DESIGN AND ENGINEERING OF STARCH-BASED POLYMER MATERIALS AS SUBSTITUTES FOR PERSISTENT NON-BIODEGRADABLE PLASTICS By Apoorva Chandrakant Kulkarni A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemical Engineering – Doctor of Philosophy 2022 ABSTRACT DESIGN AND ENGINEERING OF STARCH-BASED POLYMER MATERIALS AS SUBSTITUTES FOR PERSISTENT NON-BIODEGRADABLE PLASTICS By Apoorva Chandrakant Kulkarni Replacing carbon-carbon backbone persistent hydrocarbon plastics with biobased and biodegradable plastics offers value proposition of reduced carbon footprint and an environmentally responsible end-of-life. This work focuses on design and engineering of starch based polymeric materials as substitutes for non-biodegradable plastics. Starch foams are being used as replacement for petroleum-based foams in insulation and cushion protection applications. However, moisture sensitivity remains a problem resulting in collapse of cell structure and loss of mechanical integrity. First section of the thesis focuses on engineering high-performance starch foams with enhanced moisture resistance using reactive extrusion processing technology. Chitosan, polyvinyl butyral (PVB) and sodium trimetaphosphate (STMP) were used with water as a plasticizer and a blowing agent to make foams with desired physico-mechanical properties. The resulting foams were hydrophobic, insoluble in water, and showed improved moisture resistance. The foams were completely biodegradable as established by ASTM/ISO standards. Crosslinking of starch with STMP increased the compressive strength of the foams by three times compared to control foams. Optimization of process parameters ensured an efficient, cost-effective route towards commercialization. In the second section, our group’s chemically modified thermoplastic starch (MTPS) prepared by reactive extrusion technology was explored in three different applications. First, MTPS, was melt blended with glycol modified polyethylene terephthalate (PETG) using transesterification chemistry to synthesize MTPS-g-PETG in situ graft copolymer with 33% grafting. Second, the use of MTPS as a biobased and biodegradable nucleating agent and barrier property enhancer in polylactide (PLA) was explored. MTPS accelerated the rate of crystallization of PLA by up to 98 times at 100°C, reducing the half time for crystallization from 20 mins to less than 1 minute. Oxygen barrier properties of PLA was improved 127% without causing detrimental impact on mechanical properties or biodegradability. Third application focused on using MTPS as a carrier for iodine, which is a very effective and strong antimicrobial agent. The new MTPS-iodine complex was incorporated in various proportions to commercial fully biodegradable-compostable polyester films. The morphological, mechanical, and antibacterial properties of these films were evaluated and compared with current commercial additives used to obtain antibacterial properties. The last section focuses on the end-of-life evaluations for biobased and biodegradable plastics using kinetics approach. The effect of temperature on biodegradation of cellulose and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in an aqueous environment seeded with a biologically aggressive microbial inoculum was studied. A global equation was derived from the reparametrized Arrhenius equation and the kinetic rate law to estimate the time required for 90% removal of polymer from the low temperature ocean environment. The t90 (time required to remove 90% of the polymer carbon from the environment) for PHBV at 10°C ranged from 6.2-6.9 years. The t90 of cellulose at 10 C was found to be 1.1-1.2 years. ASTM/ISO standards for measuring and reporting ocean biodegradability is static and conducted at one temperature (30°C), whereas ocean temperatures can vary from −1.8 °C to 33.4 °C. The kinetic analysis and model developed can provide a method to estimate time for complete removal of the biodegradable polymer carbon in ocean environments. Copyright by APOORVA CHANDRAKANT KULKARNI 2022 Dedicated to my family v ACKNOWLEDGMENTS First, I would like to thank my advisor Dr. Ramani Narayan for his constant support and guidance throughout my journey in graduate school. His unique style of promoting independent research, giving students all the freedom to explore the field of their research own their own made me very confident and independent researcher. Because of his insistence on always exploring new areas and not getting stuck with just your narrow research project, I got to work on a variety of projects throughout my time here at MSU. This truly enabled me to think out of the box and look at the big picture of any project that I worked on. These qualities have proved to be of utmost importance for me in securing a job in the industry and I am sure will help me in the rest of my career as well. I would also like to thank all my committee members Dr. Robert Ofoli, Dr. Shiwang Cheng and Dr. Rafael Auras for their invaluable feedback during the course of my work. I would especially like to thank Dr. Auras and his students who were always there to help me in using their instruments. Special thanks to Aaron Walworth and Pooja Mayekar who trained me in various instruments at School of Packaging. I am also grateful to all my professors here at MSU who helped in learning the ins and outs of chemical engineering and polymers. I am also very grateful to all the collaborators – Dr. Jeffrey Catchmark, Dr. Teresa Bergholz, Dr. Shilpa Manjure, and everyone else who supported our work and provided the required facility to conduct the experiments. I would also like to thank Abby Vanderberg at the Center of Advanced Microscopy who helped me in sample preparation and microscopy imaging of all my samples. Thanks to all the members from CMSC center who helped in preparation and testing of various samples. vi I am very grateful to all the members of Biobased Materials Research Group (BMRG) including my seniors Dr. Sayli Bote, Dr. Preetam Giri and Manas Nigam who helped me in learning and understanding my group’s work and culture. I would also like to thank Dr.(very recent) Moh Alhaj for being there when additional help was required. Moh joined BMRG with me and was always there throughout my journey at MSU. Doing assignments and studies together along with some gossiping made the time at BMRG more enjoyable. I owe a big thank you to all the undergrads – especially Melissa Joslyn, Jakob Emrich and Nicole Mancina who were passionate about their research, constantly gave valuable inputs for improving the experiments and never complained about cleaning up the starch clogged extruder or doing endless amounts of titrations. It was their contribution that allowed me to finish the work in much less time. I would also like to thank and wish all the best to Shrirang, Shehla and Badal for their future work at BMRG. I would also like to take this opportunity to thank my second family away from home – Aditya (Adi), with whom I always used to pick a fight for no reason. Thank you for tolerating me. And for being there with me throughout my journey. Gouree (Gourabai) my so called ‘roommate’ who never lived with me but was always there for doing all the shopping trips, cooking, gossips and everything. Thanks to Affan (Wise man) for all your advice and for always bringing me food and trying to make me fat. Thank you Sneha (oo we don’t have a nickname for you) for making pakodas with me every day and tormenting Gouree. Thank you to Renu and Sushant for being the wise people amongst this group of jokers and keeping this crazy family together. And thank you for bringing Chintu in our life . We all love him a lot. Finally, thank you to Shalin, who gave me this amazing idea to write such acknowledgements and enjoy the writing and for always doing ‘all the work’. My Spartan life vii wouldn’t be the same without all of you. Last but not the least, I would like to thank my parents and my brother Rushi for their support through each step of my life. It was their motivation and love that helped me in pursuing this dream of PhD. Special thanks to all my grandparents (Aaji and Ajoba), some of whom are unfortunately not with me today. I know you all were always proud of me and will bless me for a great future. Thank you and apologies to everyone else whom I might have missed in this acknowledgment. viii TABLE OF CONTENTS LIST OF TABLES................................................................................................................................................. xiii LIST OF FIGURES ................................................................................................................................................. xv KEY TO ABBREVIATIONS ............................................................................................................................. xxii 1. INTRODUCTION ............................................................................................................................................ 1 1.1 INTRODUCTION ................................................................................................................................... 1 1.2 OVERALL GOALS AND OBJECTIVES ............................................................................................. 7 1.3 ORGANIZATION OF THE THESIS ................................................................................................... 9 REFERENCES ............................................................................................................................................... 13 2. DESIGN AND ENGINEERING OF INSOLUBLE, HIGH-PERFORMANCE STARCH FOAMS VIA EXTRUSION TECHNOLOGY .................................................................................................................... 15 2.1 ABSTRACT ........................................................................................................................................... 15 2.2 INTRODUCTION AND BACKGROUND ....................................................................................... 17 2.3 MATERIALS ......................................................................................................................................... 24 2.4 EXPERIMENTAL ................................................................................................................................ 25 2.5 FOAM EXTRUSION PROCESS DEVELOPMENT ...................................................................... 29 2.6 CHARACTERIZATION AND ANALYSIS ..................................................................................... 36 2.7 RESULTS AND DISCUSSION .......................................................................................................... 42 2.8 DESIGNING AN ANNULAR DIE .................................................................................................... 79 2.9 FUTURE RECOMMENDATIONS ................................................................................................... 83 2.10 CONCLUSIONS.................................................................................................................................... 84 REFERENCES .............................................................................................................................................. 86 ix 3. BIOBASED MALEATED THERMOPLASTIC STARCH (MTPS) AND MTPS-G-PETG (GLYCOL MODIFIED PET) GRAFT COPOLYMER VIA REACTIVE EXTRUSION: EVALUATIONS ..................................................................................................................................................... 92 3.1 ABSTRACT ........................................................................................................................................... 92 3.2 INTRODUCTION ................................................................................................................................ 93 3.3 EXPERIMENTAL ................................................................................................................................ 96 3.4 CHARACTERIZATION AND ANALYSIS ..................................................................................... 98 3.5 RESULTS AND DISCUSSIONS ..................................................................................................... 102 3.6 CONCLUSIONS AND FUTURE STUDY...................................................................................... 112 REFERENCES ............................................................................................................................................ 113 4. EFFECTS OF MODIFIED THERMOPLASTIC STARCH ON CRYSTALLIZATION KINETICS AND BARRIER PROPERTIES OF PLA ................................................................................... 116 4.1 ABSTRACT ......................................................................................................................................... 116 4.2 INTRODUCTION .............................................................................................................................. 117 4.3 MATERIALS AND METHODS ...................................................................................................... 121 4.4 RESULTS AND DISCUSSION ........................................................................................................ 127 x 4.5 CONCLUSIONS.................................................................................................................................. 146 REFERENCES ............................................................................................................................................ 148 5. ANTIMICROBIAL SOLID STARCH-IODINE COMPLEX VIA REACTIVE EXTRUSION AND ITS APPLICATION IN PLA-PBAT BLOWN FILMS....................................................................... 154 5.1 ABSTRACT ......................................................................................................................................... 154 5.2 INTRODUCTION .............................................................................................................................. 155 5.3 STARCH IODINE COMPLEX......................................................................................................... 157 5.4 EXPERIMENTAL SECTION .......................................................................................................... 159 5.5 CHARACTERIZATION AND ANALYSIS ................................................................................... 162 5.7 CONCLUSIONS AND FUTURE WORK ...................................................................................... 176 APPENDIX .................................................................................................................................................. 178 REFERENCES ............................................................................................................................................ 181 6. UNDERSTANDING BIODEGRADABILITY OF BIOBASED POLYMERS IN MARINE ENVIRONMENT................................................................................................................................................. 184 6.1 ABSTRACT ......................................................................................................................................... 184 6.2 INTRODUCTION AND BACKGROUND ..................................................................................... 185 6.3 BIODEGRADATION PROCESS .................................................................................................... 189 6.4 MECHANISMS OF BIODEGRADATION .................................................................................... 191 6.5 FACTORS AFFECTING AQUEOUS BIODEGRADATION ..................................................... 193 6.6 POLYHYDROXY ALKANOATES (PHAS) .................................................................................. 196 6.7 EXPERIMENTAL SECTION .......................................................................................................... 198 6.8 MATERIAL PROCESSING AND CHARACTERIZATION ...................................................... 199 xi 6.9 EXPERIMENTAL BIODEGRADATION SETUP ....................................................................... 203 6.10 RESULTS AND DISCUSSION ........................................................................................................ 208 6.11 MECHANISM OF PHBV BIODEGRADATION ......................................................................... 228 6.12 CONCLUSION AND FUTURE WORK ......................................................................................... 232 APPENDICES ............................................................................................................................................. 235 REFERENCES ............................................................................................................................................ 263 xii LIST OF TABLES Table 2.1: Extrusion runs with various additives.................................................................................. 27 Table 2.2: Screw configurations for starch foam extrusion............................................................... 30 Table 2.3: General temperature profile for extrusion ......................................................................... 32 Table 2.4: Different salt solutions for relative humidity..................................................................... 38 Table 2.5: Mineral solution composition for the test ........................................................................... 40 Table 2.6: Parameters of Peleg model for kinetics of moisture absorption of starch foams....................................................................................................................................................................... 60 Table 2.7: Physico-mechanical properties of extruded starch foams............................................ 77 Table 3.1: Results for percent grafting of glycerol on MTPS ........................................................... 102 Table 3.2: Tensile properties of PETG and MTPS-g-PETG blend ................................................... 107 Table 4.1: Summary of physico-mechanical properties of packaging polymers ..................... 118 Table 4.2: Sample name and composition .............................................................................................. 122 Table 4.3: Effect of MTPS content on tensile properties of PLA .................................................... 128 Table 4.4: Thermogravimetric analysis (TGA) of PLA and blends ................................................ 131 Table 4.5: Thermal transition temperatures and percent crystallinity of modified PLA samples ....................................................................................................................................................... 133 Table 4.6: Crystallization half times and Avrami constants for PLA samples at different temperatures .................................................................................................................................. 142 Table 5.1: Temperature and processing conditions for making blown films ........................... 161 Table 5.2: Sample name and composition .............................................................................................. 161 Table 6.1: Aqueous biodegradation test setup. Test carried out at 10ºC ................................... 199 Table 6.2: Aqueous biodegradation test setup. Test carried out at 30ºC ................................... 200 Table 6.3: Aqueous biodegradation test setup. Test carried out at 40ºC ................................... 200 Table 6.4: Buffer solutions used for aqueous biodegradation........................................................ 205 Table 6.5: Order of reaction for cellulose and PHBV biodegradation.......................................... 214 xiii Table 6.6: Parameter estimates for activation energy and pre-exponential factor using method 1, 2 and 3 ................................................................................................................................ 218 Table 6.7: Final estimated parameter values for the global equation for cellulose and PHBV............................................................................................................................................................. 219 Table 6.8: Evolution of number-average molecular weight (Mn), weight-average molecular weight (Mw), polydispersity index (Mn/Mw) and melting point (Tm)of PHBV samples during the course of biodegradation ......................................................................... 229 Table 6.9: Lifetime estimation for PHBV and cellulose at 30, 10 and 4° C................................. 251 Table 6.10: Percent carbon remaining vs time ..................................................................................... 252 Table 6.11: Literature summary ................................................................................................................ 253 Table 6.12: Ocean plastics accumulation model .................................................................................. 262 xiv LIST OF FIGURES Figure 1.1: Various biobased and fossil-based plastics and their relation to biodegradability (all the polymers below the horizontal line are fully biodegradable or compostable)[3] ............................................................................................................................................... 2 Figure 1.2: Global production capacities of bioplastics 2021[1] ........................................................ 3 Figure 1.3: Structure of starch units - a) Amylose b) Amylopectin ................................................... 4 Figure 1.4: SEM images for showing structure, size and shape of various starch granules depending on the source .................................................................................................................................... 4 Figure 1.5: Some applications of bioplastics .............................................................................................. 5 Figure 1.6: Schematic for overall summary of the work in this thesis ............................................. 8 Figure 1.7: Schematic for the summary of chapter 2 .............................................................................. 9 Figure 1.8: Schematic for summary of chapter 4 ................................................................................... 10 Figure 1.9: Schematic for summary of chapter 5 ................................................................................... 11 Figure 1.10: Schematic for summary of chapter 6 ................................................................................ 12 Figure 2.1: Structure of polyvinyl butyraldehyde(PVB) ..................................................................... 19 Figure 2.2: Schematic for polyelectrolyte complex formation ......................................................... 20 Figure 2.3: Scheme for formation of polyelectrolyte complex between chitosan and starch ...................................................................................................................................................................... 20 Figure 2.4: Phosphorylation of starch with STMP ................................................................................ 21 Figure 2.5: Experimental setup for foam extrusion .............................................................................. 26 Figure 2.6: Foams obtained from a) Screw configuration #1 b) Screw configuration #2 ..... 30 Figure 2.7: Second Screw configuration used for foam extrusion .................................................. 32 Figure 2.8: Three strand dies used for extrusion and their corresponding expansion ratios ....................................................................................................................................................................... 34 Figure 2.9: Process development with slit die ........................................................................................ 35 Figure 2.10: Effect of talc and PVOH addition on density and expansion ratio of foams ....... 43 Figure 2.11: Effect of chitosan on density and expansion ratio of foams..................................... 44 xv Figure 2.12: Effect of STMP addition on density and expansion ratio of starch foams .......... 45 Figure 2.13: Water solubility for foams with different additives .................................................... 47 Figure 2.14: Solubility of corn starch foams with glycerol ................................................................ 48 Figure 2.15: Solubility studies for potato starch foams ...................................................................... 49 Figure 2.16: Water solubility studies for foams containing PVB ..................................................... 50 Figure 2.17: Water penetration test for foams with various additives ......................................... 51 Figure 2.18: water penetration test for foams containing PVB, chitosan and STMP ............... 52 Figure 2.19: Water penetration test for foams with and without glycerol.................................. 53 Figure 2.20: Swelling characteristics of starch-chitosan-STMP foams ......................................... 55 Figure 2.21: Moisture absorption curves for starch foams in different RH conditions a)control foams (starch+water) b)Control foam(PVOH +water), c)corn starch +PVB + chitosan ,d)corn starch+STMP+chitosan .................................................................................................. 56 Figure 2.22: Sorption isotherms for different types of starch foams .......................................... 58 Figure 2.23: Shrinkage in foams with high RH of 95% a) control starch foam, b) PVOH foam, c) PVB foam d) chitosan +STMP (1.5%) ..................................................................... 59 Figure 2.24: Peleg model constants k1 and k2 for the starch foam samples .............................. 61 Figure 2.25: Moisture content at equilibrium for different foam formulations at 95% RH .................................................................................................................................................................. 62 Figure 2.26: Effect of different additives on surface hydrophobicity of foams.......................... 63 Figure 2.27: Effect of increasing percentage of chitosan and addition of STMP on surface hydrophobicity.................................................................................................................................................... 65 Figure 2.28: Effect of glycerol addition on surface hydrophobicity of starch foams ............... 66 Figure 2.29: SEM images for various additives used in foam formulations – a) talc, b) chitosan, c)STMP, d) PVB at 200x magnification; e)talc, f)chitosan, g)STMP; h) PVB at 5000x magnification ..................................................................................................................... 68 Figure 2.30: SEM images of starch foams containing a)0% talc b) 0.7% talc c)2% talc d)4% chitosan e)7% chitosan f)10% chitosan g) 1% STMP h) 1.5% STMP i) 3% STMP ...... 69 Figure 2.31: Cell size distribution for foams containing different percentages of talc ........... 70 xvi Figure 2.32: Cell size distributions for starch foams with PVB at different STMP levels a) 0% b) 1% c)1.5 % d)3%............................................................................................................................. 71 Figure 2.33: Cell size distribution of starch foams with increasing percentages of chitosan .................................................................................................................................................................. 72 Figure 2.34: Confocal laser scanning microscope (CLSM) 3D MIP images for 0%, 4%, 7% and 10% chitosan foam formulations from the bulk of the sample (a,b,c,d) and from the surface (e,f,g,h) ................................................................................................................................. 75 Figure 2.35: Compressive strength of starch foams 1)Control foams (starch+water), 2)Control foams (PVOH), 3)10% PVB,4% chitosan, 4) 10% PVB,7% chitosan, 5) 10% PVB,10% chitosan, 6) 10% PVB,4% chitosan, 1% STMP, 7) 10% PVB,4% chitosan, 1.5 STMP, 8) 10% PVB,4% chitosan, 3% STMP ............................................................................................. 76 Figure 2.36: Aqueous biodegradation curves for control starch foam, PVB containing foam and STMP containing foam ................................................................................................................. 78 Figure 2.37: Annular die design parts ........................................................................................................ 79 Figure 2.38: AUTOCAD design for the die ................................................................................................ 80 Figure 2.39: 3D printed die ............................................................................................................................ 81 Figure 2.40: CNC machined die..................................................................................................................... 81 Figure 2.41: a), b) Some annular die foams produced from the trial runs c) flat sheets formed after cutting the foam tubes ........................................................................................................... 82 Figure 3.1: Structure of maleated thermoplastic starch ..................................................................... 94 Figure 3.2: Structure of PETG ........................................................................................................................ 96 Figure 3.3: Preparation of maleated thermoplastic starch ................................................................ 97 Figure 3.4: DTG curves of MTPS, glycerol and residue showing only MTPS after Soxhlet extraction ............................................................................................................................................................ 103 Figure 3.5: TGA graphs for MTPS, PETG and MTPS-g-PETG ........................................................... 104 Figure 3.6: DTG curve of residue remaining after Soxhlet extraction ......................................... 105 Figure 3.7: Comparative FT-IR spectra of PETG, MTPS and MTPS-PETG residue .................. 106 Figure 3.8: The average stress-strain curves of PETG and MTPS-g-PETG ................................. 107 Figure 3.9: Notched Izod impact strength of PETG and MTPS-g-PETG reactive blend......... 108 xvii Figure 3.10: SEM images of a) MTPS-PETG blend after leaching of MTPS phase with HCL; b)Tensile fracture surface of PETG c) Tensile fracture surface of MTPS-g-PETG ........ 109 Figure 3.11: Experimental aqueous biodegradation setup ............................................................. 110 Figure 3.12: Biodegradation curve for MTPS-PETG for a)Initial 80 days b)Total 150 days............................................................................................................................................... 111 Figure 4.1: Modulus, tensile stress, and tensile strain graphs for PLA and blends ................ 128 Figure 4.2: Scanning electron microscopy (SEM) images of fractured tensile surfaces of (a) PLA, (b) PLA + 1% MTPS, (c) PLA + 2% MTPS, (d) PLA + 5% MTPS .................................... 129 Figure 4.3: SEM images of the blends after selective extraction of MTPS phase (a) PLA, (b) PLA + 1% MTPS, (c) PLA + 2% MTPS, (d) PLA + 5% MTPS...................................................... 129 Figure 4.4: a)TGA and b)DTG curves for PLA and PLA/MTPS blends ......................................... 132 Figure 4.5: Differential scanning calorimetry (DSC) thermograms of PLA and the blends ............................................................................................................................................................ 134 Figure 4.6: Melting curves for PLA blends after annealing .............................................................. 135 Figure 4.7: DSC melting thermograms of a)PLA and blends (b) PLA+1%MTPS, c) PLA+2%MTPS, d) PLA+5% MTPS )at various temperatures .................................................... 136 Figure 4.8: Fractional crystallinity vs. time of a)PLA and blends (b) PLA+1%MTPS, c) PLA+2%MTPS, d) PLA+5% MTPS )at various temperatures .................................................... 138 Figure 4.9: Plots of ln(ln(1-X(t)) vs ln t of a)PLA and blends (b) PLA+1%MTPS, c) PLA+2%MTPS, d) PLA+5% MTPS )at various temperatures .................................................... 139 Figure 4.10: Half time for crystallization vs isothermal crystallization temperatures for PLA and blends .......................................................................................................................................... 139 Figure 4.11: Comparing the t1/2 values of PLA/MTPS blends with other nucleating agents ............................................................................................................................................. 140 Figure 4.12: Polarized optical microscopy (POM) images of PLA/MTPS blends with MTPS content of 0%: (a–c); 1%: (d–f); 2%: (g–i); 5%: (j–l) ............................................................ 141 Figure 4.13: Effect of MTPS content on moisture permeability (WVP) and oxygen permeability (OP) of PLA films ................................................................................................................... 143 Figure 4.14: Experimental aqueous biodegradation setup ............................................................. 144 Figure 4.15: Aqueous biodegradation curves for PLA and PLA + 5% MTPS............................. 144 xviii Figure 5.1: Amylose coil structure - due to acetal linkages ............................................................. 157 Figure 5.2: Structure of amylose-iodine complex ............................................................................... 158 Figure 5.3: Production of MTPS-iodine pellets via reactive extrusion ........................................ 160 Figure 5.4: DTG curves for MTPS, MTPS-iodine and iodine ............................................................ 167 Figure 5.5: Isothermal TGA for MTPS and MTPS-iodine................................................................... 167 Figure 5.6: Weight loss and visual decay analysis for coated and uncoated strawberries . 168 Figure 5.7: Photographs of the petri plates from disk diffusion and direct inoculation test with E. coli. a) Kirby Bauer test on starch-iodine pellets showing antibacterial zone of inhibition b)-f) bacterial colonies growth on the petri plates after 24 hours b) control PLA films c) AM: Silver antimicrobial films d) SI1: starch-iodine-PLA films with 0.7% iodine and 1 mil thickness e) SI2: starch-iodine-PLA films with 0.7% iodine and 2 mil thickness f) SI3: starch-iodine-PLA films with 1.3% iodine and 2 mil thickness.................... 170 Figure 5.8: Average change in Log CFU/ml of the Escherichia coli (E. coli) strains after 24 h incubation on five different PLA film configurations (control PLA;AM film and SI1, SI2 and SI3) ....................................................................................................................................... 171 Figure 5.9: Mechanical properties for the different PLA blown film formulations in machine and trnasverse direction a) tensile strength b) Elongation at break c)Tear strength ................................................................................................................................................. 174 Figure 5.10: Visual and SEM images of the fractured surface of the films after selective extraction of MTPS-iodine phase a), b) and c) : SI1- 0.7% iodine, 1 mil; d), e) and f) : SI2 – 0.7 % iodine, 2 mil; g),h) and i) : 1.3% iodine, 2 mil ......................................................... 175 Figure 6.1: Temperature-depth Ocean water profile[11] ............................................................... 187 Figure 6.2: The different steps involved in the biodegradation process .................................... 189 Figure 6.3: Degradation mechanisms for biodegradable polymers A) Bulk erosion, B) Surface erosion[15] ................................................................................................................................... 192 Figure 6.4: Chemical structures of cellulose and PHBV .................................................................... 198 Figure 6.5: A typical biodegradation curve for %biodegradation vs time ................................. 203 Figure 6.6: Experimental biodegradation setup .................................................................................. 206 Figure 6.7: TGA and DTG curves for a) cellulose, b) PHBV; DSC curves for c) cellulose d)PHBV ......................................................................................................................................... 209 xix Figure 6.8: Average biodegradation curves of Cellulose tested at different temperature (10, 30, 40ºC). Each curve represents average of 2 replicates ....................................................... 211 Figure 6.9: Average biodegradation curves of PHBV tested at different temperature (10, 30, 40ºC). Each curve represents average of 2 replicates ....................................................... 211 Figure 6.10: C/Co as a function of time for aqueous biodegradation of a) Cellulose and b) PHBV at 10, 30 and 40°C. Points represent experimental data whereas dotted lines indicate fitting of 1st order equation ....................................................................................................... 212 Figure 6.11: Schematic for showing the comparison between standard linear regression analysis, reparametrized Arrhenius equation analysis and the one step global equation approach ............................................................................................................................. 217 Figure 6.12: Precision differences for biodegradation time estimations for the three methods ............................................................................................................................................................... 220 Figure 6.13: percent carbon remaining as a function of time for cellulose and PHBV at 30, 10 and 4 °C................................................................................................................................................... 221 Figure 6.14: Comparing the experimental rate constants to literature values for Cellulose and PHBV ......................................................................................................................................... 223 Figure 6.15:Statistical analysis of rate constants for a)cellulose and b)PHBV at 25 and 30 C ............................................................................................................................................................... 224 Figure 6.16: Model for plastics accumulation in ocean – impact of replacing a fraction of non-biodegradable plastics with biodegradable plastics ................................................................. 227 Figure 6.17: PHBV SEM and optical microscopy images at various stages of biodegradation .................................................................................................................................................. 228 Figure 6.18: a) DSC melting peaks and b) TGA degradation curves of PHBV pellets as a function of biodegradation percentage in laboratory scale aqueous biodegradation .......... 230 Figure 6.19: NMR curve for PHBV ............................................................................................................. 237 Figure 6.20: Comparing the microbial populations from wastewater and seawaters from various places ......................................................................................................................................... 240 Figure 6.21: ln(-dC/dt) vs lnC plot for cellulose at A)10 C, B) 30 C, C) 40 C ............................. 242 Figure 6.22: ln(-dC/dt) vs ln C plots for PHBV at A)10 C, B) 30 C, C)40 C ................................. 243 Figure 6.23: mmoles of carbon remaining vs time plots for cellulose at A)10C, B)30C, C) 40 C .................................................................................................................................................................. 245 xx Figure 6.24: Mmoles of carbon remaining vs time plots for PHBV at A)10C, B)30C, C) 40C ................................................................................................................................................................... 246 Figure 6.25: minitab methodology for global equation-1 ................................................................ 248 Figure 6.26: minitab methodology for global equation-2 ................................................................ 248 Figure 6.27: minitab methodology for global equation-3 ................................................................ 249 Figure 6.28: Percent carbon remaining vs time graphs (lifetime prediction) for cellulose at various temperatures ............................................................................................................. 250 Figure 6.29: ln(Co/Ct) vs time for PHBV and cellulose at 10, 30 and 40°C ............................... 260 Figure 6.30: Arrhenius plots for PHBV and cellulose......................................................................... 261 xxi KEY TO ABBREVIATIONS PLA – Polylactide PE – Polyethylene LDPE – Low density polyethylene PCL – Polycaprolactone MTPS – Maleated thermoplastic starch PETG – Polyethylene terephthalate glycol PHBV - Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PVB – Polyvinyl butyral STMP – Sodium trimetaphosphate PHAE – Polyhydroxylamino ether PBAT - Polybutylene adipate terephthalate BPA – Bisphenol A PEC – Polyelectrolyte complex PVOH - Polyvinyl alcohol MCC – Microcrystalline cellulose FITC - fluorescein isothiocyanate ISO - International Organization for Standardization SEM – Scanning electron microscopy CLSM - Confocal laser scanning microscopy TGA – Thermogravimetric analysis DSC – Differential scanning calorimetry xxii MIP – Maximum intensity projection REX – Reactive extrusion DCM – Dichloromethane FT-IR - Fourier Transform Infrared Spectroscopy ThCO2 – Theoretical CO2 PS – Polystyrene MA – Maleic anhydride WVP – water vapor permeability POM – Polarized optical microscopy OTR – Oxygen transmission rate WVTR – Water vapor transmission rate CSI – Crosslinked starch iodine TPS – Thermoplastic starch DTG – Derivative thermogravimetric graphs MD – Machine direction TD – Transverse direction PGA – poly (glycolic acid) BD – Biodegradation CI – Confidence intervals SD – Standard deviation MT – Million tones PDI – Polydispersity index xxiii 1. INTRODUCTION 1.1 INTRODUCTION Plastics and polymers have emerged as a vast market for a wide range of applications including electronics, packaging, automotive, medical etc. According to European bioplastics, the global market for plastics was about 367 million tonnes. Amongst those plastics, biobased and biodegradable plastics share is still less than one percent. But rising awareness about environmental pollution due to fossil fuel-based plastics is fueling the research and use of biobased and biodegradable plastics in various fields. The demand for bioplastics is surging. Global bioplastics production capacities are set to increase from around 2.42 million tonnes in 2021 to approximately 7.59 million tonnes in 2026[1]. European bioplastics defines bioplastics as biobased, biodegradable, or both. They have the same or similar properties as conventional plastics but offer additional benefits, such as a reduced carbon footprint, better functionalities, or additional waste management options, such as organic recycling[1]. Biodegradability is an end-of-life option that harnesses the power of microorganisms present in the selected disposal environment, to completely remove plastic products, designed to be biodegradable, from the environmental compartment via the microbial food chain in a timely, safe, and efficacious manner[2]. The property of biodegradation does not depend on the resource basis of a material but is rather linked to its chemical structure. In other words, 100 percent biobased plastics may be non- biodegradable, and 100 percent fossil-based plastics can biodegrade. Figure 1.1 shows the schematic for various biobased and fossil-based plastics and their relation with biodegradability based on their chemical structure[3]. 1 Figure 1.1: Various biobased and fossil-based plastics and their relation to biodegradability (all the polymers below the horizontal line are fully biodegradable or compostable)[3] Replacing the petro/fossil carbon with biobased carbon derived from plant/biomass resources offers the value proposition of a zero-material carbon footprint[2]. Target markets for biodegradable polymers include packaging materials like trash bags, loose-fill foam, food containers, film wrapping, laminated paper, hygiene products like diaper back sheets and cotton swabs, consumer goods like fast-food tableware and containers, egg cartons, and toys, and agricultural tools like mulch films and planters etc. Starch based bioplastics Biobased plastics are derived from various biomass sources. The most widely used resource is starch. Starch based plastics are often incorporated in various petroleum-based polymers or biobased polymers to create unique composite materials. The main advantage of using starch based polymeric materials is the ability of starch to biodegrade in almost all different environments: soil, compost and water. Hence, starch-based products are particularly ideal 2 for products when the risk of leakage in the environment after its use is high. Starch based bioplastics are not generally thought as a direct replacement for traditional plastics. Because of their peculiar properties, they present an opportunity to redesign the systems to enable the use of starch products[4, 5]. Out of the 2.42 million tons of bioplastics produced in 2021, 16.4% were starch-based plastics[6]. Figure 1.2 shows the global production capacities of bioplastics in 2021 by their material types. Figure 1.2: Global production capacities of bioplastics 2021[1] Starch is used as a starting material for a wide range of green materials. 75% of all organic material on earth is present in the form of polysaccharides. Most important industrial starch sources are crops such as corn (82%), wheat (8%), potato (5%) and cassava (5%). US is the 2nd largest producer of corn with 15.1 billion bushels produced in 2021[7]. Starch consists of two polymers – 1) amylose which is a linear polymer containing chains of α-1,4- anhydroglucose units which are mainly responsible for film-forming abilities and 2) 3 amylopectin which is a highly branched polymer containing α-1,4-anhydroglucose units and α-1,6-glycosidic branched chains [8, 9, 10]. Figure 1.3 shows the structures of the two units. n n a) Amylose b) Amylopectin(70-90%) Figure 1.3: Structure of starch units - a) Amylose b) Amylopectin The ratio of amylose and amylopectin is different in different starches [11]. The properties of the products like films, foams depend on the type of starch being used. Waxy starches contain less than 15% amylose, normal starches contain about 30% amylose whereas high amylose starches contain more than 40-50% amylose. Starch granules occur in all shapes and sizes and their dimensions range form 2-200 μm depending on the source of the starch. Figure 1.4 shows two such examples of different granule shapes and sizes for potato and corn starch. 10kV SS30 x1000 10μm 10kV SS30 x1000 10μm a) Corn starch b) Potato starch Figure 1.4: SEM images for showing structure, size and shape of various starch granules depending on the source 4 Starch in its native state is not thermoplastic. Various plasticizers such as water, glycerol, sorbitol etc. are added during processing to make the starch pseudo thermoplastic and processable. Starch is commonly blended with other biodegradable or non-biodegradable polymers such as polylactide (PLA), polyethylene (PE), Low density polyethylene (LDPE), polycaprolactone (PCL) etc. Other common applications are in loose fill packaging foams, cups, plates, films and trays. Figure 1.5 shows some of the common products available commercially which make use of bioplastics. Figure 1.5: Some applications of bioplastics In the current work we have focused on design and engineering of various starch based polymeric materials including starch foams, maleated thermoplastic starch (MTPS) and its applications as – a blend with a non-biodegradable polymer (PETG), as a nucleating agent and barrier property enhancer in PLA and as a vector for safe delivery of antimicrobial iodine in an environment without the usual hazards associated with handling solid iodine. Next part of the thesis focuses on the end-of-life assessments for such bioplastics. Biodegradation of the polymers in aqueous environment was studied in detail, impact of temperature on the 5 rate of biodegradation was analyzed, and a global kinetic analysis model was proposed for predicting the persistence of microplastics in oceans. 6 1.2 OVERALL GOALS AND OBJECTIVES This study focuses on using starch as a backbone to design various polymeric products – starch foams, thermoplastic starch, blends of thermoplastic starch with polyesters such as PLA and PETG for improving their performance properties, thermoplastic starch as a vehicle to deliver antimicrobial iodine into other polymers, plastic films, foams etc. and evaluating the biodegradability of these bioplastics at their end of life for their lifetime prediction in natural environments like ocean. This study targeted the design and engineering of high performance, water insoluble and moisture resistant starch foams. One part of the study was focused on the scale up of a starch- chitosan polyelectrolyte complex foam product prepared via microwave expansion. The target applications were use in medical field as hemostatic pads and in packaging where long-term moisture resistance and mechanical properties are required. Polyvinyl butyral (PVB) was also investigated as an additive along with chitosan and sodium trimetaphosphate (STMP) and their synergistic effects on the performance properties of foams were studied. Next part of the thesis was development and application of another value-added product from starch– Maleated thermoplastic starch (MTPS). Covalently grafted glycerol containing MTPS was prepared in a twin-screw extruder and its reactive blends to form graft copolymers with glycol modified polyethylene terephthalate (PETG) achieving in-situ grafting using transesterification chemistry were evaluated. It also targets on studying the effect of MTPS in a PLA- another biobased polymer, as nucleating agent, and barrier property enhancer. Further, A solid starch-iodine complex resin was also developed in this study as an antibacterial additive for application in active packaging, films, and foams using reactive extrusion. 7 Finally, this thesis also covers the aqueous biodegradation studies for various polymers and their kinetic analysis. The models developed here are useful for predicting the lifetime of these polymers in low temperature marine environment. Figure 1.6 shows the schematic for overall summary of the work in this thesis. Applications Amylose End of life biodegradation Starch Starch foams Iodine + KI Iodine + KI studies (aq.) Starch + Starch + (aq.) glycerol + (Aqueous glycerol + Starch + Iodine + KI (aq.) maleic maleic anhydride biodegradation) anhydride glycerol + maleic anhydride Reactive blending with Starch- Starch- Iodine PETG Iodine complex complex Starch- Modified Iodine complex Thermoplastic starch Nucleating agent and barrier property enhancer in PLA Active packagingActive packaging Filters Filters Antibacterial & antiviral Antibacterial & antiviral • Manufacturer – Natur-tec® • Air filtration systems additive for plastics and • Manufacturer – Natur-tec® • Air filtration systems additive for plastics and • Masks polymers Active packaging Filters • Masks polymers Antibacterial & antiviral • Manufacturer – Natur-tec® • Air filtration systems additive for plastics and Carrier for antimicrobial • Masks polymers iodine Figure 1.6: Schematic for overall summary of the work in this thesis 8 1.3 ORGANIZATION OF THE THESIS This thesis is divided into six chapters, each of which individually addresses the work that has been done in relation to the objectives outlined above. Chapter 2 deals with the existing starch foams technology and the optimization of the process variables such as screw configuration, temperature profile, die type and diameter, water content etc. It also covers the procedures for all characterization techniques used for all the foam formulations developed. It includes the different formulations developed using additives and reactive modifiers such as PVOH, PVB, chitosan, STMP, glycerol and talc. The impact of each of these on various properties of foam such as density, expansion ratio, contact angle, water sensitivity, TGA, cell size, water absorption, mechanical properties, biodegradation etc. is studied in detail. This chapter also includes the design of a new annular die and some preliminary trials done with that which form the basis for future work in this area. Figure 1.7 shows the summary of chapter 2. High-performance starch foams Reactive extrusion Polyvinyl butyral Chitosan and STMP (PVB) + Insoluble starch-chitosan polyelectrolyte complex Hydrophobic Water-insoluble Low density Water-insoluble Hydrophobic Increased Water absorbing compressive strength Medical, hemostatic pads Packaging, insulation Applications Figure 1.7: Schematic for the summary of chapter 2 9 Chapter 3 covers the synthesis and characterization of maleated thermoplastic starch (MTPS) and its use for making reactive blends with PETG. Precent grafting calculations for both MTPS and MTPS/PETG blends are performed. The changes in mechanical and thermal properties of PETG and the effect on biodegradation due to addition of MTPS for are compared and contrasted with neat PETG. Chapter 4 studies the effect of addition of MTPS in PLA as a nucleating agent and barrier property enhancer. The changes in crystallinity, crystallization kinetics and barrier properties of PLA due to addition of MTPS is studied. Additional properties such as thermal degradation temperatures, tensile modulus and strength and aqueous biodegradation of the blends is also evaluated. Figure 1.8 shows the summary for chapter 4. MTPS-PLA blends Increased crystallinity (tortuosity effect ) PLA MTPS (1-5%) feeder Die motor PLA-MTPS Films Fractional crystallinity 1 Neat PLA PLA+1% MTPS 0.5 PLA+2% MTPS PLA+5% MTPS 0 0 10 20 30 40 Without MTPS With MTPS Time (Min) (Nucleating agent) Increased crystallization rate Figure 1.8: Schematic for summary of chapter 4 10 Chapter 5 deals with the synthesis of a solid MTPS-iodine resin as an antibacterial and antiviral additive. The iodine release and antibacterial properties of these pellets were studied. Further, they were used as an additive for making blown films with PLA and PBAT. The mechanical properties, morphology and antimicrobial properties of these films were compared with other commercial PLA films with and without other antimicrobial additives. Figure 1.9 shows the schematic for summary of chapter 5. Characterization Application : Blown films, foams, antimicrobial additive in plastics -Iodine release studies -Antimicrobial activity Future work - Other applications in the field of foams and additive for other plastics and polymers Blown films Figure 1.9: Schematic for summary of chapter 5 Final chapter 6 covers various studies related to aqueous biodegradation of biobased polymers cellulose and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Mechanisms of biodegradation for these polymers and various factors affecting the rate of biodegradation are evaluated. Then detailed studies on the effect of temperature on the rate of biodegradation are performed at three different temperatures. A global equation using the reparametrized Arrhenius equation is developed and lifetime predictions for these polymers in low temperature ocean environments are made. Figure 1.10 shows the summary for chapter 6. 11 Biodegradation Kinetics and persistence of microplastics in the oceans Model for accumulation of plastics in ocean To be used as basis for developing new ASTM D7081 standard. Figure 1.10: Schematic for summary of chapter 6 12 REFERENCES 13 REFERENCES [1] “Materials – European Bioplastics e.V.” https://www.european- bioplastics.org/bioplastics/materials/ (accessed May 07, 2022). [2] R. Narayan, “Principles, drivers, and analysis of biodegradable and biobased plastics,” Handbook of Biodegradable Polymers, pp. 455–473, Jan. 2020, doi: 10.1515/9781501511967-016/PDF. [3] K. L. Law and R. Narayan, “Reducing environmental plastic pollution by designing polymer materials for managed end-of-life,” Nature Reviews Materials, 2021, doi: 10.1038/S41578-021-00382-0. [4] R. v. Gadhave et al., “Starch Based Bio-Plastics: The Future of Sustainable Packaging,” Open Journal of Polymer Chemistry, vol. 8, no. 2, pp. 21–33, May 2018, doi: 10.4236/OJPCHEM.2018.82003. [5] C. Bastioli, P. Magistrali, and S. G. Garcia, “Starch-based technology,” Handbook of Biodegradable Polymers, pp. 217–243, Jan. 2020, doi: 10.1515/9781501511967-008/PDF. [6] “The utility of starch-based plastics - Green Dot Bioplastics.” https://www.greendotbioplastics.com/starch-based-plastics/ (accessed Apr. 21, 2022). [7] “Corn and soybean production up in 2021, USDA Reports Corn and soybean stocks up from year earlier, Winter Wheat Seedings up for 2022.” https://www.nass.usda.gov/Newsroom/2022/01-12-2022.php (accessed Apr. 15, 2022). [8] G. Colomines, V. Ducruet, C. Courgneau, A. Guinault, and S. Domenek, “Barrier properties of poly(lactic acid) and its morphological changes induced by aroma compound sorption,” Polymer International, vol. 59, no. 6, p. n/a-n/a, Jun. 2010, doi: 10.1002/pi.2793. [9] “Starch,” Britannica. Encyclopædia Britannica, inc. Accessed: Mar. 01, 2020. [Online]. Available: https://www.britannica.com/science/starch. [10] F. Carosio et al., “Efficient gas and water vapor barrier properties of thin poly(lactic acid) packaging films: Functionalization with moisture resistant Nafion and clay multilayers,” Chemistry of Materials, vol. 26, no. 19, pp. 5459–5466, Oct. 2014, doi: 10.1021/cm501359e. [11] J. Muller, C. González-Martínez, and A. Chiralt, “Combination Of Poly(lactic) acid and starch for biodegradable food packaging,” Materials, vol. 10, no. 8, pp. 1–22, Aug. 2017, doi: 10.3390/ma10080952. 14 2. DESIGN AND ENGINEERING OF INSOLUBLE, HIGH-PERFORMANCE STARCH FOAMS VIA EXTRUSION TECHNOLOGY 2.1 ABSTRACT Insoluble, high-performance starch foams with high resistance to moisture were prepared by ZSK-30 twin-screw extruder using additives and reactive modifiers such as chitosan, polyvinyl butyral (PVB) and sodium trimetaphosphate (STMP). Under the optimized extrusion conditions, water acted as a plasticizer and a blowing agent breaking up the hydrogen bonds within the starch granules and releasing the starch polymer chains without significantly reducing their molecular weight. The pressure drop at the die led to expansion, and formation of closed cell foams. A screw configuration made up of 3 kneading sections was found to be the most effective for better mixing and foaming. It was found that properties like density, expansion ratio, compressive strength, resiliency, and cell size distribution of foams can be controlled by adjusting feed rates of starch, chitosan, and the crosslinking agent. The use of PVB and STMP was extremely effective in minimizing moisture sensitivity and made the foams hydrophobic and insoluble in water. Crosslinking of starch with STMP gave anionic mono and di-starch phosphates which formed an insoluble polyelectrolyte complex with cationic chitosan in presence of formic acid in the extruder due to electrostatic attraction. This made the foams water insoluble and water absorbing. These insoluble composite foams absorbed over 600% by weight water and formed a gel kind structure; a property which could be useful in hemostatic applications. It also increased the compressive strength of the foams by 3 times compared to the control foams. STMP, chitosan, and talc acted as nucleating agents with varying efficiencies. They reduced the cell size and gave more uniform cell size distribution. Densities of foams were found to vary from 15 21 to 51 kg/m3 for different compositions studied. A maximum expansion ratio of 38.7 was obtained for the formulation containing 10% PVB and 4% chitosan. Increased stability of the foams had no negative impact on the biodegradability and end of life for these products. With increasing drive to eliminate the carbon-carbon backbone persistent polymers, these biobased and biodegradable foams will provide a great value. 16 2.2 INTRODUCTION AND BACKGROUND Biodegradable and biobased plastics have received increased attention in recent years for replacing petroleum-based counterparts. Traditional polystyrene, polyethylene, polyvinyl chloride and polyurethane foams are not biodegradable or require a long time, even hundreds of years, to degrade completely. They also impact the soil, water or other environments negatively during this time. Therefore, the exploration of degradable plastics that can replace traditional plastics holds great practical significance. Currently, the foam packaging market is facing growing pressure from environmental and disposal regulations that call for better carbon management and new end-of-life initiatives. There is an urgent need to reduce the carbon footprint of the package and provide environmentally responsible end-of-life disposal alternatives. Biobased and biodegradable starch foams are well-known alternatives for polystyrene foams in packaging, insulation, and medical applications[1–4]. Starch based foams are prepared by various processes such as extrusion, baking, microwave expansion, freeze drying etc. Reactive extrusion has allowed for mass production of starch foams. It offers several advantages over other methods like fast reaction time, enhanced heat and mass transfer, better mixing and it does not require any solvents[1] Food industry has been using the extrusion process for making various modified starch products including expanded snack products for several years. Starch is not thermoplastic. It does not soften or flow. The thermal degradation temperature of starch is higher than its melting temperature. Various plasticizers such as water, glycerol, sorbitol etc. are added to make the starch processable and flow like a thermoplastic. The plasticizers such as water disrupt the intermolecular hydrogen bonding between the starch molecules and destroy its crystalline structure. Starch foam extrusion is a 2-step process. In the first step- starch, 17 additives and water are added to the extruder. In presence of high temperature, shear and pressure, water gets mixed with the starch and the starch granular structure is destroyed. Then as the mixture exits the die, a sudden drop in pressure causes water to evaporate and form the cellular structure of the foam. The foam formation can be divided into 3 steps- nucleation, bubble growth and stabilization. The nature of foam and cellular structure depends on various extrusion conditions such as screw configuration, starch type, temperature, water content, other additives like blowing or nucleating agents etc. Starch is an abundantly available polysaccharide which is nontoxic, easily biodegradable in almost any environment, inexpensive and also possesses many superior properties. However, starch foams have some shortcomings such as very low mechanical strength[5], poor moisture resistance, immediate solubility in water etc.[6]. They are sensitive to moisture, and they tend to shrink and lose their cell structure in presence of high moisture environments. Condensation of water on the surface of the foams tends to dissolve them and they lose their mechanical integrity in humid and wet conditions which also causes loss in mechanical properties. This limits the use of starch foams in certain applications involving packaging and insulation. Hence, significant research is being conducted on using various additives for overcoming these problems. This chapter focuses on the design and engineering of high-performance starch foams for addressing these issues. Three types of additives were used in this study either alone or in combination with each other and their impact on the properties of foams was studied. Polyvinyl butyraldehyde (PVB) Hydrophobic polymers like polyhydroxylamino-ether (PHAE), PLA, polybutylene adipate terephthalate (PBAT) and crosslinkers like glyoxal, glutaraldehyde, citric acid have been 18 investigated as an additive and were found to greatly improve humidity resistance of the foam[7]–[11]. However, production of PHAE was discontinued in 2002 and currently it is not available commercially. Also, one of the building blocks of PHAE is Bisphenol A (BPA), which is a suspected endocrine disrupter [12] and its use in “green” material negates the “green” nature of the product. Crosslinking agents like glyoxal are toxic and hazardous and hence cannot be used for making foams for medical and food contact purposes[13]. One approach that was used in the past to solve the moisture sensitivity was to employ starch esters instead of native starch [14, 15]. This approach, however, was proven to be economically unattractive because of cost constraints. In this work we have investigated the use of readily available polyvinyl butyral (PVB) as an environmentally friendly additive to improve the moisture resistance of starch foams. PVB is a thermoplastic obtained by condensation reaction between polyvinyl alcohol and butyraldehyde. Figure 2.1 shows the structure of PVB. It is hydrophobic, non-toxic and could be used in applications involving direct food contact.[16] Figure 2.1: Structure of polyvinyl butyraldehyde(PVB) Chitosan Another area where low density foams are widely being used is in biomedical applications[17]. Chitosan, a derivative of second most abundant polymer chitin, has unique physicochemical and biomedical properties which make it useful for hydrogels, films and 19 sponges which could be used in biomedical domain. Chitin when heated in alkaline media, the acetamide groups present on C-2 position get transformed to primary amino groups to get chitosan. This chitosan can become positively charged in weakly acidic aqueous solutions via protonation of the amine group.[18] When two oppositely charged polyelectrolytes are mixed, a polyelectrolyte complex (PEC) can form due to electrostatic attraction.[19] Figure 2.2 shows the schematic for polyelectrolyte formation. Since chitosan cationic polyelectrolyte, it can form crosslinking with anionic starch to form an insoluble polyelectrolyte complex.[17, 20] Figure 2.3 shows the possible scheme for formation of polyelectrolyte complex between starch and chitosan. Figure 2.2: Schematic for polyelectrolyte complex formation + Insoluble starch-chitosan polyelectrolyte complex Figure 2.3: Scheme for formation of polyelectrolyte complex between chitosan and starch 20 Chitosan in foam is expected to promote the blood coagulation and thrombosis, inhibit the growth of bacteria and fungi and promote repair of damaged tissues[21]. Therefore, this foam is expected to have good hemostatic properties and find applications in medical field in the form of surgical pads. Studies have reported such polyelectrolyte foams made using microwave expansion.[17,22]. Dang et al. also reported that, when blended with starch, chitosan migrates to the surface of the films and decreases the hydrophilicity of the materials[23]. This could be an additional advantage for improving the moisture sensitivity of starch foams. Sodium trimetaphosphate (STMP) One approach for improving the mechanical and performance properties of starch foams is crosslinking. Starch has abundant hydrophilic hydroxyl groups, and therefore composites made from it have poor moisture barrier properties[24]. The hydrophilicity of starch can be substantially decreased by blocking some of its hydroxyl groups by crosslinking. Citric acid[25], glutaraldehyde[26], and malonic acid [27] have been investigated for the crosslinking of hydroxyl groups of starch and cellulose. STMP is widely used as phosphorylating agent in food and construction industries[28]–[30]. Starch reacts with sodium trimetaphosphate to give mono and distarch phosphates as shown in Figure 2.4 – Figure 2.4: Phosphorylation of starch with STMP Phosphate crosslinked starches improve the starch stability against high temperature, pH and shear. It also improves the firmness of swollen starch granule and its textural 21 characteristics. Plus, phosphorylated anionic starch has a better tendency to form polyelectrolyte complex with cationic chitosan compared to native starch. To our best knowledge, no studies have reported the synergistic use of PVB and STMP for making high performance starch foams with chitosan by formation of polyelectrolyte complexes. Another approach for making the insoluble polyelectrolyte complexes used in this project was using potato starch instead of High amylose corn starch. Potato starch has high amylopectin content (~70%) and it also has a high amount of phosphates present naturally[31] (0.01-0.6% by wt.) compared to corn starch[32] (0.016%). Hence, these foams do not need an external addition of STMP to form polyelectrolyte complexes with chitosan. Such foams have been prepared using microwave expansion technique by Deng et al.[17] Efforts were also done to scale up those foams using extrusion technology as a part of a collaboration work between MSU and Penn State University. Extrusion technique has allowed mass production of starch foams.[9, 11, 33, 34] It offers several advantages over other methods like fast reaction time, enhanced heat and mass transfer, better mixing and it does not require any solvents [35]. In this study, water insoluble starch-chitosan polyelectrolyte complexes with and without crosslinking agent were prepared in the form of foams using the extrusion technology. Use of PVB as an additive for making the foams hydrophobic and water insoluble was also investigated. Extrusion parameters like screw configuration, temperature profile, rpm and feed ratios were optimized. Two types of starches – high amylose corn starch and potato starch, were used for making foams with different properties. Physico-mechanical properties of the foams including density, expansion ratio and compressive strength were investigated and 22 compared with the commercial starch foams. Properties like moisture sensitivity, contact angle and water absorption were also reported for the various formulations studied. Finally, the end of life of these foams in aqueous environment was investigated. These foams could find potential applications in various fields such as in making hemostatic pads in the field of wound care, in packaging or insulation require long term moisture resistance and mechanical properties. 23 2.3 MATERIALS High amylose corn starch was obtained from National starch and chemicals (Indianapolis, IN) with equilibrium moisture content of 12% (w/w). Native potato starch was obtained from Ingredion (NJ, USA) under the commercial name of PenPure®10 with equilibrium moisture content of about 12% (w/w). Water was used as both plasticizer and blowing agent. Chitosan was obtained from Primex EHF, Iceland under the trade name ChitoClear® 42010-cg110 75cp. It was obtained from Fresh North Atlantic Shrimp Shells, Pandalus borealis as raw material source. Chitosan was used to make insoluble polyelectrolyte complex foams with starch. The degree of deacetylation for chitosan was >75%. Sodium trimetaphosphate (STMP) was used for phosphorylation and light crosslinking of starch for its efficient reaction with chitosan. It was purchased from Fischer scientific (Pittsburgh, PA) and was used as it is. Polyvinyl alcohol (PVOH) was used as an additive to make control foams and was supplied from Kuraray America, Inc. under the trade name Mowiol 40-88. Polyvinyl butyral (PVB) was supplied by Kuraray America, Inc. (Houston, TX) under the trade name PVB 60HH. Other additives like polyvinyl alcohol (PVOH) (Kuraray America, Inc. (Houston, TX) and talc (magnesium silicate) used as nucleating agent was obtained from Luzenac (Ontario, Canada). These additives were used to prepare the control foams. Formic acid was used in some formulations to make chitosan cationic and increase the efficiency of polyelectrolyte formation. It was obtained from Fisher scientific (Pittsburgh, PA). 24 2.4 EXPERIMENTAL The laboratory starch foam extrusion was done with a co-rotating twin screw extruder (Century ZSK-30)(MI, USA) with an L/D of 42:1 (Figure 2.5) . A peristaltic pump (model C.P. 78017-10, Ismatec) was used for injecting water into the extruder and accurate single-screw feeders were used for feeding starch and additives. A screw speed of 180-200 rpm was used for laboratory experiments for foam noodle extrusion. Three different types of dies were used for various formulations. The strand dies used were 3.5, 4.3 and 8 mm in diameter. A Slit die and annular die were also used for making the foams in form of sheets as explained in later sections of this chapter. Both peristaltic pump and single screw feeders were calibrated for different feed rates of water and starch. Initially during start-up, the starch feed rate was kept lower (50-60%) than the final feed rate and water feed rate was kept at a higher level than the final water feed rate (about 20-25% of starch feed rate). Once the mixture starts coming out from the die, starch feed rate is increased, and water feed rate is decreased gradually till a point where the product starts foaming. Starch feed rate used for most of the runs was 9 kg/ h and it was observed that water flow rate of 6-8% of starch was optimum for consistent foam production. The starch used was not dried and hence it had 12% of equilibrium moisture content. Thus, the total water content was 18-20% on dry basis. A 1% v/v formic acid aqueous solution was used in some formulations instead of distilled water. Formic acid was added for protonation of amino groups on the backbone of chitosan in acidic medium which could form PEC with anionic starch molecules. Table 2.1 shows all the runs with various additives used for foam extrusion and their contents. 25 Typical Processing conditions Starch + chitosan + Water other additives 200 rpm feeder motor 40 60 80 90 100 110 120 130 140 140 Foam Die Temperature 0C Co-rotating twin screw CENTURY ZSK-30 extruder. Figure 2.5: Experimental setup for foam extrusion 26 Table 2.1: Extrusion runs with various additives For die mul Water or talc chitosa PVOH PVB STMP Glyce Othe die Starch type diameter atio formic acid (%) n (%) (%) (%) (%) rol r type (mm) n# Variation in talc content High amylose Stran 0 - - - - - - 1 corn starch Water d 4.3 High amylose Stran - - - - - - 2 corn starch Water 0.5 d 4.3 High amylose Stran - - - - - - 3 corn starch Water 0.7 d 4.3 High amylose Stran - - - - - - 4 corn starch water 2 d 4.3 Using PVOH and chitosan High amylose Stran - - - - - - 5 corn starch Water 10 d 4.3 High amylose Stran - 4 - - - - - 6 corn starch Water d 4.3 Variation in die diameter High amylose Stran - - - - - 7 corn starch Water 4 10 d 4.3 High amylose Stran - - - - - 8 corn starch Water 4 10 d 3.5 High amylose Stran - - 9 corn starch Water - 4 10 - - d 8 Using PVB and Variation in chitosan content High amylose Stran - 0 - - - - 11 corn starch Water 10 d 4.3 27 Table 2.1cont’d High amylose Stran - - - - - 12 corn starch Water 4 10 d 4.3 High amylose Stran - - - - - 13 corn starch Water 7 10 d 4.3 High amylose Stran - - - - - 14 corn starch Water 10 10 d 4.3 Variation in STMP content High amylose Stran - - - - 15 corn starch Water 4 10 1 d 4.3 High amylose Stran - - - - 16 corn starch Water 4 10 1.5 d 4.3 High amylose Stran - - - - 17 corn starch Water 4 10 3 d 4.3 Use of formic acid and glycerol High amylose - - - - - 18 corn starch Formic acid 4 1.5 Slit 25*1.5 High amylose - - - - 19 corn starch Formic acid 4 1.5 5 Slit 25*1.5 High amylose - - - - 20 corn starch Formic acid 4 1.5 10 Slit 25*1.5 Use of annular die High amylose Annul - - - - 1 corn starch Water 4 10 1.5 ar die Potato starch foams Potato starch - - - - - 22 (Ingredion) Formic acid 4 - Slit 25*1.5 Potato starch - - - - - 23 (Ingredion) Formic acid 4 10 slit 25*1.5 Potato starch MCC - - - - 24 (Ingredion) Formic acid 4 10 2% Slit 25*1.5 28 2.5 FOAM EXTRUSION PROCESS DEVELOPMENT First step was optimizing the various parameters for extrusion process to achieve a consistent, uniform process for foam production. The main parameters varied in this study include – screw configuration, temperature, water content, feeding ratios, screw speed, and die type and diameter. Screw Configuration Screw configuration plays an important role in the foaming process. During foaming, the crystalline structure of starch granule is disrupted using water as plasticizer. Water and additives also need to be mixed together properly to form a single phase for production of consistent quality foams. Hence the screw configuration was designed specifically to achieve these objectives. Two different screw configurations were used for production of starch foams as shown in the Table 2.2. The first configuration was mostly conveying elements and thus had less shear. It did not mix the starch, additives and water efficiently and hence the foams produced were not consistent. They also showed presence of some solid blocks in between the foam strands which indicated inadequate mixing as shown in Figure 2.6-a. Thus, the first screw configuration was not much of a success. However, it did help in forming a baseline for different parameters of extrusion. In the second screw configuration, more kneading elements were added to the screw to improve mixing and increase the torque. The feed zone contained the largest single pitch screw elements to convey the starch rapidly and avoid buildup of material in the feed zone. The pitch of the screw was then reduced to force the materials downstream. Kneading elements were introduced after regular intervals to provide efficient mixing of raw materials. This second screw configuration with more kneading elements proved to be more efficient in terms of mixing and torque and was used 29 for all further experiments. The foams produced by second configuration were much more consistent in production and had uniform structure and surface properties (Figure 2.6-b). a) b) Figure 2.6: Foams obtained from a) Screw configuration #1 b) Screw configuration #2 Table 2.2: Screw configurations for starch foam extrusion Screw Screw # config #1 config #2 1 28/14 28/14 2 60/60 60/60 3 60/60 60/60 4 60/60 60/60 5 42/42 42/42 6 42/42 28/28 28/28 28/28 7 20/20 20/20 8 20/20 20/20 9 20/20 KB 45/5/14 10 11 20/20 KB 45/5/14 12 KB 45/5/28 KB 45/5/14 13 KB 90/5/28 KB 45/5/20 14 KB 90/5/28 KB 45/5/20 30 Table 2.2 cont’d 15 KB 45/5/14 60/60 KB 45/5/14 42/42 16 28/28 28/28 17 28/28 28/28 18 28/28 20/20 19 20 20/20 KB 45/5/42 21 20/20 KB 45/5/42 22 20/20 KB 45/5/42 23 20/20 KB 45/5/42 24 20/20 60/60 20/20 42/42 25 20/20 28/28 26 KB 45/5/14 28/28 27 KB 45/5/14 20/20 28 29 KB 90/5/28 KB 45/5/14 30 KB 90/5/28 KB 45/5/14 31 28/28 KB 90/5/28 32 28/28 KB 90/5/28 33 20/20 60/60 34 42/42 42/42 35 42/42 42/42 42/42 28/28 36 37 42/42 28/28 38 42/42 28/14 39 28/28 20/20 40 28/28 KB 90/5/28 41 20/20 20/20 42 20/20 43 20/20 31 Figure 2.7: Second Screw configuration used for foam extrusion Temperature profile Gelatinization is a process of breaking down the intermolecular bonds of starch molecules in presence of water and heat. High amylose corn starch gelatinizes at higher temperatures above 90-100°C. Hence, efforts were done to keep the initial zones at lower temperatures and the last 4 or 5 zones at temperatures higher than the gelatinization temperatures of starch. This allowed the gelatinization and cross-linking with chitosan to occur in the end zones just before foaming. For other additives like PVOH and PVB, the temperatures were adjusted according to their melting temperatures. But most of the times the temperature profile given in Table 2.3 worked well for all the formulations with minor adjustments. Table 2.3 shows the optimized temperature profile used for extrusion. Sometimes, the initial 2 heat zone temperatures were increased to 80°C to get the screws moving properly. This was due to the LDPE purge material used in the extruder. A higher temperature was required to soften the LDPE present in the extruder and get the screws moving. Efforts were made to keep the temperature of the initial zones below 90°C to avoid the evaporation and loss of water from the feeding zone. Table 2.3: General temperature profile for extrusion Zone 1 2 3 4 5 6 7 8 9 die Temperature (°C) 40 60 80 90 100 110 120 130 140 140 32 Die type and diameter Strand die The density and expansion ratio of the foams depends on the pressure developed the end of the extruder. Apart from the screw configuration and screw speed, one more factor that affects the pressure at the end of the extruder is die type and diameter. Smaller the die diameter, larger is the pressure developed at the die face. This causes more extreme drop in the pressure and the expansion ratio increases. Hence, the effect of die diameter on density and expansion ratio of the foams was studied with 3 strand dies of 3.5 mm, 4.3 mm and 8 mm diameter. The foam formulation contained high amylose corn starch, 10 wt% PVOH and 4 wt % chitosan. The figure below shows the dies, the foams produced form them and their respective expansion ratios. Expansion ratios for strand die foams are calculated as the ratio of cross section area of the foam vs cross section area of the die. As expected, it was observed that the smallest die yielded foams with lowest density and highest expansion ratio. Density increased and ER decreased on increasing the die diameter. But the smallest strand die with 3.5 mm diameter was also prone to causing more blockages at the die face. The small opening of the die was causing it to get plugged with starch more often making the runs done more unstable. The runs with 4.3 mm die were more consistent and yielded a uniform product. Hence, 4.3 mm die was used on most of the runs after that. The largest strand die yielded foams with highest density and lowest ER. So, it was not used further. 33 Die diameter : 3.5 mm 4.3 mm 8 mm Expansion ratio: 11.4 ± 1.1 15.1 ± 1.2 6.5 ± 0.2 Figure 2.8: Three strand dies used for extrusion and their corresponding expansion ratios Slit Die The shape of the foams is also determined from the type of the die used. Second part of this work was to develop starch foam sheets for using as hemostatic pads. For this purpose, slit die and annular die were used for those formulations. The foams obtained from slit die were not completely flat. They showed presence of a ridge in the middle due to the rheology of the flow material coming out from the die. Efforts were done to flatten these foams by immediately passing them through a roller press when they were hot. Figure 2.9 shows the slit die foams with ridges and after they were flattened with the press. The details for design and use of annular die are covered in chapter 3. 34 a) Slit die b) Foam sheets with ridges c) Roll press for pressing foam d) Pressed foam sheets noodles and sheets Figure 2.9: Process development with slit die Optimized water content of 6-8%, starch feeding rate of 150g/min and screw speed of 180- 200 rpm was used for all the experiments. These values were based on the previous research done in our group on starch foams [8, 9, 11]. These parameters were qualitatively adjusted to give optimum properties, but detailed quantitative studies were not done. 35 2.6 CHARACTERIZATION AND ANALYSIS All the samples were conditioned for at least 72 hours at 50% relative humidity and 23°C as per ASTM D4332 before performing any testing. Density: The density of the foams was calculated from the mass to volume ratio of the samples according to ASTM D3575. The dimensions of the sample were measured using a Vernier caliper graduated to an accuracy of ± 0.01 mm. For the cylindrical foams (noodle), samples were collected at various time intervals from each run and 10 measurements were taken for each sample to determine an average diameter and density. Expansion ratio: The expansion ratio of the cylindrical foam was calculated by dividing the cross-sectional area of the foam by the cross-sectional area of the die in mm2. The result was the mean of ten samples of each formulation. Water solubility test: 0.5 g of starch foam was kept in water and stirred. Pictures were taken at various times like 1min, 5 min, 30 min, 3 hours and after 2 days to see whether the foams dissolved in water or not. Surface wetting test: Foam sheets or noodles were dipped into water, removed and left undisturbed for five hours. After five hours, photographs were taken of the foam sheets to determine the degree of disintegration. Solution uptake measurements: For the samples which did not dissolve readily in water, solution uptake measurements were performed. Swelling experiments were performed using the method described by Deng et al. (2014). Swelling ratio (S) was calculated for the samples in DI water at 25ºC. Approximately 0.5 g of samples were cut and immersed in DI water for predefined time intervals. After that, the swollen samples were taken out and 36 weighed after excess liquid on the sample surfaces was absorbed by paper towels. This was continued till a set time interval of 120 h was reached. Swelling ratio is calculated as: Equation 2.1 𝑀𝑠 − 𝑀𝑑 𝑆% = × 100 𝑀𝑑 Where Ms is the weight of swollen sample and Md is the weight of dry sample before immersing in water. Moisture sorption analysis: 3 samples of each foam formulation were kept in 95% relative humidity chamber at 25oC. Weight and dimensions of the samples were monitored and measured at regular intervals until a steady state value was achieved. Scanning electron microscopy (SEM): A JOEL 6610 LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) was used examine cell size distributions and surface morphology of all the samples. Foam samples were sectioned with a razor blade, mounted on aluminum stubs, and coated with sputter coater before observing in scanning electron microscope. ImageJ software was used to determine the average cell size and cell size distribution for the foams. Absorption isotherms: For studying moisture adsorption isotherms of starch foams under different relative humidity conditions, 6 different saturated salt solutions were prepared and placed at the bottom of desiccators to obtain the relative humidity ranging from 11 to 95 %. The starch foams were dried in an oven at 90 C for 2 days, weighed and then placed in the closed desiccators for a definite amount of time. Change in the moisture content of the starch foams as a function of time for different foam samples were reported to create the adsorption isotherms. Table 2.4 shows the salt solutions used for creating different humidity environments. 37 Table 2.4: Different salt solutions for relative humidity Salt RH Lithium chloride 11 % Magnesium chloride 33 % Magnesium nitrate 52 % Sodium chloride 75 % Potassium Nitrate 95 % Confocal microscopy: The chitosan containing foam samples were visualized using Fluoview FV1000 CLSM inverted type microscope (Olympus, Japan). A fluorescein isothiocyanate (FITC)/water solution (50mg/ml) was prepared and the starch foam samples were soaked in it for about 1 minute. Then the samples were taken out, washed with distilled water to remove any excess dye and were plated on glass slides for observation. A UPLFLN objective with 10X magnification and numerical aperture of 0.3 was used for capturing the images. The chitosan was labelled with FTIC for getting green fluorescence [23]. Excitation at 488 nm was provided using argon lasers. Emission filter SDM560 and BF505-525 were used for collecting green fluorescence in channel 1. Then a Z series was collected over the thickness of 388 um with z step size of 6.93 microns. Maximum intensity projection image (MIP) was generated and saved using Olympus FLUOVIEW 4.2 software. Kalman average of 4 images was used for reducing the background noise in the image. The foam samples were analyzed for 2 positions in the sample – surface and middle part of the foam. Contact angle measurements: Contact angle measurements were carried out using a contact angle goniometer DSA30S (Krüss GmbH, Germany) at 25°C. Water droplets were dropped carefully on the surface of the foams with a micro syringe and contact angles were measured with the help of Advance software (Krüss GmBH, Germany). An average of 4 to 5 different measurements were taken for a single sample at different positions. The measured values 38 were reported 30 sec after the deposition of water droplets. Change in the contact angle after 1 min of deposition was also observed. Compressive strength and resiliency: Compressive strength of the foam noodle specimens was measured according to the test method described by Tatarka et al.[36]. using an Instron testing machine. The cylindrical foam samples were placed between the compression plates and an initial load of 0.5 N was applied. Then the samples were pressed at a rate of 19 mm/min for a deformation of 13% or yield point, whichever occurs first. The maximum compression load was recorded. Compressive strength was recorded by dividing the maximum compression load by cross section area. Then the plate was returned to its original position and a second relaxation load was measured after 60 sec. Percent resilience was calculated as the force required for the second compression divided by the first. All the readings were obtained as an average of 5-7 samples for each formulation. Biodegradability: The biodegradability of 3 foam samples (formulation 7, 12 and 16) was tested in an aqueous environment. These foams contained 1. PVOH and chitosan, 2. PVB and chitosan 3. PVB, STMP and chitosan. All the tests were performed in an aerobic environment at 30 °C. A respirometric mineralization test system for calculating CO2 evolution was set up based on International Standard ISO 14852. The system comprised blank, positive reference (cellulose) and the test materials for all the runs. All the samples, blanks, and references were run in duplicates. An optimized test medium containing all the nutrients and buffers was prepared according to the ISO standard. Table 2.5 gives the detailed composition of the mineral solution prepared for all the tests. 39 Table 2.5: Mineral solution composition for the test 1 L Mineral Solution g Solution A KH2PO4 (anhydrous) 3.75 Na2HPO4·2H2O 8.73 NH4Cl 0.2 Solution B MgSO4·7H2O 2.25 Solution C CaCl2·2H2O 3.64 Solution D FeCl3·6H2O 0.025 g Wastewater inoculum (ml) 50 Distilled water (ml) Remaining Wastewater inoculum obtained from a local waste water treatment plant was added to all the flasks to obtain the concentration of 5% v/v in the test medium as described in ISO 14852. Then the foam samples were added to these flasks, and they were subjected to the test conditions. A solution of 1 N NaOH was used for trapping the CO2 generated from test flasks. CO2 trapping is a two-step process as shown below: NaOH + CO2 → NaHCO3 NaHCO3 + NaOH → Na2CO3 1 g of sample was taken from each of the 50 mL NaOH trapping solution and titrated with 0.1 N standardized hydrochloric acid (HCl) solution to find the amount of CO2 trapped. The reactions are as follows: 1st end point: NaOH + HCl → NaCl + H2O Na2CO3 + HCl → NaHCO3 + NaCl 2nd end point: NaHCO3 + HCl → NaCl + H2O + CO2 40 The titrations were done with the help of an auto titrator to get the volumes of HCl, V1, and V2 required for reactions 1 and 2 respectively. The amount of HCl consumed can be used to calculate the mmoles of CO2 evolved using the following Equation: Equation 2.2 (𝑉2 − 𝑉1 ) ∗ 𝑁𝐻𝑐𝑙 ∗ 𝑉𝑁𝑎𝑂𝐻 𝑀𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝐶𝑂2 = 𝑊𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 The percentage biodegradation (% B) was further calculated by the following Equation: Equation 2.3 ∑(𝐶𝑂2 )𝑠𝑎𝑚𝑝𝑙𝑒 − ∑(𝐶𝑂2 )𝑏𝑙𝑎𝑛𝑘 %𝐵 = × 100 𝑇ℎ𝐶𝑂2 ∑(𝑪𝑶𝟐 )𝒔𝒂𝒎𝒑𝒍𝒆 is the amount of carbon dioxide that evolved in a test flask between the start of the test and time t; ∑(𝑪𝑶𝟐 )𝒃𝒍𝒂𝒏𝒌 is the amount of carbon dioxide that evolved in a blank flask between the start of the test and time t; ThCO2 is the theoretical amount of carbon dioxide that evolved from the test material. All the values were expressed as mmoles of CO2. The samples were replaced every 2–3 days in the starting phase when the rate of biodegradation was expected to be maximum and weekly or biweekly in the end [43,44]. Plots of cumulative CO2 evolution for all the samples and blanks and % biodegradation vs. time were made for all the samples and compared for any differences between the foam samples. 41 2.7 RESULTS AND DISCUSSION Effects of various additives on density and expansion ratio Talc Figure 2.10 shows the effects of talc and PVOH on density and expansion ratios of starch foams. While optimizing the processing conditions of extrusion for making starch foams talc was used as an additive. It was observed that the density of starch foams increased, and the expansion ratio reduced with increasing talc content from 0-2%. The control foams containing only starch and water showed density of 40.7 ± 8.9 kg/m3 and expansion ratio of 24.2 ± 4.2. This was higher as compared to the values reported by Nabar et. al[9]. Control starch foams without any other additives were highly inconsistent in nature and very brittle which could explain the higher standard deviation in the measurements. Increasing talc content to 0.7 and 2% yielded increased densities of 42.3 ± 7.3 kg/m3 and 49.7 ± 5.4 kg/m3 and expansion ratios of 17 and 17.4 respectively. Similar nucleating effect of talc on density and ER has been observed in many studies[39][8]. Besides increasing density and expansion ratio, talc addition also helped in increasing the formation of large number of smaller cells. And the diameter of the cells reduced. These results were quantified further in the SEM studies as explained in section 2.7.7. 42 60 50 Density (kg/m3) 40 30 Expansion Ratio 20 10 0 0% talc 0.7 % talc 2% talc PVOH Density Expansion ratio Figure 2.10: Effect of talc and PVOH addition on density and expansion ratio of foams Polyvinyl alcohol It was found that by adding PVOH and chitosan to the formulation, the density was reduced (39.4 ± 4.7 kg/m3) but the expansion ratio remained almost the same (16.7 ± 1.0). Addition of PVOH made the foams much more consistent, less brittle and made the surface smoother. This could be attributed to the fact that polyvinyl alcohol is a high molecular weight molecule and highly soluble in water. So, addition of PVOH reduced the diffusivity of water and led to a more controlled expansion and hence lower expansion ratios [8]. Polyvinyl butaral (PVB) Next additive used was PVB. Polyvinyl butyraldehyde is a resin used in automotive applications for safety shields, in adhesives, for increasing flexibility and toughness. It is prepared by reaction between polyvinyl alcohol and butyraldehyde. Studies have reported that use of polyvinyl butyraldehyde (PVB) increased the water barrier properties of paper boards and starch films [40, 41]. Hence it was added to the starch foams to study its effect on water solubility and moisture resistance. It was observed that PVB also acted as a blowing 43 agent and increased the expansion ratio and decreased the density of starch foams significantly to 38.7 ± 2.1 and 21.1 ± 1.7 kg/m3 respectively. This might be due to the hydrolysis of PVB in presence of water to yield butaraldehyde which evaporates to form gas and causes more expansion of foams[42]. Chitosan The effect of chitosan % on density and expansion ration of foams was also studied. Density of foams was found to increase from 21.1 to 36.2 kg/m3 by increasing the chitosan content from 4 to 10 % whereas expansion ratio reduced from 38.75 to 26.05 as shown in Figure 2.11. Addition of chitosan showed a similar nucleating effect as talc. The detailed studies on cell size and cell size distribution are covered in in the SEM section 2.7.7. 45 40 35 Density (kg/m3) 30 25 Expansion Ratio 20 15 10 5 0 4% chitosan, 10% 7 % chitosan, 10% chitosan, PVB 10% PVB 10% PVB Density Expansion ratio Figure 2.11: Effect of chitosan on density and expansion ratio of foams Sodium trimetaphosphate STMP was added to the foams as a crosslinking agent to form mono and di-starch phosphates which can then form a polyelectrolyte complex with cationic chitosan as shown in Figure 44 2.3[17]. Crosslinking of starch is also known to improve the mechanical properties and water resistance of the starch which are important characteristics for some applications like packaging and high humidity applications. 1, 1.5 and 3 % of STMP was added to the formulations and it was found that the density increased, and expansion ratio decreased steadily with increasing degree of crosslinking as expected (Figure 2.12). STMP acted as a crosslinker for starch increasing its molecular weight and making it stiffer restricting the expansion due to loss of flexibility. 60 50 Density (kg/m3) 40 30 Expansion Ratio 20 10 0 0% STMP 1% STMP 1.5% STMP 3% STMP Density Expansion ratio Figure 2.12: Effect of STMP addition on density and expansion ratio of starch foams 45 Water solubility tests Corn starch foams, potato starch foams and control foams with PVOH and talc additives were tested for their water solubility. Control foams and PVOH containing foams dissolved almost immediately in water within a few minutes. When chitosan was added to the foams, a gelling effect was observed. For 4% chitosan containing foams with corn starch, they did not dissolve completely even after a day. Instead, they formed a gel kind of structure as shown in Figure 2.13. This suggested that a polyelectrolyte complex was starting to form. However, for efficient formation of polyelectrolyte complex, it is necessary that chitosan should be positively charged, and starch should have a greater number of anionic phosphate groups which can electrostatically get attracted to the positive chitosan. Formic acid and STMP are necessary for this to happen. These were not added in corn starch + chitosan 4% formulation (#6 form Table 2.1). Hence, the polyelectrolyte formation was not complete and efficient, and these foams did not hold their shape well. When formic acid solution was used for making the foams along with addition of 1.5 % STMP (formulation #18 Table 2.1) it was in fact seen that the foams did not dissolve in water even after 5 days, they held their shape in water and absorbed water as shown in Figure 2.13. This confirmed that addition of formic acid and STMP resulted in better electrolyte complex formation. However, these foams started to lose their shape after day 5 and were completely dissolved in water by day 30 (images not included). This demonstrated that the interaction between starch and chitosan and formic acid can keep the morphological structure of foams intact for a few days making them more stable as compared to control foams. But this interaction is reversible and effective only for limited time. Similar results were observed by Zhang et. al 2020[43], when they studied starch chitosan foams for solubility in water and other pH solutions. 46 Corn starch + water Corn starch + talc 2% Corn starch + PVOH 10% T=0 hr T=1 hr T=0 min T=30 min T=0 hr T=1 hr 20 min Dissolved within 1 hour Corn starch + Chitosan 4% Corn starch + Chitosan 4%+ STMP 1.5% + formic acid T=0 hr T=1 hr T=0 T= 5 days Did not dissolve; started forming gels Figure 2.13: Water solubility for foams with different additives Once the parameters for insoluble foams were optimized, the effect of glycerol addition on the water solubility was also studied (Figure 2.14). The foams containing 5 and 10% of glycerol were more flexible compared to the foams with no glycerol. These foams also contained 1.5% wt./wt. STMP. So, it was expected that they should form electrolyte complexes. This was confirmed when they did not dissolve in water for a day. But it was observed that the glycerol containing foams did start to break a little after a day in water. And finally, they dissolved after 5 days. Addition of glycerol made the corn starch foams more flexible but more soluble in water. This could be attributed to the hydrophilic nature of glycerol. 47 Corn starch + Chitosan 4%+ STMP 1.5% Corn starch + Chitosan 4%+ STMP 1.5% Corn starch + Chitosan 4%+ STMP 1.5% + formic acid (0 % glycerol) + formic acid (5 % glycerol) + formic acid (10 % glycerol) T=0 T= 5 days T=0 T= 1 day T=0 T= 1 day Dissolved after 5 days Figure 2.14: Solubility of corn starch foams with glycerol Similar studies were also done with potato starch foams. It was observed that all the potato starch foams were insoluble in water even without the addition of STMP. This might be due to the fact that native potato starch contains more number phosphates groups naturally compared to corn starch[32]. Hence, addition of STMP was not necessary for formation of polyelectrolyte complex with chitosan. Surprisingly, opposite results were observed in case of these foams with glycerol addition. Both the 0% and 10 % glycerol foams were insoluble in water. But the 10% glycerol foams (formulation 23-Table 2.1) maintained the structure better as compared to 0% glycerol foams (formulation 22). The reason for this observation was not clear. It might be that there was some processing issue encountered during production of these foams due to which the polyelectrolyte complex was not formed properly in the 0% glycerol foams. In case of potato starch foams one more formulation was also made with addition of microcrystalline cellulose (MCC)(formulation #24-Table 2.1). This was done to see if cellulose acts as a biobased nucleating agent in foams as well and make the cell size smaller and more uniform. MCC has been used in polymers like PLA as a nucleating agent[44]. However, in case of water solubility studies, it was found that the MCC containing foams were much more brittle compared to other potato starch foams and the foams could not hold their shape. These foams dissolved in water completely after a few hours. 48 Potato starch + Chitosan 4% + Potato starch + Chitosan 4% + Potato starch + Chitosan 4% + formic formic acid (0 % glycerol) formic acid (10 % glycerol) acid + 2% MCC (10 % glycerol) T=0 hr T= 2 hr T=0 hr T= 2 hr T=0 hr T= 2 hr Formed gel but did not maintain Formed gel and maintained Formed gel but did not maintain structure structure better structure Figure 2.15: Solubility studies for potato starch foams Another additive which had a significant impact on foam solubility in water was PVB. It was observed that the foams containing only PVB or chitosan and PVB or chitosan and PVB and STMP did not dissolve in water as shown in Figure 2.16 . This could be due to the hydrophobic nature and film forming and binding properties of PVB. PVB helped in binding the foam structure together and hence foams did not dissolve in water. However, without STMP crosslinking, the foams containing only PVB could not maintain their cell structure for long after immersing in water. Crosslinking of starch with STMP and its polyelectrolyte complex with chitosan, made the foams more rigid and cell structure strong. So, these foams did not lose their cell structure in water (formulation 15-17 -Table 2.1). Instead, they absorbed water and formed a gel kind of structure without getting dissolved in the water. This could be an important property for making the use of these foams in making absorbent, hemostatic pads. 49 Corn starch + 10 % PVB Corn starch + 10 % PVB + 4% chitosan Corn starch + 10 % PVB + 4% chitosan + 1.5% STMP T= 0 T= 2 days T= 0 T= 2 days T= 0 T= 2 days Did not dissolve and hold shape after 5 days Figure 2.16: Water solubility studies for foams containing PVB In summary – the formulations containing chitosan, PVB and STMP showed a synergistic effect (formulation 15 to 17-Table 2.1) and the best water insolubility characteristics while maintaining their shape. 50 Water penetration test As a qualitative test for water penetration, starch foam sheets or noodles were dipped in water, taken out and kept undisturbed for 5 hrs. Photographs were taken before and after to see the effect of water on the sheets and noodles. It was found that the control foams with no additives and foams with talc and PVOH as additive were affected the most by dipping in water. They disintegrated and shrunk the most. Whereas, addition of chitosan, PVB and STMP seemed to increase the water penetration time for the sheets and noodles. There was not much disintegration or shrinkage observed in these foams (Figure 2.17) Before Starch + talc + Starch Starch+ Starch+ PVB Starch +water water +chitosan PVOH+chitosan +water +water +water After 5 hours Starch + talc + Starch Starch+ Starch+ PVB Starch +water water +chitosan PVOH+chitosan +water +water +water Figure 2.17: Water penetration test for foams with various additives Figure 2.18 shows all the foams with varying content of PVB and/or chitosan with or without STMP. All of these foams showed minimum surface disintegration upon contact with water. This was in agreement with the previous studies of water solubility discussed in section 2.8. 51 Before 10% PVB, 10% PVB, 10% PVB, 10% PVB, 10% PVB, 10% PVB, 4% chitosan 7% chitosan 10% 4% chitosan, 4% chitosan, 4% chitosan, chitosan 1% STMP 1.5% STMP 3% STMP After 5 hours 10% PVB, 10% PVB, 10% PVB, 10% PVB, 10% PVB, 10% PVB, 4% chitosan 7% chitosan 10% 4% chitosan, 4% chitosan, 4% chitosan, chitosan 1% STMP 1.5% STMP 3% STMP Figure 2.18: water penetration test for foams containing PVB, chitosan and STMP Similar studies were also done with corn and potato starch foams with varying content of glycerol (Figure 2.19). It was observed that the foams with 0% glycerol showed least surface disintegration after contact with water. The foam surface became much more hydrophilic and sensitive to water after addition of hydrophilic glycerol. It was also interesting to note that addition of MCC which made the potato starch foams brittle and more soluble in water, also made the foam surface more sensitive to water. These foams were almost disintegrated completely after 5 hours. This made them a poor candidate for use in the intended application of moisture resistant foams. 52 Before Corn starch, Corn Starch, Corn starch, Potato st, Potato st, Potato st, 0 % glycerol 5% glycerol 10% glycerol 0% glycerol 10% glycerol 10% glycerol, 2% MCC After 5 hours Corn starch, Corn Starch, Corn starch, Potato st, Potato st, Potato st, 0 % glycerol 5% glycerol 10% glycerol 0% glycerol 10% glycerol 10% glycerol, 2% MCC Figure 2.19: Water penetration test for foams with and without glycerol 53 Solution uptake measurements for foams The foams which did not dissolve in water for more than 1 day and formed insoluble polyelectrolyte complexes, were tested for solution uptake measurements. These included the corn starch formulations containing STMP and PVB and STMP (formulation #15-20) and foams containing glycerol. Figure 2.20 shows the swelling ratios for all the corn starch formulations. The solution uptake by all the corn starch foams with PBV and STMP was similar regardless of STMP content. Equilibrium solution uptake ratio of about 600% was reached in 80-100 hours. Theses foams maintained the structure well and formed gels after absorbing water. There was little to no disintegration observed for these foams even after 120 hours. For foams containing glycerol (0, 5 and 10%) and 1.5 % STMP, it was observed that the addition of glycerol made these foams more hydrophilic and soluble in water. Their initial solution uptake was faster than the PVB containing foams to reach between 450-500% by weight. After that they started disintegrating in water and that caused mass loss for the foams and reduction in the swelling ratios. This testing was not continued after 72 hours due to continued mass loss in these foams. Hence, it could be concluded that the binding abilities of PVB were helping these foams in 1st case to retain their shape and structure together properly. In absence of PVB, the polyelectrolyte complex between starch and chitosan was more unstable and lead to loss of structure in water more easily. These studies were only performed in distilled water in this project. As a part of the collaboration project with Penn state university, these foams were further tested in different buffer solutions for their solution uptake characteristics. That data also showed that the STMP and PVB containing 54 foams were performing better compared to other foams in different pH solutions. That data is not shown here. 700 600 500 Swelling ratio % 400 300 200 100 0 0 20 40 60 80 100 120 140 Time (hours) 1% STMP, 10% PVB 1.5% STMP, 10% PVB 3% STMP, 10% PVB 0% glycerol, 1.5% STMP 5% glycerol, 1.5 % STMP 10% glycerol, 1.5 % STMP Figure 2.20: Swelling characteristics of starch-chitosan-STMP foams 55 Moisture sensitivity The moisture absorption curves of different starch foams prepared using various additives are as shown in Figure 2.21 at room temperatures under different relative humidity conditions. Control Foam (Starch + water) a) 35 30 25 Moisture % 20 15 10 5 0 0 20 40 60 80 100 Time (h) RH 11% RH 33 % RH 52 % RH 75% RH 95% PVOH 10% + chitosan 4 % 35 b) 30 25 Moisture % 20 15 10 5 0 0 20 40 60 80 100 Time (h) RH 11% RH 33 % RH 52 % RH 75% RH 95% Figure 2.21: Moisture absorption curves for starch foams in different RH conditions a)control foams (starch+water) b)Control foam(PVOH +water), c)corn starch +PVB + chitosan ,d)corn starch+STMP+chitosan 56 Figure 2.21 cont’d PVB 10% + chitosan 4 % c) 35 30 25 Moisture % 20 15 10 5 0 0 20 40 60 80 100 Time (h) RH 11% RH 33 % RH 52 % RH 75% RH 95% PVB 10% + chitosan 4 % + STMP 1.5 % d) 35 30 25 Moisture % 20 15 10 5 0 0 20 40 60 80 100 Time (h) RH 11% RH 33 % RH 52 % RH 75% RH 95% For all the samples, the foams exhibited typical adsorption behavior. Figure 2.22 shows the adsorption isotherms for the different foam samples. All sorption isotherms were of sigmoidal shape therefore can be classified as a type II according to Branauer classification. This type of isotherms is common for starch-rich products. 57 Starch + water Starch + PVOH + water 40 30 30 % Moisture % Moisture 20 20 10 10 0 0 0 20 40 60 80 100 0 50 100 Relative Humidity % Relative Humidity % Starch + PVB + chitosan + water Starch + PVB + chitosan + STMP 30 30 % Moisture % Moisture 20 20 10 10 0 0 0 20 40 60 80 100 0 50 100 Relative Humidity % Relative Humidity % Figure 2.22: Sorption isotherms for different types of starch foams It was observed that the rate of moisture absorption was fast initially, but it decreased gradually with time and an equilibrium was achieved after about 40 hours. The foams became brittle when placed in low RH environments like 11 and 33 %. When kept in high RH environment of 95%, a noticeable shrinkage occurred in control foams without additives and foams with PVOH (40 and 45 % of their original dimensions) as shown in Figure 2.23- a and b. Foams with PVB and STMP showed much better dimensional stability (15 and 10% loss in dimensions respectively) and did not shrink as much (Figure 2.23- c and d). Further quantitative analysis of the adsorption isotherms can be done using models such as GAB (Guggenheim-Anderson-de Boer). However, that was not focused on in this study. 58 Loss in the original dimensions 45% 40% 15% 10% Figure 2.23: Shrinkage in foams with high RH of 95% a) control starch foam, b) PVOH foam, c) PVB foam d) chitosan +STMP (1.5%) From the shape of the moisture absorption curves, it was apparent that the moisture absorption was directly proportional to the RH. The moisture sorption kinetics was described using the well-known Peleg model [45],[46] given as: Equation 2.4 𝑡 𝑀(𝑡) = 𝑀0 + 𝑘1 + 𝑘2 𝑡 Where M(t) is the moisture content at any time t and Mo is the initial moisture content of the sample. The major advantage of Peleg model is it helps in saving time by predicting the moisture sorption kinetics for samples containing equilibrium moisture content using short time experimental data. Here, k1 is the constant related to mass transfer rate. The lower the k1, the higher the initial water absorption rate. k2 is the constant related to maximum water absorption capacity i.e. the lower the k2, the higher the maximum water absorption capacity [45]. The parameters for Peleg model and the goodness of fit (R2) were estimated using MATLAB and are shown in Table 2.6. 59 Table 2.6: Parameters of Peleg model for kinetics of moisture absorption of starch foams Sample RH k1 k2 R2 Control foam 0.444 0.249 0.984 PVOH 10%, chitosan 4% 0.460 0.270 0.988 LiCl= 11% PVB 10%, chitosan 4% 0.615 0.279 0.987 PVB 10%, chitosan 4%, STMP 1.5 % 0.842 0.299 0.993 Control foam 0.114 0.131 0.998 PVOH 10%, chitosan 4% 0.302 0.141 0.991 MgCl2= 33% PVB 10%, chitosan 4% 0.147 0.136 0.994 PVB 10%, chitosan 4%, STMP 1.5 % 0.212 0.133 0.997 Control foam 0.100 0.093 0.997 PVOH 10%, chitosan 4% 0.410 0.095 0.996 MgNO3 = 52% PVB 10%, chitosan 4% 0.352 0.100 0.994 PVB 10%, chitosan 4%, STMP 1.5 % 0.466 0.097 0.994 Control foam 0.129 0.050 0.994 PVOH 10%, chitosan 4% 0.181 0.066 0.884 NaCl = 75 % PVB 10%, chitosan 4% 0.185 0.075 0.998 PVB 10%, chitosan 4%, STMP 1.5 % 0.168 0.069 0.998 Control foam 0.070 0.037 2.662 PVOH 10%, chitosan 4% 0.215 0.035 0.999 KNO3 = 95% PVB 10%, chitosan 4% 0.206 0.039 0.995 PVB 10%, chitosan 4%, STMP 1.5 % 0.223 0.038 0.998 The data from table 5 indicates that the k1 and k2 values were smallest for the control starch foams indicating their fast water absorption and high-water absorbing capacity. Figure 2.24 shows the change in k1 and k2 values for the different foam samples. It was observed that the k1 values increased for foams containing PVB, chitosan and STMP, which indicated that the rate of moisture absorption was less for these foams compared to the control foams. On the contrary, k2 values for all the samples remained almost the same. This showed that the maximum water absorption capacity for the foams remained the same. Thus, even though these foams showed a delayed moisture absorption, after some time a breakthrough will be 60 achieved and the foams will absorb water. This was a very important property for the end of life of the foams. k1 values 1.000 0.800 k1 (h%-1) 0.600 0.400 0.200 0.000 Control foam PvOH PVB PVB, Chitosan, STMP RH 11% RH 33% k RH 52% RH 75% RH 95% 2 values 0.300 k2 (%-1) 0.200 0.100 0.000 Control foam PvOH PVB PVB, Chitosan, STMP RH 11% RH 33% RH 52% RH 75% RH 95% Figure 2.24: Peleg model constants k1 and k2 for the starch foam samples Addition of PVOH and chitosan increased k1 and k2 values by small amounts which is probably due to the hydrophobic effect of chitosan [47, 48]. Addition of PVB to minimize the water sensitivity was found to be extremely effective. It also made the foams insoluble in water as opposed to the control foams and the foams made with starch and PVOH. This was 61 also observed in the water penetration tests (2.7.3 Water penetration test) done on the foams where it was observed that control foams and PVOH containing foams disintegrated in 5 hours whereas similar foams extruded with PVB remained intact. It was also observed that addition of STMP as crosslinking agent made the foams even more hydrophobic in most of the cases. Both rate of water absorption and maximum moisture absorption capacity (k1 and k2) were found to reduce. Figure 2.25 shows the equilibrium moisture content achieved by these foams after 80 hours when kept in a 95% RH environment. It was found to reduce from 29% by weight to 24% and 25% for PVB and STMP foams. 30 29 28 Moisture % 27 26 25 24 23 22 Control 1 (no Control 2 (with PVB 10 % chitosan + additives) PvOH) STMP 1.5 % Figure 2.25: Moisture content at equilibrium for different foam formulations at 95% RH 62 Contact angle measurements Starch foam’s surface hydrophobicity water was studied with contact angle measurements. Figure 2.26 shows the contact angles of water droplets on different starch foam surfaces with various additives after 30 sec and after 1 min. Contact angle Contact angle Formulation after 30 sec after 60 sec Corn Starch + water (no 0 0 additives) Corn Starch + water + talc (2%) 62 49.5 Corn Starch + water + PVOH (10%) 80.5 72.5 Corn Starch + water + Chitosan (4%) 83.7 80.5 Corn Starch + water + Chitosan (4%)+ PVB (10%) 82 80.3 Figure 2.26: Effect of different additives on surface hydrophobicity of foams For the control corn starch foams with just starch and water, it was observed that the water droplet did not stay on the surface. It was absorbed inside the foam almost immediately due to hydrophilic starch surface (Figure 2.26). Addition of talc increased the hydrophobicity of the foam surface. This might be attributed to the hydrophobic nature of talc[49]. However, previous tests of water solubility and moisture absorption showed that it was not sufficient 63 to make the foams moisture resistant. Addition of PVOH made the surface of foams smoother as compared to control foams. So, it was observed that the water droplet did stay for longer time on PVOH containing foams starting with a contact angle of 80°. However, the contact angle did decrease rapidly with time and the droplet was absorbed inside after ~3 mins. With addition of chitosan, similar, slightly higher contact angle was observed (82.4°). But with chitosan, the water droplet did stay for much longer time on the surface of foam and did not get absorbed. With addition of chitosan the contact angle and the surface hydrophobicity increased. Interaction between the amino groups of chitosan and OH groups of starch may have resulted in stabilization of hydrophilic starch matrix and reduced the number of polar groups available to bond with water[50]. With increasing chitosan content from 4 to 10 %, a steady increase in contact angle to 101° was observed (Figure 2.27). This might be due to the same reason of hydrogen bonding between polar groups of chitosan and starch which reduced the polar groups available on the surface of foam for contacting with water. 64 Contact angle Contact angle Formulation after 30 sec after 60 sec Corn Starch + water+ chitosan (7%) + PVB (10%) 98 94 Corn Starch + water + chitosan 10% + PVB (10%) 101.5 100.2 Corn Starch + water+ chitosan (7%) + PVB (10%)+ STMP 1% 105 107 Corn Starch + water+ chitosan (7%) + PVB (10%)+ STMP 1.5% 102 100.8 Corn Starch + water+ chitosan (7%) + PVB (10%)+ STMP 3% 113.6 112.2 Figure 2.27: Effect of increasing percentage of chitosan and addition of STMP on surface hydrophobicity Interestingly, starch-PVB-chitosan-STMP foams demonstrated much better hydrophobicity. Increase in hydrophobicity of starch due to cross linking has been observed by some authors including Shuzhen et. al. (2018) [51] and Shah et al. (2016)[52]. The contact angles were found to increase to almost 123° for 1.5 % STMP containing foams. Chitosan, PVB and STMP had a synergistic effect on making the foams dense and foam surface smoother which might have caused the highest contact angles for these foams. Corn and potato starch foams with and without glycerol were also compared for the contact angle measurement. Addition of glycerol made both potato and corn starch foams more 65 hydrophilic. The contact angle reduced from Potato starch foams were found to be less hydrophilic in general than corn starch foams (71.3° for potato starch foam vs 65.7° for corn starch foam). Contact angle Contact angle Formulation after 30 sec after 60 sec Corn Starch + formic acid+ chitosan (4%) + STMP 1.5% + 65.7 59.6 Glycerol 0% Corn Starch +formic acid+ chitosan (4%) + STMP 1.5% + 0 0 Glycerol 5% Corn Starch + formic acid+ chitosan (4%) + STMP 1.5% + 0 0 Glycerol 10% Potato Starch + formic acid+ chitosan (4%) + Glycerol 0% 71 63.8 Potato Starch + formic acid+ chitosan (4%) + Glycerol 10% 0 0 Figure 2.28: Effect of glycerol addition on surface hydrophobicity of starch foams Also, they absorbed the water droplet slowly compared to similar formulations with corn starch. We suspect that this contact angle variation is due to differences in amylose and amylopectin contents and the size of the starch molecules and the degree of polymerization, of the two starches. Potato starch had higher amount of amylopectin compared to corn starch and its particle size was also much higher than the corn starch which was observed in the SEM studies as explained in chapter 1. [53]. 66 Scanning electron microscopy Cell size and cell size distribution are important parameters which affect the physico- mechanical properties of the foams. SEM images were taken for all the different foam formulations to study the effect of different additives and processing conditions on the cell size and morphology of the foams. Effect of various additives Without any additives, the starch foams produced were non uniform in nature. The cell sizes were very large, and the foams were brittle. Different additives used in the foams for improving various performance properties included – talc, PVOH, chitosan, STMP and glycerol Additives such as talc, chitosan and STMP, acted as nucleating agents and helped to reduce the cell size of the foams and made them more uniform. According to the qualitative guidelines by McClurg [54], ideal nucleating agents have uniform size, geometries and surface properties and they are easily dispersible. Different additives used here showed different efficiencies in reducing the cell sizes of the foams. As studied by Leung et al[55] the geometry the nucleating agent affects the efficiency of nucleation. Hence the shapes of various nucleating agents were examined using SEM. Figure 2.29 shows the SEM images for talc, chitosan and STMP at 200 and 5000x magnifications. Talc particles were the smallest in size and had a scaly structure compared to all the other nucleating agents which might be a reason that makes them one of the most efficient nucleating agents (Figure 2.29-a and e). Figure 2.30 shows the SEM image for different foams with various additives. It can be observed that increasing the talc content from 0-2% reduced the cell size of the foams and made the cell size distribution more uniform (Figure 2.30-a-c). The cell size distributions 67 were also quantified using image analysis and plotted as normalized graphs as shown in Figure 2.31. It was clearly observed that with increasing percentage of talc, the number of smaller cells increased, and the cell distribution became more and more narrow. SEI 10kV SS30 X200 100µm SEI 10kV SS30 X200 100µm SEI 10kV SS30 X500 50µm SEI 10kV SS30 X200 100µm a) b) c) d) SEI 10kV SS30 X5000 5µm SEI 10kV SS30 X5000 5µm SEI 10kV SS30 X5000 5µm SEI 10kV SS30 X5000 5µm e) f) g) h) Figure 2.29: SEM images for various additives used in foam formulations – a) talc, b) chitosan, c)STMP, d) PVB at 200x magnification; e)talc, f)chitosan, g)STMP; h) PVB at 5000x magnification 68 a) b) c) SEI 7kV SS30 X20 1mm SEI 7kV SS30 X20 1mm SEI 7kV SS30 X20 1mm d) e) f) SEI 7kV SS30 X15 1mm SEI 7kV SS30 X15 1mm SEI 7kV SS30 X15 1mm g) h) i) SEI 7kV SS30 X20 1mm SEI 7kV SS30 X20 1mm SEI 7kV SS30 X20 1mm Figure 2.30: SEM images of starch foams containing a)0% talc b) 0.7% talc c)2% talc d)4% chitosan e)7% chitosan f)10% chitosan g) 1% STMP h) 1.5% STMP i) 3% STMP 69 0% talc 0.7% talc a) 40 b) 40 Percentage % Percentage % 30 30 20 20 10 10 0 0 Cell size (mm) Cell size (mm) 2% talc c) 40 Percentage % 30 20 10 0 Cell size (mm) Figure 2.31: Cell size distribution for foams containing different percentages of talc Even with SMTP, the shapes of the particles were very uniform and small (Figure 2.29- c and g). Hence, a greater number of nucleation sites were available in the foams and STMP proved to be efficient as a nucleating agent. One more theory that can be used for explaining the impact of STMP is the crosslinking of starch by sodium trimetaphosphate. Starch reacts with sodium trimetaphosphate to give mono and distarch phosphates as shown in Figure 2.4. Studies have reported the effects of crosslinking on the growth of cells[56, 57]. Foaming and crosslinking reactions compete with each other and the foamed structure can be controlled by adjusting the process conditions[56]. The cell sizes of foams with increasing percentages of STMP were monitored and plotted as a function of % of cells (Figure 2.32). It was observed that the cell sizes became narrower and the distribution became more uniform as the percentage of STMP increased from 0 to 3%. Due to increasing crosslinking of starch with 70 STMP, the cell sizes go on reducing giving a narrower cellular size distribution as shown in Figure 2.29-g, h,i . Furthermore, strain hardening is a necessary requirement for the foaming process. A lower extensional viscosity is necessary for expansion of the bubbles whereas a high extensional viscosity is needed to stabilize the bubbles. Crosslinking of starch with STMP increases the melt viscosity of the mixture making the bubbles more stable and cell distribution more uniform, thus giving the nucleating effect. a) STMP 0% b) STMP 1% 30 30 Percentage % Percentage % 20 20 10 10 0 0 0-0.25 0-0.25 0.25-0.5 0.25-0.5 0.5-0.75 0.5-0.75 0.75-1 0.75-1 1-1.25 1-1.25 1.25-1.5 1.25-1.5 1.5-2 1.5-2 2-2.5 2-2.5 2.5-3 2.5-3 3-3.5 3-3.5 3.5-4 3.5-4 4-4.5 4-4.5 4.5-5 4.5-5 5-7 5-7 6-7 6-7 Cell size (mm) Cell size (mm) c) STMP 1.5% d) STMP 3% 30 30 Percentage % Percentage % 20 20 10 10 0 0-0.25 0 0.25-0.5 0.5-0.75 0-0.25 0.25-0.5 0.75-1 1-1.25 0.5-0.75 0.75-1 1.25-1.5 1.5-2 1-1.25 1.25-1.5 2-2.5 2.5-3 1.5-2 2-2.5 3-3.5 3.5-4 2.5-3 3-3.5 4-4.5 4.5-5 3.5-4 4-4.5 5-7 6-7 4.5-5 5-7 6-7 Cell size (mm) Cell size (mm) Figure 2.32: Cell size distributions for starch foams with PVB at different STMP levels a) 0% b) 1% c)1.5 % d)3% Addition of increasing amounts of chitosan showed a similar nucleating effect as talc although the effect was not as drastic (d, e, f). This might be due to the fact that chitosan particles were much larger and more non uniform compared to talc or STMP as observed from their SEM (Figure 2.29-b and f). Va ́zquez et al. studied chitosan as nucleating agent for thermoplastics foams and they found that, for every nucleating agent there is a ‘critical 71 concentration’ at which the cell size and distribution is most uniform. And the critical concentration depends on the size of the particles. Larger the particle size, higher the percentage required for nucleating effect[58]. So, it might be possible that higher concentrations of chitosan were needed in the formulation to have more impact on the nucleation and cell size which were not tried in this study. Figure 2.33 shows the cell size distribution of starch foams containing 4, 7 and 10% chitosan respectively. Higher amounts of chitosan were required for achieving similar nucleating effect as talc. a) 4% Chitosan b) 7% Chitosan 30 30 Percentage % Percentage % 20 20 10 10 0 0 0-0.25 0-0.25 0.25-0.5 0.25-0.5 0.5-0.75 0.5-0.75 0.75-1 0.75-1 1-1.25 1-1.25 1.25-1.5 1.25-1.5 1.5-2 1.5-2 2-2.5 2-2.5 2.5-3 2.5-3 3-3.5 3-3.5 3.5-4 3.5-4 4-4.5 4-4.5 4.5-5 4.5-5 5-7 5-7 Cell size (mm) Cell size (mm) 10% Chitosan c) 30 Percentage % 20 10 0 0-0.25 0.25-0.5 0.5-0.75 0.75-1 1-1.25 4-4.5 1.25-1.5 1.5-2 2-2.5 2.5-3 3-3.5 3.5-4 4.5-5 5-7 Cell size (mm) Figure 2.33: Cell size distribution of starch foams with increasing percentages of chitosan PVB acted as a foaming agent and increased the cell size and expansion ratios of the foams considerably as compared to control foams and foams with PVOH. The reason for that could be the hydrophobic nature of PVB. It is very sensitive to hydrolysis in presence of water[42] to form polyvinyl alcohol and butyraldehyde. The boiling point of butyraldehyde (74.8°C) is 72 lower than the extrusion processing temperatures. Hence it might be generating extra vapor apart from the steam generated from evaporation of water and act as a chemical blowing agent to increase the cell size and reduce the density of resulting foams. Confocal microscopy Confocal laser scanning microscopy (CLSM) was applied to determine the distribution of chitosan in starch foams. Several studies have reported on the characterization of chitosan stained with different types of fluorescent labels, such as fluorescein isothiocyanate (FITC) (Onishi & Machida, 1999) and rhodamine B isothiocyanate (RBITC) (Ma et al., 2008) using CLSM technique (Ma et al., 2003, Ma et al., 2008, Onishi and Machida, 1999). Here FTIC was used for green fluorescent staining of chitosan. Some studies have reported the migration of chitosan from the bulk of the material to the surface[23]. The migration of chitosan to the surface of the foams can be one of the reasons for increased hydrophobicity of the foams. Hence the difference between the chitosan distribution in the bulk of the foam (middle part) and the surface of the foams was observed using CLSM. The foams were cut in thin slices from two parts- the inner bulk and the surface. Then they were stained and mounted on the slides as per the method explained in section 2.4 and compared for amount of chitosan present in the bulk vs the surface of the foam. Figure 2.34 shows the 3D MIP images for 0%, 4%, 7% and 10% chitosan foam formulations from the surface of the sample and from the bulk. Dang et. al 2016, have confirmed that chitosan gets distributed on the surface of the films, forms hydrogen bonds with the starch molecules and reduces the surface hydrophobicity of the films[23]. Similar results were observed with the foams as well. It was clearly observed for all the samples that more chitosan was present on the surface of the foams as compared to the bulk. Chitosan chains might have oriented towards the surface of 73 the foams during cooling of the foams after coming out from the die or during the storage of the foams. This also explains the increased hydrophobicity of the foam surface as observed from contact angle measurements. One more possible explanation for this hydrophobicity is - during gradual drying of a polyelectrolyte it is possible for relatively non-polar polymers or their segments to migrate to the air-water interface (Khavet and Essalhi 2015). Such orientation achieves an arrangement that is thermodynamically favorable – providing a relatively low-energy surface facing the air. In this case, most of the polar groups, including both the amine and hydroxyl groups, will face inwards or parallel to the surface. The formation of hydrogen bonds might be expected to drive such a reorientation during drying. The presence of non-polar groups on the surface of the foams could account, at least in part, for the often-observed relatively hydrophobic nature of chitosan containing foams. To confirm this, the foam surfaces can be scratched and the contact angles can be measured again to see if more hydrophilic groups, then get exposed and change the hydrophobicity of the surface. 74 0% chitosan 4% chitosan 7% chitosan 10% chitosan a) b) c) d) Bulk samples g) h) e) f) Foam surface samples Figure 2.34: Confocal laser scanning microscope (CLSM) 3D MIP images for 0%, 4%, 7% and 10% chitosan foam formulations from the bulk of the sample (a,b,c,d) and from the surface (e,f,g,h) Compressive strength Compressive strength is the measure of ability of foam to deform under load. Figure 2.35 shows the effect of various additives on the compressive strength. The detailed results for compressive testing of starch foam samples are listed in Table 2.7. Denser foams tend to have thicker walls and hence resist the deformation better and thus have a higher compressive strength. Since addition of PVB reduced the density of foams considerably as explained previously, the compressive strength was also found to be less for these foams. (0.04 MPa vs 0.08 MPa for control foams). However, addition of STMP as crosslinking agent made the foams stronger, less resilient and increased the compressive strength to 0.215 MPa; 3 times that of the control foams. Similar results were observed by Hassan et al. 2020[25] where crosslinking of starch by citric acid increased the compressive strength to double the value. 75 The compressive strain at maximum stress is also an indicator of sample’s resilience and elastic limit. It was found to increase from 8% for control foams to 12% for foams with PVB which indicated increase in resilience. Crosslinking of starch with STMP reduced this strain to around 9% which was still higher compared to control foams. 0.250 0.200 Compressive strength (MPa) 0.150 0.100 0.050 0.000 1 2 3 4 5 6 7 8 Figure 2.35: Compressive strength of starch foams 1)Control foams (starch+water), 2)Control foams (PVOH), 3)10% PVB,4% chitosan, 4) 10% PVB,7% chitosan, 5) 10% PVB,10% chitosan, 6) 10% PVB,4% chitosan, 1% STMP, 7) 10% PVB,4% chitosan, 1.5 STMP, 8) 10% PVB,4% chitosan, 3% STMP 76 Table 2.7: Physico-mechanical properties of extruded starch foams Compres sive strain at Density Expansion Compressive maximu Sample (kg/m3) ratio strength (MPa) m compres sive load (%) Control foam 40.7 ± 8.9a 46.6 ± 7.9a 0.076 ± 0.01a 7.95 PVOH 10%, chitosan 4% 45.7 ± 2.3 a 27.5 ± 0.9b 0.114 ± 0.01b 7.15 PVB 10%, chitosan 4% 21.1 ± 1.7b 74.5 ± 3.9c 0.040 ± 0.005c 12.08 PVB 10%, chitosan 7% 35.0 ± 3.8c 51.1 ± 5.5d 0.0614 ± 0.008d 11.14 PVB 10%, chitosan 10% 35.7 ± 3.0c 50.1 ± 5.7d 0.0800 ± 0.005a 10.63 PVB 10%, chitosan 4%, 37.0 ± 3.8c 50.3 ± 5.3d STMP 1 % 0.181 ± 0.017e 9.82 PVB 10%, chitosan 4%, 43.9 ± 4.0a 43.9 ± 4.0e STMP 1.5 % 0.178 ± 0.017e 9.54 PVB 10%, chitosan 4%, 51.6 ± 3.7e 51.6 ± 3.7d STMP 3 % 0.215 ± 0.022f 12.12 Different superscript letters within the same column indicate significant differences among formulations (p < 0.05). Aqueous biodegradability studies Various moisture resistant and water insoluble starch foams were prepared in this project. It was important to check the impact of these additives and modifiers on the end of life of foams. Hence 3 foam samples – 1. control foam containing PVOH, 2. Foam with 4% chitosan, and 10% PVB and 3. Foams with 4% chitosan, 10% PVB and 1.5% STMP, were studied for their aqueous biodegradation according to ISO 14852 at 30°C. The CO2 evolved from all the samples was subtracted from the cumulative CO2 of the blanks to calculate % biodegradation as explained in section 2.4. Figure 2.36 shows the graphs of percent biodegradation vs time for the 3 foams. 77 100 80 % biodegradation 60 40 20 0 0 20 40 60 80 Time (days) Control foam (starch + water) PVB foam PVB + Chitosan + STMP foam Figure 2.36: Aqueous biodegradation curves for control starch foam, PVB containing foam and STMP containing foam It was observed that there was not much difference in the rates of biodegradation of the 3 foams. Even in presence of PVB and STMP, the foams reached ~90% biodegradation in 80 days which was a positive result. Similar results were obtained by Merino et. al 2019 [59] where they observed that neither crosslinking of starch by phosphorylation nor chitosan addition has any significant impact on the soil biodegradation of the starch films. Thus, even if the starch foams were made more resistant to moisture using PVB, chitosan and STMP, their end of life was not impacted much due to these modifications and these foams still remained readily biodegradable in aqueous environment. Thus, in case of inadvertent leakage of these products in the marine environment, they will be completely removed within a reasonable timeframe and may cause minimum impact to the environment. 78 2.8 DESIGNING AN ANNULAR DIE In order to get rid of the ridges occurring in the foams made with slit die, another approach of using an annular die is being studied. The idea is to make the foams in the form of hollow tubes from this annular die which can be then cut to open up the tubes and get flat sheets. The first step was making a preliminary die design using AUTOCAD with all the measurements suitable for our extruder. Die design A preliminary design for an annular die was developed to fit the Century ZSK-30 twin screw extruder. The die was designed in 2 parts. The outer part included the die plate and a tube- like structure and a cylindrical mandrel was designed to fit inside this part so as to produce the foams in the form of tubes/cylinders. These cylinders can then be cut using blades to open up the tubes and yield flat sheet foams. The 2 parts are as shown in Figure 2.37. Figure 2.37: Annular die design parts The detailed design using AUTOCAD is shown in Figure 2.38. 79 Figure 2.38: AUTOCAD design for the die Next, this die design was 3D printed using polymer composite to check for all the measurements and also perform a few trial runs before getting the die CNC machined. Figure 3 shows the composite 3D printed die. It did match with all the measurements for extruder die plate. However, the polymer composite could not withstand the high temperatures of 140-150°C which were used for the extruder and the die started to melt. Hence, it could be used for the trial runs. However, this step was useful to ensure the dimensions of the die were appropriate and the die was fitting perfectly on the extruder die head. 80 Figure 2.39: 3D printed die The next step was getting the die CNC machined. We have recently acquired the metal die from a manufacturer, and we are in the process of testing the die for trial runs to make the sheets. Figure 2.40 shows the CNC machined die. Figure 2.40: CNC machined die Trial runs A couple of trial runs were carried out using this die for making the foams. Processing conditions similar to strand and slit die were also used for this die as a starting point. Figure 2.41- a and b show the foams produced from this die. These foam tubes were cut and 81 flattened to make the sheets as shown in Figure 2.41-c. The formation of ridges was very less using this kind of die. There are still some problems with making the runs smoother and optimizing the processing conditions and design for this die. a) c) b) Figure 2.41: a), b) Some annular die foams produced from the trial runs c) flat sheets formed after cutting the foam tubes 82 2.9 FUTURE RECOMMENDATIONS ➢ The cell size for the foams prepared was in the range of mm in this study. Efforts should be made to make this cell size smaller and more uniform. That might affect the moisture sensitivity as well. ➢ The chemistry of chitosan-starch polyelectrolyte complex and STMP-starch crosslinking and degree of substitution etc. was not studied in detail in this project. The formation of insoluble foams itself was taken as an indication that polyelectrolyte complex was forming, or crosslinking was taking place. Further quantitative NMR or other studies can be done to get a better idea of these mechanisms. ➢ The stability of PEC could be affected by many factors, including density of charges, degree of ionization, pH of reaction medium, concentration of polyelectrolytes, distribution of ionic groups, molecular weight, mixing ratio, order of reacting polyelectrolytes, and drying process, etc. these factors could be studied in detail to make these foams more or less stable as required. ➢ A design of experiments approach can be used to study the effect of multiple factors on the properties of the foams. ➢ The foams sheets need to be completely flat and of uniform thickness to make their use in hemostatic pads. First steps in this direction have been taken by design of the annular die and the trial runs as a proof of concept. Further optimization in die design and processing conditions is required to calibrate this process. 83 2.10 CONCLUSIONS Cylindrical and sheet starch foams were prepared in ZSK-30 twin screw extruder using various reactive modifiers and additives such as PVB, chitosan, STMP and glycerol. Water and formic acid aqueous solutions were used as a plasticizer as well as blowing agent. The extrusion conditions were optimized for each formulation to obtain lowest density consistent foams with controlled expansion. Various extrusion parameters such as temperature, screw configuration, water content and feeding rates were optimized for process development of foam extrusion. Both slit and strand dies were used for making foams in different shapes. A roller press was used for making foams as flat sheets. Talc, chitosan and STMP acted as nucleating agents and reduced the cell size of the foams and made the cell size distribution more uniform. With addition of increasing percentage of talc, chitosan and/or STMP, the density increased, and expansion ratio reduced steadily. PVB seemed to act as a blowing agent and resulted in foams with the lowest density and highest expansion ratio of 21 kg/m3 and 38.7 respectively. Solubility testing of starch foams showed that the control starch foams without any additives and with PVOH as additive dissolved immediately in water. When chitosan was added to the foams, a polyelectrolyte complex was formed, and the foams formed gel structures in water. Addition of formic acid for protonation of amino groups and addition of STMP for making anionic starch increased the efficiency and stability of polyelectrolyte complex in water. Addition of PVB proved to be extremely efficient in improving the moisture resistance of starch foams. PVB and STMP together made the cell structure strong and improved the humidity resistance of the foams. These foams showed the best performance in terms of water insolubility, dimensional stability and surface hydrophobicity. Addition of glycerol made the foams more flexible but also more 84 hydrophilic and susceptible to moisture. Starch foams were kept in different relative humidity environments to study the rate and moisture absorption capacity using Peleg model. CLSM imaging showed that chitosan from the foams was preferentially migrating to the surface of the foams making the surface hydrophobic. Crosslinking of starch with STMP and forming its polyelectrolyte complex with chitosan also improved the mechanical properties of the foams upto 3 times compared to control foams. Efforts were also done to design an annular die to make the foams as flat sheets. Further optimization is required to fine tune the design and runs for the annular die. 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Alvarez, “In-soil Biodegradation Behavior of Chitosan-Coated Phosphorylated Starch Films,” Res. Artic., vol. 2019, no. 12, pp. 907–912. 91 3. BIOBASED MALEATED THERMOPLASTIC STARCH (MTPS) AND MTPS-G-PETG (GLYCOL MODIFIED PET) GRAFT COPOLYMER VIA REACTIVE EXTRUSION: EVALUATIONS 3.1 ABSTRACT This study reports on using reactive extrusion (REX) modified thermoplastic starch for making in situ MTPS-PETG (glycol modified Polyethylene terephthalate) graft copolymers. Reactive blends of maleated thermoplastic starch (MTPS) and PETG in 30:70 ratio (wt/wt) were prepared using a ZSK-30 twin-screw extruder. 80% glycerol was grafted on the starch during the preparation of MTPS as determined by soxhlet extraction with acetone. 30% of added PETG was grafted on MTPS backbone was obtained by soxhlet extraction with dichloromethane (DCM). The results were confirmed by TGA and FT-IR analysis of residue and extracts. The tensile and impact properties of graft copolymer were analyzed and compared with the properties of PETG. Scanning electron microscopy (SEM) images showed a uniform, microdispersion of MTPS in PETG matrix. Aqueous biodegradation for these blends did now show any improvement in the biodegradability of PETG. 92 3.2 INTRODUCTION Replacing petro-fossil carbon with bio-based carbon in polymers offers a reduced material carbon footprint and managed end of life [1–3]. This has given rise to the area of biopolymers i.e. synthesis of polymers from renewable resources. Starch is an abundant, inexpensive, 100% bio-based, and completely biodegradable polymer. As explained in chapter 1, the ratio of amylose and amylopectin is different in different starches [4]. The properties of the products like films and foams depend on the type of starch being used. When amylose content is more, the film is stronger and more flexible whereas when amylopectin content is more it leads to poor mechanical properties like brittleness. Another application of starch-based bioplastics is in the form of thermoplastic starch. The melting temperature of pure starch is above its decomposition temperature. Therefore, it does not flow on thermal processing [5]. To make starch processable, plasticizers such as water, glycerol, sorbitol are used[6] . This thermoplastic starch can then be blended with other biodegradable or non-biodegradable polymers for use in several applications such as food packaging, films, disposable utensils, pharmaceutical etc. The cost reduction due to use of starch and increased biobased content are additional benefits. However, such thermoplastic starches (TPS) have poor dimensional stability and reduced mechanical properties with time. More problematic is the leaching of the plasticizer (glycerol for example) over time contributing to brittleness and making the film surface tacky and unusable[7]. In our group, we have synthesized a maleated thermoplastic starch (MTPS) using reactive extrusion (REX) in which the glycerol plasticizer is covalently bonded to the starch, thereby eliminating glycerol migration and maintaining good processability[8–11]. 93 The structure of the glycerylated starch polymer is shown below in Figure 3.3 and described in our earlier papers. Figure 3.1: Structure of maleated thermoplastic starch It has been shown that MTPS has a high reactivity towards polyesters[11]. It can undergo transesterification reaction with other polyesters to yield graft copolymers (US patent 7629405). Hence, it can be blended with hydrophobic polymers to reduce its hydrophilic nature and improve mechanical properties. [9] Glycol modified PET (PETG) is a modification of Polyethylene Terephthalate (PET). PETG is synthesized by replacing a portion of glycol component of PET with a cyclic diol called cyclohexanedimethanol (CHDM). It is a highly hydrophobic polymer with excellent tensile strength and elongation properties. PETG is used in wide range of industrial applications and is one of the important polymers in packaging. The main disadvantage of PETG is in terms of its end of life- it is not biodegradable in aqueous or composting environment. Increasing pollution of aquatic and terrestrial environments by plastics has fueled the research on biobased and biodegradable plastics. Various studies claim that addition of biodegradable polymers or additives like starch, polylactide (PLA), polycaprolactone (PCL) increase the biodegradation of non-biodegradable polymers like polyethylene (PE), PET etc.[12–15]. This hypothesis was tested in this study. Neat PETG itself is not biodegradable. Our hypothesis 94 was - by incorporating 30% of thermoplastic starch in the blend, the biodegradability of this compatibilized blend will be much higher. The SEM and Soxhlet extraction studies confirmed the presence of a well dispersed MTPS phase in PETG matric and in-situ formation of a graft copolymer. Aqueous biodegradation studies of these pellets showed that only the MTPS portion of the blend degraded rapidly in water leaving the PETG portion as it is. Addition of starch did not affect the biodegradability of PETG. Mechanical and thermal properties of the blends were also analyzed and compared with neat PETG. This study provides a good example of false and misleading claims made for accelerated biodegradation of non- biodegradable polymers and refutes the many claims of biodegradability of non- biodegradable polymers by the addition of starch and similar additives in the marketplace. 95 3.3 EXPERIMENTAL 3.3.1 Materials High amylose corn starch was obtained from National Starch (NJ, USA) with equilibrium moisture content of about 12% (w/w). Glycerol was obtained from J.T. Baker (NJ, USA) and was used as received. Maleic anhydride (MA) and 2,5-bis(tert-butyl-2,5-dimethylhexane, tech. 90% (Luperox 101) were obtained from and Sigma–Aldrich (WI, USA). Glycol modified PET (PETG) was purchased from the Eastman Chemical Company (TN, USA) and was used as it is. Figure 3.2 shows the structure of PETG. Figure 3.2: Structure of PETG 3.3.2 Preparation of maleated thermoplastic starch (MTPS) MTPS was prepared in a twin screw co-rotating CENTURY ZSK-30 extruder. The screw diameter was 30 mm and transport length was 1260 mm with L/D ratio of 42. Moisture is not preferred in the reactive extrusion experiment because it can interfere with the reactivity of glycerol and can cause foaming of the extrudate. So, the corn starch was dried for 48 hours in oven at temperature of 65°C to reduce its moisture content to 0.1%. MA (20g) was ground to fine powder using a mortar and pestle and was premixed with dry starch (800g). Luperox (1.1 g) was mixed with glycerol (200g) and the mixture was then pumped into the extruder directly via peristaltic pump. The feeder was calibrated to get the ratio of 80:20 (starch: glycerol). [8] The temperature profile was set as 70/90/110/120/130/140/150/150/150/140 from the feed port to the die. The screw speed was set at 100 rpm and melt temperature was 150°C. 96 The vent port was kept open to remove the moisture formed during the reaction. The extrudate coming out of the extruder was air cooled and pelletized simultaneously using Scheer Bay pelletizer as shown in Figure 3.3. The resulting MTPS pellets were dried overnight in oven at 65°C and then stored in vacuum sealed bags before using for any further characterization. Figure 3.3: Preparation of maleated thermoplastic starch 3.3.3 Preparation of MTPS-PETG graft copolymers MTPS-PETG graft copolymers were prepared via transesterification reaction mechanism. MTPS with more than 80% grafting was dried for at least 24 hours to remove excess moisture. Similarly, PETG was also dried to remove moisture. The dried MTPS and PETG were mixed in 30:70 w/w ratio and fed through the single screw feeder. The feed rate was maintained at 100 g/min or 6 kg/h for the mixture by calibrating it. The temperature profile was set at 90/110/120/130/140/150/160/160/160/150. The screw speed was set at 100 rpm and melt temperature was about 140°C. The resulting MTPS-PETG was pelletized in line after cooling in water bath. The pellets were dried overnight in an oven at 65°C before storing. 97 3.4 CHARACTERIZATION AND ANALYSIS Initial thermal characterization of the raw materials- corn starch, PETG and glycerol was done to determine the degradation and melting temperatures of the raw materials. After preparing the blend in the extruder, the extent of reaction or grafting was measured using soxhlet extraction and was further confirmed using thermogravimetric analysis. FT-IR spectroscopy was used to provide further validation of transesterification reaction. The mechanical properties of the MTPS-PETG graft copolymer were determined and compared with neat PETG properties. Scanning Electron Microscopy (SEM) was used to study the dispersion of MTPS in PETG phase. Aqueous biodegradation of the reactive blend was also studied using ISO 14852. 3.4.1 Soxhlet extractions using acetone and dichloromethane Selective solubility of glycerol in acetone was used to establish and determine percent covalent grafting of glycerol[8], [10]. The MTPS pellets prepared were ground to a fine powder and about 5 g of sample was put in a pre-dried and pre-weighed cellulose extraction thimble. The thimbles were then inserted in the soxhlet extractor connected to a 500 mL round bottom flask containing around 200–250 mL acetone. The flasks were heated, and the solvent was allowed to reflux. The extraction was continued for 72 h. After the extraction, the thimbles were removed; residue and extract were separated and dried overnight at 70°C. The dried thimble with residue was weighed again and the weight change in the residue was calculated. The reproducibility of the results was confirmed by testing three replicates for each sample. It was expected that the covalently grafted glycerol will not get extracted in acetone and there will be a weight gain in the residue. Percent grafting was calculated from the mass balance as shown in the following equation- 98 Equation 3.1 |𝑊1 − 𝑊2 | % 𝑔𝑟𝑎𝑓𝑡𝑖𝑛𝑔 = × 100 𝑊1 where, W1 is the weight of glycerol present in the sample originally and W2 is the glycerol in the extract after 72 h. i.e., free glycerol. Similar Soxhlet analysis was used to determine % grafting of PETG on MTPS backbone. In this the solvent used was Dichloromethane (DCM) which selectively dissolves PETG. The residues and extracts of all the samples were analyzed using TGA in order to confirm the results from Soxhlet analysis. 3.4.2 Thermal analysis The degradation temperature of samples was obtained by thermogravimetric analysis (TGA). TGA measurements of all the samples were conducted under an inert atmosphere of nitrogen using a TGA Q50 (TA Instruments, New Castle, DE, USA). The general sample weight used was 5–7 mg. The sample was placed in an aluminum pan and was heated to 600 °C at the rate of 10 °C/min. The weight loss (%) of a sample as a function of temperature (°C) was obtained from this analysis. Also, the glass transition temperature of PETG and the blend were obtained by using a differential scanning calorimeter (DSC). The sample was heated to 200 °C in DSC Q20 (TA Instruments, New Castle, DE, USA) with a heating rate of 10 °C/min and held for 5 min to erase thermal history. The sample was then cooled back to 20 °C and heated again to 200 °C with a heating rate of 10 °C/min. The glass transition temperature (Tg) was calculated using TA universal analysis 2000 software. Since PETG is amorphous polymer, further analysis of melting point and crystallinity was not performed. 3.4.3 Tensile testing The injection molded test bars were prepared using a tabletop DSM 15 cc mini extruder (DSM Research B. V., Sittard-Geleen, The Netherlands) and 3.5 cc mini-injection molder 99 (DACA Instruments, Santa Barbara, CA, USA). The injection pressure was set as 140 psi and the cylinder and mold temperatures were 210 and 65 °C, respectively. The samples were stored for 2 days at 25 °C in a humidity chamber with RH of 50% before any analysis. Tensile testing was performed using an Instron model 5565-P6021 (Instron, Norwood, MA, USA) with a 5 kN load cell and grip separation speed of 12.5 mm/min as per ASTM D882. Data from five samples of each formulation were averaged and compared with the properties of neat PETG. 3.4.4 Impact Testing Injection molded test bars required for notched Izod impact testing were also prepared by a DSM 15 cc mini extruder (DSM Research B. V., Sittard-Geleen, The Netherlands) & 3.5 cc mini injection molder (DACA Instruments, Santa Barbara, CA, USA). The injection pressure was set as 140 psi and the cylinder and mold temperatures were 210 and 65°C respectively. The samples were tested with Ray-Ran RR-IMT (Warwickshire, UK) pendulum impact tester as per ASTM D256-10(2018). The samples with a dimension 64 mm* 12.7 mm* 4 mm were notched using a Tinius Olsen Model 22-05-03 Motorized Specimen Notcher (Pennsylvania, USA). The notch marked was 2.54 mm deep and six replicates were used per sample. 3.4.5 Fourier Transform Infrared Spectroscopy (FT-IR) FT-IR spectra of pure PETG, MTPS and of MTPS-PETG residue after Soxhlet extraction were recorded on Shimadzu IRAffinity-1 spectrometer (Columbia, USA) equipped with MIRacle ATR attachment. The spectra were recorded between the wavelength on 500-4000 cm-1 in absorption mode. 100 3.4.6 Scanning Electron microscopy A JOEL 6610 LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) was used to study the dispersion of MTPS in PETG in the pellets. They were treated with 6 N HCl for 12 h to remove the MTPS phase from the samples and air-dried for 12 h in a fume hood. Then they were mounted on aluminum stubs using high vacuum carbon tabs, and coated with gold using a sputter coater and examined using JOEL at 3000× magnification at 10 kV. A set of fractured tensile bars was also examined to look at the fracture morphology of the samples. The bars were immersed in liquid nitrogen for ~2 min and then fractured. Fracture surfaces were mounted on aluminum stubs and examined using JOEL at 3000× magnification at 10 kV. 3.4.7 Aqueous biodegradation The biodegradability of MTPS-PETG samples was tested in an aqueous environment. All the tests were performed in an aerobic environment at 30 °C. A respirometry mineralization test system for calculating CO2 evolution was set up based on International Standard ISO 14852. The system comprised blank, positive reference (cellulose) and the test material (MTPS- PETG) for all the runs. All the samples, blanks, and references were run in duplicates. An optimized test medium containing all the nutrients and buffers was prepared according to the ISO standard as explained in section 2.4 101 3.5 RESULTS AND DISCUSSIONS 3.5.1 MTPS analysis: Soxhlet extraction Soxhlet analysis provided percent covalent grafting of glycerol to the starch backbone. Acetone was used as the extraction solvent. In this, the covalently bonded glycerol is not extracted, and only free, ungrafted glycerol is extracted by acetone. The results are shown in Table 4. The removal of free glycerol by acetone was further confirmed from the results of TGA analysis as shown in Figure 3.4. A decrease in the peak corresponding to glycerol was observed in the residue after Soxhlet extraction whereas the TGA of the extract showed a peak only for glycerol with no MTPS indicating that acetone extracts only free glycerol and no MTPS. Results indicated that 79% of added glycerol was chemically grafted on the starch backbone. Similar results have been observed in the previous studies from our group[10], [11] Table 3.1: Results for percent grafting of glycerol on MTPS Sample 1 2 3 Weight (g) 5.009 5.015 5.003 Starch (g) 4.007 4.012 4.002 Glycerol (g) 1.002 1.003 1.001 Extract (g) 0.214 0.221 0.201 Residue (g) 4.794 4.793 4.801 %Grafting 78.58 77.87 79.90 Average Grafting (%) 78.7 ± 0.7 102 2.5 MTPS 2 Extract (glycerol) Derivative Wt % Residue 1.5 1 0.5 0 0 200 400 600 Temperature (°C) Figure 3.4: DTG curves of MTPS, glycerol and residue showing only MTPS after Soxhlet extraction 3.5.2 PETG-MTPS analysis: Soxhlet extraction PETG/MTPS (70/30) resins prepared by reactive extrusion were analyzed using Soxhlet extraction and TGA. Figure 3.5 shows the comparative TGA graphs for MTPS, PETG and MTPS-g-PETG reactive blend. It was found that the degradation temperature for the reactive blend was in between that of pure MTPS and PETG. It showed 2 degradation stages. The first one corresponding with MTPS and the second with PETG. These PETG pellets were analyzed for % grafting using Soxhlet extraction as explained in section 3.4. PETG is soluble in DCM and MTPS is not. Hence, DCM was used for Soxhlet extraction to find the % grafting of PETG on MTPS. To confirm that the grafting reaction has occurred, the residues and extracts of the resin after Soxhlet extraction were compared with a control. The control here was a physical mixture of PETG and MTPS. PETG and MTPS pellets 103 were mixed in the same ration of 70:30. The mixture was then extracted with DCM for 72 hours. It was found that, entire PETG gets extracted with DCM in 72 hours and none of the MTPS gets dissolved in DCM. Thus, the residue consists of pure MTPS and the extract is pure PETG. There results were confirmed by TGAs of residue and extract. When PETG-g-MTPS extruded resins were extracted with DCM, it was found that some PETG remains in the residue even after 72 hours. Since DCM dissolves only PETG, it was expected that after extraction the extract TGA should show a peak only for PETG and the residue should only show MTPS if no grafting reaction had occurred. However, it was observed that, the residue showed a distinct peak for PETG accounting for 25% of total weight as shown in Figure 3.6. Considering that 70% of PETG was introduced while making the blend, it was concluded from the mass balance that around 33% of added PETG was grafted on the MTPS backbone. 120 100 80 Weight % PETG 60 MTPS 40 MTPS-g- 20 PETG 0 50 150 250 350 450 550 Temperature (°C) Figure 3.5: TGA graphs for MTPS, PETG and MTPS-g-PETG 104 Figure 3.6: DTG curve of residue remaining after Soxhlet extraction 3.5.3 PETG-MTPS analysis: FTIR To confirm the grafting of PETG on MTPS, FT-IR spectra of PETG, MTPS and residue of MTPS- PETG copolymer were recorded as shown in Figure 3.7 . The MTPS spectra clearly showed presence of broad OH peak between 3100-3600 cm-1. PETG spectrum shows presence of C=O and C-C-O bonds at 1740 cm-1 and 1240 cm-1. The MTPS-g-PETG residue showed presence of both MTPS and PETG characteristic peaks. The presence of PETG in the residue was confirmed from the characteristic peaks of the ester linkage at 1741 and 1240 cm-1. 105 MTPS-g-PETG Residue PETG MTPS Transmittance C=O C-C-O 3500 2500 1500 500 Wavelength (cm-1) Figure 3.7: Comparative FT-IR spectra of PETG, MTPS and MTPS-PETG residue 3.5.4 PETG-MTPS analysis: Mechanical properties Tensile properties of PETG and MTPS-g-PETG (30:70) graft copolymer were analyzed on a universal testing machine (UTS). This data was compared to the tensile properties of PETG. The results are as shown in Figure 3.8. It was observed that inclusion of 30% MTPS in PETG caused only a 15% reduction in the tensile stress of the sample whereas the elongation properties decreased by almost 40%. However, the resulting elongation is still significant looking at the brittle nature of MTPS. The resulting blend still has acceptable mechanical properties for many applications. The results of the data are summarized in Table 3.2. 106 Figure 3.8: The average stress-strain curves of PETG and MTPS-g-PETG Table 3.2: Tensile properties of PETG and MTPS-g-PETG blend Modulus Tensile Tensile strain Tensile Elongation at (MPa) stress at at yield stress at break (%) yield (MPa) (mm/mm) break (MPa) PETG 632.7±45.7a 53.8±0.8a 2.2±0.1a 41.9 ±0.8a 215 ± 10.6a MTPS- 483.68 ± 44.5 ± 1.2b 1.5 ± 0.1b 22.9± 1.5b 120.7 ± 15.5b g-PETG 29.3b Impact properties of MTPS-g-PETG (30:70) graft copolymer were also analyzed and compared with neat PETG as shown in Figure 3.9. Around 40% reduction in notched Izod impact strength was observed for the MTPS-g-PETG reactive blend. Impact strength testing for neat MTPS could not be performed because of its extremely brittle nature. 107 7 Notched Izod Impact Strength 6 5 4 (kJ/m2) 3 2 1 0 PETG MTPS-g-PETG Figure 3.9: Notched Izod impact strength of PETG and MTPS-g-PETG reactive blend 3.5.5 MTPS-PETG analysis: Morphology Figure 3.10-a shows the morphology of the MTPS-g-PETG blends produced by extrusion after selective removal of the MTPS phase. Cavities represent the spaces occupied by MTPS particles before their selective removal by dissolution in concentrated HCL. The proportion of cavities was close to 30% which correlated with the percentage of MTPS added in the blend. Figure 3.10-a shows micron size distribution of MTPS particles evenly distributed in PETG matrix. It was observed that the particle size for MTPS was between 1-5 μm, which indicated good compatibilization between MTPS and PETG. The reduction in the mechanical properties of the blend as observed in section 3.4.3 might be due to the high amount of MTPS added and the inherent brittle nature of MTPS. The morphological behavior of neat PETG and the blend was analyzed using SEM images of tensile fracture surfaces. The representative images are as shown in Figure 3.10: b and c. The SEM images of tensile fractured surfaces of PETG showed stretch marks indicating a ductile 108 fracture whereas for the reactive blend, rougher surface was observed indicating comparatively brittle fracture. a) b) c) WD 14mm 11kV SS30 500x 50μm WD 14mm 11kV SS30 500x 50μm Figure 3.10: SEM images of a) MTPS-PETG blend after leaching of MTPS phase with HCL; b)Tensile fracture surface of PETG c) Tensile fracture surface of MTPS-g-PETG 3.5.6 MTPS-PETG analysis: Biodegradability testing Figure 3.11 shows the test setup for aqueous biodegradation according to ISO 14852. The system was kept in a dark, temperature-controlled room maintained at 30 °C. The test flasks were agitated throughout the run with the help of magnetic stirrers. Air inlet was passed through NaOH solution to get CO2-free air. This air was then divided and passed through flowmeters for each bioreactor at a constant flow rate. The CO2 evolved from the flasks was collected in NaOH solution and titrated with HCl to determine the CO2 that evolved from the samples and % biodegradation as described in Section 3.4.7. 109 Figure 3.11: Experimental aqueous biodegradation setup The initial curve obtained for aqueous biodegradation till day 80 is as shown in Figure 3.12- a. It was observed that during the first 80 days, the slope for this curve was linear and increasing. It was continued till the biodegradation reached about 30%. However, once it reached a biodegradation of 30% the biodegradation curve reached a plateau, and no further biodegradation was observed in Figure 3.12-b. This might be due to the fact that this blend contained only 30% of MTPS, which is the component that is readily biodegradable. PETG is not biodegradable in aqueous or composting environments as observed in our another study (data not shown here). Hence, once the MTPS present in the blend was consumed by the microbes, no further biodegradation was observed. From the SEM images it was confirmed that the dispersion of MTPS in PETG was uniform. So PETG was as accessible to the micro- organisms as MTPS. 110 What it looks like … b) Reality.. a) 30 100 80 % Biodegradation % Biodegradation 20 60 40 10 20 0 0 0 20 40 60 80 0 50 100 150 Days Days MTPS-PETG blend MTPS-PETG blend Increasing biodegradation with linear slope till day 80 After reaching 30% biodegradation, the curve becomes flat We should be able to extrapolate this to 100% ?? Only the 30% MTPS part biodegraded No further biodegradation observed after that. Figure 3.12: Biodegradation curve for MTPS-PETG for a)Initial 80 days b)Total 150 days Even then, only MTPS was selectively consumed leaving non-biodegradable PETG. This showed that blending of a biodegradable polymer like starch with a non-biodegradable polymer like PETG does not improve the biodegradability of the PETG. Many studies claim that addition of biodegradable polymers to non-biodegradable polymers like LDPE accelerate the biodegradation of LDPE[15], [18]. However, that is not the case. The increased biodegradation that is observed in these blends is only due to the consumption of the biodegradable polymer. The non-biodegradable part of the blend is still expected to last in the environment for a long time and form microplastics. Thus, we should be aware of the invalid the claims made for accelerated biodegradability of such plastics. 111 3.6 CONCLUSIONS AND FUTURE STUDY Maleated thermoplastic starch (MTPS) was successfully prepared by reacting glycerol with corn starch using maleic anhydride as a promoter. It was found that 79% of added glycerol was grafted on the starch during reactive extrusion. Further, this MTPS was melt blended with PETG to undergo transesterification to form in situ MTPS-g-PETG graft copolymers. It was observed that, although addition of starch reduces the strength and percent extension of the copolymer, it is still significant and hence, this polymer can be used for many applications. Currently, the grafting of PETG on MTPS is about 30% which is quite less. Hence, efforts should be done to improve the percent grafting and the effect of that on the properties of the reactive blend should also be studied. 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EFFECTS OF MODIFIED THERMOPLASTIC STARCH ON CRYSTALLIZATION KINETICS AND BARRIER PROPERTIES OF PLA 4.1 ABSTRACT This study reports on using reactive extrusion (REX) modified thermoplastic starch particles as a bio-based and biodegradable nucleating agent to increase the rate of crystallization, percent crystallinity and improve oxygen barrier properties while maintaining the biodegradability of PLA. Reactive blends of maleated thermoplastic starch (MTPS) and PLA were prepared using a ZSK-30 twin-screw extruder; 80% glycerol was grafted on the starch during the preparation of MTPS as determined by soxhlet extraction with acetone. The crystallinity of PLA was found to increase from 7.7% to 28.6% with 5% MTPS. The crystallization temperature of PLA reduced from 113 °C to 103 °C. Avrami analysis of the blends showed that the crystallization rate increased 98-fold and t1/2 was reduced drastically from 20 min to <1 min with the addition of 5% MTPS compared to neat PLA. Observation from POM confirmed that the presence of MTPS in the PLA matrix significantly increased the rate of formation and density of spherulites. Oxygen and water vapor permeabilities of the solvent-casted PLA/MTPS films were reduced by 33 and 19% respectively over neat PLA without causing any detrimental impacts on the mechanical properties (α = 0.05). The addition of MTPS to PLA did not impact the biodegradation of PLA in an aqueous environment. Significance: Given the increasing demand for novel, nature compatible materials, this chapter presents a completely biobased and biodegradable nucleating agent with a green end of life option and potential for reduced cost. 116 4.2 INTRODUCTION Replacing petro-fossil carbon with bio-based carbon in polymers offers a reduced material carbon footprint and managed end of life [1–3]. The most widely studied and commercial bioplastic is polylactide (PLA) polymer. It is manufactured commercially by NatureWorks LLC, MN, USA (https://www.natureworksllc.com/) (Accessed 2nd June 2021) (150 kton plant in Blair, Nebraska), and Total Corbion (https://www.total-corbion.com/) (Accessed 22nd September 2021) (a total capacity of 175 kton with plants in Thailand, and France). PLA is 100% bio-based and at its end-of-life recyclable [4] or industrially compostable. However, several property deficiencies in PLA have restricted its use in many packaging applications— primarily a low percentage of crystallinity and slow rate of crystallization. Nucleating agents like talc, nanocrystalline cellulose, hydrazine, PDLA, and other molecules have been used for increasing the crystallization rate and percent crystallinity of neat PLA [5–12]. PLA has mechanical and barrier properties comparable to polystyrene (PS) and thermal properties similar to polyethylene terephthalate (PET). The water vapor permeability of PLA films is low (1−4 × 10−14 kg.m2/s.m.Pa) [13–16] because of its hydrophobic nature. However, the oxygen permeability of PLA is very high as compared to PET, which limits its use in many packaging applications [17–19]. Table 4.1 shows a comparison of the literature values of mechanical, thermal, barrier, and tensile properties of some commonly used polymers in packaging including PLA, Polyethylene terephthalate (PET), Low density polyethylene (LDPE), polystyrene (PS), polypropylene (PP) and starch. 117 Table 4.1: Summary of physico-mechanical properties of packaging polymers PLA PET LDPE PS PP Starch Ref. Tensile Strength (MPa) 50–80 50–70 10–20 40 44 <6 11, 12 Glass Transition Temperature (°C) 60 70 −130 170 100 _ 11 Melting Temperature (°C) 160–170 260 110 160 222 120–150 (Td) 16, 17 Oxygen permeability 7.9–52 1.18 98–453 98.5–171 35–377 0.35–2.19 17–19 (cm³mm/m²datm) * 4, 13– Water permeability (cm3cm/cm2 139–617 130 68 123–600 35 533–3300 15, smmHg (×1010)) * 19–20 * Values from the literature have been converted to the same units for ease of comparison. 118 REX offers several advantages over traditional batch and flow reactors (CSTR, PFR) like fast reaction time, enhanced heat and mass transfer, better mixing and does not require any solvents [18]. Starch based films have shown some desirable properties like high barrier to oxygen and CO2 which is useful in packaging [27,28]. The oxygen permeability of starch films ranges between 0.4–2.5 × 10−13 cm3/m.s.Pa. Because of these advantages, different types of starch are often blended with PLA to reduce its cost and improve properties. However, pure starch and PLA blends are thermodynamically immiscible due to the hydrophobic nature of PLA and the hydrophilic nature of starch. Hence, the resulting system shows reduced strength and ductility compared to neat PLA. Several strategies have been tried to improve the compatibility by modifying either PLA or starch [29–31]. Studies have also shown the effect of starch and thermoplastic starch as a completely bio-based and biodegradable nucleating agent for PLA as opposed to inorganic talc [24]. Sun et al. studied the crystallization kinetics of PLA and starch composites and found that the addition of 1% of starch increased the crystallization rate considerably [25]. Jang et al. studied the thermal properties and morphology of PLA/starch blends using MA as compatibilizer and it was found that MA modified starch was much more compatible with PLA than pure starch [26]. Starch is hydrophilic and highly water sensitive. However, encapsulating the starch within the hydrophobic PLA matrix can mitigate this issue. This is in fact observed in several starch- based blends with various polyesters [35,36]. Multilayer films of starch and PLA have higher oxygen and moisture barrier compared to neat PLA [37–39]. There are no reports on the compatibilized blends of maleated thermoplastic starch and PLA and their effect on the properties like crystallinity, crystallization rate, barrier, thermal, mechanical, and biodegradability. 119 In this paper, we report on using inexpensive, REX modified thermoplastic starch particles in the PLA matrix to increase the rate of crystallization and percent crystallinity of PLA. The MTPS-filled PLA polymer films were found to improve oxygen and water vapor permeability without any effect on biodegradability. Crystallinity, crystallization kinetics, and barrier properties were studied and compared with neat PLA. Mechanical and thermal properties as well as morphology of the MTPS-filled thermoplastic PLA were also analyzed. This MTPS could be used as bio-based and biodegradable nucleating agent with a responsible end of life option and a replacement for talc of inorganic origin. 120 4.3 MATERIALS AND METHODS 4.3.1 Materials High amylose corn starch with an initial moisture content of 12.8% (w/w) was obtained from National Starch (NJ, USA). Glycerol was obtained from J.T. Baker (NJ, USA) and was used as received. 2,5-bis(tert-butyl-2,5-dimethylhexane), 90% (Luperox 101), and Maleic anhydride (MA) were obtained from and Sigma–Aldrich (WI, USA). IngeoTM biopolymer 3001D, a commercially available semi-crystalline grade of polylactide (PLA) was supplied from NatureWorks LLC (MN, USA). It had a molecular weight Mw of 128,000 Da and polydispersity of 1.52. It was prepared from the polymerization of L-lactide and had a meso content of 9%. 4.3.2 Preparation of and Polylactide (PLA)/MTPS Blends MTPS was prepared in a co-rotating twin-screw CENTURY ZSK-30 extruder (MI, USA) as explained in chapter 3. Next step was using this MTPS in PLA as additive. Both polylactide and MTPS quickly absorb moisture from the atmosphere. Therefore, they were dried at 55 °C for 12 h before reactive extrusion. Then, MTPS and PLA pellets were mixed in various proportions of 1–10 wt. % in an aluminum tray before feeding. The detailed compositions are listed in the table below (Table 4.2) 121 Table 4.2: Sample name and composition Sample PLA wt. % MTPS wt. % PLA 100 - PLA-1 99 1 PLA-2 98 2 PLA-5 95 5 PLA-10 90 10 The temperature profile used on the extruder going from the feed section to the die is as follows: 150/160/165/170/180/180/175/175/160/155 °C. These temperatures were selected based on the processing temperatures required for semicrystalline PLA. The screw speed and throughput were 100 rpm and 130 g/min. The extrudate was quenched in a water bath and was then pelletized. The resulting pellets were dried overnight in an oven at 50 °C and then stored in vacuum-sealed bags before using for any further characterization. 4.3.3 Soxhlet extraction Selective solubility of PLA in dichloromethane (DCM) was used to establish and determine percent covalent grafting of PLA[23,24]. About 5 g of sample was put in a pre-dried and pre- weighed cellulose extraction thimble. The thimbles were then inserted in the soxhlet extractor connected to a 500 mL round bottom flask containing around 200–250 mL DCM. The flasks were heated, and the solvent was allowed to reflux. The extraction was continued for 72 h. After the extraction, the thimbles were removed; residue and extract were separated and dried overnight at 70 °C. The dried thimble with residue was weighed again and the weight change in the residue was calculated. The reproducibility of the results was confirmed by testing three replicates for each sample. It was expected that the covalently grafted MTPS will get extracted in DCM and there will be a weight gain in the extract. Percent grafting was calculated from the mass balance as shown in Equation (1). 122 Equation 4.1 |𝑊1 − 𝑊2 | % 𝑔𝑟𝑎𝑓𝑡𝑖𝑛𝑔 = × 100 𝑊1 where, W1 is the weight of MTPS present in the sample originally and W2 is the weight of residue in the thimble after 72 h. i.e., free MTPS. 4.3.4 Thermal analysis The degradation temperature of samples was obtained by thermogravimetric analysis (TGA). TGA measurements of all the samples were conducted under an inert atmosphere of nitrogen using a TGA Q50 (TA Instruments, New Castle, DE, USA). The general sample weight used was 5–7 mg. The sample was placed in an aluminum pan and was heated to 600 °C at the rate of 10 °C/min. The weight loss (%) of a sample as a function of temperature (°C) was obtained from this analysis. Also, the thermal transitions of the samples were obtained by using a differential scanning calorimeter (DSC). The sample was heated to 200 °C in DSC Q20 (TA Instruments, New Castle, DE, USA) with a heating rate of 10 °C/min and held for 5 min to erase thermal history. The sample was then cooled back to 20 °C and heated again to 200 °C with a heating rate of 10 °C/min. The glass transition temperature (Tg), melting temperature (Tm), the crystallinity of samples (%Xc), enthalpy of melting (ΔHm), and enthalpy of cold crystallization (ΔHc) were calculated using TA universal analysis 2000 software. The % crystallinity of PLA samples was calculated from the formula given by Bher et al., 2017 [40][41]. Equation 4.2 ∆𝐻𝑚 − ∆𝐻𝑚 𝐶𝑟𝑦𝑠𝑡𝑎𝑙𝑙𝑖𝑛𝑖𝑡𝑦 (%) = × 100 ∆𝐻𝑜 (1 − α) 123 where, ΔHm and ΔHc are enthalpies of melting and crystallization respectively. α is the weight fraction of MTPS in the blends and ΔHo is the enthalpy of melting for 100% crystalline PLA which was obtained from the literature as 93.1 J/g [29, 33, 41]. 4.3.5 Isothermal Crystallization Analysis To study the isothermal crystallization kinetics, the samples were heated to 200 °C and maintained for 5 min at that temperature to remove any thermal history. Then they were cooled to the desired crystallization temperatures (90, 95, 100, 105 and 110 °C) at a rate of 20 °C/min and held at that temperature till crystallization was complete, then heated again to 200 °C to obtain the melt temperature and final crystallinity after annealing. 4.3.6 Polarized Optical Microscopy (POM) POM observation was performed on an Olympus BH-2 microscope (Olympus corp., Japan) with crossed-polarizers, equipped with a digital camera system and a Mettler Toledo FP82 (Ohio, USA) hot stage. All the samples were first inserted between two microscope coverslips and squeezed at 200 °C to obtain a thin slice. The films were held at 200 °C for 2 min to achieve thermal equilibrium. This was followed by rapid cooling to the selected crystallization temperature of 105 °C. The polarized optical micrographs during isothermal crystallization were recorded after every 90 s to monitor the formation and growth of crystallites. 4.3.7 Mechanical Properties The injection molded test bars were prepared using a tabletop DSM 15 cc mini extruder (DSM Research B. V., Sittard-Geleen, The Netherlands) and 3.5 cc mini-injection molder (DACA Instruments, Santa Barbara, CA, USA). The injection pressure was set as 140 psi and the cylinder and mold temperatures were 200 and 65 °C, respectively. The samples were 124 stored for 2 days at 25 °C in a humidity chamber with RH of 50% before any analysis. Tensile testing was performed using an Instron model 5565-P6021 (Instron, Norwood, MA, USA) with a 5 kN load cell and grip separation speed of 12.5 mm/min as per ASTM D882. Data from five samples of each formulation were averaged and compared with the properties of neat PLA. 4.3.8 Scanning electron microscopy A JOEL 6610 LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) was used to study the dispersion of MTPS in PLA using the tensile fracture surfaces of all samples. The tensile bars were immersed in liquid nitrogen for ~2 min and then fractured. Fracture surfaces were mounted on aluminum stubs using high vacuum carbon tabs and coated with gold using a sputter coater. A different set of bar specimens were also treated with 6 N HCl for 12 h to remove the MTPS phase from the samples and air-dried for 12 h in a fume hood. Then they were mounted on aluminum stubs as explained before and examined using JOEL at 500× magnification at 10 kV. 4.3.9 Barrier properties The barrier properties were measured using MOCON instruments (OX-TRAN Model 2/21 and PERMATRAN-W Model 3/33). All the measurements were undertaken at 50% RH for oxygen and 100% for water vapor. Circular films of 3.14 cm2 area were used. The thickness of the samples was measured using a micrometer (TMI) and was used to calculate the permeability to oxygen and moisture. Water vapor permeability (WVP) is given as: Equation 4.3 𝑊𝑉𝑇𝑅 𝑊𝑉𝑃 = × 𝑇ℎ ∆𝑃 125 Oxygen permeability (OP) was calculated from oxygen transmission rate (OTR) data using Equation (4): Equation 4.4 𝑂𝑇𝑅 𝑂𝑃 = × 𝑇ℎ ∆𝑃 where, WVTR is water vapor transmission rate, OTR is oxygen transmission rate, Th (m) was the thickness of the sample and ΔP was the pressure difference between both sides of the sample (Pa) [34]. 4.3.10 Aqueous biodegradability Testing The biodegradability of neat PLA and PLA + 5% MTPS samples was tested in an aqueous environment using ISO 14852 as explained in chapter 2. All the tests were performed in an aerobic environment at 30 °C. 126 4.4 RESULTS AND DISCUSSION MTPS and all the PLA/MTPS blends were prepared in a Century ZSK-30 twin screw extruder (MI, USA). MTPS was first characterized to ensure sufficient grafting of glycerol and then it was used for blending with PLA. 4.4.1 Percent Grafting for MTPS-PLA blends Soxhlet extraction was carried out for the blends of PLA and MTPS as well using DCM as extraction solvent. It was found that the weights of residues and extracts did not change after the extraction. Entire PLA was extracted in the solvent and MTPS remained in the residue. There were no additional peaks in residue or extract TGA which could represent any sign of reaction between PLA and MTPS. Hence, it was concluded that there was no reaction between MTPS and PLA. 4.4.2 Mechanical Testing and Phase Morphology Figure 4.1 illustrates the modulus, tensile stress, and strain for neat PLA and the various blends. Table 4.3 summarizes all the data of tensile stress at yield, tensile stress at break, modulus, and strain at break with averages and standard deviations. The analysis of the tensile testing of neat PLA samples reveals a characteristic brittle behavior of PLA with tensile strength values of ~82 MPa and elongation at break ~8%. The results indicated that there was no significant change in the modulus, tensile stress, and strain after addition of 1% and 2% MTPS (p > 0.05). Only for 5% MTPS containing blends, the modulus increased by 15% indicating they were stiffer than neat PLA whereas tensile stress reduced by 12%. Increasing MTPS content in PLA increased the brittleness of PLA bars. Similar results were observed by Wootthikanokkhan 2012 et al. [45] where the modulus of the samples was found to increase with an increasing per-centage of starch at the expense of elongation and 127 tensile toughness. For PLA + 10% MTPS, the samples became brittle and broke after loading on the tensile machine. Hence, the readings for modulus, stress, and strain were not recorded. Because of the reduction in mechanical properties, they were not considered for any further analysis. This reduction in mechanical properties could be due to the reduced compatibilization and increased particle size of the MTPS particles in the PLA matrix. This was observed in the SEM images of the samples as explained in Figure 4.2 and Figure 4.3 Table 4.3: Effect of MTPS content on tensile properties of PLA Tensile Stress at Tensile Strain at Tensile Stress Modulus Materials Yield Break at Break (MPa) (MPa) (mm/mm) (MPa) PLA neat 1796.45 ± 110.08 a 82.10 ± 1.14 a 0.077 ± 0.004 73.30 ± 2.74 PLA-1 1825.34 ± 47.76 a 80.67 ± 1.60 a 0.073 ± 0.005 72.76 ± 2.01 PLA-2 1748.27 ± 44.03 a 78.210 ± 0.65 a 0.068 ± 0.002 73.63 ± 2.17 PLA-5 2059.88 ± 47.09 b 72.38 ± 1.14 b 0.066 ± 0.005 67.77 ± 3.97 Different superscript letters within the same column indicate significant differences among formulations (p < 0.05) 2.5 100 Tensile stress at break 2 80 Modulus (GPa) 1.5 60 1 (MPa) 40 0.5 NA 20 NA 0 0 PLA neat PLA-1 PLA-2 PLA-5 PLA-10 PLA neat PLA-1 PLA-2 PLA-5 PLA-10 Samples Samples 10% Tensile strain at break (%) 8% 6% 4% 2% NA 0% PLA neat PLA-1 PLA-2 PLA-5 PLA-10 Samples Figure 4.1: Modulus, tensile stress, and tensile strain graphs for PLA and blends 128 Figure 4.2: Scanning electron microscopy (SEM) images of fractured tensile surfaces of (a) PLA, (b) PLA + 1% MTPS, (c) PLA + 2% MTPS, (d) PLA + 5% MTPS Figure 4.3: SEM images of the blends after selective extraction of MTPS phase (a) PLA, (b) PLA + 1% MTPS, (c) PLA + 2% MTPS, (d) PLA + 5% MTPS 129 The morphological behavior of neat PLA and the blends was analyzed using SEM images of tensile fracture surfaces. The main results are reported in Figure 4.2. The tensile fracture surface of neat PLA shows a smooth and featureless surface which is an indicator of typical brittle behavior. Figure 4.2-b, c and d show presence of small spherical MTPS particles (shown with dotted yellow circles) with good interfacial adhesion with PLA. They showed a smooth surface and were well wetted by the PLA. The size of MTPS particles seemed to increase with increasing content of MTPS (Figure 4.2-b vs d). However, the number of MTPS particles did not increase much and their effect on thermal properties was also negligible. Figure 4.3 shows the morphology of the PLA matrix and the PLA/MTPS blends produced by extrusion after selective removal of the MTPS phase. Cavities represent the spaces occupied by MTPS particles before their selective removal by dissolution in concentrated HCl. The proportion of cavities was well correlated with the percentage of MTPS added in the particular blend. Figure 4.3-b to d all show micro size distribution of MTPS particles in the PLA matrix. The size of the cavities increased with increase of MTPS content which suggested a reduction in compatibilization of MTPS. The particle size for MTPS increased from 4.1 ± 1.5 um for 1% MTPS to 9.5 ± 3.5 um for 5% MTPS blends. An increase in the domain size is indicative of less compatibilization between PLA and MTPS. This could explain the reduction in mechanical properties for 5% and 10% MTPS containing blends. The 1% and 2% MTPS (Figure 4.3-a,b) had smaller size and more spherical particles which indicated greater compatibilization, and hence no reduction in tensile strength was observed compared to neat PLA. These particle sizes of 4–9 um were much smaller as compared to the 30 um size observed by Clasen SH et al. (2015) in the PLA-TPS blends without any compatibilizer[35]. 130 4.4.3 Thermal analysis Thermogravimetric (TGA) and derivative (DTG) graphs of the blends containing 1%, 2%, 5%, and 10% MTPS along with neat PLA are shown in Figure 4.4. With increment in MTPS until 5%, the weight loss in the first part of the curve (< 200°C), was almost the same and was found to be less than 0.5% which is a significant feature since it represents the range and possible process temperatures for the blends after production. With 10% MTPS, the weight loss until 200 °C increased to 0.7%. Thermal stabilities of the blends were also characterized by the temperatures at which 5% (T5%), 10% (T10%), and peak wt. loss (Tpeak) occurred. Increasing the percentage of MTPS caused a steady decrease in 5% and 10% wt. loss temperatures. The peak degradation temperatures shifted towards the lower temperature with increase in the percentage of MTPS. The results are summarized in Table 4.4. Table 4.4: Thermogravimetric analysis (TGA) of PLA and blends Sample T5% (°C) T10% (°C) Tpeak (°C) PLA 337.4 347.7 377.5 PLA-1 333.4 342.7 371.9 PLA-2 327.1 336.9 371.0 PLA-5 308.4 320.9 343.3 PLA-10 295.68 310.7 342.6 131 120 a) 100 Neat PLA PLA+1% MTPS 80 PLA+2% MTPS PLA+5% MTPS Wt % 60 PLA+ 10% MTPS 40 20 0 0 200 400 600 Temperature (°C) 3.5 b) 3 Neat PLA PLA+1% MTPS 2.5 derivative wt % PLA+2% MTPS 2 PLA+5% MTPS PLA+ 10% MTPS 1.5 1 0.5 0 100 200 300 400 500 600 Temperature (°C) Figure 4.4: a)TGA and b)DTG curves for PLA and PLA/MTPS blends The glass transition temperature Tg (°C), crystallization temperature Tc (°C), melting temperature Tm (°C), enthalpy of crystallization (J/g), enthalpy of melting (J/g), and crystallinity (%) data of PLA and modified PLA pellets were determined from the DSC analysis and are given in Table 4.5. All the thermal properties were obtained from the second 132 heating curve (Figure 4.5). Thermal degradation and mechanical properties of PLA with 10% MTPS (explained in Section 4.4.2) were significantly lower as compared to neat PLA. Hence, that sample was not used for further testing of isothermal crystallization, barrier properties, etc. Figure 4.5 shows the second heating curves for PLA and PLA/MTPS blends. Tg was found to decrease negligibly with addition of MTPS to PLA whereas Tc reduced from 113.8 to 103.1 °C. In our opinion, this decrease might be due to the migration of some glycerol from the MTPS phase to the PLA phase. This might lead to the formation of plasticized PLA with lower Tg. Table 4.5: Thermal transition temperatures and percent crystallinity of modified PLA samples % ΔHm % Crystallinity Tg (°C) Tm (°C) Tc (°C) ΔHc (J/g) (J/g) Crystallinity After Annealing PLA neat 63.30 171.50 113.86 40.94 33.74 7.74 43.18 PLA + 1% 62.30 171.00 111.40 40.72 31.13 10.42 47.28 MTPS PLA + 2% 61.38 170.57 108.46 45.45 32.55 14.15 48.20 MTPS PLA + 5% 59.50 161.40 103.10 45.03 19.71 28.66 50.61 MTPS 133 Heat flow (J/g) exo up 30 80 130 180 Temperature (°C) PLA neat PLA+1% MTPS PLA+2% MTPS PLA+5% MTPS PLA + 10% MTPS Figure 4.5: Differential scanning calorimetry (DSC) thermograms of PLA and the blends From DSC of neat PLA, it seemed that there was just a little endothermic peak on the curve of pure PLA and the crystallinity was about 7.74%. After adding MTPS, an exothermic peak appeared on the heating curve. In the course of heating, more crystals were formed and hence the crystallinity of PLA increased with increasing concentration of MTPS suggesting its function as a nucleating agent similar to starch [24]. The final crystallinity of the samples was also calculated after annealing of the samples as explained in the isothermal crystallization analysis. Figure 4.6 shows the melting curves for annealed samples. The final crystallinity of the samples after annealing increased to 50.6% with 5% MTPS compared to 43% for neat PLA. 134 Heat flow (J/g) exo up 130 150 170 190 Temperature (°C) Neat PLA PLA +1 % MTPS PLA+ 2% MTPS PLA + 5% MTPS Figure 4.6: Melting curves for PLA blends after annealing 4.4.4 Isothermal Crystallization Kinetics The isothermal crystallization isotherms of PLA and the blends obtained by cooling the molten polymer to the selected crystallization temperature (Tc) are as shown in Figure 4.7. The shape of the exotherm was dependent on Tc. The time required for crystallization was found to be minimum at 100 °C. Above and below that temperature, the isotherm became flatter, and the time required for complete crystallization increased. A similar effect was observed for the PLA/MTPS blends as well. Fractional crystallinity Xt vs time is the ratio of the area of the endotherm until time t divided by the total area of the endotherm, as shown in Equation (7). 135 Equation 4.5 𝑡 𝑑𝐻𝑐(𝑡) 𝑋𝑐 (𝑡) ∫ ∫0 𝑑𝑡 𝑑𝑡 𝑋𝑡 = = 𝑋𝑐 (𝑡∞ ) ∞ 𝑑𝐻𝑐(𝑡) ∫0 𝑑𝑡 𝑑𝑡 where Hc is the heat flow at time t, and 𝑡∞ is the end time for complete crystallization. a) 0.4 b) 0.4 Neat PLA PLA+1% MTPS 0.3 Heat flow (J/g) 90°C 95°C 0.3 Heat flow (J/g) 90°C 95°C 100°C 105°C 0.2 110°C 0.2 100°C 110°C 0.1 0.1 0 0 0 10 20 30 40 0 10 20 30 40 Time (Min) Time (Min) c) 0.4 d) 0.4 PLA+2% MTPS PLA+5% MTPS 0.3 0.3 Heat flow (J/g) Heat flow (J/g) 90°C 95°C 90°C 95°C 100°C 105°C 100°C 105°C 0.2 110°C 0.2 110°C 0.1 0.1 0 0 0 10 20 30 40 0 10 20 30 40 Time (Min) Time (Min) Figure 4.7: DSC melting thermograms of a)PLA and blends (b) PLA+1%MTPS, c) PLA+2%MTPS, d) PLA+5% MTPS )at various temperatures Typical crystallization isotherms showing the degree of crystallinity X(t) vs time were plotted for PLA and the blends as shown in Figure 4.8. It was observed that the rate of crystallization increased with an increasing amount of MTPS addition and was fastest for neat PLA as well as all the blends for the temperature of 100 °C. The Avrami Equation (Equation 4.6) was used for studying the isothermal crystallization behavior of PLA and the blends. It is often written in logarithmic form as shown in Equation 4.7. 136 Equation 4.6 𝑋(𝑡) = 1 − 𝑒𝑥𝑝(−𝑘𝑡 𝑛 ) Equation 4.7 𝑙𝑛 [− 𝑙𝑛(1 − 𝑋(𝑡)] = 𝑛𝑙𝑛𝑡 + 𝑙𝑛𝑘 where X(t) is the fractional crystallinity at time t, k is the overall kinetic rate constant, and n is the Avrami exponent, which depends on the mechanism of nucleation and the form of crystal growth. The rate constant k contains the nucleation and growth parameters for crystallization.It was observed that the total crystallization time was reduced significantly (<8 min for 5% MTPS) by the addition of MTPS as compared to neat PLA (~20 min at 100 °C). The half time (t1/2) was calculated and reported for all the temperatures in Table 4.6. Avrami plots of ln[−ln(1−X(t))] versus ln(t) were plotted to obtain the values of k and n as shown in Figure 4.9. The crystallization rates (k) were much higher for the blends containing MTPS as compared to neat PLA at all temperatures indicating that MTPS increased the crystallization rate of PLA. Also, from Avrami analysis results, two-dimensional crystal growth was observed as n values were around 2 [5]. Thus, it can be concluded that MTPS was acting as a nucleating agent for PLA. The half crystallization time, t1/2, the time in which 50% of the total crystallinity is achieved, was calculated using Equation 4.8. Equation 4.8 2 1/𝑛 𝑡1/2 = 𝑙𝑛 (𝑘) 137 a) 1.2 b) 1.2 Neat PLA Fractional crystallinity PLA+1% MTPS Fractional crystallinity 1 1 0.8 90°C 90°C 95°C 0.8 95°C 0.6 100°C 0.6 100°C 0.4 105°C 110°C 0.4 0.2 0.2 0 0 0 20 40 0 20 40 Time (Min) Time (Min) 1.2 d) 1.2 c) PLA+ 5% MTPS Fractional crystallinity PLA +2% MTPS Fractional crystallinity 1 1 90°C 0.8 90°C 0.8 95°C 95°C 0.6 0.6 100°C 100°C 105°C 0.4 105°C 0.4 0.2 0.2 0 0 0 20 40 0 20 40 Time (Min) Time (Min) Figure 4.8: Fractional crystallinity vs. time of a)PLA and blends (b) PLA+1%MTPS, c) PLA+2%MTPS, d) PLA+5% MTPS )at various temperatures Saddle-shaped curves were obtained by plotting Tc vs. t1/2 for PLA and its blends with MTPS as shown in Figure 4.10. As the MTPS content increased, t1/2 values decreased and the rates for crystallization k were found to increase. The minimum t1/2 for neat PLA was observed as 6.83 min at 100 °C and 1.66 and 0.94 min for 2% and 5% of MTPS at 100 °C. 138 a) 3 b) 3 Neat PLA PLA +1% MTPS 1 1 -1 -1 90°C 90°C ln(-ln(1-Xt)) ln(-ln(1-Xt)) -3 95°C -3 95°C -5 100°C -5 105°C 100°C -7 -7 110°C 110°C -9 -9 -11 -11 -2 0 2 4 -2 0 2 4 ln(t) ln(t) c) d) 3 3 PLA + 2% MTPS PLA+5% MTPS 1 1 -1 90°C -1 90°C ln(-ln(1-Xt)) ln(-ln(1-Xt)) -3 95°C -3 95°C -5 100°C -5 100°C 105°C 105°C -7 110°C -7 110°C -9 -9 -11 -11 -2 0 2 4 -2 -1 0 1 2 ln(t) ln(t) Figure 4.9: Plots of ln(ln(1-X(t)) vs ln t of a)PLA and blends (b) PLA+1%MTPS, c) PLA+2%MTPS, d) PLA+5% MTPS )at various temperatures 24 20 16 t 1/2 (min) 12 8 4 0 85 95 105 115 Temperature °C Neat PLA PLA +1% MTPS PLA +2% MTPS PLA+5% MTPS Figure 4.10: Half time for crystallization vs isothermal crystallization temperatures for PLA and blends 139 The values for t1/2 obtained for PLA/MTPS blends were compared with several nucleating agents including starch, talc, CNC, wood flour, polyoxymethylene, etc. [5,7–9,11,12,33,46,47] from the literature. Figure 4.11 shows the comparison for t1/2 values of other nucleating agents compared to MTPS. The t1/2 values for MTPS were found to be lower compared to all the other nucleating agents except talc. Talc is one of the most effective nucleating agents for PLA. 1–2% of talc is commonly added to decrease the t1/2 of PLA to less than one minute [25]. Thus, MTPS, although not as effective as talc, could be a completely bio-based and biodegradable nucleating agent as opposed to talc of inorganic origin. Figure 4.11: Comparing the t1/2 values of PLA/MTPS blends with other nucleating agents The effect of MTPS on crystal morphology and size was studied using polarized optical microscopy. Figure 4.12 shows the morphology of crystals for all the compositions after crystallization at 105°C for 6 min. As expected, neat PLA showed larger size spherulites and 140 the rate of formation of spherulites was less as compared to other samples. As shown in Figure 4.12-b, neat PLA started forming the spherulites well after holding it at 105°C for 3 minutes, whereas, for all other PLA/MTPS samples we could see a good number of spherulites by that time (Figure 4.12-e, h and k). For PLA+5% MTPS, this was even faster, and the crystals were visible within 90 sec (Figure 4.12- j). This agreed well with the k values obtained by Avrami analysis for the samples which indicated 98-fold faster crystallization for PLA+ 5% MTPS at 100°C. This can be attributed to the nucleation effect of MTPS, which provides much more heterogeneous nuclei, reduces the spherulite size, and speeds up the crystallization process. Neat PLA PLA + 1% MTPS PLA + 2% MTPS PLA + 5% MTPS Figure 4.12: Polarized optical microscopy (POM) images of PLA/MTPS blends with MTPS content of 0%: (a–c); 1%: (d–f); 2%: (g–i); 5%: (j–l) 141 Table 4.6: Crystallization half times and Avrami constants for PLA samples at different temperatures Temperature Sample t1/2 (min) n ln k k (°C) 90 20.03 2.520 −7.919 3.6410-4 95 8.36 1.890 −4.380 1.2510-2 Neat PLA 100 6.83 2.320 −4.825 8.0310-3 105 7.32 2.420 −5.184 5.6110-3 110 12.21 2.240 −5.972 2.5510-3 90 8.12 2.198 −4.969 6.9510-3 PLA + 1% 95 4.19 2.127 −3.412 3.3010-2 MTPS 100 2.03 1.797 −1.641 1.9410-1 110 2.91 2.084 −2.593 7.4810-2 90 7.54 2.619 −5.657 3.4910-3 95 3.83 1.964 −3.004 4.9610-2 PLA + 2% 100 1.66 1.811 −1.289 2.7610-1 MTPS 105 2.10 1.946 −1.809 1.6410-1 110 2.55 1.958 −2.200 1.1110-1 90 2.94 1.960 −2.479 8.3810-2 95 1.06 1.410 −0.447 6.4010-1 PLA + 5% 100 0.94 2.010 −0.242 7.8510-1 MTPS 105 0.82 1.780 −0.018 9.8210-1 110 1.12 1.740 −0.566 5.6810-1 4.4.5 Permeability Studies Starch based films have demonstrated their good oxygen barrier properties in previous studies [13,42,37–39]. Figure 4.13 summarizes the effect of MTPS on WVP and OP of PLA films. A decrease of 33% and 27% in oxygen permeability was observed by adding 5% and 1% MTPS, respectively. This improvement can be attributed to increased crystallinity. Crystalline regions in PLA form the impermeable regions which create a tortuous path for the diffusion for permeants, which leads to lower permeability [48–52]. Also, high oxygen barrier properties of starch might also be helpful in reducing the OP of the films with blends. WVP and OP show a significant reduction for the addition of 1% MTPS, whereas it becomes less significant as the concentration is increased to 2% and 5%. This could also be explained 142 by the particle size of MTPS observed from the SEM analysis. The 1% MTPS blends had an average particle size of 4.1 ± 1.5 um whereas, for 5% MTPS, the size was almost double: 9.5 ± 3.5 um. More small particles could have caused a greater number of tortuous paths leading to reduced permeability. The water vapor permeabilities of the blends did not show any significant increase even after addition of hydrophilic MTPS. This might be due to the morphology of the blend in which the MTPS particles were observed to be surrounded by hydrophobic PLA matrix thus shielding it from water (Figure 4.3 and Figure 4.2). Figure 4.13: Effect of MTPS content on moisture permeability (WVP) and oxygen permeability (OP) of PLA films 4.4.6 Biodegradability Studies Figure 4.14 shows the test setup for aqueous biodegradation according to ISO 14852. The system was kept in a dark, temperature-controlled room maintained at 30 °C. The test flasks were agitated throughout the run with the help of magnetic stirrers. Air inlet was passed through NaOH solution to get CO2-free air. This air was then divided and passed through flowmeters for each bioreactor at a constant flow rate. The CO2 evolved from the flasks was 143 collected in NaOH solution and titrated with HCl to determine the CO2 that evolved from the samples and % biodegradation as described in chapter 2. Figure 4.14: Experimental aqueous biodegradation setup Figure 4.15: Aqueous biodegradation curves for PLA and PLA + 5% MTPS 144 The average % biodegradation curves for cellulose and PLA and PLA + 5% MTPS at 30 °C are as shown in Figure 4.15. It was observed that neat PLA and PLA + 5% MTPS have almost the same biodegradation curve. Both showed negligible biodegradation in aqueous environment (<10%) at the end of the test. Therefore, it can be concluded though MTPS acted as a nucleating agent and increased the crystallinity and crystallization rate of PLA, it was well embedded in the hydrophobic PLA matrix and hence did not change the biodegradation properties of PLA. Poor biodegradability of PLA was attributable to the temperature at which the aqueous biodegradation test was carried out: 30 °C. The glass transition temperature of PLA is 58 °C. Below this temperature, PLA does not biodegrade easily due to the polymer segments behaving as a glass with little or no mobility of the polymer chains. Hence, no difference could be observed at lower temperature studies. Similar to any chemical reaction the rate of biodegradation depends on temperature and is expected to increase as temperature increases. A higher biodegradation rate is expected in the high temperature composting environment testing which is the ideal environment for PLA biodegradation. PLA reaches 80–90% biodegradation within 60–90 days in composting environment at temperatures of 58 °C as observed in several studies [53–55]. These compatibilized blends with maleic anhydride are also expected to have higher biodegradability as compared to PLA and pure starch blends in the composting environment [34,56]. During composting, the presence of MA might lead to the formation of an acid group due to its reaction with water. That can accelerate the chain scission in PLA resulting in faster biodegradation [47]. Further testing for biodegradation in composting environment needs to be done to validate this hypothesis. 145 4.5 CONCLUSIONS Maleated thermoplastic starch (MTPS) was successfully prepared by reacting glycerol with corn starch using maleic anhydride as a promoter. It was found that 79% of added glycerol was grafted on the starch during reactive extrusion. The dual effect of MTPS as a nucleating agent and barrier property enhancer for PLA was studied. MTPS increased the rate of crystallization of PLA significantly (98-fold at 100 °C). A decrease in glass transition and thermal degradation temperature was observed with increasing concentration of MTPS. Percent crystallinity of PLA increased from 7.7% to 28.6% by addition of 5% MTPS whereas total crystallinity of the blends was as high as 50.6% after annealing. SEM images of tensile fractured samples showed good interfacial adhesion and wetting between MTPS and PLA. The 1% MTPS showed the best compatibilization with domain sizes of 4.1 ± 1.5 um as observed from the SEM images of tensile bars after selective extraction of the MTPS phase. Melt isothermal crystallization kinetics using Avrami analysis showed a drastic reduction in half crystallization time t1/2 from 20 min to less than 1 min with addition of 5% MTPS. An increased crystallization rate was also confirmed by POM images of neat PLA and the blends. More number of smaller spherulites were observed with an increasing percentage of MTPS in the blends. MTPS was found to be more effective as compared to many other nucleating agents used for PLA such as starch, CNC, wood flour, polyoxyethylene, etc. Oxygen permeability values of PLA were reduced by 27% by the addition of just 1% MTPS whereas water vapor permeability values remained constant. No significant change in the mechanical properties of the blends was observed as opposed to neat PLA until 5% addition of MTPS by weight. A small decrease in tensile stress and elongation at break was observed after addition of 10% MTPS which could be explained by the brittle nature of MTPS. There was no 146 significant increase in the aqueous biodegradability of these blends compared to neat PLA, which suggested that the MTPS particles were well embedded in the hydrophobic PLA matrix. It is expected that the presence of starch and MA will enhance the biodegradability of these blends in a composting environment. Further studies need to be undertaken to obtain the actual experimental data supporting this hypothesis. These MTPS/PLA blends have demonstrated improved barrier and crystallization properties and similar mechanical, thermal and biodegradation properties to neat PLA. These blends have a potential for reduced cost due to use of an inexpensive, naturally abundant, completely bio-based, and biodegradable nucleating agent and can find applications in several food contact packaging purposes. 147 REFERENCES 148 REFERENCES [1] R. Narayan, Degradable Polymers and Materials: Principles and Practice (2nd Edition), vol. 1114. Washington, DC: American Chemical Society, 2012. doi: 10.1021/bk- 2012-1114. [2] R. Narayan, “Biobased & biodegradable plastics: Rationale, drivers, and technology exemplars,” ACS Symposium Series, vol. 1114, pp. 13–31, Nov. 2012, doi: 10.1021/bk-2012- 1114.ch002. [3] K. L. Law and R. Narayan, “Reducing environmental plastic pollution by designing polymer materials for managed end-of-life,” Nature Reviews Materials, 2021, doi: 10.1038/S41578-021-00382-0. [4] R. Narayan, “Opinion Missing the point about biodegradability,” Bioplastics Magazine, pp. 38–39, 2020. [5] M. Nofar, W. Zhu, C. B. Park, and J. 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Thermogravimetric (TGA) and isothermal TGA analysis of the pellets showed that there was no significant loss of iodine due to sublimation during reactive extrusion. The antifungal and antibacterial activity of the solid pellets was analyzed using dip coating on strawberries and Kirby Bauer test. These pellets were further blended with PLA-PBAT film formulations in different percentages to make blown films. Iodine containing blown films showed superior antibacterial activity against E. coli compared to PLA control films and the commercial silver antimicrobial containing films. SEM analysis of the films showed a non-uniform dispersion of starch-iodine complex in the film. Tensile strength and elongation at break in machine direction for the starch-iodine containing films was comparable to the control films in machine direction (MD) whereas tear strength was found to be better for the starch-iodine containing films in transverse direction (TD). 154 5.2 INTRODUCTION Iodine is a well-known antimicrobial agent, used for more than a century in the pharmaceutical/medical industry[1, 2]. It has a broad spectrum of antimicrobial activity with efficacy against bacteria, mycobacteria, fungi, protozoa and viruses and can be used to treat both acute and chronic wounds. Tincture of iodine is a mixture of iodine and sodium/potassium iodide in water and ethanol and has been widely used in sterilization. Various patents and papers also describe incorporation of iodine into fabrics, polymers, and other materials for the sole purpose of antibacterial and antiviral activity[3–6]. The main disadvantage associated with iodine is its instability. A unique feature of iodine is its ability to bind with various polymeric materials[1]. Recently, a study was shown that povidone- iodine (PVP-I), a widely used antiseptic for skin disinfection, can be used to rapidly and effectively eliminate SARS-CoV-2 at a reduction rate >99.99% with a contact time of 30 s[2]. However, PVP-I is known for its toxicity at higher doses, and it has found limited uses in applications (automotive, packaging, etc.) outside the medical field[7]. As a biobased, biodegradable material, starch is a naturally occurring resource which is biocompatible and safe for humans. Amylose in starch is known to form complexes with iodide polyanions (blue colored complex). Various studies for structures of amylose-iodine complex have shown that amylose exists in the form of a helical spiral with each turn of the helix containing 6 glucose units and the iodine molecules get arranged along the spiral as triiodide ions[8–10]. Cross-linked starch iodine (CSI) has been studied for its antibacterial, antiviral activity[1112]. Reactive extrusion has allowed mass production of thermoplastic starch (TPS) and maleated thermoplastic starch (MTPS)[13]. This TPS and MTPS is often added to other polymers like PLA, PBAT, PCL to improve their biobased content, certain 155 performance properties like oxygen barrier or crystallization, biodegradation etc., and to reduce the cost of the blends[14–16]. Iodine can be added during thermoplastic starch production in the extruder to make these thermoplastic starch-iodine complex pellets in solid form which can be used as a vehicle to introduce iodine in an environment, without the usual hazards associated with handling solid iodine. Targeted applications of this product could be - (1) As an additive for film to be applied in active packaging for food-safe packaging, (2) As an additive to make foams or filters to be used in air filtration systems for deactivating airborne viruses, including SARS-CoV-2, and (3) In pellet form as an additive to provide antibacterial, antiviral properties in other plastics and polymers. In the present study, solid thermoplastic starch-iodine complex was prepared in the form of pellets in ZSK 30 twin screw extruder. These pellets were characterized for iodine content and their antimicrobial activity. Further, this complex was added in various proportions to PLA-PBAT blown films and the morphological, mechanical, thermal, and antibacterial properties of these films were evaluated. 156 5.3 STARCH IODINE COMPLEX As explained in the previous chapters, starch is a mixture of amylose and amylopectin. In the Amylose unit all monomers are D-glucose which have -1,4 linkages. Because of the bond angles and arrangement of these acetal linkages, amylose forms a spiral spring like structure as shown in the Figure 5.1 [17] Figure 5.1: Amylose coil structure - due to acetal linkages Iodine is not very soluble in water. Iodine is dissolved in water in presence of potassium iodide (KI) to form a linear triiodide (I3-) complex that is soluble in water. This triiodide molecule then slips up inside the hollow amylose helix. Water molecules interact with iodide to form a charge transfer complex which causes the blue color of the complex as shown in the Figure 5.2. 157 Figure 5.2: Structure of amylose-iodine complex 158 5.4 EXPERIMENTAL SECTION Materials High amylose corn starch with an initial moisture content of 12.8% (w/w) was obtained from National Starch (NJ, USA). Glycerol was obtained from J.T. Baker (NJ, USA) and was used as received. 2,5-bis(tert-butyl-2,5-dimethylhexane), 90% (Luperox 101), and Maleic anhydride (MA) were obtained from and Sigma–Aldrich (WI, USA). Molecular Iodine and Potassium iodide were obtained from Thermo Fisher Scientific (MA, USA). Strawberries were bought from a local grocery store and were washed with distilled water and dried before use. The bacterial strain E. Coli (TB0484) was used for bacterial cultivation and antimicrobial activity tests. Preparation of MTPS-Iodine pellets The MTPS-iodine pellets were prepared in co-rotating twin-screw CENTURY ZSK-30 extruder (MI, USA) using a process similar to described in Kulkarni;2021[15] . The optimum quantity of iodine and potassium iodide to be added in the high amylose corn starch was calculated as shown in the appendix 1. For 1 kg of total masterbatch, Potassium Iodide 26.9 gm (0.162 mol) was dissolved in 40 ml of water. To this solution, 53.2 g (0.419 mol) of solid Iodine was added with stirring. The result was a black/blue colored solution of iodide anions in water. Pre-dried high amylose corn starch (800g) was mixed with glycerol (200g) to make 1000 gm of starch glycerol mixture. To this mixture, 20 gm of maleic anhydride and 2 gm of Luperox 101 were added. Then the Iodide solution was added to form a deep blue colored powder. This powder was then extruded in a Century ZSK-30 co-rotating twin screw extruder using temperature profile as 60/80/100/110/120/120/130/140/140/130. Other screw speed, feed rate and vent conditions were the same as that used for MTPS [15] The 159 extrudate coming out of the extruder was air-cooled and pelletized simultaneously using Scheer Bay pelletizer as shown in Figure 5.3. Iodine + KI Starch + (aq.) glycerol + maleic anhydride Starch- Iodine complex Active packaging Filters Antibacterial & antiviral • Manufacturer – Natur-tec® • Air filtration systems additive for plastics and • Masks polymers Figure 5.3: Production of MTPS-iodine pellets via reactive extrusion Preparation of PLA-PBAT-Starch-Iodine films The MTPS-iodine masterbatch pellets were shipped to Natur-tec® (MN, USA) for production of blown films. The MTPS-iodine pellets were mixed in 10 and 18% by wt. with commercial PLA and PBAT film formulations (detailed formulation not disclosed here) to yield films with 0.7% and 1.3 % iodine in the resulting films. A LabTech LE20-30/C (Thailand) extruder with 20mm diameter screw with L:D of 30:1 and LabTech LF-250 blown film frame (Thailand) with die diameter of 2 inches were used for making these films. The detailed temperature and processing conditions for making the films were as shown in Table 5.1.Three different films with different thickness and iodine content were prepared as shown in the Table 5.2 160 and their properties were compared with neat PLA blown films and a commercial antimicrobial film supplied by Natur-tec® with silver as the antimicrobial agent. Table 5.1: Temperature and processing conditions for making blown films Upper Lower Die Zone Zone Zone Screw Upper Lower Motor Die Die Adapter 3 2 1 RPM Pull Pull Amps Temp Temp Temp (°C) (°C) (°C) Rate Rate (°C) (°C) (°C) (ft/min) (ft/min) 170 170 165 160 154 148 100 10 10.5 77 Table 5.2: Sample name and composition Formulation code Iodine content Film thickness SI1 0.7 % 1 mil (25.4 μm) SI2 0.7% 2 mil (50.8 μm) SI3 1.3% 2 mil (50.8 μm) PLA film - 1 mil (25.4 μm) AM film Silver Antimicrobial present 1 mil (25.4 μm) 161 5.5 CHARACTERIZATION AND ANALYSIS Thermogravimetric analysis (TGA) The MTPS-iodine pellets, solid iodine and neat MTPS pellets without any iodine were analyzed using TGA. TGA measurements of all the samples were conducted under an inert atmosphere of nitrogen using a TGA Q50 (TA Instruments, New Castle, DE, USA). The general sample weight used was 5-7 mg. The sample was placed in an aluminum pan and was heated to 600 °C at the rate of 10 °C/min. The weight loss (%) of a sample as a function of temperature (°C) was obtained from this analysis. Then, to determine the Iodine content in MTPS-iodine pellets, isothermal TGAs were also run on neat MTPS and MTPS-iodine samples. Sample was heated to 160 C and maintained at that temperature for 30 mins. Then it was heated further to 550°C. Same was done with the control (neat MTPS) for comparison. Antimicrobial Properties: Antifungal test As a simple qualitative way for testing the antifungal properties of the synthesized starch- iodine pellets, it was dissolved in water and applied to fresh strawberries as a coating[18]. The solution was prepared by dissolving 1g and 2 g of starch-iodine pellets in water to yield 1% and 2 % solutions. Fresh strawberries were obtained from Meijer grocery store, East Lansing, MI. USA. The strawberries were washed carefully using distilled water and were air dried for 1 hour before use. Then 15 strawberries were selected based on uniform size, color, and absence of any physical or pathological damage. The fruits were evaluated for visual decay and weight loss. Then 5 strawberries each were coated with 1% and 2% solutions of starch-iodine and 5 were kept uncoated. All the strawberries were kept in 75% relative humidity chamber for 11 days and were tested for visual decay, fungal growth, and weight loss immediately after coating (day 0) and at day 2, 4, 6, 8 and 11. 162 Visual decay and weight loss of strawberries Photographs of the strawberries were taken every day and were observed for signs of lesion, brown spots, or fungal growth. Weight loss of strawberries was expressed as percentage loss based on initial weight measured after coating[18]. Weight loss was calculated using the equation below: Equation 5.1 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 − 𝑓𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑊𝑒𝑖𝑔ℎ𝑡 𝑙𝑜𝑠𝑠 = × 100 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 Antimicrobial properties The antimicrobial properties were tested using the disk diffusion assay and direct inoculation assay following Shojaeiarani et. al, 2020[19]. Escherichia coli (E. coli) strain (TB0484) was streaked on nutrient media from a -80°C freezer stock dissolved in glycerol. One colony was picked and used to inoculate 5 ml nutrient (TSB) broth. The bacteria were left to grow at 37°C for 18 hours. Antibacterial (Kirby-Bauer Test) test The disk diffusion test was performed on the synthesized MTPS-iodine pellets with iodine content of 6.7% by weight. 100 ml of the 18-hour broth culture was used to inoculate nutrient media plates, and a single pellet was kept in the middle. These plates were left to grow for 20 hours. Three replicates of plates were used for each pellet type. After 20 hours, zone of inhibition was indicative of the measure of antimicrobial property. Antimicrobial properties: antibacterial (direct inoculation) test The direct inoculation test was performed as per “ISO 22196- Measurement of antibacterial activity on plastics surfaces”. PLA control films, silver antimicrobial film (AM Film) and the 163 PLA films containing different concentrations of iodine were cut in sterile squares of 5 cm X 5 cm. The stomacher bag was cut in 4cm X 4cm, which was used to cover the films. 2 ml of 18-hour grown culture was centrifuged, the pellet was washed with 1XPBS (phosphate buffer saline) suspension buffer, centrifuged once again, and later dissolved in 2ml 1X PBS, 100 μl of which was placed on top of every PLA film in a petri plate, covered with the stomacher bag film and placed in the 37°C incubator for 24 hours. The enumeration of E. coli from PLA control films were taken at the time of inoculation and after 24 hours, whereas readings from all the test films were taken after 24 hours. To enumerate E. coli, the inoculated film sandwich was dropped with a set of sterile forceps in small sterile Whirlpack bags with 10 ml of 1X PBS buffer, mixed for 2 minutes, diluted at different concentrations, and plated on to nutrient media. Three technical replicates were tested for all the experiments. More than three biological replicates were tested for each film type. Mechanical Properties The tensile strength, tear strength and penetration strength of PLA control film supplied by Natur-tec® were compared with the Starch-iodine-PLA film samples SI1, SI2 and SI3. The samples were stored for 24 hours at 25 °C in a humidity chamber with RH of 50% before any analysis. Tensile testing was performed using an Instron model 5544 (Instron, Norwood, MA, USA) with a 100 N load cell and grip separation speed of 20 inch/min as per ASTM D882-10. Data from five samples of each formulation were averaged and compared with the properties of control PLA films. Tear strength test was performed using Thwing-Albert Elmendorf tear tester (NJ, USA) as per ASTM D1922. Five film samples were prepared and tested in the machine direction (MD) and transverse direction (TD) for each group. 164 Scanning Electron Microscopy A JOEL 6610 LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) was used to study the dispersion of MTPS-iodine in PLA films using the fracture surfaces of all samples. The films were treated with 6 N HCl for 24 h to remove the MTPS phase from the samples and air-dried for 12 h in a fume hood. Then the films were immersed in liquid nitrogen for ~2 min and then fractured. Fracture surfaces were mounted on aluminum stubs using high vacuum carbon tabs and coated with gold using a sputter coater and examined using JOEL at 500× magnification at 10 kV. 165 5.6 RESULTS AND DISCUSSION Iodine content evaluation During the MTPS-iodine pellets preparation, iodine and KI were added to the starch glycerol mixture to give a total iodine content of 6.7 %. However, during extrusion there is a possibility of iodine sublimation at the higher temperatures. Hence, to calculate the final content of iodine retained in the pellets after cooling, thermogravimetric analysis was used. From the derivative thermogravimetric graphs (DTG) of neat MTPS (without iodine), iodine and MTPS-iodine the peak degradation temperatures for each of the compounds were determined (Figure 5.4). It was found that MTPS and MTPS-iodine start degrading above 180-200°C, whereas the complete degradation of iodine is completed by 150°C, with peak degradation temperature at 133°C. Hence, to calculate % iodine present in the MTPS-iodine sample, it was heated to 160°C and maintained at that temperature for 30 mins to ensure all the iodine present in the sample is degraded. Then it was heated further to 550° C. Same was done with the control (neat MTPS) for comparison. This was done in triplicate to get an average iodine percentage. Figure 5.5 shows the isothermal TGA curves for MTPS and MTPS- iodine. For neat MTPS, the average weight loss from 25 to 200°C was 3.52% which might be due to the moisture present. For MTPS-iodine samples, the average weight loss from 25 to 200°C was found to be 10.18%. So, it was concluded that the difference in the weight loss between these two samples corresponded to the amount of iodine present in the MTPS- iodine sample which was calculated as 6.66 %. Thus, the total amount of iodine added during the pellets’ preparation (6.7% by weight) and the iodine present in the final pellets after cooling down (6.66% by weight) was similar and no significant iodine loss happened during the extrusion step. 166 5 133.55°C 4 Derivative wt % 3 303.86°C 2 257.15°C 1 0 0 100 200 300 400 500 Temperature (°C) Iodine MTPS MTPS-iodine Figure 5.4: DTG curves for MTPS, MTPS-iodine and iodine 100 80 Weight % 60 40 20 0 0 100 200 300 400 500 600 Temperature (°C) MTPS MTPS-iodine Figure 5.5: Isothermal TGA for MTPS and MTPS-iodine 167 Antifungal properties: Changes in weight loss and visual decay To determine the effectiveness of starch-iodine for antifungal properties, weight loss and visual decay of strawberries were observed every other day and were compared to the control (uncoated) samples. Figure 5.6 shows the summary of the procedure followed and the results for this testing. The effect of coating the strawberries with starch-iodine solution on weight loss is shown in the weight loss analysis graph. It was observed that the weight loss increased with increasing storage period for all the treatments. There was no significant difference between the weight loss of control and 1% starch-iodine coated samples. Control and 1% starch-iodine coated samples showed higher weight loss compared to the 2% starch- iodine coated samples. Visual fungal decay started quickly on the control samples on day 4 of the storage. Coated samples started showing visual decay on day 6. For 2% starch-iodine coated samples the visual decay was even less, and it started on day 8 of the storage. Thus, the starch-iodine coating showed great potential in reducing the fungal growth and visual decay for the strawberries for extended time. MTPS-Iodine pellets Visual decay after day 11 Stored at RT and 75% RH for 11 days 30 25 Aqueous Wt loss (%) 20 solution 15 Distilled 10 water 5 0 0 2 4 6 8 11 Day control 1% starch-I solution 2% Starch-I solution Applied on fresh Weight loss analysis strawberries Figure 5.6: Weight loss and visual decay analysis for coated and uncoated strawberries 168 This was a good preliminary test as a proof-of-concept for establishing the antifungal- antimicrobial properties of the starch-iodine pellets. In case of its use for actual food applications, further detailed studies for checking the compatibility of this formulation with human body, studies on toxicity of iodine, edibility of this coating will all have to be done. Those were not focused on in this project. Antimicrobial activity studies on the pellets and films Two separate assays were employed to evaluate the antimicrobial activity of starch-iodine pellets and the blown films. First, a disk diffusion method was used for starch-iodine pellets and films. A clear zone of inhibition was observed in the bacterial lawn around the pellets (Figure 5.7-a). For the films, there was no growth on the surface of the films, no clear zone of inhibition was observed for them (pictures not shown). It could be due to factors such as diffusibility of iodine from the film. In a separate approach, a slightly modified direct inoculation method “ISO 22196- Measurement of antibacterial activity on plastics surfaces” was used, where E. coli cells were directly inoculated on the surface of the films. The bacterial counts were observed on PLA control and test films after the 24 h of inoculation in comparison to the bacterial counts observed on the PLA control films at the time of inoculation (Figure 5.7 b-f). The differences in the average change in log CFU/ml were calculated. Figure 5.8 shows the average results for CFU/ml for all the samples. 169 a)starch-iodine pellet b) Neat PLA c) AM film d) SI1 – 0.7%, 1 mil e) SI2 – 0.7%, 2 mil f) SI3 – 1.3%, 2 mil Figure 5.7: Photographs of the petri plates from disk diffusion and direct inoculation test with E. coli. a) Kirby Bauer test on starch-iodine pellets showing antibacterial zone of inhibition b)-f) bacterial colonies growth on the petri plates after 24 hours b) control PLA films c) AM: Silver antimicrobial films d) SI1: starch-iodine-PLA films with 0.7% iodine and 1 mil thickness e) SI2: starch-iodine-PLA films with 0.7% iodine and 2 mil thickness f) SI3: starch-iodine-PLA films with 1.3% iodine and 2 mil thickness 170 Reduction in 24 hours compared to PLA at 0 hours Control PLA SI1 (0.7% I, 1 SI2(0.7% I, 2 SI3-1.3% I, 2 film AM film mil) mil) mil) 0 -1 log CFU/ml reduction -2 -3 -4 -5 -6 Film types Figure 5.8: Average change in Log CFU/ml of the Escherichia coli (E. coli) strains after 24 h incubation on five different PLA film configurations (control PLA;AM film and SI1, SI2 and SI3) It was observed that survival of E. coli was negatively impacted by all the films during incubation compared to the control films. All the iodine containing films (SI1, SI2 and SI3), showed a greater negative impact on survival of E. coli compared to the commercial antimicrobial film supplied by Natur-tec® (AM film). It should be noted that, these values represented in the graph are not an actual representation of all the replicates. During these studies, for multiple biological replicates a limit of detection was reached. i.e., no growth of bacteria was observed on any plates for that dilution (for example- Figure 5.7 d and f). Log CFU/ml values for those replicates were not calculated and were not included while plotting the graph. Considering this limit, it was observed that SI3 showed the best performance. The limit of detection was reached for the highest number of replicates for this sample followed by SI1 and SI2. 171 Mechanical Properties and morphology of the films Mechanical properties like tensile strength, extension at break and tear strength were measured for the PLA-MTPS films (SI1, SI2 and SI3) and were compared with the control PLA films supplied by Natur-tec®. Tensile strength in machine direction (MD) for SI1 and SI2 was comparable to the control PLA films (Figure 5.9-a). In TD, a significant reduction in the tensile strength and elongation at break was observed (Figure 5.9). This might be due to the fact that the dispersion of MTPS-iodine complex in the PLA-PBAT blends used for blown film was poor. This was also observed in the morphology studies done on the fractured surfaces of the films (Figure 5.10). Two images for the same sample represent different areas on the films fractured surfaces. The cavities represent the MTPS-iodine phase that was leached out with concentrated HCL. It was observed that there were some areas where the MTPS-iodine phase was very concentrated. Large size cavities observed in Figure 5.10 b, e and h represent the agglomeration of MTPS-iodine phase in the films. There were also some areas which had very less concentration of MTPS-iodine (Figure 5.10- c,f and i). This confirmed that the MTPS-iodine was not uniformly distributed in the films. This agglomeration was more pronounced in SI1 and SI2. SI3 samples showed comparatively smaller size of the agglomerates and they were also distributed more uniformly compared to SI1 and SI2. This maybe be the reason why the mechanical properties of SI3 were comparable, or even better in some cases (for example – tear strength in TD) than SI1 and SI2, even though the MTPS-iodine content was almost double in these films. Better distribution of the additives helps in enhancing the mechanical properties of the films. This was also observed visually by looking at the films themselves. As shown in Figure 5.10-a and d, the SI1and SI2 have visual stripes in the films in some places indicating a poor dispersion 172 of the additive. For SI3, the run was smoother, and we could not see any visual difference in the dispersion of the additive (Figure 5.10-g). Starch is hydrophilic and polyesters are hydrophobic. So inherently it is difficult to disperse starch or starch-based compounds in PLA or other hydrophobic polyesters. Using modified thermoplastic starch like MTPS improves the compatibility as shown in chapter 3 and 4 [16, 20]. However, it was observed that the dispersion of MTPS-iodine in blown films was not uniform in this case. One way to improve the dispersion could be to make graft polyester of MTPS-iodine with PBAT as studied by Hablot et al. [16] and then blend that with the blown film’s formulation. 173 40 a) 35 Tensile strength (MPa) 30 25 20 15 28 30 26 27 23 10 5 11 12 11 0 Control PLA SI1 SI2 SI3 film Tensile Strength (MPa) MD Tensile Strength (MPa) TD 600 b) 500 Extension at break (%) 400 300 464 200 291 100 188 200 185 115 120 96 0 Control PLA SI1 SI2 SI3 film Extension at Break (%) MD Extension at Break (%) TD Figure 5.9: Mechanical properties for the different PLA blown film formulations in machine and trnasverse direction a) tensile strength b) Elongation at break c)Tear strength 174 Figure 5.9 cont’d 1.8 c) 1.6 1.4 Tear strength (N) 1.2 1.0 0.8 1.468 0.6 0.921 0.898 0.4 0.755 0.804 0.657 0.639 0.2 0.447 0.0 Control PLA SI1 SI2 SI3 film Tear Strength (N) MD Tear Strength (N) TD a) d) g) Figure 5.10: Visual and SEM images of the fractured surface of the films after selective extraction of MTPS-iodine phase a), b) and c) : SI1- 0.7% iodine, 1 mil; d), e) and f) : SI2 – 0.7 % iodine, 2 mil; g),h) and i) : 1.3% iodine, 2 mil 175 5.7 CONCLUSIONS AND FUTURE WORK Solid MTPS-iodine complex was prepared in a ZSK-30 twin screw extruder. The masterbatch pellets contained 6.7% of iodine by weight. TGA analysis of these pellets confirmed that there was no significant loss of iodine due to sublimation during the reactive extrusion process. This MTPS-iodine complex was dissolved in water and was coated on strawberries to check its antifungal properties. The weight loss studies, and visual decay observations established that these pellets have great potential in reducing the fungal growth and visual decay for the strawberries for extended time. Antibacterial properties for the pellets against E. Coli were also analyzed using Kirby Bauer test. The presence of zone of inhibition clearly indicated the antibacterial activity of pellets against E. Coli. These pellets were further used as antimicrobial additive to make three PLA-PBAT blown films with two different thicknesses (1 mil and 2 mil) and 2 different iodine concentrations (0.7% and 1.3% by weight). The antimicrobial activity of the films against E. Coli was analyzed using ISO 22196 method. Colony forming units were counted and Log CFU/ml reduction values were reported for all the films. It showed that the films with 2 mil thickness and 1.3 % iodine concentration (SI3), were the most effective against E. coli followed by the films with 0.7% iodine and 1 mil thickness (SI1) and the films with 0.7% iodine and 2 mil thickness (SI2). In general, all the films containing iodine showed superior antimicrobial activity vs E. Coli as compared to the control PLA films and the commercial silver antimicrobial (AM film) containing films. SEM imaging of the fractured surfaces of the films showed poor dispersion of MTPS-iodine complex in the blown films. This also explained the loss of tensile strength and elongation at break in transverse direction for the SI1 and SI2 blown films compared to control PLA films. This might be due to the hydrophilic nature of starch which makes it difficult to blend with 176 hydrophobic polymers like PLA and PBAT. One way to improve the dispersion might be to make graft copolyesters of MTPS-iodine and PBAT similar to the work done by Hablot et. al., 2013[16] and then use that as an additive in the blown film formulation. This solid MTPS-iodine complex can be used as an easy and effective way to introduce iodine in an environment, without the usual hazards associated with handling solid iodine. One of the applications of this complex- an additive in the PLA-PBAT blown films was studied in detail in this project. Other applications of this product could be - as an additive to make foams or filters to be used in air filtration systems for deactivating airborne viruses, including SARS-CoV-2, in pellet form as a biobased additive to provide antibacterial, antimicrobial properties in other plastics and polymers. The antibacterial activity of pellets and films against E. Coli was studied in this project. Other gram positive and gram negative bacteria as well as antifungal and antiviral studies of these pellets and the products could be useful in establishing the broad spectrum activity of this product and widen the scope for possible applications in many fields. 177 APPENDIX 178 APPENDIX Calculation for iodine loading Based on several studies done on structure and chemistry of amylose-iodine complex, it has been postulated that amylose in the complex assumes a 6-fold helical conformation and the iodine molecule slips inside the amylose coil. Iodine, by itself is not very soluble in water. Hence KI is added. Together, they form polyiodide ions in the form In-. These facts were used to calculate the total theoretical quantity of iodine that can be added to the high amylose starch used in this project. According to Baily et.al, 6 glucose units are required for 1 iodine atom. So, for a tri-iodide ion, 18 glucose units are required Molecular wt. for 1 anhydro glucose unit à 162 g/mol Hence for 18 glucose units à 162* 18 = 2918 ~ 3000 g/mol The high amylose starch used in this project has amylose content of 60-70% as given by the supplier. An average of 65% amylose content was assumed for these calculations. For making 1 kg of MTPS-iodine batch, the formulation was – 179 800 𝑔 𝑠𝑡𝑎𝑟𝑐ℎ + 200 𝑔 𝑔𝑙𝑦𝑐𝑒𝑟𝑜𝑙 + 20𝑔 𝑀𝐴 + 𝑐𝑎𝑡𝑎𝑙𝑦𝑠𝑡 𝑜𝑢𝑡 𝑜𝑓 800𝑔, 65 % 𝑖𝑠 𝑎𝑚𝑦𝑙𝑜𝑠𝑒 800 × 0.65 = 520 𝑔 𝑎𝑚𝑦𝑙𝑜𝑠𝑒 1 ℎ𝑒𝑙𝑖𝑥 𝑢𝑛𝑖𝑡 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑖𝑛𝑔 1 𝑡𝑟𝑖𝑖𝑜𝑑𝑖𝑑𝑒 𝑖𝑜𝑛 = 3000 𝑔/𝑚𝑜𝑙 520 ℎ𝑒𝑛𝑐𝑒, = 0.173 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑎𝑚𝑦𝑙𝑜𝑠𝑒 ℎ𝑒𝑙𝑖𝑥 𝑢𝑛𝑖𝑡𝑠 𝑎𝑟𝑒 𝑝𝑟𝑒𝑠𝑒𝑛𝑡 3000 𝑓𝑜𝑟 1: 1 𝑟𝑎𝑡𝑖𝑜 𝑜𝑓 𝑎𝑚𝑦𝑙𝑜𝑠𝑒 ℎ𝑒𝑙𝑖𝑥 𝑢𝑛𝑖𝑡 𝑡𝑜 𝑡𝑟𝑖𝑖𝑜𝑑𝑖𝑑𝑒, 0.173 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑡𝑟𝑖𝑖𝑜𝑑𝑖𝑑𝑒 𝑤𝑖𝑙𝑙 𝑏𝑒 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 This corresponds to a total of 65. 74 g of triiodide (43.96 g of iodine, and 29.9 g of iodide). 65.7 = 8.21% 𝑏𝑦 𝑤𝑡 𝑜𝑓 𝑠𝑡𝑎𝑟𝑐ℎ 800 Assuming that there will be some loss in handling and processing, 10-12% extra iodine was added in the batch. 180 REFERENCES 181 REFERENCES [1] S. 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Bayer, “Antibacterial bioelastomers with sustained povidone-iodine release,” Chemical Engineering Journal, vol. 347, no. April, pp. 19–26, 2018, doi: 10.1016/j.cej.2018.04.091. [6] Richard J. GreffIan N. AskillConnie C. Lee, “EP1028848A1 - Prepolymer compositions comprising an antimicrobial agent - Google Patents,” EP1028848A1, Aug. 23, 2000 Accessed: Apr. 30, 2022. [Online]. Available: https://patents.google.com/patent/EP1028848A1/en. [7] S.-H. Li, Y. Wang, H.-B. Gao, K. Zhao, Y.-C. Hou, and W. Sun, “Experimental study on the toxicity of povidone-iodine solution in brain tissues of rabbits,” International Journal of Clinical and Experimental Medicine, vol. 8, no. 9, p. 14863, 2015, Accessed: Mar. 28, 2022. [Online]. Available: /pmc/articles/PMC4658857. [8] W. Saenger, “The Structure of the Blue Starch-Iodine Complex,” Naturwissenschaften, vol. 71, pp. 31–36, Jan. 1984, Accessed: Mar. 28, 2022. [Online]. Available: https://link.springer.com/article/10.1007/BF00365977. [9] X. Yu, C. 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Nabar, M. Srinivasan, B. Y. Shin, R. Narayan, and P. Dubois, “Maleated thermoplastic starch by reactive extrusion,” Carbohydrate Polymers, vol. 74, no. 2, pp. 159– 169, 2008, doi: 10.1016/j.carbpol.2008.01.027. [14] J. M. Raquez, Y. Nabar, R. Narayan, and P. Dubois, “In Situ Compatibilization of Maleated Thermoplastic Starch / Polyester Melt-Blends by Reactive Extrusion,” Polymer Engineering & Science, vol. 48, no. 9, pp. 1747–1754, Sep. 2008, doi: 10.1002/pen. [15] A. Kulkarni and R. Narayan, “Effects of Modified Thermoplastic Starch on Crystallization Kinetics and Barrier Properties of PLA,” Polymers (Basel), vol. 13, no. 23, Dec. 2021, doi: 10.3390/POLYM13234125. [16] E. Hablot et al., “Reactive extrusion of glycerylated starch and starch-polyester graft copolymers,” European Polymer Journal, vol. 49, no. 4, pp. 873–881, Apr. 2013, doi: 10.1016/j.eurpolymj.2012.12.005. [17] “Starch and Iodine - Chemistry LibreTexts.” https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Modules_(B iological_Chemistry)/Carbohydrates/Case_Studies/Starch_and_Iodine (accessed Apr. 25, 2022). [18] I. Khan, C. N. Tango, R. Chelliah, and D. H. Oh, “Development of antimicrobial edible coating based on modified chitosan for the improvement of strawberries shelf life,” Food Science and Biotechnology, vol. 28, no. 4, pp. 1257–1264, 2019, doi: 10.1007/s10068-018- 00554-9. [19] J. Shojaeiarani, D. S. Bajwa, N. M. Stark, T. M. Bergholz, and A. L. Kraft, “Spin coating method improved the performance characteristics of films obtained from poly(lactic acid) and cellulose nanocrystals,” Sustainable Materials and Technologies, vol. 26, p. e00212, 2020, doi: 10.1016/j.susmat.2020.e00212. [20] J. Wootthikanokkhan et al., “Effect of blending conditions on mechanical, thermal, and rheological properties of plasticized poly(lactic acid)/maleated thermoplastic starch blends,” Journal of Applied Polymer Science, vol. 124, no. 2, pp. 1012–1019, Apr. 2012, doi: 10.1002/APP.35142. 183 6. UNDERSTANDING BIODEGRADABILITY OF BIOBASED POLYMERS IN MARINE ENVIRONMENT 6.1 ABSTRACT The use of biobased and biodegradable polymers instead of carbon-carbon backbone polymers promises to reduce plastics accumulation in natural environment. For most of the polymers, the biodegradation process is highly temperature dependent. We present a novel approach to study the effect of temperature on the biodegradability of cellulose and PHBV in an aqueous environment seeded with a biologically aggressive microbial inoculum. The testing was done at three different temperatures using ISO 14852. We use a global equation derived from reparametrized Arrhenius equation and the kinetic rate law to estimate the time required for 90% removal of polymer from the low temperature ocean environment. The time required for 90% biodegradation of PHBV at 10°C ranged from 6.2-6.9 years whereas it was found to be 1.1-1.2 years for cellulose. This model can be used to estimate the persistence of plastics in ocean in case of their inadvertent leakage. The progress of biodegradation was also monitored for any morphological, structural and chemical modifications to analyze the mechanism for PHBV biodegradation. Surface erosion by SEM and optical imaging, slight decrease of molecular weight by GPC and crystallinity and melting point changes by DSC all suggested the presence of enzymatic surface erosion mechanism for PHBV. 184 6.2 INTRODUCTION AND BACKGROUND Need for lifetime prediction and end of life analysis The end-of-life of plastics, particularly packaging and disposable products is raising serious concerns worldwide and driving regulations on plastics waste management in the USA and around the world. Ocean plastics pollution is of particular concern and articles in print and e-media appear daily on the subject. The global plastics pollution statistics indicate plastic pollution of 300 million tons/year out of which only 10% is recycled whereas 4.8-12.7 million tons enter into the ocean every year[1, 2]. “Petroleum-based polymers such as polyethylene, polypropylene, polystyrene, aromatic polyesters, and polyamides with strong C-C or C- heteroatom backbone are considered to be the main sources of environmental pollution with microplastics. In contrast, conventional biodegradable polymers (aliphatic and aliphatic– aromatic polyesters), such as polylactic acid (PLA), polycaprolactone (PCL), lactide– glycolide copolymers, have not attracted the main attention as sources of microplastics. Probable reasons could be the limited use of these polymers and the common misconception that they would degrade under any environmental conditions. However, biodegradable polymers degrade only under certain conditions (temperature, humidity, light, oxygen availability, and microorganisms).”[3] Complete biodegradation of plastics occurs when all of the original polymer is broken down to carbon dioxide, methane and water. All the biodegradation standards including ISO 14852, ASTM D6691, ISO 14851, ASTM D6400 etc. are static, and they generate the data for percent biodegradation data at a single temperature. However, for most of the polymers this process is temperature dependent. For example, some plastics like PLA labelled as ‘biodegradable’ biodegrade only in industrial composting 185 conditions with temperatures of 58°C [4–6]. Some studies have shown upto 97% biodegradation of polymers based on PBAT in marine environment within 1 year [7, 8] However, these studies were done at a temperature of 30°C which hardly represent the temperatures of ocean. In another study by Atsuyoshi Nakayama et al. [9] biodegradation of aliphatic polyesters was studied in seawater at 2 different temperatures, and they found that the rate of biodegradation was almost twice at 27°C as compared to the rate at 10°C. This suggests that the temperature has a very strong impact on the activity of micro-organisms which control the rate of biodegradation. Another study has reported that temperature and rate of biodegradation soil environment follow Arrhenius relationship [10]. To our knowledge, no systematic efforts have been done till now to quantify the effect of temperature on the rate of biodegradation and predict the expected time for which the polymer will linger in the marine environment. The ocean water is divided into 3 parts- the surface layer, then a boundary layer called the thermocline and the deep ocean as shown in Figure 6.1. 90% of total volume of ocean is found below the thermocline in deep ocean. And most of this deep-water temperature is between 0-4°C [11] 186 Figure 6.1: Temperature-depth Ocean water profile[11] Thus, it is expected that the rate of biodegradation at such temperatures will be very low and they will exist in the ocean environment much longer than observed in lab studies done at 30°C. During this time the plastics will impact marine life and habitats. Therefore, unqualified “ocean biodegradability” claims and certifications based on room temperature respirometry analysis is misleading. This paper provides a method based on kinetic analysis to estimate the time required for 90% removal of the polymer at low temperatures through microbial metabolism. In this the biodegradation of so-called biodegradable polymers (PHBV) and cellulose was studied in aqueous environment at 3 different temperatures of 10, 30 and 40°C according to ISO 14852. A relation between the rate of biodegradation and temperature was found out using reparametrized Arrhenius equation and using that, the time required for complete biodegradation of PHBV and cellulose at actual sea temperatures i.e., 4°C was calculated. This 187 simulation-based approach is useful in estimating the potential environmental buildup of the polymer in low temperatures of marine environments. Further, the biodegradation mechanism for PHBV was also investigated. For this purpose, initial properties of the pellets (TGA, DSC, Molecular weight, pellet size and morphology) were characterized and the changes in these properties was monitored over the course of biodegradation. The percent biodegradation values were combined with other indicators like morphological, structural, and chemical modifications induced on the surface of the PHBV to for confirming surface erosion as the mechanism of PHBV biodegradation. 188 6.3 BIODEGRADATION PROCESS Lifetime estimation for biodegradable polymers starts with the basic understanding of the biodegradation process, mechanisms of hydrolysis, factors affecting hydrolysis and the methods used for measuring the percent biodegradation. Complete biodegradation of plastics occurs when all of the original polymer is broken down to carbon dioxide (aerobic biodegradation) and/or methane (anaerobic biodegradation) and water. In general, the biodegradation process is divided into four steps as shown in Figure 6.2 - i) biodeterioration, ii) depolymerization iii) bio assimilation, and iv) mineralization By extracellular enzymes Fragments/microplastics Oligomers and monomers Plastic Biomass CO2 H2O Mineralization By Microbes Figure 6.2: The different steps involved in the biodegradation process Biodeterioration is the formation of a biofilm on the surface of the polymer or plastic. Different microorganisms like bacteria, fungi, protozoa, algae can develop consortia on and/or inside the surface of the material. They can either adhere physically to the surface 189 with a secreted glue kind of material or can enter inside the surface via chemical deterioration [12, 13]. Formation of biofilm increases the size of pores and promotes formation of cracks on the surface of the material reducing the resistance and durability. Polymer is a very high molecular weight compound and hence unable to enter the cell wall or cytoplasmic membrane of microorganisms. Depolymerization or biofragmentation is the process of conversion of high molecular weight polymer to oligomers, dimers and monomers. Then in the step of assimilation, microorganisms use this as a substrate for growth and reproduction. Once inside cells, the molecules are oxidized through catabolic pathways like glycolysis, oxidative phosphorylation and electron transport to generate energy in the form of adenosine triphosphate (ATP) and other metabolites of cells [5, 13]. These metabolites are then mineralized to CO2, CH4, H2O, N2 etc. and are released back to the environment. The release of CO2 or other end products or consumption of O2 is a measure of complete or ultimate biodegradation which is generally estimated by standardized respirometric methods (ISO 14852, ISO 14855, ASTM 6691, ASTM 6400 etc.) in various environments. 190 6.4 MECHANISMS OF BIODEGRADATION Hydrolysis is a process of chemical breakdown of a compound due to reaction with water. In this, the polymer bonds sensitive to water break leading to chain scission and molecular weight reduction. It is a major pathway for chemical degradation of many polymers containing heteroatoms like polyesters, polysaccharides, polyamides and their copolymers[13]. This reaction can either be acid or base catalyzed called chemical hydrolysis or enzyme catalyzed called enzyme hydrolysis. For example- hydrolysis of some polymers like polylactic acid (PLA) or poly(lactic-co-glycolic) acid is accelerated under acidic or basic conditions. For some polymers like PHBV, the rate of chemical hydrolysis is very less as compared to enzyme hydrolysis [14]. In natural environment, enzymes play an important role in hydrolysis. Various microbes secrete different extracellular enzymes which act as catalysts and reduce the activation energy barrier required for polymer hydrolysis. Different enzymes can degrade specific bonds. Hydrolysis can occur via either surface or bulk erosion mechanism. Laylock et.al has described these erosion mechanisms in detail[12]. Bulk erosion occurs when the rate of diffusion of water exceeds the hydrolysis reaction rate. Water enters inside the polymer and the degradation occurs uniformly inside the thickness of the material. Hence, there is a significant reduction in the molecular weight of the polymer before any mass occurs. Bulk erosion is found to be more dominant mechanism in hydrolysis of polyesters like PLA, poly(glycolic acid) (PGA), polycaprolactone (PCL) etc. 191 Bulk erosion Surface erosion Figure 6.3: Degradation mechanisms for biodegradable polymers A) Bulk erosion, B) Surface erosion[15] Surface erosion mechanism is generally found to be dominant in enzyme hydrolysis reactions. Enzymes are high molecular weight compounds and hence they cannot penetrate easily to the polymer bulk. Surface erosion can also occur when the rate of hydrolysis exceeds the rate of water diffusion in the bulk. This could be due to hydrophobicity, high crystallinity or high glass transition temperature of the polymer. In this, mass is lost form the material surface but the core of the polymer still retains its high the molecular weight [5]. Several studies have shown PHAs as surface degrading polymers [12, 14, 16, 17]. A large number of PHB depolymerases from various bacteria and fungi have been isolated and purified. Enzyme hydrolysis is a heterogenous reaction between water insoluble polymer and water soluble depolymerase enzyme. PHAs are high molecular weight compounds and hence they cannot pass through microbial membranes. Hence, the microbes secrete extracellular enzymes like depolymerase which diffuse out and get adsorbed on the solid surface of the polymer forming enzyme substrate complex. These enzymes catalyze the hydrolysis reaction on the surface and the resulting oligomers diffuse out into the solution. They are then absorbed by the cells for further assimilation and mineralization. 192 6.5 FACTORS AFFECTING AQUEOUS BIODEGRADATION Some factors that affect the rate of biodegradation significantly are as follows: Shape and surface area Rate of biodegradation is expected to increase as the surface area to volume ratio increases. Most of the studies on PHA biodegradation are performed on films (Deroiné et al. 2015a; Doi, Kanesawa, Kunioka, and Saito 1990; Greene 2012; Hoshino et al. 2003; K. ichi Kasuya et al. 1995; Kumagai 1992; Thellen et al. 2008). A few studies used injection molded dogbone samples [23, 24] whereas some studies used polymer pellets or powder as it is [17, 25, 26]. Modelli et al. (1999) studied the biodegradation of PHBV and PCL films and powder in soil and showed that increasing the surface area increases the rate of biodegradation drastically. Powdered samples reached >90% BD very fast compared to the films of the same sample. Crystallinity Not many studies study crystallinity as a controlling factor for biodegradation. However, it has been generally found that the amorphous regions are first attached by the enzymes during biodegradation. Hence, the crystallinity values are found to increase with time. Doi et al. 1992b studied the aqueous biodegradation of various PHB films with different crystallinities (53-73 %) using isolated PHB depolymerase from Alculigenes faecalis. They found that the polymer enzymatic erosion was strongly dependent on the crystallinity of PHB films. Weight loss of polymer decreased with increasing crystallinity. Static vs dynamic water conditions It has been found that biodegradation of plastics varies with the lab or field conditions used during the experiment. Thellen et al. 2008 studied the biodegradation of PHBV under static and dynamic water conditions. PHBV samples were immersed in seawater in lab under static 193 conditions at temperature of 21°C while for dynamic conditions the samples were incubated in aquarium tanks with continuous flow of water with varying water temperatures according to the season from 12-22°C. Weight loss as a function of time was recorded for both the tests and it was found that the weight loss was less by 40-50% for the dynamic environment. It was argued that the dynamic water flow had lower and constantly changing temperatures. The nutrient supply was also limited as compared to the static water which represents more realistic conditions of marine environment. Temperature Temperature has a significant influence on the hydrolytic reactions and hence rate of biodegradation. All the studies which studied the relationship between temperature and biodegradation supported the idea that the rate of biodegradation is higher at higher temperatures. K. ichi Kasuya et al. 1995 studied the kinetics of enzyme hydrolysis as a function of temperature (25-37°) and pH (6.0-8.0)[28, 29]. Rate of enzyme hydrolysis was found to be strongly dependent on increasing temperature. Most of the aqueous biodegradation studies did not use extreme ranges of temperatures. More data for biodegradation at two extremes was taken from soil biodegradation studies. In a study by Nakayama, Yamano, and Kawasaki 2019, biodegradation of aliphatic polyesters was studied in seawater at 2 different temperatures and they found that the rate of biodegradation was almost twice at 27°C as compared to the rate at 10°C[9]. Similar results were observed in a study by Mergaert et al. 1995 where the biodegradation of PHB samples was studied at 15, 28 and 40°C in soil environment. The weight loss was much more rapid at 40°C compared to 15 or 28°C due to increased enzymatic hydrolysis. However, no systematic studies for actual 194 lifetime prediction of polymers specifically PHBV, at different environmental temperatures were found. 195 6.6 POLYHYDROXY ALKANOATES (PHAS) PHAs are bacterial derived polyesters which are largely used for production of bioplastics. More than 150 different monomers in this family can be combined to give various co- polyesters with a range of properties [30]. A random copolymer of 3-hydroxybutyrate (HB) and 3- hydroxyvalerate (HV) is produced from bacterial strains like Ralstonia eutropha and Alcaligenes eutrophus using propionic acid as carbon substrate. This P(2-HB-co-3-HV) also called PHBV has been commercialized by several companies like Enmat, Eureka and Imperial Chemical Industries using fermentation process. The 3-HB unit imparts stiffness whereas 3- HV unit is responsible for the flexibility of the polymer. Thus, PHBV shows a range of physical and thermal properties which depend on the copolymer compositions[27]. PHBV is expected to be degradable in the environment via enzymatic and/or hydrolytic degradation processes [14, 17, 27, 29, 31]. However, a detailed understanding of its estimated lifetime in a specific environment like ocean is required to make informed decisions about their environmental impact. A biodegradable polymer should also mean that it should biodegrade in the natural environment in a reasonable timeframe. ASTM and ISO standards specify that a reasonable time period for biodegradation of plastics should be around 180 days which corresponds to one growing season. It is seen from the biodegradation studies that often harsh (high temperatures, low or high pH) or environmentally unrealistic (isolated microorganisms or pure enzymes, optimum temperature, and other conditions) are used in order to get accelerated biodegradation results. However, these conditions often do not represent the ‘real’ environmental conditions. For example – A study done at 30oC in aqueous environment has shown 97% biodegradation of polymers based on PBAT in marine environment within one year. Several studies from Deroiné et al. 2014; Doi et al. 1990, 1992; Kumagai, Kanesawa, 196 and Doi 1992 studied the degradation of PHAs and PHBV in aqueous environment using isolated PHB depolymerase from Alcaligenes faecalis and/or at higher temperatures which do not represent actual environmental conditions. The rate of biodegradation under non ideal environmental conditions is expected to be lower and hence, they will exist in the ocean environment much longer than observed in lab studies done at 30°C. Therefore, even if they are termed as “biodegradable polymers” they can contribute to environmental contamination and caution must be taken before making any false and misleading claims of biodegradability. 197 6.7 EXPERIMENTAL SECTION Materials Two polymer materials were selected to assess their biodegradation in aqueous environment at various temperatures. Cellulose as a positive reference and polyhydroxy- butyrate-co-valerate (PHBV): a bacteria-derived biobased copolymer. PHBV was supplied by TianAn Biologic Materials Co. Ltd., China under the trade name Y100P. The weight average molecular weight (Mw) of the copolymer was 350,000 da. as provided by the manufacturer. The hydroxyvalerate (HV) content was 2% as determined by NMR given in supplementary materials (Appendix 1). Microcrystalline cellulose was obtained from ACROS Organics™ with particle size of less than 90 μm and was used as a positive control material in all biodegradation tests. Same batch of PHBV and cellulose was used for all the tests. All other chemicals used were analytical grade and were purchased from Sigma Aldrich. Error! Reference source not found. shows the chemical structures for Cellulose and PHBV Figure 6.4: Chemical structures of cellulose and PHBV 198 6.8 MATERIAL PROCESSING AND CHARACTERIZATION Elemental analysis The carbon content of test and reference materials determined by elemental analysis using a PerkinElmer 2400 Series II CHNS/O Elemental Analyzer (Shelton, CT, USA) and is shown in Table 6.1, Table 6.2 and Table 6.3. The amount of sample to be added in each flask for the biodegradation test was adjusted according to the carbon % of the polymer so as to get equal number of carbon millimoles for each replicate. The theoretical amount of CO2 evolved by the total oxidation of test material (ThCO2) is given by equation Equation 6.1. Equation 6.1 44 𝑇ℎ𝐶𝑂2 (𝑔) = 𝑚 × 𝑋 × 12 where m is the mass of the test material added in biodegradation flask. X is the fractional carbon present in the test material, determined from the chemical formula of the polymer or calculated from an elemental analysis; 44 and 12 are the molecular weights of carbon dioxide and carbon, respectively. This ThCO2 is used to calculate percent biodegradation during biodegradation test. The results are shown in Table 6.1, Table 6.2 and Table 6.3. Table 6.1: Aqueous biodegradation test setup. Test carried out at 10ºC Test Amount %C Total C in Theoretical Inoculum material of test test CO2 added material material (mmoles) (ml) added (mg) (mg) Blank - - - 50 Blank - - - 50 Cellulose 4002 43.58 1744 145.3 50 Cellulose 4003 43.58 1745 145.4 50 PHBV 4001 55.40 2217 184.7 50 PHBV 4005 55.40 2219 184.9 50 199 Table 6.2: Aqueous biodegradation test setup. Test carried out at 30ºC Test Amount %C Total C in Theoretical Inoculum material of test test CO2 added material material (mmoles) (ml) added (mg) (mg) Blank - - - 50 Blank - - - 50 Cellulose 4541.8 43.58 1979.3 164.9 50 Cellulose 4540.9 43.58 1978.9 164.9 50 PHBV 3629.8 55.40 2010.9 167.9 50 PHBV 3635.8 55.40 2014.2 167.8 50 Table 6.3: Aqueous biodegradation test setup. Test carried out at 40ºC Test Amount %C Total C in Theoretical Inoculum material of test test CO2 added material material (mmoles) (ml) added (mg) (mg) Blank - - - 50 Blank - - - 50 Cellulose 2626 43.58 1144.4 95.4 50 Cellulose 2602 43.58 1133.9 94.5 50 PHBV 3628 55.40 2009.9 167.6 50 PHBV 3628 55.40 2009.9 167.8 50 Thermal analysis PHBV and cellulose were characterized for glass transition temperature (Tg), melting temperature (Tm) and degradation temperature as a measure of initial properties. The degradation temperature of samples was obtained by thermogravimetric analysis (TGA) using a TGA Q50 (TA Instruments, USA). A sample (5-10 mg) was heated to 600°C with a heating rate of 10°C /min. The weight loss (%) of a sample as a function of temperature (°C) was obtained from this analysis. Also, the thermal transitions of the samples were obtained by using a differential scanning calorimeter (DSC). The sample was heated to 200°C in DSC 200 Q20 (TA Instruments, USA) with the heating rate of 10ºC/ min to remove any thermal and stress history. The sample was then cooled back to 20°C and heated again to 200ºC with the heating rate of 10°C/ min. The samples were used as it is for the biodegradation experiments without any thermal processing. Hence, the transitions appearing on the first heating cycle were recorded for analysis of % crystallinity, Tg, Tm and degradation temperature. The crystallinity of samples was calculated using Equation 6.2. Enthalpy of melting (ΔHm) and enthalpy of cold crystallization (ΔHcc) was recorded from DSC in W/g. The enthalpy of melting for 100 % crystalline PHBV was obtained as 146 J/g from the literature[32] . Equation 6.2 ∆𝐻𝑚 − ∆𝐻𝑐 𝐶𝑟𝑦𝑠𝑡𝑎𝑙𝑙𝑖𝑛𝑖𝑡𝑦 (%) = × 100 ∆𝐻𝑜 Determination of particle size of pellets The size of the pellets for PHBV was determined by considering them as cylinders for PHBV. The dimensions of 15-20 pellets were measured using a Keyence VHX-6000 digital microscope. Microcrystalline cellulose was purchased from Thermo Fisher Scientific Inc. in powdered form and was used as it is. Scanning electron microscopy A JOEL 6610 LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) was used to study the surface erosion of all the PHBV pellets. The stubs were mounted on the SEM stands using high vacuum carbon tabs and coated with gold using a sputter coater. The stubs were then placed in SEM stands and examined using JOEL at 2000× magnification at 10 kV. Molecular weight analysis The PHBV samples dissolved in chloroform at a concentration of 5 mg/ml. PHBV samples needed to be heated to 50 °C and agitated for 1–2 h until complete dissolution could be 201 achieved. Resulting solutions were then filtered through a Whatman Rezist 30 mm 0.20μ PTFE syringe filter. Molecular weights were measured by GPC (Waters system) at 35 °C using a Agilent PLgel MIXED-C 7.5x300 mm 5 micron HPLC column (PL1110-6500) column and Wyatt Technology TREOS II (LS detector) and Optilab T-rEX (RI detector). HPLC grade chloroform was used as an eluent at a flow rate of 1 ml/min. The number-average (Mn) and weight-average (Mw) molecular weights were calculated using a calibration curve from polystyrene standards using ASTRA 7.3.2 software. The polydispersity index (PDI) was calculated from the following equation Equation 6.3 𝑀𝑛 𝑃𝐷𝐼 = 𝑀𝑤 where Mn and Mw are the initial number-average molecular weight and the number-average molecular weight at a given time of biodegradation. Microbial inoculum The microbial inoculum used in all the tests was a sample of activated sludge collected from a well operated wastewater treatment plant handling predominantly domestic sewage at East Lansing, MI, USA. The sample was mixed well, kept under aerobic conditions, and was used within 48 hours of collection. metagenomic analysis of the activated sludge was done to map the microbial population present initially in the sludge. it was compared with the seawater population from various locations to check for any similarities. The detailed procedure and results for metagenomic analysis are given in Appendix 3. 202 6.9 EXPERIMENTAL BIODEGRADATION SETUP A typical biodegradation curve for percent biodegradation vs time is as shown in Fig. 1a. The basic reaction for aerobic biodegradation of a polymer is: Equation 6.4 Carbon present in the polymer is first converted to biomass which is then mineralized to CO2. There are typically 3 different phases involved in the biodegradation reaction - lag phase, activation phase, and plateau phase as shown in the Figure 6.5. Plateau phase % Biodegradation Biodegradation phase Lag phase Days Figure 6.5: A typical biodegradation curve for %biodegradation vs time Out of those, the activation phase is the phase where majority of the biodegradation takes place. For the two polymers- cellulose and PHBV, considered in this study, these reactions will be - 203 Equation 6.5 (C6 H10 O5 )n + 6O2 → 6CO2 + 6H2 O Equation 6.6 [COCH2 CH(CH3 )O]m [COCH2 CH(C2 H5 )O]n + 11O2 → 9CO2 + 7H2 O Similar to other chemical reactions, the rate of biodegradation can be calculated using either consumption of reactants which in this case is carbon present in the polymer or the oxygen consumed or using the appearance of products which is CO2 evolution in this case. A reliable method to measure the biomass is still not available. For the assessment of % biodegradation, the mineralization % is commonly used. When the evolved CO2 is used to calculate the biodegradability, the method is called respirometric method and biodegradation % is given as the ratio between evolved CO2 and theoretical CO2 as shown in following equation. Equation 6.7 𝐶𝐸𝑣𝑜𝑙𝑣𝑒𝑑 𝑎𝑠 𝐶𝑂2 𝐵𝑖𝑜𝑑𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 % = 𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 × 100 𝐶𝑂2 𝑃𝑜𝑙𝑦𝑚𝑒𝑟 A respirometric mineralization test system for calculating CO2 evolution was setup based on International Standard ISO 14852 as shown in Figure 6.6. The system comprised of blank, positive reference(cellulose) and the test material (PHBV) for all the runs. All the samples, blanks and references were run in duplicates. An optimized test medium containing all the nutrients and buffers was prepared according to ISO standard. Table 6.4 gives the detailed composition of the mineral solution prepared for all the tests. Wastewater inoculum was added to all the flasks to obtain the concentration of 5% v/v in the test medium as described in ISO 14852. Then the polymer samples were added to these flasks, and they were subjected to the test conditions as described in Table 6.4. 204 Table 6.4: Buffer solutions used for aqueous biodegradation 1 L mineral solution g Solution A KH2PO4 (anhydrous) 3.75 Na2HPO4·2H2O 8.73 NH4Cl 0.2 Solution B MgSO4·7H2O 2.25 Solution C CaCl2·2H2O 3.64 Solution D FeCl3·6H2O 0.025 g Wastewater inoculum (ml) 50 Distilled water (ml) Remaining The system comprised of six flasks in total. Two for blank, two for positive reference cellulose and two for each the PHBV samples. The system was kept in a dark, temperature-controlled room maintained at a temperature specific for that run. The test flasks were agitated throughout the run with the help of magnetic stirrers. Air inlet was passed through NaOH solution to get CO2 free air. This air was then divided and passed through flowmeters for each bioreactor at a constant flow rate. 205 Figure 6.6: Experimental biodegradation setup A solution of 1N NaOH was used for trapping the CO2 generated from test flasks. CO2 trapping is a two-step process as shown below: NaOH + CO2 → NaHCO3 NaHCO3 + NaOH → Na2CO3 1 g of sample was taken from each of the 50mL NaOH trapping solution and titrated with 0.1N standardized hydrochloric acid (HCl) solution to find the amount of CO2 trapped.[33] The reactions are as follows: Na2CO3 + HCl → NaHCO3 + NaCl NaHCO3 + HCl → NaCl + H2O + CO2 The titrations were done with the help of autotitrator to get the volumes of HCl, V1 and V2 required for reaction 1 and 2 respectively. The amount of HCl consumed can be used to calculate the mmoles of CO2 evolved using following formula: 206 Equation 6.8 (𝑉2 − 𝑉1) ∗ 𝑁𝐻𝑐𝑙 ∗ 𝑉𝑁𝑎𝑂𝐻 𝑀𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝐶𝑂2 = 𝑊𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 The percentage biodegradation (% B) was further calculated by the following equation: Equation 6.9 ∑(𝐶𝑂2 )𝑠𝑎𝑚𝑝𝑙𝑒 − ∑(𝐶𝑂2 )𝑏𝑙𝑎𝑛𝑘 %𝐵 = × 100 𝑇ℎ𝐶𝑂2 ∑(𝐶𝑂2 )𝑠𝑎𝑚𝑝𝑙𝑒 is the amount of carbon dioxide evolved in test flask between the start of the test and time t; ∑(𝐶𝑂2 )𝑏𝑙𝑎𝑛𝑘 is the amount of carbon dioxide evolved in blank flask between the start of the test and time t; ThCO2 is the theoretical amount of carbon dioxide evolved by the test material. All the values were expressed as mmoles of CO2. The samples were replaced every 2-3 days in the starting phase when the rate of biodegradation was expected to be maximum and weekly or biweekly in the end.[18, 34] These samples were tested at 3 different temperatures of 10, 30 and 40°C. Table 6.1, Table 6.2, and Table 6.3 show a summary the different tests performed, the materials used in each test and other information like carbon % and theoretical CO2 evolution expected from each sample. Biodegradation kinetics analysis and other statistical analysis was done using Microsoft excel and Minitab. 207 6.10 RESULTS AND DISCUSSION This section first presents results for initial characterization of the test materials and various graphs for percent biodegradation vs time for all the polymers at different temperatures. Using that data kinetics study was done for cellulose and PHBV with the help of Arrhenius equations. Experimental results were compared with several values from the literature and a model was suggested for predicting the rates of biodegradation of Cellulose and PHBV in marine environment. Finally, the mechanism for biodegradation of PHBV was also studied by monitoring various chemical, morphological, and thermal properties of PHBV. Initial characterization of polymer pellets Average pellet diameter and height for PHBV was found to be 1.97±0.10 mm and 2.7 ± 0.04 mm and average surface area for the pellets was 23.00±1.48 mm2. Microcrystalline cellulose with 60um size was used as positive control. DSC and TGA curves for cellulose and PHBV are shown in Figure 6.7. The thermal degradation temperatures for cellulose and PHBV were found to be 342.1 C and 278 °C respectively. DSC curves show the melting points for cellulose and PHBV at 104.6 and 175.9 °C. 208 Figure 6.7: TGA and DTG curves for a) cellulose, b) PHBV; DSC curves for c) cellulose d)PHBV 209 Biodegradation: CO2 evolution and mineralization In all tests, the percent biodegradation of cellulose was according to the standard ISO 14852 requirement (i.e. % Biodegradation > 60% at the end of the test). Hence, the inoculum was considered active, and the tests were valid even at the lowest test temperature of 10°C. The values of cumulative mmoles of CO2 evolved from each sample were divided by the maximum theoretical CO2 mmoles possible to be generated by the sample to obtain percent biodegradation as shown in Equation 6.9. Theoretical CO2 was calculated from the elemental analysis of percent carbon present in the polymer. The comparative biodegradation curves for PHBV and cellulose for 3 temperatures are shown in Figure 6.8 and Figure 6.9. It was observed that the rate of biodegradation was strongly dependent on the temperature. It took cellulose almost 200 days to reach 80 % mineralization at 10°C whereas, at 40°C, it was achieved in less than 25 days. Similar results were obtained for PHBV polymer as well. shows the normalized graphs for change in concentration of carbon with time for cellulose and PHBV. The experimental percent biodegradation curves were re-plotted in terms of – carbon remaining (Cremaining) against time (days) by subtracting the Cremoved from the environment from the initial carbon added to the added in the form of polymer (Cinitial)( Figure 6.10 – a and b). The detailed calculations and the derivation for Cremaining is given in Appendix 2. Biodegradation kinetics study was done to further quantify this dependence of biodegradation rate on temperature. 210 100 80 % Biodegradation 60 Cellulose 40°C 40 Cellulose 30°C Cellulose 10°C 20 0 0 50 100 150 200 Time (Days) Figure 6.8: Average biodegradation curves of Cellulose tested at different temperature (10, 30, 40ºC). Each curve represents average of 2 replicates 100 80 % Biodegradation 60 PHBV 40°C 40 PHBV 30°C PHBV 10°C 20 0 0 50 100 150 200 Time (Days) Figure 6.9: Average biodegradation curves of PHBV tested at different temperature (10, 30, 40ºC). Each curve represents average of 2 replicates 211 1.0 Cellulose 10 C 0.8 Cellulose 30 C 0.6 Cellulose 40 C C/C0 0.4 0.2 0.0 0 50 100 150 Time (days) 1.0 0.8 PHBV 10 C 0.6 PHBV 30 C C/C0 0.4 PHBV 40 C 0.2 0.0 0 50 100 150 Time (days) Figure 6.10: C/Co as a function of time for aqueous biodegradation of a) Cellulose and b) PHBV at 10, 30 and 40°C. Points represent experimental data whereas dotted lines indicate fitting of 1st order equation 212 Biodegradation kinetics: Parameter estimation and order of reaction To calculate the order of the reaction for overall biodegradation reaction of PHBV and cellulose, the general rate law was used. Equation 6.10 −𝑑𝐶 = 𝑘𝐶 𝑛 𝑑𝑡 This nth order kinetic equation can be solved for calculating the order and rate constant of the biodegradation of cellulose and PHBV at various temperatures using either differential method or Non-Linear Least-Squares Analysis Differential analysis. Taking natural log of Equation 6.10 on both sides we get Equation 6.11 −dC 𝑙n ( ) = lnk + n ln C dt Plot of ln(-dC/dt) vs ln C gives order of reaction and rate constant. Where Slope → n = order of reaction And Intercept → ln k = rate constant Non-Linear Least-Squares Analysis Integrating Equation 6.10 gives the rate law equation for nth order reaction as Equation 6.12 1 𝐶 = [𝐶0 (1−𝑛) − (1 − 𝑛)𝑘𝑡]1−𝑛 Where - k → rate constant, n → order of the reaction, t→ time C0 → mmoles of carbon added initially in the biodegradation flask C → mmoles of carbon remaining at any time t 213 Assuming some initial values for k and n, a predicted value for C → Cpredicted can be calculated for each day. Then calculate the root mean square of the errors, i.e., difference between the predicted values and the actual value of C remaining Minimize that using excel solver to get the optimized values of n and k. Both these methods were applied to Cellulose and PHBV biodegradation data to calculate and compare the order of reaction, n, and rate constant, k. Table 6.5 gives the values for order of the biodegradation reaction for cellulose and PHBV at various temperatures calculated using both differential method and RMSE method. The detailed calculations and graphs for each temperature and polymer are given in Appendix . Table 6.5: Order of reaction for cellulose and PHBV biodegradation Method Differential RMSE Differential RMSE Differential RMSE Polymer → method method method Temperature (°C) → 10 30 40 Cellulose n 1.045 1.291 1.018 1.14 0.876 0.95 PHBV n 0.989 1.09 1.038 1.19 1.094 1.234 As expected, the higher the temperature faster the rate of biodegradation. Biodegradation being a microbial reaction, is expected to be sensitive to temperatures. At temperatures higher than 40 or 50°C, it is possible that the rate of reaction might reduce due to inactivation of microbes and denaturation of microbial enzymes resulting in decreased rate of biodegradation. However, for the temperature range considered in this study, no such affect was observed. The order of reaction for both PHBV and cellulose was found to be near one for all the temperatures used in this study. Hence, a first order kinetics equation was used for further analysis. 214 Biodegradation as a first order reaction For a first order reaction, the rate law is Equation 6.13 d[C] 𝑅ate = − = k[C] dt where [C] is the moles of carbon consumed by the micro-organisms to yield CO2, k is the rate constant and t is the time. [33] 𝐶 𝑡 𝑑[𝐶] ∫ − = ∫ 𝑘[𝐶] 𝐶0 𝑑𝑡 0 ln[𝐶] − ln[𝐶0 ] = −𝑘𝑡 ln[𝐶] = ln[𝐶0 ] − 𝑘𝑡 Where [Ct]- moles of carbon remaining at time t and [C0] is initial moles of carbon present. Equation 6.14 C𝑡 C𝑜 ln = −kt or ln = kt or 𝐶𝑡 = 𝐶𝑜 𝑒 −𝑘𝑡 C𝑜 C𝑡 Where Co is initial mmoles of carbon present in added polymer sample calculated from the elemental analysis and Ct is the mmoles of carbon remaining in the sample at any given time t. Millimoles of carbon remaining (Ct) were found by subtracting the mmoles of carbon evolved from the initial mmoles of carbon added in the test (Co). 215 Parameter estimation: Arrhenius equation In this study, the lifetime prediction for PHBV and Cellulose in aqueous environment was done using Arrhenius equation expressed as: Equation 6.15 𝐸 𝑘 = 𝐴𝑒 −𝑅𝑇 Where k is the rate constant for the reaction, A is called the pre-exponential or frequency factor, R is the universal gas constant (8.314 J.mol-1. K-1) and E is the activation energy. The obtained datasets of temperature vs rate constant were analyzed in 3 different ways to calculate the range for activation energy and pre-exponential factor. First, we applied logarithmic transformation followed by linear regression as commonly followed in the literature. Second method was non-linear regression using reparametrized Arrhenius equation. Reparameterization of Arrhenius equation was done to estimate more accurate range for Ea and avoid the high correlation between Ea and A. This was done by introducing a reference temperature Tref and the corresponding rate constant kref so that the temperature is centered about the mean value 𝑇𝑟𝑒𝑓 of the temperatures to be studied. The resulting Arrhenius equation is as shown in Equation 6.16 Equation 6.16 Ea 1 1 𝑘 = 𝐴′ exp − ( − ) R 𝑇 𝑇𝑟𝑒𝑓 These two are 2-step methods in which first we find the rate constants and then the Arrhenius parameters. This 2-step way of analysis is statistically insufficient because too much information is lost on the way. Hence, in the 3rd method, a one-step approach of 216 incorporating the reparametrized Arrhenius equation in kinetic rate law was used. The rate constant k was replaced by Arrhenius equation in the 1st order kinetic rate law as follows Equation 6.17 Thus, we bypass the estimation of rate constants, and we have more data points and more degrees of freedom which yield in more accurate estimation. A schematic for showing the comparison between standard linear regression analysis, reparametrized Arrhenius equation analysis and the one step global equation approach is given in Figure 6.11: Schematic for showing the comparison between standard linear regression analysis, reparametrized Arrhenius equation analysis and the one step global equation approach The results using these 3 methods of analysis are as shown in Table 6.6. The details for calculating these estimates for are given in Appendix 217 Table 6.6: Parameter estimates for activation energy and pre-exponential factor using method 1, 2 and 3 Cellulose Parameter Method Estimate Standard Deviation 95% CI 3.25 ‐ 12.54 1 7.9 4.64 (A =25.7 – 268337) ln A 2 10.79 0.25 10.41 ‐ 11.02 3 9.53 0.04 9.49 ‐ 9.57 1 29.29 11.53 17.76 ‐ 40.83 Ea (kJ) 2 36.64 20.36 20.72 ‐ 61.44 3 33.15 2.82 30.33 ‐ 35.97 PHBV Parameter Method Estimate Standard deviation 95% CI 1 23.41 7.97 15.22 ‐ 31.38 ln A 2 13.69 0.4 13.06 ‐ 14.04 3 9.9 0.06 9.83 ‐ 9.96 1 70.62 19.79 50.83 ‐ 90.42 Ea (kJ) 2 45.66 30.35 23.10‐ 83.80 3 61.67 4.87 56.51 ‐ 66.26 Note: Method 1, linear regression of Arrhenius equation; method 2, nonlinear regression of reparametrized Arrhenius equation; method 3, one step method of incorporating reparametrized Arrhenius equation in kinetic rate law The results obtained from the analysis using three different methods clearly showed the difference in the range of estimates. For the first method, the precision obtained was disappointing. Even though the standard errors were not so bad, the 95% confidence intervals (CI) were very large due to small number of degrees of freedom. After reparameterization, the 95% CI for A’ was much better than its untransformed counterpart A. This clearly proved the advantage of reparameterization. However, the reparameterization did not improve the estimation ranges for Ea. When one step global equation was used by incorporating the Arrhenius equation in first order reaction rate law 218 as shown in Equation 6.17, the 95% CI for A’ and Ea obtained were much more precise and statistically useful (Table 6.6). It should be noted that the nonlinear regression analysis had difficulties in finding estimates especially when using second method. Only when initial values close to the final values were supplied, a solution was found. This might be due to the limited number of data points (for 3 temperatures in this case) and the strong correlation Overall, the final parameters and models developed for lifetime predictions of cellulose and PHBV are as shown in Table 6.7. This model is valid for a temperature range between 10 to 40°C. However, an estimate for the time required for biodegradation at temperatures lower than 10°C could also be made by extrapolation of these models. A comparative analysis for the estimated time ranges given by the 3 methods was performed and the results are as shown in Figure 6.12. The model was also extrapolated to find the estimated time range for biodegradation at the ocean temperature of 4°C. A graphical representation illustrating the precision differences between method 1,2 and 3 is shown in Figure 6.12. Details for the calculations are given in appendix 6. Table 6.7: Final estimated parameter values for the global equation for cellulose and PHBV Parameters Cellulose PHBV A’ 0.023 0.0082 Ea 33.14 61.68 Tref 26.5 26.5 𝐶𝑡 𝐸𝑎 1 1 Global equation = exp (−𝐴′ × exp ((− ) × ( − )) × 𝑡 𝐶𝑜 𝑅 𝑇 𝑇𝑟𝑒𝑓 219 Figure 6.12: Precision differences for biodegradation time estimations for the three methods The results showed that the time ranges estimated using method 3 were much narrower as compared to method 1 and 2 for all the temperatures. It suggests that even though the polymers like cellulose and PHBV biodegrade readily at 30°C (0.5 -0.6 years for cellulose and 1.2 – 1.5 years for PHBV), they will take much longer time at lower temperature of 10°C (1.4 -1.5 years for cellulose and 7.4 -8.3 years for 99% biodegradation of PHBV). Further, if this model is extrapolated to the actual marine temperature of 4°C, this time will be even higher. Even a readily biodegradable polymer like cellulose is estimated to last for almost 2 years at low ocean temperatures whereas PHBV is estimated to last for 12 -15 years (Figure 6.13). Hence, the standard ASTM or ISO tests for aqueous or marine biodegradation which are carried out at temperatures of 25-30°C might not give an accurate representation of polymer’s biodegradability in ocean. A plot of percent carbon remaining as a function of time (years) was plotted from these values of rate constants (Figure 6.13). A straight line was assumed to represent any non-biodegradable polymer. The details for obtaining this graph are given in Appendix . 220 100 % Carbon remaining 80 Cellulose 4°C 60 Cellulose 10° C Cellulose 30 C 40 LDPE 20 0 0 1 2 3 4 Years 100 % Carbon remaining 80 PHBV 4°C 60 PHBV 10° C PHBV 30 C 40 LDPE 20 0 0 5 10 15 20 25 30 Years Figure 6.13: percent carbon remaining as a function of time for cellulose and PHBV at 30, 10 and 4 °C 221 Biodegradation data compilation To validate the model further, an extensive literature review was done to compare the rates of biodegradation from different studies to our experimental data. Biodegradation data for aerobic aqueous biodegradation of cellulose and PHBV was identified through a search of peer reviewed literature. Appendix 8 shows a summary for selected articles using several methods for assessing biodegradation of cellulose and PHBV in aqueous environment. The studies using CO2 evolution or O2 consumption for determining the % biodegradation were used further to calculate the rate constants for biodegradation. Biodegradation rate was either obtained directly or calculated assuming first order kinetics for each study. Some authors have also used weight loss and other visual inspection techniques for estimating biodegradation. Appendix 8 also provides other relevant information for biodegradation such as the size and shape, molecular weights, time and other characterization methods used. This data was used further for comparison of our experimental data with literature values. Rate constants for the biodegradation were either used directly as given in the paper or were calculated by digitizing the graphical % biodegradation vs time data using Webplot digitizer and assuming a first order kinetics. Arrhenius graph (ln k vs 1/T) as well as temperature vs rate constant graphs were plotted for both PHBV and cellulose and were compared with our experimental data and the predicted data using our model (Figure 6.14). It was observed that the data from literature agrees well with our experimental data. The average activation energy values calculated using the literature rate constants and the values obtained in this study were very similar. For cellulose the activation energy obtained was 33.14 kJ whereas from literature the average activation energy was found to be 35.5 kJ. For 222 PHBV this difference was even less – calculated 61.68 kJ vs average of 61.98 kJ from literature. 4.E-02 3.E-02 Rate constant (day-1) 2.E-02 1.E-02 0.E+00 -1.E-02 -2.E-02 -3.E-02 0 5 10 15 20 25 30 35 40 45 50 Temperature ( C) predicted k using our model Literature values Experimental k values Figure 6.14: Comparing the experimental rate constants to literature values for Cellulose and PHBV 223 A statistical analysis was done to determine the variation between all the rate constant values obtained from various papers. It was observed that, for a particular temperature, 70% of the obtained rate constant values from various literature lie within ±1 SD. The type of polymer used, the physical state of the sample (powder, film or injection molded articles), the microbial inoculum used etc. resulted in small variations, but the final rates of biodegradation were similar for most of the cases. Figure 6.15 below represents various datapoints for cellulose and PHBV at 25°C and 30°C and their distance from the mean value. Considering that all these studies were done in various parts of the world, with different samples and different aqueous conditions by different authors, this similarity in the results looks very promising. a) Cellulose 0.07 0.06 Rate constant k (day-1) 0.05 0.04 1SD 0.03 0.02 1SD 0.01 0 20 25 30 35 Temperature (°C) Figure 6.15:Statistical analysis of rate constants for a)cellulose and b)PHBV at 25 and 30 C 224 Figure 6.15 cont’d b) PHBV 0.030 0.025 Rate constant k (day-1) 0.020 0.015 1SD 1SD 0.010 0.005 0.000 20 25 30 35 Temperature (°C) 225 Accumulation of plastics in ocean Plastic pollution is a planetary threat affecting nearly all the ecosystems in the world including marine. 8 MT of plastic enters the ocean every year worldwide. United States generates the most solid waste the most solid waste than any country in the world thus contributing to enormous amounts of plastic waste to the environment including oceans[1], [2], [35]. However, all these claims lack a quantitative model that could predict a measurable reduction in ocean plastic accumulation by replacing the petroleum-based plastics by biodegradable plastics. Here, we present a model to evaluate how replacing a certain percentage of non-biodegradable plastics by biodegradable plastic would reduce the plastic accumulation in ocean by 2030. We assume the current benchmark of 8 MT annual ocean plastic pollution and impact of replacing the plastics in that waste by a percentage of biodegradable plastics. The calculations are done by using the rate constant k and Ea obtained for PHBV at temperature of 10°C (0.00161 day-1 and 61.68 kJ respectively). The detailed model and calculations can be found in Appendix 8 Our results show that the plastic accumulated in ocean in 10 years can reduce by 77.5 % (from 80 Mt to 17.95 Mt) if 100% plastics are replaced by bioplastics with biodegradation rate similar to PHBV. Out the plastics accumulated in sea at the end of 10 years, 8 MT addition will be from the year 10. 92% of the plastics remaining would be from year 6-10 whereas there would be hardly any plastic remaining from year 1-6 as shown in the Figure 6.16. The results indicate that replacement of non-biodegradable plastics by 20, 40, 60 and 80% biodegradable plastics will result in 15.5, 31.0, 46.5 and 62.0 % reduction in ocean plastic accumulation respectively. Thus, even though the biodegradable plastics will take longer time to biodegrade in the low 226 temperature ocean environment, they won’t last there for hundreds of years like the non- biodegradable counterparts. 90 Plastic remaining in ocean after 10 years ( 80.00 80 8% 70 67.59 60 55.18 50 8% 42.77 40 45% 14% MT) 30.36 30 20 17.95 25% 10 0 0% 20% 40% 60% 80% 100% Year 1 Year 2 Year 3 year 4 year 5 Percentage of Plastic waste in ocean replaced by biodegradable year 6 year 7 year 8 year 9 year 10 plastic Figure 6.16: Model for plastics accumulation in ocean – impact of replacing a fraction of non-biodegradable plastics with biodegradable plastics Following formula was derived for estimating the amount of bioplastics accumulated in ocean at any time – Equation 6.18 𝑓 𝑃𝑓 = 𝑊(1 − 𝑥) ∗ 𝑓 + ∑ 𝑊 × 𝑥 × exp[−𝑘(𝑡𝑓 − 𝑡𝑛 ) × 365) 𝑛=1 Where, Pf = plastic remaining in ocean after ‘f’ years (MT) W = amount of plastic leaking in ocean every year in MT (for eg 8MT) x = fraction of plastic waste replaced by biodegradable plastic (0.2, 0.4, 0.6 etc.) k = rate constant of the biodegradable plastic at a particular temperature (as determined from kinetics studies for that polymer) in day-1 227 6.11 MECHANISM OF PHBV BIODEGRADATION To follow macroscopic modifications occurring on the surface of the PHBV pellets, optical microscopy and SEM images of the pellets were taken at different time intervals. Figure 6.17 shows the optical microscopy and SEM images and the corresponding level of biodegradation at that time obtained from the CO2 evolution measurements. It was observed that the degradation occurred on from the surface of the pellets and the size of the pellets decreased with time. These pellets became porous with time with formation of eroded structures on the surface. This formation of porous structures could be due to faster degradation of the amorphous phases compared to crystalline phase of PHBV as generally reported in literature about PHBV[36, 17]. This clearly indicated enzymatic surface erosion mechanism. 0% BD 15% BD 62% BD 72% BD Figure 6.17: PHBV SEM and optical microscopy images at various stages of biodegradation Changes in the molecular weight of PHBV (Mn and Mw) were also monitored as a function of biodegradation. Table 6.8 shows the Mn, Mw and PDI values for the PHBV samples at various stages of biodegradation. 228 Table 6.8: Evolution of number-average molecular weight (Mn), weight-average molecular weight (Mw), polydispersity index (Mn/Mw) and melting point (Tm)of PHBV samples during the course of biodegradation % BD Mn Mw PDI Tm(°C) 0 2.52E+05 3.73E+05 1.48 176.25 15 1.71E+05 2.44E+05 1.43 175.90 62 1.33E+05 1.81E+05 1.36 172.40 A linear decrease in Mn was observed as % biodegradation increased. 30% loss in Mn was observed after 15 % biodegradation. Afterwards the rate in decrease of molecular weight was reduced. A molecular weight of 133000 was maintained even after 72% biodegradation. Polydispersity index also reduced slightly from 1.48 to 1.36. These results matched with other PHBV degradation studies in the literature [17, 24, 37]. These results suggested that the chain scission via hydrolytic degradation is not the mechanism for PHBV degradation. It is due to enzymatic activity on the surface. The slight change in molecular weight might be due to the loss of oligomers from the film surface which might be small enough to diffuse in the aqueous medium. This loss of small Mn molecules might also be the reason for decrease in PDI[36]. Next step was analysis of the thermal properties of the PHBV pellets. Figure 6.18 shows the DSC and TGA measurements for the PHBV pellets at various stages of biodegradation. 229 0 a) -0.4 Heat Flow (W/g) -0.8 Neat PHBV -1.2 PHBV 15% BD PHBV 62 % BD -1.6 PHBV 72% BD -2 100 120 140 160 180 200 Temperature (℃) 120 b) 100 PHBV neat 80 PHBV 15% BD Wt % 60 PHBV 62% BD 40 PHBV 72% BD 20 0 0 200 400 600 Temperature (°C) Figure 6.18: a) DSC melting peaks and b) TGA degradation curves of PHBV pellets as a function of biodegradation percentage in laboratory scale aqueous biodegradation The DSC analysis of PHBV at various stages of biodegradation revealed that the crystallinity remained almost the same till 72% biodegradation. This suggested that both amorphous and crystalline stages are equally biodegraded from the enzymes as observed by Salomez et al. It 230 was also observed that the PHBV pellets showed presence of two melting peaks evolving conversely over time. the enthalpy of firs peak decreased, and the second melting peak increased with time. This suggests that there was a reorganization of the crystalline phase of the polymers during aqueous biodegradation conditions. There was a gradual decrease observed in melting temperatures for PHBV which was supported by the corresponding reduction in molecular weights as shown in Table 6.8. 231 6.12 CONCLUSION AND FUTURE WORK In conclusion, this study was aimed at studying the temperature dependence of biodegradation of the two well-known polymers cellulose and PHBV. All the current ASTM and ISO standards are static approaches and they do not focus on the rate of biodegradation as a function of temperature. Aqueous biodegradation of these polymers was studied under 3 different temperatures of 10, 30 and 40°C according to ISO 14852. The rate of biodegradation was found to increase with increase in temperature following the Arrhenius relationship. Order of the reaction for biodegradation were calculated using the general nth order kinetic equation by differential and RMSE analysis. All the orders of the reactions for both cellulose and PHBV were found to be close to one. Hence, a first order kinetics was used for further analysis. The values for rate constants at different temperatures for cellulose and PHBV, activation energy for the reaction and the pre-exponential factors were calculated using three statistical approaches. It was observed that the logarithmic linearization of Arrhenius equation gave statistically insignificant values for Ea and A with large standard deviations and large confidence intervals. Reparameterization of Arrhenius equation was found to reduce the error in pre-exponential factor, but it did not improve estimation of the activation energy Ea. The third approach of incorporating reparametrized Arrhenius equation in the first order rate law and solving the resulting non-linear global equation in one step gave the most accurate and statistically useful results with narrow confidence intervals. Based on our model, the activation energies for cellulose and PHBV were calculated to be 33.1 and 61.6 kJ/mol. These values matched very well with the average literature values for activation energies. Further, this model was used to estimate the time required for 90% 232 removal of the polymer from ocean environments at low temperatures through microbial metabolism. The time required for 90% biodegradation of PHBV at 10°C ranged from 6.2-6.9 years whereas it was found to be 1.1-1.2 years for cellulose. Thus, it was confirmed that the polymers will last for much longer in low temperature ocean environment than in the lab studies done at 30°C. It underlined the need for a new ASTM standard which needs to take temperature effects in account. Based on these results, a model for plastics accumulation in ocean was also developed. It was calculated that, if 100% of the plastics accumulating in ocean every year were replaces by a biodegradable plastic with a rate of biodegradation similar to PHBV, the accumulation after 10 years would decrease from 80 MT to 17.9 MT of plastics. Moreover, 45% of that plastic will be due to fresh addition of plastic in year 10; less than 8% of the total plastic would be from the first six years. Thus, we could say that, even if these biodegradable plastics leaked into the ocean inadvertently, they will be removed from the environment within a few years. They won’t remain in the ocean for hundreds of years like the non-biodegradable counterparts. The last part of the study was to investigate the mechanism of biodegradation for PHBV. The percent biodegradation values were combined with other indicators like morphological, structural and chemical modifications induced on the surface of the PHBV to for confirming surface erosion as the mechanism of PHBV biodegradation. SEM and optical imaging were powerful tools in assessing the erosion and morphological changes in the pellets throughout the course of biodegradation. High molecular weights of the pellets till the end of biodegradation were one more indication pointing towards enzymatic surface erosion 233 mechanism. Lastly, the changes in the thermal properties of the pellets such as melting points and enthalpies of the melting peaks suggested a recrystallization phenomenon. It should be noted that all this work was done for two well-known and easily biodegradable polymers- cellulose and PHBV. Both of them have many degraders present in the natural environment. The situation might change for other polymers. More detailed studies might be required for the specific case. 234 APPENDICES 235 Appendix 1: NMR analysis of PHBV The poly(3HB-co-3HV) samples were characterized by proton nuclear magnetic resonance spectroscopy (H1 NMR). About 10 mg of polymer sample was dissolved in chloroform-d (CDCl3) (99.8%; Cambridge Isotope Laboratories) and recorded using Agilent DDR2 500 MHz spectrophotometer (MSU, Michigan). The conditions were as follows- 45° pulse, 2.04 sec acquisition time, 8012.8 sweep width and 64 scans. The data was processed using MestReNova software. The characteristic peaks at 0.87 ppm and 1.27 ppm were assigned to the resonance absorption of methyl (CH3) from hydroxyvalerate (HV) unit and methyl (CH3) from hydroxybutyrate (HB) unit respectively. (Liu et al 2010). The peaks at 2.47, 2.60 and 5.26 ppm were identified as -CH2 (HV side), -CH2 (HB side) and -CH (for both HB and HV side). 236 Figure 6.19: NMR curve for PHBV The characteristic peaks at 0.87 ppm and 1.27 ppm can be used for determining the mole % of HV in poly(3HB-co-3HV) using following equation. Equation 6.19 𝐴𝑟𝑒𝑎 (−𝐶𝐻3) 𝐻𝑉 𝐻𝑉 𝑚𝑜𝑙 % = 𝐴𝑟𝑒𝑎 (−𝐶𝐻3)𝐻𝑉 + 𝐴𝑟𝑒𝑎 (−𝐶𝐻3) 𝐻𝐵 It was found that HV composition is 2 mol %. 237 Appendix 2: Carbon removal against time curves Re-plot the experimental percent biodegradation curves in terms of - carbon removal against time (days) Percent carbon removal or C/C0 vs time (days) 1 mole of C → 1 mole CO2 Number of moles of CO2 produced = number of moles of carbon consumed from the sample CO2(sample-blank) = Cconsumed And Cremaining = Cinitial - Cconsumed where Cinitial can be calculated theoretically from the % C content and the weight of the polymer added at the start of the test. = CO2(theoretical) i.e. Cremaining = 𝐶𝑂2 (𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙) - CO2(sample-blank) 238 Appendix 3: Metagenomic analysis Activated sludge from east lansing wastewater treatment plant was used as microbial inoculum in all the tests. It was analyzed for the microbial communities present using metagenomic analysis. Illumina sequencing was performed for the 16S rRNA gene region to assess the bacterial community at Research Technology Support Facility (RTSF, Michiagan State University). The sequencing were analyzed using the QIIME2 database to generate taxonomic/phylogenetic data for statistical analysis. The results were compared with the various seawater microbial communities from literature. Although there are some differences in the quantitative microbial communities in water obtained from different sources, the overall microbial population on family level was more or less the same. Hence, a wastewater sample can be used as an easily obtainable source of inoculum which could be used as a representation for seawater samples. 239 Metagenomic analyis - comparison with seawater 100% 80% 60% 40% 20% 0% activated Seawater 1* Seawater 2* Seawater Seawater Seawater sludge 3** 4** 5** proteobacteria bactereoidetes actinobacteria firmicutes choroflexi cyanobacteria Planctomycetes Verrucomicrobia acidobacteria Figure 6.20: Comparing the microbial populations from wastewater and seawaters from various places * - Rehman, Z. U., Ali, M., Iftikhar, H., & Leiknes, T. (2019), Water Research (Oxford), 149, 263- 271. ** - Greer, Charles & Wyglinski et al., (2014). Natural Attenuation Potential for Oil in Ice in the Canadian Arctic Marine Environment 240 Appendix 4: Order of reaction and rate constant determination using nth order kinetic equation 1. Differential method: The general rate law equation is Equation 6.20 −𝑑𝐶 = 𝑘𝐶 𝑛 𝑑𝑡 Where, [C] is the moles of carbon consumed by the micro-organisms to yield CO2, k is the rate constant, n is the order of the reaction and t is the time. Taking natural log on both sides Equation 6.21 −𝑑𝐶 𝑙𝑛 ( ) = 𝑙𝑛 𝑘 + 𝑛 𝑙𝑛 𝐶 𝑑𝑡 If we plot ln(-dC/dt) vs ln C, the slope of the line should give n – order of the reaction And the intercept should give ln k – rate constant for the reaction These plots were made for all cellulose samples at 3 different temperatures as follows. It should be noted that only the biodegradation phase of the curves was considered for calculating the rate constants and order of the reaction. Lag phase and the plateau phase of the biodegradation graph was not considered in these calculations. 241 A) Cellulose 10 C 1 0.5 y = 1.0455x - 4.6984 R² = 0.98 0 ln(-dC/dt) -0.5 -1 -1.5 -2 2.5 3.5 4.5 5.5 ln C Cellulose 30 C B) 1.5 1 y = 1.0188x - 4.141 R² = 0.95 ln(-dC/dt) 0.5 0 -0.5 2.5 3.5 4.5 5.5 ln C Cellulose 40 C C) 2 1.5 y = 0.8764x - 2.6264 1 R² = 0.94 ln(-dC/dt) 0.5 0 -0.5 -1 -1.5 1.5 2.5 3.5 4.5 5.5 ln C Figure 6.21: ln(-dC/dt) vs lnC plot for cellulose at A)10 C, B) 30 C, C) 40 C 242 PHBV 10C A) -1.3 -1.4 y = 0.9892x - 6.7069 R² = 0.96 ln(-dC/dt) -1.5 -1.6 -1.7 -1.8 5 5.1 5.2 5.3 ln C PHBV 30 C B) 1.5 1 y = 1.0384x - 4.5621 R² = 0.98 ln(-dC/dt) 0.5 0 -0.5 -1 2.5 3.5 4.5 5.5 ln C PHBV 40 C C) 1.5 y = 1.0947x - 4.4785 1 R² = 0.83 ln(-dC/dt) 0.5 0 -0.5 3.5 4 4.5 5 5.5 ln C Figure 6.22: ln(-dC/dt) vs ln C plots for PHBV at A)10 C, B) 30 C, C)40 C 243 2. RMSE method The general rate law is ‐ Equation 6.22 −𝑑𝐶 = 𝑘𝐶 𝑛 𝑑𝑡 Integrating gives the rate law equation for nth order reaction as ‐ Equation 6.23 1 𝐶 = [𝐶0 (1−𝑛) − (1 − 𝑛)𝑘𝑡]1−𝑛 Where, C → moles of carbon left at any time t Co → initial moles of carbon added to the biodegradation flask k → rate constant n → order of the reaction Assuming some initial values for k and n, a predicted value for C – Cpredicted was calculated for each day. Then the root mean square of the errors, i.e. difference between the predicted values and the actual value of C remaining were calculated and was minimized using excel solver to get the optimized values of n and k. Equation 6.24 ∑𝑛𝑖=1(𝑦̂𝑖 − 𝑦𝑖 )2 𝑅𝑀𝑆𝐸𝐸𝑟𝑟𝑜𝑟𝑠 = √ 𝑛 Following graphs show the Cpredicted vs Cactual graphs and the calculated n and k values for cellulose at 3 different temperatures. 244 A) Cellulose 10°C 160 mmoles of C remaining n = 1.29, k = 3.02 10-3 day-1 120 80 40 0 0 50 100 150 200 Time (days) Cellulose 10 C Cellulose 10 C predicted Cellulose 30°C B) 200 mmoles of C remaining n = 1.14, k = 10.85 10-3 day-1 160 120 80 40 0 0 50 100 150 Time (days) Cellulose 30 C Cellulose 30 C predicted Cellulose 40°C C) 120 mmoles of C remaining n = 0.95, k = 60.0 10-3 day-1 80 40 0 0 50 100 150 Time (days) Cellulose 40 C Cellulose 40 C predicted Figure 6.23: mmoles of carbon remaining vs time plots for cellulose at A)10C, B)30C, C) 40 C 245 PHBV 10°C A) 200 mmoles of C remaining 160 n = 1.09, k = 0.83 10-3 day-1 120 80 40 0 0 50 100 150 Time (days) PHBV 10 C PHBV 10 C predicted PHBV 30°C B) 200 mmoles of C remaining n = 1.19, k = 5.42 10-3 day-1 160 120 80 40 0 0 50 100 150 Time (days) PHBV 30 C PHBV 30 C predicted C) PHBV 40°C 200 mmoles of C remaining n = 1.234, k = 7.005 10-3 day-1 160 120 80 40 0 0 50 100 150 Time (days) PHBV 40 C PHBV 40 C predicted Figure 6.24: Mmoles of carbon remaining vs time plots for PHBV at A)10C, B)30C, C) 40C 246 Appendix 5: Using global equation for estimating Ea and A’ values Use non-linear regression analysis of the global kinetic rate law equation and plot percent or fraction of polymer carbon removal against time graphically The reparametrized Arrhenius parameters is put into the rate law equation to generate the global kinetic rate law equation. Equation 6.25 𝐶 𝐸𝑎 1 1 = exp (−(𝐴′ exp(− ( − ))𝑡) 𝐶𝑜 𝑅 𝑇 𝑇𝑟𝑒𝑓 Use this equation to calculate the values of A’ and Ea along with their 95% confidence intervals. (you can use any statistical program like minitab or matlab etc. for getting these values) Here, C/C0 values vs time will be obtained from experimental data. You need at least 3 temperature datasets (preferably in replicates) for using this global equation. Tref is generally taken as average of the testing temperatures. The method shown here is using Minitab software. First select the Stat option from main menu bar and go to ‘Regression’ → ‘Nonlinear regression’ 247 Figure 6.25: minitab methodology for global equation-1 Input the global equation described below in the ‘edit directly’ dialogue box. Set some initial values for Ea and A’ and run the regression. Figure 6.26: minitab methodology for global equation-2 248 We get the values of the A’ (shown as kref in the picture) and Ea. Figure 6.27: minitab methodology for global equation-3 Then you can use these values to calculate the time required for reaching any % biodegradation at any temperature (T) between the studied experimental range using the global equation given above. 249 100 90 80 % Carbon remaining 70 Cellulose 4°C 60 Cellulose 10° C 50 Cellulose 30 C 40 30 20 10 0 0 1 2 3 4 Years Figure 6.28: Percent carbon remaining vs time graphs (lifetime prediction) for cellulose at various temperatures 250 Appendix 6: lifetime prediction Using the values of activation energy and pre-exponential factors obtained from the three methods, time required for 50, 90 and 99 % biodegradation of PHBV and cellulose at temperatures of 10, 30 and 40°C was calculated. An estimation for the time required for biodegradation at 4°C was also made by extrapolation of this model at lower temperatures Table 6.9: Lifetime estimation for PHBV and cellulose at 30, 10 and 4° C Time range estimation (years) (95% confidence interval) Polymer -3 k range ( 10 ) Ea range 90% 99 % -1 Temperature Method (day ) (kJ/mol) 50% Biodegradation Biodegradation Biodegradation 1 0.86 -2.1 50.83- 90.42 0.90 -2.19 3.00 -7.28 6.0 -14.5 2 1.4 -2.4 23.11- 84.07 0.77 - 1.33 2.56 -4.42 5.14 -8.84 10 3 1.52 -1.68 56.51 – 66.26 1.12 - 1.25 3.74 -4.15 7.48 - 8.31 1 0.37 -1.32 50.83- 90.42 1.44 - 5.04 4.79 -16.75 9.58 - 33.50 PHBV 2 0.66 -1.81 23.11- 84.07 1.04 - 2.87 3.48 - 9.56 6.97 - 19.2 4 3 0.81 -1.00 56.51 – 66.26 1.89 -2.32 6.29 - 7.71 12.59 - 15.44 1 8.75- 10.9 50.83- 90.42 0.17 - 0.21 0.58 -0.72 1.15 - 1.44 2 4.28 -15.0 23.11- 84.07 0.12 - 0.44 0.42 - 1.47 0.84 - 2.95 30 3 8.23 -10.0 56.51 – 66.26 0.19 - 0.23 0.63 - 0.76 1.25 - 1.53 1 8.13 - 13.7 17.75 - 40.83 0.18 - 0.23 0.46 - 0.78 0.92 - 1.55 10 2 5.98 - 8.48 20.71 - 61.44 0.22 - 0.32 0.74 - 1. 06 1.49 -2.11 3 8.61 - 8.89 30.33 - 33.15 0.21 -0.22 0.71 - 0.73 1.42 -1.46 1 5.58 - 11.6 17.75 - 40.83 0.16 - 0.34 0.54 - 1.13 1.09 - 2.26 Cellulose 4 2 3.40 - 6.97 20.71 - 61.44 0.27 - 0.56 1.04 - 1.86 1.81 -3.72 3 6.19 - 6.72 30.33 - 33.15 0.28 - 0.31 0.94 -1.02 1.88 - 2.04 1 22.5 - 25.5 17.75 - 40.83 0.07 - 0.08 0.25 - 0.28 0.49 -0.56 30 2 15.1 - 33. 5 20.71 - 61.44 0.06 - 0.13 0.19 - 0.42 0.38 - 0.84 3 20.8 - 23.6 30.33 - 33.15 0.08 - 0.09 0.29 - 0.30 0.53 - 0.57 251 Appendix 7: Table for obtaining %C remaining vs time graphs Table 6.10: Percent carbon remaining vs time %C Cellulose 10 Cellulose 30 Cellulose 4 PHBV 10 PHBV 30 PHBV remaining C C C C C 4C 100 0.000 0.000 0.000 0.000 0.000 0.000 90 0.028 0.011 0.037 0.149 0.026 0.263 80 0.058 0.023 0.079 0.316 0.056 0.558 70 0.093 0.037 0.126 0.505 0.090 0.891 60 0.133 0.053 0.181 0.724 0.128 1.277 50 0.181 0.071 0.246 0.982 0.174 1.732 40 0.239 0.094 0.325 1.298 0.230 2.290 30 0.314 0.124 0.427 1.705 0.302 3.009 20 0.420 0.166 0.570 2.280 0.404 4.022 10 0.601 0.237 0.816 3.261 0.578 5.755 5 0.782 0.309 1.061 4.243 0.752 7.487 1 1.203 0.474 1.632 6.523 1.156 11.509 252 Appendix 8: Literature summary Literature summary for selected articles using several methods for assessing biodegradation of cellulose and PHBV in aqueous environment Table 6.11: Literature summary environme Sampl form size Mn Mw time Tempe % Referenc nt e rature Biodegradation e (°C) 1 Deroine distilled PHBV solid 180*10 172600 39880 12 25, 30, not studied et al., water pelletes *4 mm 0 mont 40, 50 2014[24] hs 2 Deroine marine PHBV PHBV 200*12 40000 180 10-20 C 36 % wt loss et al., water (8% films 0 mm* 0 days 2015[17] (natural) HV) 200 μm 3 deroine PHBV PHBV - 259000 450,00 1 year 25 90% et al., (8% powder 0 2015 HV) 4 Doi et al., distilled PHB , 10*10*0. 10 mm Hydrol 20% wt loss 1990[14] water PHBV 04 mm dia * ytic - (hydrolysis (HV 0.04- 55 ) 45%) 0.07 mm thick Enzymatic 20 h Enzym (aqueous) atic - 37 5 Doi et al., aqueous P-3- films 10*10* - 20400 19 hr 37 20-25% wt loss 1992[27] HBV 0.04 0 mm 253 Table 6.10 cont’d 4.5-6 19400 60-70% wt loss mg wt 0 6 Ken-ichi seawater PHB, film 10 mg , 186000 48000 28 25 100% wt loss Kasuya,” PHBV, thickne 0 days Ko-ichi PHBB ss 0.1 Takagi et mm al., 1997[29] 80% acc to BOD 7 Yamada, Enzymatic PHB 37000- 3-4 hr 25-37 Doi (aqueous) 60000 1995[28] 8 Greene et marine Mirell film 1g 6 30 38 al., 2200 mont 2012[21] PHA hs Mirel film 1g 7 30 45 4100 mont PHA hs Micro powder 1g 8 30 33 cellulo mont se hs 9 Ho, Gan river water PHA film 15*15 86 28 71% wt loss et al., mm days 2002[38] 10 Lo Wing Aqueous PHBV discs, 15 mm 40000 10-15 20, 30 , not studied Hong, Jian dia, 0.1 0 days 37 Yu[39] mm thick, 18-23 mg 254 Table 6.10 cont’d 11 Lotto et soil PHBV powder 15000 24, 46 al., 0 2004[25] 12 Mergaert soil PHBV injection 200 15, 28 st al., molded days and 40 1992[40] dog bone pieces 13 Sashiwa, seawater PBHH pellets 30 mg 280000 55000 28 28 30% Nakayam 0 days a; 2018[26] 14 Thellen et seawater PHBV films 100 30 80-85 % al., days 2008[22] 15 Tsuji, static PHB films 3*30*5 765000 15300 70 25 Suzuyosh seawater 0 um 00 dyas i; and 2002[41] 18*30* 25 um 17 Wang, seawater PHBH sheet 5*5*1 446203 Ambien PHBV sheet - Laydon et (7.1% mm t temp 55.3+_38.3 148- al; hexan (20 -25 195 2018[42] oate) C) days flakes 50670 PHBV flakes - 88.6 18 Volova et seawater PHB flim disc 73 mg, 40-42 % for al.; (south 30 mm both PHB and 160 2011[37] china sea) dia, 28-30 PHBV disc days 0.1mm thick 255 Table 6.10 cont’d PHBV molded 300 mg, 38% for PHB (11 solids 10 mm and 13 % for mol % dia, 5 PHBV for HV) mm molded solids thick 19 Nanthini Mangrove PHB film 1cm * 600000 56 30 ~ 70% Sridewi, environme 1cm days Kesaven nt Bhubalan, Kumar Sudesh; 2006[43] PHBV 680000 similar for all 3 PHBH 460000 20 Doi, Seawater P-3- solvent 50-150 variab variabl not given Kanesawa HB- casted um le e from , co- and melt (5*10 13 to Tanahash 3HV extruded cm in 26 i; films size) 1992[31] P-3- 2.05- HB- 2.10 co- mm 4HV 21 Shang, Distilled PHBV films 0.2 mm _ _ 50 37 20% for without Logan, et water and (9.5 % thick , days enzyme in 50 al; enzyme HV) 2cm2 days, ~ 70% 2011[44] hydrolysis area with lipase enzyme 256 Table 6.10 cont’d 22 Greene J, Ocean PHA bag - - - 180 30 ASTM 6691 2018[45] water days 23 Wang seawater cellulo powder 150 25 90% et al., se days 2018[42] 24 Greene et marine Micro powder 1g 8 30 33 al., cellulo mont 2012[46] se hs 25 Iggui, Aqueous Micro 28 20 85% Moigne et crysta dyas al., 2015 lline cellulo se 26 Vorgele Aqueous Cellul powder 300 25 >90% gt von Jan ose days P. Eubeler 27 U. aqueous Mater 50-60 25 betn 60-92 % Pagga et Bi days al.; 2001 starch blend 28 Fa, Wang Aqueous filter 60 25 85% et al.; paper days 2015 29 V. Aqueous Cellul 150 RT averaage 83% Mezzanot ose days (assum tea, e 25) , M. Tosin et al.; 2004 257 Table 6.10 cont’d 30 Puccini, Aqueous filter 365 20 78% Sandra et paper days al.; 2017[47] 31 Thellen et Seawater cellulo Powder 100 30 90% al.; 2008 se days 32 CiCLO Seawater Cellul 498 25 78 -80 % additive ose days technolog y; 2020 33 Tosin M, Seawater Filter - - - 250 RT 78% Weber M, paper days (Assum et. al., e 25) 2012[48] 34 Greene J., Seawater Cellul Powder - - - 180 30 50% 2018[45] ose Days 258 Appendix 9: Method 1 calculations Method 1 For a first order reaction, the rate law is 𝒅[𝑪] 𝑹𝒂𝒕𝒆 = − = 𝒌[𝑪] 𝒅𝒕 where [C] is the moles of carbon consumed by the micro-organisms to yield CO2, k is the rate constant and t is the time. [33] 𝐶 𝑡 𝑑[𝐶] ∫ − = ∫ 𝑘[𝐶] 𝐶0 𝑑𝑡 0 ln[𝐶] − ln[𝐶0 ] = −𝑘𝑡 ln[𝐶] = ln[𝐶0 ] − 𝑘𝑡 Where [Ct]- moles of carbon remaining at time t and [C0] is initial moles of carbon present. 𝐶𝑡 𝐶𝑜 𝑙𝑛 = −𝑘𝑡 𝑜𝑟 𝑙𝑛 = 𝑘𝑡 𝐶0 𝐶𝑡 Millimoles of carbon remaining (Ct) were found by subtracting the mmoles of carbon evolved from the initial mmoles of carbon added in the test (C0). A plot of ln[C0/Ct] vs t was made for the PHBV and cellulose samples at different temperatures . 259 A) 1.8 1.6 y = 0.016x 1.4 R² = 0.96 1.2 y = 0.0132x ln(C0/C) 1 R² = 0.99 0.8 0.6 y = 0.0013x 0.4 R² = 0.97 0.2 0 0 50 100 150 Time (days) 10 C 30 C 40 C ln(Co/Ct) vs time for PHBV at 10, 30 and 40 C B) 4.5 4 y = 0.0444x 3.5 R² = 0.98 3 ln(C0/C) 2.5 2 y = 0.018x y = 0.0108x 1.5 R² = 0.96 R² = 0.99 1 0.5 0 0 50 Time (days)100 150 10 C 30 C Figure 6.29: ln(Co/Ct) vs time for PHBV and cellulose at 10, 30 and 40°C The natural log of rate constants at various temperatures were plotted against reciprocal of temperature to obtain Arrhenius plot. 260 The biodegradation rate values are reported as positive values. However, in reality, they should be considered as negative, as they are rates of consumption. PHBV 0 y = -8493.6x + 23.409 -2 R² = 0.961 -4 ln k -6 -8 -10 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 1/T (K-1) Cellulose 0 y = -3523.7x + 7.8992 -2 R² = 0.9255 ln k -4 -6 -8 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 1/T (K-1) Figure 6.30: Arrhenius plots for PHBV and cellulose Where k is the rate constant; T is the temperature in K; E is the activation energy and R is the ideal-gas constant (8.314 J/K.mol). The energies of activation were calculated as 70.6 kJ/mol and 29.29 kJ/mol respectively. 261 Appendix 10: Ocean plastic accumulation model Table 6.12: Ocean plastics accumulation model Assume if it is BD PHBV Temperature 10 C day- rate constant 0.00161 1 100 % replaced by Plastic remaining from biodegradable plastic like PHBV amount of plastic added every year year Year Year Year Year Year Year Year Year Year (MT) Year 1 2 3 4 5 6 7 8 9 10 1 8 8 2 8 4.445 8.000 3 8 2.470 4.445 8.000 4 8 1.372 2.470 4.445 8.000 5 8 0.762 1.372 2.470 4.445 8.000 6 8 0.424 0.762 1.372 2.470 4.445 8.000 7 8 0.235 0.424 0.762 1.372 2.470 4.445 8.000 8 8 0.131 0.235 0.424 0.762 1.372 2.470 4.445 8.000 9 8 0.073 0.131 0.235 0.424 0.762 1.372 2.470 4.445 8.000 10 8 0.040 0.073 0.131 0.235 0.424 0.762 1.372 2.470 4.445 8 Total plastic remining after 10 years 17.95258455 262 REFERENCES 263 REFERENCES [1] K. L. Law and R. 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