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I.) it‘s-I: . . ‘rtrai.. 3.5.3.9) . to l. :2“). :fibil€.:s.xrt.;)vuuutn. 1 {33531111, 5.. ssiiigr!’ It... 1 cud-Iv!" Iv :13... c (I lift: (2...)! it. to .F LU.) (Fit.€€l...3.!y¢0 g..tl{9l.lv.t¢}r!lu .r . than.“ Bani; inn": rt... 3.1021633: l .31... . . an... itch”: i... WW“?! 5 t ' l .. (can; . . mi afinfigin .bfidlsré. (I . o , ..li.:.z..fiu#pfiaJ.JLfi!-lt Cf... aovfini.l:ll.. . (1 i! A F . F l I ~ n irr’av aflubVOO" mum”? ‘Mwmmmw L... ‘7 I lzl‘flé‘lowclloo 6583 Michigan State University A This is to certify that the thesis entitled THE DEVELOPMENT OF A STIRRED TANK REACTOR SYSTEM FOR THE PRODUCTION OF LIGNIN PEROXIDASE BY PHANEROCHAETE CHRYSOSPORIUM presented by Frederick Car] MicheT Jr. has been accepted towards fulfillment of the requirements for . M.S. degree in CHE Major professor Date 06*! ‘0‘6 1"! \QK‘ 0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE w l . w ,__J — J , I MSU Is An Affirmative Action/Equal Opportunity Institution TI! DIVILOPIIIT 0' A ITIRIID 21'! 2310103 SYSTEM ’0! run PIODUCTIOI OI LIGIII’PIIOXIDIBI DY Frederick Carl Michel Jr. I 138818 Submitted to Michigan State University in partial fulfillment of the requirements for the degree of IIBTBI 0’ SCIENCE Department of Chemical Engineering 1988 TEE DEVELOEIEE! OE I.STIREED Ill! REACTOR SYSTEM E03 TEE PRODUCTIOI OE LIGHIE’PEEOXIDISE by Frederick C. Michel Jr. Lignin Peroxidaeee (ligninasee) produced by the white rot fungus Engngzggnggtg‘ghzygggpgginn have several important potential industrial applications. A bench scale stirred tank reactor system for the production of lignin peroxidases was developed. Scale-up studies included the optimization of a low-cost media, examination of the mechanism of pellet formation, and the effects of agitation in shake flask cultures. Higher levels of lignin peroxidase activity were obtained with acetate buffer compared to 2,2-dimethy1 succinate buffer and other buffers. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activities in this medium. The agitation rate affected pellet size, the number of pellets formed and enzyme activity. A model was proposed for the production of lignin peroxidase in shake flask cultures. In a baffled stirred tank reactor high lignin peroxidase titres were observed when the agitation rate, oxygen dispersion and foaming were closely controlled. I wish to sincerely thank Dr. Eric A. Grulke and Dr. C.A. Raddy for the encouragement, support and expertise they shared with me during this project. I also wish to thank the Michigan Biotechnology Institute and the Department of Chemical Engineering for financial assistance. iii TIBLI 0' CONTENTS page List of tables ...................................... vi List of Figures .................................... vii Chapter I: Introduction .............................. 1 Chapter 11: Literature Review ........................ s 1. Lignin Biodegradation ............................ 5 2. Lignin Peroxidases from Rnangzgghagtg OOOOOOOCOOOOOOOOOOOOOOOOCOOOOOOOOO... 7 1. Strains of 2‘ gh:ygggpgz19n,.................. 10 2. Characteristics of lignin peroxidase ......... 10 3. Factors affecting lignin peroxidase production.................................... 13 3. mgalbiocata1YSts OOOOOIOOOOOOOOOOOOO0.0.0.0... 17 1. Fungal pellet reactor systems ................ 17 2. Fungal film reactor systems .................. 18 4. Research objectives ............................ 20 cupt.r III: nt.r1‘1. “d “.thOd. O O O O O O O O O O O O O O O O O O 21 1. culture CharQCteristicseeeeeeeeeeeeeeeeeeeeeeeeee 21 2. Solution and Media preparation .................. 21 1. Stocx salutions OOOOOOOOOOOOOOOOOOO0.00.0.0... 21 2. Media preparation ..................... ....... 24 3. Culture inoculation methods 26 1. Spore inoculum preparation ................... 26 2. Starter culture preparation .................. 28 3. Agitated culture inoculation ................. 29 4. Stirred tank reactor inoculation ............. 29 ‘. cultur. conditions OOOOOOCOOOOOCOOOOOOIO000...... 3o 1. Flask culture conditions ..................... 30 2. STR culture conditions ....................... 30 iv So ”say, OOOOOOOOOOOOOOOOOOOOO0.0..0.0.0...00...... 32 1. Lignin peroxidase ............................ 32 2. Protein ...................................... 33 3. pH measurement ............................... 35 4. Dry weight measurement ....................... 35 5. Pellet size measurement ...................... 35 6. Protein Characterization by FPLC ................ 36 cn.pt.r 1': mt. OOOCOOCOOOOOOOOOOOOOOOOOOOOOOOOOO 37 1. Media optimization ...............................37 1. Strain selection ............................. 37 2. Buffer selection ............................. 37 3. Optimization of spore inoculum concentration ................................ 37 4. Optimization of Tween 80, veratryl alcohol and basal medium concentration ............... 42 5. Culture characteristics using optimized media ........................................ 44 2. Biocatalyst pellets ............................. 48 l. Pellet formation mechanism ................... 48 2. Pellet growth ................................ 51 2. Agitation effects ............................ 51 3. Lignin peroxidase production in a Stirred tank reactor (STR) ...................... 6O 1. Development of operating conditions for a stirred tank reactor system................... 60 2. Lignin peroxidase production by the STR system ................................... 63 3. Product recovery and characterization......... 63 Chapter V: Discussion ............................... 67 1. Significance OOOOOOIOOOOOOOOOOIOOOO0.00.00.00.00. 67 2. Comparison with other systems ................... 67 3. Future directions ............................... 69 APPENDIX 0.000.0.00...00.00.000.000...II...0.0.0.0.... 72 Appendix A: Proposed model for lignin peroxidase production .................. 72 Appendix B: Trickle bed reactor system ............. 82 Appendix C: Tabulated data ......................... 85 anuoam OOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 10° List of tables table title page 3.1 Culture media compositions .................. 25 3.2 Culture inoculation and conditions ...... ..... 27 4.1 Optimized culture media for lignin peroxidase production ................. 45 4.2 Culture characteristics for 8 day old cultures of nggnzygggpgzinn,at three agitation rates...56 5.1 Comparison of the characteristics of various reactor systems for the production Oflignian1'021da“......................... 68 11.1 Summary of model equations ................... 78 A1.2 Model equation symbols and parameter values... 79 vi List of Figures title page Typical structure of lignin [44].............. 6 Time course of growth, substrate utilization and lignin peroxidase activity of agitated cultures of z‘ughzysggpgrinn_[26]............. 9 The mechanism of the biodegradation of a lignin model compound [46].................... 12 Comparison of 2;,gnzygggpgzinn strains SC26 ( D ) and BKM-P-l767 ( v ) for the production of lignin peroxidase............... 38 Comparison of various buffers for the production of lignin peroxidase. . . . . . . . . . . . . . . 40 Time course of lignin peroxidase activity for four different spore inoculum concatratiom...’0.0.0.0....00...... ......... 41 Time course of lignin peroxidase activity as affected by: Tween 80 concentration (A), veratryl alcohol concentration (8) and basal medium concentration (C)...................... 43 Characteristics of agitated cultures grown in optimized media...................... 46 Semi-log plot of the number of hyphal agglomerations (n) as a function of time...... 49 Schematic representation of the mechanism of pellet formation by 2‘ Enzygggpgrigm observed using a light microscope...... ....... 50 P... W pellet size as a function of culture age....................... 52 vii A1.1 A1.2 A2.1 Photo micrograph of an eight day old mycelial pellet of z‘mghzyggfipgzinn........... 53 Effect of the agitation rate on lignin peroxidase activity........................... 55 Photographs of mycelial pellets of 2‘ formed at three different agitation rat“OCOOOOOOOOOOO0.0.0.0....0.0.... 57 Correlation of the Sauter mean pellet diameter (On) and the agitation rate (RPM)............. 59 Stirred tank reactor system for the production or lignin ”taxi“..eeeeeee.eeeeeeeeeeeeeeeeeeee61 Lignin peroxidase activity and total extracellular protein concentration in a stirred tank reactor system.............. ..... 64 FPLC profile of heme-proteins in six day old stirred tank reactor system extra- cellular fluid, four days after inoculation... 66 Medal for growth, substrate utilization and lignin peroxidase production in batch cultures...OOOOOOOOOOOOOOOOOOOOO000......COOO. 79 Model predicted media effects on the production of lignin peroxidase in batch mltura‘OOOOCOOOOOOOOOOOOOOOCOOOOOO0.0.0.....0 80 Design of a trickle bed reactor for the production of lignin peroxidase. . . . . . . . . . . . . . . 83 viii CIIPTII I: IIIIDDUCIIOI Lignin Peroxidasesu'“ are extracellular, hydrogen peroxide-dependent heme proteins which catalyze the oxidation of the Ca-Cp linkages in lignin, a major constituent of all vascular plants and the second most abundant renewable organic polymer in the biosphere“. These enzymes are produced during idiophase in nitrogen or carbon limited cultures of certain white rot fungi, as exemplified by the basidiomycete Phangrggnagtg W”. Lignin peroxidases have several potential industrial applications including biopulping, detoxification of recalcitrant organo-pollutants such as DDT, dioxins and PCB's"'7’"'22 degradation of pulp and paper mill effluentsu'“ and pretreatment of lignocellulosic biomass for efficient bioconversion to feeds, fuels and chemicals‘z'“. Unfortunately, the low levels of lignin peroxidases produced under current culture conditions limit the study of lignin peroxidases and their applications. Procedures for large scale production have not been adequately developed. Lignin peroxidases were first produced in the laboratory in shallow stationary cultures of L, W“. This filamentous fungi grows in the form of a mycelial mat or mycelial pellets and requires a pure oxygen,environment for optimal lignin 2 peroxidase production. Enzyme production is induced during secondary metabolism by lignin or lignin metabolites such as veratryl alcohol. Initial attempts to produce lignin peroxidase in submerged cultures were unsuccessful due to the organism's sensitivity to agitatiod”. Recently it has been shown that the addition of surfactants, particularly Tween 80, enables £3,9nxyggspgzinn to produce lignin peroxidase under mildly agitated conditions”. This made the production of lignin peroxidase using biocatalyst pellets feasible in a stirred tank reactor system. The development of a stirred tank reactor system for lignin peroxidase production however has not been described to date. This type of reactor is readily available at both the laboratory and commercial scale. The hypothesis of this study was that lignin peroxidases could be produced in a stirred tank reactor system from mycelial biocatalyst pellets of 2‘ ghzysgapgzinm grown in a low-cost media. A three part study was undertaken to test this hypothesis. First a low-cost culture medium was optimized for the production of lignin peroxidase in shake flask cultures. Next, the mechanism of biocatalyst pellet formation, growth and the effects of agitation were studied. Using the optimized media and an understanding of the characteristics of the pellet biocatalysts, a stirred 3 tank reactor system for the production of lignin peroxidases was developed. This system produced the lignin peroxidases at levels comparable to shake flask cultures. This thesis is divided into five chapters, including the introduction (Chapter 1). The literature review (Chapter 2), describes work previously undertaken to understand the lignin degrading system in the white rot fungus zp‘gnrygggpgxign and the factors that affect the production of the lignin peroxidase enzymes by this organism. Also described are reactor systems that have been developed for lignin peroxidase enzyme production. In Materials and Methods (Chapter 3), the techniques, and procedures used in this thesis are described. In the Results (Chapter 4), the findings of the three part study, as described above, are presented, primarily through the use of figures and tables. The major findings and future directions are explored in the Discussion (Chapter 5). Finally, in the appendix, an unsteady state model for the batch culture production of lignin peroxidases is described. This model used the data from optimization experiments and from other sources". It accounted for the growth, substrate utilization, effects of media components and product formation in lignin peroxidase producing cultures of 2‘ ghzysggpgriun. Also presented in the appendix are the 4 description of a trickle bed reactor system which was developed but produced no detectable lignin peroxidase (Appendix B). The tabulated data used in this thesis are presented in Appendix C. CEIPTIR II: LITIIIIUII RIVIII 2.1 Lignin biodegradation Lignocellulosics, 25% to 35% of which are lignin, represent a vast renewable resource for the production of fuels and chemicals. Lignin exists in the cell wall of vascular plants to provide structural rigidity and protection from pathogenic organisms“. It is a chemically and structurally complex aromatic biopolymer (Figure 2.1). Lignin is formed by the dehydrogenative polymerization of cinnamyl alcohol derivatives of which coumaryl alcohol, sinapyl alcohol and coniferyl alcohol are the primary monomers“. Certain micro-organisms, particularly the white-rot 11'“ . Lignin fungi, are known to metabolize lignin however, does not serve as a growth substrate for these cells". The primary function of lignin metabolism by the micro-organisms appears to be to free plant polysaccharides protected by lignin which can serve as growth substrates. The filamentous fungus Rnangzgghaetg ghryfigspgrign has been shown to be particularly capable 14 of degrading lignin based on the conversion of C radio labeled lignin to CO ”, as well as the ability to 2 produce the extracellular enzyme lignin peroxidase“. This organism displays rapid growth, metabolism of Figure 2.1 Typical structure of lignin [44]. lignin, prolific conidiation (spore production), a high temperature optimum of 40° C and the ability to grow on a range of media”. In addition to degrading lignin, 2. W has been shown to degrade environmental pollutants such as DDT, polychlorinated biphenyls and °'7"'”'”'”. These pollutants have chemical dioxins structures similar to lignin metabolites and may be degraded by the same mechanism as lignin. 2.2 Lignin Peroxidases from zhangxgghggtg_gnrzgggpgxinl Lignin peroxidases are produced during the stationary phase (idiophase) by 2‘_gn:y§g§pgzinn,when growth is limited by carbon or nitrogen”. Under shallow stationary conditions 2,,ghzygggpgrinn grows in the form of a mycelial mat”. Under the influence of agitation, 2‘ ghxygggpgzinn grows in the form of mycelial pelletsu’3°'5°. L, W has been shown to produce optimal ligninolytic activity in a buffered media (pH - 4.5) consisting of a nitrogen source and a carbon source (one of which must be limiting), trace elements, basal elements and the vitamin thiamine”. Both carbon and nitrogen limited cultures of 2‘ gnzysggpgzinn produce lignin peroxidases. Nitrogen limited conditions more closely approximate the natural environment encountered by white rot fungi (the nitrogen content of wood is low)“; Therefore, nitrogen limitation is generally used in the laboratory to study lignin peroxidase production. Carhon limited cultures have been shown to only transiently degrade lignin and to autolysefll However, despite this finding, many researchers have successfully used carbon limited cultures to produce high lignin peroxidase titres . ”M'” The time course of lignin peroxidase production and the growth of 2‘ gnzyggspgzinn in nitrogen limited cultures is presented in Figure 2.2. Cultures grow rapidly and reach the stationary phase during the second day. During this period, the nitrogen in the medium is depleted and becomes incorporated into the growing mycelium“. At the same time, mycelial dry weight increases and glucose is steadily depleted. The mycelial dry weight continues to increase after the second day due to the production of an extracellular polysaccharide. Glucose is consumed both in the growth and the stationary phase, declining linearly throughout the fermentation. Lignin peroxidase activity first appears 5 days after inoculation and reaches a maximum on the sixth or seventh day. After maximum activity is reached, lignin peroxidase activity declines. The decline in activity may be due to enzyme degradation by proteases. Myceliol Dry Weight, mg 1 J IO" Glucose . mM s 9 Cuiture Age. days Figure 2.2 Time course of growth, substrate utilization and lignin peroxidase activity of agitated cultures of £1 shrxsgsesfium [26]- Totol Myceliol N per Culture, m9 10 24.1 strain- of 2. Wins various strains of 2,,ghrysgspgzinnlexist which have been used to study lignin peroxidase production. The wild type strains and-1767”“ (arcc 24725) and It!-446"’""1 (ATCC 34541) have been predominantly used. Of the wild type strains, BKM-F—1767 has been shown to be the most rapid lignin degrader and produces the highest lignin peroxidase activities”'”. Two mutant strains have also been studied: SC26°°'33, derived from axon-lune? and Ina-12"“. Strain sczs was developed for the production of lignin peroxidase in a rotating biological contactof”. It produces higher lignin peroxidase activities than the wild type33 and more easily adheres to surfaces. Strain INA-12 has produced the highest lignin peroxidase activities to datez:h1 carbon limited cultures but is not available. 2.2.2 Characteristics of Lignin Peroxidase Tien and Kirk‘“, and Gold et alm first separated and characterized the extracellular lignin degrading enzyme from six day old cultures of 2; ghzysggpgzigm. This enzyme non-specifically catalyzed several oxidations in the alkyl side chains of lignin related compounds. It was a heme containing glyco-proteinw'that required hydrogen peroxide for activity”. The enzyme belonged to a group of isozymes which catalyzed the oxidative depolymerization of lignin”. Collectively 11 these isozymes were called "ligninases”. They share the ability to catalyze the oxidation of veratryl alcohol to veratryl aldehyde in the presence of 3202, have a MW of between 39,000 and 43,000”, and cross react with polyclonal antibodies raised against 88 one of the predominant lignin peroxidase isozymes”. Further studies”'“ have shown that these extracellular enzymes have a pH optimum of 2.5 to 3.0 and a 915°C. between 3.5 and 4.65. The Michaelis-Menten constant, Km, for H202 and veratryl.alcohol was found to be 80 uM and 60 pM respect ive 1y“ . First thought to be oxygenases, ligninases are now known to be typical peroxidase enzymes“ and are now referred to as lignin peroxidases. The key reaction is the one electron oxidation of aromatic nuclei to produce unstable cation radicals”. The evidence supporting this finding is that chemically catalyzed one electron oxidation of lignin model compounds gives the same reaction products as lignin peroxidases” and UV/Spectroscopic studies which have shown that lignin peroxidases change in the same way as other peroxidases during reaction”. The lignin model compounds have been shown to be degraded by lignin peroxidase via the following mechanismz’"""-“ (Figure 2.3). First, the lignin peroxidase heme forms an oxo-iron (IV) porphyrin cation radical with hydrogen peroxide. This removes an 12 cos, ocu Lignin tonal mu m 0.6 and and u. on 0‘ o C c”. OCH: . b + + u D gas-u” . 2W "“"\°. M eta: " F 4 H $61.4 WW Figure 2.3 The mechanism of the biodegradation of a lignin model compound [46]. 13 electron from an oxygenated aryl ring of a lignin model compound to form.an aromatic cation radical (A to B). Cleavage of a lignin cg-Cb bond follows resulting in two products (C and D). One of these products (C) may incorporate atmospheric oxygen forming a peroxy radical (E) which oxidizes to an aldehyde (M) or other products (G). The other cation product (F) may be oxidized by water forming an alcohol. 2.2.3 Factors Affecting Lignin Peroxidase Production The production of lignin peroxidases by cultures of Eiqghrysgspgxiun is influenced by many factors. These include oxygen tension, strain, buffer, the presence of enzyme inducers“, agitation“'", trace element concentration, and the presence of surfactants. Oxygen Partial Pressure. The partial pressure of oxygen in cultures of 2‘ gnzyfigspgzinn has a profound affect on the production of lignin peroxidase. Barlev and Kirk}, found that in shallow stationary cultures of 2‘ ghzygggpgzinn, lignin was degraded to CO2 faster in a pure oxygen environment than in air. In these cultures growth was unaffected by the oxygen partial pressure. However, the oxygen partial pressure did affect both the amount of the ligninolytic system produced and the rate of lignin oxidation. Faison and Kirk” measured a twenty fold increase in lignin peroxidase activity in cultures grown in pure oxygen 14 compared to cultures grown in air. Leisola et al" found that in agitated cultures with a 100% oxygen atmosphere, lignin peroxidase activity increased ten fold compared to cultures with an air atmosphere. The mechanism by which oxygen stimulates lignin peroxidase production is not known. Buffer and pl. A pa of 4.5 has been shown to be optimal for the expression of ligninolytic activity in L, W”. The choice of buffer in the culture medium, however, can affect the activity of the ligninolytic system. Cultures buffered with polyacrylic acid have been shown to more rapidly degrade lignosulfones than cultures buffered with the widely used 2,2-dimethyl succinate buffer”. Acetate buffer has been reported to be toxic to L, W32 however other investigators have mentioned that acetate buffered cultures produce higher lignin peroxidase activities than 2,2-dimethyl succinate buffered cultures. Compared to phthalate buffered cultures, aconitate and tartrate buffered cultures mineralized lignin more slowly”; Some buffers (succinate and citrate) may be used as carbon sources by the growing organism. The buffer, 2,2- dimethyl succinate, is commonly used by investigators but is expensive. A more effective and less expensive buffer would greatly reduce media costs for lignin peroxidase production. 15 lnsyme Inducers. Lignin peroxidase activity has been shown to be inducible by lignin and by products of lignin degradation, particularly veratryl alcohol (3,4- dimethoxybenzyl alcohol). Faison and Kirk”) found that birch, spruce and synthetic lignins, the lignin monomers syringyl and vanillyl alcohol as well as veratryl alcohol, veratryl aldehyde and veratrylglycerol increase the lignin peroxidase activity of stationary nitrogen limited cultures. Of these, veratryl alcohol gave the greatest increase in lignin peroxidase activity. Further study“ also revealed increases in some extracellular lignin peroxidase proteins when veratryl alcohol was added. Leisola et ah” detected no lignin peroxidase activity in agitated nitrogen limited cultures without veratryl alcohol. With the addition of veratryl alcohol lignin peroxidase activity appeared and reached a maximum 10 to 12 hours later. These results indicate that veratryl alcohol is an enzyme inducer. It may affect messenger RNA (mRNA) transcription. Surfactant Addition. Agitated cultures of 2‘ anysgfipgzinn initially produced little or no lignin ”'33. Jager et al“ demonstrated that peroxidase agitated cultures could produce lignin peroxidase at levels greater than or equal to stationary cultures when the surfactants Tween 80, Tween 20 or 3-[(3- 16 colamidopropyl) dimethylammonio] 1-propanesulfonate (CHAPS) were added. Tween 80 and Tween 20 can supply fatty acids to the fungus however CHAPS cannot. Thus a mechanism other than fatty acid supplementation is thought to affect lignin peroxidase production. Asther et al? found that the addition of oleic acid emulsified in Tween 80 further increased lignin peroxidase production. They measured lipase activity in the extracellular fluid during the primary growth phase and found that fatty acids emulsified in Tween 80 were depleted by the organism. The mechanism by which surfactants enhance lignin peroxidase activity has not been established. Surfactants may modify the cell membrane affecting the uptake and release of compounds. Effect of Agitation. It has been reported that inCreased agitation reduces lignin peroxidase production in agitated cultures of L W15”. In shallow agitated cultures of strain INA-12 Asther et al? found that at agitation speeds greater than 50 rev min-1, lignin peroxidase activity markedly decreased. Leisola et at" have found that pellet size and concentration affects lignin peroxidase activity although they did not quantify pellet size. Wase et al" have reported an analogous result in disc turbine agitated cultures of cellulase-producing mutants of Aapgzgillng‘niggz. They found that increasing 17 the agitation rate from 100 to 300 rev min-1 caused a decrease in enzyme activity. They also found an increase in extracellular protein with increased agitation. They implied that, “Mycelium was being disrupted by shear forces generated by the impeller, liberating protein.', and measured low levels of protease activity in the liberated protein. The effect of agitation on the production of lignin peroxidase by 2‘,ghzy§9§pgzinn has been overcome by the use of surfactants (see abovef". The mechanism by which agitation affects lignin peroxidase production by 2‘,gnzy§g§pgzinn is not known. 2.3 Fungal biocatalysts Fungal biocatalysts are used industrially for the production of organic acids“, antibiotics, enzymes and the biotransformation of pharmaceuticals‘Jm‘Lflh 2.3.1 Fungal pellet reactor systems Many filamentous fungi including, 2‘ gnxysggpgzium form pellets in submerged culture“. Pellets are formed by the aggregation of growing hyphae. Their formation is influenced by the rate of agitation, medium composition, strain, inoculum concentration and other factors"*”. Fungal pellets provide a convenient means of immobilizing the biomass within a reactor. Once formed pellets can serve as biocatalysts for the production of secondary metabolites. Fungal pellets have been used in reactor systems 18 for the production of lignin peroxidase. A 1.5 liter air-lift reactor has been developed which produced lignin peroxidases from mycelial pellets. In this reactor pellets were formed by air circulation however the enzyme yields of this reactor were low (60 U 1.1)". A 78 ml column reactor using preformed mycelial pellets has been reported” which produced lignin peroxidases continuously (172 U 1-1). The development of a stirred tank reactor system for the production of lignin peroxidases has not been described. 2.3.2 Fungal film reactor systems Filamentous fungi generally exhibit a strong affinity for surfaces. In fungal film bioreactors, mycelia attaches to immobilization support material submerged in the culture medium. Suitable support materials must not be toxic to the organism, encourage mycelial attachment and be steam sterilizable. Kirk et al developed a rotating biological contactor to produce lignin peroxidase from an adsorbed fungal film of strain SC26”. In this reactor disks of polyethylene were slowly rotated (1 rev min-1) through a cylinder half filled with medium. This reactor produced lignin peroxidase activities comparable to stationary flask cultures (130 U l-1 ). A 1-liter Biostat M fermenter (designed for tissue cultures) has also been used to produce lignin peroxidases from fungal films“§ In this 19 reactor system the wild type strain BKH-P-l767 was immobilized on silicone tubing wrapped around stainless steel rods. Lignin peroxidase activities of 200 U 1'1 were achieved. Kirkpatrick and Palmer“ developed a method to produce lignin peroxidase semi-continuously using polyurethane foam immobilized mycelia in a 1 liter shake flask. very recently, a 10 liter bioreactor system was developed in which the mycelia was immobilized on nylon web carrier”. This system produced over 700 U l"1 of lignin peroxidase activity in carbon limited medium. The drawbacks to the scale up of fungal film reactors are that free mycelia foul sensors and clog lines and high culture viscosity hinders oxygen transport. In industrial scale fermentations the immobilization support material must be cleaned following each fermentation. 20 2.4 Research Objectives 1. The currently used media for lignin peroxidase production are costly. This is due primarily to the use of the expensive buffer 2,2-dimethyl succinate. The first objective of this study is to optimize a low cost defined medium for the production of lignin peroxidase using a less costly buffer. Pellet formation, growth and the effects of agitation on lignin peroxidase production by 21 ghzygggpgzinn are not well understood. Experiments will be conducted to gain an understanding of the mechanism of pellet formation and to determine the consequences of varying the agitation rate. Existing reactor systems which have been developed for the production of lignin peroxidases can not be readily scaled up. The primary objective of this study is to develop a stirred tank reactor system to produce lignin peroxidases from biocatalyst pellets whamm- CEIPTII.IIIS IISIIIILI I'D IITIDDI 3.1 Culture Characteristics All experiments were conducted in nitrogen limited cultures of zhangzgghaggg,ghzygggpgzinm. Strains BKM-F- 1767 (ATCC # 24725) and SC26 (obtained from Dr. T.K.Kirk, U.S.Porest Products Laboratory, Madison, WI) were used. The culture medium used was a defined low nitrogen (nitrogen limited) medium” buffered at pH 4.5, with veratryl alcohol (an enzyme inducer), and Tween 80. Cultures were initiated by a conidial inoculum. After 2 days growth in stationary Fernbach flasks, the mycelia were blended and then inoculated into Erlenmeyer flasks (125ml or 21) containing low nitrogen medium. The cultures were incubated at 39° C. Lignin peroxidase activity as measured by the oxidation of veratryl alcohol to veratryl aldehyde, in the presence of hydrogen peroxide, was generally detected on day 5. Maximum lignin peroxidase enzyme activity occured on day Ger“). 3.2 Solution and media preparation 3.2.1 Stock solutions The following stock solutions were prepared and sterilized as aliquots and stored until use at 4° C. 21 22 Deionized water (DI) was used in the preparation of all stock solutions. Basal medium was prepared by adding 20 gm KH2P04, 5 gm 119804 and 1 gm CaCl2 to a final volume of one liter. The solution was sterilized by passing it through a 115 ml 0.45u Nalgene filter sterilizer unit, (Nalge Co., Rochester M.Y., type 8 CM #245-0045). Trace element solution was prepared by first dissolving 1.5 g nitrilotriacetate in 800 ml 320 and adjusting the pH to 6.5 using 1.0 M ROM. Then 3.0 gm ugso"7n o, 0.5 gm unso4, 1.0 gm NaCl, 0.1 gm 2 F0804 732 Cuso4, 10 mg 21x(so 0, 0.1 gm CoCl , 0.1 gm znso '73 0, 0.1 gm 4 2 o, 10 mg H330 2 '12s and 10 mg 4’2 2 3 Na M00 '28 0 were added to make a final volume of one 2 4 2 liter. The solution had a faint yellow color. The solution was sterilized by passing it through a 115 m1 0.45u Malgene filter sterilizer unit. 10% Glucose (560 ml) was prepared by dissolving 100 gm glucose in a final volume of one liter. The solution was sterilized by autoclaving at 115 C for 15 min. Buffer Solutions. Acetate Buffer (0.2 M, pH 4.5) was prepared by adding 5.98 g Na-acetate'afi 0 and 3.23 ml 2 acetic acid to 800 ml DI water. The pH was checked (should be 4.5) and the solution was brought to 1 liter using DI water. Malate (0.2 M), citrate (0.1 M), 23 tartarate (0.1 x), succinate (0.1 x) and oxalate (0.1 u) buffers were prepared similarly according to the procedure of Gomori”. 2,2-Dimethyl succinate buffer (0.1 M) was prepared using 2,2-dimethyl succinic acid and 1.0 M laOH to adjust the pa. All buffer solutions were sterilized by autoclaving at 1150 C for 20 minutes. Ammonium Tartrate (0 g/l, 43.4 ml) was prepared by dissolving 8.0 gm ammonium tartrate in 800 ml DI water and then bringing the volume to one liter. The solution was sterilized by autoclaving at 1150 C for 20 minutes. Thiamine (1.0 g/l, 2.95 ml) was prepared by dissolving 0.10 g thiamine (Nutritional Biochemical Corp., Cleveland 08) in a final volume of 100 ml. The solution was sterilized by passing it through a 115 ml Malgene 0.45p filter sterilizer unit. Veratryl alcohol (400 ml) was prepared by adding 6.73 gm of veratryl alcohol (Aldrich Chem. Co. Inc., Milwaukee, WI. #D13,300-0) to DI water to a final volume of 100 ml. The solution was sterilized by passing it through a 115 ml Nalgene 0.45p filter sterilizer unit. Tween 00 (1.0 t) was prepared by adding 1.0 gm Tween 80 (Sigma Chem. Co., PO Box 14500, St.Louis, NO, #P- 1754) to 80 ml of DI water and bringing the solution to 100ml. The solution was sterilized by autoclaving at 115° c for 20 min. 24 3.2.2 Media preparation Media components were combined asceptically using autoclaved graduated cylinders, pipets and Erlenmeyer flasks. Mixtures were brought to the final volume using autoclaved DI water. Once prepared, the media were used immediately. The compositions of the three culture media used in this study are presented in Table 3.1. Culture medium #1 is a malt extract medium which was used to maintain the fungus and to produce conidia for inoculation of starter cultures. It was prepared by combining the ingredients into 1 liter of deionized water (final volume), adjusting the pH to 4.5 using 0.1 M hydrochloric acid, and autoclaving. The medium was cooled to approximately 50° C and asceptically poured into 20 ml Petri dishes to a depth of approximately 1 cm (15 ml per plate) and allowed to solidify at room temperature for 2 hours before use. Culture medium #2 was used for producing lignin peroxidase in stationary cultures and for preparing inocula for agitated cultures. Culture medium #3 was used for producing lignin peroxidase in agitated cultures. Stock solutions prepared as described above were used for making media 2 and 3 (Table 3.1).these two media. The concentrations of some media components were varied in optimization experiments. 25 Table 3.1 Culture Media Compositions Glmose 209 mtnctract 209 mums 1g AgarBacto 15g 0.1mm topi4.5 DIwater tonhmsliter mmn: mammals:- 10% (illness (autoclaved) Amman Tartrate (8 g/l stock, mtoclaved) Trace slants (filter sterilized) Thiamine (1 g/l stock, filter sterilized) Differ solution, pH 4.5 (autoclaved) Veratryl Alcdxol (400 :04 stock: filter sterilized) Basal medium (filter sterilized) (11111113 1230.1” DIwater(autoclaved) mm mmtaxmwmmcmm 10% Glucose (autoclaved) Amrnim Tartrate (8 g/l stock, mtoclaved) Trace elanents (filter sterilized) Thiamine (1 g/l stock, filter sterilized) mffer solutim, PH 4.5 (autoclaved) Veratryl Alcdiol (400 1124 stock: filter sterilized) Basal Indium (filter sterilized) mean 80 (1% stock, autoclaved) DI water (autoclaved) mnvom (*lhdiacalpaimtswhoseomoentratimsmcptimized.) 26 3.3 Culture Inoculation Methods 3.3.1 spore Inoculum.rreperation 2hanezgghnggeflghrylggpgzinl,BKM-P-1767 and SC26 were maintained through periodic transfers on malt extract agar plates as previously described”. A loop full of conidia from stock cultures maintained on malt extract agar slants (Medium #1) was added to 5 m1 of sterilized DI water. Approximately 0.5 ml of this conidial susupension was added to a Petri dish containing 10 ml of solidified culture medium #1. The conidial suspension was spread over the surface of the plate and the plate was incubated for 4 or 5 days at 39° C until white conidia covered the plate. The conidia were collected asceptically by adding 5 ml of sterilized DI water onto a plate and then stirring the surface of the plate using a bent glass rod. The conidial suspension was vacuum filtered, to remove mycelia, through sterilized glass wool into a sterile Erlenmeyer flask to remove mycelia. The concentration of the conidia in the suspension was determined by preparing a 1:10 dilution of the conidial suspension, placing it on a haemocytometer and counting the conidia using phase contrast microscopy. The optical density of a 1:100 dilution of the conidial suspension was also determined at a wavelength of 650 27 nm. An optical density of 1.0 cm'1 corresponds to approximately 5 x 10 conidia ml‘l. This conidial suspension was used to inoculate Pernbach flask starter cultures, used to inoculate additional malt extract plates or stored in 2.0 m1 cryogenic vials at -15°’C for up to 2 months for future use. 3.3.2 Starter culture preparation Starter cultures were shallow stationary cultures of 2;,gnryggspgzinn (culture medium #2) used to create biomass for inoculating agitated cultures (Table 3.2 type 1). Pernbach flasks (3.0 liter volume), plugged with cotton wool wrapped in cheese cloth, were autoclaved along with flasks for media preparation. After autoclaving for 20 minutes at 15 psig and 121° C, 75 ml of culture medium #2 was added to each Fernbach flask using a sterile graduated cylinder. The Fernbach flask was inoculated to an optical density 0.1 at 650 nm using fresh or frozen conidia prepared as described above. The starter cultures were incubated at 39° C and not disturbed for 48 hr (any disturbance invariably caused the mycelia to clump and affected lignin peroxidase activity). After 48 hr, the contents of the Fernbach flasks were blended for five minutes at the highest setting in a Sorval omni-mixer blender, using an 28 .mousuHsO Amamv uouooou xsou omuufium aw .nousuHso cofiuosooua Madame uouocou xccu omuufium In .mousuaso hosum nu3ouu use sowuoauou poaamm .Cowuouwaaumo In .mousuaso umuucun xmeau socnsumh la a :55 23 2: 3030s He c.8m 2 H a v 753 28. mfi 3H8? a... can on o.m$ % a w n T53 >3 8m «3029. E m... as mé. 2 He m2 m 5330 00.8% no To B can e. H n H - .. A A - .. - 7. r104. ”did ‘ 6 .1:0 snzumHuFu aaanacH momaaao> aflfififl- ”aflzw maze l. IflOflfldUn—OU Us BOdUIHflOOflH .Hufiudg Non 52h. 29 autoclaved blender cup (500 ml volume). Incomplete blending resulted in clumps and agglomerates which gave large irregular mycelial growths during the pellet formation process. The blender cup was immersed in a mixture of ice and water to reduce heating during the preparation of the blended mycelial suspension. After blending, the contents of the blender cup were checked to be sure that all of the mycelia had been homogenized and that no strings or clumps existed. This suspension consisted of approximately 1.1 g of dry mycelia per liter of mycelial suspension. It was used as a 10 % (v/v) mycelial inoculum for agitated cultures. 3.3.3 Agitated culture inoculation Agitated shake flask cultures were used for media optimization, and pellet formation and agitation studies (Table 3.2 type 2). About 40.5 ml of culture medium #3 was inoculted with 4.5 ml of the mycelial suspension prepared as described above. The suspension was transferred from the autoclaved blender cup to the flask using a sterilized, cotton plugged 5 ml pipet. 3.3.4 Stirred tank reactor inoculation The stirred tank reactor was inoculated with pellets which were prepared in a 2 liter shake flask culture (Table 3.2 type 3). Culture medium #3 (675 ml) was inoculated with 75 ml of blended mycelial inoculum 30 prepared as described above..After incubation at 39° C at an agitation rate of 125 rev min-1 for 48 hrs, the formed pellets and medium were transferred asceptically into a 1 liter stirred tank reactor (Table 3.2 type 4). 3.4 Culture conditions 3.4.1 flask culture conditions All cultures were incubated at 39° C. Agitated cultures were incubated in a New Brunswick Shaker bath with a 1.25 cm diameter of rotation. The 45 n1 cultures were agitated at an agitation rate of 200 rev min’1 (and other agitation rates as indicated) and the 750 ml cultures were agitated at a rate of 125 rev min-1. The rate of rotation was measured both by counting the number of cycles during a timed interval and using a phototachometer. The 750 ml cultures were also sometimes incubated in the Labline incubator shaker which has a diameter of rotation of 2.5 cm. Stationary cultures were incubated in a Lunaire environmental chamber, and were not disturbed. Shake flask cultures were oxygenated by flushing the head space of the culture flask with at least 10 flask volumes of 99.5 % oxygen. Agitated cultures were oxygenated at the time of inoculation and daily thereafter (usually for 1.0 minute at a flowrate between 31 6 and 10 liters min’l). Stationary Pernbach flask cultures were not oxygenated. A sterile Pasteur pipet plugged with cotton was used to convey the oxygen from the oxygen tank to the flask. The pipet tip was flamed repeatedly to maintain sterility. 3.4.2 STE culture conditions After 48 hrs growth, pellets grown in a 2 liter Erlenmeyer flask culture were used to inoculate a stirred tank reactor (Mew Brunswick Sci. Co. model C30, operating volume 500 ml). The stirred tank reactor had a temperature controller and a heating element which were used to control the culture temperature at 39° C Agitation was provided by a stainless steel agitator with four impeller blades mounted on a thick disk, at the bottom of the reactor vessel at a rate of 100 rev 1. The agitator was powered by a magnetic stirrer min- beneath the reactor vessel. Humidified, sterile oxygen was metered continuously through a rotameter and added via a gas dispersion tube with a fine glass frit located directly above the agitator. Antifoam (Dow Corning, Silicone Emulsion 10%) was used to reduce culture foaming. 32 3 . s assays Samples for assays were collected asceptically using Pasteur pipettes. Generally a 0.5 ml sample was collected. The samples were centrifuged for 10 minutes in a bench top centrifuge at 8000 rev min-1 to remove suspended mycelial and other culture debris. The cultures were reoxygenated after samples were taken. Lignin peroxidase activity samples were generally collected on days 3,4,5,6,7 and 8. 3.5.1 Lignin Peroxidase The lignin peroxidase activity of a culture filtrate or enzyme preparation was assayed by monitoring the increase in absorbence at 310 nm due to the oxidation of veratryl alcohol (3,4-dimethoxy benzyl alcohol) to veratryl aldehyde (3,4-dimethoxy benzylaldehyde) at room temperature“. Veratryl aldehyde absorbs strongly at a wavelength of 310 nm whereas veratryl alcohol does not. A 500 p1 volume of the supernatant was transferred to a test tube along with 400 pl of 125 mM tartarate buffer, adjusted to the enzyme's pH optimum of 2.5, and 50 pl of 40 mM veratryl alcohol. The sample was poured into a 2.0 ml quartz cuvette with a path length of 1.0 cm and the baseline was measured at a wavelength of 310 nm using a UV spectrophotometer (Varian Cary Model # 33 219). A chart speed of 10 sec cm-1 and an optical density (OD) range of 1.0 was used. To begin the enzyme reaction, 50 pl of freshly prepared 5.4 ml hydrogen peroxide was added to the sample (this saturates the available enzyme since the Km for hydrogen peroxide is 80 pM). The increase in absorbance over time at 310 nm was measured for a period of at least one minute. This value was used to quantify the units of enzyme activity. A unit of lignin peroxidase enzyme activity is defined as the oxidation of one pmole of veratryl alcohol to veratryl aldehyde per minute. Using the molar extinction coefficient of veratryl aldehyde (9300 M-1 cm'l) and the absorbance change of the sample, the units of lignin peroxidase activity per liter of sample can be calculated according to the following formula. Lignin _ Peroxidase (U 11) - Activity Absorbance change (min'l) * 1 x 10" (umol/mol) 9300 (Mfl'cmfi) * 1.0 (cm) * 0.5 (ml sample/ml assay) 3.5.2 Protein The protein concentration of culture filtrates was determined with Coomassie blue according to the method of Bradford’. 34 Samples were collected and centrifuged at 8000 rev min-1 for 10 minutes in a tabletop centrifuge to remove mycelia. A Biorad protein staining solution (100 pl) was added to 400 pl of sample in a test tube and mixed by shaking. The reaction mixture was then added to a 2.0 m1 cuvette and the absorbance at a wavelength of 595 nm was measured using a Varian spectrophotometer. This absorbance value was recorded and compared to a standard calibration curve to estimate the protein concentration of the sample. A calibration curve was constructed using bovine serum.albumin as the standard protein. A BSA solution 1 was made and stored with a concentration of 1.0 mg ml- at 0° C This solution was diluted ten fold with uninoculated media (or 1 ml Acetate pH 6.0 for FPLC samples) giving a solution with a concentration of 100 1 pg ml- . This solution, in turn, was diluted to give solutions with protein concentrations of 0.0, 2.0, 5.0, 10.0, 15.0 and 20.0 pg ml-l . Biorad protein staining solution (200 pl) was added to 800 pl of the standard solutions. The absorbance of the standard solutions was measured at a wavelength of 595 nm and the values were recorded. A least squares linear regression was used to determine the slope and intercept of the calibration curve. 35 3.5.: pm measure-sat . The p8 of culture samples was measured using a Corning digital expanded scale pH meter with a Corning general purpose pa electrode (Cat #476531). The pH meter was calibrated before each measurement with standard buffers at pH 4.01 and pH 7.00. Samples were measured at room temperature. 3.5.4 Dry weight measurement The cell dry weight was measured using an 0'haus balance. The mycelia was vacuum filtered through tared 9.0 cm diameter lhatmann GP/C grade filter paper and rinsed with deionized water. The filter cake was dried to a constant weight at 39° C and weighed. 3.5.5 Pellet sise measurement Pellet sizes and size distributions were measured using U.S.A. standard screen sieves with 2.0, 1.4, 1.18, 1.0, 0.85, 0.5 and 0.212 mm opening sizes. Samples were added directly to stacked sieve trays and rinsed with deionized water to move the pellets over the screen openings. The number of pellets in each tray from a sample of known volume was counted. During the first 24 hours pellets were too small to be counted using sieve trays so a microscope and a haemacytometer was used to count pellets. 36 3.6 Protein characterisation by PPLC The culture filtrate was collected and generally concentrated. An Amicon ultra concentrator cell (10,000 MW cut-off) was used at a temperature of 4.0° C The filter was prepared by stirring DI water over it for 30 minutes. The extracellular fluid was concentrated 100 fold using Nitrogen at 60 psi. After concentration, the fluid was dialysed against 5 mM potassium phosphate buffer (pl 6.5) overnight. Usually 4 liters of potassium phosphate solution was used to dialyze 40 ml of concentrated extracellular fluid. The concentrated fluid was stored at 4° C until used. Samples generally lost approximately half of their activity per month when stored in this manner. The concentrated and dialyzed extracellular fluid was applied to a Mono Q column (Pharmacia, Uppsala, Sweden) using a gradient of acetate buffer (pH 6.0) from 10 mM to 1.0 M'. Thirty-eight ml of dialyzed extra- cellular fluid was purified using injections of 2 ml. The sample was loaded with the acetate buffer gradient and eluted for 40 minutes. CHIP!!! IV: 2380328 4.1 Media optimization The culture media were optimized with regard to the strain and buffer used, the inoculum size, and the concentrations of various media components. Low cost acetate buffered media was found to give improved enzyme activity. All experiments were conducted in 125 ml Erlenmeyer flasks using culture medium #3 as described in Materials and Methods. 4.1.1 Strain selection Bhangxgghggtg.ghzygggpgzinn strains SC26 and BKM-F- 1767 were compared based on lignin peroxidase activity. Strain BKM-F-l767 was found to give greater maximum lignin peroxidase activity (Figure 4.1). Furthermore, strain BKM-F-l767 formed pellets more readily, was easier to culture and more consistently produced the enzyme than strain SC26. Hence, strain BKM-F-1767 was used in all subsequent experiments. 4.1.2 Acetate buffer selection A comparison was made between the widely used 2,2- dimethylsuccinate (DMS) buffer and a number of less costly buffers, based on lignin peroxidase activity in shake flask cultures. The data are from two separate experimental runs. Acetate buffer (20 mM, pH 4.5) 37 38 250 2004 150‘ 1004 Lignin Peroxidase Activity (U/l) 504 0 u ' 2 3 8 Figure 4.1 Comparison of 2; gnzyggspgzigm strains SC26 ( D ) and BKM-F-1767 ( v ) for the production of lignin peroxidase. 39 produced greater maximum.lignin peroxidase activity compared to DMS buffer (10 ml) on both occasions (Figure 4.2). The maximum activity ocurred later in acetate buffered cultures than in DMS buffered cultures. Tartrate (10 ml) and oxalate (10 ml) buffered cultures produced no detectable lignin peroxidase activity. Citrate (10 ml) and succinate (10 ml) buffered cultures gave slightly less average activities than DMS buffered cultures. The data of Jager et a1" is presented as a comparison. In DMS buffered cultures the culture pH was more stable than in other cultures. In DMS buffered cultures pH rose only slightly after 8 days whereas the culture pH rose approximately 1.0 pH unit after 8 days in all other cultures. This may be due to the metabolism of the acid component of some of the buffers causing the media pH to rise. 4.1.3 Optimisation of spore inoculum concentration The amount of conidia added to Fernbach flasks appeared to affect lignin peroxidase activity (Figure 4.3). Maximal lignin peroxidase activities were obtained 5 5 conidia ml"1 when conidial inocula of 2.5x10 to 5.0x10 were used. These conidial (spore) concentrations correspond to optical density values of 0.05 and 0.1 OD respectively. The concentration of conidia used in the following experiments was 5.0x105 spores ml-l. 40 200 -a— 10 ml DMS -*- 20 ml Acetate g: + 10 mM Citrate B + 10 ml Succinate :150 - —o— 10 mM Tartrate 3;. -+— 10 mM Oxalate 3%; + lager et a1 [26] <1 8 100 . d '6 «II a. O I- 0 On :: 50 ~ "-4 f: .U .3 0 2 3 9 Figure 4.2 Comparison of various buffers for the production of lignin peroxidase. 41 250 2001 150- 100 - / 4 5 Lignin Peroxidase Activity (U/l) C” O I I T Day Figure 4.3 Time course of lignin peroxidase activity for four different spore inoculum concentrations. ( c1 ) 2.5 x 105 spores m1.1 ( x ) 5.0 x 105 spores m1.1 ( v ) 1.5 x 10'5 spores ml.1 ( # ) 3.0 x 10" spores ml"1 42 4.1.4 Optimisatiom.of Tween 00, veratryl alcohol and basal medium concentrations The concentrations of the media components Tween 80, veratryl alcohol and basal medium which support optimal lignin peroxidase activity in acetate buffered cultures were determined. The culture conditions of Jager et a1" were used as a starting point for media optimization experiments except that acetate buffer (20 mM, pH 4.5) was substituted for DMS buffer. Tween 80, which allows lignin peroxidase production in agitated cultures, gives maximal lignin peroxidase activity at a concentration of 0.05 wt% in DMS buffered cultures.” In acetate buffered cultures, as in DMS buffered cultures, no activity was detected in the absence of Tween 80 (Figure 4.4a) and optimal enzyme levels were observed at a Tween 80 concentration of 0.05 wt%. Lignin peroxidase activity decreased when the Tween 80 concentration was increased to 0.4 wt%, but the decreases in enzyme activity observed with 0.2% and 0.025% concentrations of Tween 80 were not significant. Veratryl alcohol is known to be an inducer of lignin peroxidase activity’"”. In the absence of veratryl alcohol, little or no activity was observed whereas optimal activity was seen at 0.4 ml veratryl alcohol concentration. No significant increase in lignin peroxidase activity was observed for veratryl alcohol 43 a Lignin Peroxidase Activity (ll/l) i A ”4 .- I 5“ E a~‘ 5' t 2 3’4 5 3 gm : 6 Exam 3 0 I 2.0 +0! --|l A +2: E”. *5! 3‘ 5 31001 i C 5 glfl< fl- 2 B 50* I .3 O 2 Figure 4.4 Time course of lignin peroxidase activity as affected by: Tween 80 concentration (A), veratryl alcohol concentration (B) and basal medium concentration (C). 44 concentrations above 0.4 ml (Figure 4.4b). The effect of basal medium concentration on lignin peroxidase production is presented in Figure 4.4c. Lignin peroxidase activity was obtained even in cultures to which no basal medium was added. Maximal activity occured in cultures to which 1 or 2 times the normal amount of basal medium was added. The optimized media used in all subsequent experiments had the composition indicated in Table 4.1. 4.1.5 Culture characteristics using optimised media The time course of pH, dry cell weight and lignin peroxidase activity were determined for cultures grown in the optimized media described above (Figure 4.5). The cell dry weight increased dramatically during the first 24 hours after incubation. After culture nitrogen was depleted (24 to 48 hrs after inoculation), cell dry weight continued to increase through the sixth day. This was probably due to the formation of polysaccharides from the excess glucose available in the medium (see Kirk et a1”) . The culture pH rose steadily throughout the fermentation. After eight days, culture pH had risen from 4.5 to more than 5.6. This rise was not observed in DMS buffered cultures. The lignin peroxidase activity of cultures grown in the optimized media varied tremendously from run to run. 45 Table 4.1 Optimised culture media for lignin peroxidase production. Optimised Culture Medium #2 10% Glucose (autoclaved) 100 ml Ammonium Tartrate (8 g/l stock, autoclaved) 25 ml Trace elements (filter sterilized) 70 ml Thiamine (1 g/l stock, filter sterilized) 1 m1 Acetate Buffer solution, (0.2 M, pl 4.5) 100 ml Veratryl Alcohol (400 ml stock: filter sterilized) 1 ml Basal medium (filter sterilized) 5 100 ml Conidia 5.0x10 spores/ml DI water (autoclaved) ~700 ml TOTAL VOLUME 1 1 Optimised Culture ledium #3 10% Glucose (autoclaved) 100 ml Ammonium Tartrate (8 g/l stock, autoclaved) 25 ml Trace elements (filter sterilized) 70 ml Thiamine (1 g/l stock, filter sterilized) 1 ml Acetate Buffer solution, (0.2 M, pl 4.5) 100 ml Veratryl Alcohol (400 ml stock: filter sterilized) 1 ml Basal medium (filter sterilized) 100 ml Tween 80 (1% stock, autoclaved) 50 m1 DI water (autoclaved) ~700 ml TOTAL VOLUME 900 ml 46 2A $12 q -1 Day Figure 4.5 Characteristics of agitated cultures grown in optimized media. ( D ) Lignin peroxidase activity (U 1 * 10°) ( x ) Dry cell weight (g l ) ( v ) pl * 10 47 In some experimental runs no activity at all was detected. variation within the same experimental run however, was less significant. The average lignin peroxidase activity from over twenty cultures grown in the optimized media is plotted in Figure 4.5. For each point the standard deviation of the average lignin peroxidase activity generally exceeded half the value of the mean. Cultures in which lignin peroxidase activity was not detected were not included. A model was proposed for the growth, substrate utilization and lignin peroxidase enzyme production. It is presented in Appendix A. 48 4.2 Biocatalyst Pellets Agitation of submerged cultures of 2;,gnzygggpggign leads to pellet formation. Agitation has also been reported to inhibit ligninolytic activity in these cultures. The pellet formation mechanism and the effects of agitation on lignin peroxidase producing cultures of .21 ghrysgapgzinm have not previously been studied. 4.2.1 Pellet formation mechanism To study the mechanism of pellet formation, the number of mycelial fragments in the inoculated Erlenmeyer flasks was determined as a function of time (see methods). A semi-log plot of the number of hyphal fragments vs. time is presented for cultures in both 125 ml and 2 l Erlenmeyer flasks in Figure 4.6. The hyphal fragments which numbered approximately 1 x 107 per liter initially, aggregated to between 2.0 and 4.0 x 104 pellets per liter in less than 8 hours. This number remained constant throughout the rest of the fermentation. The number of hyphal fragments per pellet was between 2 x 102 and 6 x 103, indicating a coagulative mechanism for pellet formation. This conclusion was further supported by observations of pellet formation using a light microscope (Figure 4.7). Once formed the pellets remained intact throughout the fermentation and did not break apart even at high agitation rates. 49 17 9 _. 8 l I 1 I I l I O 3 6 9 12 15 18 21 24 Hour Figure 4.6 Semi-log plot of the number of hyphal agglomerations (n) as a function of time; ( x ) 2 liter flask culture, ( D ) 125 ml flask culture. 50 O h 4h 6h Figure 4.7 Schematic representation of the mechanism of pellet formation by 2; chrysosporium observed using a light microscope (not to scale). 51 4.2.2 Pellet growth After aggregation ceased, the pellet size increased due to cellular growth. The Sauter mean diameter” of the pellets was calculated according to the formula: Sauter Mean - D32 - (2n d3/d2)/n ( 1) Diameter 1 where D32 is the Sauter mean diameter, d is the pellet diameter as measured by screen sieves and n is the number of pellets (Figure 4.8). The Sauter mean diameter averages particle size based on the ratio of volume to surface area, which is appropriate for catalyst applications. The average pellet size was 1.29 t .21 mm after eight days of growth. The standard deviation, which indicates the range of pellet sizes, indicates that pellet size is fairly uniform. A photo-micrograph of an 8 day old mycelial pellet (Figure 4.9) reveals that pellets consisted of two regions: a dense inner region consisting of round structures (arthrospores) and branched hyphae packed closely together, and a less dense outer region consisting primarily of long hyphal filaments. 4.2.3 Agitation Effects It has been reported previously that increased agitation has a negative effect on lignin peroxidase enzyme production in agitated cultures of 2; 52 1500 14002 H H N C.) O O O O l l 1100* 1000~ 900: 800~ Sauter Mean Diameter (um) 700‘ 600 I I I I I I I Figure 4.8 2; ghrysospgrium pellet size as a function of culture age. Pellet size is expressed as the Sauter mean diameter. ( x ) STR culture, ( D ) 125 m1 flask culture. 53 I ‘ ' i O .' . 3’.s V .""v:. . .’. ‘ ’0 ‘. '- ’4 I e' ' >. \ ‘. 1 .e [to . ‘ . . V (1.5: “’9‘“; .~ -I' '. . Figure 4.9 Photo micrograph of an eight day old mycelial pellet of E; gngysosporium. 54 W”. In this study the size of pellets, the number of pellets produced, and the expression of lignin peroxidase were all affected by the rate of agitation. The time course of lignin peroxidase activity for three agitation rates is presented in Figure 4.10. Cultures agitated at a rate of 260 rev min'1 had significantly less lignin peroxidase activity compared to flasks agitated at 200 and 150 rev min-1. Some of the properties of the biocatalyst pellets produced at three different agitation rates were determined (Table 4.2). These data (Table 4.2) show that higher agitation rates result in an increase in extracellular protein, a decrease in pellet size and an increase in the pellet concentration. Pellet size was significantly affected by the agitation rate. Photographs of pellets formed in shake flask cultures agitated at three different agitation rates are presented in Figure 4.11. At a low agitation rate (<75 rev min-1), a single mycelial pellet is formed. At higher agitation rates pellet size decreases and the number of pellets increases as the agitation rate increases. Shear rate often relates to particle size in liquid- liquid and solid-liquid dispersions. A characteristic shear rate experienced by the liquid phase in a rotating flask is given by rotation rate (RPM) times the diameter 500 55 400‘ 300‘ N O o 1 1004 Lignin Peroxidase Activity (U/l) Figure 4.10 Effect of the agitation rate on lignin peroxidase activity. ( c1 ) 150 rev min‘1 ( x ) 200 rev min: (v) 260 rev minl 56 TABLE 4.2 Culture parameters for 8 day old cultures of 1,, mm at three agitation rates. 150 341 i 73 9.2 :1: 1.0 1.83 i .32 10000 :1; 7000 200 376 ‘1: 33 10.9 i 1.4 1.29 t .21 32600 i 3700 260 62 $100 11.9 1: 2.8 1.27 i .23 36500 i 8500 57 150 rev min'1 200 rev min—1 260 rev min-1 Figure 4.11 Photographs of mycelial pellets of E; chrysosporium formed at three different agitation rates. 58 of the flask at the liquid level (D), (D)*(RPl). The shear rate in a shake flask can be altered by varying either of these parameters. The Sauter mean pellet diameter for cultures agitated at seven different agitation rates from 125 to 300 rev min"1 was measured. The data was plotted using a log-log scale (Figure 4.12), and a straight line (equation 2) with a correlation coefficient of 0.70 was obtained. 0 - 2.1.1::10‘:RPHT°'52 32 ‘2’ This equation, relating pellet size to the agitation rate in shake flasks, indicates that pellet size may be inversely proportional to the square root of the agitation rate. 3500 59 I 032- 2.134 x I04(RPM)'°'52°7 ] E 3- 2000 - a o C) L. 2 0) S o o E a O B EEIOOO ' a a) 2 D h- .9 '3 O m 500 l 1 I IOO 200 300 400 Agitation Rate (rev. min I) Figure 4.12 Correlation of the Sauter mean pellet diameter (Du) and the agitation rate (RPM). 60 4.3 Lignin peroxidase production in a stirred tank reactor (in) Two reactor systens were examined for the production of lignin peroxidase by 2;,thyfigsngzinn; A trickle bed reactor with an immobilized mycelia (see Appendix C) and a stirred tank reactor with nycelial pellets. 4.3.1 Develop-ant of operating conditions for a stirred tank reactor systen A bench scale stirred tank reactor system was developed for the production of lignin peroxidase (Figure 4.13). The reactor system produced lignin peroxidase activities comparable to shake flask cultures. Several factors were critical to obtaining lignin peroxidase activity in the stirred tank reactor (STR): biocatalyst pellet formation, reactor mixing, oxygen dispersion, mycelial adsorption to reactor surfaces and foaming. Two methods for making pellet biocatalysts were used: inoculation of blended mycelia directly into the STR and transfer of pellets to the STR from 2 l shake flask cultures. Inoculation of blended mycelia to the STR did not give detectable lignin peroxidase activities. Some of the mycelia formed pellets and some adhered to reactor surfaces, forming large, irregular agglomerates. 61 sterile antifoam D. in addition T V Control sample port ________ exit gas - - — — I -J headplate CI 0 fi 0 - - medium I _ I o - 0- O 0 O O D O O D O O O O O D L 00 fi 00 liquid level o baffles W ll agitator disk Figure 4.13 Stirred tank reactor system for the production of lignin peroxidase enzymes. 62 The pellets formed under these conditions failed to produce detectable amounts of lignin peroxidase. Removing the reactor baffles reduced the adsorption of mycelia and the amount of irregular agglomerates, but gave large pellets which did not produce lignin peroxidase in detectable amounts. When 2 day-old pellets formed in a 2 l shake flask culture were used to inoculate the STR lignin peroxidases at times and levels similar to shake flask cultures were produced. The agitation rate was adjusted to 100 rev min.1 to provide adequate suspension of the pellet biocatalysts . High agitation rates in the STR (200 rev min-1) resulted in no measurable lignin peroxidase production. Oxygen transfer affected lignin peroxidase activity in the STR. In shake flask cultures, oxygen is transferred readily into the culture medium due to agitation and a high surface to volume ratio. In stirred tank reactors oxygen transfer must be accomplished by sparging. In the bench scale STR, oxygen was introduced above the agitator using a fine glass frit at a rate of 0.5 liter min-1 (1.0 v/v/min). When a glass tube was used as a sparger, no lignin peroxidase activity was detected. The small bubbles produced by the frit tube created foam which did not disperse easily. At an oxygen flowrate of 0.2 liter min-1, foam overflowed the reactor 63 while at 0.05 litermin"1 foaming was roughly static. below 0.05 liter min-1 little foam was created. To enable STR operation without foaming silicone antifoam (Dow Corning FG-lo lot emulsion) was added and an oxygen flowrate of 0.5 liter min.1 (1.0 v/v/min) was maintained. At these levels, antifoam did not inhibit culture growth or enzyme activity. 4.3.: Lignin peroxidase production by the ST! system A typical plot of the time course of lignin peroxidase activity and extracellular protein produced in an STR (agitated at 100 rev min-1, oxygenated at 0.5 liter min.1 and with 5 ml of silicone antifoam emulsion added) is presented in Figure 4.14. One day after pellet biocatalyst transfer, the extracellular fluid became brownish yellow in color. On the third day the pellets turned a dark brown color (which is generally associated with lignin peroxidase production) and lignin peroxidase activity was detected. Lignin peroxidase activity peaked on the fourth day. Six days after transfer, the pellets began to collect around the reactor components and coagulate in the top third of the operating volume. This coagulation of pellets corresponded with the appearance of long filaments growing from the pellets. 4.3.3 Product recovery and characterisation An FPLC profile of the heme proteins in 38 ml of extracellular fluid from an STR culture four days 20- 200- E B, 2 \ 3- 3 .5 Is-Vlso- .9 2‘ g I: 0 § uoquoo- 3 Q) = 8 § :9 .3 5.3 Lu 5-00..) 50F :62. g 0 a '— :1 64 l C) Figure 4.14 Lignin peroxidase activity (D) and total extracellular protein concentration ( O ) in a stirred tank reactor system. 65 after inoculation, was somewhat similar to the protein profile previously described by Kirk et all7 (Figure 4.15). Peaks at an elution volume of 9, 15, 17, 21 and 26.5 ml all had lignin peroxidase activity. The peaks at elution volume 12 to 14 ml (probably manganese peroxidases which correspond to the H3 and H4 peaks in Kirk's profile)17‘were somewhat larger in comparison. Also the peak at an elution volume of 26 ml, which corresponds to the H8 protein peak in Kirk's profile, was reduced in size. The total protein content, lignin peroxidase activity and specific activity versus the elution volume is presented in Appendix C. The protein peaks corresponding to H2 and H8 peaks described by Kirk et a1 had highest specific lignin peroxidase activities. RELATIVE PEAK HEIGHT 66 )— — L— p- - p— .- )— ELUTION VOLUME (ml) Figure 4.15 FPLC profile of heme-proteins in six day old stirred tank reactor system extra- cellular fluid, four days after inoculation. CIIPTIR'Vt DISCUBBIGI 5.1 significance In this study the mechanism and kinetics of pellet formation in agitated submerged cultures of 2‘ ghzygggpgzinl has been determined. Agitation has been found to have a pronounced effect on lignin peroxidase producing cultures of BL‘gnrysggpgzinm. A low cost acetate buffered media has been optimized for lignin peroxidase production. For the first time a STR system for the production of lignin peroxidases has been described. This reactor system utilized optimized media and biocatalyst pellets formed in a shaker flask. Lignin peroxidase was produced at concentrations comparable to that in shake flask cultures. 5.2 Comparison with other Systems A comparison of this stirred tank reactor system with fungal film and pellet biocatalyst systems previously developed is presented in Table 5.1. During the preparation of this thesis two articles appeared in the literature in which immobilized fungal films were used to obtain high lignin peroxidase activities (see Table 5.1)”"°. Another fungal film system previously developed by Kirk et al30 is presented. In these systems, nylon, silicone or polyethylene immobilization support materials were used to which the growing mycelia 67 68 TABLE 5.1 Comparison of the characteristics of various reactor systems for the production of lignin poroxiduu by L. W Volume Maximum Strain Nutrient Activity used limitation (l) (U11) Fungal Film.lioreactors 336 2.5 130 8626 Nitrogen Polyethylene disc immobilized Kirk et a1 1986 Biostat I 1.0 232 BKH Nitrogen Silicone tube immObilized Willar- shausen et a1"9 1987 Biostat H 7.6 730 BKM Carbon Nylon-web immobiliged S.Linko 1988 Pellet Biocatalyst Reactors Airlift 1.5 60 BKM Nitrogen Mycelial pellets 37 Leisola et a1 1986 Column reactor 0.078 172 BKM Carbon Y.Y.Linko et a1” Mycelial pellets 1987 STR 0.5 180 BKM Nitrogen Hycelial pellets (this work) 1988 69 adsorbed. In fungal film reactors the mycelia present in the culture fluid makes product separation difficult and frequently clogs pumps lines and filters. Cleaning of mycelia from immobilization support materials and sensor fouling also pose problems. Pellet biocatalyst reactors should offer an advantage over fungal film reactors for the large scale production of lignin peroxidase. The pellet biocatalyst reactor system developed in this work compares favorably to other small scale pellet biocatalyst reactors. The airlift reactor and developed by Leisola et al37 used a spore inoculum and produced low enzyme activities (60 U 1%). The column reactor developed by Linko et al” was a very small volume reactor (78 ml) in which continuous production of lignin peroxidases was demonstrated. Lignin peroxidase activities above 150 U l'1 were observed in this reactor. 5.3 Future Directions Many obstacles remain for further scale-up of the stirred tank reactor system for lignin peroxidase production. It is especially important to devise methods for producing pellets in the stirred tank itself and for improving the consistency of enzyme production consistency. Methods for efficient product recovery and separation must also be developed. The inoculum scale-up procedure in this study 7O consisted of inoculating a stationary culture with conidia, blending the mycelial mat which forms after 48 hours, and using the blended mycelia as a ten percent inoculum for 750 ml of medium in a 2 liter Erlenmeyer flask. This procedure gives approximately 75 ml of blended mycelial inoculum.per each 3 liter (flask volume) stationary culture. Using this same procedure to create an inoculum for a ten liter fermenter would require over 15 stationary cultures or 45 for a 30 liter fermenter. This is impractical for large scale production. An improved method of producing biomass for inoculation must be developed. This might involve using an agitated starter culture. Using agitated starter cultures a 15 fold increase in biomass could be achieved in the same total flask volume. Other investigators have used blended mycelial pellets as inocula for scale— upn. Thus it may be possible to blend growing pellets (<48 hr old) to give adequate biomass for large scale inoculations. Pellet formation in the 500 ml stirred tank reactor system was accomplished using large 2 liter shake flask cultures to produce pellets for inoculation into the STR. At a larger scale this procedure would also be impractical. If this procedure were used, the culture volume in the shake flask cultures used to produce pellets would equal the volume of the pilot scale 71 reactor. Therefore for a large scale fermentor methods of producing pellets in the reactor itself must be developed. The primary problem with producing pellets in the small STR was that the growing mycelia adsorbed to reactor components and to the agitator. It may be that moving to a larger volume, free of protrusions and uneven surfaces to which mycelia can adsorb, would minimize mycelial adsorption and allow pellet formation in the reactor itself. Intrestingly, in the bench scale STR, removing the baffles greatly increased the amount of mycelia which formed pellets when blended mycelia was used as the inoculum. Finally, the consistency with which reactor systems produce lignin peroxidase must be improved. Although the bench scale STR did produce lignin peroxidase on three separate occasions, on approximately 15 attempts, no detectable enzyme was produced. These failures were in some cases attributable to operational errors however generally they were not. More understanding of the process of enzyme production and excretion is necessary. By more closely monitoring and controlling culture parameters such as dissolved oxygen, glucose and nitrogen depletion, pH, agitation rate and biomass production the consistency of enzyme production may be improved. APPENDIX 72 Appendix A Proposed model for the production of lignin peroxidases by 2;.QIIIIQIPQI19I Model for lnsyme Production A kinetic model to describe the growth, substrate utilization and lignin peroxidase production by cultures of 2‘ ghzyfiggpgrinn has not previously been developed. Detailed structured models have been developed for the production of anti-biotics by filamentous fungi during secondary metabolism“: . A model was developed to account for substrate utilization, biomass growth and lignin peroxidase production in submerged cultures of 2; ghzyfigspgginn. In this model the data for biomass growth, lignin peroxidase activity, nutrient depletion and the effects of media components were used. This model accurately predicted the effects of the media components Tween 80, veratryl alcohol and basal medium on lignin peroxidase production. Biomass growth Changes in the cell dry weight in cultures of 2; gnzygggpgzinn occur due to growth on the limiting nutrient nitrogen, growth on glucose and cell death. The Honod equation can be used to describe microbial 73 growth due to the uptake of the limiting nutrient. In lignin peroxidase producing cultures of zgflgnzysgspgrinm, nitrogen is limiting (equation 1). In this equation N is n a ”In * u i x (1) dt Kn + N nitrogen the nitrogen source (9 1'1), x is the biomass (g l-1 ) and Umn (hr-1) and Kn (g 1-1) are the Henod constants for nitrogen. Cell growth also occurs due to the formation of polysaccharides from glucose (6)”; An additional term is used to account for this growth (equation 2). -B*G*x 3: (2) glucose The cell death rate is represented by a first order death constant (Kd). fix I - Kd * X dt (3) death Combining equations 1 to 3 gives the total rate of cell growth due to nitrogen uptake, glucose uptake and cell death (equation 4). d! g HDD_I_H * X + (B * G * X) ’ (Rd * X) 4 at Kn+N () 74 Substrate utilisation As cell growth occurs, nitrogen and glucose substrates are depleted in the medium. The rate of substrate utilization is equal to the rate of growth due to the particular substrate (equations 2 8 3) multiplied by sm--m_a_n*X*Yxn (5, dt Kn + N -—*** as B G x ng (6) proportionality constants (Yxn, ng) equivalent to the mass of substrate utilized per mass dry weight created. Product formation The production of lignin peroxidase occurs during idiophase, generally two to three days after nitrogen has been depleted. Time dependent genetically structured models have been used to describe the production of secondary metabolites°"°. In an individual cell, messenger RNA for the lignin peroxidase enzyme is transcribed only after a lag of some time (ti) (equation 7). If it is assumed that only cells which were growing at time t-ti will produce mRNA, then the production of mRNA is equal to a constant (Kr) times the growth rate can =- Kr * (3%)t-ti - (Krd * mRNA) (7) at time t-ti. Within the cell mRNA is deactivated at some rate which is modelled using a first order death 75 rate expression (Krd). The rate of production of lignin peroxidase is proportional to the rate of mRNA production in the individual cell times the total cell weght. The production of lignin peroxidase enzymes as affected by media composition is accounted for using an empirical constant: H (equation 8). Kid (hr-1) is a first fl-(H*mRNA*X) - (K1d*L) (3) dt H - media effects - f(tween, veratryl alc, other) order enzyme deactivation constant. Since the mechanism of action of tween 80, veratryl alcohol and basal medium are not well understood, an empirically determined variable (H) was used to account for the effect of these media components on the production of lignin peroxidase. The effect of veratryl alcohol concentration on the production of lignin peroxidase was modeled assuming Hichaelis-Henten kinetics. The effect of Tween 80 and basal medium concentration on lignin peroxidase production required the use of second order models since maxima were apparent. The media effects variable represents the collective effect of media components on lignin peroxidase production. H '.__JI___ * ___B_____ + kbz * V (9) kt+Tz kb1+Bz kv+‘V 76 To evaluate the model, data for glucose and nitrogen depletion, cell growth, lignin peroxidase production and mRNA.production were needed. Empirical data was readily available for the depletion of nitrogen and glucose by nitrogen limited cultures of 2‘ W“ . The growth rate and the production of lignin peroxidase are easily measured. The rate of mRNA production is not known nor is it readily measurable, however this is not essential to the model. By beginning with reasonable estimates for constant values and modifying the parameter values, the model can be made to fit the data. The data used to evaluate the model parameters were from two sources: Jager et.alJ”, and this work. Since the depletion of glucose and nitrogen was not measured in this work, the data for substrate utilization was obtained from the work of Jager et.al.26 (see Figure 2.2), in which agitated cultures identical (except for the use of DMS buffer) to the optimized cultures used in this work were used. The data for cell dry weight, lignin peroxidase activity and the effects of media components are presented in section 4.1. Euler's method was used with a time interval of 4 hours to simultaneously solve the differential equations describing growth, substrate utilization and lignin 77 peroxidase formation. Data curves were fitted by programming the equations into a PC computer using SC4 software, and modifying the model parameters until the model curves fit the data. A summary of the model equations (Table A1.1) and the model parameter values are presented (Table A1.2). The time course of nutrient depletion, lignin peroxidase activity, and cellular growth as predicted by the model for optimal conditions is presented in Figure A1.1. Compared to Figure 4.5, the model curves accurately reflect the trends of the data. The model predicted media effects are presented in Figure Al.2. These model curves closely reflect the trends in the data observed in Figure 4.4 a,b,c. 78 TABLE A1.1 Model equations for 2;,Qhrysgspgrigm growth and enzyme expression 1 ss gg - 933:! * X + (B * G ' X) - (Kd ' X) dt Kn + N Subst a tion ‘QQ . 3 e G e x e Yxs dt Gene ggpressicn mg- ntgt H — Krd'mRNA dt dt Lignin pegogidase egpgession g; - M ' mRNA ‘ X - Kld * L H ' I 2 * Q zi'kbz * V kt + T kbl + B kw + V 79 TABLE A1.2 Model Equation Symbols and Parameter Values 1 x Biomass (s l ) G '1 N Nitrogen source (a l ) B Basal medium ( - ) mRNA - V Veratryl alcohol (IN) 1 1 M Media constant (0 3 hr ) -l T Tween 80 concentration (a 100 ml ) £2£lll§1£.!ll§£l Umn Mound constant for N Kn Honod constant for N Kd Biomass death constant Yxn N Proportionality coefficient ng G Proportionality coefficient 8 Glucose constant ti time lag constant Kr mRNA production constant Krd mRNA deactivation constant Kld Lignin peroxidase degradation ktw tween effects constant kbl basal medium effects constant kb2 basal medium effects constant kv veratryl alcohol constant Glucose (s l ) -1 L (U l ) Lignin peroxidase activity Messenger RNA '1 0.009 hr -1 0.020 g l -1 0.011 hr 0.40 - 1.50 - -1 0.0018 hr 84.0 hr -1 0.30 l g '1 0 04 hr -1 0 01 hr 1 2 ‘2 0.0015 g 100ml 2.00 - 0.35 - 80 2.5 -*— N (g/l‘iO) -9— G (g/l/IO) : —A— X (g/l) 21 —$— L(U/l/100) -#— mRNA U) G éiLfid a s :0.) d o S 1 - o L’ : 5‘ o l I I I I I I o 1 2 3 4 5 6 7 a Day Figure A1.1 Model for growth, substrate utilization and lignin peroxidase production in batch cultures. 81 Tween 00 Concentration (wt. 7.) us — .08 — .“ 2 — \ I. a — .0 DI“ 3' 3 o ( 3 . new 3 m 2 e a. c "‘ 3 us 3 e - z a 4 s e 1 a Day V t I U see era :7! Alcoho (m ) _ ”I -— .4 § — n _, — 1 5|“( 3 3 o < 8 . I“) 3 m 2 e I. c “‘ 'c' is :3 . f f v v a a o e e 7 e Day Basel Medium (a norm) see — o Lignin Peroxidase Activity (0]!) i 301 Figure A1.2 Model predicted media effects on the ‘ production of lignin peroxidase in batch cultures. 82 Appendix 3 Operation of a trickle bed reactor A bench scale trickle bed reactor system for the production of lignin peroxidases was developed (Figure A2.l). Experiments showed that both strains SC26 and BKM-Fl767 would readily and completely adsorb to telleret rings composed of polyethylene and would produce small titres of lignin peroxidase in agitated shake flask cultures. Therefore, the plastic rings were used as a mycelial support material in a 1.5 l trickle bed reactor. Optimized culture media #3 was added to the lower third of the reactor volume and inoculated with blended mycelia. The inoculated media was circulated to the top of the reactor using a centrifugal pump. The temperature was controlled by a heating jacket surrounding the reactor and 99.5% oxygen was added continuously via a sparger located at the base of the reactor. Unfortunately, this reactor system was plagued by problems which could not be overcome. Initially a medium was pumped directly to the top of the reactor column through a 1/2 ” diameter steel pipe. With this sparger however, growth was limited to a thin stream of mycelium 83 sterile 0. in ! headplate l 1 exit as 1 I I _ T - 9 medium sparger fifififififififififififififi fififi 39° fifififififififififififififi fififi H.0 in aetellereteage 3:: fifiringfifififififififinfififi fifiimmebilizedm ea: fifimvceliafififififi fie? H30 oxygen out sparger media sample e——_——1 pump port I 1 _______J Figure A2.1 Design of a trickle bed reactor for the production of lignin peroxidase. 84 and no lignin peroxidase activity was detected. To improve medium.sparging, a metal sparger was designed with a cross configuration and small diamaeter medium ports. With this sparger, growth was more disperse however bits of mycelia from the liquid phase of the reactor or from the supports, would invariably break loose and clog the sparger and pump. No activity was detected when the improved sparger was used. Clogging by mycelia is a typical problem encountered in fungal film reactor configurations. This reactor configuration was abandoned in favor of a stirred tank reactor system. table A3.1 A3.2 A3.3 A3.4 A3.5 A3.6 A3.7 A3.8 A3.9 A3.10 A3.11 A3.12 A3.13 A3.14 85 Appendix c Tabulated data Tabulated Tabulated Tabulated Tabulated Tabulated Tabulated Tabulated Tabulated Tabulated Tabulated Tabulated Tabulated Tabulated tit data data data data data data data data data data data data data Is for for for for for for for for for for for for for Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 4.4 4.4 B 4.4 4.5 4.6 4.8 Lignin peroxidase activity of all control cultures. page 86 87 88 89 9O 91 92 93 94 95 96 97 98 99 86 Table A3.1 Tabulated data for strain comparison experiment. (refer to Figure 4.1) Run 1: 9/14/87 Lignin Peroxidase Activity (U/l) Day 4 s 6 7 8 Strain BKM-P-l767 181 200 55 0 0 204 205 58 0 0 194 194 145 0 SC26 0 19 100 110 13 0 65 142 85 39 0 90 155 103 68 87 Table A3.2 Comparison of various buffers for the production of lignin peroxidase. Tabulated data for Figure 4.2. values are averages of triplicate cultures. Lignin Peroxidase Activity (U/l) Buffer Day 3 4 5 6 7 10 mM DMS 0 64.25 54 76.25 17 20 mM Acetate 0 32.75 131.17 166.2 155.85 10 mM Citrate 0 o 11.5 37.5 28 10 mM Succinate 0 43.5 72.5 62 41.5 10 mM Tartrate 0 O 0 0 O Jager at al 0 0 50 132 134 10 mM Oxalate 0 O 0 0 o Table A3.3 Tabulated data for spore inoculum size optimization (refer to Figure 4.3). Run 1: 8/25/87 Ligninase Activity (U/l) Day 4 5 6 7 Inoculum OD .05 OD 0 106 138 177 90.3 170 175 0.1 OD 0 58 145 166 117 183 166 0.3 0D 0 21.9 106.4 145 45.7 122.5 138 0.6 OD 0 23 0 0 142 188 126 155 119 90 20 89 Table A3.4 Tabulated data for tween 80 optimization experiments (refer to Figure 4.4 A). Run 1: 8/25/87 Lignin Peroxidase Activity (U/l) Day 4 5 6 7 8 Tween 80% (w/v) 0.05% 0 21.9 106.4 145 119 0 45.7 122.5 138 90 0.1% 0 96 97 142 68 0 50 95 53 76 0.2% 0 45 51.6 71 45 0 54.8 90 94 56 Run 2: 3/12/88 Lignin Peroxidase Activity (U/l) Day 4 5 6 7 8 Tween 80 % (w/v) 0.025% 21.5 53 41 142 137 26.9 0 19 142 135 3 55 42 187 151 0.05% 22 48 65 169 71 28 72 71 218 26 0 53 77 187 84 0.2% 0 34 39 168 103 10 38 18 152 93 0 52 19 161 81 0.0% 0 0 0 0 0 90 Table A3.5 Tabulated data for veratryl alcohol concentration optimization experiments (refer to Figure 4.4 8). Run 1: 2/20/88 Lignin Peroxidase Activity (U/l) Day 4 5 6 7 8 Veratryl Alcohol 0.4 mM 181 400 403 331 330 129 354 306 274 242 158 300 306 218 210 1.0 mM 123 400 452 387 290 110 371 235 347 338 126 350 371 331 314 2.0 mM 155 269 434 371 395 155 242 371 403 395 0 270 419 193 338 91 Table A3.6 Tabulated data for the optimization of basal medium concentration (refer to Figure 4.4 C). Run 1: 1/22/88 Ligninase Activity (U/l) Day 4 5 6 7 8 Basal Medium 0X 20 39 70 89 0 11 45 62 101 101 1X 76 32 264 222 169 63 180 160 185 149 2X 109 201 177 222 149 77 97 150 181 125 5X 48 142 124 185 121 0 13 103 133 117 92 Table A3.7 Tabulated data for the characteristics of agitated cultures grown in optimized medium. (refer to Figure 4.5). Dry Day Weight (g/l) pH 0 1.18 4.5 0 .98 4.5 1 1.92 4.5 1 1.86 4.5 2 1.71 4.5 2 2.12 4.5 3 2.04 5.0 3 1.98 5.0 4 2.19 4.8 4 2.45 4.9 5 2.15 4.9 5 2.11 4.9 6 2.38 5.0 6 2.27 5.1 7 1.89 5.4 7 1.87 5.3 8 1.59 5.5 8 1.81 5.5 9 1.24 5.6 9 1.37 5.7 93 Table A3.8 Tabulated data for the number of hyphal agglomerations as a function of time (refer to Figure 4.6) 125 m1 flask 2 liter flask n- number of n- number of Hour pellets/l pellets/1 0 12000000 12000000 4 2400000 2600000 6 40000 160000 7.5 50000 45000 8.5 30000 40000 20 16000 59000 24 16700 43700 48 23000 41000 94 Table A3.9 Pellet size as a function of culture age. Tabulated data for Figure 4.8. 125 I1 Bhlk. £1383 culture Sieve Number of pellets per tray size (mm) 24 hr 49 hr 73 hr 96 hr 140 hr 2 0 l 0 7 7 1.4 0 23 61 115 107 1.18 47 86 69 70 56 1 84 66 38 51 27 .85 18 15 12 14 1 .5 l7 l7 7 10 4 Sauter Mean 1046.215 1159.398 1238.367 1320.730 1363.847 Diam.(um) STR culture Number of pellets per tray 24 hr 49 hr 73 hr 96 hr 140 hr 2 0 0 0 0 1.4 0 14 20 114 1.18 70 55 116 150 1 223 104 172 210 .85 134 52 65 86 .5 320 79 90 85 Sauter Mean 925.6399 1034.862 1059.837 1147.482 Diam.(um) Table A3.10 Effect of the agitation rate on lignin 95 peroxidase activity. Tabulated data for Figure 4.10. Run 1: 12/1/87 Lignin Peroxidase Activity (U/l) Day 4 s 6 7 8 Agitation Rate 150 RPM 13 65 387 419 395 90 129 354 225 258 0 71 323 427 371 200 RPM 155 269 434 371 395 155 242 371 403 395 0 270 419 193 338 260 RPM 0 0 39 73 185 0 0 0 0 0 0 0 0 0 0 Run 2: 3/28/88 Lignin Peroxidase Activity (U/l) Day 4 s 6 7 a Agitation Rate 125 RPM 0 0 0 0 0 200 RPM 27 68 32 44 62 100 34 24 260 RPM 34 54 8 15 38 32 8 5 96 Table A3.11 Sauter mean pellet diameter as a function Sauter mean of the agitation rate. Tabulated data for Figure 4.12. RPM pellet diam. Ln (RPM) Ln (D32) D32 (mm) 150 2 5.010635 7.600902 150 1.620529 5.010635 7.390508 150 1.860691 5.010635 7.528703 200 1.257833 5.298317 7.137146 200 1.302042 5.298317 7.171689 200 1.301004 5.298317 7.170892 260 1.191148 5.560682 7.082673 260 1.319476 5.560682 7.184990 260 1.296639 5.560682 7.167531 125 1.708593 4.828314 7.443425 125 1.711006 4.828314 7.444837 125 1.911837 4.828314 7.555820 125 1.944835 4.828314 7.572933 165 1.361629 5.105945 7.216437 165 1.287960 5.105945 7.160815 165 1.409698 5.105945 7.251131 165 1.456255 5.105945 7.283623 210 1.146687 5.347108 7.044632 210 1.232677 5.347108 7.116944 210 1.137920 5.347108 7.036957 260 1.012458 5.560682 6.920136 260 .9753344 5.560682 6.882780 260 .9800289 5.560682 6.887582 260 .7886938 5.560682 6.670378 300 1.410078 5.703782 7.251400 300 1.384071 5.703782 7.232784 300 1.373099 5.703782 7.224825 300 1.314032 5.703782 7.180855 97 Table A3.12 Lignin peroxidase activity and total extracellular protein in a Stirred tank reactor system. Tabulated data for Figure 4.13. Lignin peroxidase Total extracellular Day activity (U/l) protein (ug/ml) 2 0 7 3 29 11 4 156 14 5 141 13 6 180 7 63 8 43 98 Table A3.13 Protein concentration and lignin peroxidase activity of STR FPLC heme-protein elution volumes. elution OD Total Lignin volume 595 nm Protein Peroxidase (ml) (ug/ml) Activity (U/l) 5 0.446 0.775 0 6 0.501 4.936 0 7 0.521 6.449 0 8 0.522 6.524 0 9 0.535 7.508 0 10 0.715 21.126 88.69 11 0.637 15.225 0 12 0.544 8.189 0 13 0.743 23.244 0 14 0.726 21.958 0 15 0.745 23.396 0 16 0.605 12.804 24.2 17 0.68 18.478 0 18 0.665 17.343 0 19 0.605 12.804 0 20 0.725 21.883 0 21 0.78 26.044 44.34 22 0.71 20.748 0 23 0.683 18.705 0 24 0.83 29.826 0 25 0.659 16.889 0 26 0.865 32.474 24.18 27 0.815 28.692 84.7 28 0.89 34.366 0 29 0.741 23.093 0 30 0.901 35.198 0 31 1.124 52.069 0 32 0.833 30.053 0 99 Table A3.14 Lignin peroxidase activity of all control flasks. Lignin Peroxidase Activity (U/l) Day 3 4 5 6 7 Date 8/25/87 0 0 106 138 177 0 0 90.3 170 175 9/14/87 0 0 181 200 55 0 0 200 205 58 0 0 194 194 145 11/3/87 0 0 136 116 116 0 0 149 171 206 12/3/87 0 155 269 434 371 0 0 242 371 403 0 155 270 419 193 1/23/88 0 76 32 264 222 0 63 180 160 185 2/17/88 0 30 87 100 80 0 0 90 87 86 3/6/88 0 22 48 65 169 0 28 72 71 219 0 0 53 77 187 3/30/88 0 27 68 32 44 0 62 100 34 24 0 37 56 16 2 Average 0 32.75 131.165 166.2 155.85 SD 0 48.37015 75.17486 123.5244 104.6514 + one SD 0 81.12015 206.3399 289.7244 260.5014 - one SD 0 0 55.99014 42.67562 51.19856 BIBLIOGM 1. 100 Anderson, 3.6. I'Immobilized Cell and Film Reactor Systems for Filamentous Fungi", in The Filamentous Fungi:Fungal Technology, eds. J.E. Smith, D.R.Berry, B. Kristiansen. 1983. Edward Arnold, publ. London. pp.146-169. Asther, M., G. Corrieu, R. Drapron, E. Odier. 1987. Effect of Tween 80 and oleic acid on ligninase production by'Ehanerechaete.chrxseenerium INA-12- Enzyme Microb. 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