 102 Figure 2 . 33 Comparison of dynamically - assessed global warming impacts over 100 years associated with the six end - of - life t reatments for PLA. The bars on the left side present production impacts of the resin (cradle - to - gate) for comparison purposes , a dapted from R ossi et al. [315] . Likewise, Figure 2.34 shows the non - weighted scores of the PLA production and end - of - life scenarios for each midpoint category, in which for most impact categories MR was the least - burdening option. On the other hand, IC and LF were the least favorable options for most impact categories. 103 Figure 2 . 34 Comparison of end - of - life options for PLA for each midpoint category , a dapted from R ossi et al. [315] . However, the authors emphasize that those conclusion s are only valid under the stated hypotheses since other factors may lead to different conclusions. Additional s tudies are needed to ensure that this preliminary f i nding can be translated to other boundar y conditions and can be applie d to other regions. 2.9 Final Remarks The range of PLA applications for consumer durable and non - durable goods has increased significantly since industrial methodologies , such as polycondensation and ROP , allowed the production of high M w PLA to reach the market . At present, the main producer of the commercial ly available high M w PLA derived from corn is NatureWorks L LC. Additional producers ( e.g. , Corbion ® ) are expected to reach the market with PLA 104 derived from plant - based materials and/or biomass waste, which should increase its availability and further commercial applications. Additional research has been focused o n understanding and enhancing the physical and mechanical properties of PLA by , for example , deriving commercial PLA from rac - lactide. E xtensive work has been conducted on blend ing PLA with biodegradable and non - biodegradable polymers and on using fillers at the micro and nano scales to create blends and composites with optimal properties, lower cost , and less environmental footprint. All these new variation s of PLA - based materials target enhance d performance of PLA while sometime s at the expense of losing the biodegradability of the polymer matrix and reducing its industrial commercial recover y . Thus, new materials should be produced while keeping in mind that they need to be recover ed by the more preferred route s of the waste management hierarchy ( i.e., source reduction, recycling, composting, incineration with energy recovery, and landfill) . The methods used for PLA mass production are well - established polymer - manufacturing techniques ( i.e., extrusion, injection molding, blow molding, t hermoformi ng, foaming, and spinning) . T herefore, PLA has found extended applications such as fiber s , textile s , plasticulture, serviceware, and pack aging containers via established processing technologies. However, use of different PLA structures may complicate the p erformance and/or use of these methods. Additional research may be needed in the production of the new rac - lactide derived PLA. One of the main value proposition s for PLA is its intrinsic degradation, which can be triggered when PLA is exposed to different environments. Thus, degradation of PLA can be seen as an advantage or disadvantage depending on the application. Extensive 105 research has been conducted on the degradation of PLA in human, processing , and composting environment s . However , additional research is needed to assess the degradation of PLA and its modification when in contact with different solvent s and simulants. Furthermore, the increasing awareness of sustainability is highly influencing consumer preferences towards bio - bas ed polymers, and PLA has the potential to become one of the major commercialized polymers . PLA can be derived from renewable resources , such as regular crops, plant - based materials , and biomass waste, and it could be treated in all levels of the waste mana gement hierarchy. In this regard , however, there are still limitations due to the lack of suitable infrastructure for sorting, recycling , and/or composting PLA products at their end of life. So, efforts should be centered on working with industries, commod ity groups, industry associations, and government groups to improve the recovery rate of PLA. Finally, l ife cycle assessment has been used to evaluate the environmental footprint of PLA , providing useful information about the environmental impacts that PL A may have during raw material acquisition, production, use , and disposal. Robust data exists about the PLA resin production from one producer , NatureWorks LLC . However, sufficient information is missing regarding the use and end - of - life scenario s of PLA p arts. In conclusion , PLA has transcended from a minor bio - based polymer player in the market of commercial fossil - based polymers to be considered as part of a new solution for an increasingly recognized new bio - based economy. 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Insights on the aerobic biodegradation o f polymers by analysis of evolved carbon dioxide in simulated composting conditions , Polymer Degradation and Stability , 137 (2017) 251 271. 138 3.0 Abstract The development of novel biodegradable polymers as a way to create sustainable materials has required the development of methodologies to evaluate and understand the ir biodegradation. In this work , we first provide a critical summary of selected biodegradation tests performed in the last fifteen years for a number of biodegradable materials, providing relevant information about the materials tested, characteristics of the compost used and the method for testing. Then, we report a comparative anal ysis of the results obtained from eight different biodegradation tests performed in simulated composting conditions by analysis of evolved CO 2 and carried out in an in - house built direct measurement respirometer . The materials evaluated for biodegradation were cellulose, starch, glycerol, polyethylene, and poly(lactic acid). Our results along with the information provided in the literature allowed us to identify that one of the main issues of biodegradation test ing is the low reproducibility d ue to the numb er of variables involved in the biodegradation process . It is difficult to provide fair comparisons of samples th at are not within the same test. Therefore , we provide a critical over view of the different factors affect ing the biodegradability, biodegradat ion rate, and biodegradation mechanisms of polymeric materials. Furthermore, we share the experiences and insights gained during the per formance of the different biodegradation tests and identify areas of opportunity for i mproving biodegradation testing th rough evolved CO 2 . This information should create a common knowledge platform for people interested i n studying the biodegradation of materials . 139 3.1 Introduction B iodegradable polymer s represent a prom ising way to reduce the amount of plastic waste disposed in landfills , with composting the preferred alternative for their disposal . Many biodegradable polymers have been developed in the last two decades with the desired performance properties [1 7] for replacing conventional polymers for applications where plastics are highly contaminated and are difficult to re cover through recycling such as agricultural films and single - use products like packaging and disposable cutlery [8,9] . Thus, a long with the development of the se novel materials, evaluation and understanding of their biodegradation performance and their environmenta l impacts have become germane [8 11] . Different analytical techniques have been used to evalua te biodegradation of polymers in composting using a direct or an indirect approach. Even though techniques like visual observations, weight loss measurements, changes in mechanical properties, and changes in molecular weight , can provide insight s into the degradation process of a polymer, they do not necessarily demonstrate biodegradation [12] . Therefore, respirometric methods , in which the con sumption of oxygen and/ or the evolution of carbon dioxide ( CO 2 ) is measured, have become the preferred techniq ue for such assessment . During aerobic biodegradation, microorganisms use the polymer as a source of carbon for growth and their metabolic processes yield CO 2 . The amount of CO 2 produced during metabolic reactions and the fraction of carbon that is incorporated into biomass is a function of the substr ate type and concentration, physical attributes of the environment, species - specific characte ristics of the degradative microbial population s, 140 and population dynamics within a complex community of microbes [8,10,13,14] . In respirometric methods, t he evolved CO 2 can be measured in eithe r a discrete or a continuous way by using different techniques . In cumulative measure ment respirometry (CMR) , the evolved CO 2 is trappe d in a solution , e.g. , sodium hydroxide (NaOH), throughout the test and then quantified by titration [8] . Similarly, in gravi metric measurement respirometry (GMR) , CO 2 is captured in absorption columns filled w ith pellets of NaOH, and the amount of CO 2 is quantified by the we ight increase in the columns [8] . When direct measurement respirom etry (DMR) is used, the output air is directly analyzed using either a non - di spersive infrared (NDIR) sensor or a gas chromatograph (GC) coupled with a thermal conductivity (TCD) detector to quantify the amount of evolved CO 2 [8] . In this context, s everal respirometric systems have been designed and built by different research groups around the world [15 20] f ollowing international standar ds such as ASTM D5338 and ISO 14855 [21,22] . A detaile d list and information about different available standards is provided elsewhere [23,24] . However, performing biodegradation tests is not an easy task ; it is costly, time - consuming , and requires constant attentio n to the proper funct ioning of the equipment. Moreover , due to the biological nature of the process, there are many variables that must be properly controlled and/or monitored. Table 3.1 shows the results of selected tests found in the literature using different methods for assessing biodegradation of materials in compost . The majority of the se tests used CO 2 evolution to tra ck the biodegrad ation of the materials, but some authors have used visual inspection and w eight loss for estimating biodegradation. Table 3.1 141 also prov ides information relevant for biodegradation test s such as the material shape and thickness, molecular weight, and the physicochemical characteristics of the compost used for testing - w henever provided by the authors . However, when comparing the same mate rials, e.g. , cellulose or PLA, there is large variation in biodegradation and the time to reach similar level s of biodegradation among tests . This variation makes it difficult to compare biodegradation values between and within tests. Therefore, further understanding and review of the different factors affecting biodegradation w ould be useful for conducting future biodegradation tests in which the key factors could be more strictly monitored and controlled, reducing such variability. In this work , we r eport a comparative analysis of th e results obtained from eight biodegradation tests of different materials ( i.e., cellulose powder (CP), glycerol (GC), cassava starch (CS) , poly(lactic acid) ( PLA ) , polyethylene powder (PE), and a blend of linear low densi ty polyethylene (LLDPE) and low density polyethylene (LDPE)) performed in simulated composting conditions by the analysis of evolved CO 2 using the same DMR system. The data from published work is critically reviewed and compared with the data from our eight biodegradation tests performed by the evolved CO 2 approach . Finally, w e share insights gained from the different biodegradation test s in an attempt to identify areas in need of improvement and to establish more standardized procedures for researchers interested in studying aerobic biodegradation of polymers by analysis of evolved CO 2 . 142 Table 3 . 1 Selected biodegradation tests in composting conditions reported in the literature and presented in reverse chronological order for 2015 through 1990, including information about the sample s , compost and the ma in methods for assessing biodegradation Sample Materials Form Thickness, mm M n , kDa M w , kDa PI % Biodegr adation Time, d Method for assessing biodegrad ation Characterization of the compost Ref . Dry solids, % Volatile solids, % pH C/N Tempe rature, o C PLA 4042D Film 0.04 - 0.06 150 1.7 CD 30 Visual inspection 45 - 60 4 - 8 45 - 70 [2] CAB 500 - 5 Film 0.04 - 0.06 57 CD >90 PLA/CAB 80/20 Film 0.04 - 0.06 CD 9 PLA/CAB 50/50 Film 0.04 - 0.06 CD >90 PLA/CAB/ PEG 80/20/20 Film 0.04 - 0.06 CD 90 PLA 4032D Film 0.2 217 2 100 28 Weight loss 6.5 58 [25] PLA - PEG Film 0.2 100 28 PLA - ATBC Film 0.2 100 28 PLA - PHB - PEG Film 0.2 100 35 PLA - PHB - ATBC Film 0.2 100 35 Cellulose Paper 0.35 78 115 CO 2 evolution (DMR - NDIR) 24.3 88.9 7.9 20 55 [26] Plastarch Sheet 0.48 51 115 Paper pulp + soy wax Sheet 2.14 12 115 PET + additive Sheet 0.36 1 115 143 Table 3.1 PLA 15 71 110 CO 2 evolution (CMR - Titration) 50.5 29 7.7 3.9 58 [27] LA - EG - MA 10.3 53 110 LA - EG - SA 10.8 51 110 Cellulose Powder 76 45 PHBV - 3 Film 0.01 - 0.08 404 80 110 CO 2 evolution (DMR - NDIR) 52.4 14.5 8.2 14.2 58 [3] PHBV - 20 Film 0.01 - 0.08 324 89 110 PHBV - 40 Film 0.01 - 0.08 324 91 110 PHB Film 0.01 - 0.09 240 80 110 P(3HB, 4HB) Powder 446 90 110 Cellulose Powder 83 110 PBAT (Manure compost) Film 0.04 67 45 CO 2 evolution (DMR - NDIR) 22.9 58 [28] PBAT (Yard compost) Film 0.04 34 45 47.1 PBAT (Food waste compost) Film 0.04 45 45 36 PLA 7000 D Sheet 3 60 80 CO 2 evolution (DMR - NDIR) 46.4 8.4 58 [29] Cellulose Powder 78 80 PLA60/St arch40 Sheet 3 >80 80 PLA90/St arch10 Sheet 3 ~60 80 PLA90/W ood - flour10 Sheet 3 ~50 80 144 Table 3.1 Microcryst alline cellulose Powder >70 45 CO 2 evolution (CMR - Titration) 42 - 52 48 7.6 32 [30] Industrial recycled cellulose Particle size < 2.8 mm >70 45 PLA (Biomer L 9000) Particle size < 2.8 mm 174.2 1.9 >60 80 Wheat straw Particle size < 2.8 mm >70 45 Soy straw Particle size < 2.8 mm >70 45 PLA - Wheat straw (50:50) Particle size < 2.8 mm 132.9 1.8 >60 60 PLA - Soy straw (50:50) Particle size < 2.8 mm 158.3 1.8 >60 60 PCL Particle size < 2.8 mm 171.7 1.6 >60 120 Soy meal Particle size < 2.8 mm >70 45 DDGS Particle size < 2.8 mm >70 45 PCL - DDGS (70:30) Particle size < 2.8 mm 162.3 1.6 >60 100 PCL - Soy meal (70:30) Particle size < 2.8 mm 168.2 1.6 >60 100 145 Table 3.1 Cellulose Powder 72.4 - 82.5 45 CO 2 evolution (DMR - NDIR) 51 45 7.2 58 [17] Potato starch - based tray 80 Weight loss - home composting [31] Starch - based tray with a starch/PC L laminate 80 Pressed wood pulp plate 40 Pressed silvergras s pulp c rate 80 Molded coconut fiber tray 40 Moulded recycled paper pulp tray 40 PLA tray <5 Starch/PC L - extrudate sample <5 PP with biodegrad ability additive <5 146 Table 3.1 PP compound ed with starch granules <5 Weight loss - home composting [ 31] EPI 0 72 Weight loss 45 91.7 6.2 - 8.5 27.9 >50 [ 32] Mater - Bi 27 72 Cellulose filter paper Paper 100 72 Microcryst alline cellulose Powder 74 45 CO 2 evolution (CMR - Titration) 49 28.4 7.2 14.1 58 [4] TPS Powder 73 56 TPDAS6 Powder 66 56 TPDAS30 Powder 56 56 TPDAS50 Powder 45 56 TPDAS70 Powder 26 56 TPDAS95 Powder 6 56 PLA (2002 D) Sheet 1 55 90 CO 2 evolution (CMR - Titration) 48 45.4 7.1 10.4 58 [5] TPS Sheet 1 87 90 PLA/TPS 75/25 Sheet 1 61 90 PLA/TPS/ Coir 52/17/30 Sheet 1 59 90 PLA/TPS/ MA 75/25/1 Sheet 1 57 90 PLA/TPS/ Coir/MA 52/17/30/1 Sheet 1 54 90 147 Table 3.1 PBAT 25w (white) Film 0.03 86.3 >60 120 CO 2 evolution (DMR - NDIR) 40 - 50 58 [6] PBAT 35w (white) Film 0.04 89.3 >60 120 PBAT B (black) Film 0.04 84.4 >60 120 Corn starch Powder >70 120 PLA Sheet 0.3 86 120 CO 2 evolution (CMR - Titration) 52.5 28.2 8.5 58 [33] Cellulose Powder 87 120 PLA bottle (96% L - lactide) 209.3 1.7 84 58 CO 2 evolution (CMR - Titration) 58 [34] Cellulose Powder 86 58 PLA bottle (96% L - lactide) 209.3 1.7 81 58 CO 2 evolution (GMR - MODA) 58 [34] Cellulose Powder 70 55 PLA bottle (96% L - lactide) 209.3 1.7 CD 30 Visual inspection 37 8.5 65 [35] PLA tray (94% L - lactide) 222.7 1.7 CD 30 Cellulose Paper 72 45 CO 2 evolution (DMR - NDIR) 95 63 8.7 10 58 [36] Kraft paper Paper 62 45 Mirel bag 64 45 PLA straws 61 45 148 Table 3.1 Sugar cane plate 60 45 CO 2 evolution (DMR - NDIR) 95 63 8.7 10 58 [36] Corn - based trash bag 60 45 Ecoflex bag 60 45 Polyethyle ne Sheet 2 45 Oxodegra dable bag 2 45 PCL Particle size <10 mesh 50 52 45 CO 2 evolution (CMR - Titration) 52 7.4 43 58 [7] CA Particle size <10 mesh 22 45 LDPE Particle size <10 mesh 36.4 8 45 Cellulose Powder 70 45 PCL/CA 60/40 Particle size <10 mesh 56 45 PCL/CA 40/60 Particle size <10 mesh 65 45 PLA bottle 64 63 CO 2 evolution (DMR - NDIR) 40 - 50 58 [18] PET bottle 3 63 Corn starch 72 63 149 Table 3.1 PLA bottle (96% L - lactide) 209.3 1.7 CD <30 Visual inspection 37 8.5 65 [37] PLA tray (94% L - lactide) 176.8 2 CD <30 PLA container (94% L - lactide) 215.5 1.7 CD <30 PLA/Starc h/PLA Sheet 2.19 78 45 CO 2 evolution (NS) 52.7 65.8 8 28.9 58 [38] Microcryst alline cellulose Powd er 90 45 CO 2 evolution (DMR - NDIR) 10 - 40 52 [19] Starch - polyester 87 45 Starch - PVOH 72 45 Biopol 88 45 CO 2 evolution (NS) 50 - 55 30 7 - 9 10 - 40 58 [39] Kraft paper Paper 80 45 Microcryst alline cellulose Powder 84 45 150 Table 3.1 Notes: Cells without values indicate that t he authors did not report or calculate the se values , f ilms are samples with thickness sheets are sa mples with thickness > 0.254 m m. M n : number average molecular weight, M w : weight average molecular weight, CD: complete disintegration, NS: not specified, PLA: poly(lactic acid), CAB: cellulose acetate butyrate, PEG: poly(ethylene glycol), PHB: poly(hydroxybutyrate), ATBC: acetyl - tri - n - butyl citrate, LA: la ctic acid, EG: ethylene glycol, SA: succinic acid, MA: malonic acid, PHBV: poly(hydroxybutyrate - co - hydroxyvalerate ), PBAT: poly(butylene adipate - co - terephthalate), PCL: poly ( caprolactone ) , DDGS: distillers dried grains with solubles, PP: poly ( propylene ) , E PI: environmental product Inc. containing 3% of totally degradable plastic additive , Mater - Bi: starch/hydrophilic - biodegradable resin blend, TPDAS : thermoplastic dialdehyde starch, TPS: thermoplastic starch, MA: maleic anhydride, CA: cellulose acetate, LDP E: low - density polyethylene, PET: poly ( ethylene terephthalate ) , Biopol: poly ( hydroxy butyrate ) / poly ( hydroxy valerate ) blend, PVOH: poly ( vinyl alcohol ) . 151 3.2 Materials and Methods 3.2.1 Materials C and glycerol (GC) 99+% was purchased from Sigma - Ald rich (St. Louis, MO) and c assava starch (CS) containing 25 ± 6% amylose content from Erawan Marketing Co., LTD ( Bangkok, Thailand) . P olyethylene powder (PE), l ow density polyethylene resin (LDPE 501I) and linear low density polyethylene resin (DOWLEX 2045G) were obtained from Dow Chemical ( Houston, TX) , and poly(lactic acid) resin (Ingeo TM 2003D and 4032D ) from NatureWorks LLC. ( Minnetonka, MN) . Materials were used as received unless specified and t he s ame batch of a compound was used for all th e tests. 3.2.1.1 Material processing and characterization A 70% wt. LDPE - 30% wt. LLDPE blend film ( hereafter referred to as LDPE) was produc ed by blown extrusion with an overall thickness of 0.023 ± 0.005 mm . The number aver age molecular weight ( M n ), weight average molecular weight ( M w ) and polydispersity ( PI ) of LDPE was 20.6 kDa, 92.6 kDa, and 4. 5, respectively; and for PE 2.9 kDa, 22.0 kDa, and 7.6, respectively. Three Ingeo TM 2003D films with different molecular weights ( PLA1>PLA2>PLA3) were obtained by cast extrusion , varying the temperature of processing , with an overall thickness of 0.031 ± 0.006, 0.022 ± 0.003, and 0.034 ± 0.009 mm , respectively . Additionally, a PLA sheet (PLA4) was produced with Ingeo TM 4032D having a n overall thickness of 0.255 ± 0.021 mm. The M n , M w , and PI of the different PLA samples are presented in Table 3.2 . The carbon , hydrogen, and nitrogen content of the different test materials were determined by elemental analysis. 152 More details regarding the film processing, molecular weight determi nation a nd elemental analysis are provide d in the appendices 3A, 3B, and 3C, respectively. Table 3 . 2 Initial M n , M w , and PI of the PLA samples Sample Material M n , kDa M w , kDa PI PLA pellet 95.1 ± 5.8 180.2 ± 6.2 1.9 ± 0.1 PLA1 93.5 ± 15.6 188.9 ± 17.3 2.0 ± 0.2 PLA2 82.9 ± 6.7 170.5 ± 16.8 2.1 ± 0.1 PLA3 72.6 ± 5.7 139.7 ± 2.8 1.9 ± 0.2 PLA4 75.0 ± 1.4 134.8 ± 0.4 1.8 ± 0.03 3.2.2 Biodegradation test The aerobic biodegradation of the materials was evaluated under controlled composting conditions by analysis of evolved CO 2 using an in - house built DMR system , which uses a non - dispersive infrared gas analyzer (NDIR) for measuring the concentration of CO 2 evolved from the bioreactors. Detailed information about the equ ipment and the calculation method is provided in the Appendices 3F and 3G , and elsewhere [40] . Besides compost, CP and PLA pellets were also tested in inoculated vermiculite in the Jan14 test . Similarly, in the Nov15 test, CP, PLA1, PLA2, and PLA3 were evaluated in t hree d ifferent media: compost, inoculated vermic ul i te and uninoculated vermiculite. Additionally, analysis of the reductio n of molecular weight of PLA was performed in these experiments . Table 3.3 shows a summary of the different tests performed, the materials that were evaluated in each test and the type of media used for testing. 153 Table 3 . 3 Biodegradation test, materials , and media used for testing Test ID a Materials tested Media for testing Sep 12 Blank, CP , LDPE Commercial compost Feb 13 Blank, CP , LDPE MSU compost (A) b May 13 Blank, CP, CS, LDPE MSU compost (A) Jul 13 Blank, CP , LDPE MSU compost (A) Ja n 14 Blank, CP, CS, GC, PE, PLA 1, PLA pellets MSU compost (B) , inoculated vermiculite Jun 14 Blank, CP, CS, PE, LDPE , PLA 1 MSU compost (C) Nov 14 Blank, CP, PLA 2, PLA4 MSU compost (C) Nov 15 Blank, CP, PLA 1, PLA2, PLA3 MSU compost (C) , inoculated vermiculite, uninoculated vermiculite a T est ID refers to the month and year in which the test was performed b The compost ID (A, B, C) indicates that the initial compost was obtained from the same compost batch 3.2.2.1 Compost source For th e Sep12 test , Earthgro ® organic humus and manure from Scotts Miracle - Gro (Marysville, OH) was us ed. For all the other tests , manure - straw compost prepared at the MSU Composting Facili ty (East Lansing, MI) was used . Detailed information about the preparation of this compost is provided in the Appendix 3E. In all cases, the compost was sieved on a 10 mm screen and preconditioned at 58°C for a period of 3 d ays before use . Deionized water was added to increase the moistu re content to about 50%. Saturated vermiculite premium grade (Sun Gro Horticulture Distribution Inc., Bellevue, WA) was added to the compost ( 1:4 parts, dry wt . compost) to provide better aeration . 154 3.2.2.2 Compost characterization S amples of the compost from the different tests were sent to the Soil and Plant Nutrient Laboratory at Michigan State University (East Lansing, MI , USA) for deter mination of the physicochemical parameters. The d ry solids (DS) were o btained af ter drying the compost sample at about 105°C to constant mass . The v olatile solids (VS) were o btained by the loss - on - ignition method, in which the residues after incineration at 550°C are subtracted from the total DS . The pH was d etermined i n a 1 :5 compost - to - water suspension. The t otal organic carb on (TOC) was d etermined by calculation from the VS since carbon i s typically considered to comprise about 58 % of the VS [41] . The t otal nitrogen content was obtained by the D umas method [42] , the a mmonium (NH 4 - ) concentration by the salicylate method [43] , and the n itrate (NO 3 - ) concentration by the cadmium reduction method [44] . Subsequent moisture content measurements were done in a moisture analy zer , model MX - 50 from A&D Engineering, Inc. ( San Jose, CA ). 3.2.2.3 Preparation of inoculum solution The solu tion used for inoculation of vermiculite was prepared by combining compost extract with a mineral solution ( Table 3.4 ) at a 1:1 ratio [2 2] . C ompost extrac t was prepared by mixing dry compost with deionized water (20% wt . /vol. ), stirring and let ting sit for 30 minutes followed by filtration through a sieve with 1 mm mesh . 155 Table 3 . 4 Detailed c omposition of 1 L of mineral solution 1 L of Mineral solution KH 2 PO 4 , g 1 MgSO 4 , g 0.5 CaCl 2 (10% sol) , mL 1 NaCl (10% sol) , mL 1 Trace - element solution , mL 1 1 L of trace - element solution , mg H 3 BO 3 500 Kl 100 FeCl 3 200 MnSO 4 400 (NH 4 ) 6 Mo 7 O 24 200 FeSO 4 400 3.2.2.4 Biodegradation in compost The bioreactors were loaded with either 50 0 g (wet wt .) of compost (first experiment) or 4 00 g of compost (subsequent experiments) and mixed thoroughly with 8 g of polymer sample . Film samples were cut to 1 cm 2 pieces and t riplicates of each test material were analyzed. Additionally, triplicates of blank bioreactors (with compost only) were evaluated. To simulate composting conditions, the bioreactors were placed in an environmental chamber set at a constant temperature of 58 ± 2 o C . W ater - saturated CO 2 - free air was provided to each bioreactor with a flow rate of 40 ± 2 sccm (cm³/min at standard temperature and pressure ) . The bio reactors were incubated in the dark for at least 45 d or until the ev olved CO 2 reached a plateau. 3.2.2.5 Biodegradation in vermiculite B iodegradation test s were also carried out with inoculated and uninoculated vermiculite during the Nov15 test in an attempt to avoid the priming effect , which is discussed in s ection 3.3 .3.6 , and to decouple biotic and abiotic degrada tion during the 156 biodegradation test of PLA . In this case , vermicul i te was mixed in a proportio n of 1:4 ( wt .) with the inoculum solution described in section 3. 2.2.3 , and with distilled water , respectively. T he bio reactors were loaded with 4 00 g (wet wt .) of either inoculated or uninoculated vermiculite and mixed thoroughly with 8 g of the polymer . T he bioreactors were then subjected to the t esting conditions described in section 3. 2.2.4 . 3.3 Resul ts and Discussion T his section first present s the physicochemical characteristics of the media (compost and v ermic ulite) that are relevant for the biodegradation test in composting conditions . Then , it provide s a comparison of the results obtained by the analysis of evolved CO 2 approach in the eight tests with the different sample materials . T o better understand and interpret the results of the biodegradation test s , a discussion of each of the factors affecting the biodegradation rate and the biodegradability of the materials is also presented . Likewise, some recommendations based on the literature and on our own experiences gained during the performance of these eight different biodegradation tests and more than 10 years of testing biodegradation of samples are provided to in form future testing . Finally, a case study is presented to gain additional understand ing on the biodegradation mechanism of PLA , one of the most popular commercial compostable biobased polymer s . 3.3.1 Biodegradation: CO 2 evolution and mineralization A n in - house built DMR system (as shown in Figure 3 . 21 ) was used to perform the e ight different biodegradation tests in which temperature, RH, air flow rate, CO 2 concentration , and time were continuously monitored and measured ( Figure 3 . 1 ) . Temperature and pH were stable at 58 o C and 7, respectively. The flow rate of air 157 passing in each bioreactor was adjusted to 40 sccm throughout the testing period. Moisture content was measured periodically in a control bioreactor to determine the amount of water required for adjustment. Detai led discussion of the effect of each physical parameter is presented in section 3. 3 . 2 . Figure 3 . 1 Example of biodegradation test parameters as a function of time. Fitted lines ( ) are included for visual guidance only. Air flow rate, temperature, moisture and pH monitored as a function of time in the May13 test during the first 60 days of testing. 3.3.1.1 Biodegradation in compost The cumulative CO 2 and % mineralization curves obtained from the different b iodegradation tests in compost are presented in Figure 3 . 2 to Figure 3 . 6 . For the data analysis (Appendix 3F) , the amount of CO 2 evolved from each bioreactor was calculated first ( Eq. 3F. 3 ) ; subsequently, the average cumulative CO 2 (Eq. 3F. 5) and % mineralization (Eq. 3F. 7) of each test material was de termined and plotted as a function of time. T he % mineralization represents the relationship between the a mount of CO 2 evolved from the test material and the theoretical amount of CO 2 that can be evolv ed 158 from the same test material; e.g. , in the case of CP , with a 42.5% carbon content ( Table 3 . 8 ) and the introduction of 8 g into a bioreactor, the theoretical ly possible CO 2 evo lution from this material is 12.5 g (denominator of Eq. 3F. 7) . Looking at the CO 2 evolution plots of the different materials , it seems that in general, the samples from the May13, Jul13, and Jun14 tests produced the highest amount of CO 2 and the samples from the Sep12 and Jan14 tests produced the least amount of CO 2 over time. The blank bioreactors produced an amount of CO 2 rang ing from 9.7 to 23.9 g after 60 days of testing , with the Jun14 test having the highest variability ( Figure 3 . 2 ) . Even though all tests were performed under the same conditions, and in most of the cas es using the same type of compost (except Sep12 ) , there is significant difference i n the production of CO 2 between some of the tests. The compost of the Sep12 test produced the lowest amount since it was a different kind of compost and due to the experienced drying conditions as explained in s ection 3.3.2.4 . Figure 3 . 2 Cumulative CO 2 evolution of blank bioreactors in the different biodegradation tests showing large variation of the CO 2 evolved although they were run under the same experimental conditions. 159 T he CO 2 evolved from the blank bio reactors represents the background, so their average is later subtracted from the amount of CO 2 produced by the test sample bioreactors to determine the mineralization , as shown in Eq. 3F. 7 of the Appendix 3F . T he background and the variability value of evolved CO 2 between the blank rep licates have a large influence o n the final mineralization values. For example, looking at the average values of the CO 2 evolution and % mineralization of cellulose ( Figure 3 . 3 ), the May13 and the Jul13 tests produced almost the same amount of CO 2 ; but the calculated mineralization in the Jul13 test was much higher than in the May13 test due to the CO 2 evolution from the blank . Similar ly, the cellulose in the Jun14 test produced a much greater amount s of CO 2 than in the Jan14 test , but the average min eralization values were not very different from each other. Figure 3 . 3 Cumulative CO 2 evolution (a) and mineralization (b) of cellulose bioreactors in the different biodegradation tests. While similar or different CO 2 values were observed, the % mineralization is highly driven by the evolved CO 2 values for the blank test. 160 Ove rall, we can also state that e xcept for the Sep12 and the Jan14 tests the behavior and amount of CO 2 evolved from most of the blank bioreactors is fairly similar (27.5 33.7 g at day 60). W hen accounting for the total background production, the % mineralizat ion varied from 61.8 to 100.8% . These results are comparable to the ones reported in the literature ( Table 3 . 1 ) with % mineralization of cel lulose ran ging 70 100% between 45 120 days. The decrease i n the mineralization curves of the Jan14 and the Jun14 tests indicates that the cellulose biore actors were no longer producing more CO 2 than the blank bioreactors ; a similar behavior was observ e d with the CS , being even more pronounced ( Figure 3 . 4 ). Figure 3 . 4 Cumulative CO 2 evolution (a) and mineralization (b) of CS in the different biodegradation tests. Biodegradation tests show a fas t increase in the mineralization during the first 10 days of testing. A possible explanation of the behavior observed in the mineralization curve of starch ( Figure 3 . 4 b ) is that a t the beginning of the test there is a rapid large increase in the microbial population since materials like starch are readily or easily available for 161 microbial assimilation, but once the se resources are depleted and/or limited, a decrease in the mineralization curve is observed . It should be considered that microorganisms do not only use carbon for generation of energy but also for grow ing [13] . Figure 3 . 4 shows that t he CO 2 evolved from the CS bioreactors ranged from 19.1 to 26.5 g at day 60, while the % mineralization varied from 28.1 to 68.3. Table 3 . 1 shows results for t wo corn starch tests (70 and 72% mineralization after 120 and 63 days, respectively) [6,18] , and two for TPS tests (73 and 87% mineralization after 56 and 90 days, respectively) [4,5] ; however, those sample s did not show the decline in the mineralization curve behavior , as reported in Figure 3 . 4 , based on the figures presented in the respective papers. In some cases, especially in polymers that are not biodegradable like LDPE, negative mineralization values have been reported ( Figure 3 . 5 ) . P hysically the s e values make no sense , but they are possible since they are generated as an artifact when the blank bioreactors produce more CO 2 than the LDPE bioreactors [14] . These negative mineralization values could be attributed to a physical barrier offered by the polymer film , which limits the availability and/or the distribution of carbon and other nutrients for basic microorganism functions. In the Jan14 and Jun14 tests, PE was evaluated to determine if this material, which is in the form of powder and with M n = 2.9 kDa, is more susceptible to biodegradation than the LDPE film. Figure 3 . 5 shows that t he amount of CO 2 evolved from the LDPE bioreactors vari ed from 8.9 to 25.1 g at day 60 , while the maximum mineralization was 6.8 ± 4.8 % . T he amount of CO 2 evolved from the PE bioreactors varie d from 10.6 to 24.5 g at day 60 , while the maximum mineralization was 3.7 ± 2.5 %. 162 Therefore, no significant increase in the biodegradability or mineralization of this material was found. The maximum mineralization of LDPE reported from the literature in Table 3 . 1 was 8% after 45 days. Similarly, Esmaeili et al. (2013) reported a minera lization of 7.6% after 126 days in soil and 15.8% in soil inoculated with a mixed culture of Lysinibacillus xylanilyticus and Aspergillus niger after the same period of time [45] . However, these mineralization values may be attributed to the microbial assimilation of organic ca rbon present in the samples used to modify the material or degradation products formed during oxidation reactions [14] . The influence of the chemical structure, form, and molecular weight of the materials on biodegr adation is further discussed in Section 3. 3.4. 163 Figure 3 . 5 Cumulative CO 2 evolution and mineralization of LDPE (a & b) and PE (c & d) bioreactors in different biodegradation tests. Negative values of mineralization are observed in many tests. Figure 3 . 6 shows t he biodegradation results of the PLA1, PLA2, and PLA3 films , indicating that the initial molecular weight of biodegradable polymer s is highly influential on the biodegradation of PLA . The amount of CO 2 evolved from the PLA1 bioreactors varied from 19.5 to 30.8 g at day 60 , while the mineralization varied from 47.4 to 68%. However, the production of CO 2 and mineralization increased as the molecular weight of PLA decreased, reaching a maximum mineralizati on of 109.1% with the lowest molecular weight . M ineralization over 100% is an in dication of the priming effect, which 164 is attributed to the over - degradation of the indigenous organic carbon present in the compost when testing materials like glucose and its polymers [46] , further discussed in Section 3. 3 . 3 .6 . Figure 3 . 6 Cumulative CO 2 evolution (a) and mineralization (b) of PLA bioreactors in the different biodegradation tests; solid, dashed and dotted lines represent PLA1 (93.5 kDa), PLA2 (82.9 kDa), and PLA3 (72.6 kDa), respectively. T he zero mineralization (or negative in some cases ) at the early stage of the PLA test corresponds to the lag time, i.e., th e period in which the polymer chains are hydrolyzed - cleaved by the presence of water - until a certa in degree of degradation has be en reached and the degradation products become wate r soluble and available for microbial a ssimilat ion [47] . The specific biodegradation mechanism of PLA will be discussed in more detail in s ection 3. 3 . 5. The results shown in this section indicate that the reproducibility between different tests is low, even if the tests were performed in the same equipment, using the same procedures, the same batches of materials, and excluding technical failures. The intrinsic variability in the s e biologi cal tests makes it difficult to provide a fair comparison 165 of samples that are not within the same test , and therefor e to compare the results obtained between and within research groups . Hoshino et al. (2007) performed a round robin test for studying the ae robic biodegradation of PCL and PLA by the gravimetric method in seven countries, and they found that even though the method is effective for testin g compostability of materials on a laboratory scale test there is variation in the results which was mainly attributed to the compost [48] . Other researchers have reported th at the inoculum quality is a source of variability that can affect the results of biodegradation tests [49] . Therefore, all the physicochemical characteristics of the compost must be reported since they may influence the efficiency and the rate of the biodegradation process . Based on the literature review and data provided in Table 3 . 1 , we observed that in some papers, including previous papers from our research group, authors have reported only 3 or fewer parameters of th e compost. W ithout a more extensive reporting of these parame ters the final results and conclusions may be in complete or misleading . In this context, it would be relevant to further understand the different factors affecting the biodegradation rate and biodegradability of the sample materials, so in future biodegradation tests such factors can be strictly monitored and controlled in an attempt to impro ve the reproducibility of the test results. The physicochemical characteristics of the media used in the eight different biodegradation tests are shown and discussed in more detail in section 3. 3 . 3 . 3.3.1.2 Biodegradation in vermiculite The biodegradation of CP and PLA samples was evaluated in inoculated vermiculite in the Jan14 and the Nov15 tests, and also uninoculated vermiculite in the Nov 15 test. The 166 results are shown in Figure 3 . 7 and Figure 3 . 8 . T he production of CO 2 from the blank bio reactors was very low , allowing better detection of the CO 2 signal from the sample bioreactors. During the Jan14 test ( Figure 3 . 7 ) , the mineral solution described in Table 3 . 4 was not provided in the compost extract used for inoculation ( s ection s 3. 2 .2.3 and 3. 2 .2.5 ), and PLA was tested in the form of pellet s as rec eived from NatureWorks LLC . The PLA pellet bioreactors produced 5.6 ± 0.4 g of CO 2 and reached 34.5 ± 2.8 % mineralization after 60 days of testing, while in compost they produced 19.0 ± 0.8 g of CO 2 and reached 39.2 ± 5.5 % mineralization in the same period of time. Similarly, cellulose bioreactors produced 4.9 ± 0.5 g of CO 2 and reached a mineralization of 35.3 ± 3.9 % after 60 days of testing, while in compost cellulose produced 18.7 ± 0.7 g of CO 2 and reached 44.3 ± 5.9 % mineralization in the same period of time. Figure 3 . 7 Cumulative CO 2 evolution and mineralization of CP and PLA tested in inoculated vermiculite in the Jan14 test. Lower values of evolved CO 2 are seen when compared with compost tests, as expected. The PLA films with three different molecular weights ( Table 3 . 2 ) were evaluated in the Nov15 test ( Figure 3 . 8 ) in which the inoculation of vermiculite was perform ed as 167 described in s ection s 3. 2.2.3 and 3. 2.2.5 . In this case, the cellulose bioreactors produced 7.3 ± 0.4 g of CO 2 and reached a mineralization of 60.2 ± 3.3 % after 60 days of testing, while in compost they reached 95.7 ± 12.1 % mineralizati on in the same period of time. PLA1, PLA2, and PLA3 produced 5.2 ± 0.6, 8.6 ± 0.9, and 7.2 ± 0.3 g of CO 2 , and reached mineralization of 34.6 ± 4.4, 58.3 ± 5.8, and 48.5 ± 1 .8%, respectively , after 60 days of testing . The mineralization in compost of these test materials was found to be 63.3 ± 6.7 , 67.6 ± 7.1 , and 91.5 ± 7.0 % at day 60. N o significant CO 2 evolution was found from the samples tested in uninoculated vermiculite , as expected. Figure 3 . 8 Cumulative CO 2 (a) and mineralization (b) of CP, PLA1, PLA2, and PLA3 in the Nov15 test. Solid line, dashed line, and dotted line represent compost, inoculated vermiculite and uninoculated vermiculite, respectively. Large difference in CO 2 production can be observed bet ween evolved CO 2 in inoculated and uninoculated vermiculite. Even though t he biodegrad ation in inoc ulated vermiculite seems to be s lower , the evolved CO 2 from the background is much lower and more stable. The use of 168 inoculated vermiculite has proven to be an excellent way to test biodegradation although with un realistic estimated biodegradation times. F or example, the mineralization values of PLA1 and PLA2 ( Figure 3 . 8 ) are basically the same in either compost or vermiculite towards the end of the test ( 130 d). T he % mineralization of PLA3 in vermiculite looks more similar to that of PLA1 and PLA2; while in compost it was believed to exhibit a priming effe ct since the mineralization was over 100%. Furthermore, the higher mineralization reached by cellulose in the Nov15 test when compared with Jan14, could be due to additional supplementation of a mineral solution in the Nov15 test , which provides the basic nutrients required by the microorganisms to grow and multiply efficiently. In the case of PLA, other factors like molecular weight also play a role in the biodegradation process as discuss ed in s ection 3. 3 .4 . 3.3.2 Environment - related factors affecting biodegradation The purpose of biodegradation as a disposal route of polymers is the total breakdown of the ir molecular structure and their complete assimilation back into the environment by the action of naturally occurring microorganism s like bacteria, fungi, and algae in a reasonable time frame (months to a few years) [14] . However, environment al conditions such as temperature, oxygen , and water availability play a crucial role in the biodegradat io n rate and biodegra dation mechanism of a material . 3.3.2.1 Microorganisms The amount and type of microorganisms present in the compost play a crucial role in the biodegradation of materials. As previously mentioned, environmental conditions like temperature, ox ygen, water, pH, and nutrients can affect the kind of microorganisms 169 prese nt in the media and strongly influence their metabolic pathways, grow th and survival [50] . In industrial composting, the microorganisms that predominate are mesophiles and thermophiles depending on the composting stage [51] , while in laboratory controlled composting conditions the microorganisms that prevail are mo stly thermophiles since the temperature is usually kept constant at 58 ± 2 o C. The microbial community in the compost is m ainly formed by bacte ria, fungi and possibly archaea and viruses. B acteria are thought to be the major microbial domain responsible for the biodegradation process and bacteria belonging to the Bacillus species are more predominant in the thermophilic stage of composting [5 1] . A number of studies have been conducted to i dentify the microbial consortia present in the compost environment [52 54] , and some have report ed the isolation and identification of several species capable of the b iodegradation of PLA [55 63] , and other polymers [45,64 71] . The isolation of thes e bacteria ha s been done using selective enrichment and clear zone formation, in which the specific polymer was provid ed as the sole source of carbon. Further classification and identification of the isolated microbial strains has been performed by 16S r RNA sequence analysis [72,73] . However, few studies have use molecular ecological techniques and next ge neration sequencing which allows the identification of the vast microbial diversity present in the compost including the uncultured microorganisms that may also play a crucial role in the biodegradation of polymers [72] . For example, terminal restri ction fragment length polymorphism (T - RFLP), a cultivation independent technique used for comparative community analysis, can be used to monitor changes in complex microbial communities over time [74,75] . R ecent studies have also shown the potential of metaproteomics to provide direct 170 information about the microbial activity and the metabolic pathways occurring during the composting process [54] . The refore, the se novel techniques could be used along with biodegradation tests to gain insight into polymer biodegradation mechanisms and metabolic pathways. The structure and diversity of microbial communities present in the soil are not likely to be the same in different regions of the world [50] , which in turn may lead to different results when testi ng biodegradation of materials. Guo et al. (2010) have suggested the use of a specific microbial community to evaluate material biodegradability in a shor ter period of time and improve the reproducibility of the results; such a community contain ing 20 selected microbial strains capable of degrading at least 14 types of bio degradable materials including among them starch, PLA, PCL, PHBV, and PVOH [73] . However, f urther studies with solid med ia, e.g. , vermiculite, in composting conditions are required to prove the improved reproducibility of the results. 3.3.2.2 Temperature D epending on the temperature, the microbial population s present in the media can be predominantly mesophilic or thermophilic. Usu ally, temperatures in the range of 54 - 60 °C are considered optimal for composting since this favors the thermophilic compost microorganisms. Moreover , elevated t emperatures can accelerate reactions like hydrolysis. Temperatures above 60 °C would kill several microbial species and contribute to a faster drying of the compost , l imiting the biodegradation rate [51,76] . Even though s ome authors have performed biodegradation studies using temperature profiles to simulate real composting, the recommendation is to keep it constant at 58 ± 2 o C if the purpose is to reduce the amount of time required for testing [39] . Figure 3 . 1 171 shows that the temperature is one of the simplest par ameters to control in simulated compost ing tests . 3.3.2.3 Oxygen availability D epending on the oxygen avai lability, biodegradation can be aerobic or anaerobic [23,76] . Composting is a predominantly aerobic process in which microorganisms use oxygen to oxidize the carbon from the organic materials and produce CO 2 , water, compost and heat [12,23] . Therefore, a continuous flow of air must be provided to ensure that aerobic conditions are maintained within the bioreactors [22,77] . I t is recommended to set the air provided to each biorea ctor to an optimal value; if the air flow rate is too low, oxygen becomes a limiting factor slowing down the biodegradation process. Conversely , high air flow rates can also be problematic in that it contribute s to faster drying and cooling of the compost that also slows down the biodegradation process by decreasing water availability and temperature [76,77] . To determine the optimal air flow rate, it should also be considered that increasing the air flow rate decreases the concentration of CO 2 in the respired air stream and therefore the air flow rate for the test should be establish ed as the one that allows the CO 2 concentration to be within the limits of the NDIR sensor [17] . In our system , the optimal air flow rate was found to be 40 sccm , and it was determine d after a series of trial tests in which different known concentrations of CO 2 were injected into the bioreactors and different air flow rates were use d for measurement of CO 2 with the NDIR sensor , as shown in the Appendix 3G. 172 3.3.2.4 Water availability W ater availability is essential for the biodegradation process and usually moisture contents between 50 and 60% are preferable [51] . W ater is a distribution medium for m icroorganisms and nutrients ; it infl uences the microbial development and metabolic activity ; and it is an important factor affecting the biodegradation rate [76] . For example in the Sep12 test ( Figure 3 . 9 ) cellulose produced ~ 16 g of CO 2 ( Figure 3 . 9 a ) and reached 59% mineralization ( Figure 3 . 9 b ) after 45 days of composting, and in the Nov15 test the same amount of cellulose produced about 26 g of CO 2 ( Figure 3 . 9 c ) and reached 93% mineralization ( Figure 3 . 9 d ). T he difference in the biodegradation rate of the samples tested in the Sep12 and the Nov15 tests was mainly attributed to the water availability, assuming that the type of compost did not greatly influence this particular behavior. The effect of the compost characteristics is explained in Section 3. 3.3 . In the case of the Sep12 test ( Figure 3 . 9 a and Figure 3 . 9 b ) , water was not added at the beginning of the test, i.e. , the moisture content depended only on the availability of water on the water - saturated a ir supplied to the bioreactors. A fter day 60 , when t he compost experienced considerable drying, distilled water was injected into each bioreactor every three days , clearly increasing the biodegradation rate and allowing the reestablishment of a healt hy microbial population, as suggested by the Birch effect . Birch (1964) demonstrated that alternate drying and rewetting of s oil results in stimulated mineraliz ation of the soil organic matter ( i.e., higher release of CO 2 ) due to a rapid increase of the microbial activity in response to the water availability [78,79] . 173 Figure 3 . 9 Cumulative CO 2 of blank and cellulose and % mineralization of cellulose of two different tests Sep12 test (a & b) and Nov15 test (c & d), respectively. The biodegradation process of cellulose was more homogeneous and more efficient in the test in which water was added t wice a week seeing as a high % mineralization in a short period of time. The a ddition of water is therefore necessary throughout the testing period ; water - saturated air helps to prevent excessive drying of the com post, but it is in general not sufficient b y itself t o maintain the moisture content at t he level required for the test . Thus, in the case of the Nov15 test ( Figure 3 . 9 c and Figure 3 . 9 d ), as well as all the other tests, water was added from the beginning of the test to each bioreactor every 174 three days . The amount of water added was determined by first measuring the moisture content of the compost in the control bioreactors with a moisture analyzer and then calculating the amount of water required to increase the moisture content to 50%, based on the in itial dry weight of the compost . C urrently, a soil moisture sensor has been integrated in to a bioreactor for constant monitoring and easier determination of compost moisture . Other researchers have determined the amount of water required by weighing each b ioreactor and then adding enough water to restore the initial weight [15] . Likewise, other researchers have collected the water condensate from each bioreactor and returned it to t he bioreactor to keep the moisture level s constant [17] . H owever, these methods can be complicated for some equipment settings or when there is a large number of bioreactors. W ater is vital for the function of the composting process; however, excessive water leads to a reduction of the airspace within the compost matrix causing oxygen limitation or anaerobiosis [77 ] . In this context, it has been recommended that inorganic structural materials like vermiculite to be added to the compost, to provide increased porosity and help maintain aerobic conditions [22] . Furthermore, it is recommended that bioreactors are regularly shaken ( e.g. , every three days) to homogenize the contents and to prevent the compost sticking together and clogging [17,19] . For example, if water is added to a bioreactor without mixing, then it is likely to have moisture variability throughout the compost that would result in zones with limited wate r for the biodegradation process. Some authors have also found that the addition of water and shaking ( material mixing ) help restore 175 favorable conditions for biodegradation increasing the biological activity of the compost [80] . 3.3.3 Inoculum - related factors affecting biodegradation T he composition o f the compost plays an important rol e in the biodegradation rate since, besides the microorganisms, it should provide the essential nutrients required for the microorganisms to grow and efficiently multiply. Previous research ers have shown that d ifferent r aw materials such as manure, yard, and food wa ste have different physicochemical parameters and also different microbial activity , consequently producing different amounts of evolved CO 2 [28,49,81 83] . The media used in the Sep12 test was commercial compost that according to the manufacturer was made of 90% organic materials (humus) and 10% manure, while the media for the other tests was taken from different piles at the MSU Compo sting Facility , comprised of a 1:1 mixture of manure and straw. Therefore , the lower evolved CO 2 in the Sep12 test ( Figure 3 . 2 ) could also be attribut ed to the type of com post as previously demonstrated [28] . Table 3 . 5 shows the physicochemical characteristics of the several compost media used in the different biodegradation tests in compariso n with the values recommended by the ISO 14855 - 1:2005 standard [22] , which in turn are mostly based on quality standards and guidelines for compost maturity and stability found elsewhere [84,85] . 176 Table 3 . 5 Characteristi cs of the compost samples for each te st and requirements according to ISO 14855 standard Parameters ISO b Sep12 Feb13 May13 Jul13 Jan14 Jun14 Nov14 Nov15 Dry solids, % 50 - 55 57.4 54.9 46.3 N/A 53.3 52.7 41.5 60.9 Volatile solids, % <30 23.1 67.6 44.6 N/A 26.4 44.3 43.2 39.1 pH 7 - 9 7.6 8.9 9.1 N/A 7.8 7.9 8.5 7.4 Total Carbon, % N/A a 13.4 39.2 25.9 N/A 15.3 25.7 25 .1 22.7 Total Nitrogen, % N/A a 1.1 2.3 1.1 N/A 0.9 2.4 2.4 2.1 C/N ratio 10 - 40 12. 4 17 .0 22.9 N/A 17.4 10.8 10 .3 10.9 Compost activity c 50 - 150 49.6 35.7 73.2 N/A 39.0 81.1 63.0 62.5 a Not applicable or not available b Values based on ISO 14855 - 1:2005 standard c Average values measured in mg of CO 2 per g of VS in the first 10 days 3.3.3.1 Dry solids and volatile solids The DS of the compost used in the different tests varied from 41.5 to 60.9%, which means that in most of the cases the initial moisture content was within a reasonable range [51,86] . Likewise, the VS of the compost used in the different tests varied from 23.1 to 44.6% of the DS, except in the Feb13 test in which the VS were particularl y hi gher (67.6%) perhaps because the compost was not mature enough at the time or because in this particular test the analysis of the compost was done before mixing with vermiculite. From Table 3 . 1 , considering the tests in which the biodegradation of cellulose was more efficient and in which the physicochemical parameters of the compost were provided, the DS and VS range d from 49 - 52% and 28 - 48%, respectively. The VS are an indication of the organic matter (OM) present in the compost, considering that other non - organic compounds ( e.g. , carbonates and structural water) may be los t after ignition at 550 o C; the portion of organic c arbon is typically considered to be 50 - 58% of the VS [51,87,88] . T he usual recommendation i s to keep the VS low since a high amount of O M may favor the priming effect , or the microorganisms may 177 prefer it over the test material especially when testing more resistant materials like hydrophobic polyesters [49] . 3.3.3.2 pH In all of t he tests , the pH was within the range 7 - 9 suggested by the ISO 14855 standard [22] . O ther composting guidelines tolerate broader initial pH ranges (5.5 - 9) due to the natural buffering capacity of the compost and the wide range of microorganisms inv olved in the process [51,86] . However, a neutral pH is preferred for the survival and full activity of the microorganisms [76] . Lauber et al. (2009), showed that the microbial community diversity is highest in soils with neutral pH [50] . A n acidic pH c an cause inhibition while an alkaline pH is usually associated with loss of nitrogen as ammonia (NH 3 ) and odor problems [51,88] . From Table 3 . 1 , considering the tests in which the biodegradation of cellulose was more efficient and in which the physicochemical parameters of the compost were provided , the pH ranged from 7.2 to 7.7, mostly neutral. 3.3.3.3 C/N T he C/N of the compost used in our different biodegradation tests ranged from 10.3 to 22.9. The values reported in the literature ( Table 3 . 1 ) , considering also the tests in which the biodegradation of cellulose was more efficient and in which the physicochemical parameters of the compost were provided, varied between 14 and 43 ; wh ich are basically within the wide range suggested by the ISO 14855 standard [22] . In our case, we obtained good results when using the compost piles with C/N of ~10 and ~23; h owever, other authors and composting guidelines have suggested different C /N, e.g., Bernal et al. suggested the C/N to be below 12 [89] , Daryl et al. 178 below or equal to 25 [85] , the Woods End Research Laboratory Incorporate below 17 [88] , the Ontario Compost Quality Standard below 22 [86] , the California Compost Quality Council below or equal to 25 but ideal of 10 [87] , Stoffella et al. mentioned a reasonable range of 20 - 40 and a preferred range of 25 - 30 due to the large variation depending on the starting feedstock materials of the compost [51] . A list of the C/N of the different feedstock materials can be found elsewhere [51,82] . Despite the difference in C/N suggested by different sources, all agreed that high C/N slow ed down the biodegradation rate since N was assumed to beco me a limiting factor for microbial growth while a low C/N cause d excess N to be converted to NH 3 and to volatize , which is also not desirable as discussed earlier [51,88] . Huang et al. studied the effect of C/N on composting and found that a pile with an initial C/N o f 30 had a more efficient composting process by ach ieving maturity faster than one with C/N of 15 [90] . Figure 3 . 10 shows that the C/N of the compost vs. time in the blank bioreactors of the Nov1 4 test slightly decreased, though not significantly. 179 Figure 3 . 10 C/N of the compost as a function of time during the Nov14 test. The fitted Besides the mineralization of carbon, o ne of the mos t important microbial processes is the mineralization of nitrogen [91] ; microorganisms require nitrogen for their cell matter [54] . U nder aerobic conditions, organic nitrogen is transformed into NH 3 or NH 4 + during ammonification , subsequently into n itrites ( NO 2 - ) and finally into NO 3 - during nitrification [52,54,91,92] . In this context, nitrogen mineralizat ion has been proposed to be used as a bio - indicator to evaluate the impact of biodegradable polymers in soil by measuring the concentrations of NH 4 + , NO 2 - and NO 3 - during the biodegradation process [91,92] . Mature compost is expected to have app reciable amounts of NO 3 - [88] . The concentrations of NH 4 + and NO 3 - as a function of time during the Nov14 test are show n in the Appendix 3I. 3.3.3.4 Compost activity The ASTM D5338 and ISO 14855 standards recommend the compost to produce between 50 and 150 mg of CO 2 per gram of VS over the first 10 days as a measure of 180 the compost microbial activity [21,22] . Figure 3 . 11 shows the production of CO 2 per gram of VS of the compost media used in the different biodegradation tests. Figure 3 . 11 Microbial activity of the compost measured as the production of CO 2 per gram of VS. Variation between 30 and 80 mg of CO 2 per gram of VS is seeing at 10 d. From Figure 3 . 11 , the compost used in the Sep12 test was in the lower limit at 10 days, while the compost from the F eb13 and the Jan14 tests did not produce the 50 mg minimum until about 13 days. The compost from all other tests (May13, Jun14, Nov14, and Nov15 tests) produced an amount of CO 2 within the suggested range ; and based on Table 3 . 5 , these active compost s had similar amount s of VS (39.1 44.6%), amount s of carbon (22.7 25.9%), C/N (10.3 10.9), and pH (7.4 8.5), except that the May13 tes t compost had a higher C/N and pH (22.9 and 9.1, respectively). However, e ven though the Feb13 and the May13 tests belong to the same compost pil e A, they display a different activity. A similar situation occurs with the Jun14, the Nov14, and the Nov15 tes ts that belong to compost pile C. 181 W hile the compost activity (CO 2 production in the first 10 days) is only a recommendation in the ASTM D5338 - 15 standard, it is required in the ISO 14855 - 1:2005 for the validity of the results. This criteria seems to be based on the composting standards and guidelines for determination of compost stability, which is the rate or degree of OM decomposition [85] . Ge et al. (2006) state that for compost to be considered stable, it should have a CO 2 evolution rate less than or equal to 4 mg of carbon in the form of carbon dioxide per gram of VS per day (mg CO 2 - C g - 1 VS d - 1 ) [85] ; the California Compost Quality Council requires the compost to produce 2 - 8 mg CO 2 - C g - 1 VS d - 1 [87] ; the Woods End Research Laboratory Incorporated classifies compost stability based on the mg of CO 2 per gram of VS per day produced as follows: high (<1), medium - high (1 - 4), medium (4 - 8), medium - low (8 - 13), and low (>13) [88] . In this context, it is important to mention that starting a biodegradation test with a large num ber of samples requires considerable resources and preparation time . Considerable loss is incurred if the experiment is discarded because the compost does not produce the 50 - 150 mg of CO 2 per gram of VS over the first 10 days as required b y the ISO 14855 - 1:2005 standard. In this scenario , it is more important to consider if the compost is stable enough so the blank bioreactors, which are the background, do not produce large amounts of CO 2 that can hinder measurement of the CO 2 evolved from the bioreactors containing the test materials . I n fact, a low production of CO 2 from the background is desired to improve the sensitivity of the measurement. 3.3.3.5 Other nutrients In general, it is assumed that with a reasonable C/N , all other nutrients require d by the microorganisms are available in sufficient quantities [51] . Table 3 . 6 shows the 182 physicochemical characteristics and the total nutrient analysis of the three different media used in the Nov15 test: compost, inoculated vermiculite, and uninoculated vermiculite. The physicochemical parameters of compost and vermiculite media are quite different. Vermiculite is a clay mineral with excellent water holding capacity, while compost is a more heterogeneous and complex matrix which contains additional organic compounds t hat can be assimilated by the microorganisms other than the test material [93,94] . According to Table 3 . 6 , the amount of VS in vermiculite is very low as expected. The pH in inoculated vermiculite is lower due to the mineral solution used, while the C/N is higher in uninoculated vermiculite since no extra source of nitrogen was provided. In any case, the C/N is expected to increase when the test material is added to the media. Other element concentrations are similar between inoculated and uninoculated vermiculite, except sodium and sulfur due to the mineral solution used for inoculation. The high concentration of aluminum was expected in the vermiculite media. 183 Table 3 . 6 Physico chemical parameters and total nutrient analysis of different media used in the Nov15 test Parameter Compost Inoculated v ermiculite Uninoculated v ermiculite Dry solids, % 60.9 98 98.6 Volatile solids, % 39.1 2.0 1.4 pH 7.4 6.8 8.0 C/N ratio 10.9 7.2 27.1 Carbon, % 22.7 1.2 0.8 Nitrogen , % 2.08 0.16 0.03 Phosphorus , % 0.55 0.13 0.11 Potassium , % 2.48 4.32 4.26 Calcium , % 9.43 0.49 0.69 Magnesium , % 2.06 8.63 8.99 Sodium , % 0.40 0.15 0.03 Sulfur , % 0.42 0.05 0.01 Iron , ppm 15080 45330 47700 Zinc , ppm 163 80 86 Manganese , ppm 503 450 447 Copper , ppm 107 155 154 Boron , ppm 33 4 3 Aluminum , ppm 5955 42880 44200 Note: The total nutrient analysis was performed by inductively coupled plasma atomic emission spectroscopy ( ICP - OES ). 3.3.3.6 Priming effect The p riming effect is the over - degradation of the indigenous organic carbon present in the compost when testing materials like glucose and its polymers [46] . Figure 3 . 12 shows an excellent example of the priming effect . I n the Jan14 test, the GC curve displayed an unusual high production of CO 2 in comparison with CP and CS ( also readily biodegradable materials ) , and a mineralization near 200%, w hich physically makes no sense; the additional carbon converted to CO 2 is coming from the compost and not from the sample material. 184 Figure 3 . 12 Cumulative CO 2 evolution (a) and mineralization (b) of CP, CS, and GC in the Jan14 test. Mineralization values larger than 100% are observed for G C. I t has been demonstrated that vermiculite is a good microbial carrier allowing the survival and full activity of the microorganisms, and it can be used as the solid media in biodegradation tests for avoiding the priming effect [46] . It has also been suggested that v ermiculite increases reproducibility and aids in recovery of the by - products released during the degradation process , which is useful for determination of carbon balances [ 38,94] . 3.3.4 Material - related factors affecting biodegradation The physicochemical characteristics of the test materials such as chemical structure, hydrophilicity, crystallinity, molecular weight, shape, and surface area, among others, are also factors affecting the biodegradation rate and the biodegradability of the materials . 3.3.4.1 Chemical structure and properties T he intrinsic characteristics of the polymer such as mobility, tacticity, crystallinity, molecular weight, glass transition temperature ( T g ) , functional groups, plasticizers, an d 185 additives highly influence its biodegradabi lity [12] . T he unique chemistry of the polymer also dictates that the microorganisms should have metabolic pathways capable of targeting the polymer for biodegradation [95] . Figure 3 . 13 shows the CO 2 evolution and mineralization of different materials fro m the Jun14 test , where there were two main groups of polymers tested . The first group, which include d PE and LDPE polymers, did not show any meaningful mineralization (3.7 ± 1.6 % for PE) while the second group, consisting of CP , CS , and PLA , reached a maximum miner alization of 61.7 ± 9.3 , 48.0 ± 4.5 , and 47.4 ± 9.8 %, respectively. Figure 3 . 13 CO 2 evolution (a) and mineralization (b) of dif ferent materials in the Jun14 test. Large difference of % Mineralization is observe d for the different materials. The different behavior between these two groups is due t o the difference in the intrinsic characteristics of the polymer . On one hand, polymers like LDPE are not easily degradable due to their h ydrophobic characteristics and relatively high stability [95] , provided by the presence of single bonds between carbon atoms in the polymer chain that are especially difficult to break [96] . On the other hand, polymers like cellulose and 186 starch tend to interact strongly with water due to their hydrophilic characteristics [96] , and their biodegr adat ion occu rs relatively quickly . In the case of hydrolytically degradable polymers , the degradation rate is highly dependent on the nature of the functional groups comprising the polymer ; som e examples of functional groups contained in degradable polymers ar - hydroxy - - hydroxy - esters), - caprolactone), and poly(carbonates). A complete list of the different functional groups and their reactivity is provided elsewhere [47] . F or example, polymers containing - hydroxy - esters) , like PLA, tend to show lag periods due to the initial diffusion of water into the polymer matrix and the subsequent break down of the polymer into oligomers and monomers before actual biodegradation can take place. The biodegradation mechanism of PLA is discussed in more detail in Section 3. 3 . 5. In this context, Mezzanote et al. (2005) pointed out the question of whether it is correct or not to use cellulose as reference material since the microorganisms that are able to biodegrade materials like cellulose or starch are ubiquitous and perfo rm cellulolytic activity, but it is not certain if they are equally able to perform esterase activity , which is required for the efficient and fast biodegradation of other materials like polyesters [49] . They have also suggested using biodegradable polyester s such as PCL as reference material besides cellulose. Similarly, other authors have proposed using PLA powder or PCL powder as reference materials for biodegradation tests [97,98] . Even if the test materials are the same , differences in composition and properties can highly influence their biodegradation rate. For example, Figure 3 . 14 shows the biodegradation test results of two types of PLA evaluated in the Nov14 test. The PLA2 film is Ingeo TM 2003D while PLA4 film is In geo TM 4032D; t he main difference between 187 these two types of PLA is their composition in terms of the L - Lactide and D - Lactide content , which in turn affect s the cryst allinity of the material. PLA2 has a crystallinity of 6.14 ± 0.08% as measured by different ial scanning calorimetry (DSC) (data not shown), while PLA4 was found to be completely amorphous due to its higher amount of D - Lactide. PLA4 produced more CO 2 than PLA2, especially at the early stage of the test. Likewise, the mineralization of PLA4 was hi gher than 100% towards the end of the test, indicating a priming effect. Figure 3 . 14 Cumulative CO 2 (a) and mineralization (b) of CP, PLA2, and PLA4 in the Nov14 test. PLA 4032D shows faster and larger mineralization than PLA 2003D. These results are in agreement with the literature, in which other researchers have found that PLA with greater D - Lactide content presented higher and faster initial chemical hydrolytic degradation [99,100] . Besides, Tsuji and Miyauchi (2001) found that enzymatic hydrolysis , in which enzymes facilitate the cleavage of bonds , occurs mainly in the amorphous regions and on polymer chains with free ends [10 1] . However, these results may be also influenced by other factors such as molecular weight and thickness. PLA4 has lower M n than PLA2 ( M n = 82.9 ± 6.7 and 75.0 ± 1.4 kDa, for PLA2 and PLA4 188 respectively; on the other hand, PLA4 is much thicker than PLA2 ( 0.022 ± 0.003 and 0.255 ± 0.021 mm, for PLA2 and PLA4 , respectively). 3.3.4.2 Concentration ASTM D5338 - 15 and ISO 14855 - 1:2005 recommend the ratio of the dry mass of the compost to the dry mass of the test mate rial be 6:1 ; for example, 600 g of dry solids of the inoculum mixed with 100 g of dry solids of the sample. However, in our case we found this rati o not to be the most convenient because most of our samples are tested as films (1 cm x 1 cm squares) and not as powder , and the volume and area occupied by thin films is too large for good exposure when mixed with the compost. For example, in our bioreactors we can only fit 400 g wet weight of compost; considering that the initial moisture content was adjusted to 50% and that 20% of that weight was vermiculit e, t hen each bioreactor contained 160 g dry weight of comp ost. I f we follow ed the compost - to - material rati o recommended by the standards the weight of the sample should be 26 g; the inconvenience with this amount is that 26 g of films with a thickness of 0.002 54 cm is too large ( i.e., density of PLA=1.24 g/cm 3 , V=21 cm 3 , A film = 8,252 cm 2 ) to be fit in to the bioreactor and p roperly mixed with the compost. Moreover, if 26 g of cellulose were added to the bioreactor the production of CO 2 would be very high and fall outside the limits of the NDIR sensor. A fter many trial tests , we found that the optimal amoun t of material for our DMR system is 8 g or a 20 :1 ratio (5% wt.) . Again, similar to setting up the air flow rate mentioned earlier, the concentration of material should be adjusted in a way that the concentration of CO 2 is within the limits of the sensor and the production of CO 2 by the reference and the blank bioreactors are clearly differentiated . 189 Figure 3 . 15 shows the cumulative CO 2 and % mineralization of CP (5%) and PLA pellets with two different concentrations ( 5% and 15% ) where 15% is closer to the 6:1 ratio suggested by the standards . As exp ected, the production of CO 2 in the 15% was higher than the one in the 5% since the amount of available carbon is higher , but the % mineralization was not affected . T he PLA pellets in both concentrations reach the same mineralization by the end of the test . Figure 3 . 15 Cumulative CO 2 and % mineralization of CP and PLA pellets with two different concentrations: 5% and 15%, in the Jan14 test. % Mineralization was not affected regardless of the initial amount of PLA. 3.3.4.3 Shape B iodegradation is usually, but not alway s, a surface erosion mechanism. Thus , materials in the form of powder usually degrade more easily since the area/volume ratio is maximized [8,76] . In this context, some researchers have suggested that the biodegradation test can be accelerated if the sample material is provided as a powder or small particles. For example, the plastic materials can be converted into very thin films and then fragmented via cryogenic milling [102] . 190 F igure 3 . 16 shows the cumulative CO 2 evolution and % mineralization of CP and of PLA provided in different forms, i.e. , pellet and film , with surface area - to - volume ratio of ~12 and ~790, respectively . The production of CO 2 , in this case , was not significantly different considering that the degradation of PLA via hydrolysis is a combi n ation of both surface and bulk erosion [47] . F igure 3 . 16 Cumulative CO 2 evolution (a) and mineralization (b) of CP and of PLA provided in different forms: pellet and film, in the Jan14 test . % Mineralization was not extensively different regardless of the shape of the material. 3.3.4.4 Comparison among different biodegradation tests Even though it has been stated that comparing the results between different tests in a direct fashion would not be fair due to the many variables involved in the process, a possible way to perform such comparison would be to normalize against the mineralization of the positiv e reference, as suggested by ASTM D5338 - 15, in which the percentage of biodegradation relative to the positive r eference ( e.g. , cellulose) at the end of the test should be reported. In this context , if the t i /t T ratio, where t i is the time at which the measurement was taken, and t T th e total time of the test, is plotted vs the ratio 191 of material mineralization and cellulose mineralization , a possible way to compare the results could be envisioned ( Figure 3 . 17 ), assuming that the biodegradation behavior was similar in the tests being compared. Figur e 3 . 17 Comparison of the mineralization values obtained for PLA1 in the Jun14 and the Nov15 tests (a), and the mineralization values obtained for PLA2 in the Nov14 and the Nov15 tests (b). The mineralization ratio when adjusting the time span of the test seems to be similar when comparing the same test material . Figure 3 . 17 shows that PLA1 reached a maximum mineralization ratio of 0.77 and 0.70 at a time ratio of 0.55 and 0.54 for the Jun14 test and the Nov15 test, respectively. Similarly , PLA2 reached a maximum mineralization ratio of 0.87 and 0.79 at a time ratio of 0.70 and 0.61 for the Nov14 test and the Nov15 test, respectively. This represents a difference of 9% between the mineralization ratio values in both cases. This approach to comparison should be further explored. 192 3.3.5 Study Case: Biodegradation of Poly(lactic acid) In the case of natural polymers like cellulose and starch, t he biodegradation process is relatively fast and starts with the depol ymerization of the material by the action of microbial extracellular enzymes that reduce the polymer to a size that is water soluble and able to be transported through the cell wall for subsequent assimilation by the microbial metabolic pathways [ 26,103] . Figure 3 . 18 a shows the biodegradation of cellulose. Figure 3 . 18 Biodegradation of CP (a) and PLA2 (b) during the Nov14 test. The black, red, blue, and green lines represent cumulative CO 2 , mineralization, evolved CO 2 per measurement, and M n red uction, respectively. The dashed blue line represents the evolved CO 2 per measurement of the blank bi oreactors. The green line indicates a fitting of an equation of the form M n = M n0 exp ( - kt), where M n0 is the initial M n , k is the rate constant and t is the time. The black dash - dot lines are used as reference to indicate the beginning and e nd of the biode gradation phase, and the M n at which the biodegradation phase gets started. Different lag phase s and biodegradation phase s were observed for CP and PLA2. 193 M icroorganisms drive the biodegradati on but other abiotic processes ( oxidative, thermal, chemical or photodegradation ) may also take pla ce before or in parallel, such a s in the case of PLA , where biodegradation is we ll known to involve abiotic hydrolysis ( Figure 3 . 18 b ) [104] . D uring the first step of PLA degradation , cleavage of ester linkages occurs due to their high susceptibility to water producing a significant reduction in the molecular weight of the polymer [103] . During the second step, the microorganisms are able to assimilate the low molecular weight lact ic acid oligomers and monomers. We have found that the second step starts once the molecular weight is 10 k Da , as shown in Figure 3 . 18 b . Figure 3 . 18 shows that there are three phases in the b iodegradation process: lag, biodegradation , and plateau phase s . In the case of natural polymers like cellulose, the biodegradation phase starts almost immediately since fragmentation occurs quickly and the lag phase is assumed to occur due to the acclimatization of the microorganisms to the environment. In the case of polymers like PLA, there is an extended lag phase due to the relatively slow fragmentation of high molecular we ight polymer chains. The b iodegradation phas e only occurs when enough low molecular weight oligomers and monomers become water soluble and are available for microbial assimilation [47] . The hydrolysis rate of the ester bonds in PLA can change depending on different factors such as water availability, pH, presence of ions, T g , crystallinity, and molecular weight [47] . As prev iously mentioned , hydrolysis occurs mainly in the amorphous regions . As a result, during degradation , an increase in crystallinity can be observed . Furthermore , in the early stage of degradation , as T g decreases from 64.0 ± 0.8 o C, as measured for PLA by DSC (data not shown), to temperatures below the test 194 temperature (58 ± 2 o C ), the oligomers and monomers have been reported to crystallize inside the PLA matrix since the polymer chains have sufficient mobility to rearrange into a more stable configuration [100,105] . Some researchers have suggested that during the early hydrolysis step no microorganisms are involved [103,106,107] , while others consider that enzymatic hydrolysis plays an important role along with abiotic hydrolysis [108] . T herefore, to decouple abiotic and biotic degradation, PLA films with three different molecular weights ( Table 3 . 3 ) were evaluated in inoculated vermiculite and uninoculated vermiculite during the Nov1 5 test and the results are show n in Figure 3 . 19 and Figure 3 . 20 . It is expected for polymers like PLA that both mechanisms c ompete against each other , with the fastest process the one that controls the initial degradation mechanism . Figure 3 . 19 shows that t here was no significant production of CO 2 from the samples tested in uninoculated vermiculite. On the other hand, the PLA1, PLA2, and PLA3 produced 5.2 ± 0.6, 8.6 ± 0.9, and 7.2 ± 0.3 g of CO 2 , respectively after 60 days of testing, and reached a mineralization of 34.6 ± 4.4 , 58.3 ± 5.8 , and 48.5 ± 1.8 %, respectively, in the same period of time. 195 Figure 3 . 19 Cumulative CO 2 (a) and m ineralization (b) of CP, PLA1, PLA2, and PLA3 in the Nov15 test. Solid line s and dotted line s represent inoculated vermiculite and uninoculated vermiculite, respectively. Figure 3 . 20 shows the M n as a function of time for the three PLA films tested in the three different media during the N ov15 test. T he molecular weight decreased during the first three weeks and a first order reaction relationship was fitted to the experimental data . The M n reduction of each sample material was not significantly different regardless of the testing media ( i.e. , compost, inoculated vermiculite, and uninoculated vermiculite), indicating that the abiotic step or hydrolysis is the main contribution to the degradation process of PLA in the early stage of degradation , and therefore it is a limiting factor for the subs equent biodegradation of PLA [102,103,107 ] . Figure 3 . 20 shows that t he initial molecular weight also affects the hydrolysis rate and therefore the overall biodegradation . The PLA with high er M n has longer polymer chains and more bonds to be cleave d, while the one with lower M n has more polymer chains with free ends that can be cleaved producing more oligomers and monomers that are available for microbial assimilation. 196 Figure 3 . 20 Molecular weight reduction as a function of time for PLA 1, PL A2, and PLA 3 in compost (solid line), inoculated vermiculite (dashed line), and uninoculated vermiculite (dotted line), in the Nov15 test . Lines indicate fitting of a first order reaction of the form M n = M n0 exp ( - kt), where M n0 is the initial M n , k is the rate constant and t is the time . In our experiment, the rate constants ( k ) w ere not significantly different. However, it could be possible that the hydrolytic degradation of PLA occurs faster in an abiotic environment due to the accumulation of by - products , such as oligomers and monomers of lactic acid, which in turn reduce the pH of the media. This lower pH can cause even more hydrolytic degradation due to an autocatalytic effect [47] . On the other hand, in a biotic environment the microorganisms ideally assimilate those by - products continuously and the pH does not change significantly , perhaps assisted by the natural buffering capacity of the compost [51] . 197 3.4 Final Remarks W e have provided a comparative analysis of the results obtained from eight different biodegradation tests for cellulose, starch, glycerol, polyethylene, and poly(lactic acid). These test s were carried out in the same in - hou se built DMR system following the analysis of evolved CO 2 approach. T he results along with the critical analysis of the information provided in the literature allowed us to identify low reproducibility as one of the main issues for this kind of evaluation , caused mainly by the difficult to control variables in measurements of the biodegradation process. In order to further understand such source s of variability a critical review of the literature regarding the different factors affecting biodegradation was also provided. This analysis allowed us to identify some key parameters that can be more strictly monitored and controlled for a n efficient biodegradation test, and therefore, to improve the current testing methodology . Among the factors producing high variability is the quality and characteristics of the compost; therefore , a stricter control on moisture content, organic matter, and C/N should be required for the test, and a ll physicochemical parameters of the compost should be reported ; otherwise , the interpretation of the results would not be complete. pH was found to be one of the easiest parameters to maintain due to the natural buffer capacity of the compost. If the test material is suspected to produce a priming effect, then biodegradation test ing in vermiculite is recommended; also, in cases where recovering of the by - products or determination of carbon balances is relevant for the study. Amendment of vermiculite with a mineral solution is recommended since it provides many of the nutrients require d by the microorganisms. 198 Regarding environmental factors, tempera ture was the easiest parameter to control while water content was the most difficult and crucial. M aintain ing the moisture content of the compost constant throughout the composting period is vital for the survival and reproduction of the microorganisms and other processes like hydrolysis. The optimal flow rate and optimal material concentrations should be found for each specifi c system in a way that allows proper measurement of CO 2 by the sens or and a clear differentiation between the background and the sample material. If the test material is not expected to be readily biodegradable or t o follow a similar behavior to cellulose, then the use of a n additional positive control should be recommen ded ( e.g. , a standardized PCL or PLA powder ) . In hydrolytically degradable materials l ike PLA, the shape did not show any significant difference since degradation occurs simultaneously in the surface and the bulk. For other types of materials, the usual re commendation is to use powder or small particles to increase the area/volume rat io and the biodegradation rate. Other polymer characteristics such as chemical structure, glass transition temperature, crystallinity, molecular weight, and functional groups h ighly influence the biodegradability and biodegradation rate of the material. The biodegradation of PLA requires prior hydrolytic degradation , which breaks down the polymer chai n into lactic acid oligomers or monomers that are easily assimilated by microorganisms. Thus, abiotic hydrolysis is the main contribution to the degradation process of PLA in the early stage of degradation and becomes a limiting factor for the subsequent biodegradation of this material . However , the hydrolysis rate is also dependent on the specific PLA properties like crystallinity and initial molecular weight. 199 T he CO 2 evolution test can always provide valuable information about the biodegradability of a test material. However, if the purpose of the study is not o nly to evaluate and/or certify a material as biodegradable or compostable, but also to understand its biodegradation mechanism and environmental impacts , then additional tests are required such as determination of carbon balanc es, ecotoxicity tests, and molecular ecological techniques, among others. Further biodegradation tests in composting conditions using different standardized reference materials and more strictly controlled inoculum (compost and/or vermiculite) characterist ics and testing parameters could be performed in d ifferent labs around the world ( e.g. , round robin test) by the analysis of evolved CO 2 in a DMR system in an attempt to unify and to im prove this testing methodology. 200 APPENDICES 201 APPENDIX 3A: Material processing LDPE film production: The LDPE/ LLDPE blend (30% LLDPE wt.) film was processed by using a Killion KLB 100 blown film extruder (Davis - Standard LLC, Pawcatuck, CT) with a screw diameter of 25.4 mm, screw L/D of 24, and annular die diameter of 5 cm. The temperature profile was 216, 216, 216, 213, 213, 210, 204 ºC for barrel z ones 1, 2, 3, clamp ring, adapto r, die 1 , and die 2 , respectively. The screw speed was 14 rpm and take up speed was 3 m per minute. PLA film production: The In geo TM 2003D films were obtained by using a Microextruder model RCP - 0625 (Randcastle Extrusion Sys tems, Inc., Cedar Grove, NJ) with a screw diameter of 15.9 mm, screw L/D of 24, and volume of 34 cm 3 . The PLA pellets were dried at 60 o C for 24 h under vacuum (85 kPa) prior processing. The temperature profile and screw speed of the films is shown in Table 3A. 1 . Table 3A. 1 Temperat ure profile and screw speed used for the production of the PLA films PLA1 PLA2 PLA3 Zone 1, o C 193 193 193 Zone 2, o C 213 249 249 Zone 3, o C 216 249 249 Transfer tube, o C 216 249 249 Die, o C 210 216 216 Screw speed, rpm 49 33 28 202 APPENDIX 3B: Elemental analysis The carbon , hydrogen, and nitrogen content of the different test materials were determined by using a PerkinElmer 2400 Series II CHNS/O Elemental Analyzer (Shelton, CT, USA), and values are presented in Table 3B.1 . Table 3B. 1 Carbon, hydrogen, and nitrogen content of the tested materials Material ID % Carbon a % Hydrogen a % Nitrogen a Cellulose powder CP 42.50 ± 0.34 6.53 ± 0.05 0.04 ± 0.01 Cassava starch CS 41.75 ± 0.35 6.69 ± 0.08 0.01 ± 0.00 Glycerol b GC 39.12 8.77 0.00 Polyethylene powder PE 85.76 ± 0.29 15.13 ± 0.09 0.01 ± 0.01 LDPE/LLDPE film LDPE 86.83 ± 0.10 14.84 ± 0.04 0.00 ± 0.00 Ingeo TM 2003D pellet PLA pellet 50.40 ±0.28 5.65 ± 0.05 0.00 ± 0.01 Ingeo TM 2003D film PLA1 50.05 ± 0.05 5.65 ± 0.02 0.01 ± 0.01 PLA2 49.93 ± 0.11 5.56 ± 0.02 0.01 ± 0.01 PLA3 49.99 ± 0.05 5.60 ± 0.01 0.01 ± 0.01 Ingeo TM 4032D sheet PLA4 50.00 ± 0.08 5.61 ± 0.01 0.04 ± 0.02 a Percentage by weight b Theoretical values based on chemical structure 203 APPENDIX 3C: Molecular weight determination The M n , M w , and PI of PLA samples before and during composting were determined with a gel permeation chromatography (GPC) system from Waters Inc. (Milford, MA). The system is equipped with a Waters 1515 isocratic pump, a Waters 717 autosampler, a series of three columns (HR 2, HR3, and HR4 Waters Styragel ® ), and a Waters 2414 refractive index detector interfaced with a Waters Breeze software. For each PLA sample, 20 mg were dissolved in 10 cm 3 of tetrahydrofuran (THF) and filtered with a hydrophobic polytetrafluoroethylene (0 cm 3 /min, a run time of 45 min, and a temperature of 35°C. A third - order polynomial universal calibration curve was obtained from polystyrene standards ranging 1.37 2,480 kDa. The Mark - Houwink constants, K= 0.000174 dL/g and 0.736, for PLA when dissolved in THF at 30°C [109] , were used to obtain the a bsolute molecular weight values. 204 APPENDIX 3D: Compost source The compost prepared at the MSU Composting Facili ty (East Lansing, MI) was produced by mixing dairy manure and straw in a proportion of 1:1. Manure, the fecal and urinary excretion of livestock , is rich in nitrogen content; hence , i t was mixed with straw, a source of carbon, to achieve the desired carbon - nitrogen ratio ( C/N ) for the compost samples. The mixture was introduced into bays, turned 2 - 3 times a week. The material was allowed to naturally h eat to about 55°C (132 °F) for 72 hours and turned at least three times. Then, the mixture was pulled out of the bay, and a pile was built up on an asphalt pad. After the active composting phase, a curing period of 6 to 12 months was required to finish the process and allow the compost to develop the desired characteristics. After curing, the compost was screened to remove large debris and inert materials [110] . 205 APPENDIX 3E: Respirometric system An enhanced direct measurement respirometric (DMR) system was built at the School of Packaging (SoP) in Michigan State University (MSU), East Lansing, MI, based on the system reported by [18] . This DMR system was designed to operate simultaneously with up to 95 bioreactors, and it is able to simulate different testing conditions by varying te mperature and relative humidity (RH). A computer application was developed for controlling the system, as well as for measuring and recording the test variables. A non - dispersive infrared gas analyzer (NDIR), model LI - 820 from LI - COR Inc., Lincoln, NE, was used to measure the concentration of CO 2 evolved from the bioreactors. Figure 3E.1 shows a schematic diagram of the DMR; detailed information of the equipment can be found elsewhere [40] . 206 Figure 3E. 1 Schematic diagram of a direct measurement respirometric system, reproduced from Castro - Aguirre, E. [40] . CO 2 from the incoming air is scrubbed by passing through a series of canisters containing soda lime. This CO 2 - free air enters a water tank, located inside the environmental chamber at 58 o C, to get humidified; then, CO 2 - free water - saturated air is provided to the bioreactors with an upward flow direction. The respi red air stream exits the bioreactors and the environmental chamber passing through a water trap, a mass flow controller (MFC) and a NDIR - CO 2 sensor for CO 2 concentration measurement. Temperature, relative humidity (RH), air flow rate, time and CO 2 concentr ation are measured and recorded by a data acquisition system (DAS). 207 APPENDIX 3F: Calculation method In order to calculate the cumulative CO 2 evolved from the bioreactors and the mineralization of the sample materials, a number of parameters and variables are acquired by the DMR control software. It records the bioreactor number, the time stamp, the CO 2 concentration (ppm), the standard air flo w rate (sccm), the temperature (°C), the relative humidity (%), the date (mm/dd/yyyy), and the time (hh:mm:ss), every 2 seconds during the last 30 seconds of each cycle time; i.e., 15 measurements of each var iable are recorded every cycle [40] . The cycle time is the period, set by the user, in which the solenoid va lv e of the selected bioreactor is opened by the control software allowing the respired air to flow through the NDIR sensor at a specified flow rate. This time is estimated according to the information presented in the Appendix 3G. All the information prese nted below regarding the calculation method can be found in more detail elsewhere [40] . T he actual CO 2 concentration of each measurement is determined by multiplying the response CO 2 concentration by the calibration factor (Eq. 3F. 1). ( Eq. 3F. 1 ) where [ C ] is the actual concentration of CO 2 of each sample (ppm), c the response concentration of CO 2 as measured by the NDIR analyzer (ppm), and k the calibration factor explained in the Appendix 3G. T he time (min) at which each measurement was done, relative to the starting time, is determined by E q. 3F. 2. ( Eq . 3F. 2) 208 where t n is the time at which each measurement was done (min), ts n is the time stamp at time t n , and ts o is the time stamp at time t o corresponding to the time at which the experiment started. T he average time (min), average concentration (ppm), average flow rate (sccm), average temperature (°C), and average RH (%) are calculated since 15 measurements of each variable are recorded every cycle and only a representative value per cycle is used for the CO 2 evolution calculation. The concentration of CO 2 (ppm) is converted to mass of CO 2 (g) evolved from each bioreactor in the period of time between measurements (measurement interval ) as described by the Eq. 3F. 3. ( Eq . 3F. 3) where E(CO 2 ) is the mass of evolved CO 2 (g), F the flow rate ( sccm ), T the measurement interval, C the concentration of CO 2 evolved during the measurement interval, 22414 the volume of 1 mol of gas in cc at STP, 44 the molecular weight of CO 2 (g/mol), an d 10 6 the ppm conversion factor [18] . If the time is plotted against the concentration, as shown in Figure 3F.1 , then the area under the curve for a specific measurement interval represents the product C X T of the Eq. 3F. 3 a nd it is determined by Eq . 3F. 4. ( Eq . 3F. 4) where A t n the time in which the measurement was done (min), t n - 1 the time in which the previous measurement was done (min), [C] n 209 the concentration of CO 2 (ppm) at time t n , and [C] n - 1 is the concentration of CO 2 (ppm) at time t n - 1 . Figure 3F. 1 Time vs. Concentration Plot , reproduced from C astro - Aguirre, E. [40] T he cumulative amount of evolved CO 2 i n each bioreactor for each measurement interv al is calculated using Eq. 3F. 5 . ( Eq . 3F. 5) where C(CO 2 ) is the cumulative mass of CO 2 (g), E(CO 2 ) n is the mass of CO 2 (g) evolved from the sample at time t n , and C (CO 2 ) n - 1 is the cumulative mass of CO 2 (g) until the previous measurement (at time t n - 1 ). S ince the cumulative mass of CO 2 of the blank has to be subtracted from the cumulative mass of CO 2 of each sample at the same time interval for further calculating the percen ta ge mineralization, t he time is converted from minutes to days, and an interpolation of CO 2 values is performed with time intervals of one day using Eq. 3F. 6. ( Eq . 3F. 6) where t I (d) is the time interval, t L (d) is the immediate lower value of the time interval, t H (d) is the immediate higher value of the time interval, I(CO 2 ) (g) is the interpolated 210 cumulative mass of CO 2 at time t I , C(CO 2 ) L (g) is the cumulative mass of CO 2 at t ime t L , and C(CO 2 ) H (g) is the cumulative mass of CO 2 at time t H . Once the cumulative mass of CO 2 of each bioreactor i s obtained, the percentage minerali zation of each bior eactor is calculated using Eq. 3F. 7 , expressing the relationship between the actual amount of CO 2 evolved from the test material and the theoretical amount of CO 2 that can be evolved from the same test material . ( Eq . 3F. 7) where % Mineralization is the percent carbon molecules converted to CO 2 , (CO 2 ) T is cumulative mass of CO 2 (g) evolved from a sample bioreactor , (CO 2 ) B the average cumulative mass of CO 2 (g) evolved from the blank bioreactors , M TOT the mass of test material (g), C TOT is the prop ortion of total organic carbon in the total mass of test material (g/g) , 44 the molecular weight of carbon dioxide, and 12 the atomic weight of carbon. 211 APPENDIX 3G: Determination of the calibration factor, optimal flow and cycle time In this case, c alibration refers to the process of establishing the relationship between the CO 2 analyzer signal (measured CO 2 concentration) and the known injected concentration of pure CO 2 . Thus, when a measurement is made by the CO 2 analyzer, the signal measurement is multiplied by the calibration factor ( k ) to yield the actual concentration of CO 2 evolved from a sample (Eq. 3F. 1) [40] . The calibration of the system was performed at 58 ± 2°C and 50 ± 5% RH. K nown amounts of pure CO 2 gas (1, 5, 10 , and 20 cc) were injected through a septum to e mpty bioreactors . Additionally, three air flow rates (20, 40, and 60 sccm) were used for determining the optimal flow. The actual concentration of CO 2 in the bioreactor was calculated using Eq. 3G.1 , being 507, 2535, 5070, and 10140 ppm the corresponding a ctual concentration for 1, 5, 10, and 20 cc injected volume, respectively . ( Eq . 3G.1 ) The response CO 2 concentration was measured by the NDIR and recorded along with the time every 2 seconds. Then, t he calibration curve was determined by plotting the peak response concentrations ag ainst the actual concentrations as shown in Figure 3G.1 [40] . 212 Figure 3G. 1 Calibration curve a t 58 ± 2°C and 55 ± 5% RH. A linear relationship of the form [ C ] = c*k was fitted to the data, where [ C ] is the actual CO 2 concentration , c the response CO 2 concentration as measured by the NDIR analyzer, and k the calibration factor . Figure 3G. 2 shows that the response con centration is not affected when using different air flow rates, as long as the CO 2 concentration is allowed to reach its maximum value. Figure 3G.3 sho ws that time to reach the maximum concentration was the lowest when using the highest air flow rate and vice versa. The small differences observed in the time among replicates of the same air flow rate are due to the different injected volumes of CO 2 . 213 Figure 3G. 2 Response CO 2 concentration obtained when using different injection volumes and air flow rates. The maximum concentration can be achieved in each case regardless the air flow rate used. Fitted lines of the form y= x+ are included for visual guidance only, with = 0, and = 507, 2535, 5070, and 10140, corresponding to the different injection volumes used. 214 Figure 3G. 3 Time to reach the maximum CO 2 concentration when using different air flow rates. The longest time to reach the maximum concentration was observed when using the lowest air flow rate. The above findings are relevant when setting the parameters for performing the real biodegradation test in which the objective is to reach the CO 2 concentration at steady - state (instead of the maximum CO 2 concentration during calibration) before recording the measurement for a particular bioreactor. For example, if using a specific cycle time during the test, the bioreactors with the lower flow may show lower concentrations by the end of the cycle (me asuring time), because there is no enough time to reach the steady - state. Therefore, it is important to select the appropriate air flow rate to be used during testing and based on that to determine t he cycle time that would include the highest CO 2 concentration that could be expected from the testing materials. Figure 3G.4 shows that the time to reach steady - state is depending on both CO 2 concentration and air flow rate. 215 Figure 3G. 4 Response concentration and time required for a selected bioreactor to rea ch the peak concentration for different injection volume s and air flow rates. The longest time was observed with the highest CO 2 concentration and the lowest air flow rate. 216 APPENDIX 3H: Compost physicochemical characteristics before and after the test Table 3H.1 shows the compost physicochemical characteristics determined before and after the test for the Feb13 and the Nov14 tests. The carbon content decreased in both cases due to the mineralization of carbon, as expected. The C/N was also decreased being more pro nounced in the compost with a higher initial C/N which is in agreement with the results obtained by Bernal et al. [89] . Table 3H. 1 Physicochemical characteristics of the compost from the Feb13 a nd the Nov14 tests determined before and after the test Parameter Feb13 Nov14 Before After Before After Dry solids, % 54.9 50.8 41.5 82.6 Volatile solids, % 67.6 42.7 43.2 38.2 pH 8.9 7.8 8.5 8.1 Total Carbon, % 39.2 24.8 25.1 22.2 Total Nitrogen, % 2.3 2.6 2.43 2.17 C/N ratio 17.0 9.5 10.3 10.2 217 APPENDIX 3I: Compost nitrate and ammonium concentration Figure 3I.1 shows the concentrations of NH 4 + and NO 3 - as a function of time of the compost in the blank bioreactors and the compost in the CP bioreactors during the Nov14 test. Other researchers have fou nd previously that during the composting process the NH 4 + concentration decreases while the NO 3 - concentration increases due to the nitrification process [89,92] . Figure 3I. 1 Concentration of NO 3 - (left - black axis) and NH 4 + (right - red axis) as a function of time of the compost in blank bioreactors (a) and CP bioreactors (b) during the Nov14 test. 218 APPENDIX 3J: Summary of the results obtained from the eight different biodegradation tests A summary of the results obtained from the eight different biodegradation tests is provided in Table 3J.1 . Table 3J. 1 Summary of the results obtained from the eight different biodegradation tests Test Sample Media g CO 2 at 60 d %Min. at 60 d Max. %Min. Days to achieve max. %Min. Sep12 Blank Compost 9.7 ± 0.2 N/A N/A N/A CP Compost 17.8 ± 2.2 67.0 ± 18.4 77 139 LDPE Compost 8.9 ± 0.5 - 3.3 ± 1.9 N/A N/A Feb13 Blank Compost 18.3 ± 0.7 N/A N/A N/A CP Compost 27.5 ± 4 67.7 ± 30.8 67.9 55 LDPE Compost 16.7 ± 1.5 - 8.7 ± 6.2 N/A N/A May13 Blank Compost 23.9 ± 4.1 N/A N/A N/A CP Compost 33.6 ± 2.8 78.9 ± 23.1 79.5 52 CS Compost 26.5 ± 2.5 22.0 ± 21.0 28.1 43 LDPE Compost 25.1 ± 2.7 4.6 ± 4.8 6.8 20 Jul13 Blank Compost 20.8 ± 2.3 N/A N/A N/A CP Compost 33.7 ± 2.6 100.3 ± 20.5 100.8 65 LDPE Compost 22.2 ± 1.2 5.5 ± 4.8 6.1 68 Jan14 Blank Compost 13.3 ± 0.2 N/A N/A N/A CP Compost 18.7 ± 0.7 44.3 ± 5.9 65.7 34 CS Compost 19.1 ± 1.2 56.2 ± 11.2 68.3 26 PE Compost 10.7 ± 10.3 N/A N/A N/A GC Compost 36 ± 2.6 194.5 ± 22.1 201.3 83 PLA pellets (5%) Compost 19 ± 0.8 39.2 ± 5.5 62.9 87 PLA pellets (15%) Compost 30.3 ± 1.6 38.8 ± 3.6 62.6 87 PLA1 Compost 19.5 ± 1 42.4 ± 6.6 53.2 87 219 Table 3J.1 Test Sample Media g CO 2 at 60 d %Min. at 60 d Max. %Min. Days to achieve max. %Min. Jan14 Blank Inoculated vermiculite 0.5 ± 0.1 N/A N/A N/A CP Inoculated vermiculite 4.9 ± 0.5 35.3 ± 3.9 41.7 79 PLA pellets Inoculated vermiculite 5.6 ± 0.4 34.5 ± 2.8 41.7 79 Jun14 Blank Compost 23.9 ± 1.8 N/A N/A N/A CP Compost 31.4 ± 1.5 60.6 ± 12.2 61.7 45 CS Compost 24.9 ± 2.1 9.1 ± 19.1 48 8 PE Compost 24.5 ± 0.6 2.2 ± 2.5 3.7 26 LDPE Compost 20.7 ± 3.4 - 13.0 ± 13.5 N/A N/A PLA1 Compost 30.8 ± 1.6 46.6 ± 11.0 47.4 55 Nov14 Blank Compost 17.8 ± 0.5 N/A N/A N/A CP Compost 28.1 ± 1.1 85.2 ± 9.0 87 82 PLA2 Compost 28 ± 1.2 73.3 ± 8.7 74.2 69 PLA4 Compost 35.1 ± 5.1 117.4 ± 35.0 118.9 68 Nov15 Blank Compost 17.8 ± 1.1 N/A N/A N/A CP Compost 29.3 ± 1.5 95.7 ± 12.1 98.2 94 PLA1 Compost 27.1 ± 1.0 63.3 ± 6.7 68 72 PLA2 Compost 27.7 ± 1.0 67.6 ± 7.1 76.9 86 PLA3 Compost 31.2 ± 1.0 91.5 ± 7.0 109.1 120 Blank Inoculated vermiculite 0.0 ± 0.0 N/A N/A N/A CP Inoculated vermiculite 7.3 ± 0.4 60.2 ± 3.3 75.6 120 PLA1 Inoculated vermiculite 5.2 ± 0.6 34.6 ± 4.4 65 120 PLA2 Inoculated vermiculite 8.6 ± 0.9 58.3 ± 5.8 74.7 120 PLA3 Inoculated vermiculite 7.2 ± 0.3 48.5 ± 1.8 75.1 120 N / A: Not applicable Min= Mineralization 220 REFERENCES 221 R EFERENCES [1] T. 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Three different nanoclays were studied due to their different surface characteristics but similar chemistry: organo - modified montmorillonite (OMMT), Halloysite nanotubes (HNT), and Laponite ® RD (LRD). Additionally, the organo - modifier of Cloisite ® 30B, methyl, tallow, bis - 2 - hydroxy ethyl, quaternary ammonium (QAC), was studied. PLA and PLA bio - nanocomposite (BNC) films were produced, characterized, and used for biodegradation evaluation with an in - house built direct measurement respirometer (DMR) following the analysis of evolved CO 2 approach. A biofilm formation essay and scanning electron microscopy were used to evaluate microbial attachment on the surface of PLA and BNCs. The results obtained from four different biodegradation tests with PLA and its BNCs showed a significantly high er mineralization of the films containing nanoclay in comparison to the pristine PLA during the first three to four weeks of testing, mainly attributed to the reduction in the PLA lag time. The effect of the nanoclays on the initial molecular weight during processing played a crucial role in the evolution of CO 2 . PLA - LRD5 had the greatest microbial attachment on the surface as confirmed by the biofilm test and the SEM micrographs, while PLA - QAC0.4 had the lowest biofilm formation that may be attributed to t he inhibitory effect also found during the biodegradation test when the QAC was tested by itself. 233 4.1 Introduction Biodegradable polymers like poly(lactic acid) (PLA), poly(butylene adipate - co - terephthalate) (PBAT), and thermoplastic starch (TPS), have great potential to replace fossil - based polymers, avoid landfill disposal of most non - recyclable polymers, and help reduce environmental impacts. However, these materials have some properties and processing shortcomings that have limited their use in many applic ations, for example, brittleness, water sensitivity, low heat distortion temperature, medium to high gas permeability, and low melt viscosity [1,2] . Therefore, the creation of bio - nanocomposites (BNCs) in which the reinforcements have at least one dimension in the nanoscale dim ension and the matrix is a biodegradable polymer, preferably a bio - based polymer, has garnered attention [1,3,4] . Ideally, BNCs could be recycled or treated together with other organic wastes in composting facilities and produce compost, a valuable soil conditioner and fertilizer [5] . One particularly useful class of nanofillers used to produce BNCs is inorganic la yered silicate minerals, or nanoclays, due to their commercial availability, low cost, significant property enhancement and relatively simple processability [3] . Natural nanoclays, such as montmorillonite (MMT) with chemical structure [Na 0.38 K 0.01 ][Si 3.92 Al 0.07 O 8 ][Al 1.45 Mg 0.55 O 2 (OH) 2 ]·7H 2 O, and synthetic nanoclays, such as Laponite ® RD (LRD) with chemical structure Na 0.7 [(Si 8 Mg 5.5 Li 0.3 ) O 20 (OH) 4 ] 0.7 , and halloysite nanotubes (HNT) with chemical structure Al 2 (OH) 4 Si 2 O 5 (2H 2 O), offer a unique route for enhancing the mechanical, physical and barrier properties of biodegradable polymers at lo w levels of loading (<5% wt.), especially when the nanoclay particles are well dispersed in the polymer matrix [2,6] . However, the dispersion of hydrophilic 234 nanofillers in a polymer matrix is challenging. Organophilization, or organic modification, is a technique that improves clay compatibility with organic pol ymers by reducing the surface energy between the clay layers. Increasing the clay inter - gallery spacing facilitates the intercalation and exfoliation of the clay in the polymer matrix [2,3] . The which the interlayer inorganic ions are exchanged with organic cations [4,7] . The most broadly studied organo - modifiers are ammo nium alkyls. When the clay inorganic ions are exchanged with these organic cations, the inter - gallery spacing increases due to the bulkiness of the alkyl - ammonium ions [7] . For example, organo - modified montmorillonite (OMMT), in which its inorganic ions ( e.g. , Na + , K + , Ca 2+ and Mg 2+ ) have been replaced by organic alkyl - ammonium ions improving the wetting with the polymer chains [1,3] . Several researchers have reported improvement in the properties and performance of PLA with addition of OMMT. For example, Ray et al., through a series of papers, demonstrated that the addition of montmorillonite has a significant effect in the improveme nt of PLA properties (in both solid and melt states), crystalline behavior, and biodegradability in comparison with pristine PLA. Among the different mechanical properties that have been improved are storage modulus, flexural modulus, flexural strength, te nsile modulus and elongation at break [8 10] . Additional benefits in performance have been reported such as increased glass transition and thermal degradation temperatures [3,11] . Another reported advantage, other than enhancement of the mechanical and thermal properties, is improvement in the barr ier properties due to the enhanced tortuous path provided by the silicate layers to gases like oxygen [9,12,13] . The decreased transparency is a minor disadvantage of these 235 BNCs [3] . Other researchers have found significant improvement in thermo - mechanical and barrier properties of BNCs based on PLA and OMMT [14,15] . Halloysit e is another type of nanoclay that has received great attention as filler for polymer/clay nanocomposites due to its biocompatibility, natural abundance, and relatively low cost. HNT has almost no surface charge and does not require organic modification fo r adequate dispersion [16,17] . However, functionalized HNT has shown improved dispersion during processing and enhanced mechanical and thermal properties [18,19] . HNT has been used as filler for several polymers like poly(propylene) (PP), vinyl ester, polyamide (PA), poly(vinyl chloride) (PVC), epoxy, and natural rubber for enhancing properties such as mechanical, thermal, crystallinity, and fire resistance [18,19] . Researchers have found that PLA - HNT nanocomposites exhibited improvement in properties like tensile strength, Young modulus, impact properties, flexural properties, and storage modulus, but no significant modification in the thermal properties in comparison with pure PLA [16,20 22] . The addition of HNT promotes crystalliz ation and formation of different crystalline phases [21,22] . HNT was also found to slightly increase water absorption [23] . However, other researchers found increased thermal and flame retardant properties besides improvem ent in mechanical properties [19] . Esma et al. also found enhanced thermal properties but in their case mechanical properties were not significantly improved [24] . Similarl y, Kim et al. found decreased tensile strength with clay loading higher than 5% wt. but enhanced rheological properties [17] . Laponite ® (LRD), another clay that might lead to novel properties, has not been widely investigated for the development of PLA - based nanocomposites. LRD is an entirely synthetic hectorite clay that belongs to the group of smectite phyllosilicate 236 minerals, and it has great capacity for swelling and exfoliation [25,26] . The advantage of using synthetic clays like LRD is the high structural regularity, single layer dispersions of nanoparticles, and low level of impurities ( e.g., silica, iron oxides, and carbonates). Due to its gelation properties, LRD has been used for different pharmaceutical and cosmetic applications; for example, toothpastes, creams, and glazes [27 30] . Zhou et al. studied PLA - LRD composite films and foun d improvement in the thermal stability, tensile strength and hydrophilicity of PLA, especially when the LRD content is below 0.2% wt. [31,32] . Similarly, Tang et al. studied nanocomposites based on starch, poly vinyl alcohol (PVOH), and LRD and found that an increase in LRD content (0 20%) enhanced tensile strength and decreased water vapor permeability [26] . Besides performance limitations, one of the drawb acks of some biodegradable polymers, like PLA, is that they do not biodegrade as fast as other organic wastes during composting, which in turn affects their general acceptance in industrial composting facilities [33] . Therefore, increasing their biodegradation rate in the composting environment should facilitate and encourage their disposal through these facilities by degrading in a time frame comparable with other orga nic materials. Several researchers studied the effect of OMMT on the biodegradation of biodegradable polymers like polycaprolactone (PCL) [34] , poly(3 - hydroxybutyrate - co - 3 - hydroxyvalerate) (PHBV) [35] , TPS [36] , and PLA [10,33,37 45] . Their results indicated that, in general, these BNCs biodegraded faster than their respective pristine polymer. T herefore, the incorporation of nanoclays into a biodegradable polymer matrix represents a promising approach not only for enhancing the polymer performance but also for increasing its biodegradation rate in composting conditions. However, the effect 237 of dif ferent nanoclays and organo - modifiers on the abiotic and biotic degradation of PLA is still unclear and needs further investigation. Even though it is well known that the biodegradation mechanism of PLA involves chemical hydrolysis, the role of microorgani sms and how they are affected by the presence of nanoparticles is still not well understood [44] . Thus, this study aimed to understand the biodegradation mechanisms of BNCs made of PLA and compounded with OMMT, HNT, and LRD, and to identify the main fact ors contributing to their biodegradation rate such as those related to the polymer structure and also those related to the soil/compost environments or to the microbial populations that could be impacted by the presence of nanoparticles. 4.2 Materials and Methods 4.2.1 Materials Poly(lactic acid) resin (Ingeo TM 2003D) was obtained from NatureWorks LLC. (Minnetonka, MN) with 3.8 - 4.2% D - LA, number average molecular weight ( M n ) of 121.1 ± 7.5 kDa, polydispesity index ( PDI ) of 1.9 ± 0.1, and melt flow index (MFI) of 6 g/10 min (HNT) were purchased from Sigma - Aldrich (St. Louis, MO). Organo - modified montmorillonite (OMMT) (Cloisite ® 30B) and Laponite ® RD (LRD) were acquired from BYK Add itives Inc. (Gonzales, TX). Additionally, TomamineTM Q - T - 2 (QAC) with 60 - 70% purity of a methyl, tallow, bis - 2 - hydroxyethyl, quaternary ammonium, the organo - modifier of Cloisite ® 30B, was obtained from Air Products and Chemicals Inc. (Butler, IN). Tetrah ydrofuran (THF) was obtained from Pharmco - AAPER (North East, CA). The composition per liter of the R2 broth (R2B) used was 0.5 g yeast extract, 0.5 g proteose 238 peptone #3, 0.5 g casamino acids, 0.5 g dextrose, 0.5 g soluble starch, 0.3 g sodium pyruvate, 0. 3 g dipotassium phosphate, and 0.05 g magnesium sulfate. The composition per liter of the M9 minimal medium was 12.8 g Na 2 HPO 4 ·7H 2 O, 3 g KH 2 PO 4 , 0.5 g NaCl, 1 g NH 4 Cl, and 1 g of 1 mM MgSO 4 , 1 mM CaCl 2 , 3x10 - 9 M (NH 4 ) 6 Mo 7 O 24 ·4H 2 O, 4x10 - 7 M H 3 BO 3 , 3x10 - 8 M CoCl 2 ·6H 2 O, 1x10 - 8 M CuSO 4 ·5H 2 O, 8x10 - 8 M MnCl 2 ·4H 2 O, 1x10 - 8 M ZnSO 4 ·7H 2 O, 1x10 - 6 M FeSO 4 ·7H 2 O. All the chemicals and reagents were commercial products of the highest available grade. 4.2.2 Processing of the PLA bio - nanocomposites PLA - BNCs (PLA - OMMT, PLA - LRD, and PLA - HNT) were produced in a two - step process. First, masterbatches were prepared in a ZSK 30 twin - screw extruder (Werner Pfleiderer, NJ) and pelletized. Second, PLA - BNC films (1 and 5% wt. nanoclay) were produced in a cast film microextruder model RCP - 0625 (Randcastle Extrusion Systems, Inc., Cedar Grove, NJ). Two PLA - QAC films (0.4 and 1.5% wt. organo - modifier) were produced in a similar fashion. Three PLA films (PLA1, PLA2, and PLA3) with different molecular weight were ob tained by varying the processing conditions, and used as control films. In all cases, the materials were dried at 60°C for 8 h under vacuum (85 kPa) prior to processing. The thickness of the films was measured using a digital micrometer (Testing Machines I nc., New Castle, DE). More details regarding the film processing are provided in Table 4A. 1 of the Appendix 4A . 4.2.3 Characterization of the PLA bio - nanocom posites To evaluate the presence and dispersion of the nanoclays in the PLA matrix, X - ray diffraction (XRD) and transmission electron microscopy (TEM) were performed. PLA and BNC films were embedded in paraffin blocks and microtomed in 100 nm sections 239 for bright field imaging using an Ultramicrotome MYX (RMC Boeckeler Instruments, Tucson, AZ). TEM micrographs were obtained using a JEOL 2200FS transmission electron microscope (JEOL USA, Inc., Peabody, MA) operating at an acceleration voltage of 200 kV. X RD analysis was conducted in a Rigaku Rotaflex Ru - 200BH X - ray diffractometer equipped with a Ni - [46] . The carbon, hydrogen and nitrogen content, as well as the amount of nanoclay present in each BNC film was determined by elemental analysis (CHN) and are reported in Table 4B. 1 of the Appendix 4B . Additional methodologies, such as differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), moisture isotherm, electrical conductivity, and contact angle, used for characterization of the BNCs are provided in the Appendix 4B. 4.2.4 Biodegradation evaluation The aerobic biodegradation of PLA and BNCs was evaluated through a series of experiments ( Table 4. 1 ) by analysis of evolved CO 2 under controlled composting conditions (at 58°C), using an in - house built direct measurement respirometer (DMR) with a CO 2 non - dispersive infrared gas analyzer (ND IR). Manure compost from the MSU Composting Facility (East Lansing, MI) was used. The compost was sieved on a 10 mm screen and preconditioned at 58°C for 3 days prior to use. Deionized water was incorporated to adjust the moisture content to about 50%. Sat urated vermiculite premium grade (Sun Gro Horticulture Distribution Inc., Bellevue, WA) was mixed with the compost (1:4 parts, dry wt. compost) for better aeration. Compost samples were sent to the Soil and Plant Nutrient Laboratory at Michigan State Unive rsity (East 240 Lansing, MI, USA) for determination of the physicochemical parameters (dry solids, volatile solids, C/N ratio, and pH) and are reported in Table 4C. 1 of the Appendix 4C . Detailed information about the methods used for compost characterization can be found elsewhere [47] . Table 4. 1 Key for biodegradation test and labels of the samples Test ID Samples tested I Blank, Cellulose, OMMT, HNT, LRD, PLA1, PLA - OMMT5 II Blank, Cellulose, OMMT, OMMT5, QAC, QAC5, PLA1, PLA - OMMT1, PLA - OMMT5, PLA - OMMT7.5 III Blank, Cellulose, PLA2, PLA - OMMT1, PLA - OMMT5, PLA - HNT1, PLA - HNT5, PLA - LRD1, PLA - LRD5, PLA - QAC1.5, PLA - QAC0.4 IV Blank, Cellulose, PLA1, PLA2, PLA3, PLA - OMMT5, PLA - QAC0.4 The bioreactors were loaded with 400 g of compost (or vermiculite) and mixed thoroughly with 8 g of polymer sample (unless otherwise specified). Film samples were cut to 1 cm 2 pieces and triplicates of each test material were analyzed. Additionally, triplicates of blank bioreactors (with compost or vermiculite only) were evaluated. To simulate composting conditions, the bioreactors were placed in an environmental chamber set at a constant temperature of 58 ± 2°C. Water - saturated CO 2 - free air was provided to each bioreactor with a flow rate of 40 ± 2 sccm (cm³/min at standard temperature and pressure). The bioreactors were incubated in the dark for at least 45 d or until the evol ved CO 2 reached a plateau. For all the biodegradation studies, the results are presented as average ( n =3) and standard deviation. 241 4.2.5 Size Exclusion Chromatography (SEC) The number average molecular weight ( M n ), weight average molecular weight ( M w ), and polydi spersity index (PDI) of PLA and BNCs before and during composting were determined by SEC with a system from Waters Inc. (Milford, MA) as previously described [47] . Shortly, 20 mg of films were dissolved in 10 cm 3 of THF and filtered with sample solution were i njected. A third - order polynomial calibration curve was obtained from polystyrene (PS) standards ranging 0.5 2,480 kDa, and the Mark - Houwink constants, K = 0.000164 dL/g and = 0.704, for PS were used. 4.2.6 Microbial attachment Biofilm Assay: The biofilm formi ng ability of microorganisms on the surface of PLA and BNCs was assessed with a biofilm assay in 24 - well polystyrene plates as described elsewhere [48,49] . For this test, sterilized PLA films and BNC films were added to the wells of a microtiter plate (24 wells). The films were sterilized by rinsing with 70% ethanol, followed by irradiation with ultraviolet light for 5 min prior to testing. Four of compost extract (CE), which was prepared by vigorously mixing dry compost with deionized water (1:2 wt./vol.) on vortex for 2 min. The mix was allowed to settle for 20 minutes and then the supernatant was passed through a sieve with 1 mm mesh. A sterile compost extract (SCE) was prepared for a control by passing the CE twice filter. The inoculated plates were incubated for 48 hours at 58°C gently shaking at 100 rpm. Pseudomonas aeruginosa (PA) strain PAO1, a biofilm producing bacterium, was used as a positive control at 23°C, and uninoculated wells 242 were considered as a negati ve control. To determine the level of biofilm formed on the surface of PLA and BNCs after incubation, the films were transferred to clean Eppendorf tubes and treated in parallel with the microtiter plates. The broth was removed from the plates and the well s and films were gently washed with water three washing three times with water. After the plates and films had air - acetic acid was added, followed by incubation for 15 min. Measurements were done VT) at 600 nm directly on the wells and following decantation of the films. Decanted acetic acid from films was transferred into clean microtiter plates for absorbance measurement at 600 nm. The biofilm formation was quantified by subtracting the average absorbance of the cognate controls from the average absorbance of the inoculated samples. Scanning Electron Microscopy (SEM): Similar to the biofilm test, sterilized PLA films and PLA - LRD5 films were added to an Erlenmeyer flask containing R2B (2x) and an overnight culture of the compost extract (CE) on R2B at 58°C (3:1 vol.). The samples were incubated for 48 hours at 58°C. The films were removed from the flasks, gently washed with water three times, and air - dried. The samples were mounted on aluminum stubs using high vacuum carbon tabs (SPI Supplies, West Chester, PA), and coated with osmium. SEM micrographs were obtained at va rious magnifications using a JEOL 6610LV (tungsten hairpin emitter) scanning electron microscope (JEOL Ltd., Tokyo, Japan) operating at a voltage of 10 kV to observe the biofilm formation. 243 4.2.7 Statistical Analysis All statistical analyses were performed using Minitab18 software (Minitab Inc., State College, PA) by analysis of variance (one - way ANOVA), and Tukey test with a p - value threshold of 0.05 as for level of significance. Data are reported as mean and standard deviations. 4.3 Results and Discussion 4.3.1 Character ization of the PLA bio - nanocomposites Figure 4. 1 and Figure 4. 2 show the XRD spectra and TEM micrographs of the BNCs, respectively. The se methods were used to evaluate the presence and dispersion of the nanoclays in the PLA matrix. Depending on the degree of dispersion, a layered silicate nanocomposite can be either intercalated or exfoliated. Intercalation occurs when the polymer chains penetrate into the interlayer regions of the clay, while exfoliation is observed when the clay layers are delaminated and randomly dispersed in the polymer matrix [3] . As observed in Figure 4. 1 a , in the case of PLA - OMMT5 film, OMMT is not fully exfoliated but intercalated in the PLA matrix, which is represented by the shift of the peak to the left, i.e., the increase in the interlay er distance from 1.85 nm, for the pristine OMMT, to 3.42 nm, for the OMMT present in the film. The organic modification of the MMT through exchange of cationic ions allows for better dispersion and exfoliation of the silicate layers into the PLA matrix [1,3,7] . However, in the case of PLA - OMMT5 it was not enough to obtain a fully exfoliated B NC. This was confirmed by the TEM micrograph ( Figure 4. 2 a ), which shows some small agglomerations. However, it seems that the OMMT is evenly distribut ed in the PLA matrix. PLA - OMMT1 showed a better dispersion of the OMMT in the polymer matrix, but in general, full exfoliation is difficult to 244 achieve, and most nanocomposites are a mixture of both structures, which is usually referred to as disordered mor phology or orderly exfoliated morphology [4] . Figure 4. 1 XRD spectra of the different nanoclays, PLA1, and ( a) OMMT, ( b) HNT, and ( c) LRD bio - nanocomposite films. Figure 4. 2 TEM micrographs of ( a) PLA - OMMT5, ( b) PLA - HNT5, and c) PLA - LRD5 bio - Similarly, Figure 4. 1 b and Figure 4. 1 c . show the XRD spectra of HNT and LRD nanocomposites, respectively. In both cases, the profiles showed broad peaks around a [50,51] . HNT is an alumina - silicate clay with an elongated hollow tubular structure consisting of an external surface composed of siloxane (Si - O - Si) groups and an inner side and edges consisting of (Al - OH) groups [16,24,52] . In the XRD spectrum of the HNT nanoclay ( Figure 4. 1 b ), 245 observed, corresponding to the basal d - spacing of 0.75, 0.45, and 0.36 nm, respectively. Similar diffraction patterns are reported elsewhere [24,53 57] . In the case of PLA - served. The small shift to the right, from the 12.02 of the pristine HNT, indicates a reduction in the d - spacing. This behavior has been observed by other researchers, and was attributed to the formation of a micro - filled composite [24,54] . The disappearance of the other peaks, such in the case of PLA - HNT5 and PLA - HNT1, has been explained as due t o the interaction of the polymer chains with the nanotubes, and also due to the preferential orientation of nanotubes during processing of the film [19,24] . It was also observed that the intensity of the char acteristic peaks depends on the level of loading of nanoclay [53,54] . LRD particles have a disk - like shape with two external tetrahedral silica sheets that present continuous corner - shared tetrahedral SiO 4 units arranged in hexagonal rings, and a central octahedral magnesia sheet which is composed of bivalent or trivalent cations sharing the edges coordinated to hydroxyl groups. The excess negative charge is compensated by the presence of Na ions between the silicate layers [25,27 29] . In th e XRD spectrum of the LRD nanoclay ( Figure 4. 1 c ), the presence of the e basal d - spacing of 0.45 nm. Similar diffraction patterns are reported for LRD elsewhere [25,26] . In the XRD spectra of the PLA - LRD, no diffraction peaks were observed. This behavior has been attributed, in the literature, to separated LRD platelets dispersed 246 individually in the polymer matrix [25] . The n anoclay dispersion was also confirmed by TEM. Figure 4. 2 b and Figure 4. 2 c show the TEM micrographs of HNT and LRD nanocomposites, respectively. In the case of PLA - HNT5, Figure 4. 2 b shows the presence of big agglomerations indicating that HNT was not evenly distributed in the PLA matrix. Similar observations have been reported in the literature for PLA - HNT nanocomposite s [20,53] . A similar distribution was also found for the PLA - LRD5 film ( Figure 4. 2 c ). Other factors influencing the nanoclay dispersion in the PLA matrix are the level of loading and the size of the nanoparticles [26] . For example, HNT and LRD are bigger particles than MMT. While MMT has layer s with 1 nm thickness and tangential [1,3,7] , HN T has inner and outer diameters of the tube ranging from 10 to 40 nm and 40 to 70 nm, respectively, while the length [16,24,52] . LRD usually has dimensions around 25 - 30 nm in diameter and 1 nm in thickness [26,27,29] . 4.3.2 Biodegradation evaluation The DMR system was used to perform four diff erent biodegradation tests in which the CO 2 evolved from each bioreactor was measured with controlled temperature, RH, and air flow rate. For the data analysis, the average cumulative CO 2 and % mineralization of each test material was calculated and plotte d as a function of time. Detailed information about the concepts and calculations is provided elsewhere [47,58 60] . The blank bioreactors contain the solid media only ( i.e. , compost or vermiculite). In all cases, cellulose powder was used as a positive reference material since it is a well - known 247 easily biodegradable material. The cumulative CO 2 and % mineralization curves obtained from the different biodegradation tests for t he evaluation of PLA and PLA - BNCs, as well as the different nanoclays and surfactant, are presented in Figure 4. 3 to Figure 4. 11 . To evaluate the effect of the nanoclays on the compost microbial population, the three different nanoclays were tested in the powder form as received. Figure 4. 3 shows the CO 2 evolved from the bioreactors containing the three different nanoclays. A significant difference between the CO 2 evolved from cellulose and the one from the nanoclays was observed. During the first 40 days of the test, OMMT and LRD bioreactors produced a significantly higher amount of CO 2 than the blank indicating that there was no inhibition. On the contrary, the H NT bioreactors produced equal or less CO 2 than the blank, especially after 35 days, indicating some kind of inhibition in which HNT may limit the availability and/or the distribution of carbon and other nutrients for basic microorganism functions. Figur e 4. 3 CO 2 evolution of the three different nanoclays (Test I in compost) . 248 Figure 4. 4 shows the CO 2 and % mineralization of the pristine PLA film and PLA - OMMT5. The typical PLA biodegradation behavior with the presence of a lag time of around 25 days was observed [47, 61] . The lag time observed in the biodegradation of PLA has been explained by the low diffusion rate of the byproducts formed during the hydrolytic degradation and present inside the sample [62] . Cellulose reached a maximum mineralization of 65.7% after 34 days while PLA and PLA - OMMT5 reached 53.2 and 59.6% after 87 days, respectively. T he decrease in the mineralization curve of cellulose indicates that these bioreactors were no longer producing more CO 2 than the blank bioreactors. This behavior may be explained by a rapid and large increase of the microbial population at the beginning of the test when there are plenty of resources easily available for microbial assimilation. Then, a decrease in the mineralization curve is observed when these resources are depleted and/or limited [47] . Even though by the end of the test, the mineralization of PLA and PLA - OMMT5 was not significantly different, it was clearly observed that the lag phase of the pristine PLA was longer than the PLA - OMMT5. The mineralization of PLA - OMMT5 was significantly higher before day 60. Among the different explanations for this accelerated biodegradation due to OMMT found in the literature is the relatively hig h hydrophilicity of the nanoclay, which improves the diffusion of water into the PLA polymeric matrix and in turn promotes hydrolytic degradation [33,37,38,44,62] . Another reason is that th e presence of terminal hydroxyl groups in the silicate layers and in some organo - modifiers promotes the hydrolytic degradation of PLA [10,44,63] . However, the molecular weight of the PLA - OMMT5 films and the thickness can play a cru cial role and influence the observed results [47] . 249 Figure 4. 4 ( a) CO 2 evolution and ( b) % Mineralization of PLA and PLA - OMMT5 films (Test I in compost) . To evaluate the effect of clay loading on the biodegradation of PLA, three films with different loadings of OMMT (1, 5, and 7.5% wt.) were tested. Figure 4. 5 shows the CO 2 evolution and % mineralization of PLA and PLA - OMMT films. Cellulose reached a maximum mineralization of 61.7% after 45 days of testing. The biodegradation behavior of the pristine PLA and PLA - OMMT1 was similar, again with a typical lag time at the beginni ng of the biodegradation test. The negative mineralization values observed in Figure 4. 5 b are generated as an artifact when the blank bioreactors prod uce more CO 2 than the sample material bioreactors. This effect might be caused because of the physical barrier offered by the polymer film at this early stage of the test, contrary to the PLA - OMMT5 and PLA - OMMT7.5 in which their biodegradation phase starte d much earlier. The observed shorter lag time of PLA - OMMT5 is in agreement with the previous test results, but in this case the average mineralization was significantly higher than the PLA control. It seems that PLA - OMMT7.5 has the highest average minerali zation and the fastest biodegradation rate in which the lag time was only around 5 days. However, 250 mineralization values above 100% indicate the presence of a priming effect, in which the additional carbon converted to CO 2 , is not coming from the sample mat erial but from the over - degradation of the indigenous organic carbon present in the compost [47,64] . Again, the initial molecular weight of the films should influence the observed results. It is important to mention that during the processing of the films, with different nanocla y loading, the resulting molecular weight was affected even though, in this case, the same processing conditions were maintained, with the higher clay loading corresponding to the lower molecular weight. Furthermore, Roy et al. analyzed the water - soluble d egradation products by electrospray ionization - mass spectrometry (ESIMS), and their results indicated a catalytic effect of MMT in hydrolysis of PLA since shorter lactic acid oligomers were formed in the case of PLA/MMT composites [41] . Some researchers have att ributed a plasticizing effect to the degradation byproducts ( i.e., lactic acid oligomers and monomers), represented by a decrease in the T g of PLA and BNCs. In this context, faster biodegradation of the PLA and BNC could also be induced by the increased se gmental mobility of backbone chains and the expanded amorphous regions of the polymeric matrix [44,62,65] . Another factor infl uencing the biodegradation rate of the BNCs is the crystallinity of the material. The presence of nanoclays could affect the degree of crystallization of PLA ( Table 4B. 2 ), with the amorphous parts preferentially biodegrading [47] . 251 Figure 4. 5 ( a) CO 2 evolution and ( b) % Mineralization of PLA and PLA - OMMT films with three different levels of loading (1, 5, and 7.5%) (Test II in compost) . The effect of the amount/concentration of clay and surfactant on the c ompost microbial populations was evaluated and the results are shown in Figure 4. 6 . In this case, OMMT refers to 8 g of the tested sample material whi le OMMT5 refers to the theoretical amount of nanoclay contained in 8 g of PLA - OMMT5 film. Similarly, QAC refers to 8 g of the tested sample material and QAC5 to the theoretical amount of surfactant contained in 8 g of PLA - OMMT5 film. Regardless of the conc entration of either OMMT or QAC, the CO 2 evolution was always significantly lower than the blank, indicating that there was clear inhibition of the microbial activity when these materials were present by themselves. 252 Figure 4. 6 CO 2 evolution of OMMT nanoclay and QAC surfactant (Test II in compost) . Figure 4. 7 shows the results of a different biodegradation test in which the PLA - OMMT and the PLA - QAC films were evaluated. Cellulose reached a mineralization of 85.5% after 38 days of testing, while the PLA control reached 74.2% after 69 days. As in the previous test, there was no significant difference between the pristine PLA a nd the PLA - OMMT1 films ( Figure 4. 7 b ). However, PLA - OMMT5 had significantly higher mineralization and a shorter lag time than the PLA control. A primin g effect was observed with mineralization values over 100%. The PLA films containing the surfactant (QAC) also showed reduced lag time and a significantly higher amount of evolved CO 2 than the PLA control, and in both cases a priming effect was observed ( Figure 4. 7 d ). This may be due to the lower initial molecular weight of these films. In Chapter 3 , it was demonstrated that the PLA film with the lowest M n presented a priming effect when tested in compost, but it was not observed in inoculated vermiculite, having mineralization values closer to the other two tested PLA films with higher M n [47] . PLA - OMMT5 and PLA - QAC0.4 were also tested in inoculated and uninoculated vermiculite, 253 and the results are later shown in Figure 4. 11 . Similarly, the priming effect was not observed in this case. Figure 4. 7 CO 2 evolution and % Mineralization of PLA - OMMT films (a & b) and PLA - QAC films (c & d) (Test III in compost) . Figure 8 shows that the mineralization of PLA - HNT films was not significantly different from the PLA control by the end of the test (90 days). However, it can be clearly observed that with both levels of loading the lag time was reduced and the mineralizat ion was significantly different before day 45. A higher variability and also a priming effect were observed in the biodegradation of PLA - HNT1 film. PLA - HNT films 254 reached their maximum mineralization after 50 days of testing with an average of 86.9 and 74.6 % for PLA - HNT1 and PLA - HNT5, respectively. Figure 4. 8 ( a) CO 2 evolution and ( b) % Mineralization of PLA - HNT films (Test III in compost) . As observed in Figure 4. 9 , PLA - LRD5 showed significantly higher mineralization than the pristine PLA and the PLA - LRD1 films. In this case, the lag time was not reduced but the PLA - LRD5 showed a priming effect. PLA - LRD films reached their m aximum mineralization by the end of the test with an average of 82.5 and 112.5% for PLA - LRD1 and PLA - LRD5, respectively. 255 Figure 4. 9 (a ) CO 2 evolution and ( b) % Mineralization of PLA - LRD films (Test III in compost) . To avoid the priming effect observed in the previous tests, a specific new biodegradation test was performed in three different solid environments (compost, inoculated vermiculite, vermiculite) as described elsewhere [47] . When tested in compost ( Figure 4. 10 ), there was no significant difference in the mineralization of these materials by the end of the test (132 days). However, it seems that the mineralization of PLA - OMMT5 was significantly higher than the PLA during the first 45 days of te sting. Similarly to the previous tests, PLA - OMMT5 showed a reduced lag time and a priming effect could be occurring due to the low molecular weight of both films. The maximum average mineralization for PLA and PLA - OMMT5 was 110.4 and 100.2%, respectively. 256 Figure 4. 10 (a ) CO 2 evolution and ( b) % Mineralization of PLA and PLA - OMMT5 films (Test IV in compost) . The biodegradation test with inoculated vermiculite should avoid the priming effect as previously demonstrated [47,64,66] . Figure 4. 11 shows that there was no significant difference in the mineralization of the tested materials at the end of the test (132 days). However, both PLA - OMMT5 and PLA - QAC0.4 showed significantly higher mineralization than the PLA control before 70 days of testing , and a much shorter lag time. The PLA control reached 77.7% mineralization after 132 days while PLA - OMMT5 reached the same mineralization after 83 days of testing and a maximum average mineralization of 83.3%. PLA - QAC reached a mineralization of 77.3%. It is important to mention that longer testing times were expected in this case since the biodegradation in inoculated vermiculite occurs at a slower rate than in compost. Even though the initial molecular weight of the films has a strong effect on their min eralization and priming effect, it seems that the addition of OMMT also accelerated the initial degradation of the samples. As previously mentioned, this behavior may be explained by the improved diffusion of water into PLA due to the high hydrophilicity o f the nanoclay, which in turn promotes hydrolytic degradation [33,37,38,44,62] . 257 Figure 4. 11 (a ) CO 2 evolution and ( b) % Mineralization of PLA, PLA - OMMT5, and P LA - QAC0.4 (Test IV in inoculated vermiculite (dashed lines) and uninoculated vermiculite (dotted lines)) . Figure 4. 11 also shows the results when testi ng with uninoculated vermiculate. As expected, there was no significant evolution of CO 2 in the abiotic degradation test, and there was no significant difference in the mineralization values. For the biodegradation test III, film samples were taken at diff erent periods of time in order to track the changes in the molecular weight and the results are explained in section 4. 3.3. 4.3.3 Molecular Weight Figure 4. 12 shows the initial molecular weight distribution (MWD) of the PLA film and BNCs. As previously mentioned, the addition of nanoclay resulted on a reduction of the M n during processing. This red uction in M n was more pronounced in the case of PLA - OMMT5, PLA - QAC1.5, and PLA - QAC0.4. More detailed information about the initial M n , M w , and PDI, of PLA and BNCs films is provided in Table 4D. 1 of the Appendix 4D. 258 Figure 4. 12 Initial molecular weight of PLA and BNCs . Figure 4. 13 shows the decrease of molecular weight of the PLA control film as function of time during the biodegradation test III, represented by the shift of the peak to the left. This behavior was prev iously reported in the literature during the hydrolytic degradation of PLA, and was attributed to the chain scission preferentially occurring in the bulk of the polymer matrix rather than the surface [67] . The broadening of the peaks over time indicates an increase in the PDI due to the fragmentation of the PLA chain s. The change in the MWD from monomodal to multimodal after day 14 has also been previously observed during hydrolytic degradation of PLA and was attributed to the formation of crystalline residues due to the rearrangement of the new shorter polymer chains into a more stable configuration ( i.e., crystalline structures) [5 1,67] . The 259 formation of more defined and higher peaks, as observed at days 42 and 56, has been attributed to the predominant degradation of the amorphous regions [68] . During the biodegradation tests a whitening effect in PLA and BNC was observed. It has been reported that this effect indicates increased crystallinity and opacity due to the beginning of the hydrolytic degradation phase of the biodegradation process [44,45,62] . The whitening effect occurs because a change in the refraction index of the polymer is induced by the absorb ed water and/or the byproducts, e.g., carboxylic end - groups that are able to catalyze ester hydrolysis [45,62] . Figure 4. 13 Change in molecular weight of PLA2 film (Test III in compost) . Figure 4. 14 shows the changes in the MWD of the BNCs as function of time until day 28 since it was not possible to collect samples for SEC analysis after that period of time (except for PLA control as sh own in Figure 4. 13 ). Similarly to the PLA control, the BNCs showed multimodal peaks after day 14, although more evidently after day 21. In general, th is behavior was less pronounced for PLA - OMMT1, PLA - LRD1, and PLA - LRD5, and it may be attributed to a slower formation of crystalline residuals. From Figure 4. 14 , it can be observed that the reduction of molecular weight was slower for 260 PLA - OMMT1 and PLA - LRD1, in comparison with the pristine PLA. Similarly, the MWD of PLA - OMMT5 and PLA - QAC15 have a similar trend with an evident multimodal peak at day 21, while the reduction of molecular weight of PLA - HNT5 and PLA - LRD5 films seems to be slower than PLA control. 261 Figure 4. 14 Change in molecular weight of ( a) PLA2, ( b) PLA - OMMT1, ( c) PLA - OMMT5, ( d) PLA - QAC0.4, ( e) PLA - QAC1.5, ( f) PLA - HNT1, ( g) PLA - HNT5, ( h) PLA - LRD1, and ( i) PLA - LRD5 films (Test III in compost). 262 Deconvolution of the peaks was performed due to the multimodal MWD observed in the previous results, followed by kinetics analysis ( Appendix 4 D ). The M n reduction rate ( k ) constant was calculated for PLA and the BNCs, fitting of a first order reaction of the form M n / M n0 = exp( - kt ), where M n0 is the initial M n , and t is the time. The results ( Figure 4D. 3 and Table 4D. 2 ) show that the BNCs, especially PLA - LRD films, have a lower M n reduction rate than the PLA contro l ( k = 0.1008 ± 0.0037) until day 28. Ray and Okamoto analyzed the molecular weight of PLA and PLA nanocomposites and found that the reduction was almost the same for all the samples [10] . In contrast, Paul et al. found that the M n of PLA decreased ~40% with respect to its initial value while for the PLA nano composites M n decreased 70 - 80% [38] . In this case, even though the M n reduction rate of the BNC was the same or lower than the PLA control, a higher evolution of CO 2 from the bioreactors supplemen ted with the BNC was generally observed during the biodegradation tests. Therefore, it is also relevant to understand the role of the microorganisms and how they are affected by the presence of these nanoclays. For example, Annamalai et al. suggested that the clay nanoparticles improve the absorption of UV light and promote polymer photo - oxidation due to the catalytic effect of metal ion impurities. That increased oxidation at the surface of the nanocomposites could favor the adhesion, accumulation and grow th of the microorganisms [69] . 4.3.4 Microbial attachment Biofilm assays were performed to evaluate the ability of the microorganisms present in the compost to attach to the surface of PLA film and BNCs ( i.e., PLA - OMMT5, PLA - QAC0.4, PLA - HNT5, and PLA - LRD5). Even though biofilm formation does not 263 necessarily mean that the material is biodegraded by the attached populations [7 0] , it is an important aspect of microbial performance and survival [71] . When biofilm - forming microorganisms release exopolymeric substances (EPS) ( e.g. , carbohydrates, nucleic acids, and proteins) such resources become available for other microorganisms, including secreted enzymes that degrade PLA and der ivatives. Secreting extracellular digestive enzymes after forming a biofilm would localize the effect of extracellular digestion and increase the benefit to biofilm - forming strains. Biofilm production is a common trait among microorganisms living in soil w hich are usually exposed to low moisture conditions. Biofilms can contribute to water retention in the soil matrix, prevent microorganisms from being washed out, and confer tolerance to other environmental stressors [71] . An initial test of the biofilm assay is shown in the Appendix 4E . Figure 4. 15 , Table 4E. 3 and Table 4E. 4 show the results of the biofilm test. A positive control was performed using Pseudomonas aeruginosa (PA) strain PAO1, a high biofilm forming strain, at 23°C [72,73] . Looking at the control with PA at 23°C (Figure 15.a), it is observed that the control wells (R2B only) have an absorbance (A600 nm) of 1.226 - 1.332, with uninoculated control wells ranging from 0.060 to 0.065, which is in agreement with the values reported by Satti e t al. [49] . The wells containing PLA, PLA - QAC0.4, PLA - HNT5, and PLA - LRD5 were approximately the same as the control lacking any film (R2B only). However, the wells containing PLA - OMMT5 showed significantly more biofilm formation (average 2.042), suggesting th at the OMMT had an indirect stimulation on biofilm formation by PA. For the biofilm formed on the surface of the films by PA at 23°C, PLA ranged from 0.501 to 0.752, which is also in agreement 264 with the values previously observed [49] . In this case, the values of PLA - OMMT5 and PLA - HNT5 were significantly different from PLA - QAC0.4. PLA - HNT5 had one of the highest average values with 1.254. Looking at the total biofilm formation, PLA - OMMT5 and PLA - QAC0.4 were significantly different from pristine PLA and the rest of the BNCs with the highest (2.917) and lowest (1.107) values, respectively. The total average biofilm values (wells + film) for PA at 2 3 °C in descending order are as follows PLA - OMMT5 >PLA - HNT5 >PLA >PLA - LRD5 >PLA - QAC0.4. Regarding the biofilm estimates with CE at 58°C ( Figure 4. 15 b ), the sterile controls (SCE) have values that are between 0.101 and 0.124, which are slightly greater than what was seen with low nutrient media at 23°C. This is probably due to significant amounts of humic material in the CE. The control wells (CE only) have values of 0.381 - 0.588. These values are less than the ones for PA at 23°C which is expected since PA is a well - known biofilm former and because microbial growth and survival is generally more challenging at 58°C and CE contains a diverse collection of microbial populations, many of which do not form biofilm under these conditions. The wells supplemented with PLA and BNCs ranged from 0.122 - 0.603 with no statistically significant difference among them. Biofilm formation was observed on the surface of PLA and BNCs with CE at 58°C. PLA - LRD5 has significantly higher value (0.519) than the rest of the BNCs. The lowest average values were observed for PLA - QAC0.4 and PLA with 0.113 and 0.090, respectively. In this case, the total biofilm was also not significan tly different among the sample materials. In general, the PLA - LRD5 biofilm was the largest among the different samples, indicating that population in CE have a preference for PLA - LRD5 at 58°C. In contrast, a 265 pure culture, Pseudomonas aeruginosa , clearly p referred PLA - OMMT5 at 2 3 °C. Overall the biofilms at 58°C are smaller than the biofilm at 2 3 °C. At both temperatures, PLA - QAC0.4 was the film producing the lowest average amount of biofilm which may be attributed to inhibition due to the surfactant. This is supported by the biodegradation test where the surfactant was tested alone. Further investigation is recommended to understand which are the specific microbial strains present in the compost that bind to and preferentially degrade PLA and the BNCs. Figu re 4. 15 Absorbance (600 nm) of ( a) PA at 2 3 °C, and ( b) CE at 58°C for second biofilm test. Columns with the same letter within a group ( i.e., wells, films, or total) are . Due to the significant differences between pristine PLA and PLA - LRD5 found in the biofilm formed on the surface of the films during the test at 58°C with CE, several SEM micrographs were taken from samples coated with osmium. Figure 4. 16 shows the difference in microbial attachment between pristine PLA and PLA - LRD5 at a magnification of 1000x. It can be clearly observed that the surface of PLA - LRD5 is much mor e heavily populated by microorganisms, in agreement with the biofilm test results ( Figure 4. 15 b ). 266 Figure 4. 16 SEM micrographs of ( a) PLA and ( b) PLA - LRD at 1000x before incubation, ( c) PLA and ( d) PLA - LRD5 after incubation for 48 h at 58°C with compost extract in R2B. 4.4 Final Remarks The effect of three different nanoclays, OMMT, HNT, and LRD, as well as the OMMT or gano - modifier (QAC) on the biodegradation of PLA was evaluated with an in - house built DMR system following the analysis of evolved CO 2 approach. The results obtained from four different biodegradation tests along with the study of microbial attachment on t he surface of PLA and its BNCs show that the biodegradation phase of the films containing nanoclay started earlier than that for pristine PLA. This behavior was confirmed by the results obtained from different tests for PLA - OMMT5, even when 267 tested in inocu lated vermiculite. The tests performed in vermiculite allowed untangling the observed priming effect even though longer testing times were required. The effect of the nanoclays on the initial molecular weight during processing played a crucial role in the biodegradation studies, also since a lower M n0 to the priming effect in compost. Further investigation is recommended using PLA and BNCs with the same initial molecular weight and thickness, a task not easy to achieve in la b settings. When the different nanoclays and surfactant were tested alone, it was observed that HNT, OMMT, and QAC showed some inhibition regardless of the amount introduced in the bioreactors. PLA - LRD5 showed a priming effect with mineralization values ex ceeding 100%. This behavior may be explained by the lower initial molecular weight and by the results observed during the microbial attachment tests, in which PLA - LRD5 showed the greatest biofilm formation on the surface as confirmed by the SEM micrographs . PLA - QAC0.4 had the lowest biofilm formation, which may be attributed to the inhibitory effect also found during the CO 2 evolution test when QAC was tested alone. Under the experimental conditions used to investigate biofilm formation, it was noted that s ignificant biofilm was established in only 48 hours; however, the timing may be different in composting conditions. Further investigation is required on the specific microbial strains that are capable of biodegrading PLA and its BNCs and how they can affec t the biodegradation rate. Disposable products like packaging would greatly benefit from the biodegradable features of PLA since it would allow its disposal along with other organic wastes in composting facilities. 268 APPENDICES 269 APPENDIX 4A: Material processing Masterbatch (MB) production: The PLA - BNCs ( PLA - OMMT, PLA - HNT, and PLA - LRD) masterbatches (15 20 % nanoclay wt. ) were prepared in a ZSK 30 twin - screw extruder (Werner Pfleiderer, NJ) and pelletized. A PLA - QAC masterbatch (10% QAC wt.) was prepared in a similar fashion. Pristine PLA was processed in the twin - screw extruder and used for the processing of PLA1 control film. Table 4A. 1 shows the general MB processing conditions. F ilm production: All films were produced by using a Microextruder model RCP - 0625 (Randcastle Extrusion Sys tems, Inc., Cedar Grove, NJ) with a screw diameter of 15.9 mm, screw L/D of 24, and volume of 34 cm 3 . Table 4A. 1 shows the processing conditions of the films and their thickness as measured with a digital thickness micro with the digital micrometer may not be the best approach due to the presence of the nanoclay. The thickness of PLA - OMMT1 and PLA - OMMT5 was measured from the SEM cross - section of th e films and it was found to be 0.020 ± 0.004, and 0.010 ± 0.002 mm, respectively. 270 Table 4A. 1 Processing conditions of the sample materials Material Conc., wt% Type Temp. range, ° C rpm Thickness, mm PLA 0% MB 146 - 186 130 N/A PLA - OMMT 20% MB 146 - 186 130 N/A PLA - QAC 10% MB 148 - 189 130 N/A PLA - HNT 15% MB 159 - 181 40 N/A PLA - LRD 15% MB 159 - 181 40 N/A PLA1 0% Film 194 - 216 49 0.031 ± 0.006 PLA2 0% Film 193 - 249 33 0.022 ± 0.003 PLA3 0% Film 193 - 249 28 0.034 ± 0.009 PLA - OMMT 1% Film 193 - 243 18 0.044 ± 0.007 PLA - OMMT 5% Film 193 - 248 18 0.073 ± 0.014 PLA - OMMT 7.5% Film 193 - 243 18 0.089 ± 0.013 PLA - QAC 0.4% Film 143 - 173 31 0.039 ± 0.008 PLA - QAC 1.5% Film 143 - 173 31 0.036 ± 0.011 PLA - HNT 1% Film 193 - 216 23 0.037 ± 0.007 PLA - HNT 5% Film 193 - 216 23 0.050 ± 0.006 PLA - LRD 1% Film 193 - 216 23 0.064 ± 0.013 PLA - LRD 5% Film 193 - 216 23 0.127 ± 0.011 N/A: Not applicable 271 APPENDIX 4B: Material characterization Elemental Analysis (CHN) : The carbon , hydrogen, and nitrogen content of the different test materials was determined by using a PerkinElmer 2400 Series II CHNS/O Elemental Analyzer (Shelton, CT, USA), and values are presented in Table 4B. 1 . The amount of filler present in each of the films was co nfirmed by CHN, considering the theoretical chemical structure of PLA and each of the components. Table 4B. 1 Carbon, hydrogen, and nitrogen content of the test ed materials Material ID % Carbon a % Hydrogen a % Nitrogen a Cellulose powder Cellulose 42.50 ± 0.34 6.53 ± 0.05 0.04 ± 0.01 Ingeo TM 2003D film PLA1 50.05 ± 0.05 5.65 ± 0.02 0.01 ± 0.01 PLA2 49.93 ± 0.11 5.56 ± 0.02 0.01 ± 0.01 PLA3 49.99 ± 0.05 5.60 ± 0.01 0.01 ± 0.01 Cloisite ® 30B OMMT 19.22 ± 0.06 3.84 ± 0.02 0.99 ± 0.00 Laponite ® RD LRD 0.18 ± 0.01 1.19 ± 0.04 0.02 ± 0.01 Halloysite HNT 0.09 ± 0.02 1.83 ± 0.05 0.01 ± 0.00 Tomamine TM QAC 59.28 ± 0.60 12.28 ± 0.05 2.55 ± 0.02 PLA - OMMT 1% a PLA - OMMT1 49.49 ± 0.07 5.54 ± 0.04 0.03 ± 0.02 PLA - OMMT 5% a PLA - OMMT5 48.76 ± 0.07 5.49 ± 0.02 0.07 ± 0.01 PLA - OMMT 7.5% a PLA - OMMT7.5 47.75 ± 0.11 5.43 ± 0.01 0.09 ± 0.00 PLA - HNT 1% a PLA - HNT1 49.67 ± 0.12 5.60 ± 0.06 0.70 ± 0.34 PLA - HNT 5% a PLA - HNT5 48.22 ± 0.10 5.44 ± 0.01 1.68 ± 0.43 PLA - LRD 1% a PLA - LRD1 49.58 ± 0.17 5.54 ± 0.05 2.43 ± 0.42 PLA - LRD 5% a PLA - LRD1 47.70 ± 0.11 5.39 ± 0.06 6.43 ± 1.82 PLA - QAC 0.5% PLA - QAC0.5 49.98 ± 0.08 5.55 ± 0.02 0.01 ± 0.00 PLA - QAC 1.5% PLA - QAC1.5 50.55 ± 0.04 5.78 ± 0.02 0.05 ± 0.01 a Percentage by weight Differential Scanning Calorimetry (DSC) : The g lass transition ( T g ) and melting ( T m ) temperatures of PLA and BNCs films were determined using a DSC model Q - 100 ( TA Instruments, New Castle, DE) and the TA Instruments Universal Analysis 2000 software (Version 4.5A) . The testing temperature was from 5°C to 210°C with a ramping rate of 10°C/min. The results are shown in Figure 4B. 1 and Table 4B. 2 . 272 Figure 4B. 1 DSC of the PLA and BNCs films (1 st cycle) . Thermogravimetric Analysis (TGA) : The d egradation temperature ( T d ) of the PLA and PLA - OMMT films was measured with a TGA model Q50 from Thermal Analysis Inc. (New Castle, DE). The testing temperature was from 23°C to 600°C with a ramping rate of 10°C/min. The results are shown in Figure 4B. 2 and Table 4B. 2 . Figure 4B. 2 TGA of the PLA and PLA - OMMT films . 273 Table 4B. 2 Thermal properties of the PLA and BNCs Sample T g , °C T c , °C T m , °C T d , °C % X c PLA1 63.3 N / A 152.0 349 .0 25.0 PLA2 54.4 107.1 146.4 N/A 4.6 PLA - MMT1 59.8 109.8 154.9 389.2 1.6 PLA - MMT5 57.8 101.5 154.6 355.0 4.3 PLA - MMT7.5 57. 9 90.3 152.8 391.5 12.3 PLA - HNT1 56.8 103.1 154.7 N/A 4.0 PLA - HNT5 56. 7 103.7 153.0 N/A 4.5 PLA - LRD1 57.9 92.0 154.2 N/A 11.6 PLA - LRD5 55.6 109.7 155.5 N/A 2.7 N/A: Not available Moisture sorption isotherm : The moisture sorption isotherms of the nanoclays, PLA, and BNCs films were examined by gravimetric analysis using an SGA - 100 from VTI Corp. (Hialeah, FL). The samples (5 - 10 mg) were exposed to relative humidity (RH) between 0 and 95 ± 2% with RH steps of 10, at 23 ± 0.1 o C. The results are shown in Figure 4B. 3 . Figure 4B. 3 Moisture sorption isotherms of the nanoclays, PLA and BNCs films . Electrical conductivity : The measurements were carried out using an e lectrochemical impedance spectroscopy (EIS) system (Gamry Instruments, 274 Warminster, PA) for 2.54 cm 2 film samples. Copper f oil t ape with c onductive a dhesive was located on the surface of the film from both sides, and the electrodes were attached to each extreme of the tape. The Gamry Framework software was used for the analysis using the Potentiostatic EIS mode. The conductivity was measured over a frequency range of 1 x 10 5 to 0.1 Hz with an applied potential of 20 mV at room temperature ( 2 3 °C ). The resistivity values presented in Table 4B. 3 were calculated using the impedance (Z) value at a frequency of 0.1 Hz. Table 4B. 3 Resisitivity of the PLA and BNCs Sample Resistivity PLA2 3.96E+13 ± 1.47E+11 AB PLA - OMMT5 3.31E+13 ± 5.05E+12 B PLA - QAC0.4 3.77E+13 ± 1.00E+12 AB P LA - HNT5 3.61E+13 ± 4.21E+12 AB PLA - LRD5 4.15E+13 ± 6.20E+11 A Note: - Kramer Test. Contact angle : Sur face wettability of the PLA and BNCs films was evalu ate d by contact angle measurements using a goniometer (Drop Shape Analysis System, DSA10 Mk2 , Krüss GmbH, Hamburg, Germany), equip ped with a diffuse light source and a CCD camera, at room temperature (2 3 ° C ). A drop of HPLC grade water (3 L) was deposited on the film surface and a magnified image of the drop profile was conveyed to a computer. The contact angle was measured with the Drop Shape Analysis Software using the tangent method. Ten measurements pe r film were performed and the values reported in Table 4B. 4 are the average of contact angles measured on both sides of the drop. 275 Table 4B. 4 Contact angle of the PLA and BNCs measured with water at room temperature Sample Contact angle PLA2 71.6 ± 2.1 D PLA - OMMT5 96.4 ± 4.2 A PLA - QAC0.4 83.3 ± 4.5 C PLA - HNT5 93.2 ± 2.5 B PLA - LRD5 85.6 ± 3.9 C Note: - Kramer Test. 276 APPENDIX 4C: Physicochemical characteristics of the compost Samples of the compost used in the different biodegradation tests were sent to the Soil and Plant Nutrient Laboratory at Michigan State University (East Lansing, MI, USA) for determination of the physicochemical parameters (dry solids, volatile solids, C/N ratio, pH, and microbial activity) as shown in Table 4C. 1 . Detailed information about the methods used for compost characterization can be found elsewhere [47] . Table 4C. 1 Physicochemical c haracteristics of the compost used in the different biodegradation tests Parameters ISO b I II III IV Dry solids, % 50 - 55 53.3 52.7 41.5 60.9 Volatile solids, % <30 26.4 44.3 43.2 39.1 pH 7 - 9 7.8 7.9 8.5 7.4 Total Carbon, % N/A a 15.3 25.7 25.1 22.7 Total Nitrogen, % N/A a 0.9 2.4 2.4 2.1 C/N ratio 10 - 40 17.4 10.8 10.3 10.9 Compost activity c 50 - 150 39.0 81.1 63.0 62.5 a Not applicable or not available b Values based on ISO 14855 - 1:2005 standard c Average values measured in mg of CO 2 per g of VS in the first 10 days 277 APPENDIX 4D: Molecular weight determination Initial molecular weight : The number average molecular weight ( M n ), weight average molecular weight ( M w ), and polydispersity index ( PDI ) of the samples before and during composting were determined by size exclusion chromatography (SEC ) with a system from Waters Inc. (Milford, MA), equipped with a Waters 1515 isocratic pump, a Waters 717 autosampler, a series of three columns (HR2, HR3, and HR4 Waters Styragel ® ), and a Waters 2414 refractive index detector interfaced with Waters Breeze software [47] . Table 4D. 1 shows the initial M n , M w , and P D I of the samples as measured before each of the different biodegradation tests. 278 Table 4D. 1 Initial M n , M w , and P D I of the PLA samples Biodegradation test Sample M n , kDa M w , kDa PDI I PLA1 113.1 ± 0.1 A 208.0 ± 0.8 A 1.8 ± 0.0 B PLA - OMMT5 59.8 ± 1.1 B 118.9 ± 0.9 B 2.0 ± 0.0 A II PLA1 113.1 ± 0.1 A 208.0 ± 0.8 A 1.8 ± 0.0 A PLA - OMMT1 82.9 ± 2.2 B 157.3 ± 1.7 B 1.9 ± 0.0 A PLA - OMMT5 59.8 ± 1.1 C 118.9 ± 0.9 C 2.0 ± 0.0 A PLA - OMMT7.5 37.5 ± 2.3 D 76.7 ± 1.3 D 2.1 ± 0.2 A III PLA2 88.8 ± 0.9 A 172.0 ± 1.3 A 1.9 ± 0.0 C PLA - OMMT1 82.9 ± 2.2 ABC 157.3 ± 1.7 B 1.9 ± 0.0 C PLA - OMMT5 52.8 ± 0.7 D 116.1 ± 0.3 D 2.2 ± 0.0 A PLA - HNT1 91.4 ± 3.3 A 171.1 ± 1.2 A 1.9 ± 0.1 C PLA - HNT5 79.7 ± 3.8 BC 153.0 ± 2.5 B 1.9 ± 0.1 BC PLA - LRD1 84.2 ± 1.7 AB 155.5 ± 1.4 B 1.8 ± 0.0 C PLA - LRD5 75.3 ± 0.9 C 139.0 ± 0.7 C 1.8 ± 0.0 C PLA - QAC0.4 43.5 ± 3.8 E 88.7 ± 1.6 F 2.0 ± 0.1 ABC PLA - QAC1.5 45.0 ± 2.4 E 96.7 ± 1.3 E 2.2 ± 0.1 AB IV PLA1 119.0 ± 11.3 A 234.4 ± 16.9 A 2.0 ± 0.1 B PLA2 101.1 ± 11.8 AB 206.2 ± 23.1 A 2.0 ± 0.1 AB PLA3 84.8 ± 6.9 B 167.4 ± 3.2 B 2.0 ± 0.2 B PLA - OMMT5 45.5 ± 5.8 C 108.6 ± 11.6 C 2.4 ± 0.2 A PLA - QAC0.4 54.5 ± 9.5 C 118.2 ± 5.8 C 2.2 ± 0.3 AB Note: Values with the same letter within the same group ( i.e., biodegradation test) and - Kramer Test. Molecular Weight Reduction during Biodegradation : Due to the observed multimodal MWD in the results presented in Section 4. 3.3, deconvolution of the MWD peaks was necessary for conducting kinetics analysis, in which the M n reduction rate ( k ) constant was calculated for PLA and the BNCs. Therefore, a curve fitting and data analysis program, Fityk version 1.3.0, developed by Marcin Wojdyr [74] , was used for deconvolution using a log normal function as was used by Perejon et al., which is more a ppropriate to fit asymmetrical functions [75] such as the ones observed for the MWD ( Figure 4. 13 and Figure 4. 14 ). Figure 4D. 1 shows an example of the deconvolution of the PLA control peaks at day 7, 14, 21, and 28. To confirm whether deconvolution of a 279 peak wa s necessary or not, and which are the main peak s of the MWD , the a rea fraction was used. Figure 4D. 2 shows the PLA control as an example of th e methodology used. Figure 4D. 2 a shows the M n calculated from the different deconvoluted peaks as f unction of time while Figure 4D. 2 b shows the area fraction of those different peaks, in which the first peak has the main contribution until day 21. For PLA control on days 28 and 42, it seems that the first and second peaks may have similar contribution in some cases and the contribution of the other peaks is minimal. In the case of PLA control for day 56 a single peak was observed. This analysis was performed for all the BNCs in a similar fashion and the main peaks were sele cted case by case for the determination of k . In most cases, no deconvolution was required for days 0, 3, and 7. 280 Figure 4D. 1 Deconvolution of the PLA2 peaks at days ( a) 7, ( b) 14, ( c) 21, and ( d) 28 (Test III in compost) . 281 Figure 4D. 2 (a ) M n and ( b) area fraction as function of time for PLA2 film (Test III in compost) . Figure 4D. 3 and Table 4D. 2 show the M n reduction as a function of time for PLA and PLA - BNCs. The dashed lines indicate fitting of a first order reaction of the form M n / M n 0 = exp( - kt ), where M n 0 is the initial M n , k is the rate constant and t is the time. It can be observed that the initial molecular weight has a real effect on the biodegradation rate, especially until day 21, in which the abiotic degradation ( i.e., hydrolysi s) takes place, and therefore the overall biodegradation. A material with low M n has more polymer chains with free ends that can be cleaved , thus producing more oligomers and monomers that are available for the microorganisms in comparison with one of high er Mn [47] . Figure 4D. 3 also shows that for each of the BNCs the film with 1% and 5% filler loading follow a similar p attern. PLA - HNT films ( Figure 4D. 3 c ) are the ones with the closest initial molecular weight to the PLA control and they follow a very similar pattern, especially after the third day. PLA - HNT and PLA - LRD films seem to have a lo wer rate than the PLA control, which is in agreement with previous results. 282 Figure 4D. 3 Molecular weight reduction as function of time for PLA2 and ( a) PLA - OMMT, ( b) PLA - QAC, ( c) PLA - HNT, and ( d) PLA - LRD films (Test III in co mpost). Dashed l ines indicate fitting of a first order reaction of the form M n / M n0 = exp ( - kt ), where M n0 is the initial M n , k is the rate constant and t is the time. 283 Table 4D. 2 Initial molecular weight and reduction rate of PLA and BNCs as estimated by the first order reaction of the form M n = M n0 exp( - kt ) Sample M n0 , kDa K , d - 1 PLA2 86 .0 ± 1 .5 A 0.1008 ± 0.0037 A PLA - OMMT1 80.0 ± 3 .5 ABC 0.0616 ± 0.0058 C PLA - OMMT5 54 .1 ± 1 .8 E 0.0815 ± 0.0057 B PLA - HNT1 83 .4 ± 3 .3 AB 0.1037 ± 0.0078 A PLA - HNT5 77 .2 ± 1 .3 C 0.0824 ± 0.0029 B PLA - LRD1 79 .6 ± 1 .8 BC 0.057 ± 0.0027 C PLA - LRD5 70 .7 ± 1 .9 D 0.0628 ± 0.0034 C PLA - QAC04 44 .9 ± 1 .3 F 0.0711 ± 0.0045 BC PLA - QAC15 42 .8 ± 1 .4 F 0.0828 ± 0.0056 B Note: Values with the same letter within the same column are not significantly different - Kramer Test. 284 APPENDIX 4E: Biofilm formation Figure 4E. 1 , Table 4E. 1 and Table 4E. 2 s how the results of the first iteration of the biofilm test. Looking at the co ntrol with PA at 2 3 °C ( Figure 4E. 1 a ), the control wells (R2B No polymer ) have an absorbanc e (600 nm) of 1.628 - 2.029 ( uninoculated control wel ls ranged from 0.065 to 0.067). There was no significant difference in the wells supplemented with PLA and BNCs ( Table 4E. 1 ) . The wells supplemented with PLA - LRD5 had the highest average value of 2.028. A t 2 3 °C , P. a eruginosa did form biofilm on the surface of the films. The quantitation of biofilm on PLA ranged f rom 0.409 to 0.966, which is in accordance with the values observed by Satti et al. [49] . There was no significant difference between PLA and BNCs. However, PLA - HNT5 and PLA - LRD5 showed the highest average values of 1.105 and 1.137, respectively. Then, viewing the total biofilm formed by PA ( i.e. , wells plus films) , PLA - LRD5 had the highest average total of 3.165 while the total average f or the pristine PLA was 2.390. Figure 4E. 1 Absorbance (600 nm) of ( a ) PA at 2 3 °C, and ( b) CE at 58°C first iteration . 285 Table 4E. 1 Absorbance (600 nm) of a ) PA at 2 3 °C first iteration Sample Wells Films Total w/o PLA 1.829 ± 0.201 A N/A 1.829 ± 0.201 A PLA 1.703 ± 0.467 A 0.688 ± 0.279 A 2.390 ± 0.544 A PLA - OMMT 1.889 ± 0.363 A 1.035 ± 0.108 A 2.924 ± 0.379 A PLA - QAC 2.012 ± 0.850 A 0.764 ± 0.214 A 2.776 ± 0.876 A PLA - HNT 1.541 ± 0.351 A 1.105 ± 0.397 A 2.646 ± 0.530 A PLA - LRD 2.028 ± 0.325 A 1.137 ± 0.353 A 3.165 ± 0.480 A Note: Values with the same letter within the same column are not significantly different - Kramer Test. Regarding the test with CE at 58°C ( Figure 4E. 1 b ), the sterile controls (SCE) have values that are between 0.54 and 0.57, which are low values considering that the CE still contains humics and other compounds that can bind to polystyrene. The control wells (CE No polymer ) have values of 0.231 - 0.449. These values were less than the ones for PA at 2 3 °C which is expected since PA is a pure culture of good biofilm former. The wells supplemented with PLA and BNCs look consistent overall in biofilm with values ranging f rom 0.087 - 0.312 and no statistically significant difference among them ( Table 4E. 2 ) . In this case, the control well showed the highest average value of 0.340. PLA - LRD5 has an average value of 0.194. Biofilm formation was detected on PLA and BNCs with CE at 58°C. In this case, PLA - LRD5 has significantly higher value (0.277) than the rest of the BNCs. PLA showed an average value of 0.130 while the lowest average value (0.034) was observed with PLA - QAC0.4. The total biofilm ( i.e., wells plus films) was not significantly different among the sample materials. 286 Table 4E. 2 Absorbance (600 nm) of a ) CE at 58°C first iteration Sample Wells Films Total w/o PLA 0.340 ± 0.109 A N/A 0.341 ± 0.109 A PLA 0.216 ± 0.095 A 0.130 ± 0.179 A 0.346 ± 0.203 A PLA - OMMT 0.185 ± 0.060 A 0.099 ± 0.082 A 0.284 ± 0.102 A PLA - QAC 0.237 ± 0.098 A 0.034 ± 0.069 A 0.271 ± 0.120 A PLA - HNT 0.192 ± 0.105 A 0.050 ± 0.021 A 0.242 ± 0.107 A PLA - LRD 0.194 ± 0.047 A 0.277 ± 0.072 A 0.471 ± 0.086 A Note: Values with the same letter within the same column are not significantly different - Kramer Test. Similarly, Table 4E. 3 and Table 4E. 4 show the results of the biofilm test discussed in s ection 4. 3.4 and Figure 4. 15 , with PA at 2 3 °C and CE at 58°C. Table 4E. 3 Absorbance (600 nm) of PA at 2 3 °C during the biofilm test Sample Wells Films Total R2B only 1.279 ± 0.053 B N/A 1.279 ± 0.053 CD PLA 2 1.376 ± 0.160 B 0.626 ± 0.125 AB 2.002 ± 0.204 BC PLA - OMMT 5 2.042 ± 0.243 A 0.875 ± 0.089 A 2.917 ± 0.259 A PLA - QAC 0.4 0.977 ± 0.180 B 0.131 ± 0.040 B 1.107 ± 0.185 D PLA - HNT 5 1.044 ± 0.061 B 1.254 ± 0.539 A 2.258 ± 0.542 AB PLA - LRD 5 1.078 ± 0.301 B 0.639 ± 0.097 AB 1.717 ± 0.316 BCD Note: Values with the same letter within the same column are not significantly different - Kramer Test. Table 4E. 4 Absorbance (600 nm) of CE at 58°C during the biofilm test Sample Wells Films Total R2B only 0.485 ± 0.103 A N/A 0.485 ± 0.103 A PLA 2 0.479 ± 0.124 A 0.090 ± 0.030 B 0.569 ± 0.128 A PLA - OMMT 5 0.360 ± 0.238 A 0.175 ± 0.073 B 0.536 ± 0.249 A PLA - QAC 0.4 0.338 ± 0.201 A 0.113 ± 0.032 B 0.451 ± 0.204 A PLA - HNT 5 0.367 ± 0.161 A 0.201 ± 0.014 B 0.568 ± 0.161 A PLA - LRD 5 0.384 ± 0.118 A 0.519 ± 0.054 A 0.903 ± 0.130 A Note: Values with the same letter within the same column are not significantly different - Kramer Test. 287 REFERENCES 288 R EFERENCES [1] A.P. Kumar, D. Depan, N.S. Tomer, R.P. Singh, Nanoscale particles for polymer degradation and stabilization Trends and future perspectives, Prog. Polym. Sci. 34 (2009) 479 515. doi:10.1016/j.progpolymsci.2009.01.002. [2] s and strategies, Trends Food Sci. Technol. 22 (2011) 611 617. doi:10.1016/j.tifs.2011.01.007. [3] H.M.C. De Azeredo, Nanocomposites for food packaging applications, Food Res. 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Wojdyr, Fityk: A general - purpose peak fitting program, J. Appl. Cryst allogr. 43 (2010) 1126 1128. doi:10.1107/S0021889810030499. [ 75 ] A. Perej n, P.E. S nchez - Jim nez, J.M. Criado, L.A. P rez - Maqueda, Kinetic Analysis of Complex Solid - State Reactions. A New Deconvolution Procedure, J. Phys. Chem. B. 115 (2011) 1780 1791 . doi:10.1021/jp110895z. 295 CHAPTER 5 ENHANCING THE BIODEGRADATION RATE OF POLY(LACTIC ACID) FILMS AND PLA BIO - NANOCOMPOSITES IN SIMULATED COMPOSTING THROUGH BIOAUGMENTATION A version of this chapter is published as: Castro - Aguirre, E., Auras, R., Selke, S., Rubino, M., Marsh, T. Enhancing the biodegradation rate of poly(lactic acid) films and PLA bio - nanocomposites in simulated composting through bioaugmentation, Polymer Degradation and Stability , 154 (2018) 46 54 . 296 5.0 Abstract Biodegradable polymers provide an opportunity to divert plastic waste from landfills, with composting as an alternative disposal route. However, some biodegradable polymers, such as poly(lactic acid) (PLA), do not biodegrade as fast as other organic wastes during composting , affecting their general acceptance in industrial composting facilities. Bioaugmentation, the addition of specific microbial strains, is a promising technique to accelerate the biod egradation of compostable plastics, so that they biodegrade in comparable time frames with other organic materials. In this study , we evaluated the effect of bioaugmentation on the biodegradation of PLA and PLA bio - nanocomposites (BNCs) in simulated compos ting conditions. PLA , PLA with 5% organo - modified montmorillonite ( PLA - OMMT5 ), and PLA with 0.4% surfactant ( PLA - QAC0.4 ) films were produced and fully characterized. PLA - degrading bacteria were isolated through an enrichment technique with PLA as the sole carbon source at 58 o C. Isolates were identified as Geobacillus using 16S rRNA gene sequencing and the NCBI database, and further used to study the effect of bioaugmentation on the biodegradation rate of PLA and BNCs in solid environments. The biotic and abiotic degradation was assessed in compost, inoculated vermiculite, and un inoculated vermiculite at 58ºC by analysis of evolved CO 2 using an in - house built direct measurement respirometer. Size exclusion chromatograph y was also used to measure and to monitor the change in molecular weight of the film s amples retrieved every week during the biodegradation test. The m icrobial attachment on the surface of PLA of the isolated microbial strain and other microorganisms prese nt in the compost was evaluated by a biofilm forming assay in wells incubated at 58°C. Bioaugmentation with Geobacillus increased the evolution of 297 CO 2 and accelerated the biodegradation phase of PLA and BNCs when tested in compost and inoculated vermiculit e with compost mixed culture. Bioaugmentation could commercially be used to accelerate the biodegradation of PLA in compost environment s . 5.1 Introduction Plastics represent 1 2.9 % of the 25 8 million tons of municipal solid waste (MSW) generated in 2014 in the U SA , from which only 9.5 % was recovered ( i.e., recycling and composting) , the recycled plastics were mostly polyethylene and polyethylene terephthalate . Hence, most plastic waste ( 25.1 million tons) ended up accumulating in landfills, which is a major env ironmental concern [1] . Compostable polymers, like poly(lac tic acid) (PLA), represent a promising way to divert plastic waste from landfills , replac ing conventional polymers for some applications, especially for non - durable goods and single - use products like packaging, disposable plates and cutlery, and contaminated plastic waste ( e.g. , food packaging and agricultural mulch films ) [2] . However, such benefit is only accomplished if PLA - based products reach the desired disposal system at their end of life. The ideal scenario is one in which these products / packaging would be collected and sent along with the organic wastes to commercial composting facilities . However, one of the main current challenges for PLA - based materials to be widely accepted in composting facilities at their end of life is that their biodegradation in c omposting is usually slower than that for other organic wastes [3] . In Chapter 4 , we studied the impact of different nanoclays ( e.g. , organo - modified montmorillonite , OMMT , and its organo - modifier , methyl, tallow, bis - 2 - hydroxyethyl, quaternary ammonium , QAC ) on the bio degradation rate of PLA bio - nanocomposites 298 (BNCs) in diff erent solid environments ( i.e., compost and vermiculite), and on microbial mixed culture It was found that the presence of OMMT seem s to accelerate the initial phase of biodegradation , but not the final mine ralization phase , and to promote microbial attachment in comparison to the pristine PLA under certain conditions [3] . Therefore, there is interest in knowing which specific microbial strains present in the compost can bind to PLA and preferentially biodegrade PLA and its BNCs, and whether they can be purposely used to accelerate the biodegradation mechanism. Some researchers have identified the microbial consortia present in the compost environment [4 7] , and others have reported the isolation and identification of several species capable of biodegrading PLA [8 15] , and other polymers [16 24] by 16S rRNA sequence analysis . In this con text, bioaugmentation ( i.e., the addition of specific microbial strains ) is a promising technique that can be studied and used to accelerate the biodegradation of compostable plastics, so that they biodegrade in comparable time frames with other organic wa stes. Increasing the biodegradation rate of PLA should facilitate its disposal through composting since PLA - based products and organic materials could biodegrade in a similar period of time. This study aimed f irst to isolate and to identify the microbial strain s present in the compost capable of bio degrading PLA , and second, t o evaluate the effect of introducing such microbial strains on the biodegradation rate of PLA and its BNCs in simulated composting conditions ( i.e., in a solid environment at 58 ° C), since most of the studies found in the literature have been performed in liquid media, which do not necessarily represent real composting conditions. 299 5.2 Materials and Methods 5.2.1 Materials Ingeo TM 2003D resin, p oly(lactic acid) , was acquired from NatureWorks LLC . (Minnetonka, MN , USA ). Cellulose powder with particle size was purchased from Sigma - Ald rich (St. Louis, MO , USA ) . Cloisite ® 30B , o rgano - modified montmorillonite ( OMMT), was obtained from BYK Additives Inc. (Gonzales, TX , USA ). Tomamine TM Q - T - 2 (QAC) with 60 - 70% purity of a methyl, tallow, bis - 2 - hydrox yethyl, quaternary ammonium , t he organo - modifier of Cloisite ® 30B , was obtained from A ir Products and Chemicals Inc. (Butler, IN , USA ). The composition per liter of the R2 broth (R2B) used was 0.5 g yeast ext ract , 0.5 g proteose peptone #3, 0.5 g casamino acids , 0.5 g dextrose , 0.5 g soluble sta rch , 0.3 g sodium pyruvate, 0.3 g dipotassium phosphate , and 0.05 g magnesium sulfate . Additionally, GELRITE® gell an gum ( CP Kelko, Inc., San Diego, CA , USA ) was used to produce R2 A plates. Th e composition per liter of the M9 minimal medium was 12.8 g Na 2 HPO 4 .7H 2 O , 3 g KH 2 PO 4 , 0.5 g NaCl , 1 g NH 4 Cl , and 1 g of 1 mM MgSO 4 , 1 mM CaCl 2 , 3x10 - 9 M (NH 4 ) 6 Mo 7 O 24 .4H 2 O, 4x10 - 7 M H 3 BO 3 , 3x10 - 8 M CoCl 2 .6H 2 O , 1x10 - 8 M CuSO 4 .5H 2 O , 8x10 - 8 M MnCl 2 .4H 2 O , 1x10 - 8 M ZnSO 4 .7H 2 O , 1x10 - 6 M FeSO 4 .7H 2 O. All the materials were used as received unless indicated. 5.2.1.1 Material processing and characterization PLA pellets were dried prior to processing for 8 h under vacuum (85 kPa) at 60°C . PLA - OMMT 5 (5% wt . OMMT ) and PLA - QAC 0.4 ( 0.4 % surfactant ) films were processed in two s teps : 1) masterbatches were pr o cessed in a ZSK 30 twin - screw extruder (Werner Pfleiderer Co. , Ramsey, NJ , USA ) with temperature range of 146 - 186 °C and screw 300 speed o f 130 rpm ; 2) the films were extruded in a RCP - 0625 m icroextruder model (Randcastle Extrusion Systems, Inc., Cedar Grove, NJ , USA ) , screw diameter of 1.5 9 cm, L/D rati o of 24 , and volume of 34 cc . The processing temperature range for the PLA - OMMT film w as 193 2 48 °C with a screw speed of 18 rpm, and for the PLA - QAC film w as 143 173°C with a speed of 3 1 rpm. A pristine PLA film was produced in the same film extruder with processing temperature range of 193 2 49 °C and screw speed of 28 rpm . The thickness e s w ere 0.073 ± 0.014 , 0.039 ± 0.008, and 0.034 ± 0.009 m m for PLA - OMMT 5 , PLA - QAC 0.4 and PLA films, respectively. The extruded films were fully characterized, as described in Chapter 4 [3] . The carbon , hydrogen, and nitrogen content s of the different films w ere obtained with a CHNS/O Elemental Analyzer , PerkinElmer 2400 Series II (Shelton, CT, USA), and values are presented in Table 5.1 . Table 5. 1 Carbon, hydrogen, and nitrogen content of the tested materials Material % Carbon a % Hydrogen a % Nitrogen a Cellulose 42.50 ± 0.34 6.53 ± 0.05 0.04 ± 0.01 PLA 49.99 ± 0.05 5.60 ± 0.01 0.01 ± 0.01 PLA - OMMT5 48.76 ± 0.07 5.49 ± 0.02 0.07 ± 0.01 PLA - QAC0.4 49.98 ± 0.08 5.55 ± 0.02 0.01 ± 0.00 a Percentage by weight 5.2.2 Isolation of PLA - degrading microbial strain The isolation of PLA - degrading microbial strains was performed through a serial enrichment technique. Compost (1 g) w as inoculated in 25 mL of fresh M9 minimal medi a in a 100 - mL Erlenmeyer flask containing twenty PLA pellets as sole carbon source and incubated in a shaker at 58 ° C and 70 rpm . After a week, five PLA pellets were aseptically transferred to fresh M9 medium containing twenty new PLA pellets along with 100 L of the previous culture and incubated for 7 days under the same 301 conditions . T his procedure was repeated for six more consecutive transfers to capture potential PLA - degrad ing microbial strains. The final enrichment (100 mL) was spread across R2A plates using seria l dilution techniques and incubated at 58 ° C . Six isolated colonies were selected and purified using the streak - plating method after three consecutive transfers that were performed every three days. Purified strains were stored in 20% glycerol at 80 ° C for further analyses . 5.2.3 I dentification of PLA - degrading microbial strain Microbial i solates w ere identified using 16S rRNA gene sequencing. Overnight broth cultures of isolates were used for genomic DNA extraction of culture Alkali ne - PEG lysis reagent [25] and incubat ing for 5 min at 55°C and then store at - 20°C . DNA extractions ( 2 L ) were used as template s for polymerase chain reaction ( PCR ) amplification of 16S rRNA using bacterial primers 27F (50 - AGAGTTTGATCCTGGCTCAG - 30) and 1389R (50 - ACGGGCGGTGTGTACAAG - 30). Each PCR reaction mix contained 25 L of master mix (GoTaq ® Master Mix, P romega, Madison, WI , USA ) , 2 L of template, 1.2 L of each primer and the rest was Milli Q ® water to adjust the volume to 50 L . The PCR amplifications were p erformed using an Applied Biosystems TM 2720 Thermal Cycler ( Thermo Fisher Scientific Inc., Wilmington, DE , USA ) with the following reaction conditions: initial denaturation of DNA at 94 ° C for 5 min followed by the amplification cycle with denaturation at 94 ° C for 45 s, annealing at 55.5 ° C for 45 s , and extension at 72 ° C for 1 min. The cycling concluded with a n extension at 72 ° C for 7 min , and then it was kept at 4 ° C . The PCR products were purified using QIAquick ® PCR Purification Kit (50) (Qiagen Sciences Inc., Germantown, MD , USA ) following the instructions from the manufacturer. The concentration of DN A in 302 the purified samples w as obtained with a NanoDrop® ND - 1000 spectrophotometer and ND - 1000 V3.1.8 software ( Thermo Fisher Scientific Inc., Wilmington, DE , USA ). The purified PCR products were mixed with the 27F or 1389R primer ( ~60ng of PCR product with 30 pmoles of primer ) and sent for Sanger sequencing at the Research Technology Support Facility (RTSF) at Michigan State University (MSU) . Identification of the microbial strains was performed using the Seq uence Match function of the Ribosomal Database Project (RDP) from the Center for Microbial Ecology at MSU (rdp.cme.msu.edu) , based on the National Center for Biotechnology Information (NCBI) taxonomy [26] . 5.2.4 Biodegradation evaluation 5.2.4.1 Preparation of the compost an d vermiculite The aerobic biodegradation of the PLA , PLA - OMMT 5, and PLA - QAC0.4 films was evaluated by CO 2 analysis i n simulated composting at 58 ° C, using the direct measurement respirometer ( DMR ) and methodology described elsewhere [27] . In brief, compost was obtained from the Composting Facili ty at MSU (East Lansing, MI , USA ) and then mixed with s aturated vermiculite (P remium g rade , Sun Gro Horticulture Distribution Inc., Bellevue, WA , USA ) using a 1:4 ratio of dry w eight compost . The moisture content of the mix was increased to ~50% by adding d eionized water . Biodegradation tests were also carried out with un inoculated and inoculated vermiculite to study abiotic degradation and bioaugmentation. In th e case of inoculation with a mixed culture ( i.e., microbial consortia extracted from compost) , vermiculite was mi xed in a proportion of 1:4 (wt.) with the inoculum solution prepared by combining compost extract with a mineral solution at a 1:1 ratio . Detailed information about the preparation 303 of the mineral solution and the compost extract can be found elsewhere [27,28] . In the case of bioaugmentation studies in vermiculite , the inoculum solution was prepared by combining the mineral solution with a pure culture of the PLA - degrading microbial strain . The initial physicochemical parameters of the c ompost and vermiculite were determined in the Soil and Plant Nutrient Laboratory at MSU (East Lansing, MI , USA ) and shown in Table 5. 2 . A complete list of the compost and vermiculite total nutrient analysis is provided in the Appendix 5A ( Table 5A. 1 ) . Table 5. 2 Initial physicochemical parameters of the compost and vermiculite used for biodegradation tests Parameters ISO b Biodegradation Test Type of media c C C IV V Dry solids, % 50 - 55 51.8 21.4 18.9 Volatile solids, % <30 41.3 2.8 3.1 pH 7 - 9 7.9 6.8 8.2 Total Carbon, % N/A a 24.0 1.6 1.8 Total Nitrogen, % N/A a 2.4 0.2 0.03 C/N ratio 10 - 40 9.9 10.2 59.9 a Not applicable or not available b Values based on ISO 14855 - 1:2005 standard for compost c C= compost; IV= inoculated vermiculite; V= uninoculated vermiculite 5.2.4.2 Bioreactor setup The bioreactors were filled with a mixture of 400 g (wet wt.) of media ( either compost, inoculated or uninoculated vermiculite ) and 8 g of film sample s ( 1 cm x 1 c m pieces). T riplicates of each sample material , positive control s (cellulose powder), and blanks (media only) were analyzed . The bioreactors were provided with w ater - saturated CO 2 - free air at a flow rate of 40 ± 2 sccm (standard cubic centimeters per minute ) and incubated in the dark at 58 ± 2 ° C. Detailed information o n the testing conditions using the DMR system can be found elsewhere [27] . 304 5.2.4.3 Bioaugmentation For the bioaugmentation studies, all media were inoculated with a pure culture of the PLA - degrading strain . First , the purified strain w as inoculated in 25 mL of R2B in a 100 - mL Erlenmeyer flask and incubated overnight at 58 ° C in a n Innova TM 4300 shaker (New Brunswick Scientific Co., Edison, NJ , USA ) . Then, this culture was used as inoculum for a 500 mL culture, also in R2B , and incubated fo r 48 hours in the shaker at 58 ° C. Direct c ell counting was performed t o determine the number of microorganisms in the culture using disposable counting slides (Nexcelom Bioscience LLC., Lawrence, MA , USA ) following the instructions of the manufacturer. Cells were counted on a Nikon Eclipse E600 microscope ( Nikon Instruments Inc., Melville, NY , USA ) using phase - contrast at 1000x final magnification. This pure culture was used to inoculate the bioreactors by adding cells equivalent to 1% of t he total community in a bioreactor , assuming that there was approximately 1 X 10 8 bacteria/g in compost . The 48 - hour culture was diluted with either water ( for compost) or the inoculum solution ( for vermiculite) to reach the 1% target. 5.2.5 Biofilm formation T he microbial attachment o f the PLA - degrading strain on PLA , PLA - OMMT5, and PLA - QAC0.4 was evaluated through a biofilm formation test using a standard microtiter plate assay (24 - well Corning ® , Corning Inc., Corning, NY , USA ) as previously described [3] . In brief , sterilized film f an overnight grown culture of the purified strain and incubated in a shaker (100 rpm) at 58 ° C for 48 hours (4 replicates tested). As explained in the previous study, Pseudomonas aeruginosa (PA) , strain PAO1, was u sed as a positive control at 23 ° C , 305 and u n inoculated wells as a negative control [3] . After incubation, wells and fil ms were gently rinsed with water , , and resolubilized for measurement in 30% acetic acid for 15 min . The a bsorbance at 600 nm was determined with an (BioTek Instruments, Inc., Winooski, VT , USA ). 5.2.6 Size Exclusion Chromatography ( SEC ) The molecular weight distribution ( MWD ) and number average molecular weight ( Mn ) of the PLA , PLA - OMMT5 and PLA - QAC0.4 film samples collected at different periods of time during the bioaugmentation test were obtained by SEC as previously described [3,27] . In brief, 1 0 mg of sample were dissolved in 5 mL of tetrahydrofuran (THF) and filtered prior to injection ( 1 cm 3 /min flow rate for 50 min at 35°C ) in a gel permeation chromatography system ( Waters Inc. , Milford, MA , USA ) . P o lystyrene standards (0.5 2,480 kDa ) were used for the calibration of the system. 5.2.7 Statistical Analysis Minitab18 software (Minitab Inc., State College, PA, USA) was used to conduct analysis of variance (one - way ANOVA) and Tukey - Kramer test s with p 0.05. All the results show mean and standard deviation. 5.3 Results and Discussion 5.3.1 Isolation and identification of PLA - degrading bacteria A serial enrichment technique was used with M9 minimal media and PLA as the sole carbon source , and compost as the only source of microorganisms (initial stage) to determine the microbial strains able to biodegrade PLA . The incubation temperature was set at 58 ° C to simulate an active composting phase. This procedure was followed 306 by isolation through the streak - plating method, and identification using 16S rRNA Sanger sequencing . T he results from the MSU - RDP Seq uence Match based on the NCBI database ( Table 5. 3 ) showed that the isolates were Geobacillus , closest to G. thermoleovorans . The sequence matching function was configured so it only retrieved the strains from the NCBI database closest to the isolated strains (KNN = 1). Geobacillus thermoleovorans , refer red to from now on, as Geobacillus only, were further used to study the effect of bioaugmentation on the biodegradation rate of PLA in solid environments, and biofilm formation on the surface of the PLA film and BNCs . Geobacillus spp. can be found in terrestrial an d marine environments and are capabl e of surviving i n extreme environments like high temperature and limited resources [29,30] . They can grow under low nitrogen and low oxygen conditions [31] . They are Gram - positive , thermophilic, motile , rod - shaped , spore - forming bacteria [29 32] . Geobacillus spp. have optimal growth temperatures ranging from 55 to 65 ° C and pH ranging from 6.0 to 8.5 [30 32] . These attributes are consistent with conditions found in composting e nvironments. Moreover, it has been reported that Geobacillus are able to utilize a variety of sugars, carboxylic acids and hydrocarbons [30] , and t hey can grow on R2A broth, lactose , lactat e, and C13 - C20 n - alkanes [31] . A complete list o f current valid species for the genus Geobacillus can be found elsewhere [33] . 307 Table 5. 3 Identification of the microbial isolates using the MSU - RDP Sequence Match and the NCBI database Isolate ID No. bases of sequence Sab Score Closest strain GenBank A ccession number EC - 1 1406 0.978 Geobacillus themoleovorans MH183210 EC - 2 1406 0.989 Geobacillus themoleovorans MH183211 EC - 3 1406 0.977 Geobacillus thermoleovorans MH183212 EC - 4 1406 0.985 Geobacillus themoleovorans MH183213 EC - 5 1406 0.962 Geobacillus themoleovorans MH183214 EC - 6 1406 0.994 Geobacillus themoleovorans MH183215 5.3.2 Biodegradation Test The biodegradation of PLA, PLA - OMMT5, and PLA - QAC0.4 was evaluated by analysis of evolved CO 2 with the DMR system at 58 ° C. The experiments were conducted using different types of solid media: compost , inoculated vermiculite , and uninoculated vermiculite. In the following biodegradation results, cellulose is the reference material , and b lank refers to t hose bioreactors without polymer films. Figure 5. 1 a , shows that regardless of testing in compost or vermiculite inoculated with the mixed cu lture , all samples produced significantly higher amount s of CO 2 than the ir respective blank s , so no inhibition was observed due to the presence of the films . Figure 5. 1 b , shows that PLA - OMMT 5 and PLA - QAC 0.4 have shorter lag times than the pristine PLA, initiating the biodegradation phase earlier in both types of media. Biodegradation in inoculated vermiculit e is slower than in compost, so longer testing times were expected . The high mineralization values reached in PLA - QAC0.4 are an indication of the priming effect occurring in the compost media, which was clearly avoided when testing in inoculated 308 vermiculit e as shown in Figure 5. 1 b . As expected, neither significant CO 2 production nor mineralization was observed from the bioreactors with un inoculated vermiculite since there are no microorganisms present. The degradation in this case is mostly attributed to an abiotic hydrolytic process . Th e results of this initial test using a mixed culture from the compost showed that vermiculite is an excellent media for testing bioaugmentation using a single purified strain like Geobacillus . Figure 5. 1 (a ) CO 2 evolution and ( b) % Mineralization of cellulose, PLA, and PLA - OMMT5, and PLA - QAC0.4 in compost (solid lines) , inoculated vermiculite with mixed culture (dashed lines), and uninoculated vermiculite (dotted lines) ; a dapted from Castro - Aguirre et al. [3] . Figure 5. 2 shows the cumulative CO 2 and % mineralization of PLA and PLA - OMMT5 in compost with and without the inoculation of Geobacillus . Figure 5. 2 a shows that the compost alone (solid line) and the compost with Geobacillus (dashed line) did not produce significantly different amount s of CO 2 . However, when an additional source of carbon was introduced ( i.e., cellulose or PLA ), there was significantly higher production of CO 2 in the presence of Geobacillus at the early stage of the test ( <25 d ) . 309 This behavior has been attributed to the synergistic effect of Geobacillus with the other microbial strains present in the compost and confirmed by the bioaugmentation test in vermiculite. Furthermore, Figure 5. 2 b shows that in all cases the lag time was reduced with the presence of the Geobacillus (dashed lines) , indica ted by the shift of the curve to the left, meaning that the biodegradation phase started earlier in comparison to the samples without Geobacillus (solid lines) . When comparing by material ( Figure 5. 2 b ) , the lag time is shorter in the PLA - OMMT 5 film than in the PLA film . Th is in agreement with previous report ed results in which faster biodegradation was attributed mostly to the initial lower molecular weight of the PLA - OMMT5 films, but also to the higher initial hydrolytic degradation promoted by the presence of OMMT [3] . Figure 5. 2 (a ) CO 2 evolution and ( b) % Mineralization of cellulose , PLA , and PLA - O MMT5 in compost without Geobacillus (solid lines) and with Geobacillus (dashed lines). Figure 5. 3 and Figure 5. 4 show the results of biodegradation tests with bioaugmentation in vermiculite with 4 different levels of inoculation: 1) un inoculated vermicul i te, 2) vermiculite inoculated with Geobacillus only , 3) vermiculite inoculated 310 with the mixed culture from the com post extract, and 4) vermiculite inoculated with mixed culture and Geobacillus . Similar to the results observed in compost, the samples in vermiculite inoculated with mixed culture and Geobacillus , produced a statistically significant higher amount of CO 2 than the samples in vermiculite inoculated with the mixed culture only , especially PLA - OMMT5 ( Figure 5. 3 c ), in which the lag time was reduced almost by half ( from ~20 d to ~10 d). In all cases, the same mineralization levels were reached towards the end of the test. As previously mentioned , n o s ignificant production of CO 2 was expected in the un inoculated bioreactors . Surprisingly , Geobacillus by itself did not produce as much CO 2 as when it was together with the mixed culture . T here was n o significant CO 2 production or mineralization when Geobacillus was inoculated alone. The observed behavior can be attributed to the Geobacillus metabolic activity and to the synergistic effect with other microorganisms. Some researchers have reported that Geobacillus spp. have high extracellular esterase a nd lipase activity [31,32] , and are able to utilize a wide range of sugars, carboxylic acids, lactose , lactat e, and even hydrocarbons ( e.g., C13 - C20 n - alkanes) [30,31] . Esterases , which break down organic molecules with ester bonds, are extracellular enzymes present o n the surface and/or within the biofilm [34] . Thus, the extracellular enzymes produced by Geobacillus become resources that may be available for other microorganism s contributing to their growth and activity. Tomita et al. , suggested that esterases secreted by Geobacillus thermocatenulatus are involved in PLA degradation [15] . Similarly, Sakai et al. , showed that degradation of PLA was related to the esterases secreted by Bacillus smithii [35] . Moreover, it has been observed that some Geobacillus spp. are involved in symbiotic relationships with other microorganisms 311 providing metabolites from cell lysis [32] . PLA - degrading bacteria may not be limited to the genus Geobaci llus ; other researchers have found members of the family Bacillaceae being dominant degraders during composting [7,36] . F urther investigation is nee ded to understand the metabolism and synergistic behavior of Geobacillus with other microbial consortia present in the compost for the optimal biodegradation of PLA. Figure 5. 3 % Mineralization of ( a) Cellulose, ( b) PLA, and ( c) PLA - OMMT5 in vermiculite with different levels of inoculation . Figure 5. 4 % Mineralization of cellulose, PLA, and PLA - OMMT5 in ( a) compost (same as Figure 5. 2 b ) , ( b) vermiculite inoculated with mixed culture, and ( c) uninoculated vermiculite . S olid lines represent samples without Geobacillus while dashed lines represent samples inoculated with Geob acillus . 312 5.3.3 Molecular Weight Samples of the different materials were retrieved at different time intervals during the biodegradation test to evaluate the change in molecular weight . Figure 5. 5 show s the reduction of M n of the film samples as function of time . The observed M n reduction is typical of a bulk degradation mechanism , which can be best represented by a first order reaction fitting with the equation: M n = M n 0 exp( - kt ), where M n 0 is the initial M n , t is the time , and k is the M n reduction rate constant [37,38] . Table 5B. 1 of the Appendix 5B shows that t he rate constant s w ere not significantly different for the samples tested in Geobacillus only, mixed culture , and mixed culture with Geobacillus . In Chapter 4 , we showed that looking at M n may not be the best approach to studying the molecular weight reduction, and that looking at the changes in the MWD may provide more in sights about the biodegradation behavior [3] . Figure 5. 5 Molecular weight reduction as function of time for ( a) PLA and ( b) PLA - OMMT 5 in vermiculite with different levels of inoculation. L ines represent the fitting of the equation M n = M n0 exp ( - kt ), where M n0 is the initial M n , k is the rate constant and t is the time. 313 Figure 5. 6 shows the MWD of the PLA film as function of time during the biodegradation in vermiculite with the different levels of inoculation. In all cases, a shift of the MWD peak to the left represen ts a decrease of the molecular weight due to bulk hydrolysis of the film while the broadening of the peak represents a higher polydispersity index ( PI ) caused by chain fragmentation [39] . The observed change in the MWD from single peak to mul tiple peaks, e specially after day s 14 and 21, has been attributed to the rearrangement of the newly formed short polymer chains into crystalline structures [39,40] . The presence of higher and sharper peaks at day 28 indicates that the amorphous regions are being preferably degraded [41] . Therefore, increased crys tallinity can be expected during and after the initial degradation [42 44] . In this case ( Figure 5. 6 ), all the samples have the same initial MWD ( black line ). Regardless of the level of inoculation, all the samples seem to have similar behavior until day 7, where the peak shifted to the left. At day 14 , the peak of uninoculated vermiculite became broader and showed the presence of more than one peak. The peak s of m ixed culture and mixed culture with Geobacillus show similar behavior. For the day s 21 and 28, the broadening of the peaks and the presence of a multimodal peak is more evident . I n all the cases, the biggest reduction of the molecular weight happened between days 14 and 21 , with an increase in the lower molecular weight tail . Similar observations were previously reported for pristine PLA and PLA bio - nanocomposite films in simulated composting conditions [3,27] . Moreover , it can be noticed between day s 21 and 28 that there was not a significant shift of the peak to the left, instead, the peaks became higher and sharper indicating degradation in the amorphous zone and the consumption of the low molecular weight chains like monomers and oligomers of lactic acid by the 314 microorganisms . This finding is supported by the significant increase on the production of CO 2 and on the mineralization observed during these days . Figure 5. 6 d shows that the MWD of the mixed culture with Geobacillus remains around the same position between days 21 and 28, but the low molecular weight tail disappears . A similar ob serv ation was found for PLA - OMMT5 films during biodegradation in vermiculite with different levels of inoculation ( Figure 5B. 1 of the Appendix 5B ). However, this behavior between days 21 and 28 was not observed in the PLA and PLA - OMMT5 samples tested in compost only during our previous work [3] . This may indicate that bioaugmentation with Geobacillus promotes the rapid microbial assimilati on of low molecular weight PLA chains and agrees with the results from the biodegradation test in which the degree of mineralization of PLA and PLA - OMMT5 significantly increased with the presence of Geobacillus in both compost and inoculated vermiculite ( i.e., with mixed culture). 315 Figure 5. 6 MWD of PLA samples in vermiculite with different levels of inoculation ( a) un inoculated, ( b) Geobacillus only, ( c) m ixed culture, ( d) m ixed culture and Geobacillus . 5.3.4 Biofilm test Table 5. 4 and Table 5. 5 , and Figure 5C. 1 and Figure 5C. 2 of the Appendix 5 C , show the Geobacillus biofilm formation test results . In Table 5. 4 , the positive control (R2B wells ) with PA at 23°C showed an absorbance (A 600 ) of 1. 0 16 - 1. 1 00 , w hile the negative control ( uninoculated wells ) had an A 600 of 0.0 59 - 0.06 7 . These values are simi lar to previously reported values [3,45] . The wells containing PLA and BNCs show no statistically significant dif feren ce from the control lacking any film (R2B only). In the case of the test with PA at 23°C, the biofilm formation individual values ranged from 316 0. 013 to 0. 155 for PLA , from 0.177 to 0.554 for PLA - OMMT5 , and from 0.005 to 0.084 for PLA - QAC0.4 samples . A statistically significant difference was found between the PLA - OMMT5 and PLA - QAC0.4 absorbance values. For the total biofilm formation (wells + film) , the same behavior was observed in which the average biofilm values were as follows PLA - OMMT5 PLA PLA - QAC0.4 ( Figure 5C. 1 a ) . These observations are in agreement with the results previously reported, and in which it was suggested that the OMMT may have an indirect stimulation on biofilm formation by PA , while QAC may have an inhibitory effect ( Figure 5C. 2 a ) [3] . Some researchers have reported that QACs are toxic to microorganisms with significant inhibition of growth of soil microbes at higher concentrations by affecting microbial pr ocesses , such as dehydrogenase activity and nitrification [46] . Furthermore, the use of Q ACs ha s been stud ied as a way to inhibit and reduce the attachment of microorganisms in tissues [47] . A list of t he minimum inhibitory concentration (MIC) and non - inhibitory concentration (NIC) values of different QACs and different microorganisms can be found elsewhere [48] . Further research is needed to better understand the effect of QACs o n the compost microbial consortia. Table 5. 4 Absorbance (600 nm) of biofilm formation samples with Pseudomonas a eruginosa ( PA ) at 2 3 °C Sample Wells Films Total R2B only 1. 058 ± 0.0 42 A N/A 1. 058 ± 0.0 42 B PLA 1. 237 ± 0. 252 A 0. 084 ± 0. 071 AB 1.321 ± 0.2 62 AB PLA - OMMT 5 1.488 ± 0. 388 A 0. 366 ± 0. 1 89 A 1.853 ± 0. 431 A PLA - QAC 0.4 1.021 ± 0.1 27 A 0. 040 ± 0.04 4 B 1. 060 ± 0.1 34 B Note: Values with the same letter within the same column are not statistically significantly different at p 0.05 with Tukey - Kramer t est. 317 Table 5. 5 Absorbance (600 nm) of biofilm formation samples with Geobacillus at 58°C Sample Wells Films Total R2B only 0. 231 ± 0. 008 A N/A 0. 231 ± 0. 008 A PLA 0. 151 ± 0. 067 A 0.0 61 ± 0.03 6 A 0. 212 ± 0. 076 A PLA - OMMT 5 0. 199 ± 0. 060 A 0. 090 ± 0.0 46 A 0. 289 ± 0. 076 A PLA - QAC 0.4 0. 169 ± 0. 042 A 0. 048 ± 0.032 A 0. 216 ± 0. 053 A Note: Values with the same letter within the same column are not statistically - Kramer test. Table 5. 5 shows the biofilm values with Geobacillus at 58°C . The control wells ( R2B only) have individual values ranging from 0. 223 to 0. 240 . Th is absorbance is significantly lower than the absorbance for PA , a known biofilm forming strain, at 23°C . We have observed similar behavior in our previous work with compost extract that w as attributed to more challenging conditions for microbial growth and survival at 58°C [3] . However, biofilm abundance also depends on the bacterial strains present [24,49] . The wells supplemented with PLA , PLA - OMMT5 and PLA - QAC0.4 had an average absorbance of 0.151, 0.199, and 0.169, respectively . N o statistically significant difference was found between the samples . Biofilm formation by Geobacillus at 58°C was observed on the su rface of all the films ( Figure 5C. 1 b ) . However, no statistically significant difference was found among the sample materials. The same behavior was obs erved for the total biofilm formation (wells + film). Although Geobacillus w as able to attach to the surface of the different films at 58°C , it did not show any preference towards a specific material. In contrast, the pure culture of PA clearly preferred PLA - OMMT5 at 2 3 °C. The fact that the biofilm formation by Geobacillus was lower than that by the compost extract at 58°C ( Figure 5C. 2 b ) agrees with the results from the biodegradation test, in which the mineralization was significantly lower when Geobacillus was tested alone. However, t here may be other environmental conditions 318 that are more stimulatory to Geobacillus biofilm . For example, the formation of biofilms by G eobacillus spp. ha s been studied in the dairy industry since they are heat - resistant spore - forming bacteria. Some researchers found that Geobacillus preferentially form biofilms on surfaces at air - liquid interfa ces rather than on submerged surfaces [50] . Moreover, the presence of cations may influence the structural integrity and cohesion of biofilms formed by Geobacillus spp., affecting not only the surface - biofilm interaction, but also the metabolism and physiology of these microorganisms [51] . E ven though biofilm formation is not a direct indication of biodegradation, it plays an important role i n the microbial performance and survival [11,52] . Synergistic effects are commonly observed in which some microbial strains release resources that become available for other microorganisms ( e.g. , extracellular digestive enzymes able to degrade PLA and derivatives ) [11,52] . Furthermore, b iofilms are beneficial for other microorganisms since they ret ain water in the compost , provide tolerance to environmental stressors , and prevent microorgan isms from being washed out [52] . Further investigation is still needed to fully understand the synergistic behavior of Geobacillus with other populations present in the compost for the biodegradation of PLA - based films. 5.4 Final Remarks This study aimed to isolate and to identify PLA - degrading microbial strain s present in compost, and t o evaluate the effect of introducing such microbial strains on the biodegradation rate of PLA and PLA bio - nanocomposites in simulated composting conditions. Geobacillus t hermoleovorans w as identified as the microbial strain present in the compost capable of degrading PLA at 58 o C. Geobacillus was further used to study bioaugmentation in compost and vermiculite with different levels of inoculation. 319 B ioaugmentation with Geobacillus increased the evolution of CO 2 and shortened the lag p hase when tested in compost and vermiculite with mixed culture . Geobacillus inoculated alone in vermiculite did not produce significant mineralization of either PLA or PLA - OMMT5 films . Further investigation is recommended to understand this behavior. The l ag time was shorter in the PLA - OMMT5 than in the PLA, results that agree with previous results . In all cases the lag time was reduced by the presence of the Geobacillus, so more CO 2 was produced at th e early stage of biodegradation. Geobacillus was able to form biofilm and attach to the surface of PLA , but in amount s less than the compost - derived mixed culture at 58 o C and PA at 25 o C . Moreover, PLA - OMMT5 provided a surface more readily colonized by P. aeruginosa and Geobacillus , suggesting that p olymer modification may provide a way to enhance colonization and therefore degradation of polymers. I f the biodegradation rate of PLA and PLA - based products can be accelerated and/or tailored, it could greatly benefit their general use and acceptance in i ndustrial composting facilities. So, using bioaugmentation to enhance the biodegradation rate of these compostable polymers can create a novel method to fast track the ir biodegradation, so that they can be easily accepted and biodegraded with other compost able organic materials. In turn, if more solid wastes can be disposed through composting, the amount of waste disposed in landfills c ould be reduced along with the social and environmental impacts associated with landfilling . 320 APPENDICES 321 APPENDIX 5A: Compost and vermiculite nutrient analysis Table 5A. 1 shows the physicochemical characteristics and the total nutrient analysis of the different media used: compost, inoculated vermiculite, and uninoculated vermiculite. Table 5A. 1 Physicochemical parameters and total nutrient analysis of different media used in the biodegradation test Parameter Compost Inoculated v ermiculite Uninoculated v ermiculite Dry solids, % 51.8 21.4 18.9 Volatile solids, % 41.3 2.8 3.1 pH 7. 9 6.8 8.2 C/N ratio 9.9 10 .2 59.9 Carbon, % 2 4.0 1. 6 1 .8 Nitrogen , % 2. 42 0.16 0.03 Phosphorus , % 0. 72 0. 21 0.1 3 Potassium , % 2. 56 4. 20 3.40 Calcium , % 7.69 0. 62 0. 57 Magnesium , % 2.0 8 8. 74 8. 33 Sodium , % 0.4 9 0.1 4 0.0 4 Sulfur , % 0. 50 0.05 0.01 Iron , ppm 5542 51300 4 8010 Zinc , ppm 206 72 6 6 Manganese , ppm 404 4 18 374 Copper , ppm 10 2 222 207 Boron , ppm 40 3 1 Aluminum , ppm 2640 4 0280 37930 Note: The total nutrient analysis was performed by inductively coupled plasma atomic emission spectroscopy ( ICP - OES ). 322 APPENDIX 5B: Molecular weight Table 5B. 1 Molecular weight reduction rate of PLA and PLA - OMMT5 in vermiculite with different levels of inoculation as estimated by the first order reaction of the form M n / M n0 = exp( - kt ) Level of inoculation k PLA , d - 1 k PLA - OMMT5 , d - 1 Un inoculated 0.136 ± 0.008 A 0.095 ± 0.005 A Geobacillus only 0.128 ± 0.004 AB 0.107 ± 0.008 A Mixed culture 0.117 ± 0.003 B 0.095 ± 0.005 A Mixed culture+Geobacillus 0.117 ± 0.006 B 0.092 ± 0.008 A Note: Values with the same letter within the same column are not significantly different - Kramer Test. Figure 5B. 1 shows the molecular weight distribution ( MWD ) of the PLA - OMMT5 film as function of time during the biodegradation in vermiculite with the different levels of inoculation. PLA - OMMT5 follows a similar behavior as pristine PLA with higher and sharper peaks on days 21 and 28 attributed to the assimilation of the low molecular weight chains by the microorganisms and the re maining of the newly formed crystalline structures. 323 Figure 5B. 1 MWD of PLA - OMMT5 samples in vermiculite with different levels of inoculation ( a) uninoculated, ( b) Geobacillus only, ( c) mixed culture, ( d) mixed culture and Geobacillus . 324 APPENDIX 5C: Biofilm Test Figure 5C. 1 Absorbance (600 nm) of ( a) PA at 23 ° C, and ( b) Geobacillus at 58 ° C of biofilm formation samples. Bars with the same letter within a group ( i.e. , wells, films, or total) are not stat - Kramer test. Figure 5C. 2 Absorbance (600 nm) of ( a) PA at 23 ° C, and ( b) mixed culture at 58 ° C for biofilm test. 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Microbiol. 18 (2016) 2732 2742. doi:10.1111/1462 - 2920.13331. 331 CHAPTER 6 CONCLUSION S AND RECOMMEND ATIONS 6.0 Conclusion s P oly(lactic acid) (P LA ) is likely the most popular polymer derived from renewable resources . It is recyclable and biodegradable under composting conditions , thus providing an alternative disposal route . PLA is already used in different applications for the medical, textile, plasticulture , and packaging industries with products manufactured via established polymer - processing techniques ( i.e., extrusion, injection molding, blow molding, thermoforming, foaming, and spinning) as critically reviewed in Chapter 2 . The range of applications of PLA keeps increasing with the development of novel materials in which PLA is blended with other polymers an d/ or compounded with different fillers to achieve the desired performance properties. Therefore , with the development of these new PLA - based materials , there is also a need for methodologies to evaluate their biodegradability and understand the ir biodegradation mechanisms if composting is their intended end of life. In this context, c hapter 3 presented a summary of the literature with the different method s that have been used to test the biodegradation of several materials. This information along with a comparative analysis of the results obtained from our own biodegradation tests ( performed by analysis of evolved CO 2 ), allowed us to identify some key factors that should be more strictly controlled for an efficient biodegradation test , especially those related to the characteristics of the compost ( e.g., organic matter, carbon - nitrogen ratio, and pH). Regarding environmental factors, temperature was the easiest parameter to control throughout the testing period while water content was the 332 most cruc ial and difficult to adjust . Chapter 3 also discussed the biodegradation of PLA as a study case based on our experiments . The results advocated that abiotic hydrolysis is the main contribution to the degradation process of PLA in the early stage of degradation and becomes a limiting factor for the subsequent biodegradation of this material , with the degradation rate depend ing also on the specific properties of the material ( e.g. crystallinity and initial molecular weight ). Throughout this work, we em phasized that o ne of the current limitations for composting as PLA end - of - life scenario is that this material does not biodegrade as fast as other organic wastes which affects its general acceptance in industrial compos ting facilities. Two approaches were proposed and studied to accelerate the biodegradation rate of PLA : the addition of nanoclays to the polymer matrix and bioaugmentation ( i.e., the addition of selective PLA - degrading microbial strain s ) . In chapter 4, we evaluated t he effect of three differe nt nanoclays and a surfactant ( OMMT, HNT, LR D, and QAC ) on the biodegradation rate of PLA . The results suggested that the biodegradation phase of the films containing nanoclay started earlier than that for pristine PLA. However, the initial molecular weight and thickness of the samples played a crucial role in the se biodegradation studies . When the different nanoclays and surfactant were tested alone, it was observed that HNT, OMMT, and QAC presented some inhibition regardless of the amount introduced in the bioreactors. The effect of nanoclays on the microbial attachment was also evaluated with a biofilm formation assay. The results showed that PLA - LRD had the greatest biofilm formation (as confirmed by the SEM micrographs ) . On the other hand, PLA - QAC had 333 the lowest biofilm formation, which was attributed to an inhibitory effect also observed during the biodegradation test when QAC was tested alone. Further investigation on the specific microbial strains capable of degrading PLA and on how they can affect the biodegradation rate of PLA was presented in Chapter 5 . Geobacillus thermoleovorans was identified as the PLA - degrading microbial strain present in the compost at 58 o C , and it was used to study bioaugmentation in simulated composting conditions. The results showed that b ioaugmentation with Geobacillus increased the evolution of CO 2 and shortened the lag phase of PLA and PLA - OMMT when tested in compost and vermiculite inoculated with a compost - derived mixed culture. Geobacillus inoculated alone in vermiculite did not produce significant mineralization of either PLA or PLA - OMMT films. Microbial attachment was also investigated in C hapter 5. Geobacillus was able to form biofilm and attach to the surface of PLA, but in lower amounts than the compost - derived mixed culture at 58 o C and P seudomonas aeruginosa (PA) a t 2 3 o C. The results also suggested that PLA - OMMT provided a surface more readily colonized b y PA and Geobacillus , indicating enhance d colonization . In general, i f the biodegradation rate of PLA and PLA - based materials ( e.g. BNCs) can be accelerated and/or tailored, it will greatly benefit their general use and acceptance in ind ustrial composting facilities. Therefore, incorporating nanoclays on the PLA matrix and/or using bioaugmentation with specific microbial strains have been proved to be eff ective methods for enhanc ing the biodegradation rate of PLA , so PLA products can be easily biodegraded along with other compostable organic materials. 334 6.1 Recommend ations The results presented in this work from our different biodegradation tests along with the information provided in the literature allowed us to identify that one of t he main issues of biodegradation testing is the low reproducibility due to the number of variables involved in the biodegradation process , making it difficult to provide fair comparisons of samples that are not within the same test. We have recommended per forming a biodegradation test in different labs around the world ( e.g., round robin test) by the analysis of evolved CO 2 in a DMR system using different standardized reference materials ( e. g. , cellulose, PLA, and PCL), and more strictly controlled compost physicochemical characteristics and testing parameters , in an attempt to unify and to improve this testing methodology. Regarding the studies made with PLA bio - nanocomposites, we have identified that the incorporation of nanoclays affected the initial mole cular weight and thickness of the PLA films even when maintaining the same processing conditions as the control films. The results suggested that these two factors ( i.e., initial molecular weight and thickness) played a crucial role in the evolution of CO 2 . Therefore, it is recommended for future biodegradation testing to produce samples with no significant difference in molecular weight and thickness , even though it can be challenging. During the bioaugmentation studies, we observed that the presence of Ge obacillus significantly increased the evolution of CO 2 when tested in compost and vermiculite inoculated with a compost - derived mixed culture . However, when Geobacillus was inoculated alone in vermiculite , no significant mineralization of the samples was o bserved . Therefore, f urther stud ies are recommended to understand this behavior , as well as the different interactions of Geobacillus with other microbial strains 335 present in the compost that may have a synergistic effect on the biodegradation of materials. Future work should also concentrate on studying other microbial strains that have been reported in the literature and that are able to assimilate PLA and its degradation by - products. In this context, most of the studies found in the literature were performed in liquid media, so it is essential to understand the biodegradation behavior in solid environments such as compost and vermiculite. It would also be relevant to study the changes in the phylogenetic composition and the different microbial interactions during composting using molecular ecological techniques ( e.g. next generation sequencing an d metaproteomics ) . For future biodegradation testing, and especially for bioaugmentation studies, we recommend the use of vermiculite. This media has been proven to be an excellent solid environment to simulate composting conditions in a more controlled manner , e.g., avoidin g priming effect produced by some testing materials , the incorporation of specific microbial strains , and the possible recovery of degradation by - products that can be potentially used for a complete carbon balance analysis. Future work should continue focu s on finding approaches to tailor the biodegradation rate of PLA. If such rate is accelerated, PLA and PLA - based products can be accep ted in industrial composting facilities and treated together with other organic wastes, which is the ideal end - of - life sce nario for this type of products. In turn, i f more solid wastes can be disposed through composting, the amount of waste disposed in landfills could be reduced along with the social and environmental impacts associated with landfilling. 336 Finally, in a wider p erspective , future work need to be done so PLA - based products can reach their intended end - of - life scenarios ( i.e., recycling and composting) . Currently, there are still limitations due to the lack of suitable infrastructure for collecting post - consumer PLA products , sorting, recycling, and/or composting . So, efforts should be centered on active collaboration with industries, commodity groups, industry associations, and government groups to improve the recovery rate of PLA.