"Applied Science, Faculty of"@en . "Chemical and Biological Engineering, Department of"@en . "DSpace"@en . "UBCV"@en . "Yu, Jian"@en . "2011-03-02T03:12:08Z"@en . "1991"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Anaerobic methanation of lactose, the main component of cheese whey, is an attractive alternative for the disposal of this wastewater because of its added benefit of recovering energy as methane. Two advances in this technology have been the separation of the bacteria in two reactors according to their substrates and the retention of the anaerobic bacteria in the form of bacterial biofilms on solid supports. The mass transfer rate of the substrates within the active biofilms must be known for the determination of an optimum biofilm thickness. A prerequisite for the investigation of substrate mass transfer rate in an active biofilm is the knowledge of intrinsic kinetics of the substrate utilization by the embedded bacterial cells. Start-up of symbiotic methanogenic biofilms was also investigated on inert supports.\r\nIn recycled reactors, mesophilic (35 \u00B0C) acid-producing bacteria or methane-producing bacteria, cultured with lactose at a pH of 4.6 for the former or cultured with mixed acids (acetate, propionate and butyrate) at a pH of 7.1 for the latter , attached onto PVC sheets, forming thin acidogenic or methanogenic biofilms, respectively. After the external mass transfer resistance had been eliminated by increasing the recycle rate and the internal mass transfer resistance was minimized by using thin biofilms, the intrinsic kinetics of lactose acidogenesis and methanogenesis of organic acids were investigated in the newly formed acidogenic and methanogenic biofilms, respectively. The lactose digestion rate as well as the production of two main products, acetate and butyrate, can be described by Michaelis-Menten equations. The production of propionate, as a minor product, was depressed in the culture environment. The digestion of acetate could also be modelled by a Michaelis-Menten equation while the dissimilation of propionate\r\n\r\nand butyrate was affected by propionate concentration, the propionate digestion being promoted but the butyrate digestion being inhibited at high propionate concentrations. Two models have been proposed for their utilization.\r\nSubstrate mass transfer in the active biofilms was investigated with a diffusion cell. After symmetric biofilms formed on the two membrane filters of the cell, the substrate concentrations on the biofilm surfaces and inside the cell were measured at steady state. The effective diffusivities of substrates in the active biofilms were estimated by numerically\r\nsolving the diffusion-reaction equations using the intrinsic kinetics and the substrate concentrations as the boundary conditions. The effective diffusivity of lactose in an acidogenic\r\nbiofilm was about 65.3 % of its diffusivity in water, and the diffusivities of acetate, propionate and butyrate in a methanogenic biofilm were reduced to about 30.2 % of the values in water. Comparing the fractional void volumes in the two types of biofilms showed that the methanogenic biofilm, which grew more slowly, had a more tortuous structure of channels than did the acidogenic biofilm.\r\nStudies on the build-up of symbiotic methanogenic biofilms were conducted using supports of wood, ceramic rings, PVC and stainless steel, which gave a range of water contact angle from 0\u00B0 to 99.7\u00B0. The accumulation of acetate-, propionate- and butyrate-degrading bacteria on the supports were monitored by measuring the substrate utilization rates of each bacterial group in a standard batch culture which was seeded by the supports with the attached biofilms. The three types of bacteria had different preference to a hydrophilic support surface,\r\nbutyrate degrader > acetate degrader > propionate degrader\r\nBased on the analysis of the process of biofilm formation, a model has been proposed. The parameters which depict the attachment of free cells onto clean surfaces were found to have a linear relationship with the water contact angles of the support surfaces."@en . "https://circle.library.ubc.ca/rest/handle/2429/31900?expand=metadata"@en . "TWO-PHASE METHANATION OF LACTOSE IN BIOFLLM REACTORS By Jian Y u B. Sc. Zhejiang Institute of Technology, 1982 M . Sc. Zhejiang University, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES CHEMICAL ENGINEERING We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1991 \u00C2\u00A9 Jian Yu, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of tmrtteA^ &l&2Aje&Z24tfr The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Anaerobic methanation of lactose, the main component of cheese whey, is an attractive alternative for the disposal of this wastewater because of its added benefit of recovering energy as methane. Two advances in this technology have been the separation of the bacteria in two reactors according to their substrates and the retention of the anaerobic bacteria in the form of bacterial biofilms on solid supports. The mass transfer rate of the substrates within the active biofilms must be known for the determination of an optimum biofilm thickness. A prerequisite for the investigation of substrate mass transfer rate in an active biofilm is the knowledge of intrinsic kinetics of the substrate utihzation by the embedded bacterial cells. Start-up of symbiotic methanogenic biofilms was also investigated on inert supports. In recycled reactors, mesophihc (35 \u00C2\u00B0 C ) acid-producing bacteria or methane-producing bacteria, cultured with lactose at a p H of 4.6 for the former or cultured with mixed acids (acetate, propionate and butyrate) at a p H of 7.1 for the latter , attached onto P V C sheets, forming thin acidogenic or methanogenic biofilms, respectively. After the external mass transfer resistance had been eliminated by increasing the recycle rate and the internal mass transfer resistance was minimized by using thin biofilms, the intrinsic kinetics of lactose acidogenesis and methanogenesis of organic acids were investigated in the newly formed acidogenic and methanogenic biofilms, respectively. The lactose digestion rate as well as the production of two main products, acetate and butyrate, can be described by Michaelis-Menten equations. The production of propionate, as a minor product, was depressed in the culture environment. The digestion of acetate could also be modelled by a Michaelis-Menten equation while the dissimilation of propionate n and butyrate was affected by propionate concentration, the propionate digestion being promoted but the butyrate digestion being inhibited at high propionate concentrations. Two models have been proposed for their utilization. Substrate mass transfer in the active biofilms was investigated with a diffusion cell. After symmetric biofilms formed on the two membrane filters of the cell, the substrate concentrations on the biofilm surfaces and inside the cell were measured at steady state. The effective diffusivities of substrates in the active biofilms were estimated by numeri-cally solving the diffusion-reaction equations using the intrinsic kinetics and the substrate concentrations as the boundary conditions. The effective diffusivity of lactose in an acido-genic biofilm was about 65.3 % of its diffusivity in water, and the diffusivities of acetate, propionate and butyrate in a methanogenic biofilm were reduced to about 30.2 % of the values in water. Comparing the fractional void volumes in the two types of biofilms showed that the methanogenic biofilm, which grew more slowly, had a more tortuous structure of channels than did the acidogenic biofilm. Studies on the build-up of symbiotic methanogenic biofilms were conducted using supports of wood, ceramic rings, P V C and stainless steel, which gave a range of water contact angle from 0\u00C2\u00B0 to 99 .7 \u00C2\u00B0 . The accumulation of acetate-, propionate- and butyrate-degrading bacteria on the supports were monitored by measuring the substrate utilization rates of each bacterial group in a standard batch culture which was seeded by the supports with the attached biofilms. The three types of bacteria had different preference to a hydrophilic support surface, butyrate degrader > acetate degrader > propionate degrader Based on the analysis of the process of biofilm formation, a model has been proposed. The parameters which depict the attachment of free cells onto clean surfaces were found to have a linear relationship with the water contact angles of the support surfaces. iii A c k n o w l e d g e m e n t The author expresses here his sincere appreciation to Dr. K. L. Pinder for his advice, direction and support throughout this research project. He is also grateful to Dr. R. M . R. Branion and Dr. K . V . Lo, two members of his supervision committee, for their constructive criticisms and review of this thesis. The author is obliged for financial support of this research to the Foundation of Pao Zhao-Long and Pao Y u - G a n g Scholarship and the Natural Science and Engineering Research Council of Canada. Special thanks go to his family and parents for their understanding and support. iv T a b l e of C o n t e n t s A b s t r a c t i i A c k n o w l e d g e m e n t iv Lis t of Tables xi Lis t of F igures x iv 1 I n t r o d u c t i o n 1 1.1 Utilization of Lactose in Whey 1 1.2 Anaerobic Methanation of Lactose 3 1.3 More Considerations on Two-phase Methanation of Lactose . . 6 1.3.1 Kinetics in Each Phase Reactor 8 1.3.2 Mass Transfer in the Biofilms 9 1.3.3 Start-up of Anaerobic Biofilms 11 1.4 Research Objectives and Scope 12 2 T h e o r e t i c a l A s p e c t s a n d P r e v i o u s Studies 14 2.1 Process Biochemistry and Microbiology 14 2.1.1 Lactose Acidogenesis 15 2.1.2 Methanogenesis of Fatty Acids 24 2.1.3 Acetogenesis of Fatty Acids . . 28 2.2 Start-up of Anaerobic Biofilms 32 2.2.1 Mechanism of Build-up of Bacterial Biofilms 32 v 2.2.2 Startup of Methanogenic Biofilms 36 2.3 Mass Transfer in Anaerobic Biofilms 39 2.3.1 Structure of Anaerobic Biofilms 41 2.3.2 Mass Transfer within Biofilms 43 2.4 Intrinsic Kinetics of Substrate Utihzation in Anaerobic Biofilms 47 2.4.1 Expression of Intrinsic Kinetics 47 2.4.2 Ehmination of External and Internal Mass Transfer Resistances . 49 2.4.3 Kinetics of Acidogenesis and Methanogenesis 51 3 Experimental Conditions and Setup 54 3.1 Experimental Conditions 55 3.1.1 Culture Temperature 55 3.1.2 Culture p H 56 3.1.3 Growth Nutrients 57 3.2 Experimental Setups 59 3.2.1 General Setup 60 3.2.2 Reactor 62 3.3 Biofilm Supports . . 66 3.3.1 Supports for Kinetic Studies 66 3.3.2 Supports for Mass Transfer Studies 67 3.3.3 Supports for Studies of Biofilm Start-up 69 3.4 Experimental Analysis 70 3.4.1 Sohd Sample Analysis 70 3.4.2 Liquid Sample Analysis 72 3.4.3 Gaseous sample analysis 73 vi 4 Intrinsic K i n e t i c s of Lactose U t i l i z a t i o n 74 4.1 Experiments on Lactose Acidogenesis 74 4.2 Development of an Acidogenic Biofilm 78 4.3 Utilization Rate of Lactose 84 4.4 Production of Organic Acids 89 4.5 Responses of Acidogenic Biofilms to Disturbances 101 5 Star t -up of S y m b i o t i c M e t h a n o g e n i c B i o f i l m s on Inert Surfaces 104 5.1 Wettability of a Support Surface 104 5.2 Experimental Steps 107 5.3 A Kinetic Model of Biofilm Start-up I l l 5.3.1 Attachment on Clean Surfaces I l l 5.3.2 Growth of Attached Bacteria 113 5.3.3 Attachment on Fixed Biomass 113 5.3.4 Detachment 114 5.4 Estimation of Model Parameters 116 6 Intrinsic K i n e t i c s of M e t h a n o g e n e s i s of O r g a n i c A c i d s 128 6.1 Experiments of Organic Acid Methanogenesis 129 6.2 Development of Methanogenic Biofilms 136 6.3 Interaction of Organic Acids 140 6.3.1 Influence of Acetate Concentration 140 6.3.2 Influence of Propionate Concentration 141 6.3.3 Influence of Butyrate Concentration 142 6.4 Utihzation Rates of Organic Acids 145 6.4.1 Propionate Utihzation Rate 146 6.4.2 Utihzation Rate of Butyrate 150 vii 6.4.3 Acetate Utilization Rate 154 6.5 Distribution of Bacterial Groups in Balanced Biofilms 158 7 M a s s Transfer i n Biof i lms 166 7.1 Diffusivities of Substrates in Water 166 7.2 Principle of Mass Transfer Measurement within Biofilms 168 7.3 Experimental Setup 173 7.4 Diffusivity of Lactose in Acidogenic Biofilms 175 7.5 Effective Diffusivities of Organic Acids in Methanogenic Biofilms 181 7.5.1 Formation of Symbiotic Methanogenic Biofilms 181 7.5.2 Effective Diffusivities of Propionate and Butyrate in the Biofilms . 182 7.5.3 Effective Diffusivity of Acetate in Methanogenic Biofilms 187 7.5.4 Effect of Support Properties on the Measurement of Diffusivities . 191 7.6 Influence of Biofilm Structure on Diffusivities 196 8 C o n c l u s i o n s a n d R e c o m m e n d a t i o n s 203 8.1 Conclusions 203 8.2 Recommendations 208 8.2.1 Concentration Effect 208 8.2.2 Controlling Biofilm Thickness 208 8.2.3 A Compromised Physical Method 211 B i b l i o g r a p h y 213 A p p e n d i c e s 230 A F e r m e n t a t i o n P a t h w a y s 230 A . l Four Fermentation Pathways of Glucose to Pyruvate 230 vm A . 2 Formation of Organic Acids from Pyruvate 231 B Analysis Methods 241 B. l Determination of Lactose 241 B . l . l Reagents and Apparatus 241 B.1.2 Procedure 242 B.2 Determination of Lactate 242 B.2.1 Reagents and Apparatus 243 B.2.2 procedure 244 B.3 Determination of Volatile Fatty Acids and Ethanol 244 B.3.1 Reagents and Apparatus 245 B.3.2 Procedure 246 B.4 Gas Composition 247 B.4.1 Apparatus 247 B.4.2 Procedure 249 B.5 Determination of Carbon Content in a Liquid Sample 251 B.5.1 Sample Preparation 251 B.5.2 Reagents and Apparatus 252 B.5.3 Procedure 252 B.6 Analysis of a Biofilm 253 B.6.1 Dry Biomass and Carbon Content of a Biofilm 253 B.6.2 Ash Content of a Biomass 253 B.6.3 Water Volume. Density and Thickness of a Biofilm 253 C Results of Lactose Acidogenesis 255 D Results of Buildup of Methanogenic Biofilms 257 ix E Resul ts of O r g a n i c A c i d M e t h a n o g e n e s i s 259 F D e r i v a t i o n of U t i l i z a t i o n R a t e M o d e l s of P r o p i o n a t e a n d B u t y r a t e 261 F . l Utihzation Rate Model of Propionate 261 F . 2 Utihzation Rate Model of Butyrate 262 G N u m e r i c a l M e t h o d s 265 G . l Direct Search Method 265 G.2 Runge-Kutta-Fehlberg Method 266 G.3 The Computer Programs in Pascal Language 269 x List of Tables 1.1 Major components of a typical whey 2 1.2 Pilot- and full-scale plants of two-phase anaerobic treatment of wastewaters 8 2.3 Main genera of acid-producing bacteria imphcated in first phase 18 2.4 Classification of seven genera of methanogenic bacteria 24 2.5 Currently known mesophilic obligate proton-reducing bacteria 30 2.6 Growth rate of obhgate H^-producing actogens 30 2.7 Ratios of effective diffusivities measured in inactive biomass to diffusivities in water . 46 2.8 Growth constants of anaerobic cultures at 35 \u00C2\u00B0C 52 3.9 Chemical components and concentrations in culture medium 59 3.10 Comparison of experimental conditions for each phase 60 3.11 Properties of biofilm supports for studies of biofilm formation 70 4.12 Experimental conditions of lactose acidogenesis 79 4.13 Maximum lactose digestion rate and half velocity concentration 87 4.14 Reactions of Lactose Conversion 90 4.15 Rate parameters of the products in lactose fermentation 94 4.16 Comparison of production rates of organic acids at two p H levels 101 5.17 Results of screening kinetic models of biofilm start-up 118 5.18 Linear relations between specific attachment rates and water contact angles 125 xi 6.19 Methanogenic reactions of organic acids 128 6.20 V F A distribution in anaerobic digestion process 129 6.21 Experimental conditions for organic acid methanogenesis 136 6.22 Properties of methanogenic biofilms 140 6.23 Methanogenesis of organic acids 157 6.24 Fractional mass of bacterial groups and utihzation rates of fatty acids . . 165 7.25 Diffusivities in water at 35 \u00C2\u00B0C 168 7.26 Effective diffusivity of lactose within acidogenic biofilms at 35 \u00C2\u00B0 C . . . . 180 7.27 Sensitivity of lactose effective diffusivity to experimental error 181 7.28 Effective diffusivities of propionate and butyrate 187 7.29 Effective diffusivity of acetate in methanogenic biofilms 190 7.30 Distribution of bacterial species in biofilms forming on nitrocellulose mem-branes 194 7.31 Effect of bacterial species distribution on the measurement of diffusivities 195 7.32 Comparison of acidogenic and methanogenic biofilms 201 8.33 Organic carbon fraction of each bacterial group in methanogenic biofilms on P V C and nitrocellulose membrane filter supports 207 C.34 Accumulation of acidogenic biofilms on removable P V C slides 255 C. 35 Results of lactose acidogenesis 256 D . 36 Accumulation of acetate-degrading bacteria on inert supports 257 D.37 Accumulation of propionate-degrading bacteria on inert supports 258 D . 38 Accumulation of butyrate-degrading bacteria on inert supports 258 E . 39 Accumulation of methanogenic biofilms on removable P V C shdes 259 xii E.40 Methanogenesis of acetate, propionate and butyrate 260 E.41 Gas production rate and composition of organic acid methanation . . . . 260 xm List of Figures 1.1 Illustration of configurations of anaerobic filter (A), fluidized/expanded bed (B) and upflow anaerobic sludge blanket ( C ) 5 1.2 Two-phase process of anaerobic digestion of organic wastes 7 2.3 Illustration of lactose structure 15 2.4 Illustration of two types of glycosidic links 16 2.5 Structure of lac operon 17 2.6 Bacterial fermentation products of pyruvate. Pyruvate formed by the catabolism of glucose is further metabolized by pathways which are charac-teristic of particular organisms. End products of fermentations are shown in boxes and intermediates in dash boxes. \" - \" refers to consumption and \"+\" to production 22 2.7 Structure of biofilms 43 2.8 Illustration of substrate concentration profiles in biofilms 50 3.9 Illustration of temperature influence on growth rate of microbes 56 3.10 Illustration of general experimental setup. 1-feed tank, 2-pump, 3-head tank, 4-microvalve, 5-rotameter, 6-break tube, 7-reactor, 8-biofum sup-port, 9-refrigerated bath, 10-heat exchanger, 11-pump, 12-thermostat bath, 13-pH controller, 14-pH adjusting solution, 15-gas collector, 16-thermoregulator 61 3.11 Illustration of reactor configuration 63 3.12 A removable sampling slide 63 xiv 3.13 Effect of recycle ratio on reactor's behavior. A-recycle ratio 3.8, x-recycle ratio 7.6 65 3.14 P V C biofilm support for kinetic studies of lactose or organic acids fermen-tation 67 3.15 A thin biofilm (the black up-layer) on a P V C support, magnified by 100 times 68 3.16 A biofilm support device for mass transfer studies of lactose or organic acids in biofilms 68 4.17 External mass transfer resistance on lactose utihzation in biofilms is tested by changing recycle rate 77 4.18 Lactose digestion rate of whole reactor changes with time 78 4.19 Specific lactose digestion rate changes with biofilm thickness 79 4.20 Biomass distribution in the fermenter 80 4.21 Biomass on removable shdes increases with time 81 4.22 Carbon content in the biomass 82 4.23 Total carbon content developed on the supports. The hne was calculated from Equation 4.16 83 4.24 Effect of lactose concentration on lactose digestion rate. The hne is calcu-lated from Equation 4.20 85 4.25 Influence of lactose concentration on acetate production rate. The hne is calculated from Equation 4.24 and the parameter values in Table 4.15 . . 91 4.26 Influence of lactose concentration on butyrate production rate. The hne is calculated from Equation 4.24 and the parameter values in Table 4.15 92 4.27 Influence of lactose concentration on ethanol production rate. The hne is calculated from Equation 4.24 and the parameter values in Table 4.15 . . 92 xv 4.28 Influence of lactose concentration on propionate production rate. The hne is calculated from Equation 4.25 and the parameter value in Table 4.15 . 93 4.29 Influence of lactose concentration on lactate production rate. The hne is calculated from Equation 4.25 and the parameter value in Table 4.15 . . 93 4.30 Pathways of acidogenesis of lactose. Sign (-) refers to consumption, and (+) refers to production. The intermediates are in dash boxes and products in sohd boxes 95 4.31 Production rate of acetate versus digestion rate of lactose 98 4.32 Production rate of butyrate versus digestion rate of lactose 99 4.33 Production rate of ethanol versus digestion rate of lactose 99 4.34 Production rate of propionate versus digestion rate of lactose 100 4.35 Production rate of lactate versus digestion rate of lactose 100 4.36 Responses of the acidogenic biofilm reactor to flow rate change. The sohd hne represents lactose concentration and the dash hne lactate concentration. 103 5.37 Schematic illustration of a spreading drop of liquid in contact with a solid surface, showing the relations between the contact angle, 6, and three interfacial free energies 105 5.38 Illustration of calculating contact angle, assuming the drop being a spher-ical segment 107 5.39 Organic acids degradation in a batch culture, o - acetate, A - propionate, x - butyrate 110 5.40 Build-up of acetate-degrading bacteria in the biofilms. The lines are cal-culated from the model. Surfaces: o - wood, x - ceramic, A - P V C , \u00E2\u0080\u00A2 -steel 120 xvi 5.41 Build-up of propionate-degrading bacteria in the biofilms. The lines are calculated from the model. Surfaces: o - wood, x - ceramic, A - P V C , \u00E2\u0080\u00A2 - steel 121 5.42 Build-up of butyrate-degrading bacteria in the biofilms The lines are cal-culated from the model. Surfaces: o - wood, x - ceramic, A - P V C , \u00E2\u0080\u00A2 -steel 122 5.43 Ease of attachment of bacteria on substrata as shown by model parameter k 123 5.44 Attachment factor, k, as a function of water contact angle of surface. Line is the least square fit. o-butyrate degrading bacteria, A-acetate degrading bacteria, X-propionate degrading bacteria 124 5.45 Spreading factor, s, as a function of the water contact angle of the surfaces for all bacteria 126 6.46 Influence of recycle rate on acetate utihzation 133 6.47 Influence of recycle rate on propionate utihzation 134 6.48 Influence of recycle rate on butyrate utihzation 134 6.49 Influence of biofilm thickness on acetate digestion rate 135 6.50 Biomass on slides increases with culture time 138 6.51 Total organic carbon on slides increases with culture time 138 6.52 Relationship between organic carbon and dry biomass of biofilms . . . . 139 6.53 Effect of acetate concentration on propionate digestion (HPr=880-996 mg/l)141 6.54 Effect of acetate concentration on butyrate digestion (HBu=48-66 mg/1) 142 6.55 Effect of propionate concentration on acetate utihzation (HAc=700-1000 mg/1) . : 143. xvn 6.56 Effect of propionate concentration on butyrate utilization (HBu=50-80 mg/1) 143 6.57 Effect of butyrate concentration on acetate digestion (HAc=1400-3000 mg/l)144 6.58 Effect of butyrate concentration on propionate digestion (HPr=700-900 mg/1) 145 6.59 Dependence of propionate digestion rate on propionate concentration. The dash hne represents Equation 6.54. The sohd hne is calculated from Equa-tion 6.56 147 6.60 Dependence of butyrate digestion rate on butyrate concentration. The dash hne is calculated from the Michaelis-Menten equation 6.60; The points ( x ) are calculated from Equation 6.62 151 6.61 Influence of butyrate concentration on butyrate digestion (HPr = 690-900 mg/1) 152 6.62 Dependence of acetate utihzation rate on acetate concentration. The hne is calculated from Equation 6.65 155 7.63 Substrate concentration distribution within a biofilm at steady state, The elemental volume has a thickness Al and an area A 170 7.64 Influence of De on substrate concentration distribution within a biofilm, D'e > D\" > D\u00E2\u0084\u00A2 171 7.65 The biofilms symmetrically fixed on the two membrane filters of the diffu-sion-measuring cell 174 7.66 A steady-state lactose concentration inside the cell was established after a dilution. The lactose concentration outside the cell ranged from 1750 to 1900 mg/1 177 7.67 A controlled change in the lactose concentration of bulk culture solution . 178 xvm 7.68 Dynamic response of lactose concentration inside the cell to the controlled change in the outside lactose concentration 178 7.69 Effect of recycle rate on the lactose concentration inside the cell. The lactose concentration in the bulk solution ranged from 1857 to 1750 mg/1 179 7.70 Establishment of a steady state propionate concentration inside the cell (o) after a dilution. \u00E2\u0080\u00A2 - propionate concentration outside the cell . . . . 183 7.71 Establishment of a steady state butyrate concentration inside the cell (o) after a dilution. \u00E2\u0080\u00A2 - butyrate concentration outside the cell 184 7.72 Effect of recycle rate on propionate concentration inside the cell (o), \u00E2\u0080\u00A2 -porpionate concentration outside the cell 185 7.73 Effect of recycle rate on butyrate concentration inside the cell (o), \u00E2\u0080\u00A2 -butyrate concentration outside the cell 185 7.74 Effect of recycle rate on the acetate concentration inside the cell (o), \u00E2\u0080\u00A2 -acetate concentration outside the cell 189 7.75 Establishment of a steady state acetate concentration inside the device (o) after a dilution, \u00E2\u0080\u00A2 - acetate concentration outside the cell 189 7.76 Top view of a methanogenic biofilm, (x 400) 197 7.77 Experimental determination of biofilm void fraction 199 7.78 Straight vertical channels (along the black and white boundary) in a sec-tion of an acidogenic biofilm (x 100). The down black part is biofilm and P V C support 200 8.79 Illustration of an optimum operation of biofilm reactors. No back- mixing of medium is assumed 209 8.80 Illustration of a process of three stages of cheese whey treatment 211 xix A.81 E M P pathway of glucose conversion to pjTuvate. Key to the enzymes: E C 2.7.1.1: hexokinase; E C 5.3.1.9: glucosephosphate isomerase; E C 2.7.1.11: phosphofructokinase; E C 4.2.1.13: fructosebisphosphate aldolase; E C -5.3.1.1: triosephosphate isomerase; E C 1.2.1.12: glyceraldehyde 3-phosph-ate dehydrogenase; E C 2.7.2.3: phosphoglycerate kinase; E C 2.7.5.3: phos-phoglyceromutase; E C 4.2.1.11: Enolase; E C 2.7.1.40: pyruvate kinase. Source is [52] 232 A.82 Schematic representation of the cyclic (pentose shunt) and non-cyclic na-ture of the H M P pathway. Source is [52]. . 233 A.83 H M P pathway of glucose utihzation. S 7-P: sedoheptulose 7-phosphate; G A 3 - P : glyceraldehyde 3-phosphate; D H A P : dihydroxy-acetonephosphate; E 4-P: erythrose 4-phosphate; F 6-P: fructose 6-phosphate; F D P : fruc-tose 1,6-bisphosphate. Key to the enzymes: EC2.7.1.1: hexkoinase; E C 1.1.1.49: glucose 6-phosphate dehydrogenase; E C 3.1.1.17: gluconolac-tonase; E C 1.1.1.44: 6-phosphogluconate dehydrogenase; E C 5.1.3.1: ribu-losephosphate 3-epimerase: E C 5.3.1.6: ribose 5-phosphate isomerase; E C 2.2.1.1: transketolase; E C 4.1.2.13: fructose bisphosphate aldolase; E C 3.1.3.11: hexose diphosphatase; E C 5.3.1.9: glucose 6-phosphate iso-merase. Source is [52] 234 A.84 E D pathway of glucose utihzation. The abbreviations as in H M P path-way. Key to the enzymes: E C 2.7.1.1: hexokinase; E C 1.1.1.49: glucose 6-phosphate dehydrogenase; E C 3.1.1.17: gluconolactonase; E C 4.2.1.12: phosphogluconate dehydratase; E C 4.1.2.14: phospho-2- keto-3-deoxy-gluconate aldolase; E C 2.2.1.1: transketolase; E C 2.2.1.2: transaldolase; E C 5.1.3.1: ribulosephosphate 5- phosphate isomerase. Source is [52]. . . 235 xx A.85 P K pathway of hexose and pentose utihzation. Key to the enzymes: E C 2.7.1.1: hexokinase; E C 1.1.1.49: glucose 6-phosphate dehydrogenase; E C 3.1.1.17: gluconolactonase; E C 1.1.1.44: phosphogluconate dehydroge-nase; E C 5.1.3.1: ribulose phosphate 3-epimerase; E C 2.7.1.15: ribokinase; E C 5.3.16: ribosephosphate isomerase; E C 5.3.1.3: arabinose isomerase; E C 2.7.1.47: ribulokinase; E C 5.3.1.5: xylose isomerase; E C 2.7.1.17: xy-lulokinase; E C 4.1.2.9: phosphokeitolase; E C 2.7.2.1: acetokinase. Source A.86 Formation of acetate from pyruvate. 1, Degradation of fructose via the Embden-Meyerhof-Parnas pathway; 2, pyruvate-ferredoxin oxidoreductase; 3, phosphotransacetylase plus acetate kinase; 4, formate dehydrogenase; 5, formyl-tetrahydrofolate synthetase; 6, methenyl-tetrahydrofolate cyclo-hydrolase; 7, methylene- tetrahydrofolate dehydrogenase; 8, methylene-tetrahydrofolate reductase; 9, tetrahydrofolate: B i 2 methyltransferase; 10, C O dehydrogenase; 11, acetyl-CoA-symthesizing enzyme; [CO], enzyme-bond. Source is [54] 237 A.87 Butyrate formation from pyruvate by Clostridia. Key to the enzymes: 1, pyruvate-ferrodoxin oxidoreductase; 2, acetyl-CoA-acetyl transferase; 3, 3-hydroxybutyryl-Co A dehydrogenase; 4, 3-hydroxyacyl-CoA hydrolyase; 5, butyryl-CoA dehydrogenase; 6, fatty acid C o A transferase. Source is [52]. 238 A.88 Formation of mixed products (ethanol, lactate, etc.). 1, enzymes for the E M P pathways; 2, lactate dehydrogenase; 3, pyruvate- formate lyase; 4, formate-hydrogen lyase; 5, acetaldehyde dehydrogenase; 6, alcohol dehy-drogenase; 7, phosphotransacetylase; 8, acetate kinase; 9, P E P carboxy-lase; 10, malate dehydrogenase, fumarase and fumarate reductase. Source is [52] 236 is [54] 239 xxi A.89 Formation of propionate from pyruvate or lactate. 1, lactate dehydroge-nase; 2, pyruvate-ferredoxin oxidorecuctase; 3, phosphoacetyl transfrase; 4, acetate kinase; 5, Ds-methylmalonyl- CoA-pyruvate transcarboxylase; 6, malate dehydrogenase; 7, fumarase; 8, fumarate reductase; 9, succinyl-C o A transferase; 10, L R - methylmalonyl-CoA mutase; 11, methylmalonyl-C o A racemase; 12, pj^ruvate-phosphate dikinase; 13, PEP-carboxytrans-phosphorylase. Source is [52] 240 B.90 Standard curve of lactose concentration vs absorbance at 480 nm, The line is calculated from the equation: S i a c t 0 j , e = 120.3369 'absorbance' - 0.07461. 243 B.91 Standard curve of lactate concentration vs absorbance. The line is calcu-lated from the equation: S i a c t a t e = 12.0048 'absorbance 4 - 0.005522. . . . 245 B.92 A typical calibration curve of acetate 247 B.93 A typical calibration curve of propionate 248 B.94 A typical calibration curve of butyrate 248 B.95 A typical volatile fatty acid chromatogram. Residence time (min): ethanol (0.20); acetic acid (0.47); propionic acid (1.03); iso-butyric acid (2.10); butyric acid (3.01) 249 B.96 A typical chromotogram of gaseous samples. Residence time (min): air (0.26); methane (0.38); carbon dioxide (1.86) 250 G.97 Flow chart of the direct search method 267 xxn C h a p t e r 1 I n t r o d u c t i o n Lactose, a major constituent of cheese whey, influences the technologies apphed to treat-ment of whey. Whey is the residual watery portion of milk after removal of the curd (cheese) during the cheese-making process. It is an opaque, greenish-yellow hquid with about 6 to 6.5 percent total sohds and a biological oxygen demand (BOD) of 32000 mg/1 or higher [1]. Treatment of this by-product is a really challenging problem to cheese manufacturers considering that 9 kilograms of whey are produced for each kilogram of cheese and over 277 million kilograms of cheese were produced in Canada and over 3 billion kilograms in North America in 1987 [2]. In recent years, growing concern about environmental pollution has forced the manufacturers to give up the conventional disposal methods such as direct discharge into receiving waters [3], landfills [4], municipal sewage system [5] and adopt other more suitable methods. Obviously, the most satisfactory method is to re-use the organic components to reduce or ehminate the cost of treatment and disposal of whey. 1.1 U t i l i z a t i o n of Lactose i n W h e y As shown in Table 1.1, lactose is the principal component of whey, and consequently, utihzation of whey actually becomes the utihzation of lactose although processes for obtaining milk proteins by ultrafiltration techniques to allow their utihzation have been developed [6]. 1 Chapter 1. Introduction 2 Table 1.1: Major components of a typical whey Component Composition (w/w %) water 93.1 lactose 4.9 protein 0.9 lacto-globuhn lacto-albumin lummune-globuhns fat 0.3 lactic acid 0.2 ash 0.6 oxides of K Na Ca Mg Fe phsophrous pentoxide chlorine oxide sulfur trioxdde total solids 6.9 Source is [7]. Whey powders are produced by drying whey [8]. Also lactose can be separated from whey by the processes of concentration and crystallization. These products can be used as feed for animals or as substrate for biological processes (e.g. penicillin production) [9]. Obviously, a great amount of energy is consumed for the removal of about 93 percent water. Use of whey as a substrate for fermentations can upgrade the raw material into useful products such as yeast, alcohol, organic acids, vitamins, alcohohc beverages and vinegar by employing microorganisms that are able to utilize lactose [1]. A variety of procedures have been suggested for the growth of yeast (Saccharomyces fragilis) in whey [10], producing food- and feed-grade yeast or yeast-whey products which, if properly produced, are highly nutritious, nontoxic sources of protein and vi-tamins and under the right circumstances can find apphcation in human and animal Chapter 1. Introduction 3 nutrition. Whey has also been used to grow microorganisms which produce useful en-zymes, including /3 \u00E2\u0080\u0094 galactosidase [11]. Anaerobic growth of lactose utilizing yeast (Kluyveromyces species) in whey converts lactose into ethanol [12], but a simple fer-mentation of whey produces a solution containing only ca 2% of ethanol, which is un-economic to distill. Some dried whey permeate has been added to liquid whey giving a high concentration of lactose and thus of ethanol. It is clear that only economical reasons prevent the use of these approaches to treat whey. First of all, not all the whey produced is available for the uses mentioned above, since much cheese is made in small factories at scattered locations, making it impractical to transport the bulky whey to a centralized plant for processing. Moreover, for recovery systems the capital and operation costs are comparatively high because of the low con-centration of lactose. Finally, the supply of whey for re-use often exceeds the demand. This situation has left many smaller cheese plants in an insecure position with regard to whey disposal, because economically acceptable alternatives to the re-use options have not been identified [13]. 1.2 Anaerobic Methanation of Lactose Anaerobic methanation is a biological digestion process in which various anaerobic bacte-rial species cooperate to convert organic materials (carbohydrates, fats, proteins, etc) to organic acids and finally a gaseous mixture of methane and carbon dioxide in the absence of molecular oxygen. In the past, broad scale applications of this process, a well known process in waste management area, have been largely with the treatment of municipal sewage sludge and animal residues to achieve waste stabilization and solid reduction. Compared with the processes of whey fermentation to products such as alcohol, yeasts, as mentioned above, the most distinctive advantage of whey anaerobic methanation is Chapter 1. Introduction 4 that the final product, methane, naturally separates from the hquid waste to save energy in downstream processing and is also useable in large quantities as a fuel without major modification to existing furnaces in the plants. Other advantages include no requirement for sterilization of the wastewater, low nutrient requirement etc. Compared to the aerobic treatment, lactose utihzation by anaerobic sludge also offers several distinctive advantages including: 1. lower sohds/sludge production, 2. highly stabilized waste biological sludge that, as a rule, can be easily dewatered, 3. no energy requirement for aeration, 4. production of methane, a useful end product. However, the conventional anaerobic processes suffer from a slow digestion rate and poor process stability (unstable performance, susceptibility to shock loading) and rela-tively long periods of time required to start up the process or recover from an unstable condition. To overcome these problems encountered in a convertional process of anaerobic di-gestion, the key role of cell immobihzation has been recognized and it was found that success of a biological treatment was directly related to the efficiency of cell immobihza-tion and/or retention because of the higher cell concentration which resulted in a lower reactor volume requirement and better stability. The use of biomass immobihzation has led to development of different configurations of anaerobic digesters such as anaerobic filter [14], fluidized/expanded bed reactor [15], and the upflow anaerobic sludge blanket (UASB) digester [16] as illustrated in Figure 1.1. The bacteria attach and grow on an inert support material which is static in anaer-obic filters (e.g. rocks, plastics) and movable in fluidized/expanded bed reactors (e.g. Chapter 1. Introduction 5 gas static supports^ -effluent particles influent effluent recycle influent (A) granules \u00E2\u0080\u00A2>> .1 (B) effluent (C) Figure 1.1: Illustration of configurations of anaerobic filter (A), fluidized/expanded bed (B) and upflow anaerobic sludge blanket (C) Chapter 1. Introduction 6 sands), forming slime bacteria films on the support and resulting in a long retention time and a high concentration of bacteria in the digesters. In U A S B reactors, under favorable chemical and physical conditions the bacteria flocculate to form granules with superior settling characteristics, which gives a high concentration and a long retention time of bacteria in the reactors. For anaerobic methanation of whey or lactose, Switzenbaum and Danskin [17], Boeing and Larsen [18] utilized the fluidized or expanded bed reac-tors. Dehaast et al [19], van den Berg and Kennedy [20], Wildenauer and Winter [21] applied upflow or downflow fixed film reactors. Reynolds and Colleran [22] used a hybrid sludge-bed/fixed-bed reactor which was actually a combination of a U A S B reactor and an anaerobic filter. Further understanding of the microbiology and biochemistry involved in the anaer-obic methanation of soluble organic materials which will be discussed in the following chapter in detail has led to a two-phase process in which acid-producing bacteria and methane-producing bacteria are cultured in separated reactors because it is believed that the acidogens and methanogens have different biological properties and thus culture re-quirements. Also in a two-phase process, more opportunities are offered for controlling the flow of the substrates between the distinct groups of bacteria, and thus, producing a better match between them [23]. 1.3 More Considerations on Two-phase Methanation of Lactose In a two-phase process, the first phase reactor contains acid-forming bacteria which ferment lactose to a mixture of mainly acetic, propionic and butyric acids. They are fast-growing bacteria (minimum doubling time around 30 min). In the second phase reactor, the bacteria can be divided into two groups, acetate-forming bacteria which convert propionic and butyric acids to acetate, and methane-forming bacteria which Chapter 1. Introduction 7 utilize acetate and H2/CO2 to produce methane. Al l of them grow very slowly, the former having minimum doubhng times of 1.5-4.0 days and the latter of 2-3 days [24]. Figure 1.2 illustrates the concept of two-phase anaerobic digestion of wastes. Gas \" ( C H 4 + C 0 2 ) Influent (whey, lactose) Acid-forming Reactor Intermediates (HAc, H B u , HPr) Methane-forming Reactor Effluent Figure 1.2: Two-phase process of anaerobic digestion of organic wastes A number of researchers have investigated the two-phase processes. Some efforts were focused on the methods of separating methanogenic from acidogenic species and on achieving and characterizing the resulting environment in each phase. Pohland and Ghosh [23] washed the methanogens out of the first phase reactor using kinetic controls. Investigations [25,26,27,28,29] have indicated that the environmental requirements of aci-dogenic and methanogenic (acetogens plus methanogens) phases are quite different from each other in terms of p H and oxidation-reduction potentials (ORP) . After comparing the performance of a conventional single-phase and a two-phase anaerobic digestion of soft-drink waste, Ghosh et al [30] indicated that phase separation could improve the op-eration stabihty as well as quality of product gas. Table 1.2 hsts the apphcations of the two-phase process in the treatment of various wastewaters. In design and operation of a two-phase biofilm process of anaerobic digestion of whey, Chapter 1. Introduction 8 Table 1.2: Pilot- and full-scale plants of two-phase anaerobic treatment of wastewaters Year Industry Location Type Capacity (kg C O D / d a y ) 1977 Distillery Belgium Pilot 180 (enzyme, alcohol) 1980 Beet sugar West Germany Pilot 45 1980 DistiUery Belgium Pilot 135 (yeast, alcohol) 1981 Beet sugar Belgium Pilot 170 1981 Citric acid West Germany Pilot 120 1981 Beet sugar West Germany Pilot 45 1980 Flax retting Belgium Full-scale 350 1982 Starch-to-glucose West Germany Full-scale 20,000 1983 Yeast-alcohol Netherlands Full-scale 20,000 1984 Baker's yeast France Full-scale 7,000 Source is [13,30,31]. further consideration are needed on several points, especially, the fermentation kinetics in each phase, mass transfer of substrates in the biofilms and build-up of anaerobic biofilms. 1.3.1 Kinetics in Each Phase Reactor When steady state operation in a two-phase system is estabhshed, the production of organic acids in the first phase reactor must be balanced by their consumption in the second phase reactor, otherwise, accumulation of these acids would lead to a \"sour\" process if the p H is uncontrolled. In this case more caustic solution will be consumed for controlling the p H of the second phase reactor in a narrow neutral range with more organic acids appearing in the effluent in the form of salts. Therefore, the knowledge of yield and consumption rates of organic acids are essential for process development. Furthermore, the effluent from a cheese plant, as is well known, has a variable flow rate and B O D strength [3]. This requires the process have an operation range over which it Chapter 1. Introduction 9 can withstand the normal feed fluctuations. The response of each phase reactor to feed fluctuations may be used for the determination of this operation range. Most of the experiments conducted to date on the anaerobic methanation of lactose or whey were designed to evaluate the performance of the process [17,18,19,20,21,22,32, 33,34,35]. Detailed biochemical kinetic information is virtually non-existent. For other substrates, some kinetic results of anaerobic methanation are available. However, most of them were obtained in single-phase processes which differ greatly from two-phase pro-cesses considering the effects of phase separation which results in a constant production of organic acids (e.g. propionic and butyric acids) in the first phase, and correspondingly, a healthy population of obligate hydrogen-producing acetogens ( O H P A ) and associated hydrogen-oxidizing methanogens ( H O M ) which ensured the rapid assimilation of those acids in the second phase reactor [36]. 1.3.2 Mass Transfer in the Biofilms As discussed above, the biomass must be retained in reactors to make a long solid reten-tion time (SRT) possible even at short hydrauhc retention times (HRT) and to maximize biomass concentrations in the reactors, which has been fulfilled by developing three ma-jor types of reactor configurations as shown in Figure 1.1. As biomass accumulates in the form of biofilms (anaerobic filters and fluidized/expanded beds) or granules (UASB) in a reactor of specified volume, two problems may arise. One is that the biomass will form a solid phase in which the substrates are gradually consumed as they diffuse into the biomass, and so, the bacterial cells embedded in the film see different substrate concentra-tions. The combination of the consumption and the diffusion rates determines a effective thickness of biofilm beyond which no substrate is available to the bacteria. Secondly, since the living bacteria are continuously growing with the consumption of substrates even though the growth rate is relatively slow in anaerobic processes, the actual H R T Chapter 1. Introduction 10 majr be reduced to impractically low values due to the accumulation of active biomass (within an effective thickness) and/or dead biomass which reduce the available reactor volume. Because of these considerations, a knowledge of mass transfer within the acidogenic and methanogenic biofilms is necessary for the estimation of the most effective biofilm thickness for maximizing the biomass concentration as well as a long term stable opera-tion. Compared with fluidized/expanded bed and U A S B , an anaerobic filter is more affected by mass transfer limitations because the biomass can be almost completely retained. This problem has been recognized by literature reports of phenomena caused by plugging and channehng in laboratory scale anaerobic filters [37,38]. In a full scale downflow anaerobic reactor which contained three layers of clay blocks with square vertical channels as supports, treating an effluent from a dairy plant (1500 to 1800 m 3 /day, 2000-3000 kg B O D 5 or 4000-6000 kg C O D per day), the C O D removal decreased from a range of 60-80 % after the start-up period to 50 % and less at the end of 2 years when it was found that the active reactor volume had declined by 80 percent [3]. Based on observations of the long term performances of an anaerobic filter and a U A S B reactor and their abilities to prevent the accumulation of excess biomass, a hybrid reactor configuration (combination of these two types of reactor) was developed in Wastewater Technology Centre, Canada, in order to minimize the excess biomass accumulation and its impact on reactor hydraulics [39]. Obviously, a growing biofilm should be controlled at its optimum thickness so as to maintain effective and long term stable operation in an anaerobic digestion process. Knowledge of mass transfer rates of substrates in anaerobic biofilms is essential to es-timate this optimal biofilm thickness. The majority of the studies on mass transfer in biomass, however, has been concerned with aerobic microbial aggregates, because it was Chapter 1. Introduction 11 easy to recognize that O2 supply is limiting due to its low solubility in water. Few works have been conducted on anaerobic bacterial aggregates and to the author's knowledge, no report on the transport of lactose and organic acids in anaerobic microbial aggregates exists. 1.3.3 Start-up of Anaerobic Biofilms One disadvantage of anaerobic digestion processes is that a relatively long time is needed for start up due to the very slow growth rate of anaerobic bacteria, especially the ace-togens and methanogens. A mature reactor is characterized by a well developed, immo-bilized biomass (granules or biofilms). During the process of biofilm formation the first step is attachment of bacteria onto inert solid supports, which is affected by many factors such as bacterial strain, limiting nutrients, physio-chemical properties of the solid sur-faces. Then the attached bacteria proliferate forming microcolonies, and a biofilm by the merging of these colonies. Start-up of an anaerobic process to a great extent nowadays still relies on industrial experience because of the complexity of the process. In a two-phase system, the acidogens grow fast and also form a biofilm more easily. The formation of a methanogenic biofilm (including acetogens), thus, becomes the rate-limiting step. Furthermore, it would also be interesting if the properties of solid supports would affect the relative attachment rates of each type of bacteria (acetate-, propionate-, and butyrate-degrading bacteria) when the supports are immersed in the mixture of these microorganisms. Choice of a solid supporter would then in part depend on the knowledge of its effect on the ease with which each group of bacteria attach on it. A number of studies on methanogenic film development have been conducted. Most of them were based on the total consumption rate of organic carbon [40], methane pro-duction rate [41], or total protein content of a biofilm [42], which can not answer the Chapter 1. Introduction 12 question whether or not and how the surface properties influence the attachment of var-ious bacterial types in a mixed culture. Some researchers [43] used a single substrate (acetate) to investigate the influence of sohd surfaces on biofilm development. This ap-proach can only give partial information because it neglects the symbiotic relationship between bacteria. 1.4 Research Objectives and Scope The two-phase anaerobic methanation of whey has been, as indicated above, demon-strated as a potentially successful process. However, some further studies are needed, especially on the three topics discussed above, if it is to become an economic, reliable, and stable alternate for the treatment of high B O D wastewaters. Kisaahta, Lo and Pinder [44,45] have studied the acidogenesis of lactose in a contin-uous stirred reactor ( C S T R ) in which the bacteria were suspended. They found that the dilution rate and p H would affect the acidogenic product distribution which was assumed to be caused by a shift in the bacterial population. The present research is an extension of their effort to introduce the two-phase method for whey treatment and will be mainly focussed on immobihzed acidogenic and methano-genic bacteria. Specifically, the present study includes the following major topics: 1. Intrinsic kinetics of lactose acidogenesis and methanogenesis Under selected conditions (temperature, p H , nutrients) which are based on the results of Kisaahta [46] and others, investigate the utihzation rate of lactose and production rates of organic acids in a fixed-film acidogenic reactor, and conduct studies on the utilization rates of organic acids in a fixed-film methanogenic reactor. These results should be obtained under conditions where there is negligible effect Chapter 2. Introduction 13 of external and internal mass transfer resistance. Also, some studies will be made on the responses of these two reactors to feed fluctuations. 2. Mass transfer of lactose in acidogenic biofilms and organic acids in methanogenic biofilms Mass transfer within active biofilms is a very comphcated process because it involves both mass transfer and reactions. Some researchers have described the influence of mass transfer on reaction with macro-kinetics which included both intrinsic kinetics as well as mass transfer and gave an overall reaction of half order [47]. Some studied a simplified biofilm which was deactivated to eliminate the influence of reaction on mass transfer. The present research will use a speciaUy designed device to measure directly the substrate concentration drop within living biofilms. The diffusivities of substrates (lactose, acetate, propionate and butyrate) in their corresponding biofilms will be found by using a reaction-diffusion model which describes the mass transfer process of these substrates in anaerobic biofilms. 3. Start-up of symbiotic methanogenic biofilms Considering that many factors can effect the start-up of biofilms, this research will concentrate on the effect of sohd surfaces, in terms of their water contact angles (0 - 1 0 0 \u00C2\u00B0 ) , on the attachment of acetogenic and methanogenic bacteria groups. Some efforts will be made to estabhsh a model to describe the start-up of a symbiotic methanogenic biofilm. Chapter 2 Theoretical Aspects and Previous Studies 2.1 Process Biochemistry and Microbiology Anaerobic digestion is usually considered to be a two-phase process consisting of acid-formation (acidogenesis) and methane-formation (methanogenesis). It has been known that at least three large, physiologically different, bacterial populations must be present for the overall conversion of organic matter to methane and carbon dioxide to occur. In the first phase, organic matter (proteins, hpids, carbohydrates) are converted by a heterogeneous group of microorganisms into fatty acids by hydrolysis and fermentation. In the second phase, the end-products of the first phase fermentation are converted to methane and carbon dioxide by strict obligate anaerobic bacteria (methanogenic bacteria) with the assistance from another group of bacteria termed acetogenic bacteria. The latter can convert the compounds the methanogenic bacteria cannot digest into acetate, dihydrogen and carbon dioxide which can be utilized by methanogenic bacteria. If the hydrolysis of biopolymer molecules is the slowest step (e.g. cellulose hydrolysis) in the whole process, the fatty acids produced in the first phase would not be accumu-lated in the culture medium due to their being digested by methanogenic bacteria. If the organic matter, however, is readily hydrolyzed (e.g. lactose which can be directly transported into microbial cells), the slowest step becomes the methanogenesis of fatty acids. It would then be possible that the fatty acids may not be digested rapidly enough by methanogenic bacteria and their accumulation in the culture medium would lead to 14 Chapter 2. Theoretical Aspects and Previous Studies 15 HO-CH2 HO-CH2 6(1 -4 ) D-Galactose D-Glucose Figure 2.3: Illustration of lactose structure an undesirable 'sour' process and no gas is produced [46]. 2.1.1 Lactose Acidogenesis Lactose, a reducing disaccharide, is constructed from a galactose residue and a glucose residue. The glycosidic hnk is 8(1 \u00E2\u0080\u0094\u00E2\u0080\u00A2 4), which means that the OH group on the anomeric carbon atom of galactose is in /3 position as shown in Figure 2.3. Although the anomeric atom on galactose residue is involved in the glycosidic bond, the anomeric carbon atom (Cl) on the glucose residue can still participate in forming a free aldehyde group and thus the free carbonyl oxygen can reduce a number of substances, for example, divalent copper (Cu + + ) to the monovalent species (Cu +). This reducing capacity of lactose has been used in this study to measure its concentration in a culture medium. The /3(1 \u00E2\u0080\u0094> 4) glycosidic hnk may have a significant effect on the properties of lactose, which can be projected by comparing the properties of cellulose and amylose (a type of starch having straight carbon chains). Cellulose is constructed from repeating units of D-glucose held together by 3(1 \u00E2\u0080\u0094> 4) glycosidic bonds and amylose by a(l \u00E2\u0080\u0094> 4) glycosidic Chapter 2. Theoretical Aspects and Previous Studies 16 a (1-4) \u00C2\u00A3 (1\u00E2\u0080\u00944) Figure 2.4: Illustration of two types of glycosidic links bonds (Figure 2.4). This subtle difference, however, results in a significant difference in the physical prop-erties of the two types of polysaccharides; starch, a storage form of cellular fuel which is quite easily digested by mammals, yeasts or bacteria, and cellulose, a structural element of plant cells which cannot be hydrolyzed by most mammals, including humans. The microorganisms capable of secreting the enzyme cellulase can hydrolyze the 8(1 \u00E2\u0080\u0094> 4) link of cellulose into D-glucose. Breakage of the lactose 8(1 \u00E2\u0080\u0094* 4) glycosidic bond involves a special enzyme, 8 -galactosidase, coded and controlled by a segment of D N A in bacteria termed lac operon. Studies of the structure of the lac operon in Escherichia coli clearly demonstrated the whole process for producing the enzymes required for lactose transport and metabolism. A diagram of the lac operon is shown in Figure 2.5 [48]. Chapter 2. Theoretical Aspects and Previous Studies 17 control i sil .es i i P o structural genes a section of prokaryotic D N A I- Lactose operon L, Operator gene: binds repressor protein 1\u00E2\u0080\u0094Promotor site: binds R N A polymerase ' \u00E2\u0080\u0094 Regulatory gene: codes for repressor protein Figure 2.5: Structure of lac operon The lac operon consists of three distinct parts: the regulatory gene (i), the operator gene (o) and the structural genes (z, y, a). The structural genes (z, y, a) code for three separate enzymes involved with lactose metabolism. The 'z' gene codes for (3 \u00E2\u0080\u0094 galactosidase, the enzyme that hydrolyzes lactose into glucose and galactose for further use in energy production. CHaQH Galactosidase 1 HzO Lactose CH2OH ChfepH HO ^ 6 Galactose Glucose The 'y' gene codes for galactoside permease, the enzyme that mediates the transport of lactose into the cell, which means that the hydrolysis of lactose occurs in the cell's cytoplasm rather than in the culture medium. The 'a' gene codes for galacoside acetylase (function unknown). A textbook [48] should be consulted for how the E coli cell mediates the transport and metabolism through the regulatory and operator genes. The glucose Chapter 2. Theoretical Aspects and Previous Studies 18 Table 2.3: Main genera of acid-producing bacteria implicated in first phase Genus Substrate Products Aerobes Pseudomonas nutritionally highly Micrococcus versatile starch lactate Facultative Bacillus starch maltose lactate Anaerobes Lactobacillus numerous sugars acetate Escherichia numerous sugars Clostridia succinate, acetate Obligate Ruminococcus cellulose, cellobiose ethanol, hydrogen Anaerobes Bacteroides hemicellulose, pectin formate Butyrivibrio starch butyrate, lactate Megasphera lactate, glucose branched V F A , hydrogen Selenomonas sugars acetate, propionate Desulfobibrio lactate, malate acetate Bifidobacteria proteins V F A Propionibacterium amino-acids propionate Anaerovibrio Source is [49]. and galactose in the bacterial cells undergo further various reactions depending on the bacterial species present. The bacteria isolated from the anaerobic digestion mixture of the first phase include a wide range of physiological groups as shown in Table 2.3. Whether all the different isolates obtained by various workers are physiologically sig-nificant in the digestion process still remains to be determined. However, the presence of various genera in a mixed culture can supply various enzymes crucial to the hydrolysis and fermentation of various organic materials. This is especially desirable in a process for wastewater treatment which may contain many kinds of organic wastes. Moreover, whether a bacterial species can survive and dominate in a digester depends greatly on the environmental conditions (temperature, p H , nutrients, etc.) and its efficiency of extract-ing energy from the environment. The more energy it is able to get from the nutrients, Chapter 2. Theoretical Aspects and Previous Studies 19 the faster it will grow. Presence of aerobes and facultative anaerobes in anaerobic di-gesters was explained by McKinney [50] by the ease of growth of the facultative bacteria which gives them an edge over the strict anaerobes and so the acid-formers are made up predominantly of facultative bacteria with a few strict anaerobes. The oxygen needed for growth of facultative anaerobic bacteria may come from the dissolved oxygen in the feed solution. After they developed a method to enumerate obhgate anaerobic acidogenic bacteria in digesters, Toerien et al [51] reported 3 9 x l 0 7 to 1 5 x l 0 9 obhgate anaerobic non-methanogenic and 8 x l 0 5 to 1x10 s aerobic and facultative anaerobic bacteria per ml in several digesters. The anaerobic counts were usually 100 or more times greater than the aerobic counts. Post et al [52] also reported the presence of a large obhgate anaerobic bacterial population in anaerobic digester. While so many bacterial species are present in a digester, glucose and its epimers (galactose, mannose etc.) are catabolized to pyruvate anaerobicaUy by four currently recognized routes [53]. ct Embden-Meyerhof-Parnas ( E M P ) pathway. \u00E2\u0080\u00A2 Hexose monophosphate (HMP) pathway, o Enter-Doudoroff (ED) pathway. \u00E2\u0080\u00A2 Phosphoketolase (PK) pathway. Through the use of 1 4 C tracers, McCarty et al [54] concluded that both E M P glycolysis and hexose phosphate pathways probably mainly occurred in anaerobic digesters receiv-ing carbohydrates. Among the four pathways, the E M P pathway provides bacteria with the greatest amount of A T P under anaerobic condition [53]. Glucose + 2 A T P + 2NAD+ \u00E2\u0080\u0094 \u00E2\u0080\u00A2 2Pyruvate + 4 A T P + 2 N A D H + 2H+ Chapter 2, Theoretical Aspects and Previous Studies 20 But it does not produce the important precursors for purine, pyrimidine, D N A and R N A biosynthesis. In contrast, the H M P pathway produces all the precursors necessary for these important biomolecules, giving half the amount of A T P . Microorganisms growing on simple defined media such as glucose (or lactose) plus mineral salts must use other pathways such as the H M P pathway if they are not supplied with growth factors. The ra-tio of usage of the E M P and H M P pathways can vary greatly depending on environmental conditions. Biological activities of living cells involve catabolic and anabobc pathways. Catabolic pathways are the degradative reactions through which nutrient molecules are degraded into simpler molecules, releasing chemical energy. Anabolic pathways are biosynthetic processes in which various cellular constituents are synthesized from simpler precursor molecules and require the input of chemical energy. The whole process is accomplished by what are known as coupled reactions usually catalyzed by enzymes. The released chemical energy is trapped in the form of an energy-rich intermediate and used to drive a second reaction. Adenosine triphosphate ( A T P ) is the primary energy carrier in all life forms. As nutrients are broken down, some of the free energy contained in those molecules is conserved in the form of A T P . When cells must synthesize various molecules, free energy is required to make the new bonds; this energy is supplied by the A T P molecules. Specifically, A T P hydrolyses into adenosine diphosphate ( A D P ) and inorganic phosphate, releasing a significant amount of free energy. A T P + H 2 0 \u00E2\u0080\u0094 \u00C2\u00BB A D P + Pi ( A G 0 = - 7 . 3 kcal/mol) The standard conditions specified in this thesis are; one atmosphere for each gaseous components, all other compounds in aqueous solution at 1 M activity at p H of 7.0 and 25 \u00C2\u00B0C. Details of the four routes can be found in Appendix A . The other points of importance Chapter 2. Theoretical Aspects and Previous Studies 21 in the sequence of the E M P glycolysis are emphasized here because its product, pyruvate, is the key component for further reactions in acidogenesis. \u00E2\u0080\u00A2 Glucose has first to be activated and converted to a phosphated fructose by con-suming two A T P molecules. \u00E2\u0080\u00A2 After the fructose is split into two triosephosphates, the oxidation of the trioses is coupled to the reduction of a electron/hydrogen carrier, nicotinamide adenine dinucleotide ( N A D ) o In the subsequent steps to pyruvate, a total of 4 moles of A T P are formed. The reduced N A D + must be regenerated by some mechanism, which to a great extent determines the final fermentation product distribution. In addition to 2 moles of pyruvate and A T P , the glycolysis gives 2 moles of reduced N A D + ( N A D H ) which must be reoxidized under anaerobic conditions for repeated use. N A D + + H 2 ^ N A D H + H+ Depending on the mechanisms by which they oxidize the reduced N A D , the acidogenic bacteria give rise to a diversity of products by further metabolism of pyruvate (see Fig-ure 2.6). These reactions permit an overall oxidation-reduction balance to be preserved under anaerobic conditions. The change in free energy during the reoxidation of N A D H into N A D + and H 2 be-comes negative only when the partial pressure of hydrogen drops below 1 0 - 3 atmospheres [180]. Thus a low hydrogen partial pressure can pull the overall metabohsm towards the formation of H 2 , C 0 2 and acetate. On the other hand, higher values of hydrogen par-tial pressure will inhibit the oxidation of N A D H and then it must be reoxidized by the reduction of pyruvate or acetyl-CoA, forming reduced products such as lactate, ethanol, Chapter 2. Theoretical Aspects and Previous Studies 22 Acrylate \\u00C2\u00BB Lactate -2H Pyruvate A Acetaldehyde j Oxaloacetate j -2H ! Malate j -2H I Succinate j Propionate -2H Ethanol ! Formate i -f I Acetyl C o A i 4 -H , Acetone IButyryl C o A l --f A T P Butyrate Figure 2.6: Bacterial fermentation products of pyruvate. Pyruvate formed by the catabohsm of glucose is further metabolized by pathways which are characteristic of par-ticular organisms. End products of fermentations are shown in boxes and intermediates in dash boxes. \" - \" refers to consumption and \"+\" to production. Chapter 2. Theoretical Aspects and Previous Studies 23 propionate and butyrate. Among them, some products (acetate, butyrate) give rise to additional energy by substrate-level phosphorylations, e.g. the conversion of an acetyl-C o A derivative to free acid, C H 3 C O - S C 0 A + A D P + Pi \u00E2\u0080\u0094 \u00E2\u0080\u00A2 C H 3 C O O H + A T P + S C o A C H 3 C H 2 C H 2 C O - S C o A + A D P + P; \u00E2\u0080\u0094> C H 3 C H 2 C H 2 C O O H + A T P + S C o A Some reactions (formation of lactate, ethanol, etc), however, can not produce extra A T P and only keep oxidation-reduction in balance by oxidizing the reduced N A D + . C H 3 C O C O O H + N A D H + H+ \u00E2\u0080\u0094 \u00E2\u0080\u00A2 C H 3 C H O H C O O H + N A D + The effect of environments (pH, temperature, etc.) on the distribution of fermenta-tion products is also quite significant. As the culture p H drops, it becomes more and more difficult to reoxidize N A D H from the point of view of energetics. Therefore, the production of acetate as sole end product would not be satisfied, and bacteria will utihze pyruvate not only to form acetate but also butyrate, a weaker acid than acetate. If the p H drops below 4, some neutral compounds are produced such as butanol and acetone. If the environmental conditions are not favorable to a bacterial species, its characteristic product from the fermentation of pyruvate apparently would not occur. Hungate [56] first expressed the idea that hydrogen production and utihzation could profoundly influence the course of a fermentation in anaerobic ecosystems. The regulatory role of hydrogen in the course of anaerobic fermentations is effected by interspecies hydro-gen transfer, induced by hydrogen-trophic organisms (methanogenic bacteria), to pull the reactions toward the direction in which more hydrogen is produced. In a two-phase pro-cess, the acid-formation and methane-formation phases are physically separated and the hydrogentrophic methanogens are not present in the first phase reactor because of some Chapter 2. Theoretical Aspects and Previous Studies 24 Table 2.4: Classification of seven genera of methanogenic bacteria Order Genus Morphology Gram Substrate I Methanobacterium long rods to filaments + H2/C02, formate M ethanobrevibcter short rod to lancet cocci + H2/C02, formate II M ethanococcus irregular and small - H2/CO2, formate III M ethanomicrobium short rods - H2/CO2-, formate M ethanogenium irregular small cocci - H2/C02, formate Methanospirillum short to long wavy spirilla - H2/CO2, formate M ethanosarcina pseudosarcina + H2/C02, methanol acetate, methylamine Source is [57]. deliberately chosen conditions (low p H , low hydraulic residence time etc.). Therefore, it is essential to investigate the rate and product distribution of lactose fermentation in a separated acidogenic biofilm reactor without effects from hydrogentrophic methanogens. 2.1.2 Methanogenesis of Fatty Acids Methanogenic bacteria are members of a very distinct group of bacteria with respect to their physiology and ecology. They are present in extremely anaerobic environments like the rumens, sewage sludge digesters and anoxic muds and sediments. They are the terminal organisms in the anaerobic transformation of the substrates available in such environments. A taxonomy, to replace an old one based on bacterial morphology, has been recently adopted which is based on the structure of their 16S ribosomal R N A (see Table 2.4). This molecule has changed very slowly during evolution, so any significant differences that are observed must indicate a very long evolution history. Chapter 2. Theoretical Aspects and Previous Studies 25 Table 2.4 indicates that, based on the structure of 16S r R N A , there is a very great diversity among the methanogens. They are not a homogeneous and closely related group of bacteria. The unifying characteristic of this bacterial group is that all mem-bers are able to reduce carbon dioxide into methane as final product of their ener-getic metabohsm. Growth on CO2 as carbon source is autotrophy, but the autotrophic growth of the methanogens is totally different from that of virtually all phototrophs and chemoautotrophs because it does not involve the ribolase bisphosphate (Calvin) cycle. As a consequence, they have been classified into a group of bacteria called archbacteria which is distinct from the \"Classical\" procaryotes. In this group, extreme halophiles (Halobacteriaceae ) and thermoacidophiles (Sulfolobus) have also been placed [58]. The methanogens are strict obhgate anaerobes, but they are capable of existing well in close association with other classes of bacteria which are capable of removing oxygen. Kiener and Leisinger investigated the sensitivity of five methanogenic bacte-ria (belonging to four genera) to oxygen [59]. The death curves of Methanobacterium thermoautotrophicum , Methanohrevibacter arboriphilus and Methanosarcina barkeri were biphasic. For these strains, exposure to air for 10 to 30 hours was without effect on their survival. Up to a critical time of contact, oxygen apparently caused no or only reversible damage on these organisms. For the two methanococci species ( M . voltae and M. vannielii), however, the number of surviving cells upon exposure to air dropped ex-ponentially without lag. The three comparatively robust strains were originally isolated from sludge digesters, i.e. ecosystems which are periodically subjected to oxygen stress and therefore may favour the development of air-tolerant methanogenic bacteria. In con-trast to them, the two Methanococci strains were highly sensitive to oxygen. Sea and lake sediments, the natural habitats of Methanococci , are not exposed to oxygen thereby providing a safe environment for these highly oxygen sensitive methanogens. This infor-mation is very useful for judging the extent to which strictly anaerobic conditions should Chapter 2. Theoretical Aspects and Previous Studies 26 be kept in handling methanogens. A l l methanogenic bacteria are capable of extracting energy from the oxidation of dihydrogen under anaerobic conditions using carbon dioxide as electron acceptor. 4 H 2 + H C O J + H+ \u00E2\u0080\u0094> C H 4 + 3 H 2 0 ( A G \u00C2\u00B0 = - 1 3 8 . 9 kJ) (2.1) The initial reduction of carbon dioxide to the level of formaldehyde is thermodynamically unfavorable whereas further reduction of formaldehyde to methane is very favorable, and the standard free energy change of the reaction would theoretically support the forma-tion of two molecules of A T P . The dihydrogen-dependent reduction of C 0 2 requires an anaerobic electron transport pathway. The exact nature of the electron carriers involved in this pathway has not yet been determined. A methyl carrier, coenzyme M , has been detected in all methanogenic bacteria and appears to be unique to this group of organ-isms [60]. A n early labelled reduction product of 1 4 C 0 2 that can be detected in whole cells or in the extract of methanogenic bacteria is methyl-coenzyme M . It is believed that this compound is the substrate for the final step in methane production catalyzed by methyl -CoM reductase [61]. C H 3 - S C o M + H 2 \u00E2\u0080\u0094 \u00E2\u0080\u00A2 C H 4 + H S C o M Methanogens differ from other autotrophs (organisms that proliferate with C 0 2 as the sole carbon source) in that their C 0 2 metabohsm involves both fixation to cell carbon and reduction to methane. At present, httle is known about the initial reactions involved in C 0 2 reduction to methane and fixation into cellular intermediates. The biochemical mechanism of methane formation from organic acids (the major products of the acid-formation phase) and its couphng to A T P synthesis is virtually unknown. Recently, a fluorescent electron transfer coenzyme (coenzyme F 4 2 0 ) has been found in all methanogens but has not been detected elsewhere. F 4 2 0 participates as an electron carrier in the nicotinamide adenine dinucleotide phosphate ( N A D P ) hnked Chapter 2. Theoretical Aspects and Previous Studies 27 hydrogenase and formate dehydrogenase systems in methanogens [62]. Thus, formate and hydrogen are essentially equivalent sources of electrons for the reduction of carbon dioxide to methane. In anaerobic digesters, the major source of methane appears to be acetate rather than carbon dioxide or formate even though only a few methanogenic species are able to use acetate as their energy and carbon source. Estimations based on carbon isotope labelling indicate that 67 to 75 per cent of the methane is derived from acetate [63,64]. Methanosarcina barkeri, one of a few species having a wider substrate spectrum, has shown its ability to use acetate, methanol and methylated amines as sole carbon and energy source instead of H 2 / C 0 2 . It has been confirmed by labelling acetate that its methyl group during the fermentation is transferred intact into methane; there is no evidence of the acetate being oxidized first to C 0 2 [65,66]. C H 3 C O O H \u00E2\u0080\u0094 \u00E2\u0080\u00A2 C * H 4 + C 0 2 ( A G \u00C2\u00B0 = - 3 2 kJ/mol) (2.2) Conservation of the protons in the methyl moiety strongly suggests that methane produc-tion from acetate was attained via a single reduction step by a single organism. Under standard conditions, the acetate conversion to methane can only offer limited energy. Therefore, acetate-fermenting methanogens would be inherently slow growing, and it would be questionable if fermentation of acetate alone would sustain the energy require-ments for growth. Values reported for the free energy of A T P hydrolysis have been estimated to range from -34.4 to -52.4 kJ /mol at physiological conditions. Normal ef-ficiencies of energy transfer in bacteria are 30 to 50 per cent. Thus, the reactions that yield less than -49 kJ /mol at standard conditions makes the formation of an energy-rich compound via substrate level phosphorylation unhkely and is insufficient for cell growth [67]. \"Highly purified\" cultures of Methanosarcina barkeri and Methanococcus species showed a very slow acetate fermentation [68]. Isotopic tracer studies by Zeikus et al [69] indicated that hydrogen was required for the metabolism of acetate to methane by Chapter 2. Theoretical Aspects and Previous Studies 28 several pure cultures. Comparing the energy formed per mole of methane produced from reactions 2.1 and 2.2 shows that approximately four times as much energy is available from respiration of hydrogen than that from acetate fermentation. Adding hydrogen to pure acetate fermentation cultures not only raises the actual free energy released from the acetate fermentation due to decreased CO2 concentration but also supplies more free energy for bacterial growth from the reduction of carbon dioxide. Alternatively, The methanogenic species which are capable of utilizing acetate may possess a mechanism through which sufficient free energy can be accumulated from the fermentations of more than one acetate molecule and then the energy is used to produce A T P . As a result, up to 70 per cent methane is produced from the reaction of acetate fermentation. In mixed cultures, more complexly, the acetate utilization was affected by many factors such as ions strength, redox potential of the culture [71] and even the presence of sulfate-reducing species [70] 2.1.3 Acetogenesis of Fatty Acids As discussed above, the substrate spectra of methanogenic bacteria are very narrow, only including H 2 / C 0 2 , formate, methanol, methylamide and acetate, while the end products from the first phase (acids-formation) include a large number of organic acids containing more than two carbon atoms (propionate, butyrate, etc.) which can not be directly utilized by methanogenic bacteria. A third group of bacteria must exist as a bridge between the acidogenic and the methanogenic microbes. These obligate hydrogen-producing bacteria, acetogens, are able to convert the chemical compounds (ethanol, propionate, butyrate, etc.) that the methanogens cannot utilize to acetate, C 0 2 and H 2 . C H 3 C H 2 O H + H 2 0 \u00E2\u0080\u0094-> C H 3 C O O - + H+ + 2 H 2 ( A G 0 = +9.6 kJ) C H 3 C H 2 C O O - + 3 H 2 0 \u00E2\u0080\u0094> C H 3 C O O - + H+ + 3 H 2 + H C O J (Ac7\u00C2\u00B0 = +76.1 kJ) Chapter 2. Theoretical Aspects and Previous Studies 29 C H 3 C H 2 C H 2 C O O - + 2 H 2 0 \u00E2\u0080\u0094> 2 C H 3 C O O - + H+ + 2 H 2 ( A G 0 = +48.1 kJ) Thermodynamically, however, these reactions are unfavorable under standard conditions ( A G 0 > 0) and move towards the right only under a very low partial pressure of hydrogen (below 10~4 atmospheres) [72]. This can be accomplished by the action of hydrogen-trophic bacteria such as methanogens or sulfate-reducing bacteria. Thus, the acetogens and methanogens constitute a symbiotic association; the former needs the latter to re-move H 2 to create a favorable environment and the latter can obtain their substrates (acetate, H 2 / C 0 2 ) from the metabolic products of the former. This advance in the knowledge of anaerobic digestion goes back to the classical studies of Bryant, Wolin and Wolfe [73] who reported in 1967 that \"Methanobacillus omelianskii''\ a species capable of producing methane from ethanol, was actually a symbiotic association of two different species. Microscopic observations suggested that M. omelianskii consisted of two organ-isms of different shape. When the organisms were separated, one, a methanogen later named Methanobacterium bryantii, grew on hydrogen and carbon dioxide but no other substrates. The other species, S organism oxidized ethanol to acetate and hydrogen. The electrons generated from this oxidation were transferred to a pyridine nucleotide carrier ( N A D + ) and ultimately used to reduce protons to molecular hydrogen. The sig-nificance of this study is their speculation that alcohols other than methanol and fatty acids other than acetate and formate are not catabobzed by methanogens, but by an-other group of nonmethanogenic bacteria. Later, Mclnerney et al (1981) [75] obtained Syntrophomonas wolfei, an anaerobic butyrate-oxidizing bacterium in co-culture with either a hydrogenotrophic sulfate reducer or a hydrogenotrophic methanogen. A syn-trophic association of an anaerobic propionate oxidizer in co-culture with a hydrogen-trophic organism was reported by Boone and Bryant (1981) [76]. Table 2.5 lists the currently known mesophilic acetogens. Chapter 2. Theoretical Aspects and Previous Studies 30 Table 2.5: Currently known mesophilic obligate proton-reducing bacteria Organisms Co-culture with Habitat Substrate Syntrophobacter Desulfovibrio Gil or digester propionic acid wolinii M. hungatei 4 - D. Gil Syntrophomonas Desulfovibrio Gil, digester, monocarboxylic saturated wolfei M. hungatei sediments fatty acids (14-8 C) Syntrophus Desulfovibrio Gil, digester, Benzoic acid buwellii M. hungatei + D. Gil sediments Clostridium Desulfovibri E70, digester, monocarboxylic fatty bryantii M. hungatei M1H sediments acids (to 11 C) Strain Gra I val Desulfovibrio E70 marine isovaleric acid sediment Strain Go I val Desulfovibrio digester isovaleric acid Strain SF-1, NSF-2 M. hungatei or digester monocarboxylic saturated Desulfovibrio sp. fatty acids (4-6 C) Strain BZ-2 Desulfovibrio PS-1 digester benzoic acid Methanosporillum sp. Source is [74]. A direct cell count of co-cultures of Syntrophomonas wolfei with Methanospirillum hungatei fed with butyrate indicated that the former made up 30 per cent of the popula-tion versus the latter 70 per cent [78]. The growth rate of acetogens is very low as shown in Table 2.6 with or without sulfate-reducing bacteria (SRB) as co-culture. Table 2.6: Growth rate of obligate H 2 -producing actogens Organism Substrate p, (day l ) with SRB p (day l ) without SRB Reference Syntrophobacter wolinii propionate 0.192. 0.103 [76] Syntrophomonas wolfei butyrate 0.307 0.185 [75] Syntrophus buswillii benzoate 0.127 0.101 [77] Chapter 2. Theoretical Aspects and Previous Studies 31 Obviously, the sulfate-reducing bacteria can promote the growth of obhgate Re-producing acetogens because of a much faster removal of the hydrogen released at the expense of some energy and carbon source ( H 2 , acetate, etc.) consumed by sulfate-reducing bacteria. Hardly any information is presently available on the biochemical pathways in ace-togens, as pure cultures are not yet available. Tracer studies with enrichment cultures that converted propionate to methane and acetate [79] showed that the 1 4 C - l a b e l e d car-boxyl group exclusively appeared in carbon dioxide whereas the accumulated acetate was practically free of labeled carbon. Both [2- 1 4 C] and [3- 1 4C]-propionate lead to the production of radioactive acetate. The methyl group and the carboxyl group of the ac-etate produced were equally labeled, regardless of whether [2- 1 4 C] or [3- 1 4C]-propionate had been used. These observations suggested that propionate was degraded through a randomizing pathway. Of the three reactions mentioned above, propionate oxidation is thermodynamically the most unfavorable, and so, propionate is not easily digested in the methanogenesis phase and occurs in the effluent in quite high concentrations, especially when the acidogenesis phase is producing a large amount of propionate due to some disturbance caused by changes in load, p H , temperature etc.. The anaerobic oxidation of butyrate in S. wolfei has been postulated to go via 3-oxidation [75], but detailed labehng studies are lacking. However, the observation that the conversion of normal monocarboxylic saturated four to eight carbon fatty acids yields H 2 and acetate, or H 2 and acetate plus propionate is consistent with a /3-oxidation mechanism for the degradation of fatty acids by Syntrophomonas wolfei. Selective lysing of cells of Syntrophomonas wolfei recently allowed Mclnerney and co-workers [78] to demonstrate the presence of enzyme activities consistent with a /3-oxidation pathway for butyrate degradation. Chapter 2. Theoretical Aspects and Previous Studies 32 2.2 Start-up of Anaerobic Biofilms The operational principle of anaerobic biofilm reactors is based on the fact that attach-ment of anaerobic bacteria which grow very slowly, to support surfaces prevents them from being washed out of the reactor. The startup of anaerobic biofilm reactors, however, is generally time consuming and often difficult partly because of the low growth rate of anaerobes, especially the methanogenic and acetogenic bacteria, and also due to the lack of understanding of the mechanisms underlying the process and the factors affecting the attachment of bacteria onto support surfaces. The present status of the understanding of the start-up of an anaerobic biofilm reactor is more experimental than theoretical [80]. 2.2.1 Mechanism of Build-up of Bacterial Biofilms Biofilm formation is the net result of depositional, metabohc and removal processes [81], including: 1. Cellular particles transport from bulk hquid to support surfaces. Bacteria, as particles (d = 1 pm) suspended in culture medium, can be transported to a surface by any one, or a combination of, the following mechanisms: motihty and chemotaxis; gravity; molecular diffusion (in laminar flow); and eddy diffusion (in turbulent flow). It is doubtful, in a nutrient-rich fermenter, that chemotaxis and gravity would play a significant role in transport of bacterial cells which have a density (1.05-1.1 g/cm 3 ) very close to that of the medium. A n d so, molecular dif-fusion and/or eddy diffusion may be the dominating mechanisms for cell particles transport in a laminar or turbulent flow situation [82,83]. The fluid flow velocity has two principal effects on the formation of biofilms; the shear stress which in-creases with increased velocity and a change in the flux of cells/nutrients across the boundary layer or viscous sub-layer which is compressed as the velocity increases. Chapter 2. Theoretical Aspects and Previous Studies 33 Moreover, the concentration of cells in the bulk aqueous phase may also play a role to some extent. Bryers and Characklis [86] reported that under turbulent flow the deposition rate increased by a factor of 4.5 for a 5 times increase in cell mass concentration. 2. Microbial adhesion. Once bacterial cells are close to or on the surface, they adhere reversibly and then irreversibly. Zobell (1943) [84] observed that bacterial attachment was of-ten reversible-that bacteria which appeared to have settled on a substratum could be washed off by a stream of water, and that firm attachment appeared to occur only after a bacterium had remained settled for several hours. The investigations of Marshall et al (1971) [85] supported this two-stage attachment theory, called 'reversible' and 'irreversible sorption' respectively. Reversible sorption is the initial stage of bacterial attachment when the bacterium, exhibiting Brownian motion, is only weakly held at the surface while irreversible sorption was a time- dependent stage, which is due to the synthesis of exocellular polymers bridging the bacterial and substratum surfaces. The fixed bacterial cell does not exhibit Brownian motion and is not removed by washing. 3. Colonization of the surface by growth of the organism and formation of a biofilm. After becoming firmly attached, the cells multiply and are joined by additional attaching cells, which leads to the formation of microcolonies. These isolated points of growth eventually unite to form a continuous layer of microbial mass, i.e. a biofilm. 4. Detachment of the microbial film due to shear force and sloughing. Chapter 2. Theoretical Aspects and Previous Studies 34 Generally, sloughing and/or biomass decay more likely occur in a thick biofilm due to lack of nutrients in deeper layers while in the period of startup of biofilm or of a thin biofilm, the removal caused by shear force may predominate [87]. A dynamic equilibrium is maintained when steps 3 and 4 are occurring at the same rate. The attachment of bacteria to sohd surfaces in aqueous solution is affected by many factors since it involves three components: the bacterial surface, the sohd substratum, and the surrounding liquid phase. A factor which can directly or indirectly affect one of them would play a significant role in the process of attachment. The influence of bacterial cell surface on attachment, for example, is reflected in the fact that the attach-ment depends upon the growth conditions of the cells, such as the medium components [85], the hmiting nutrients [88], the dilution rate in a continuous culture [89] and growth phase in batch culture [90]. Some factors can influence more than one component, such as electrolyte concentration [85], presence of dissolved substances which are adsorbed onto the surfaces modifying the surface properties [91], p H of the medium [92] as well as temperature [90]. Obviously, the intrinsic properties of the substratum and the bac-terial surface have significant influence on attachment. The physicochemical properties (surface charge, surface free energy) of a potential substratum have been imphcated in influencing attachment [93]. Recently, the hydrophobicity of bacterial cell surfaces has been studied [95,96] as an overall parameter for the measurement of the adhesion po-tential to sohd surfaces, because in low-ion-strength environments the microbial surface hydrophobicity and charges are of greater importance [97] while most previous studies on bacterial attachment were conducted in a marine environment [98]. Initial attempts were made to model the adhesion phenomenon on the basis of colloid stabihty theory ( D E L V O theory) since the small size, low derisity and net negative charge of bacterial cells are similar to those of colloidal particles. This approach has achieved Chapter 2. Theoretical Aspects and Previous Studies 35 some success in the explanation of general features and 'long range' interactions between cells and surfaces [99]. A n alternative phenomenological approach involving the study of free energies employs an analysis of the thermodynamic potential for cell adhesion on the basis of known interfacial tensions [100] and has also been in some measure successful in modeling cell-surface contact events in terms of decreases in surface free energies resulting from adhesion [102]. The weakness of the physico-chemical models lies in their failure to take account of biological reactions since the cells are treated as solid inert particles rather than as bving systems. Marshall [101] proposed a two-stage model based on experimental observations. The first stage involves cell deposition in which the bacterial cell is held a small but finite distance from the solid surface, and this situation is maintained by the existence of an energy barrier. The second stage occurs when the energy barrier is breached allowing intimate surface contact and permanent adhesion. Three bridging mechanisms have been suggested: short-range physico-chemical forces such as chemical bonds or hydrophobic bonding [100]; specialized bacterial holdfasts, such as pili, fimbriae; and 'polymer bridging' through the mediation of bacterial extracellular polymers. Since most bacteria have no obvious structures for attachment, it is widely believed that the existence or 'in situ' synthesis of extracellular adhesives is required for permanent adhesion, the polymer occurring as an acidic polysaccharide [103], glycopro-teins [104] etc. Polymer bridging was the mechanism proposed by Marshall for the time dependent second stage in which irreversible sorption awaits polymer synthesis. How-ever, it has been observed that some bacteria have the ability to bind rapidly to surfaces \u00E2\u0080\u0094 this spontaneous binding suggests that bridging may depend on the compatibility of biopolymers (exuded prior to reversible adsorption) with the sohd surface. It is further observed that the initial, rapid attachment of a marine pseudomonad was mediated by a compact acidic polysaccharide followed by synthesis of a 'second' fibrous polysaccharide responsible for biofilm consolidation [103]. Chapter 2. Theoretical Aspects and Previous Studies 36 Many bacteria (Gram-positive and Gram-negative) can produce layers of polysac-charide outside their cell wall (capsules or shmes). The degree of capsulation and the production of extracellular polysaccharides by a particular organism can be markedly influenced by the growth conditions. The nature of the linkage to the underlying wall structures remains unknown. However, the biosynthesis of several capsular polysac-charides is known to require the participation of lipid intermediates [105]. In terms of structure, capsules and shme layers are in general highly hydrated. Capsules of Klebsiella aerogenes strains A l and A3 were reported to give a viscous solution containing only 1 to 2 per cent polysaccharide material [106]. Therefore, a bacterial cell can be imagined as a small ball around which a layer of hydrated adhesive exists. 2.2.2 Startup of Methanogenic Biofilms In the anaerobic treatment of cheese whey, the methanogenic and acetogenic bacteria grow much more slowly than do acidogens. Therefore, the start-up of the methanogenic biofilm is more time consuming than that of acidogenic biofilm. Moreover, in the methanogenic phase a symbiotic community is involved which includes methanogens and acetogens. It is essential to know whether the properties of support surfaces would affect the attachment of each bacterial type \u00E2\u0080\u0094 that is whether or not the attached biomass would have the same bacterial composition as that in the bulk culture medium. Shapiro and Switzenbaum [107] investigated the initial anaerobic biofilm development in a mixed culture fed with sucrose. The methanogen accumulation was monitored by F 4 2 o fluorescence content of the biofilm material. The ratio of methanogens to total anaerobic biomass was found to remain constant throughout 25 days for high organic space loadings. Longer solid residence time (SRT), higher concentrations of anaerobic microorganisms and higher organic space loadings were beneficial to the startup of anaerobic biofilms. Many investigations have been conducted on the effect of support surface on startup Chapter 2. Theoretical Aspects and Previous Studies 37 of methanogenic biofilms. Murray and van den Berg [43] studied the development of methanogenic fixed films on pieces of polyvinyl chloride ( P V C ) plastic, etched glass and baked clay and the results showed that support material markedly affected the rate of attachment and growth of bacteria converting acetic acid to methane. Scanning electron micrographs showed that the film of bacteria attached to clay was thick and uniform, while the bacterial film attached to P V C plastic was thin but still uniform. Attachment to etched glass was spotty. That the clay was superior was attributed to its rough porous surface and the presence of minerals in it. particularly iron which is known to stimulate methanogenesis and growth. More supports were tested by van den Berg and co-workers [109]. Comparing methane production rates of anaerobic biofilms on four supports, needle punched polyester, red draintile clay, gray potters clay and P V C , they found that needle punched polyester was one of the most effective in developing an active biomass film even though it could not offer the microbes extra nutrients like clay. A series of packing materials, non porous and porous, were compared by Huysman et al [110] in a mixed anaerobic biofilm reactor in order to elucidate the most impor-tant factors for biofilm formation. Their results indicate that surface roughness and total porosity plus pore size have the largest effect on the colonization velocity, and a reticulated polyurethane foam appeared an excellent colonization matrix. However, the study of attachment of methanogenic bacteria to solid supports with various porosities performed by Kuroda et al [40] revealed that methanogens adhered to moderately rough surfaces that had pores measuring a few tenths of a micron in diameter more than to pol-ished and rough surfaces, and also preferably adhered to solid supports made of carbon material. Obviously, the roughness of a support surface is playing a significant role in the development of biofilms because a rough surface either offers more area for microbes to attack or entrap the fixed biomass by preventing them from being detached by shear stress of fluid flow. In this sense, the porosity of a surface is a common factor to all Chapter 2. Theoretical Aspects and Previous Studies 38 bacterial attachment (not only beneficial to methanogens and acetogens). Furthermore, it should be controUed because in thick biomass entrapped in the channels of supports (e.g. foam) the resistance to mass transfer would become significant, resulting in more dead volume in digesters. A fundamental study was conducted by Verrier and Albagnac [111] to investigate adhesion of methanogenic bacteria onto inert sohd surfaces, P V C and glass shdes. Again the experiments showed that bacterial attachment on glass shdes was very spotty, and remained always low, while it was higher and increased constantly with time on P V C shdes. It was calculated that 7 x l 0 3 bacteria adhered per m m 2 of P V C per day. The predominant bacteria were filaments with septa and irregular surfaces tentatively iden-tified to be Methanothrix soehngenii. A strong influence of calcium concentration on bacterial adhesion was observed with an optimal effect at about 2 m M of C a + + . Above this concentration a negative effect appeared due to bacterial aggregation. Sodium addi-tion was less effective. Verrier and co-workers conducted more research on the adhesion of methanogens onto surfaces [112]. This time, 4 pure methanogenic cultures were used to investigate their initial adhesion on polymeric surfaces with different hydrophobic-ities. Methanothrix soehngenii adhered preferentially to hydrophobic polymers while Methanospirillum hungatei preferred hydrophilic surfaces and Methanobrevibacter arbo-riphilicus adhered onto all the supports. These results suggest that initial adhesion may influence the start-up rate of anaerobic fixed film reactors and is a function of the sup-port hydrophobicity. It is interesting that Methanosarcina rnazei, an acetate-degrading species, did not adhere to any support even though Methanosarcina spp can always be found in large amounts in methanogenic biofilms, which may imply that some symbiotic function is involved when a mixed bacterial population attaches onto support surfaces. The importance of support materials on anaerobic biofilm formation was also pointed out by Switzenbaum et al [113], but their effort to enhance the rate of initial anaerobic Chapter 2. Theoretical Aspects and Previous Studies 39 biofilm accumulation by precoating support media with denitrifying bacteria biofilms (biological precoating) and various polymers (chemical precoating), failed. Recently, Wollersheim [42] investigated the adhesion and biofilm development of methanogenic biofilms on glass. The experiments carried out with an undefined cul-ture as well as with a propionate-degrading anaerobic culture revealed that the primary adhesion was a passive process while the following biofilm development was an active one, which was accompanied by excretion of polysaccharides. The primary adhesion of bacteria may be controlled by the hydrophobicity and the surface charge of the support-ing material. The adsorption of acetogenic and methanogenic bacteria to wood chips, studied by Moo-Young and co-workers [114], has been assessed in terms of adsorbed par-ticulate organic nitrogen (PON) representing a total amount of symbiotic biomass. It was found that the adsorbed P O N / m 2 of support could be related to the cell concentration in solution by a Freundlich-type isotherm. It seems that the installation of the first bacterial layer may be of major importance for further biofilm growth and, stability. To this author's knowledge, however, no study has been conducted on the effect of support materials on the bacterial composition of an attached microbial biomass in a symbiotic bacterial community (methanogens plus acetogens) because of the difficulty in differentiating the specific substrate-degrading bacterial type from a mixed culture. 2.3 Mass Transfer in Anaerobic Biofilms Once an anaerobic biofilm forms on a support surface, two major phases exist in the fermenter, an aqueous medium phase and a concentrated biomass phase. The sequence of steps occurring during substrate utihzation by the biofilm can be simplified into two important steps: (1) substrates are transported from the bulk medium solution to the Chapter 2. Theoretical Aspects and Previous Studies 40 interface between the liquid and the biofilm (external mass transfer), (2) diffusion and consumption of substrates within the biological slime (internal mass transfer plus reac-tion). These two steps are in series, and so the slower one becomes the rate-limiting step of the overall process. The transport rate of substrates from the bulk solution to the film-fluid interface can be represented by the equation below N = kL(Sb-Ss) (2.1) where N is the rate (flux) of mass transfer (mg/sec/cm 2 ) , kr, is the mass transfer co-efficient (cm/sec), Sb and Se are the substrate concentrations in the bulk solution and at the biofilm surface (mg/cm 3 ) , respectively. If the mass transfer coefficient is so large that the external mass transfer rate is much faster than the rate of internal mass transfer plus reaction, at any particular value of 5b, the overall rate will depend only on the kinetics of internal mass transfer plus reaction. This hmiting regime has been termed kinetic regime, or reaction-controlled regime [115]. It is the only case in which the actual reaction kinetics of the biological film can be directly measured, provided the internal diffusional effects are reduced to neghgible level. Frequently, the external mass transfer coefficient, kj, is incorporated in the dimension-less Sherwood number, Sh, which is predicted as a function of Reynolds number, Re, and Schmidt number, Sc, characterizing hydrodynamic conditions and molecular diffusion, respectively, Sh = f(Re,Sc) (2.2) where Sh = k^djDi,] Re = ud/v; and Sc = u/Dr,. For creeping flow (Re < 1), the theory developed by Levich [116] shows that: Sh = 0 .99#e 1 / 3 Sc 1 / 3 (2.3) Chapter 2. Theoretical Aspects and Previous Studies 41 In the range 10 < Re < 104, Sh = 0 .95#e 1 / 2 Sc 1 / 3 (2.4) More empirical or semi-empirical equations can be found for other fluid patterns [117]. Use of stagnant film theory, i.e., estimation of a liquid/solid mass transfer coefficient as the quotient of a diffusivity and diffusion layer thickness (fcf, = DW/6W) is an alternative method which gives a lower bound for ki when Re number is less than 0.01 [118]. The stagnant diffusion layer can be taken, for example, to be the ratio of the liquid volume to media surface area. Obviously, the external mass transfer coefficient is markedly affected by fluid flow pattern in a fermenter. Experimentally, the external mass transfer resistance can be minimized or eliminated by increasing the fluid velocity over the biofilm [119]. 2.3.1 Structure of Anaerobic Biofilms By using light, U V , scanning and transmission electron microscopy, Robinson et al [120] examined the structure of anaerobic biofilms sampled from eight anaerobic fixed-film digesters fed with swine waste which had been in operation for 12 months at the Univer-sity of Florida Swine Research Unit. Although the biofilm samples were collected from various support materials (different shapes of plastics and blocks of pine, cypress, oak), the biofilms did not differ significantly in microbial or overall appearance. The overall surface of the biofilms was rough and uneven. Protuberances resembling volcanoes were observed with openings of 100 to 500 /un in diameter with a smooth microbial lining. Cross-sections of biofilms revealed a high density of material containing a heterogeneous bacterial population toward the base of the biofilms. Various cell types were found in small, loosely packed congregates or microcolonies. Some microcolonies contain only one morphological type of coccus, but most microcolonies contained several different types of cells (perhaps symbiotic communities). Large aggregations of sarcina-containing cysts Chapter 2. Theoretical Aspects and Previous Studies 42 were the most prevalent microbe found in thin sections cut from the biofilms. Individual cells were enclosed by a dark-staining wall closely associated with an amorphous matrix material 180 nm thick that extended throughout the cyst. Cells on the periphery of the cyst were covered by a capsule-like material or by a honeycombed netword of dead cells. Transmission electron micrographs of thin sectioned biofilm samples fixed with ruthe-nium red, an electron dense marker which can stain the extracellular polysaccharides, revealed that at least three types of matrix material or glycocalyx existed in biofilms [121]. Around individual cells is glycocalyx type II wrhich is a flexible integral microcap-sule and glycocalyx III envelopes microcolonies containing morphologically similar sister cells. The type I, rigid peripheral glycocalyx, appears to be spatially extensive and pos-sesses interstices which have different cross sectional size. A clear demarcation was seen between the interstices and their boundary walls; suggesting that the interstices may be channels within the type I glycocalyx. Large vacant areas in some of the interstices were observed and it was attributed to dead cells which once occupied these areas and left their 'foot print' in the rigid matrix [98]. The glycocalyx also contain mineral precipitates containing Ca , P and a small amount of M g [120]. In a methanogenic biofilm on a needle-punched polyester support, the concentrations of C a and P in the extracellular matrix were 92 and 28 times higher, respectively, than in the cells [122]. In the sense of internal mass transfer, a biofilm may be described as an assemblage of bacterial cells, biological catalytic centers, that are distributed in an extracellular fibrous polysaccharide-containing matrix and connected by tortuous channels of various size as shown in Figure 2.7. Chapter 2. Theoretical Aspects and Previous Studies 43 Channel Embedded cell Figure 2.7: Structure of biofilms 2.3.2 Mass Transfer within Biofilms Because of the gelatinous character of biofilm matrix it is thought that convective trans-port contributes little to the movement of reactive constituents within biofilms and sub-strate molecules transport towards the inner layers of biofilms along the tortuous channels by a mechanism of diffusion. Simultaneously, they also transport, by some mechanisms (passive and/or active transfer etc.), into the embedded microbes (see Figure 2.7) where utihzation reactions occur. It should be pointed out here that this latter mass transfer is a part of the intrinsic kinetics of substrate utihzation by microorganisms because it involves some characteristic enzymes of the bacteria even though immobihzation of living cells may produce a layer of exocellular polymers (glycocalyx type II) around the cells which affects the mass transfer rate. It is almost impossible to determine the actual transfer mechanism and rate of diffusion within the tortuous pores of the biofilms, and therefore, some workers have defined an effective diffusivity De in such a way that the Chapter 2. Theoretical Aspects and Previous Studies 44 substrate molecules may be thought of as diffusing through an equivalent homogeneous medium as is done in the study of porous catalysts. N \u00E2\u0080\u0094 \u00E2\u0080\u0094De(dS/dx) (2.5) Unhke in homogeneous systems such as water solutions where the major diffusional resis-tance comes from the collision of solute molecules with water molecules, the diffusion of substrate molecules in biofilms will be further retarded because they must move through the gelatinous matrix. In addition to the factors which influence the diffusivity in pure water, D , such as temperature, molecule size and viscosity, the effective diffusivity, by its definition, must be influenced by the structures of biofilms. Therefore, investigation of internal mass transfer within a biofilm comes to be the measurement of the effective diffusivity, De, of substrate molecules in the biofilm. Effective diffusion coefficients of some substrates have been measured in aerobic mi-crobial aggregates (films, blocs) because the supply of oxygen to aerobes embedded in microbial aggregates is believed to be the rate-hmiting step. These are reported in several papers which have been reviewed by Libicki and coworkers (1988) [123]. In contrast, no report has been found by this writer on the internal mass transfer in fermentative biofilms. Actually, as discussed in Chapter 1, the knowledge of effective diffusion coefficients in fermentative biofilms and then, an optimum biofilm thickness, becomes essential when the anaerobic technology is apphed to industry where a long term stable and optimal operation is required. To evaluate the internal mass transfer parameter under a neghgible external transfer resistance, three approaches have been taken: (1) evaluation of intrinsic reaction rates from a separate experimental study or literature and the internal transfer rate by fit-ting them simultaneously to the reactor breakthrough data (indirect method) [124], (2) Chapter 2. Theoretical Aspects and Previous Studies 45 evaluation of internal mass transfer rate by measuring a concentration gradient through an active biofilm and coupling it with the flux of material into the biofilm [125], (3) evaluation of internal mass transfer rate from the flux across an inactive biofilm in a two chamber device (direct method) [127]. The biofilm is often killed by chemicals (mercuric chloride) or radiation (ultraviolet) [128]. In the first method by wrhich most diffusivities in active microbial aggregates have been measured, the effective diffusivities have been used as fitting parameters in substrate utilization models and it is likely that error would be introduced into their results if the reactor hydrodynamies was not exactly the same as predicted by their model. The second method is a direct method to measure effective diffusivities in active biofilms, but measuring a concentration profile within an active biofilm needs special probes which should be very small in size and able to detect the target molecules. This method has been used by Bungay and co-workers for oxygen diffu-sion into laboratory-grown [125] and actual trickling filter films [126]. The third method may be able to avoid the effects of non-ideal reactor deviations on the measurement of effective diffusivities because it focuses on the two sides of the target biofilm. But it usu-ally uses an 'inactive' biofilm to avoid the uncertainty caused by the kinetics of substrate utihzation. The measurements involve the concentration changes in the chambers with time after a pulse input is given to one chamber. Not only must it be certain that the biofilm is completely killed and so no reaction will occur [128] but also some factors of importance in a hving biofilm like gaseous products released in small bubbles, symbiotic relationship and interaction between active bacterial species are neglected. Furthermore, the process of deactivating a biofilm may introduce some changes in the structure of the biofilms. Table 2.7 summarizes some experimentally determined diffusivities in inactive biomass. Variability in these results is substantial even in this narrow sample of experiments. Chapter 2. Theoretical Aspects and Previous Studies 46 Table 2.7: Ratios of effective diffusivities measured in inactive biomass to diffusivities in water DJDW Biofilm preparation Reference Oxygen Glucose NHj NO; NO; Na+ 0.85 - 0.8-0.87 0.86 0.93-1.0 - filtered [127] 0.2-1.0 0.3-0.5 - - - - centrifuged, [128] pressed 1.2 0.15-1.2 0.7 - - - settled, filtered [129] - 0.6 - - - 0.6 grown in place [130] 0.3 - - - - - centrifuged [131] This may be due in part to the differences in biomass growth conditions but it is prob-ably also due to the different techniques that have been used to prepare the biofilms. For example, Onuma and Omura [129] have reported variance in film diffusivities with carbon to nitrogen ratio in the media in which the biomass was grown. Matson and Charackhs [128] grew mixed cultures of bacteria on glucose in completely mixed reac-tors, concentrated them by centrifugation, and formed them into films by spreading them onto a template with a spatula. The biofilm was then sandwiched between two membrane filters prior to its placement in the diffusion apparatus. It is possible that some artificial effects may be introduced by the process of preparing such a biofilm because the major difference in diffusivities in water and in hydrated biofilms is caused by the matrix made of exocellular polymers. In order to avoid the possible effects introduced by artificially treating biofilms, a nonreactive solute has been used to measure its effective diffusive permeability in intact microbial cell aggregates [123]. Because of the difficulties associated with the direct measurement of the effective diffusivity, most studies on the modehng of fixed-film reactors have used assumed values. Based on their previous work on prepared biofilms of aerobic, autotrophic, nitrifying bacteria [127], subsequent studies by Williamson, McCarty and their co-workers [132,133] Chapter 2. Theoretical Aspects and Previous Studies 47 have assumed effective diffusivities of 80 percent of the free values for a larger number of substances in various biofilms. The effective diffusivity of acetate in a methanogenic biofilm was also taken as 80 percent of the value in free water, based on that same work [134]. Harris and Hansford [135] assumed that the effective diffusivity of glucose was equal to the value in water because of the results of Atkinson and Daoud [1.36]. It is one purpose of the present study to measure the effective diffusivities of lactose in active acidogenic biofilms, and organic acids in active symbiotic methanogenic biofilms in a two-chamber device which can offer a more direct measurement. However, in situ measurement of effective diffusivities in an active biofilm needs firstly the knowledge of intrinsic kinetics of substrate utihzation by the biofilm. 2.4 Intrinsic Kinetics of Substrate Utilization in Anaerobic Biofilms The so-called intrinsic kinetics describe the relationship between the utihzation rates and substrate concentrations in biofilms without interference from external and internal mass transfer resistances. Thus, the focus is on the process by which the substrate molecules are transported into microbial cells and dissimilated in the cytoplasm of the cells rather than on the transport of substrates from bulk solution to individual cells. As discussed in Section 1, this process involves various enzymes, and each of them is responsible for the catalysis of a specific chemical reaction. Generally, the slowest reaction catalyzed by a key enzyme becomes the rate-limiting step and determines the overall rate of substrate utihzation. 2.4.1 Expression of Intrinsic Kinetics Michaehs and Menten proposed a theory of enzyme action. Enzyme E can bind to substrate 5, forming an enzyme-substrate complex (ES complex). Chapter 2. Theoretical Aspects and Previous Studies 48 E + S i ES P + E This ES complex breaks down to yield free enzyme and product (P). The and k2 are rate constants that define how fast the individual steps proceed. They further assumed that the ES complex does not easily reform from free enzyme and products, and more importantly, that the rate of formation of the ES complex was equal to its rate of degradation - that is, that the formation of the ES complex has reached a steady state. In this case, the concentration of the ES complex will be constant. Using these assumptions, Michaelis and Menten derived Equation 2.6, Ke = (k2+k_1)/ki, indicating the affinity of the substrate for the enzyme active site. This classical equation is identical to the empirical Monod equation which depicts microbial growth and has been used as a theoretical basis for the Monod equation since growth is the result of a number of enzymatically catalyzed biosynthetic steps. Another advantage of this mechanistic model is that modifying the reaction pathways can give rate equations describing some unusual phenomena such as substrate inhibition [137]. Cell growth and substrate utihzation are generally considered to be coupled reactions, i.e., substrate removal occurs because of cell growth. The proportionahty constant is the true growth yield, Yg. Intrinsic rates of biological reactions are generally expressed on the basis of a unit of biomass, e.g. the intrinsic rate of substrate removal is expressed as the mass of substrate removed per unit time per unit of biomass. The unit can be based on some biomass volumetric property, such as dry weight, carbon content, protein Chapter 2. Theoretical Aspects and Previous Studies 49 content. The substrate removal rate is related to the specific growth rate of microbes by r = p/Yg (2.7) The Monod expression for the specific growth rate is and so, rmax = p.max/Yg. These definitions assume that all substrate utihzation is channeled into cell synthesis and that cell maintenance needs are met by decay. Another approach would be to assume that a portion of the substrate was channeled directly into cell maintenance. Although there are differences in the fundamental mechanisms employed by the two models both yield the same result and can be considered to be equivalent [138]. 2.4.2 Elimination of External and Internal Mass Transfer Resistances In an active biofilm, reaction and diffusion are occurring at the same time, which creates various substrate concentration profiles within the film as illustrated in Figure 2.8. Figure 2.8 also illustrates that external mass transfer resistance, which is modeled with a diffusion layer, effects the substrate concentration at the biofilm surface. Having lower concentrations entering into the biofilm means that the reaction rate per unit biomass may dechne as the biomass is located further away from the outer biofilm surface. Indirect evidence for the importance of external mass transfer 'resistance has been obtained by observing changes in the reaction rate when the fluid velocity past a biofilm is changed. As introduced in the last section, the external mass transfer resistance can be minimized or eliminated by increasing the fluid velocity over the biofilm. By using a rotating annular reactor, LaMotta [119] found that the reaction rate increased until the velocity past the biofilm was around 0.8 m/s, but that thereafter it was constant. Chapter 2. Theoretical Aspects and Previous Studies 50 Figure 2.8: Illustration of substrate concentration profiles in biofilms Since the external mass transfer is in series with the diffusion plus reaction, whether the external mass transfer resistance is important and what velocity past the biofilm can ehminate the external mass transfer resistance depend upon the potential (intrinsic) reaction rates in the biofilm. The slower the intrinsic reaction rates, the more easily the external mass transfer resistance is minimized. In contrast to external mass transfer resistance, the internal mass transfer resistance is more difficult to ehminate because it occurs simultaneously with reaction. The dis-tribution of substrate concentration within the biofilm, as shown in Figure 2.8, depends on the relative rates of diffusion and intrinsic reaction. Fast diffusion with slow reaction gives a shallow substrate concentration profile while slow diffusion with fast consumption causes a deep profile where substrate concentration reaches zero at or before the support surface. A strategy, widely applied in reaction engineering to minimize the internal mass transfer resistance of porous catalysts, is to reduce the thickness of the biofilm so that Chapter 2. Theoretical Aspects and Previous Studies 51 the concentration drop in the biofilm is not substantial and can be represented by the concentration at the surface. Experimentally, this has been achieved by LaMotta [124] in aerobic utilization of glucose and by Shieh and Mulcahy [139] in biological denitrification with methanol as the organic carbon source. It should be noted that much faster intrinsic reactions were involved in these studies than in lactose and organic acid fermentations. Shieh and Mulcahy observed that for each feed concentration, a critical biofilm thickness existed, beyond which the observed denitrification rate, on a per unit biomass basis, decreased sharply. A n d then, the denitrification rates obtained from experimental runs in which the biofilm thickness was less than the critical value were used to determine the intrinsic kinetic parameters. To what extent a biofilm thickness should be reduced, obviously, depends upon the relative rates of diffusion and intrinsic reaction, and can only be determined by experiments. Therefore, an ideal support surface should give a uniform, thin biofilm for a relatively long period of time. Considering the effects of sup-port materials on the thickness of biofilms, as discussed in Section 2, smooth polyvinyl chloride ( P V C ) sheet may be a good support material for the studies of intrinsic kinetics of lactose and organic acid fermentations in anaerobic biofilms. 2.4.3 Kinetics of Acidogenesis and Methanogenesis Many laboratory experiments have been performed on the anaerobic methanation of whey or lactose to evaluate the performance of reactors using immobihzed bacteria [35,140,19, 18,17,33,32], but kinetic data of the process are virtually non-existent. Kisaahta [46] investigated mainly the reaction pathways of lactose acidogenesis as well as the effects of dilution rate and p H on the pathways in a suspended culture. But the growth rate expression for acidogenic bacteria was not satisfied due to considerable wall growth which would cause a more serious problem at high dilution rates. In contrast to lactose acidogenesis, more kinetic studies have been conducted on Chapter 2. Theoretical Aspects and Previous Studies 52 Table 2.8 : Growth constants of anaerobic cultures at 35 \u00C2\u00B0C Culture day\" 1 y* max g V S S / g C O D ' ' m m fl'max/Ymax g C O D / g V S S / d a y g C O D / 1 Acetic acid producing bacteria 2.0 0 .15 13 0.2 Methane producing bacteria 0.4 0.03 13 0.05 Combined culture 0.4 0.18 2 -Source is [144]. methanogenesis of organic acids. Lawrence and McCarty investigated the kinetics of methane production in cultures enriched by acetate, propionate and butyrate [142]. After a calculation was made that the specific utihzation rate of one acid was based only on the fraction of biomass which was responsible for the bioconversion of that acid, their results showed that the three bacterial groups responsible for degradation of three organic acids had the same specific growth rate, 0 . 3 7 - 0 . 4 ( d a y - 1 ) . In a suspended methanogenic culture stirred by bubbling of recycled gas, Lin et al [143] studied the kinetics of the methanogenesis of mixtures of organic acids, and expressed the Michaelis-Menten model as a function of the C O D concentration of total organic acids. Many results on the anaerobic treatment of wastewater in fixed film reactors can be found in a comprehensive review by Henze and Harremoes [144]. After reviewing the results of theoretical and experimental studies on various anaerobic systems, they proposed growth constants of anaerobic cultures as hsted in Table 2.8 . However, this author has found no report on intrinsic kinetics of lactose acidogenesis and methanogenesis of organic acids. In most of the kinetic studies, the researchers had made no effort to ehminate external and/or internal mass transfer resistance nor Chapter 2. Theoretical Aspects and Previous Studies 53 have they described clearly in their reports their handling of this problem even though microbial aggregates (biofilms, blocs) were involved in their investigations. If the possible effects of bacterial immobilization on the metabolic behavior of the attached microbes are considered, such as the exocellular glycocalyx around the wall of individual cells, it will further reduce the available information on intrinsic kinetics of lactose methanation by immobilized bacteria. From the,discussions on these topics; attachment of symbiotic communities of ace-togens and methanogens onto inert sohd supports; effective diffusivities of lactose in acidogenic biofilms and organic acids in methanogenic biofilms; intrinsic fermentation kinetics of lactose and organic acids by anaerobic biofilms, it can be seen that related information is available but no previous work has been conducted specifically on these topics. This study will concentrate on these topics and some fundamental investigations will be made which may be valuable for a better understanding of the complex process of lactose anaerobic methanation in fixed biofilms. Chapter 3 Experimental Conditions and Setup It has been indicated in Chapter 1 that this study, as an extension of studies of lactose acidogenesis in suspended cultures conducted by Kisaahta [46], will concentrate on lactose utihzation in two-phase anaerobic biofilms. Although a great deal of research has been carried out on anaerobic digestion of organic substrates, some problems still need more investigations which are crucial for an optimal design and operation of a process in which lactose is utihzed in two-phase anaerobic biofilm reactors. Among these, five have been selected as the present research objects: intrinsic kinetics of lactose utihzation and organic acids production in acidogenic biofilms, attachment and build-up of symbiotic methanogenic biofilms, intrinsic kinetics of organic acids dissimilation in methanogenic biofilms, lactose transfer in the acidogenic biofilms and organic acids transfer in the methanogenic biofilms. Mass transfer data are prerequisites for estimating and controlling the optimum biofilm thickness so that a stable operation can be achieved in a fermenter with growing biofilms. The intrinsic kinetics of substrate utihzation by anaerobic biofilms not only give fundamental knowledge on how fast the sugar and the acids can be digested by the responsible microbes embedded in the biofilms but also are needed to estimate the diffusion properties of substrates in hving biofilms. A l l the five research subtitles will involve different microbial species, substrates, cul-ture conditions and biofilm supports, which will be detailed in each of the following chapters. However, some common conditions and setups which will be used in all these 54 Chapter 3. Experimental Conditions and Setup 55 investigations are described in this chapter. 3.1 E x p e r i m e n t a l C o n d i t i o n s Experimental conditions mainly include culture temperature, culture p H , composition and concentration of growth nutrients. 3.1.1 C u l t u r e T e m p e r a t u r e Microbial growth rate, and thus substrate utihzation rate caused by the biological activ-ities of microorganisms, as with all chemical reactions, are a function of temperature. It has been found that in nature microorganisms can grow at temperatures below 0 \u00C2\u00B0C and above 93 \u00C2\u00B0C \u00E2\u0080\u0094 the primary requirement is for liquid water. According to their biological activities in different temperature ranges, bacteria can be classified into three groups, psychrophiles, mesophiles and thermophiles. There are three generalizations that can be made concerning the effect of temperature on growth rate as shown in Figure 3.9 [210]. First, growth only occurs over a 30 \u00C2\u00B0C range for any group of microorganisms; second, growth rate increases slowly with raising temperature until the maximum growth rate is reached; and third, at temperatures above the maximum, growth rate falls rapidly. In general, the maximum growth rate of each group of microorganisms increases with temperature. It can be seen in Figure 3.9 that the maximum substrate utihzation rates occur over a very narrow temperature range. Psychrophiles are able to grow at lower temperatures, but few of them are found in industrial practice due to their low biological activity, especially anaerobic microbes which have very low intrinsic growth rates. Ther-mophiles have attracted research interests recently in anaerobic treatment of wastewater because of their faster growth rates. Compared with mesophiles, however, they have two disadvantages, worse process stability and more energy is needed to maintain the Chapter 3. Experimental Conditions and Setup 56 2-i x E a. psychrophi le mesophile thermophile Temperature (degree) Figure 3.9: Illustration of temperature influence on growth rate of microbes. culture at a higher temperature. Cheese whey, when it is discharged from the plant, has a temperature which is suitable for mesophilic bacterial growth and little or no energy is needed to heat the culture. Moreover, in the temperature range of 32-38 \u00C2\u00B0 C , the mesophiles behave actively [144]. This study will adapt and use mesophilic bacteria at 35 \u00C2\u00B1 2 \u00C2\u00B0C to investigate the fermentation kinetics, mass transfer of lactose and organic acids in biofilms and build-up of methanogenic biofilms. 3.1.2 Culture pH The p H , a measure of the hydrogen ion (H+) activity, is particularly important as a parameter of microbial growth. Most microorganisms grow over a p H range of 3-4 units; this represents a 1000 to 10000 fold range of hydrogen ion concentration. However, they have an optimum biological activity around p H 6.5-7.5. Chapter 3. Experimental Conditions and Setup 57 The acidogens responsible for lactose fermentation grow in a quite wide p H range of 4-8. The studies conducted by Kisaalita et al [44] on the influence of culture p H on lactose acidogenesis revealed that the utilization rate of lactose was not significantly affected by p H in the p H range of 4-7 and the p H had a great influence on the distribution of the acids producted by the fermentation. A low p H value can not only depress the production of propionic acid which is the product most difficultly digested in the following methanogenic phase [145] but can also save caustic solution which is needed for maintaining the culture p H at a higher value due to the accumulation of acid products of lactose acidogenesis. Although the methane-producing bacteria have been found in alkaline (pH 9.7) lake sediments [146] and in acidic peat (pH 3.6) [147] , the favorable p H range of methanogenic bacteria in a digester is narrow, 6-8, as proposed by Zehnder et al [148]. In their in-vestigations on methane production from organic acids in immobilized cell bioreactors, Scharer and co-workers [114] found a narrower optimum p H range of 6.5-7.5 over which no significant differences in the rate of methane production occurred. In this study, the p H value of lactose acidogenesis will be controlled at 4.6 \u00C2\u00B1 0.2 and the p H value for methanogenesis of organic acids at 7.1 \u00C2\u00B1 0.2 . 3.1.3 Growth Nutrients In this study, the utihzation of lactose and organic acids in anaerobic biofilms were investigated separately. Two types of medium were used which contained lactose or organic acids respectively. It is desirable that only the carbon source, lactose or organic acids, is the growth limiting component in the culture medium. In addition to the carbon source, lactose or organic acids, the bacteria also need N , P, S, as well as trace elements for their growth. Ammonium is the preferred nitrogen source for methanogenic bacteria [149], although for one species of Methanobacterium it could be replaced by glutamine [150]. Nitrate is not a suitable nitrogen source for methanogens. Chapter 3. Experimental Conditions and Setup 58 In addition, it increases the redox potential of the media and may result in overgrowth of enrichment cultures by nitrate-reducing bacteria, van den Berg and Lentz [151] found a suitable C O D / N (or C / N ) ratio to be 420/7 (or 158/7) and a good N / P ratio can be considered to be 7 [152]. Sulfide has often been used as both sulphur source and reducing agent since the redox potential of the media for methanogenesis should be reduced to E h < -200 mv, but in high concentrations sulfide may inhibit growth [153]. Theoretically, it is beheved that enrichment media must usually be free of sulfate because methanogens may otherwise be outcompeted by sulfate reducing bacteria at least for the most common substrates H2 and acetate. However, assimilatory sulfate reduction was also described for methanogens by Daniels et al [154]. A n d the concentration of sulfide produced from the reduction of sulfate would not be high enough to inhibit bacterial growth. Sulfates of M g , Fe, Zn and C u added to the medium supply both the metal ions and sulphur. For mixed cultures used for acidogenic and methanogenic bacteria, a ratio of C : N : P (158:7:1) was required for balanced biological growth [152,155,156]. But little work has been done on the growth requirements for single phase systems. Into the lactose acido-genesis medium having the same C : N : P ratio as mentioned above, Kisaahta [46] added, as growth nutrients for the acidogens, the same inorganic salts which were used by Mc-Carty [157] and Speece et al [158] in their mixed cultures. Concentrations of calcium and sodium were kept at 100 - 200 mg/1 while potassium and magnesium concentrations were at 200-250 mg/1. It was beheved that under such concentrations of growth nutrients the carbon source (lactose) was the only growth hmiting component. It would be ex-pected that the inorganic salts which satisfy the growth requirements of mixed cultures of acidogenesis and methanogenesis should also supply enough nutrients to a separated culture, acidogenesis or methanogenesis. Table 3.9 hsts the chemical components and their concentrations used in this study which are similar to those used by Kisaahta. The concentrations of inorganic salts are based on a lactose concentration of 12 g/1 Chapter 3. Experimental Conditions and Setup 59 Table 3.9: Chemical components and concentrations in culture medium Component Concentration (g/1) Substrates Lactose 12 H A c : H P r : H B u 6 : 3 : 3 Macro elements N H 4 C 1 0.42 ( N H 4 ) 2 H P 0 4 0.8 M g S 0 4 . 7 H 2 0 0.35 KC1 0.7 C a C l 2 . 2 H 2 0 0.5 Micro elements F e ( N H 4 ) 2 S 0 4 0.2 Z n S 0 4 . 7 H 2 0 0.005 M n C l 2 . 4 H 2 0 0.005 C u S 0 4 . 5 H 2 0 0.002 N a B 4 O 2 . 1 0 H 2 O 0.002 N a M o 0 4 . 2 H 2 0 0.002 for lactose acidogenesis or a mixed solution of organic acids for organic acid methano-genesis where acetic acid is 6 g/1, propionic acid 3 g/1 and butyric acid 3 g/1. If the substrate concentrations were changed, the concentrations of the nutrients were also ad-justed correspondingly. No growth factors (vitamins, yeast extract etc.) were added to this chemically defined medium. Al l the chemicals were reagent grade and supphed by B D H unless otherwise indicated. Table 3.10 summerizes the experimental conditions used in each type of fermentation. 3.2 Experimental Setups Experimental setups include the general experimental setup, the configuration and opera-tion properties of the reactor, and the biofilm supports used in three types of experiments. Chapter 3. Experimental Conditions and Setup 60 Table 3.10: Comparison of experimental conditions for each phase Phase Substrate p H Temp. p H adj. soln. experiments Acidogenesis Lactose 46 35 \u00C2\u00B0 C K O H + N a O H kinetics mass transfer Methanogenesis H A c , H P r , H B u 7.1 35 \u00C2\u00B0C HC1 kinetics mass transfer build-up of biofilms 3.2.1 G e n e r a l Se tup A reactor and some auxiliary facilities are needed to keep the bacteria, acidogens or methanogens, in a defined environment and to investigate their biological behavior in the environment. Figure 3.10 illustrates the general setup which allowed the control of culture temperature, p H , feed, recycle rate etc. needed for this study. Lactose (or salts of organic acids) was mixed with the inorganic nutrient solutions (see Table 3.9) and diluted to a desired concentration with tap water. The feed was stored in the feed tank and cooled to 1-4 \u00C2\u00B0C by a refrigerated circulating bath ( N E S L A B ) . Nitrogen was bubbhng through the feed to maintain a positive oxygen-free atmosphere in the tank, which is necessary for anaerobic microbes, especially for the strict anaerobic methanogens. A Masterflex pump ( C O L E P A R M E R ) recycled the feed between the feed tank and the head tank, mixing the feed and maintaining a constant hquid head over the reactor to give a stable feed flow rate. The feed flow rates were controlled with a micro-valve and monitored with a rotameter at high flow rates or with a break tube at low flow rates by counting the feed drops over a period of time. The break tube also prevented contamination of the feed in the tanks by the microbes in the fermenter. The desired p H values of the cultures were controlled by a p H / p u m p system (Cole Parmer Series 7142). As p H adjusting solutions, an equimolar K O H and N a O H solution Figure 3.10: Illustration of general experimental setup. 1-feed tank, 2-pump, 3-head tank, 4-microvalve, 5-rotameter, 6-break tube, 7-reactor, 8-biofilm support, 9-refriger-ated bath, 10-heat exchanger, 11-pump, 12-thermostat bath, 13-pH controUer, 14-pH adjusting solution, 15-gas collector, 16-thermoregulator Chapter 3. Experimental Conditions and Setup 62 (total concentrations 0.5-2 N) was used for lactose fermentation and a HC1 solution (concentrations 0.3-1 N) for organic acids fermentation. The culture temperature was maintained at 35 \u00C2\u00B1 2 \u00C2\u00B0C by heating the recycled culture medium with hot water from a ultra thermostat ( C O L O R A ) . The temperature of the thermostat was in turn adjusted according to the culture temperature by the thermoregulator inserted in the reactor. The production rates (volume/time) of gaseous products from lactose fermentation ( C 0 2 , H 2 etc.) and from organic acids fermentation ( C H 4 , C 0 2 etc.) were measured with the gas collector. The two liquid surfaces in the collector were kept at almost the same level when the gas was collected. A layer of oil was added on the liquid surface in the scaled tube to prevent the soluble gaseous components (e.g. C 0 2 ) from absorbing into the water. 3.2.2 Reactor For studies on anaerobic biofilms, the properties of biofilms must be known during the experiments. A common practice is to measure a biofilm on a removable support slide which is identical to the biofilm supports immersed in a reactor. Unhke investigations on aerobic biofilms where a reactor is open to air and a biofilm which is attached to a removable slide can be repeatedly measured, anaerobic biofilms on a shde can not be put back into the reactor after it has been exposed to the atmosphere for a long time. Therefore it is required that the reactor can hold enough sampling slides for multiple measurements. The reactor is made of a half Plexiglass tube as shown in Figure 3.11. 35 sampling ports could be arranged on the large surface area of the top and the curved bottom minimized the accumulation of bacterial sludge in the reactor. The removable shdes were fixed in slots cut in rubber stoppers which in turn were fixed in the samphng ports in the reactor cover as shown in Figure 3.12. Recycling the culture medium by a Masterflex pump ( C O L E P A R M E R ) is another Chapter 3. Experimental Conditions and Setup 63 Figure 3.11: Illustration of reactor configuration rubber stopper slide i Figure 3.12: A removable sampling slide Chapter 3. Experimental Conditions and Setup 64 significant characteristic of this reactor, which offers a means for controlling the culture temperature by heating the recycled medium. More importantly, it was possible to reduce and even ehminate the influence of external mass transfer on the substrate utihzation rates by increasing the recychng rate, i.e. increasing the linear velocity over the biofilms without changing the feed rate. This is a critical requirement for the investigation of the intrinsic kinetics of substrate digestion in biofilms and will be explained in detail in following chapters which describe the experiments. Another important advantage of the recycle flow is that the reactor behavior approaches, and can be modelled as, a continuous stirred tank reactor ( C S T R ) . For the ideal C S T R reactor model, the concentration of substrate (temperature etc.) in the reactor is uniform and is equal to the concentration in the effluent, which greatly reduces the complexity of modehng the reactor. Generally, when the ratio of recycle rate to the fresh feed rate is above 10, the reactor can be approximated as a C S T R . A pulse test was used to confirm this behavior. In a continuous completely mixed reactor, the concentration change of tracer in the effluent with the time after a pulse input of the tracer can be described as foUows: where Co is the concentration at the beginning of measurement, V the reactor volume and F the constant flow rate. Equation 3.9 can be rewritten as, HC1 solution was used as the tracer in a flow of tap water and the p H change in the effluent should have a hnear relationship with time if the reactor could be taken as a C S T R at a high enough recycle ratio. C = C 0 e - t / ( v / F ) (3.9) - l o g C = ( l / / J P ) ( l o g e ) < - l o g C 7 0 (3.10) Chapter 3. Experimental Conditions and Setup 65 - \og[H+] = (V/F)(\og e)t - \og[H+}0 (3.11) and, pH = At - B (3.12) where A, B are constants. At a fixed recycle rate, the flow rate of tap water was changed to test the influence of the recycle ratio on mixing behavior in the reactor. Figure 3.13 indicates that as long as the recycle ratio was kept above 8, the reactor behaved like a CSTR. It was found in the kinetic tests (see Chapters 4 and 6) that the recycle rate had to be kept above 14 1/hr to minimize the effect of external mass transfer resistance on substrate utihzation in biofilms, therefore, the feed flow rate had to be kept below 1.7 Chapter 3. Experimental Conditions and Setup 66 1/hr. This requirement was satisfied in all experiments. 3.3 Biofilm Supports In addition to a controlled environment, the microbes must also be given a sohd support on which to form biofilms, only then can the biofilms be investigated in detail; fermenta-tion kinetics of substrates in biofilms, mass transfer in biofilms and build-up of biofilms. Each of these themes requires a special biofilm support because of its different focus. Three kinds of supports were designed and used in this study. 3.3.1 Supports for Kinetic Studies The investigation of the intrinsic kinetics of lactose utihzation in acidogenic biofilms or methanogenesis of organic acids in methanogenic biofilms needs as much biofilm as pos-sible so that the biofilms contain most of the biomass in the reactor and the contribution from suspended microbes may be neglected. Intrinsic kinetic data can be obtained only after both the external and internal mass transfer resistance have been greatly reduced. Generally, the internal mass transfer resistance can be minimized by using a thin biofilm. Kennedy and Droste [159] used several materials as anaerobic biofilm supports, including needle-punched polyester, polyvinyl chloride sheet ( P V C ) , glass and two types of clay, and found that P V C supported biofilms gave a relatively low methane production rate due to small mass of the biofilm (a thin biofilm) and this low methane production rate was stable and could last up to 80 days. Obviously, it is desirable for a thin biofilm to be able to last a long time during the experiments. The biofilm supports used in kinetic studies of lactose or organic acids fermentation, therefore, were made of P V C sheets as shown in Figure 3.14. The material has other advantages as well such as no physiological effect on the bacteria [43] and ease of fabrication. The total support surface had an area Chapter 3. Experimental Conditions and Setup 67 Figure 3.14: PVC biofilm support for kinetic studies of lactose or organic acids fermen-tation of 1280 cm2. The picture (Figure 3.15) shows a section of a thin and uniform biofilm on the PVC support as seen under a microscope (NIKON). 3.3.2 Supports for Mass Transfer Studies In the investigation of substrate mass transfer in biofilms, attention was focussed on a part of a biofilm in the reactor and the measurement of substrate concentration difference on the two sides of the biofilm. The configuration of the biofilm support is shown in Figure 3.16. The reasons for this design will be given in Chapter 7 on mass transfer in biofilms. Since it was made from a section of Plexiglass tube, the device had a symmetrical structure. On each side a nitrocellulose membrane filter, which has no biological effect on the bacterial growth, was sandwiched by rubber gaskets and fixed by two stainless steel flanges, forming a closed volume of 30 ml. The porosity of the filters (d=0.45 pm) kept the microbes (generally, d = 1 /zm) from entering the device while the substrate molecules Chapter 3. Experimental Conditions and Setup 68 Figure 3.15: A thin biofilm (the black up-layer) on a P V C support, magnified by 100 times. pump Figure 3.16: A biofilm support device for mass transfer studies of lactose or organic acids in biofilms Chapter 3. Experimental Conditions and Setup 69 could enter it freely. When the device was immersed in a culture medium, some biofilm formed on its two membrane filters. A higher substrate concentration outside the device than inside the device caused a substrate transfer into the device by the mechanism of molecular diffusion through the biofilms and filters. The medium inside the device was recycled with a Masterflex pump ( C O L E P A R M E R ) to promote estabhshment of a uniform concentration inside it. At steady state, the concentrations of substrates outside and inside the device were measured and used to estimate the mass transfer properties of the substrates within the biofilms. In order to avoid convective mass transfer through the biofilms, the pressure as well as the hquid levels outside and inside the cell were kept the same. The hole on the top of the cell was not only to maintain a zero pressure difference but also to ehminate any mechanical disturbance to the filters caused by the recycle. 3.3.3 Supports for Studies of Biofilm Start-up Attachment and build-up of symbiotic methanogenic biofilms were investigated on 4 types of sohd materials; wood, ceramic Rasching rings, P V C and stainless steel. These materials have a wide range of surface wettability as can be seen in Table 3.11 and also are widely used in industry. Except for the ceramic rings, which are often used as packing material in separation towers and reactors to give a large surface area, the other three materials have the same shape, like microscope shdes, and so, they could be fixed on rubber stoppers as mentioned before. Suspending a ceramic ring in the culture medium as a removable support was done by hanging the ring on a hook which was made from a thin steel wire and fixed on a rubber stopper. After the surfaces were immersed in a culture medium, the accumulation of biologi-cally active materials on these inert sohd supports was measured and used to follow the build-up of biofilms. Chapter 3. Experimental Conditions and Setup 70 Table 3.11: Properties of biofilm supports for studies of biofilm formation Substratum Size Water contact angle mm X mm X mm Degrees Wood (fir) 15 x 35 x 2 0.0 Ceramic ring (010 - c/>6) x 9 56.6 P V C 15 x 35 x 1.5 88.0 Stainless steel 15 x 35 x 1 99.7 3.4 Experimental Analysis Many samples were collected during the experiments for analysis. The samples can be classified into three groups; sohd, liquid and gas, and hence different methods of analysis were needed, which are briefly described here while the details are given in Appendix B. 3.4.1 Solid Sample Analysis Sohd samples were taken of acidogenic or methanogenic biofilms. For the studies on substrate fermentation and mass transfer in anaerobic biofilms, the properties of the biofilms such as dry weight, total organic carbon content, density and thickness of the biofilms were needed. o Dry weight of a biofilm sample: A sample of biofilm attached on a P V C shde as described in Figure 3.12 or taken from a support was dried at 70 \u00C2\u00B0 C to a constant weight. The low temperature was used to avoid any disturbance from the plastic material. After the dried biomass with the shde or in a container was weighed, it was dissolved away in distilled water which had been acidified with sulfuric acid to p H 1. The cleaned and dried shde or container was weighed again. The difference of two weights was taken as the dry weight of the biofilm sample. Chapter 3. Experimental Conditions and Setup 71 \u00E2\u0080\u00A2 Total organic carbon of a biofilm sample: The total organic carbon of the dissolved biomass, the dry weight of which had been measured, was analyzed with a carbon analyzer ( A S T R O ) . Thus, the total organic carbon content in the biofilm sample was known directly. \u00E2\u0080\u00A2 Density of a biofilm: The density of a biofilm refers to the dry weight (or carbon content) of biomass in a unit volume of the biofilm. A n intact biofilm was scraped from a support surface and put into a glass tube (d=6 mm), and then, was centrifuged at 4000 rpm to have a constant total volume (height) consisting of a sohd volume at the bottom and a clean water volume above. After the total volume, V , was measured, the sohd biomass was dried for dry weight (Wd) analysis and the T O C content (M^oc) of this biofilm sample was measured. Pa = Wd/V (3.13) Ptoc = W t o e / V (3.14) o Thickness of a biofilm: This measurement is crucial to the measurement of diffusion properties of substrates (lactose and organic acids) in biofilms. The total biomass attached on a membrane filter of the device as shown in Figure 7.65 was carefully scraped into a container and dried to measure the dry weight and T O C content. Based on the area of the filter from which the biomass was collected and the density of the biofilm, the Chapter 3. Experimental Conditions and Setup 72 biofilm thickness could be estimated as following: PdA ptocA where Wd \u00E2\u0080\u0094 total dry weight of a biofilm, Pd = biofilm density based on dry weight of biomass, Wtoc \u00E2\u0080\u0094 total organic carbon content of a biofilm, Ptoc \u00E2\u0080\u0094 biofilm density based on organic carbon content of biomass, A = filter area Obviously, this thickness is an average value over the whole surface. 3.4.2 L i q u i d S a m p l e A n a l y s i s Liquid samples were collected in all three parts of the study (kinetics, mass transfer and biofilm build-up). Analysis of these samples was done with a spectrophotometer and a gas chromatograph (GC) . The components analyzed by the spectrophotometer included lactose and lactate and those by the G C were acetic, propionic, butyric acids and ethanol. o Lactose and lactate analysis: Lactose contains a reduced group which can give an orange-yellow color when treated with phenol and concentrated sulfuric acid. Lactate in a very diluted so-lution can be converted to acetaldehyde when heated in the presence of sulfuric acid. The acetaldehyde acting with p-hydroxydiphenyl gives a purple color. At a characteristic wave length, absorption of a monochromatic light is proportional to the color strength of a solution which is placed between the light source and a hght receiver. The more lactose (lactate) in a sample solution, the stronger the color produced, and thus the more hght that is absorbed. The concentration was Chapter 3. Experimental Conditions and Setup 73 found from a standard curve which was obtained by analyzing several samples with known concentrations. \u00E2\u0080\u00A2 Organic acids and ethanol analysis: The organic acids in their free form and ethanol are easily vaporized and can be analyzed with a G C ( C A R L E ) equipped with a flame-ion detector (FID). The samples were acidified to below p H 2.5 with a H 3 P 0 4 solution since acetic acid, the strongest acid compound, is in free form below this p H value. \u00E2\u0080\u00A2 Solid carbon content in a hquid sample: The sohd carbon content in a hquid sample is needed to determine the mass of free microbes in the culture medium. Half the volume of a hquid sample was analyzed with a carbon analyzer to find the original carbon content of the sample. The rest of the sample was filtered through a membrane filter (d = 0.45 pm) to remove the suspended microbes in the medium, and then the total carbon content of the filtrate was measured under the same instrument conditions. The difference in total carbon content of these two parts reflected the free microbe content in the liquid sample. 3.4.3 Gaseous sample analysis Gaseous samples were mainly collected during the studies of the methanogenesis of or-ganic acids ( C H 4 , CO2 etc.) while much less gas was produced from lactose acidogenesis (CO2 etc.). The concentrations of the gas components are needed when a carbon balance around the reactor is made. Therefore, only methane and carbon dioxide were analyzed by using a G C ( C A R L E ) equipped with a thermal conductivity detector ( T C D ) . Chapter 4 Intrinsic Kinetics of Lactose Utilization Anaerobic digestion of lactose in a two-phase process consists of acidogenesis and metha-nogenesis which are carried out in series reactors. In the acids-forming reactor, lactose is utihzed by acidogens with the main products of acetates, propionates and butyrates. Then, these intermediates are converted to methane and CO2 by the methanogens in the following methanogenic reactor. This chapter describes the intrinsic kinetics of lactose utihzation in acidogenic biofilms. 4.1 Experiments on Lactose Acidogenesis The general experimental setup is as shown in Figure 3.10. The feed consisted of lactose as the limiting organic substrate and inorganic salts as the stimulating nutrients which are listed in Table 3.9. The lactose concentration of the feed ranged from 5 to 10 g/1 in order to control its level in the fermenter with the experimental dilution rates used. This range of lactose and nutrient concentrations made lactose the only possible limiting substrate [46]. The methods used to keep the feed from fermenting in the storage tank include: \u00E2\u0080\u00A2 Low temperature, the medium was maintained in 1 to 4 \u00C2\u00B0 C in the tank; \u00E2\u0080\u00A2 Low oxygen partial pressure, nitrogen was slowly bubbled through the medium; \u00E2\u0080\u00A2 Small amount of feed was stored at one time, enough for only 4 to 5 days operation. 74 Chapter 4. Intrinsic Kinetics of Lactose Utilization 75 The feed concentration was determined from the first and final day sample analyses during the storage of 4 to 5 days. The fermenter had a working volume of 1,000 ml. The flow rate of feed, measured from the effluent collected over a period of time (3-8 hours), was controlled at above 150 ml/hr (D = 0.15 h r - 1 ) so that the suspended bacteria were washed from the reactor and this part of the biomass, compared with the fixed bacteria, could be neglected. Henze and Harremoes reviewed the hterature on anaerobic processes and reported a maximum specific growth rate of acetic acid producing bacteria at 35 \u00C2\u00B0C of 2 d a y - 1 (or D = 0.083 h r - 1 ) [144]. The carbon content of the solids in the effluent under these conditions was found to be neghgible when the carbon contents of both the effluent and their filtrate from a membrane filter (d = 0.45/im) were analyzed and compared by using a T O C analyzer ( A S T R O ) . This indicated that no significant amount of biomass was suspended in the reactor. The p H of the culture was controlled at 4 . 6 \u00C2\u00B1 0 . 2 with 0.5 to 2 N equimolar N a O H and K O H solutions. The caustic concentration was adjusted on the basis of lactose utihzation rates to produce a neghgible influence on the dilution rates. The inoculum was obtained from the first phase reactor of a two-phase mini-plant which had been fed with cheese whey for two years. The experiment can be divided into four stages. 1. Adaptation; The fermenter was filled with a diluted lactose solution (2 g/1) and N 2 was continuously introduced to strip the dissolved oxygen. When the temperature reached 35 \u00C2\u00B0C, the fermenter was seeded (200 ml inoculum) and then operated in batch mode. After lactose had been used up, < 0.01 g/1, half the volume of the culture (500 ml) was replaced by fresh feed (4 g/1 lactose) and this was repeated five times. Chapter 4. Intrinsic Kinetics of Lactose Utilization 76 2. Build-up of biofilm; The operation, then, was shifted to continuous mode while the flow rate was controlled at less than 50 ml/hr to prevent the suspended bacteria from washout. It was observed that a white slimy biofilm gradually started to form on the grey P V C supports. 3. Steady state operation; After the build-up of biofilms, the flow rate was gradually increased to greater than 150 ml/hr. The temperature was maintained at 35 \u00C2\u00B0C, and the p H of the medium at 4.6. To investigate the influence of lactose concen-tration on the rates of lactose utilization and organic acid production, the lactose concentration in the reactor was controlled by adjusting either the feed flow rate (residence time) or the feed concentration, sometimes both of them. These two con-trol modes had the same effect in a reactor in which the bacteria were immobilized. A dynamic response caused by a change in flow rate is described and discussed in Section 4.5. At each desired concentration level, a steady state during which there was no significant lactose concentration change (< 10 %) in the effluent, was maintained more than 2 days to collect experimental data. The concentration was adjusted from a low to a high level , and after a set of desired levels had been investi-gated, the tests on the same set of levels were repeated. Since the biofilm thickness increased with time, the lactose utihzation rate at each of the concentration levels was obtained with acidogenic biofilms having different thickness. Therefore, by comparing the data, the influence of internal mass transfer resistance on lactose digestion rate could be estimated. 4. Influence of p H change; After the kinetic experiments had been completed, the cul-ture p H was gradually increased from 4.6 to 6.5 while the flowrate and temperature were maintained at 220 ml/hr and 35 \u00C2\u00B0 C , respectively. Chapter 4. Intrinsic Kinetics of Lactose UtiEzation 77 3-1 o CO 0 60 100 160 200 260 300 360 400 Recycle rate (ml/mln) Figure 4.17: External mass transfer resistance on lactose utilization in biofilms is tested by changing recycle rate. Before the intrinsic kinetic data could be collected, the influence of external mass transfer was tested by varying the recycle rate. Figure 4.17 shows that the lactose utihzation rate would not be affected by the external mass transfer resistance as long as the recycle rate was kept above 228 ml/min, corresponding to a hnear velocity over the biofilm of 3.3 cm/min. The influence of internal mass transfer is often reduced by using a thin biofilm which is available in the early stage of biofilm development. To extend the experimental time during which the effect of internal mass transfer resistance could be minimized even though the biofilms were getting thicker, the experiments were arranged in such a way that the concentration levels were adjusted from low to high for a test recycle. At higher bulk concentrations the internal concentrations were also relatively high, which would give all the bacterial cells embedded in a thick biofilm the maximum utihzation rate, Chapter 4. Intrinsic Kinetics of Lactose UtiEzation 78 3 0 0 0 n oH 1 1 1 1 1 1 1 1 1 0 10 20 30 40 60 60 70 80 90 Culture time (day) Figure 4.18: Lactose digestion rate of whole reactor changes with time C thereby, the influence of concentration distribution could be reduced. Figure 4.18 indicates that the lactose digestion rate based on the whole reactor grad-ually increased and then leveled off because of the gradually thickening biofilm, which means that more bacteria saw lower substrate concentration. Since the experiments were repeated at several concentration levels from low to high, it can be assumed that the kinetic data obtained on days 0 to 55 were not markedly affected by the internal mass transfer resistance. This becomes clear if the lactose digestion rates based on unit weight of biomass (mgCarbon) are plotted versus biofilm thickness (Figure 4.19). The experimental conditions are summarized in the Table 4.12. 4.2 Development of an Acidogenic Biofilm As discussed in the previous chapters, the presence of sohd supports helps the retention of biomass in a reactor, and so the total amount of biomass in the reactor will increase er 4. Intrinsic Kinetics of Lactose Utilization o.e-i 0.2 0.4 0.6 0.8 1 Biofilm thickness (mm) Figure 4.19: Specific lactose digestion rate changes with biofilm thickness Table 4.12: Experimental conditions of lactose acidogenesis Parameter Condition Temperature p H Lactose feed Flow rate Dilution rate Biomass Recycle rate 35 \u00C2\u00B1 2 \u00C2\u00B0C 4.6 \u00C2\u00B1 0.2 5-10 g/1 > 150 ml/hr (2.5 ml/min) > 0.15 hr-1 acidogens > 230 ml/min Chapter 4. Intrinsic Kinetics of Lactose Utihzation 80 Figure 4.20: Biomass distribution in the fermenter slowly with the digestion of substrates. To estimate the lactose utihzation rate based on unit mass of biomass, it is essential to know the total mass of biomass retained in the reactor during the experimental time. Three types of biomass existed in the reactor as shown in Figure 4.20; \u00E2\u0080\u00A2 suspended bacteria, \u00E2\u0080\u00A2 attached bacteria on the supports, \u00E2\u0080\u00A2 sludge. The suspended bacteria, compared with the fixed ones, were negligible as mentioned above. The biomass in the biofilms was the major part of the total biomass retained in the fermenter, and measured during the experiments by analyzing the biomass on the removable slides at given time intervals. The time when the first kinetic data were Chapter 4. Intrinsic Kinetics of Lactose Utilization 81 160-1 140-\u00E2\u0080\u00A2 E 120-CO 0.103 38/5.6-7.2 anaerobic sludge /dextrose, tryptone [164] 0.0917 2.0 35/6.3-8.1 fixed films /dextrose, protein [165] 0.0417 0.15 35/6.3-8.1 fixed films /dextrose [165] 0.0367 0.25 20-35/> 6 anaerobic filter [166] /starch Chapter 4. Intrinsic Kinetics of Lactose Utihzation 88 literature on the influence of immobilization on the bacterial activity and concluded that so far neither experimental nor theoretical evidence exists that immobilization of cells directly affects their metabolism. For the phenomena of decreased substrate utilization, growth rate, assimilation etc. in a community of immobilized bacteria, various explanations existed such as less cell surface available for substrate uptake [171,172], higher maintenance coefficient [172], substrate transport limitation [173], and desorption limitation [174,175]. Around fixed cells quite a lot of extracellular polysaccharides have been found under the microscope, which connects i the cells forming a network of channels as described in Chapter 2 while on free cells there is less extracellular polysaccharides (only a thin capsule if any). Therefore, the mass transfer from outside to inside of cells, which is a part of intrinsic kinetic process, has more resistance due to the extra polysaccharides on the cell wall which is excreted by the bacteria during the immobilization of the free cells. Moreover, part of a cell surface may be unavailable for the mass transfer due to very close proximity between some cells. On the basis of activity measurements on attached and free living Vibriosp., Jeffrey and Paul [171] suggested that not only the maximum substrate consumption rate but also the apparent substrate affinity of attached cells are different from suspended cells. A decrease in substrate affinity or an increase in the half saturation constant, Ks, for adhered cells has often been reported [173,176,177]. According to Bright and Fletcher [176], there are two possible explanations: (1) the difference is due to changes in the environment of the cell, or (2) the higher K\u00E2\u0080\u009E values for surface-associated cells is a reflection of a real difference in assimilation behavior. Independent experimental evidence that the results of the present study are more reliable can be obtained from the experimental data of lactose transport through active acidogenic biofilms (see Chapter 7). By using Kisaahta's kinetic data and assuming that the diffusivity of lactose in free solution is its effective diffusivity within the biofilms, the Chapter 4. Intrinsic Kinetics of Lactose Utilization 89 lactose concentration found from the reaction-diffusion model very quickly dropped to less than 1 0 - 3 at a biofilm thickness 0.5 mm while the measured lactose concentration was about 50 mg/1 at a biofilm thickness 1.3 mm (Table 7.26). If Kisaahta's kinetic data and the measured lactose concentrations on the two sides of a biofilm were used to estimate the effective diffusivity of lactose in the biofilm, the lactose effective diffusivity was found to be in a range of 15 to 25 times higher than its value in free solution. However, the estimated lactose diffusivity by the kinetic data of the present study was quite reasonable, D e / D < 1. 4.4 Production of Organic Acids At the same time the lactose digestion rate was measured, the products were also analyzed and measured. The detectable products included acetate, propionate, butyrate, ethanol and lactate, the major ones being acetate and butyrate (see Appendix C) . Some organic acids with carbon number more than 4 (Ce, Cg) were also detected in transient processes (change in dilution rate, feed concentration etc. ), but at steady state they disappeared. Table 4.14 hsts the reactions of lactose being converted to those products and their standard free energy. Although the standard free energy of the reaction for butyrate formation is the least, butyrates were produced in large amounts in this experiment. This must have been affected by the experimental conditions. A carbon balance on conversion of lactose to these products ranged from 52.1 to 67.7 percent as shown in Appendix C , a reasonable range considering the fact that the carbon converted to CO 2 and to biomass had not been included in the balance. If carbon leaving the reactor in gaseous bubbles, or dissolved in the effluent was estimated according to the reactions in Table 4.14 (e.g. 1 mole of acetate accompanied with 1 mole of H C O j ) , the carbon balance was between 85 and 102 percent. Chapter 4. Intrinsic Kinetics of Lactose Utihzation 90 Table 4.14: Reactions of Lactose Conversion Reactions A G 0 (kJ) C 1 2 H 2 2 0 i i + 9H20 - \u00C2\u00BB 4 C H 3 C O O - + 4HCO3 + 8H+ + 8H2 -412.6 C 1 2 H 2 2 0 \u00E2\u0080\u009E + 4H2 -\u00C2\u00BB 4 C H 3 C H 2 C O O - + 3 H 2 0 + 4H+ -716.2 C 1 2 H 2 2 0 n + 5H20 - \u00C2\u00BB 2 C H 3 C H 2 C H 2 C O O - + 4 H C 0 3 + 6H+ + 4H2 -50.92 C i 2 H 2 2 0 n + 5H20 - \u00C2\u00BB 4 C 2 H 5 O H + 4 H C 0 3 + 4H+ -451.8 C i 2 H 2 2 Q \u00E2\u0080\u009E + H 2 0 -+ 4 C H 3 C H O H C O Q - + 4H+ -396.6 Source is [156]. Similar to the utilization of lactose, each of the products was balanced around the reactor at a steady state. Produced from the reactions + Flow in = Flow out The 'flow in ' amount was zero because no digestion of lactose occurred in the feed tank under the measures mentioned before. So, for the ith component, at steady state, Production rate = Flow out that is, TiW = Fd (4.22) or, r< = FCJW (4.23) Where r{ is the production rate of the ith component based on the unit weight of carbon content of biomass (mg i / m g C / h r ) , F the feed flow rate (ml/hr), Ci the concen-tration of the component in the effluent (mg i/1) and W the total amount of biomass carbon content within the reactor at that time. The right side of Equation 4.23 can be calculated from the experimental data of F, Ci and W, and thus the production rate of the ith product can be found. Chapter 4. Intrinsic Kinetics of Lactose Utilization 91 0 . 1 2 ^ 0 . 1 0 -o E D) 0 . 0 8 -co \u00C2\u00AB 0 . 0 6 -k_ O) c ff 0 . 0 4 -o \" 0 . 0 2 < 0 . 0 0 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 T ? 1 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 Lacto$e concentration (mg/l) 6 0 0 0 Figure 4.25: Influence of lactose concentration on acetate production rate. The hne is calculated from Equation 4.24 and the parameter values in Table 4.15 As discussed in Chapter 2, lactose fermentation proceeds via the Embden-Meyerhof pathway and pyruvate is the key intermediate from which other products are formed. The rates of these parallel reactions were dependent on the concentration of pyruvate and thus lactose concentration. The dependence of the production rate of each product on lactose concentration is shown in Figures 4.25,4.26,4.27, 4.28,4.29. The products can be divided into two groups according to the pattern of influence of lactose concentration on their production rates; acetate, butyrate and ethanol one group (Figures 4.25, 4.26, 4.27), lactate and propionate another (Figures 4.28, 4.29). The first group can be described with a Michaelis-Menten type equation; Where rmaa.t- is the maximum production rate of the ith component (acetate, butyrate and Chapter 4. Intrinsic Kinetics of Lactose Utilization 92 Figure 4.26: Influence of lactose concentration on butyrate production rate. The hne is calculated from Equation 4.24 and the parameter values in Table 4.15 0.014 0.000 1000 2000 3000 4000 Lactose concentration (mg/l) 6000 Figure 4.27: Influence of lactose concentration on ethanol production rate, calculated from Equation 4.24 and the parameter values in Table 4.15 The hne is Chapter 4. Intrinsic Kinetics of Lactose Utihzation 93 0.010 -1 Figure 4.28: Influence of lactose concentration on propionate production rate. The hne is calculated from Equation 4.25 and the parameter value in Table 4.15 0.014-, o 0 .012-6 0 0 0 Lactose concentrat ion (mg/l) Figure 4.29: Influence of lactose concentration on lactate production rate. The hne is calculated from Equation 4.25 and the parameter value in Table 4.15 Chapter 4. Intrinsic Kinetics of Lactose Utilization 94 Table 4.15: Rate parameters of the products in lactose fermentation Tmaxj KBti k{ F test F0.95 ( m g l / m g C / h r ) (mg s/1) I P \" 6 Acetate 0.0599 89 3129 2.1 Butyrate 0.115 532 1246 2.1 Ethanol 0.0101 929 556 2.1 Lactate 2.18 1383 2.1 Propionate 1.25 705 2.1 ethanol) (mg i / m g C / h r ) , S the lactose concentration (mg/1) and KB^ the half velocity concentration (mg/1). The two parameters, rmaXti and Ksi, were estimated with the same method as for lactose utihzation rate, i.e. by a linear least-square method after a transformation of Equation 4.24 to a hnear expression hke Equation 4.21, and then by the direct search method to optimize the fit of Equation 4.24 to the experimental data. The estimated values of parameters are listed in Table 4.15 The production rates of the second group, including propionate and lactate, has a hnear relationship with the lactose concentration. r{ = hS (4.25) Where k{ is the specific reaction rate (mg i /mgC/hr)(mg/l) _ 1 which is the production rate when lactose concentration is 1 mg/1. The estimated values of ki are also listed in Ta-ble 4.15. The statistical tests (F test) indicate that the influence of lactose concentration is significant on the yield rates of these 5 products. Cohen and co-workers [178] investigated acidogenesis of glucose in a suspended culture and found that propionate and butyrate were formed by at least two different species, and thus two types of fermentation occurred under different environments: \u00E2\u0080\u00A2 butyrate type fermentation; production of mainly butyrate, acetate, CO2, H2 and Chapter 4. Intrinsic Kinetics of Lactose Utihzation 95 low E h values, \u00E2\u0080\u00A2 propionate type fermentation; mainly propionate and acetate, minor amounts of valerate, low or no gas production consisting solely of CO2, relatively high E h values. Under the present experimental conditions as summarized in Table 4.12, the butyrate type fermentation predominated. Propionate +1/3 A T P Lactose i Glucose I + 2 A T P Lactate -2H +2H -4H Propionate j Pyruvate j A Succinate j + H : Ethanol -4H + A T P 8-Acetyl C o A \ -4H ' + A T P Butyrate + A T P Acetate Figure 4.30: Pathways of acidogenesis of lactose. Sign (-) refers to consumption, and (+) refers to production. The intermediates are in dash boxes and products in sohd boxes. Chapter 4. Intrinsic Kinetics of Lactose Utilization 96 Many reaction pathways are known which lead to a great variety of fermentation products for carbohydrate anaerobic digestion as reviewed in Chapter 2. Figure 4.30 gives a simple relationship between the substrate and the products as well as production of A T P and transfer of electrons/hydrogen by which the reduced co-enzyme, N A D , can be reoxidized. The possibility of producing various products is especially high in a mixed culture because the presence of so many bacterial species offers the potential to induce a great variety of enzymes which are needed for various reactions. It is well known that het-erotrophic bacteria degrade carbohydrate for energy and carbon which are necessary for their reproduction and life maintenance. The energy released from the chemical bonds of the substrate is stored in A T P , an energy-rich molecule. The more A T P a reaction pathway can yield, the more likely it is that it would predominate. The fact that only butyrate and acetate were the main products under the present conditions implied that their formation could give more energy to the bacteria than could the other pathways. Thauer and co-workers [179] have extensively reviewed energy conservation in chemo-trophic anaerobic bacteria. It seems that, in many anaerobic bacteria, the amount of A T P formed per mole of energy supplying substrate can be regulated, thus, permitting the organism to optimize the thermodynamic efficiency of energy transformation. The formation of lactate can only give 4 moles of A T P per mole of lactose utilized, because no energy is available from the conversion of pyruvate to lactate even though it accepts the electrons/hydrogen produced. Conversion of pyruvate, however, to acetyl-C o A , the primary energy-rich intermediate, can offer more A T P , which is obtained during its further conversion to butyrate and acetate, and so the bacteria prefer the butyrate type fermentation. Furthermore, acetate, butyrate and ethanol have the same precursor, acetyl-CoA. That may be why they have the same type of production rate equation since acetyl-Co A is formed from pyruvate via pyruvate:ferredoxin oxidoreductase or pyruvate Chapter 4. Intrinsic Kinetics of Lactose Utilization 97 formate lyase [179]. As with the formation of lactate, no extra A T P is produced during acetyl-Co A conversion to ethanol, and thus, the amount of ethanol, as a minor product, had the same level as lactate in the fermenter (Figures 4.27 4.29). 8 moles of A T P are produced per mole of lactose utilized via the pathway to acetate, 6 moles A T P to butyrate and 4 moles A T P to ethanol. The preference of bacteria on these pathways are also reflected in their affinity coefficients or half velocity concentrations, KeS 0.004 i \u00E2\u0080\u00A2J 0.002 CO 0.000 0.0 rbB: 0.1 0.2 0.3 Lactose -us ing rate (mg/mgC/hr) 0.4 Figure 4.35: Production rate of lactate versus digestion rate of lactose Chapter 4. Intrinsic Kinetics of Lactose Utihzation 101 Table 4.16: Comparison of production rates of organic acids at two p H levels Relative production rate P H Acetate Propionate Butyrate Lactate 4.6 1 1 1 1 6.6 1.08 17.16 1.15 0.97 4.5 Responses of Acidogenic Biofilms to Disturbances Immobilized bacterial communities are quite stable. A change in p H of the culture medium from 4.6 to 5.1 did not affect the lactose utihzation rate nor its product distri-bution. However, when the p H of the culture medium jumped up to 6.5 and stayed at this level, it affected not only the product distribution but also the biofilm itself. The amount of biofilm attached on the support was reduced from a density of 4.1 m g C / c m 2 to 2.4 m g C / c m 2 and it was observed that the biomass on the bottom of the reactor increased and a lot of foam was produced on the medium surface. So, a dramatic change in p H could make the environment very unfavorable to some bacterial species. Unhke a suspended culture where a bacterial species will be washed out quickly if its growth rate is less than the dilution rate, these bacteria are trapped in the biofilm so that their death and lysis caused the biofilm thinning and the medium foaming. The change in the p H of culture medium gradually affected the distribution of the bacterial species and/or their fermentation mechanism, which was reflected in the change of the yield rates of organic acids, especially propionate formation. Table 4.16 compares the production rates of acetic, propionic, butyric and lactic acids at two p H levels, where the production rates at the p H of 4.6 are taken as one unit for the ease of comparison. It is clear that the propionate production rate was increased considerably. Its con-centration in the reactor effluent jumped from about 30 mg/1 to 450 mg/1. This proves that low p H depresses the formation of propionate which is generally the least desirable Chapter 4. Intrinsic Kinetics of Lactose Utihzation 102 fermentative product for the following methane-producing bacteria. A n acidogenic reactor even under normal operation can become unstable because of load changes which are caused by fluctuations in feed concentration and/or flow rate. For a fixed film reactor these two factors produce the same results, a change in substrate concentration, because of negligible washout of cells. Figure 4.36 shows the response of the biofilm reactor after a change in flow rate, from 120 ml/hr to 280 ml/hr. The input concentration of lactose was constant at 5500 (mg/1). At the first steady state (flow rate 120 ml/hr), lactose was almost completely digested (100 mg/1) with very low lactate concentration (10-20 mg/1). The flow rate jump resulted in an increase in lactose and lactate concentrations in the fermenter. After reaching a peak value where its conversion was only about 24 per cent, the lactose concentration returned to a new low steady value (400 mg/1) corresponding to the new flow rate. In parallel, the lactate concentration returned to a new steady value (20 mg/1), too. This response may be due to some kind of temporary physiological slowdown of the bacteria at high concentrations of substrates or products because the bacteria could return to another stable state after a period of time for adaptation. From the results of kinetic studies discussed in the previous sections it can be predicted that an increase in lactose concentration should result in higher production rates of butyrate, propionate, lactate etc., and thus an increase in their concentrations in the fermenter. Recently, van den Heuvel and co-workers investigated the effect of free butyric acid on the acidogenic dissimilation of glucose in anaerobic continuous cultures by adding extra acetate and butyrate to the culture medium and found that only free butyric acid inhibited bacterial activity and the inhibition was more obvious at lower p H values [181]. In the culture medium, butyrate is in equihbrium with its free molecule at steady state, C H 3 C H 2 C H 2 C O O H ^ C H 3 C H 2 C H 2 C O O - + H+ Chapter 4. Intrinsic Kinetics of Lactose Utilization 103 Legend Time (hr) Figure 4.36: Responses of the acidogenic biofilrn reactor to flow rate change. The sohd hne represents lactose concentration and the dash hne lactate concentration. The dissociation constant, K, is only a function of temperature, [CH3CH2CH2COO-}[H+] [CH3CH2CH2COOH] Lower pH values (higher [H+]) and/or higher butyrate concentration can lead to more free butyric acid. It is widely accepted that free fatty acids will pass through the plasma membrane easily and may dissociate intracellularly, thereby abohshing the transmembrane pH-gradient which is a part of proton motive force necessary for bacterial functions [182]. Chapter 5 Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces Compared with the start-up of acidogenic biofilms by which lactose is converted to or-ganic acids, the build-up of methanogenic biofilms is more time consuming because of the much slower growth rate of methanogens and acetogens. Moreover, since methane-and acetate-producing bacteria form a symbiotic microbial community and collectively convert the fermentative products from the first acid-producing phase to methane, it would be valuable to know if support materials have some kind of selective function on the attachment of various bacterial species responsible for different substrate utihzation. As discussed in Chapter 2, many properties of a support material may be able to in-fluence the attachment of bacteria. One of them is wettability or hydrophobicity of the surface which has been shown by Verrier et al [112] to affect the attachment of different methanogenic bacteria. 5.1 Wettability of a Support Surface Wettability measurement of a sohd surface is an old technique. Compared with some new surface-sensitive techniques which probe the sohd/vacuum interfaces like E S C A (electron spectroscopy for chemical analysis) and S E M (scanning electron microscopy), however, wettabihty measurements that probe the solid/liquid interface have some advantages such as great surface sensitivity (5-10 A), inexpensive, simple and compatible with most samples including organic materials and hquids [183]. When a hquid drop comes in contact with a sohd, the liquid may wet the sohd very 104 Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 105 Figure 5.37: Schematic illustration of a spreading drop of hquid in contact with a sohd surface, showing the relations between the contact angle, 6, and three interfacial free energies. well, and thus spread completely across the surface, or not so well, with a result that the drop edge expands to a maximum extent which is dependent on the strength of interaction between sohd and hquid surfaces. The contact angle, 6, as illustrated in Figure 5.37, is an important thermodynamic quantity that characterizes this interaction between a sohd and a hquid surface at the interface with gas. It is generally agreed to give the name of contact angle to the angle within the hquid, i.e. 6 as shown in Figure 5.37. This angle is an experimental representation of an important thermodynamic rela-tionship known as the Young equation, which relates the cosine of the angle, 6, to the interfacial free energies of the three interfaces, sohd-vapor (sv), sohd-hquid (si) and liquid-vapor (lv). At equilibrium, flvcos6 - f\u00E2\u0080\u009E - jai (5.26) Obviously, the contact angle, 0 can be interpreted only in terms of differences and ratios Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 106 of surface free energies rather than as a direct measure of interfacial tension or free energy between sohd and liquid surface. However, with comparisons of measurements in similar systems rather than the interpretation of absolute values obtained from only one system, this technique of contact angle measurements is applicable to the characterization of solid-liquid interfaces. Roughness of a surface also affects the \"equilibrium\" contact angles. Every groove, valley or scratch on a sohd surface acts as a capillary tube in which the liquid rises if the contact angle is less than 90\u00C2\u00B0 or descends if this angle is greater than 90\u00C2\u00B0 [184]. Hence, a rough surface usually is better wetted than a smooth surface by a well-wetting liquid, while a poorly wetting liquid should spread on a smooth surface better than on a rough one. The liquid thus acts as a sensitive probe of the surface by interacting chemically with functional groups at the surface, or physically with surface asperities. The contact angle formed by a drop on a horizontal sohd surface can be computed from the dimensions of the drop. The computation is simple if the drop is so small that it can be treated as a spherical segment. As illustrated in Figure 5.38, the angle 6 can be calculated from the volume, v, of the drop and the diameter, a, of its base. If the drop is so small as to be nearly spherical, the relation between v and a is given by the equation [184], a 3 _ 24Sm 3 fl V ~ TT(2 - ZCosO + Cos39) ^5'2 ' Four types of material have been used in this study as the supports for symbiotic methanogenic biofilms (wood, ceramic, P V C and stainless steel) because they not only are widely used in industry but also give a quite wide range of hydrophobicity. The contact angle between a clean surface and a small water drop (1 pi) was measured with a 30 power microscope. For the base length, a, of a small drop, the average of the values Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 107 V X Figure 5.38: Illustration of calculating contact angle, assuming the drop being a spherical segment. measured at 0, 45 and 90\u00C2\u00B0 was used. The water contact angle on each material which is hsted in Table 3.11 in Chapter 3, is an average of measurements on more than 10 drops. 5.2 Experimental Steps The general setup has been illustrated in Chapter 3. The film attachment experiments were conducted in the continuous flow reactor as shown in Figure 3.10. The feed was a mixture of organic acids (acetate : propionate : butyrate = 6 : 3 : 3 g/1) plus growth stim-ulating inorganic salts as hsted in Table 3.9. It was stored at 4 \u00C2\u00B0C under an oxygen-free atmosphere provided by nitrogen bubbling. The inoculum was taken from a continuous flow methanogenic reactor which was fed with the same feed. Firstly, the reactor was filled with 500 ml of feed and 500 ml of tap water and the temperature of the medium solution was adjusted to 35 \u00C2\u00B0C by the heat exchanger on the recycle hne with nitro-gen bubbhng through the solution. After the temperature was constant at 35 \u00C2\u00B0C7 and the p H of the medium was controlled at 7.1 by the automatic p H controller (with 0.2N Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 108 hydrochloric acid solution), the reactor was seeded with 400 ml of inoculum under a nitrogen atmosphere and operated in batch mode for 2 days with a recycle rate of 152 ml/min. The operation was then shifted to a continuous mode with a hydrauhc reten-tion time at 7 to 8 days controlled with the microvalve. Under this hydrauhc retention time, the recycle ratio was greater than ten and hence, the reactor could be treated as a completely mixed continuous flow reactor with uniform nutrient concentration and tem-perature profiles being maintained in the reactor. Also this long retention time gave a considerable concentration of free bacterial cells suspended in the medium, which was essential for the investigation of attachment of free cells on support surfaces. At steady state, the concentrations of acetate, propionate and butyrate in the reactor were about 1,800, 1,000 and 100 mg/1, respectively. At this time, the four support materials men-tioned above were inserted into the culture medium from the top of the reactor and the time was recorded as zero time for start-up of biofilms on the supports. A run started with a total of 32 samples which had been washed with detergent and distilled water before their being immersed into the medium. After some time (6 - 10 days) one of each type of surface was removed and the biomass accumulated on it was measured in a batch culture till all the samples were used. A key, and also the most difficult, problem in this investigation was how to discrim-inate and describe quantitatively each bacterial group responsible for special substrate degradation in a mixed bacterial community. The usual methods such as by morpho-logical difference, by protein content, or by special co-enzyme (e.g. .F420) content are not capable of discriminating the mixed bacterial species from each other. Based on a principal that the bacterial species responsible for a special substrate assimilation can be lumped into a group (such as acetate-degrading, propionate-degrading and butyrate-degrading bacteria), the shde samples with attached biomass were immersed in a \"stan-dard\" medium which was prepared by diluting the feed to one fifth and the utihzation Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 109 rates of each organic acid in the batch culture were measured. 50 ml of the standard medium which was used throughout this investigation was placed in a 150 ml Erlenmeyer flask immersed in a water bath at 35 \u00C2\u00B0C and nitrogen gas bubbled through the medium to give good mixing and an oxygen free atmosphere. Each support shde with fixed biomass was placed in a flask and the concentrations of acetate, propionate and butyrate in the medium samples taken at regular intervals were measured by gas chromatography (see Appendix B). A sample of medium (1 -5 ml) from the continuous flow reactor was treated in the same way in order to estimate the number of free cells in the fermenter. To be certain that the constant growth rate assumption would hold during the batch test, the run time of batch tests was controlled to maintain a high substrate concentration. The mass of bacteria carrying out dissimilation of each of the three organic acids was monitored by foUowing the degradation rates of the organic acids in the standard cultures. Substrate consumption is related to the growth and survival of the bacterial type using that particular substrate, [dS / dt]totai \u00E2\u0080\u0094 [dSIdrouth + [dS'/dt]maintenance (5.28) or, V[dS/dt]total = (fi/Y + m)X (5.29) where, V = volume of batch culture medium (1) t = culture time (hr) S \u00E2\u0080\u0094 concentration of a substrate in the batch culture (mg/1) p \u00E2\u0080\u0094 specific growth rate of a bacterial group ( h r - 1 ) Y \u00E2\u0080\u0094 yield coefficient of a bacterial group (mg/mgS) m = maintenance coefficient (mgS/mg hr) X \u00E2\u0080\u0094 total mass of bacteria which degrade that substrate (mg). Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 110 c o c CD O c o o \u00E2\u0080\u0094I\u00E2\u0080\u0094 60 100 Time (hr) \u00E2\u0080\u0094 i \u00E2\u0080\u0094 160 200 Figure 5.39: Organic acids degradation in a batch culture, o - acetate, A - propionate, x - butyrate. In a nutrient rich environment the microorganisms have sufficient food supply so that p is a constant. Thus the total mass of a bacterial type is proportional to the utihzation rate of that substrate. p/Y + m v ' Figure 5.39 shows that the concentrations of the three organic acids decreased linearly with culture time (i.e. [ k ^ x - A > J ^ x A where A' is the fractional area occupied by fixed colonies/films and A + A'=l (5.39) Again assuming that the forward and backward rates easily reach equilibrium because of the very slow fixation of the complex, x \u00E2\u0080\u0094 A', k3[x}A' = k_3[x-A'} (5.40) and so, [x-A'] = {k3/k-3)[x]A' (5.41) Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 114 The rate of accumulation of fixed cells due to attachment of free cells on the biomass already fixed on the sohd support is r 3 = kA[x-A'] = K[x]A' = K(l - A)[x] (5.42) where K = k3k4/k_3 (l /cm 2 .hr), signifying the ease with which bacterial cells attach onto biomass already present. Obviously, in contrast to k, K is not affected by the properties of support surfaces. 5.3.4 Detachment The mechanisms of detachment of immobihzed biomass previously has not been investi-gated throughly as was indicated in Chapter 2. The major possible reasons for detach-ment of fixed biofilms are sloughing (for thick biofilms) and shearing caused by fluid flow. In the latter case, the detachment may occur not only on the outer layer but also at some locations inside the biofilm depending on the bridging strength between the cells. The thicker the biofilm, the more connections there are between cells, and the greater is the probabihty that the connections may be broken. Therefore, the rate of detachment of fixed cells has been set proportional to the amount of biomass on the surface, r 4 - K'[xA) (5.43) The parameter K' ( h r - 1 ) is influenced by the shear stress caused by culture medium flow, by the bacterial species, and by other factors which influence the structure and homogeneity of the biofilm, e.g. the concentration of cations such as C a 2 + , and M g 2 + . The total rate of accumulation of one bacterial subgroup is the sum r = r1 + r 2 + r3 - r4 which gives, = k[x]A + K[x](l - A ) + p[xA] - K'[xA) (5.44) dt Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 115 or, ^ 1 = {k- K)[x)A + K[x] + (p- K')[xA) (5.45) dt where A , the fractional area of clean surface, is a function of time, A = 1 at t=0 and A \u00E2\u0080\u0094> 0 as t \u00E2\u0080\u0094> oo It was observed during the experiments that the bacterial colonies, rather than a uniform biofilm with one- or two-cell thickness, were scattered on the surface and had different thickness. As the fixed microorganisms grew they spread slowly over the surface thus reducing the clean surface area left. Therefore, the clean surface area should be inversely proportional to the growth rate of the fixed bacteria which is an exponential function of time in a nutrient rich environment. This can be expressed as; A = e-'1 (5.46) The parameter s ( h r - 1 ) indicates the ease with which the colonies can spread over a sur-face and may be a function of rugosity of the surface. Substitute for A in Equation 5.45, ={k- K ^ e - 1 + K[x] + {u- K')[xA] (5.47) dt Integrating with the initial conditions, [xA] = 0 at t=0 gives (5.48) Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 116 The density of fixed bacteria on a support shde, xA, and the concentration of free cells in the fermenter, x, were measured with the 'standard' batch cultures as discussed above. The left side of Equation 5.48 becomes, =A] _ Xfixed ^ volume of sample [x] XfTee area of support where the volume of sample refers to the volume of medium taken from the continuous flow reactor (1-5 ml) and the area of support is the area of the removable support sample. Using Equation 5.31, Equation 5.48 becomes [dS/dt}fixed x area = = k-K {u-K')t _ -.t] , K r>-*')< _ -n ,~ ~ Q ) [dS/dt]free x volume s+p-K'[ J p - K<1 J K ' J Now, the left side of Equation 5.50 can be experimentally determined with the data from the batch cultures as shown in Figure 5.39. 5.4 Estimation of Model Parameters Equation 5.50 is a general equation which can be apphed to each substratum; wood, ceramic, P V C and stainless steel, and also to each bacterial group; acetate-degrading, propionate-degrading and butyrate-degrading. Hence in all, 12 equations hke Equa-tion 5.50 can be written down with different parameters. Not all the parameters, however, in these 12 equations should be different since they may be independent of the surface type or the bacterial type. Since it was impossible, a priori, to determine which values were common to all equations, a direct search method [186] (see Appendix G) was used to search for a set of optimum parameters which would best fit one of the models with 13, 16, 21 and 22 parameters to the 12 curves (108 experimental data points as hsted in Appendix D) . The optimum parameters for each model were determined by comparision Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 117 of the local minimum fit errors obtained from different initial search points and by the significance of the values (e.g. the parameter values should be positive). Table 5.17 summarizes the results of screening the models by comparing the sum of absolute error between the experimental values and the values predicated by a model. In the table, K represents the attachment of free cells onto fixed biomass already on the support surfaces; K' the detachment of immobilized cells into the medium; p the specific growth rate of attached biomass; k the attachment of free cells onto clean surfaces and s the expansion of attached microbial colonies on support surfaces. The subscripts indicate the surfaces and the bacterial types Since start-up of a biofilm is the net result of several steps hke attachment, growth and detachment, a mechanistic model which describes these steps always contains more parameters than an empirical correlation. If the consideration is limited to one bacterial and one support type, 5 parameters and 4 freedoms (freedom = number of parameters -number of equations) are involved in Equation 5.48. In this case, some of the parameters may be highly correlated. However, when the number of types of bacteria and supports was increased, even though the total number of parametrs were increased, the freedom for each of bacterial and support type was decreased. Furthermore, since the steps described by the model always occur simultaneously during the build-up of a biofilm and could not be separated experimentally, changing one factor hke support surface wettabihty which would affect one of the steps (free cells attaching onto clean surfaces), and thus, the value of the parameter (k) describing this step, may be the only practicable method to investigate this complicated process. The simplest model, model 1, assumed that the surface characteristics had the same effect on the three bacterial groups, and thus there were only 13 parameters in the 12 equations. The sum of absolute errors for this model was 5.53 as compared to the experimental data. Model 2 tested whether the attachment-detachment of bacteria to Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 118 Table 5.17: Results of screening kinetic models of biofilm start-up Model 1 2 3 4 Parameters Unit 13 16 21 22 K cm/day day'1 0.001125 0.00225 . 0.000275 0.003194 K'w 0.02225 0.02225 0.02148 0.05192 K P day'1 >) 0.02938 !) >; K day'1 \u00C2\u00BB) 0.03062 )) )) K day'1 0.02819 )) )> pwa day'1 0.03444 0.03963 0.03581 0.06651 Ppa day'1 ii >) ii 0.06364 P-sa day'1 ii ;) ii 0.06118 Pea day'1 ii ii 0.06151 P'wp day'1 0.02806 0.0330 0.03415 0.05481 thp day'1 j) >) 1 ! 0.06005 day'1 )> >) 11 0.05788 Vcp day'1 ii 11 0.05192 Pwb day'1 0.03875 0.0445 0.03504 0.07236 fhb day'1 !) j) )> 0.06632 P-Bb day'1 )) >) >j 0.07156 Pcb day'1 !) 0.06757 k \"\"wa cm/day 0.05319 0.04738 0.05596 0.06729 h cm/day )) j) 0.03396 jj kwb cm/day j) 0.082 ;> kpa cm/day 0.0245 0.02794 0.03356 0.02966 kpp cm/day ii )> 0.02968 ii kpb cm/day ii >) 0.04191 ii kea cm/day 0.0210 0.02844 0.02909 0.03119 kgp cm/day )! ii 0.02628 )> ktb cm/day )) ii 0.03786 j) kca cm/day 0.03481 0.03769 0.04202 0.03234 kep cm/day J J )! 0.03071 kcb cm/day )> )) 0.06597 >) day'1 0.04731 0.06306 0.05218 0.07837 Sp day'1 0.05675 0.06788 0.08931 0.09576 Ss day'1 0.05069 0.06638 0.0908 0.1082 day'1 0.0595 0.06975 0.08536 0.05146 sum of error 5.533 5.271 2.783 4.249 w,p,s,c: surfaces of wood, P V C , stainless steel and ceramic ring a,p,b: bacteria degrading acetate, propionate and butyrate Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 119 the biomass was influenced by the substrata. This added 3 parameters and only reduced the sum of absolute error to 5.27. Model 4 tested the possibihty that the specific growth rate of each species was influenced by the characteristics of the substrata. Although the number of parameters was increased to 22, the sum of absolute error was only reduced to 4.25. Model 3 which was used to describe the results was based on the assumption that each bacterial type attached differently to each surface, but their specific growth rates were independent of the surface properties because the support materials used in this study were inert (wood is not biodegradable to any extent in the short time period used) and fixed microbes could get enough nutrients from the culture medium with no help from the sohd surfaces. Twenty one parameters in 12 equations gave a sum of absolute error of only 2.78 probably due to the reasonable assumptions which were made. Figures 5.40, 5.41 and 5.42 show how well this model fits the experimental points. Mathamatically, although 22 parameters were estimated simultaneously and the total degree of freedom (22 - 12) of the search was 10, for each of the equations the degree of freedom was less than 1 (10/12) (a hnear equation in general has one degree of freedom). Therefore, for each combination of bacterial and support type, the fit of the equation with the experimental data was made by adjusting less than one parameter, so, there was very low correlation between the parameters. In addition to the assumption mentioned above that a special bacterial group (say, acetate- degrading bacteria) has the same specific growth rates on four different supports, the three bacterial groups have almost the same specific growth rates, 0.035 ( d a y - 1 ) . Lawrence and McCarty [142] obtained a similar result in their study on the kinetics of methanogenic cultures - that is, acetate-, propionate- and butyrate-degrading bacte-ria had the same specific growth rates. Moreover, a value of specific growth rate for methanogenic anaerobes was calculated from the data given by Verrier et al [111] as 0.027 (day-1). This is good agreement considering that they used a different technique Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 120 Figure 5.40: Build-up of acetate-degrading bacteria in the biofilms. The lines are calcu-lated from the model. Surfaces: o - wood, x - ceramic, A - P V C , \u00E2\u0080\u00A2 - steel Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 121 Figure 5.41: Build-up of propionate-degrading bacteria in the biofilms. The lines are calculated from the model. Surfaces: o - wood, x - ceramic, A - PVC, \u00E2\u0080\u00A2 - steel Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 122 4-1 Time (day) Figure 5.42: Build-up of butyrate-degrading bacteria in the biofilms The lines are calcu-lated from the model. Surfaces: o - wood, x - ceramic, A - PVC, \u00E2\u0080\u00A2 - steel Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 123 10-1 WOOD CERAMIC PVC STEEL Sur face type Figure 5.43: Ease of attachment of bacteria on substrata as shown by model parameter k. to measure the growth rate on different substrates. The other two factors which were considered to be independent of surface type are K and K', the attachment and detach-ment to and from fixed biomass, respectively, since the surface involved was a biomass surface. The parameter k, defined in Equation 5.36, is a proportionality factor which takes into account the ease with which bacteria can attach and remain on a surface. Figure 5.43 shows the same data graphically. It can be found that the acetate and butyrate degrading bacteria had obvious surface preferences of, wood > ceramic > PVC > stainless steel The propionate degrading bacteria, however, attached less easily and had almost no surface preference. The spreading factor, s, was calculated for all the bacteria lumped together but for Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 124 0.10-1 120 Contact angle (degree) Figure 5.44: Attachment factor, k, as a function of water contact angle of surface. Line is the least square fit. o-butyrate degrading bacteria, A-acetate degrading bacteria, X-propionate degrading bacteria each support surface. Here it can be seen that, once attached, the ease with which the microorganisms spread on the surface by growth was in the order, stainless steel > PVC > ceramic > wood This is exactly the reverse of the ease of attachment order. Figure 5.44 is a plot of the attachment factor, k, against the water contact angles for the four surfaces tested. There appears to be a relationship between these two factors which can be fitted very well by a hnear least square method. The fitting parameters are hsted in Table 5.18. It was reported by van Loosdrecht et al [187] that on a single surface (sulfated polystyrene), attachment of 16 bacterial strains could be linearly correlated with the bacterial wall hydrophobicity (water contact angle between 15\u00C2\u00B0 and 70\u00C2\u00B0). There have been some contradictory reports on the relationship between wettabihty and attachment as reported by Dexter et al [93], and by Pringle and Fletcher [188]. Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 125 Table 5.18: Linear relations between specific attachment rates and water contact angles Bacteria Intercept Slope r- squared (cm/day) (cm/day.degree) (%) Acetate-degrading 0.05632 -2.646 X l O \" 4 99.6 Propionate-degrading 0.03422 -6 .659xl0- 5 88.1 Butyrate- degrading 0.0847 -4.546 x l O - 4 94.9 Bright and Fletcher [176] found that amino acid assimilation and respiration by attached and free-living marine Pseudomonas spp decreased with increasing water contact angle of the support surface while Fletcher and Leob [189] observed that lower hydrophobicity, i.e. smaller contact angles, promoted attachment of marine bacteria. Absolom [190] has reported that bacterial attachment depends not only on the hy-drophobicity of the substrata but also on the hydrophobicity of the bacterial wall [191]. The bacteria with hydrophilic surfaces have smaller contact angles and larger contact areas on hydrophilic support surfaces. Thus they are deformed more and may be able to remain on the surface for a longer time because more adhesive extracellular glycocalyces (capsules or s layers) are in contact with the substrate [192,100]. If bacterial cells can stay in contact with a substratum for a long time the physiological functions of the cells can produce more extracellular polysaccharide materials which bridge the cells and the substratum [85,189]. If it is assumed that the initial contact and attachment is a function of the relative hydrophobicity of the surface and the bacteria, the nature of the three bacterial types can be summarized as follows: 1. The butyrate degrading bacteria have the most hydrophilic wall surfaces since their specific attachment rate, k, decreases most rapidly with increasing hydrophobicity of the substrata. Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 126 0 . 1 0 - 1 Contact angle (degree) Figure 5.45: Spreading factor, s, as a function of the water contact angle of the surfaces for all bacteria. 2. The propionate degrading bacteria are least hydrophilic since the support surface wettability has only a small effect on their attachment. 3. The acetate degrading bacteria have somewhat hydrophihc wall surfaces. Thus, since the surface of the three bacterial types have different hydrophobicities, it should be possible to some extent to influence the composition of a methanogenic biofilm by purposely choosing a substratum. The coefficient of spread, s , defined by the film attachment model has also been shown to be affected by the substrata wettability. Figure 5.45 shows graphically this model parameter, which describes the rate of colony spreading on a surface, as a function of the wetting angle of surfaces. Fixed cells divide within a hydrated exopolysaccharide matrix so that the daughter cells are trapped in a juxtaposition that results in the formation of microcolonies as Chapter 5. Start-up of Symbiotic Methanogenic Biofilms on Inert Surfaces 127 indicated in Chapter 2. In a fluid flow environment, as found in this study, solid sup-ports offer shields for bacterial colonies fixed on them because the flow stagnates on the surface due to the fluid viscosity. However, on hydrophobic surfaces the solid surfaces do not strongly attract the water molecules and there is a greater possibility of slip. It is expected that this phenomenon will have an influence on the size of microcolonies. Flattening would result in a spreading of the microcolonies in the flow direction. Since the flow velocity approaches zero at a hydrophilic surface the microcolonies would be less flattened and the spreading would, be slower than on a hydrophobic substratum. The more hydrophobic the surface, the more rapid is the spread of bacteria. Shimp and Pfaender [193] have also observed the spreading phenomena of microbial colonies as a function of flow rate. Chapter 6 Intrinsic Kinetics of Methanogenesis of Organic Acids Flow from the acidogenic phase reactor, where lactose was converted to organic acids by the acidogens, enters the second phase reactor where the symbiotic methanogenic bacte-ria convert these intermediates into gaseous products, methane and carbon dioxide. As discused in Chapter 2, many species of methane-producing bacteria have been identified and maintained in pure culture. AU known species can utihze carbon dioxide and hydro-gen as substrates for methane generation whereas a few of them can use acetate as their substrate to produce methane. Except for these two types of substrates (CO2/H2 and acetate), the methanogens must rely on the biological activities of acetogens, who can convert other substrates such as ethanol, propionic and butyric acids to acetate, carbon dioxide and hydrogen. Table 6.19 lists the four main reactions occurring in fatty acid methanogenesis reported by Thauer et al [179]. Table 6.19: Methanogenic reactions of organic acids Reactions A G (kJ) +76.1 +48.1 -135.6 Source is [179]. However, the acetogens cannot do this job at all at high hydrogen partial pressures, 128 C H 3 C O O - + H 2 0 -> C H 4 + H C O 3 C H 3 C H 2 C O O - + 3 H 2 0 -> C H 3 C O O - + H C O j + H+ + 3 H 2 C H 3 C H 2 C H 2 C O O - + 2 H 2 0 -> 2 C H 3 C O O - + H+ + 2 H 2 4 H 2 + H C O 3 + H + - \u00C2\u00BB C H 4 + 3 H 2 0 Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 129 Table 6.20: V F A distribution in anaerobic digestion process Substrate H A c : H P r : H B u (w:w:w) Reference Primary sludge Sewage sludge Sewage sludge Sewage sludge Raw sludge Lactose (D=0.25 1/hr) Lactose (D=0.1 1/hr) 1 : 0.6 : 0.2 [201] 1 : 0.9 : 0.7 [202] 1 : 0.8 : 0.4 [203] 1 : 0.3 : 0.3 [204] 1 : 0.3 : 0.3 [26] 1 : 0.4 : 0.6 [44] 1 : 0.1 : 0.2 [44] and thus must rely on the methanogens to remove the produced hydrogen, too. In con-trast to the case of lactose acidogenesis where all the bacterial species could be lumped as a single group from the point of view of lactose consumption, the methanogenic com-munity includes at least four groups of bacteria classified by their substrates; butyrate-, propionate-, acetate-, and hydrogen-utilizing bacteria. 6.1 Experiments of Organic Acid Methanogenesis The experimental setup was the same as for lactose acidogenesis; the general setup was as shown in Figure 3.10 and the biofilm support surface was made from P V C sheets as described in Chapter 3. The feed was a mixture of acetate, propionate and butyrate in a ratio of 1 : 0.5 : 0.5 by weight. This ratio was determined from the ratios of volatile fatty acids ( V F A ) concentrations produced in the conventional anaerobic digestion process and the two-phase digestion process as hsted in Table 6.20. The studies on lactose acidogenesis described in Chapter 4 revealed that under those experimental conditions the major products were acetate and butyrate with propionate being a minor product. However, propionate was still used in this study for more informa-tion about its rate of utihzation, and its influence on the digestion of other components. Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 130 The necessary growth nutrients for the microorganisms were added as inorganic salts as shown in Table 3.9. The feed had an acetate concentration ranging from 2 to 5 g/1, and hence the acetate concentration in the fermenter could be controlled at different levels under the desired experimental dilution rates. Correspondingly, the concentrations of propionate and butyrate in the fermenter were also controlled at their own levels at the experimental dilution rates because the concentrations of propionate and butyrate in the feed had a fixed ratio to that of acetate (HAc : H P r : H B u = 1 : 0.5 : 0.5). The oxygen dissolved in the feed was stripped off with N 2 . The feed was then kept under a N 2 atmosphere by bubbling N 2 through the feed because the methane-producing bacteria are obhgate anaerobes. To avoid evaporating the volatile fatty acids the p H of the feed was adjusted to 7.0 - 7.2 by the addition of equimolar sodium hydroxide and potassium hydroxide. Every batch of feed was analyzed at least two times during the experiments to determine the feed concentrations. The fermenter had a working volume of one liter. The flow rate, measured by col-lecting effluent during a period of time (3-4 hours), was controlled at above 30 ml/hr to maintain the dilution rate, D , greater than 0.72 d a y - 1 . In their review of the anaerobic digestion hterature, Henze and Harremoes [144] concluded that the maximum specific growth rate of methanogenic bacteria at 35 \u00C2\u00B0C was 0.4 d a y - 1 . Under the chosen hy-drauhc conditions the bacterial cells suspended in the culture medium were washed out and this fraction of biomass retained in the reactor could be neglected in comparison with the biomass fixed on the supports. The neghgible amount of free bacterial biomass was confirmed by analyzing the sohd carbon content in the effluents as was done in the experiments of lactose acidogenesis. The p H of the fermenter was controlled by an automatic p H controller at 7 . 1 \u00C2\u00B1 0 . 2 with 0.3 to 1 N hydrochloric acid solutions. The HC1 concentration was adjusted according to the strength of the methanogenic activity to have as low an influence on flow rate and Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 131 local p H in the fermenter as possible. The inoculum was obtained from the secondary phase reactor of a two-phase mini-plant which had been in continuous operation for two years. The experiments on the methanogenesis of organic acids can be divided into four stages: 1. Adaptation; The feed used during this period was a mixture of acetate (3 g/1), pro-pionate (1.5 g/1) and butyrate (1.5 g/1) so that the bacterial community would have a balanced population, containing acetate-, propionate-, butyrate-, and hydrogen-utilizing groups. The fermenter was filled with the feed (500 ml) and tap water (300 ml). Nitrogen was continuously bubbling through the medium for 2 hours to remove the dissolved oxygen. When the temperature reached 35 \u00C2\u00B0 C , the fermenter was inoculated with 200 ml of inoculum and then operated in batch mode. After the three acids had been used up, half the volume of the culture (500 ml) was replaced by fresh feed and this was repeated 5 times. 2. Build-up of a symbiotic biofilm; Adaptation of the microorganisms to the mixture of fatty acids gave a balanced microorganism population which was suspended in the culture medium. At that time the supports made of P V C sheets were immersed in the medium under N 2 atmosphere. The operation, then, was shifted to continuous operation mode with the flow rate controlled below 6 ml/hr (dilution rate D < 0.15 d a y - 1 ) to avoid washout of the free cells. After about 60 days a uniform black biofilm could be observed on the grey P V C sheets. 3. Steady state operation; With build-up of the biofilm the flow rate was gradually increased to above 30 ml/hr to wash out the suspended cells. The temperature of the culture medium was kept at 35 \u00C2\u00B0C and the p H at 7.1. The fatty acid concentra-tions in the fermenter were controlled by adjusting the feed concentration and flow Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 132 rate. At each of the desired concentration levels, a steady state (effluent concentra-tion fluctuation < 10 %) was maintained at least 3 days to get experimental data. Similar to the procedures for lactose acidogenesis, after a set of concentration lev-els had been investigated, the experiments were repeated on the same set of levels again and again. Therefore, the utilization rates of fatty acids could be obtained at each of the concentration levels but on different thickness of methanogenic biofilms. By comparing these data, the effect of internal mass transfer resistance could be estimated, and only those data not affected by the biofilm thickness were taken as the intrinsic data. 4. Interaction of fatty acids; Following the kinetic studies, the interaction of fatty acids was investigated in the \"mature and balanced\" methanogenic biofilms which contained aU the bacterial types responsible for the utihzation of three organic acids. In this stage, the concentration of one acid in the feed was adjusted at different levels while those of other two acids and other conditions were unchanged. Therefore, any change in the utihzation rates of these two acids might be attributed to the effect of the acid with changed concentrations. The acetate concentration in the fermenter was adjusted from 800 to 1,100 mg/1, the propionate concentration from 0 to 2,000 mg/1, and the butyrate concentration from 100 to 1,500 mg/1. The culture temperature and p H were maintained at 35 \u00C2\u00B0C and 7.1, respectively. The intrinsic kinetics of biofilms must be investigated under neghgible external and internal mass transfer resistance. In this study the influence of external mass transfer was reduced as low as possible by increasing the recycle rate, and thus the linear veloc-ity over the biofilm, which effectively reduced the external mass transfer resistance as indicated in the studies made on lactose digestion by acidogenic biofilms in Chapter 4. It should be easier to minimize the external and internal mass transfer resistance during Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 133 I .6-1 E < x 0 1 ' ' ' \u00E2\u0080\u00A2 I I \u00E2\u0080\u00A2 ' \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 I , . i . [ . . . . , 0 60 100 160 2 0 0 260 3 0 0 360 Recycle rate (ml/min) Figure 6.46: Influence of recycle rate on acetate utilization the methanogenesis because the physiological activities of methanogens are much weaker than those of acidogens. Figure 6.46, Figure 6.47 and Figure 6.48 indicate that as long as the recycle rate was above 190 ml/min, i.e. a hnear velocity of 2.8 cm/min over the biofilm, the utihzation rates of acetate, propionate and butyrate should not be affected by external mass transfer. This conclusion was confirmed later by the relationship between these reaction rates and the bulk substrate concentrations. Grady and L i m [205] showed that the presence of external mass transfer resistance prevents the rate expression from exhibiting a Michaelis-Menten type of a dependence on the bulk substrate concentration even when the true kinetics were assumed to do so. The influence of internal mass transfer resistance is always present if the mass transfer rate, based on the mechanism of molecular diffusion, is not much greater than the con-sumption rate, and if the bulk substrate concentration is taken as representative of the internal substrate concentrations within the biofilms. Obviously, the thinner the biofilm, Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 134 Figure 6.47: Influence of recycle rate on propionate utilization Figure 6.48: Influence of recycle rate on butyrate utilization Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 135 0.20 0 . 1 6 CJ cn o\u00C2\u00BB E \u00E2\u0080\u0094 0.12 CO c O 0 . 0 8 Ui 0 . 0 4 -< 0 . 0 0 \u00E2\u0080\u00A2 T J Q r P ^ L ^ L p 0 . 1 0 0 . 1 6 0 . 2 0 0 . 2 6 0 . 3 0 0 . 3 6 0 . 4 0 0 . 4 6 Biofilm thickness (mm) \u00E2\u0080\u0094 i 1 0 . 6 0 0 . 6 6 Figure 6.49: Influence of biofilm thickness on acetate digestion rate the less the influence of internal mass transfer resistance. The kinetic data collected during the early stage of biofilm development are considered to be the result of pure bacterial reaction because a very thin biofilm had developed at that time. Figure 6.49 shows that the acetate utihzation rate per unit weight of biomass car-bon gradually decreased with an increase of the methanogenic biofilm thickness because the bacterial cells embedded in the thicker biofilms saw lower substrate concentrations. Therefore, the experiments were arranged in such a way that the experiments at low substrate concentrations were conducted first and those at high concentrations later. Corresponding to higher bulk concentrations, the concentrations inside the biofilms were also higher, and hence, all the bacterial cells embedded in a thick biofilm might have their maximum utihzation rates which was determined by the Michaelis-Menten type kinetics of the bacteria. Thereby, the effect of internal mass transfer resistance could be reduced. As shown in Figure 6.49, the internal mass transfer resistance in a biofilm Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 136 Table 6.21: Experimental conditions for organic acid methanogenesis Parameter Unit Condition Temperature \u00C2\u00B0C 35 \u00C2\u00B1 2 p H 7.1 \u00C2\u00B1 0.2 Flow rate ml/hr > 30 Dilution rate d a y ' 1 0.72 Recycle rate 1/hr 11.4 Biomass methanogenic Feed g/1 H A c : H P r : H B u for biofilm buildup 3 : 1.5 : 1.5 for kinetics 2-5.5 : 1-2.7 : 1-2.7 having a thickness less than 0.3 mm could be considered to be minimal, and hence, the intrinsic kinetic data should be collected during this period (about 100 days). Mixtures of acetate, propionate and butyrate in the ratio of 1 : 0.5 : 0.5 (weight) were used as the feeds for biofilm build-up and kinetic studies. However, investigations on the interaction between fatty acids were also conducted on the balanced biofilms with the feeds having different ratios of acetate, propionate and butyrate. The concentrations of each acid wiU be listed in the description of each test. Table 6.21 hsts the experimental conditions for fatty acid methanogenesis. The recycle ratio, the ratio of recycle rate to flow rate, was up to 380, that is, much greater than 10. Therefore, the fermenter could be taken as a completely mixed continuous flow reactor ( C S T R ) and the effluent concentration was the same as that in the fermenter. 6.2 Development of Methanogenic Biofilms The amount of biomass retained in a fermenter has a large effect on the performance of this reactor. In this study all the utihzation rates of the organic acids were based on unit weight of active biomass. Therefore, the total amount of biomass in the reactor Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 137 must be known during the experiment. As was discussed in Chapter 4 for the acidogenic biomass, the methanogenic biomass also existed in three forms: biofilms, the major part of the retained biomass; free cells, a minor fraction under the experimental dilution rates; and the sludge on the reactor walls which was taken as biofilm on the walls as shown in Figure 4.20. When the P V C biofilm supports as described in Figure 3.14 were immersed into the medium, about 30 removable P V C slides were also inserted into the fermenter. The biomass on these shdes was measured at different times and was believed to be the same as those on the supports. Each time one or two shdes were analyzed and it was observed that a uniform biofilm was forming on the supports because the two shdes randomly taken from different locations in the reactor had almost the same amount of biomass. Figure 6.50 shows that the biomass on the shdes increased gradually with the culture time. Correspondingly, the carbon content of the biomass on the shdes also increased with the culture time as shown in Figure 6.51. The carbon content of the biofilms gave a very good linear relationship with their dry biomass as shown in Figure 6.52. The slope of the straight hne in Figure 6.52 is 0.353 (mg carbon/mg dry biomass). From the carbon content of the biomass on a removable shde and the areas of the supports and the shde, the total carbon content of biofilms fixed on the supports at that time could be calculated. The biomass on the reactor walls was not easily measured. The sludge which was introduced into the fermenter as seed and had reproduced during the period of biofilm development was removed by discharging the culture medium under a N 2 atmosphere after a visible black biofilm had formed and before the kinetic data were collected. Thus the biomass left on the reactor walls was taken to be the biofilm attached on the walls. Based on the same assumption used for acidogenic biomass, this part of biomass should have the same growth rate as the biomass on the supports. After the experiments were Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 138 40 80 120 160 Culture time (day) 240 Figure 6.50: Biomass on slides increases with culture time 26-i 160 Culture time (day) 200 240 Figure 6.51: Total organic carbon on slides increases with culture time Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 139 26-| 2 0 -00 E Biomass (mg) Figure 6.52: Relationship between organic carbon and dry biomass of biofilms finished the biomass on the reactor walls was carefully collected and measured, and the initial amount of biomass was estimated by using this data and the growth rate of biofilms on the supports. The total biomass, in terms of total organic carbon, of the biofilms on the supports and reactor walls can be predicted by using the following equation. TC = 1678.9e0 0 0 9 0 t (6.51) Table 6.22 lists some analysis results of biomass samples taken from biofilms on the supports and 'sludges' on the reactor walls. The 'biofilms' on the reactor walls contained more ash and less organic carbon because more inorganic salts (metal ions) were deposited on the bottom of the reactor. That is why the total carbon content, of the biofilm, instead of the dry biomass, was used in this study. Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 140 Table 6.22: Properties of methanogenic biofilms Item Sample 1 Sample 2 Ash content (w%) biofilm sludge T O C content (w%) biofilm sludge 38.1 37.7 58 59 34.4 35.2 15.2 17.3 6.3 Interaction of Organic Acids The methanogenesis of fatty acids depends on a symbiotic microorganism community which consists of at least four groups of bacteria. Each of them has its own substrate. Generally the substrate concentration affects the biological activity of the bacterial group responsible for the utihzation of this substrate. One example is the dependence of lac-tose utihzation rate on the lactose concentration as discussed in lactose acidogenesis. However, one substrate may also affect other groups of bacteria because of the symbi-otic properties of the bacterial community. Scharer and Moo-Young [206] found that the kinetic information obtained using single substrates could not be used for predicting methane generation from a mixture of acids and when equimolar mixtures of acetate, propionate and butyrate were used, propionate was not consumed at all. This may be caused by the high substrate and/or product concentrations being toxic or beneficial to the bacterial activities, but may also be caused by inhibition or competition from other substrates in a feed of mixed organic acids. The following experiments were conducted by using the feeds with different concentrations of organic acids. 6.3.1 Influence of Acetate Concentration Methanogenesis of organic acids was investigated in immobilized cell bioreactors by Scharer et al [114]. Their results indicated that acetic acid was consumed very rapidly Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 141 0.04-1 o | * 0 .03 CO \u00C2\u00AB 0.02 c o CO CO O ) 0.01 T J w O. X 0.00 \u00E2\u0080\u00A2 X I T J 3 \u00E2\u0080\u00A2 760 8 0 0 860 900 960 1000 1060 Acetate concentrat ion (mg/l) \u00E2\u0080\u0094 i 1 1100 1150 Figure 6.53: Effect of acetate concentration on propionate digestion (HPr=880-996 mg/1) in comparison to propionic and butyric acids and the presence of acetic acid had an in-hibiting effect on the utihzation of either propionic or butyric acid. Therefore, it seems possible that acetogenesis of these two organic acids could be inhibited by their product, acetate. To test this hypothesis, the concentrations of propionate and butyrate were maintained in narrow ranges, 880-996 mg/1 and 48-66 mg/1 respectively, and only the acetate concentration was adjusted in the fermenter. Figure 6.53 and Figure 6.54 re-veal that the digestion of neither propionate nor butyrate was inhibited by the presence of acetate. This means that in a balanced bacterial community the acetogens are not inhibited by the products of their metabolic activities. 6.3.2 Influence of Propionate Concentration Propionate is the component most difficult for the bacteria to digest. Hence, it often appears in high concentrations in the effluent of an unstable digester. Its influence on Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 142 0.04 (J o> f= 0 . 03 \u00E2\u0080\u00A2\u00E2\u0080\u0094* JE ~ 0 . 0 3 O 0 . 0 2 -03 CD ai \" \u00C2\u00B0 0 . 0 1 CD X 0 . 0 0 T J B 4 0 0 8 0 0 1 2 0 0 1 6 0 0 2 0 0 0 . Propionate concentration (mg/l) 2 4 0 0 Figure 6.56: Effect of propionate concentration on butyrate utilization (HBu=50-80 mg/l) Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 144 0.20 0.16 cj O) E E \u00E2\u0080\u0094 0 .12 CO c O 0 . 0 8 CO CO O) ~ \u00C2\u00B0 0 . 0 4 O < X 0 . 0 0 m\u00E2\u0080\u0094SF\u00E2\u0080\u0094\u00E2\u0080\u00A2 X L \u00E2\u0080\u00A2 D 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 Butyrate concentration (mg/l) 1 6 0 0 Figure 6.57: Effect of butyrate concentration on acetate digestion (HAc=1400-3000 mg/l) were kept in a relatively narrow range, 1400-3000 m g / l and 700-900 m g / l respectively. Although the acetate concentrations changed from 1400 to 3000 m g / l no significant influ-ence of butyrate concentration was observed on acetate digestion as shown in Figure 6.57. This was because at high substrate concentrations acetate-digesting bacteria had reached their maximum metabolism rate and hence further increase in acetate concentration had little effect on the utihzation rate. Figure 6.58 shows no influence of butyrate concentra-tion on propionate utihzation even though the later inhibited the former's digestion. This may be an indirect evidence that conversion of these two fatty acids involves different bacterial species. The interaction of substrates or bacterial groups of a balanced microorganism com-munity can be summarized as follows: \u00E2\u0080\u00A2 Acetate has no influence on the biological activities of acetogens which are respon-sible for the conversion of propionate and butyrate to acetate and H2, and also, Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 145 0.03 o O) E O) 0.02 E CD *-< 0 . 0 4 E \u00E2\u0080\u00A2*-* to k. c o co CD 3 CO I 0 . 0 3 0 . 0 2 -0 . 0 1 0 . 0 0 ) \u00C2\u00A3 1 0 0 2 0 0 3 0 0 4 0 0 6 0 0 Butyrate concentration (mg/l) e o o Figure 6.60: Dependence of butyrate digestion rate on butyrate concentration. The dash hne is calculated from the Michaelis-Menten equation 6.60; The points (x) are calculated from Equation 6.62 Equation 6.60 was linearized to give an initial estimation of the two parameters, rMAII6 and Kb, and then, the direct search method was used, with the initial esti-mation as the start point, to further optimize the fitness of the model equation to the experimental data. fmox.b = 0.0334 mg/mgC/hr Kb = 20 mg/l Figure 6.60 shows that the butyrate utihzation rate is not predicted satisfactorily by the Michaelis-Menten type equation 6.60 represented by the dash hne because the rate is depressed at higher butyrate (propionate) concentrations. There are two explanations for this phenomenon. One is that the rate is inhibited by pro-pionate, as indicated earlier in the discussion about the interaction of substrates, Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 152 0 . 1 0 - 1 \u00C2\u00BB 0 0 8 o.oe-CD H\u00E2\u0080\u0094* <0 c O 0 . 0 4 -co CD O) \u00E2\u0080\u00A2 \u00C2\u00B0 0 . 0 2 -CO D X 0 . 0 0 2 0 0 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 i ' 1 \u00E2\u0080\u00A2 1 \u00E2\u0080\u00A2 1 \u00E2\u0080\u00A2 1 \u00E2\u0080\u00A2 1 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 4 0 0 6 0 0 Butyrate concentrat ion (mg/l) Figure 6.61: Influence of butyrate concentration on butyrate digestion (HPr = 690-900 mg/1) since the feeds, having a fixed weight ratio of fatty acids, contained also a large amount of propionate at high butyrate concentrations. It may also, on the other hand, be attributed to high butyrate concentrations, i.e., a substrate inhibition. An experiment was designed to check the latter possible mechanism. The propi-onate concentration was controlled in a narrow range, 690-900 (mg/1), the butyrate concentration in the fermenter was adjusted from 100 to 1500 (mg/1). As shown in Figure 6.61, the high butyrate concentration did not inhibit its utihza-tion by the responsible bacteria, at least in the present experimental concentration range. Therefore, the only mechanism to explain why the butyrate digestion rate is inhibited is the effect of propionate. This result also indicates that the inhibition of butyric acid in anaerobic digestion processes, as reported by van den Heuvel [181], is caused by the effect of the acid on acidogenic microbes instead of methanogenic Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 153 bacteria. It appears that an increase in propionate concentration can promote its own utihzation rate but wiU inhibit the butyrate digestion rate. The inhibition mechanism is assumed to be of the form: k h E + S ^ E S ^ P + E (1) h ESb + Sp SpESb (deactivation) (2) k-3 In addition to the main reaction (1), a Michaelis-Menten reaction, the enzyme-butyrate complex, ESb may also be attacked by a propionate molecule, S p , which results in a deactivated active enzyme center, SpESb- A reaction rate expression is derived from this mechanism (see Appendix F for detailed derivation). r = ^ (6.61) (&_! + A;2)/fci + Sb + {k3/k^)SbSp V ; Correspondingly, the overaU butyrate utihzation rate, rj,, has a similar expression, Tmax,bSb , \u00E2\u0080\u009E \u00C2\u00AB0\ r b = Kb + Sb + KrSbSp <6-62) Where rmaxb is the maximum butyrate digestion rate (mg/mgC/hr) , Kb the half velocity concentration (mg/l). Kj is a equilibrium constant of the deactivation reaction and has a unit of reciprocal of concentration (1/mg). The direct search method was used to estimate the optimum parameter values to fit the experimental data with Equation 6.62. rmax,b = 0.0473 m g / m g C / h r Kb = 27 m g / l Ki = 0.000296 1/mg Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 154 The points marked by the sign ' x ' in Figure 6.60 are predicted by Equation 6.62 with these parameters at different propionate concentrations (20-1700 mg/1). Again, the addition of the third independent parameter, Kj, into the two-parameter model 6.60 was statistically tested by calculating the coefficient of partial determi-nation, and its value was 0.53. Therefore, the introduction of Kj into the model re-duced the error sum of squares by 53 % compared with the two-parameter Michaelis-Menten equation 6.60. The F test shows that F = 241 > F(0.95: 2, 13) = 3.81, which means that the butyrate utihzation rate is significantly related with all three parameters. 6.4.3 Acetate Utilization Rate At steady state, the amount of acetate which was introduced into the reactor should be balanced by the amount consumed by the bacteria plus that in the effluent. The acetate introduced into the reactor could be divided into direct and indirect input. The direct input was the amount fed into the reactor as feed and the indirect part was the amount converted from propionate and butyrate by the acetogens. Generally one mole of propionate produces one mole of acetate and one mole of butyrate produces two moles of acetate as shown in Table 6.19. The material balance gives FSiA + W(^rp + ^ r b ) = FSe,a + raW (6.63) M \u00E2\u0080\u009E Mb and it is re-arranged as FiSi^-S^) Ma 2Ma W + M / p + M b V b (6.64) Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 155 0.16-1 Acetate concentrat ion (mg/l) Figure 6.62: Dependence of acetate utilization rate on acetate concentration. The hne is calculated from Equation 6.65 Where F is the flow rate of medium (1/hr), S i a , 5 e j a the acetate concentrations (mg/l) in the feed and effluent, respectively, W the total amount of biomass retained in the fermenter in terms of total organic carbon (mgC), Ma, Mp, Mb the molecular weight of acetate, propionate and butyrate, ra, rp, rb the utihzation rates of acetate, propionate and butyrate (mg/mgC/hr) , respectively. The items on the right side of Equation 6.64 can be determined experimentally. The rate, ra, was plotted against the acetate concentrations in the fermenter, 5 e > a , since in a completely mixed continuous flow reactor the effluent concentration equals the concentration within the reactor. Figure 6.62 indicates that the dependence of acetate utihzation rate on acetate concentration can be described quite well by a Michaelis-Menten type equation. Also it had previously been shown that the acetate utihzation rate is affected by Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 156 neither propionate nor butyrate as discussed in the section on interaction between substrates. ' a S (6.65) a Ka + S The two parameters are estimated by linearizing the non-linear Equation 6.65 as S + (6.66) f n f m n rr n 7*n and then by using the least square method to fit Equation 6.66 with the experimen-tal data. The initial estimation of the parameters was then used as the first search point from which a direct search was conducted on Equation 6.65 to optimize its fitness to the experimental data. The results are rmax,a = 0.098 m g / m g C / h r Ka = 160 mg/l . where r m a a . j a is the maximum utihzation rate of acetate and Ka is the half velocity acetate concentration which characterizes the affinity of acetate-utihzing bacteria on acetate. In order to confirm the assumption of stoichiometry of propionate and butyrate conversion to acetate, a feed containing only acetate (14828 mg/l) was used. The acetate digestion rate at steady state was 0.093 (mg/mgC/hr) and ac-etate concentration in the fermenter was 5611 (mg/l). This rate was obviously the maximum rate since it was very close to the value of rmana. Therefore, it is further confirmed (1) that the presence of propionate and butyrate has no influence on acetate-utilizing bacteria; (2) that the stoichiometry of propionate and butyrate conversion to acetate was reasonable; (3) that the dependence of the acetate uti-hzation rate on acetate concentrations follows the Michaelis-Menten equation up Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 157 to 5600 (mg/1). Table 6.23 summarizes the utilization rates and half velocity constants for the methanogenesis of organic acids obtained in this study and reported in the litera-ture. For the sake of comparison with other data, organic acids are converted to equivalent C O D content using the ratios of 1.07 (mg C O D / m g acetic acid), 1.51 (mg C O D / m g propionic acid) and 1.86 (mg C O D / m g butyric acid). Table 6.23: Methanogenesis of organic acids Substrate Culture Temp. p H r m a x K\u00E2\u0080\u009E Reference c\u00C2\u00B0 mgCOD mqV SS.hr m g C O D / 1 Acetate H A c enrichment sludge 35 7 0.36 165 [142] Propionate H P r enrichment sludge 35 7 0.6 34 [142] Butyrate H B u enrichment sludge 35 7 1.21 5 [142] Acetate H A c enrichment biofilms 35 7 0.11-0.21 [208] Acetate H A c enrichment sludge 35 7 0.45-0.54 [144] Acetate H A c enrichment sludge 38 0.045 [144] Propionate mixed sludge 33 0.26 246 [209] Mixed acids mixed biomass 35 7.2 0.712 166 [143] Mixed acids mixed biofilms 37 7 0.02-0.24 [114] Mixed acids acetate mixed biofilms 35 7.1 0.3 171 this study propionate mixed biofilms 35 7.1 0.04 77 this study butyrate mixed biofilms 35 7.1 0.22 39 this study It should be pointed out here that various measures of biomass have been used by researchers as a base for calculating the substrate utihzation rates; suspended sohd [143], suspended sohd nitrogen [142], particulate protein content [114] and total Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 158 organic carbon (this study). Moreover, the cultures had different distributions of bacterial groups in the fermenter, some containing much more acetate-degrading bacteria due to acetate enrichment, some containing a distribution determined by substrate concentrations in the feed. A n d thus the substrate digestion rate would be different if the rate is based on whole biomass. The next section will discuss this problem further. 6.5 Distribution of Bacterial Groups in Balanced Biofilms As indicated repeatedly, conversion of a mixture of acetate, propionate and bu-tyrate to methane and carbon dioxide needs contributions from at least four dis-tinct functional bacterial groups. This balanced microorganism community con-tains acetogens which convert propionate and butyrate to acetate and H2, and methanogens which utihze acetate and CO2/H2 to produce methane and carbon dioxide. Whenever the biomass retained in a fermenter is referred to as the basis for the determination of the utilization rates of these organic acids, all four groups of bacteria are included. Obviously, the distribution of these bacterial species in biofilms has a great effect on the determination of digestion rates of each acid. It is almost impossible, experimentally, to measure this distribution because so many species are involved in the biological process and no convenient method exists to identify and count them. Even in a defined mixed culture the distribution of each species would change corresponding to changes in the environment, such as avail-ability of substrates and nutrients, temperature, p H , alkahnity, dilution rate and toxic components. This may be one reason why the kinetic information obtained on a single acid could not be used to predict the digestion rates of mixtures of organic acids as found by Scharer and Moo-Young [206]. In the present study, a Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 159 mixture of three acids with a fixed ratio has been used in the adaptation of the inoculum, formation of biofilms and kinetic experiments with an attempt to have and maintain a balanced bacterial community by a sufficient supply of substrates. In this section, an effort will be made to estimate the distribution of the bacterial groups in a mixed and undefined biofilm. Lawrence and McCarty [142] used a single acid as substrate to investigate the methanogenesis of fatty acids. In the case of acetic acid, the culture was quite simple and it was believed that only acetate utihzing methanogens existed. But for propionate or butyrate, at least three functional groups were involved, propionate-or butyrate-utilizing bacteria, hydrogen removing bacteria and acetate fermenta-tion bacteria, even though they did not consider the fraction of microorganisms responsible for the generation of methane from CO2/H2. This approach might be explained by the fact that it is only recently that the important symbiotic function of this fraction of bacteria has been discovered. Their estimation of the bacteria distribution in the 'two-components' sludge was based on thermodynamic princi-ples. The more free energy available as a result of biochemical transformations of substrate, the more bacteria, growth responsible for this substrate dissimilation. More researchers, however, just took the mixed culture as a pure one, and so, their results were based on total volatile suspended solids [143,211,212], on adsorbed particulate organic nitrogen (PON) [114], or on volatile film solids [213]. Some of them only used the reactor volume as the basis for the evaluation of various types of fermenters such as upflow sludge bed reactors and fixed bed reactors [214] even though the retained biomass was the key parameter for these reactors. In a nutrient-rich environment, all bacterial species get enough food supply and grow at their intrinsic maximum growth rate, correspondingly, each substrate is Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 160 consumed at its maximum dissimilation rate, rmax^. Experimentally, this situation can be confirmed by using much higher concentrations of substrates in the fermenter than their half velocity concentrations, Ki. If this rate is based on 1 mg of biomass and only four major functional groups are considered, this one mg of biomass contains ma, m p , mb, nth mgs of bacteria responsible for the utihzation of acetate, propionate, butyrate and hydrogen, respectively. ma + mp + rnb + mh = 1 (mgC) (6.67) that is, mp mb mh ma(l + \u00E2\u0080\u0094 -f 1 ) = 1 (6.68) m\u00E2\u0080\u009E m\u00E2\u0080\u009E m\u00E2\u0080\u009E or, 1 m\u00E2\u0080\u009E = (6.69) 1 + mpjma + mb/ma + mhjma The investigation on build-up of symbiotic methanogenic biofilms revealed that the acetate-, propionate- and butyrate-utihzing bacteria had the same intrinsic maxi-mum growth rate, and this was also reported by Lawrence and McCarty [142] in their studies on single acid methanogenesis. Therefore, the distribution of these three specific bacterial groups in a biofilm mainly depends on their initial attach-ment on the biofilm supports because the initial attached ceUs simultaneously mul-tiply, forming colonies and biofilms. The attachment of each specific bacterial group was expressed as fcja:];, where [x]t- is the free cell concentration in the cul-ture medium and ki the specific attachment rate affected by the physicochemical properties of the supports. ka[x]a (6.70) Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 161 and 771f, fcfcfllf, \u00E2\u0080\u0094 = T T l 6 ' 7 1 m Q . \u00C2\u00AB a [ a ; j a If the number of bacterial cells in unit volume of medium was so high that the attachment rate became a quasi-Oth order rate, TTir) fan , _ , \u00E2\u0080\u0094 p - = (6.72) ma Ka and ^ = \u00C2\u00A3 (6.73) ma ka In the nutrient-rich environment, acetate, propionate and butyrate are being con-sumed at their maximum rates (mg/mgC/hr) ; rp = r>\u00C2\u00B0\"*Sp(l+0SP) ~ ^ ( i + 0.00045p) (6.75) -ftp ~T T\"Tnax,bSb r m a x b . \u00E2\u0080\u0094\u00E2\u0080\u009E\ n ~ Kb + Sb + KrfbSt ~ 1 + 0.00015SP 1 \" ' If propionate concentration in the environment is controlled under 1000 mg/1, but still much higher than 50 mg/1, the half velocity propionate concentration of the propionate dissimilation rate, Equations 6.75 and 6.76 simplify to, rp rmaXtp (6.77) Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 162 anc n = rmax CHA + ZH20 Therefore, the hydrogen removal rate is 3 2 rh \u00E2\u0080\u0094 ~~^~rmax,p + ~j^rmax,b (6.79) where r/, is the hydrogen removal rate by symbiotic bacteria (mmol/mgC/hr) , Mp, Alb the molecular weight of propionate and butyrate. Correspondingly, according to the conversion of CO2/H2 to methane above, the methane production rate from C 0 2 / H 2 utihzation, rma reA This equation imphes that the group which can obtain more energy from its sub-strate utihzation would predominate in the methanogenic sub-community. Replac-ing Equations 6.82, 6.83, 6.80 and 6.81 into Equation 6.84 gives rnh _ 9/MprmaXiP + 6/Mbr max.b in r\r\ ma 4/MarmaXia Put Equations 6.72, 6.73, 6.85 into Equation 6.69 and get Equation 6.86 for esti-mating the amount of biomass (mg organic carbon) responsible for acetate digestion within one mg organic carbon of biofilm in a nutrient-rich environment. 7 7 1 ( 1 = 1 4 - k Ik 4 - hulk 4 - (ZM^r o 4 - \\~U^r ^'86^ 1 ^ \"'PI ^a v Kb/ \" - o i ^ ' max,P ' Mb maxjb )/ ^' max,a Using Equations 6.72, 6.73, 6.85 and the estimated m a expression above gives the mp, mb and m/j, respectively. kp/ka 1 \"1\" k p / k a + kb/ka + ( rmax,p M;, max jo II 1 1 m o i , o h / k a 1 + kp/ka + kb/ka + ( ^rmax,p 4 - 6 M a r ,/Ar Mi, max jo 1 1 1 raoi,a V. Mp ' max,p i M b \u00E2\u0080\u00A2 max :,b ) /^ 4.Tmaxa (6.87) m b = , , / , . , ,. / , . , , 9 M \u00E2\u0080\u009E _ T - TT (6-88) _ - , r ' \" ' In O Q \ 7 7 1 / 1 ~ 1 4 - k Ik A-hulk 4 - 6 M a r .W4- ^ \u00C2\u00B0 - 0 y ' J - ~ \"-p/ > V a ~ \"\u00E2\u0080\u00A2bl \" \" a ~ ^ ^ p ' mox,p ~ ' max.o / / ^ ' max.a Table 6.24 hsts the maximum utihzation rates of fatty acids, r m a X ) t , based on the integrated biomass of a methanogenic biofilm, the fractional biomass responsible Chapter 6. Intrinsic Kinetics of Methanogenesis of Organic Acids 165 for each acid digestion in one mg of the integrated biomass and the recalculated maximum dissimilation rates of the organic acids, r'maxi based on only a fractional biomass being responsible for the specific dissimilation reaction. The hydrogen removing rate is estimated from the Equation 6.79. Table 6.24: Fractional mass of bacterial groups and utihzation rates of fatty acids Substrate ' ' raoi mi r' max (mg/mgC/hr) (mgC,i/mgC) ( g/mgC,i/hr) Acetate 0.098 0.26 0.38 Propionate 0.0162 0.22 0.074 Butyrate 0.0473 0.32 0.148 H 2 0.0035 0.20 0.018 Chapter 7 Mass Transfer in Biofilms Immobilization of microorganisms results in two distinctive phases in a fermenter, the liquid medium phase and the sohd phase of biofilms or bioflocks. Different mechanisms of substrate transfer are involved in these two phases. The hydraulics, the macro-transport of water and substrates within the hquid phase, differs very much from one reactor type to another, just as the significance of a possible hquid film layer which is adjacent to the sohd phase does. However, common to all types of reactors is the process of mass transfer within the sohd phase which is a combination of bacterial ceUs, extracellular polysaccharides and some inorganic salts trapped in the biofilms. Fick's laws of diffusion form the basis for the current theoretical approaches to the mass transfer within the biofilms. 7.1 Diffusivities of Substrates in Water Fick (1855) developed the laws of diffusion by analogy to Fourier's work and defined a one dimensional flux JA as: JA = D A B J 1 (7.90) Where JA is the flux of solute, A , through unit area across which diffusion is occurring, c is the concentration of the solute and / the distance in the solvent B. The quantity DAB, which Fick called \"the constant depending on the nature of the substance\" is the diffusion coefficient of solute A in solvent B. To estimate the diffusivities of various solutes 166 Chapter 7. Mass Transfer in Biofilms 167 in solvents, prediction methods have been developed based on a variety of theories, such as hydrodynamic theory, kinetic theory and statistical mechanics, as reported in several reviews [194,195,196]. Among them the Stokes-Einstein equation [197] RT D A B = ~\u00E2\u0080\u009E =- (7.91) 6-MIBRA has found wide application which considers the solute A as large spherical molecules diffusing in a dilute solution. Many researchers have used it as a starting point in de-veloping their correlations [197]. In Equation 7.91 T)B is the viscosity of the solvent and R 4^ is the radius of the 'spherical' solute. In the present study an old but still widely used correlation for DAB M infinite dilution, the Wilke-Chang (1955) estimation method [198], was used to estimate the diffusivities of lactose, acetic, propionic and butyric acids in the culture mediums. 7.4* lO-8(MBy'2T VBVJ where D\u00C2\u00B0AB = diffusivity of solute A at very low concentrations in solvent B, cm 2 / s = association factor of solvent B, dimensionless MB = molecular weight of solvent B, g/mole T = temperature, K TJB = viscosity of solvent B, cp VA = molar volume of solute A at its normal boiling temperature, c m 3 / m o l . Table 7.25 lists the estimated diffusivities of the concerned solutes and experimental values if available. The values of VA were calculated from Le Bas' additive method [197]. The diffusivity of sucrose is also listed in the table since to this author's knowledge the experimental value of lactose diffusivity in water solutions is unavailable while sucrose has Chapter 7. Mass Transfer in Biofilms 168 Table 7.25: Diffusivities in water at 35 \u00C2\u00B0C Solute vA D x 105 Dexp x 105 Reference (cm 3 /mol) (cm 2/sec) (cm 2/sec) Lactose 291.2 0.72 Sucrose 294.7 0.71 0.75 [199] Acetic acid 68.4 1.71 1.74 [199] Propionic acid 90.6 1.45 Butyric acid 112.8 1.27 the same molecular weight and equivalent molecular size as lactose. Equation 7.92 was also used to convert the experimental data measured at other temperatures to those at 35 \u00C2\u00B0 C . Comparing the calculated diffusivities of acetate and sucrose with those measured experimentally indicates that Wilke-Chang equation 7.92 has a satisfying accuracy. The diffusivity of a solute in infinite dilution implies that each solute molecule is in an environment of pure solvent. From an engineering viewpoint this value might be used to represent the diffusion coefficient for concentrations of solute up to 5 and perhaps 10 mole percent [197]. This reported finding may be due to the fact that the solute molecules in an aqueous solution are densely packed and strongly affected by the force fields of neighboring molecules and even in a dilute solution the major influence still comes from the water molecules. The concentrations of substrates in this study were not higher than 10 g/1. Although other components hke nutrients and products exist in the culture medium, the substrate molecules are still mostly surrounded by the water molecules, and hence the medium can be thought of as a dilute solution. 7.2 Principle of Mass Transfer Measurement within Biofilms As indicated in Chapter 2, the biofilms contain many irregular channels through which the substrates transport from outer to inner layers by molecular diffusion to replenish Chapter 7. Mass Transfer in Biofilms 169 the consumed substrates. The channels have very complicated structures, tortuous and even dead-ends. Therefore, it is almost impossible to describe them mathematically. It is common, analogous to reactions in a porous catalyst, to express the diffusion in the biofilm by a parameter, De, the effective diffusion coefficient. In this way, the transfer of substrate molecules in the biofilm can be imagined as proceeding in a homogeneous medium and still can be described by Fick's law, Compared with Equation 7.90, this modified Fick's law only replaced the diffusivity, DAB, with the effective diffusivity, De. When an active biofilm slab is immersed in a culture medium, the substrate molecules transport from the bulk solution to the outer face of the biofilm, and then, at the same time they penetrate into the biofilm, some of them are digested by the bacteria embedded in the biofilm. Finally a steady state is achieved and the substrate concentration has a distribution within the biofilm as shown in Figure 7.63. Since in an active biofilm the substrate diffusion and digestion occur simultaneously, a diffusion-reaction model could be deduced by making a material balance around the elemental volume in Figure 7.63. Usually, biofilms are quite thin with the thickness ranging from tens to several hundreds of micrometers and thus the mass transfer from the side areas is negligible. A diffusion-reaction model with one dimensional diffusion is deduced as follows; (7.93) Diffusion in = Diffusion out + Consumption by reaction ADe^(S - ^-Al) = -ADe~ + AlApcr (7.94) Chapter 7. Mass Transfer in Biofilms 170 Figure 7.63: Substrate concentration distribution within a biofilm at steady state, The elemental volume has a thickness Al and an area A . ADe\u00E2\u0080\u0094Al = AlAPcr (7.95) d2S D e \u00E2\u0080\u0094 = p c r (7.96) Where De is the effective diffusion coefficient of substrate molecules in the biofilm (cm 2/sec), S the substrate concentration (mg/cm 3 ) , pc the density of the biofilm (mgC / c m 3 ) , r the substrate utilization rate based on unit weight of biofilm (mg/mgC/sec). Al and A are the thickness and area of the elemental volume respectively. The left side of Equation 7.96 is the net amount of substrate transported into a unit volume of the biofilm and which should be digested by the bacteria embedded in this volume at steady state as represented by the right side of the equation. In situ measurement of substrate transport in active biofilms requires the knowledge of the intrinsic kinetics of substrate Chapter 7. Mass Transfer in Biofilms 171 S Figure 7.64: Influence of De on substrate concentration distribution within a biofilm, D'e > D'e' > D?. utihzation by the target bacteria. The previous two chapters have described the intrinsic kinetics of lactose digestion in acidogenic biofilms and of organic acids degradation in methanogenic biofilms. Mathematically, Equation 7.96 can be solved by a numeric method when two bound-ary conditions are determined. S = Si at / = L (biofilm surface) dS/dl = 0 at / = 0 (due to the symmetry of biofilms) Therefore, a family of curves can be obtained which describe the substrate concentra-tion distribution depending on the values of effective diffusion coefficient as shown in Figure 7.64. Theoretically, if another experimentally determined value can be found, for example, Chapter 7. Mass Transfer in Biofilms 172 Se, the substrate concentration at the center of the biofilm slab, the substrate distri-bution within the biofilm can be determined uniquely, and thus, the effective diffusion coefficient. In this sense, the effective diffusion coefficient, De, becomes a model parame-ter of the reaction-diffusion model rather than a physical parameter determined only by the properties of the solute and the pseudo-homogeneous solvent (medium plus biofilms). However, as long as the assumption of one dimensional diffusion holds for the actual pro-cesses such as substrate diffusion in thin biofilms or bioflocks, the possible error caused by the assumption can be minimized or eliminated. Putting the utihzation rate expressions for lactose and organic acids into the diffusion-reaction model, Equation 7.96, gives a second-order ordinary differential equation; d<1P=P<-K-TS (7'97) A numerical method must be used to solve this equation. If the boundary conditions at / = 0 (Se and dS/dl) are known, the boundary condition problem can be solved as an initial value problem and the value of De can be determined by fitting the experimental value of Si, the substrate concentration on the biofilm surface, with the calculated value from Equation 7.97. In this study, the Runge-Kutta-Fehlberg ( R K F ) method, was se-lected from many available numerical methods [200] to solve this initial value problem. The R K F method (see Appendix G) has the advantages of requiring fewer calculations and having the abihty to control local truncation error and its local error is less than h 6 (h is the step size). So, the key point in the present work is how to measure the substrate concentration at the center of a biofilm, Se, as well as the concentration on the biofilm surface, Si, and also how to meet the boundary condition at the center of the biofilm, dS/dl = 0. Chapter 7. Mass Transfer in Biofilms 173 7.3 Experimental Setup The general experimental setup has been described in Chapter 3, the same one as for the kinetic studies. The major difference is the support on which biofilms formed. In the kinetic experiments the support surface was made of many P V C sheets (1.5 mm thick) and thus the biomass on the supports was a major part of whole biomass retained in the reactor. In the measurement of substrate diffusivities within biofilms, however, a diffusion cell was used since the attention was focused on the biofilm growing on the membrane filters and other biomass was not very important. The device's structure has also been described in Chapter 3. On the each side of the cell a cellulose nitrate membrane filter (pore size = 0.45 /xm, Sartorius, W . Germany) was fixed by sandwiching it between two rubber gaskets, and hence, the bacteria (d = 1 pm) could not enter into the cell while the substrates could be transported into the cell through the layers of biofilm and the membrane filters. This was confirmed by the observation that there was no water leakage after replacing the membrane filters with plastic films. Biofilms (acidogenic or methanogenic ) formed uniformly on the filters and had the same thickness on each side of the cell as shown in Figure 7.65. Because no bacteria was inside the cell due to the pore size of the filters, the sub-strates which moved into the cell would not be further digested, therefore, the substrate concentration within the cell, Se, equals that on the other side of the membrane filter, S'e, if a steady state was maintained. This condition could exist only at steady state because at unsteady states, mass transfer occurred across the filter, and thus, Se ^ S'e. The medium within the cell was recycled to promote a uniform concentration as well as the establishment of steady state. Moreover, because of the symmetry of both the cell's Chapter 7. Mass Transfer in Biofilms 174 S S b = Sj Biofilm Figure 7.65: The biofilms symmetrically fixed on the two membrane filters of the diffu-sion-measuring cell structure and the biofilms formed on the two membrane filters, the substrate concentra-tion indice the divice, Se, was the concentration at the biofilm center as mentioned in the last section if the two biofilms on the membranes were thought of as the two half biofilms shown in Figure 7.63. The symmetrical characteristics of the diffusion cell also gave another boundary condition at steady state, dS/dl = 0, at each membrane filter. The two boundary conditions at 1=0 are known or experimentally available from the discussion above. The third one, the substrate concentration on the biofilm surface, Si, can be determined from the bulk concentration if the (imagined) hquid film thickness adjacent to the biofilm is known. Experimentally, the hquid velocity over the biofilm can be increased to such an extent that the hquid film or its effect is neghgible and the substrate concentration on the biofilm surface approaches that of the bulk solution. Chapter 7. Mass Transfer in Biofilms 175 7.4 Diffusivity of Lactose in Acidogenic Biofilms As in the experiments made to measure the kinetics of lactose utilization, the feed con-tains lactose as the limited organic substrate and inorganic salts as growth nutrients (listed in Table 3.9). The lactose concentration in the feed was 5-6 g/1. To prevent lactose from being digested in the feed tank, the same measures as mentioned in lac-tose acidogenesis kinetics were used, i.e. low temperature, low oxygen partial pressure and small amount of feed prepared each time. The p H of the culture was controlled at 4 . 6 \u00C2\u00B1 0 . 2 with 0.5 N N a O H solution. The medium in the reactor was recycled to produce a uniform concentration and temperature distribution in the reactor, and also, to reduce the external mass transfer resistance by increasing the recycle flow rate. The temperature was maintained at 3 5 \u00C2\u00B1 2 \u00C2\u00B0C. In situ measurement of lactose diffusivity in acidogenic biofilms was carried out in two stages, formation of biofilms on the cell's membrane and measuring lactose concentrations outside and inside the cell at steady state. The fermenter was first fed with a diluted feed (2 g lactose/1) and N 2 was introduced to strip off the oxygen dissolved in the medium. After the temperature and p H of the medium were adjusted to 35 \u00C2\u00B0C and 4.6, respectively, the diffusion device which had a capacity of 30 c m 3 was immersed in the medium. Gradually the medium penetrated into the device through the membranes. When the hquid outside and inside the cell reached the same level, recycle of the hquid within the cell was started. Analyses for lactose showed the same concentrations outside and inside the cell. Under a N 2 atmosphere the fermenter was seeded with 50 ml of inoculum collected from the effluent of lactose acidogenesis. The fermenter was then operated in batch mode. To obtain qualitative information on the formation of the biofilms, when the cell was immersed in the reactor some P V C shdes, used as in the kinetic studies, were also immersed into the medium Chapter 7. Mass Transfer in Biofilms 176 since it was believed that biofilm formation was easier on the membranes than on P V C sheets. After the lactose had been used up, half the volume of the culture (500 ml) was replaced by a fresh feed (5 g lactose/1) and this was repeated until a biofilm had formed on the membranes. After biofilms were observed on the removable P V C shdes the reactor operation was shifted to continuous mode. Every seven days the biofilm on a removable P V C shde was measured and the second stage started when a thick biofilm (ca. 1 mm) had formed. The second stage was to measure the steady-state lactose concentrations outside and inside the cell. To ensure that a steady-state concentration was being measured, a distur-bance was given to the concentration inside the ceU while the outside concentration was maintained as stable as possible. This was done by replacing 20 ml of the hquid within the cell with the same volume of distilled water which had been stripped of dissolved 0 2 by N 2 , and then the lactose concentration within the cell was monitored until there was no further change in the concentration. Figure 7.66 shows that the lactose concentration inside the device, after dilution with oxygen-free distilled water, approached a constant value, which means that a steady state was reached. To confirm this, the dynamic behavior of the device was further investigated. When the lactose concentrations inside and outside the cell were at a steady state, the feed flow rate, and thus the outside concentration was gradually reduced to a new level, and then returned to the original flow rate as shown in Figure 7.67. It was found that the inside lactose concentration, corresponding to the change in the lactose concentration of the bulk solution, also decreased to a lower value of 30 mg/ l from a steady-state of 46 m g / l and then it returned to the original level of 45 mg/ l as shown in Figure 7.68. This indicated that the dynamic response of the cell was quite good and that as long as Chapter 7. Mass Transfer in Biofilms 177 100 - i o H \u00E2\u0080\u0094 i \u00E2\u0080\u0094 i \u00E2\u0080\u0094 i \u00E2\u0080\u0094 i \u00E2\u0080\u0094 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 i \u00E2\u0080\u0094 * \u00E2\u0080\u0094 i \u00E2\u0080\u0094 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 i \u00E2\u0080\u0094 i \u00E2\u0080\u0094 i \u00E2\u0080\u0094 ' \u00E2\u0080\u0094 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 ' \u00E2\u0080\u0094 i \u00E2\u0080\u0094 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 ' \u00E2\u0080\u0094 i \u00E2\u0080\u0094 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 \u00E2\u0080\u00A2 \u00E2\u0080\u0094 i 0 40 80 120 160 200 240 Time (hr) Figure 7.66: A steady-state lactose concentration inside the cell was established after a dilution. The lactose concentration outside the cell ranged from 1750 to 1900 mg/1. a steady-state outside concentration was maintained longer than 5 hours, a correspond-ing steady-state inside lactose concentration could be obtained because under normal operating conditions the inside lactose concentration fluctuation was not more than 15 mg/1. This dynamic behavior also made it possible to use reliable operating conditions since fluctuations in the outside lactose concentration was unavoidable, however it was relatively easy to keep it at a steady state for 5 - 8 hours. Moreover, during this short period of time the biofilm thickness (see Chapter 4) would not increase markedly and a quasi-steady state biofilm thickness could be assumed. As shown in the experimental results of the kinetic studies in Chapter 4, the external mass transfer could be reduced to a negligible extent by increasing the recycle velocity, and thus the velocity over the biofilms. Under this condition the lactose concentration Chapter 7. Mass Transfer in Biofilms 178 4 0 0 0 - , Time (hr) 1 0 0 Figure 7.67: A controlled change in the lactose concentration of bulk culture solution 40 eo Time (hr) 8 0 1 0 0 Figure 7.68: Dynamic response of lactose concentration inside the cell to the controlled change in the outside lactose concentration Chapter 7. Mass Transfer in Biofilms 179 80 - i . . 70 -a -E 6 0 -c o 5 0 -l _ *-> en 4 0 -o c o 3 0 -o CD \u00E2\u0080\u00A2 OS 2 0 -o CO _1 10-0-G - -e- -o 10 12 14 16 Recycle rate (l/hr) \u00E2\u0080\u0094 i \u00E2\u0080\u0094 18 \u00E2\u0080\u0094 i \u00E2\u0080\u0094 20 22 Figure 7.69: Effect of recycle rate on the lactose concentration inside the cell. The lactose concentration in the bulk solution ranged from 1857 to 1750 mg/ l on the biofilms, S;, was very close to the concentration, Sb, in the bulk solution which was measurable. Figure 7.69 indicates that under different recycle rates the steady-state lactose concentration inside the cell did not change markedly when the outside concentration was kept within a narrow range. This fact implies: (1) that the major resistance to lactose transfer from the bulk solution to the cell was in the biofilm and (2) that the mass transfer resistance between the bulk solution and the biofilm surface was neghgible. Therefore, with a recycle rate of 220 ml /min the bulk solution, Sb, was a reasonable approximation of Si. Finally after steady-state lactose concentrations outside and inside the cell had been measured and judged to be satisfactory, the diffusion cell was taken out of the fermenter and the biofilms on the membrane filters were carefully collected and analyzed according to the procedures described in Chapter 3. The biofilms on the two membrane filters had Chapter 7. Mass Transfer in Biofilms 180 Table 7.26: Effective diffusivity of lactose within acidogenic biofilms at 35 \u00C2\u00B0C Parameter Unit Value sb mg/1 2114 Se mg/1 45 L mm 1.3 Pc m g C / m l 29.7 ^max m g / m g C / h r 0.3144 K. mg/1 x l O - 5 cm 2 /sec 201 0.47 De/D 0.653 almost the same thickness, 1.32 mm and 1.29 mm respectively, which means that the biofilms were symmetrical. Therefore, the prerequisite on the boundary conditions at I = 0 as discussed in the last section was satisfied and the conditions were reliable. By using these data and the numerical solution method, the diffusivity of lactose within the acidogenic biofilms was estimated and the results are listed in Table 7.26. Obviously, the value of lactose effective diffusivity in acidogenic biofilms estimated in this way depended on many factors even if the calculating error of the numerical method was controlled to within a reasonable range (< 10 - 4 ) . It was affected not only by the calculated values of the intrinsic lactose utihzation rate in biofilms, K\u00E2\u0080\u009E and rmax, but also by the biofilm thickness and density, L and pc, as well as the lactose concentrations inside and outside the diffusion cell, Se and Sb. Table 7.27 compares the influence of these data on the effective diffusivity, De, (the sensitivity of De to the possible error of these data) by giving one of them a 10 % fluctuation while the rest are unchanged. The most sensitive parameter was the biofilm thickness which could give a 15 % error and the least sensitive ones were concentration inside the device, Se and the kinetic parameter Ks since they gave a corresponding error less than 5 %. The error caused in the other parameters was not amplified or reduced. Therefore, experimentally, more attention was Chapter 7. Mass Transfer in Biofilms 181 Table 7.27: Sensitivity of lactose effective diffusivity to experimental error Parameter sb L Pc Tmax Ks Unit mg/ l mg/ l mm m g C / m l m g / m g C / h r mg/ l Value 45 2114 1.3 29.7 0.314 201 Error (%) +10 + 10 +10 + 10 +10 +10 De error (%) +2.9 -7.1 +15.9 +10 +10 -4.8 Error (%) -10 -10 -10 -10 -10 -10 De error (%) -3.1 +8.4 -14.9 -10 -10 +5.2 given to the measurement of the sensitive parameters like the biofilm thickness, density, and the maximum utihzation rate to reduce the possible error. 7.5 Effective Diffusivities of Organic Acids in Methanogenic Biofilms Using the same diffusion-measuring device and the fermenter as described before, the process for measuring the effective diffusivities of organic acids in methanogenic biofilms could also be divided into two stages, formation of the biofilms on the two membrane filters of the ceU and then measuring the organic acids concentrations inside and outside the cell. 7.5.1 Formation of Symbiotic Methanogenic Biofilms In order to build up balanced methanogenic biofilms which comprised acetate-, propio-nate- and butyrate-utihzing bacteria, on the membranes of the device, an inoculum was collected from a methanogenic phase reactor which was being fed with a mixture of organic acids. Also, the feed was a mixture of acetate, propionate and butyrate at concentrations of 5, 2.5, 2.5 (g/1), respectively, which could supply enough substrates to each bacterial group in the fermenter. The inorganic salts hsted in Table 3.9 were used as growth nutrients. Chapter 7. Mass Transfer in Biofilms 182 The reactor was firstly filled with 500 ml of tap water and 500 ml of culture medium and nitrogen was introduced to strip out the dissolved oxygen. The cell was immersed into the solution, and the hquid penetrated into the cell through the membrane filters. After the hquid surface inside the cell reached the same level as that of the culture medium outside the cell, recycling was started. The p H and temperature in the reactor were adjusted to 7 . 1 \u00C2\u00B1 0 . 2 , 3 5 \u00C2\u00B1 2 \u00C2\u00B0 C , respectively. Under a nitrogen atmosphere the reactor was seeded with 50 ml of the inoculum then operated in a batch mode. After the organic acids had been used up by the bacteria, 500 ml of culture medium was replaced by 500 ml of fresh feed, which was repeated till black bacterial colonies were observed on a removable P V C shde which was inserted into the reactor at the same time as the ceU was immersed into the medium. Afterwards, the reactor was shifted to continuous flow mode. The flow rate was gradually changed to and maintained at 40 ml/hr (dilution rate ~ 0.04 h r - 1 ) . At this rate the organic acids concentrations in the fermenter were kept high enough, 3 g/1, 1.5 g/1 and 0.5 g/1 for acetate, propionate and butyrate respectively, to support the bacterial growth at their maximum rates. 7.5.2 Effective Diffusivities of Propionate and Butyrate in the Biofilms As indicated previously the diffusion-reaction model, Equation 7.96, which describes a substrate concentration distribution within a biofilm can be solved numerically or ana-lytically and the model parameter, De, can be determined if three boundary conditions are measured experimentally; the substrate concentration on the biofilm surface, S{, and the substrate concentration inside the device, Se, and the concentration gradient, dS/dl, at the membrane filters. The last one (dS/dl = 0) can be assumed confidently as long as the biofilms on the two membrane filters have the same thickness, which was to a great extent dependent on the symmetrical structures of the device and the fermenter. Thickness measurements of the biofilms on the two membrane filters at the end of the Chapter 7. Mass Transfer in Biofilms 183 2 4 0 0 n 2 2 0 0 -E 2000;= ioooH 1 1 . 1 . 1 . 1 0 20 40 60 80 Time (hr) Figure 7.70: Establishment of a steady state propionate concentration inside the cell (o) after a dilution. \u00E2\u0080\u00A2 - propionate concentration outside the cell experiments confirmed the accuracy of this assumption. Using the substrate concentration inside the cell as the value at the interface between the biofilm and the membrane filter would be rehable only when a steady state throughout the biofilm was estabhshed and no microbes existed inside the cell. Estabhshment of a steady state was checked by displacing 20 ml of the medium inside the cell with the same volume of oxygen-free distilled water and then monitoring the concentration change while the outside concentration was kept as constant as possible. That the concentrations of propionate and butyrate inside the cell, after dilution with the oxygen-free distilled water, gradually approached a constant as shown in Figure 7.70 and Figure 7.71 implied that a steady state was estabhshed. This dilution operation was repeated eight times both to confirm the estabhshment of a steady state in the films and to minimize the biological activity inside the cell. It Chapter 7. Mass Transfer in Biofilms 184 800-1 7 0 0 -100-0-| . 1 \u00E2\u0080\u00A2 i ' 1 \u00E2\u0080\u00A2 1 0 20 40 60 80 Time (hr)' Figure 7.71: Establishment of a steady state butyrate concentration inside the cell (o) after a dilution. \u00E2\u0080\u00A2 - butyrate concentration outside the cell was beheved that the number of microbes inside the cell could be neglected because of the filtering action of the membrane filters (pore size 0.45 pm) and the dilution of the medium inside the dive with oxygen-free water. The bulk substrate concentrations were taken as those on the biofilm surface. This assumption, however, was reliable only when the external mass transfer resistance was small. The effect of external mass transfer resistance was to reduce the substrate con-centration on the biofilm surface and thus the substrate concentration inside the cell. If this resistance is minimized or the external mass transfer is far faster than the rates of substrate digestion and internal mass transfer within the biofilms, the substrate con-centration on the biofilm surface would be very close to the bulk concentration, and correspondingly the inside concentration would reach its maximum value. Experimen-tally this was checked by increasing the recycle rate to a value above which the inside Chapter 7. Mass Transfer in Biofilms 185 2 1 0 0 - 1 ^ 2 0 0 0 -E c o 1 9 0 0 -co \u00C2\u00A3 1 8 0 0 -c CD O O 1 7 0 0 -U 1 6 0 0 -L3--\u00E2\u0082\u00AC> 1 5 0 0 -8 10 12 14 Recycle rate (l/hr) 16 18 Figure 7.72: Effect of recycle rate on propionate concentration inside the cell (o), \u00E2\u0080\u00A2 porpionate concentration outside the cell. 8 0 0 - i 7 0 0 |\u00C2\u00BB 6 0 0 -\u00C2\u00A7 5 0 0 -CO \u00C2\u00A3 4 0 0 -c CD C 3 0 0 O O 3 200-\ m X 1 0 0 B - - B --e-8 10 12 14 Recycle rate (l/hr) -O 16 18 Figure 7.73: Effect of recycle rate on butyrate concentration inside the cell (o), \u00E2\u0080\u00A2 -butyrate concentration outside the cell. Chapter 7. Mass Transfer in Biofilms 186 concentrations leveled off. Figure 7.72 and Figure 7.73 show that when the recycle rate was raised above 11.4 l/hr, (superficial velocity = 3 cm/min), the concentrations of pro-pionate and butyrate inside the cell approached their constant values, which indicated that the external mass transfer resistance had been minimized and was not a rate limiting step. Thereby, the bulk substrate concentrations could be reasonably taken as those on ts the biofilm surface. Steady state concentrations of propionate and butyrate inside and outside the cell were averaged for four measurements which were conducted at a recycle rate of 16 l/hr. Substituting the intrinsic utihzation rates of propionate and butyrate, Equations 6.75 and 6.76, for the reaction rate on the right side of the diffusion-reaction model (Equation 7.96) gives the following equations which describe diffusion and dissimilation of these two acids in the methanogenic biofilm, n d2Sp rmaa. iP5p(l 4 - 3SP) D^~dP = * \u00E2\u0080\u0094 K p T S p \u00E2\u0080\u0094 ( 7 - 9 8 ) D ^ = P < K b + Sb + KlSpSb- ( 7 ' 9 9 ) Where the subscripts, p and 6, refer to propionate and butyrate. The reaction rates for each of the acids are based on an integrated biomass (mg/mgC/hr) , i.e., a biomass containing a mixture of bacterial groups responsible for the utihzation of various sub-strates, as discussed in Chapter 6. Correspondingly the density of the biomass, pc, has units of carbon content in unit volume of the integrated biomass (mgC/ml). The density and thickness of the biofilms on the membrane filters were measured according to the steps described in Chapter 3. For each of the equations above, three boundary conditions were known; Sp = SeiP and dSp/dl = 0, at I = 0, Chapter 7. Mass Transfer in Biofilms 187 Table 7.28: Effective diffusivities of propionate and butyrate Item Unit Propionate e Butyrate sb mg/l 1805 515 Se mg/l 1725 326 Pc m g C / m l 26.8 26.8 L mm 0.78 0.78 Tmax m g / m g C / h r 0.0162 0.0473 K. mg/l 50 27 De x l 0 5 c m 2 / s e c 0.60 0.357 DJD 0.41 0.281 sb = SEib and dSb/dl = 0, a U = 0, sP = SbiP and Sb -: sbib, at I = L. Where L is the biofilm thickness, Se

[ l + u - K > [ 1 A model with 21 parameters has been found to be able to fit 12 equations hke the equation above. For each type of bacteria, a linear relationship is found between Chapter 8. Conclusions and Recommendations 205 the model parameter, k. which is a specific attachment rate of free bacterial cells onto clean surfaces, and the wettabihty of the surfaces. If it is explained that the bacteria with more hydrophilic surfaces attach more easily to more hydrophihc supports, then the order of decreasing hydrophobicity of bacterial wall surfaces is; The rate of spread of bacterial microcolonies on support surfaces was also found to be influenced by the wettabihty of the substrata. In this case, the spreading factor, s, that is, the ease with which the microcolonies spread on surfaces, was found to increase with the hydrophobicity of the surfaces. Compared with the rate of attachment of bacteria onto a clean surface and that of fixed bacterial growth, the attachment of free ceUs onto immobilized biomass already on the surface was neghgible. The specific growth rates of the three types of bacteria were found to be the same, and therefore, the composition of a fixed biomass, i.e., the distribution of each group of bacteria in the fixed bacterial community, is to a great extent dependent on the initial attachment rate of each type of bacteria which is affected by the wettabihty of the supports. 3. Again after external and internal mass transfer resistance had been experimen-tally minimized and/or eliminated, biomethanation kinetics of organic acids in methanogenic biofilms were investigated at a p H of 7.1 and 35 \u00C2\u00B0C. The substrate was a mixture of organic acids containing acetic, propionic and butyric acids at a weight ratio of 1 : 0.5 : 0.5 (mg/l). The degradation rate of acetate can be expressed by a Michaelis-Menten equation, butyrate degraders > acetate degraders > propionate degraders rHAc = 0.0985/fA c 160 + SHAc (mg H A c / m g C / h r ) Chapter 8. Conclusions and Recommendations 206 However, the degradation of propionate and butyrate are affected by the concen-tration of propionic acid in the medium, and so, two modified Michaelis-Menten equations are used for the assimilation rates of propionic and butyric acids. For propionic acid utilization, higher propionic acid concentration promotes its degra-dation, 0.0162S H Pr(l + 0.0002Stfp r) rHPr = zrit c ( m \u00C2\u00A7 H P r / m g C / h r ) 50 + bHPT O n the other hand, the utihzation of butyric acid is inhibited by high concentrations of propionate, 0 . 0 4 7 3 S g B \u00E2\u0080\u009E = 2 7 + S g . , + 0 . 0 0 0 2 9 6 W \u00E2\u0080\u009E , ( m g H B\"/m\u00C2\u00ABC/h r> In the three equations above, the rates are based on unit weight of carbon in the methanogenic biofilms and the concentrations of fatty acids in mg/1. To check the interaction between the three organic acids, feeds having various ratios of the three organic acids were also used and it was found that only propionic acid concentration had promotional and inhibitory influences as described above. 4. A methanogenic biofilm is a symbiotic bacterial community including four fun-damental types of bacteria, acetate degraders, propionate degraders, butyrate de-graders and hydrogen-utihzing bacteria. A method has been proposed to estimate the distribution of each bacterial group in a methanogenic biofilm from the knowl-edge of the attachment rate of methanogens and acetogens on clean surfaces, the kinetics of utihzation of the organic acids, and the hydrogen and the energy balance in mature methanogenic biofilms. The compositions (mg carbon of the ith bacterial type per mg carbon of total biomass) of mature symbiotic methanogenic biofilms Chapter 8. Conclusions and Recommendations 207 Table 8.33: Organic carbon fraction of each bacterial group in methanogenic biofilms on P V C and nitrocellulose membrane filter supports Substrate Acetate Propionate Butyrate Hydrogen degraders wt% wt% wt% wt% on P V C 25.5 22.0 31.8 20.7 on membrane 26.3 16.0 37.5 20.2 on P V C supports and nitrocellose membrane filters were estimated and hsted in Table 8.33. 5. Based on the intrinsic kinetics of lactose fermentation in acidogenic biofilms and organic acids digestion in methanogenic biofilms, in situ measurements of mass transfer of lactose and organic acids were conducted in active biofilms. Effective diffusivities of lactose and organic acids, defined by a modified Fick's law and a diffusion-reaction model, were estimated from steady state concentration drops through an active biofilm by numerically solving a diffusion-reaction model. The effective diffusivity of lactose in an acidogenic biofilm is 0 . 4 7 x l O - 5 cm 2 /sec that is 65.3% of its diffusivity in pure water. After a correction was made to allow for the fact that the composition of bacterial types might be somewhat different in methanogenic biofilms fixed on both P V C and nitroceUulose filter supports, the effective diffusivities of acetate, propionate and butyrate were found to be 0.558 x l O - 5 , 0 .4374xl0 - 5 and 0.421 x l O - 5 cm 2/sec, respectively, which means that diffusivities of acetate, propionate and butyrate in methanogenic biofilms have been reduced to about 30.2 % of the values in pure water. By using these data and the experimentaUy measured fractional void volume of biofilms, tortuosity factor r , a parameter describing the structure of channels formed in biofilms, was estimated for acidogenic biofilms (r = 1.03) and methanogenic biofilms (r = 1.69) respectively. Chapter 8. Conclusions and Recommendations 208 A comparison was also made between other properties of these two kind of biofilms. 8.2 Recommendations Going back to the questions put froward in Chapter 1, two problems have been tentatively solved in this study, i.e. the measurement of the intrinsic kinetics and mass transfer of lactose and organic acids in the corresponding biofilms, and so it may be possible to calculate an optimum biofilm thickness. Further studies are, however, needed on how to control a growing biofilm at a steady state thickness so as to maintain an optimum operation for a long time. 8.2.1 Concentration Effect Obviously, an optimum biofilm thickness with which a fermenter can hold up the largest amount of active biomass without dead volume caused by excess biomass is dependent on substrate concentrations in the bulk medium and at the interface between a biofilm and a support. The former mainly depends on the flow pattern in a reactor while the latter would be determined mainly by the process designer, e.g, a concentration which gives a half maximum reaction rate, (K\u00E2\u0080\u009E). From the effect of concentration on optimum biofilm thickness, a biofilm should have a thickness which gradually decreases with the reduced substrate concentration from the inlet to the outlet. Therefore, a plug flow of medium over a biofilm with decreasing thickness as shown in Figure 8.79 may be an ideal for optimum operation. 8.2.2 Controlling Biofilm Thickness To this author's knowledge, few studies have been conducted on how to control a growing biofilm at a constant film thickness though some investigations have been conducted Chapter 8- Conclusions and Recommendations 209 S So Reactor Inlet Outlet Figure 8.79: Illustration of an optimum operation of biofilm reactors. No back- mixing of medium is assumed. on the influence of shear stress of fluid flow on biofilms. Most of investigations were concentrated on the formation of biofilms or on the possibility of biofilm technology being apphed to a special wastewater. It may be thought that a steady state biofilm would be established automatically by the action of fluid flow stress and sloughing. However, in most cases of present industrial applications, the fluid flow rates are quite low due to the long residence time required for the slow utihzation rate of substrates and so it can not be used purposely to control the biofilm thickness. In some fluidized bed reactors the biofilm on sand carriers are very thin due to strong turbulent flow, and hence, the thickness is controlled, but not kept at an optimum value. Sloughing is not a good method, either, because it occurs randomly with a result that block biomass would be lost and a bare support left. Moreover, it has been observed in this investigation that an over thick acidogenic biofilm (3-4 mm) could hold quite strongly on a membrane filter even though Chapter 8. Conclusions and Recommendations 210 the substrate concentration within the biofilm had fallen to zero at a thickness of about 2 mm. Another experimental observation which shows that sloughing does not occur easily was that anaerobic biofilms, especially the acidogenic biofilms, could stand in a pure water for a very long time (up to 7 days) without falling off. Although there are many factors which can influence attachment and formation of biofilms (genetic, physiological, environmental, chemical factors) as discussed in Chapter 2, those which can be used to control the size of microbial aggregations (thickness of biofilms, size of granules) are generally limited to chemical or physical methods. The chemical methods utilize some chemicals such as p H , chelating agents (hke E D T A ) , to redisperse the fixed biomass into the medium, or, a decrease in the amount of chemicals in a medium which are beneficial for the formation of the extracellular polymer matrix such as Ca++ could also reduce film thickness. The most striking advantage of a chemical method is that it would not cause extra back-mixing in a fermenter and thus an approach to the ideal flow pattern shown in Figure 8.79 can be attained. However, it may not be easy for an operator to use chemicals to control biofilm thickness by redispersing immobilized biomass, and also, the lysis of bacteria which have been damaged by the chemicals may introduce extra operational difficulties such as foaming in a fermenter as observed in this study. Another method is by physical means as have been utilized in fluidized bed fermenters. As indicated above, the active biofilm on supports (usually sand) can be kept very thin due to the interactions among particles and/or between particles and liquids, but it is difficult to control the biofilm thickness at a desirable value by this method. Moreover, with this method the turbulence of fluid flow and thus the back-mixing in the fermenter must be increased, which greatly decreases the thickness of an active biofilm and the hold-up of active biomass in the fermenter while the bulk medium concentration approaches the outlet concentration due to the back-mixing. Chapter 8. Conclusions and Recommendations 211 Gas Gas ,Gas III -a -a Support size Figure 8.80: Illustration of a process of three stages of cheese whey treatment 8.2.3 A Compromised Physical Method With a physical method to control the biofilm thickness, the excess biomass can be easily removed from the fermenter, thus, a long term stable operation can be estabhshed. First of all, considering the concentration effect on biofilm thickness and the complete back-mixing caused by fluid flow, it is suggested that the process of cheese whey treatment be divided into several stages connected in series, not only two stages (or two phases) and each stage can be operated with a small amount of back-mixing. Figure 8.80 shows a process of three stages. The distribution of acidogens and methanogens (with acetogens) is flexible. It may be possible for the first stage to act as the acidogenic phase and the following two stages as the methanogenic phase. One recommended support is balls of porous materials such as natural sponge or polyurethane foam which can entrap the active biomass within their pores. The size Chapter 8. Conclusions and Recommendations 212 of the balls, however, should be controlled according to the optimum biofilm thickness calculated for the special conditions in a stage. In general, the first stage has the balls with the biggest diameter, with gradually decreased ball size in the following stages. The last stage may use sand as its support if the desirable biofilm thickness is very thin. The excess biomass produced by the growing biofilms can be removed from the surface of the balls by fluidizing the reactors as with sand fluidized beds while the biomass within the balls will always be active throughout the balls because of their controlled sizes. As is well known, the microbial growth in the first one or two stages will be the fastest because of the faster intrinsic growth rate of acid-producing bacteria and the higher substrate concentrations. Therefore most of the excess biomass should be removed in these stages. Another arrangement is recommended in which the first two stages are operated in a fluidized bed mode having supports as suggested above, while the last stage is operated in a plug flow mode with stationary supports such as needle-punched P V C . The removal of excess biomass from the stationary supports may be performed periodically by recychng the medium which would add a stress on the biofilm or by adding stripping chemicals. In conclusion, a long term optimal and stable operation of fermenters using immo-bilized microorganisms depends to great extent on whether the excess biomass due to microbial growth can be removed from the reactors smoothly and continuouly to main-tain the size of microbial aggregates at an optimum value. The results of this study have offered some basic data for calculating the optimum size of the microbial aggregates, but more investigations are needed on how to control a growing biofilm at this desirable thickness. Bibliography F. Kosikowski, \"Cheese and fermental milk foods\", F . V . Kosikowski and Associates, Brooktindale, New York, p455 (1977) O C D E , \" M i l k and milk products balances in O C D E contries 1979-1987\", Head of Pubhcation Service, O E C D , France, (1989) Beak Engineering Ltd, \"Anaerobic treatment of dairy effluent\", Report E P S 3 / F P / 1 , Environmental Canada, Ontario, Canada, (1986) S.W. Watson, A . E . Peterson and R . D . Powell, \"Benefits of spreading whey on agricultural land\", Journal of Water Pollution Control Federation, 49:24-34 (1977) P . G . Muller, \"Economic evaluation of feeding whey to hvestocks, part 2 \" , Project Report for Food Research Institute, Research Branch, Agriculture Canada, (1979) R. Hirschl and F . V . Kosikowski, \" A new type of acid whey concentrate product derived from ultrafiltration\", Journal of Dairy Science, 58:793 (1975) E . H . Marth, \"Fermetation products from whey\" in \"Byproducts from milk\", edited by B . H . Webb, The Avi Pubhshing Company, Inc. , Westport, Connecticut, (1970) I. Galsmar and A . Bergmann, \"Spray drying of whey\", Journal of the Society of Dairy Technology, 20:106-110 (1967) T . A . Nickerson, \"Lactose\" in \"Byproducts from milk\", edited by B . H . Webb, The Avi Pubhshing Company, Inc., Westport, Connecticut, (1970) S. Knight, W . Smith and J .B . Mickle, \"Cheese whey disposal using Saccharomyces fragilis yeast\", Cultural Dairy Products Journal, 7(2):17-22 (1972) A. Gomez and F . J . CastiUo, \"Production of biomass and /3-D-galactosidase by Candida pseudotropicalis grown in continuous culture on whey\", Biotechnology and Bioengineering, 25:1341-1357 (1983) G . J . Mouhn, M . Guillaume and P. Galzy, \"Alcohol production by yeast in whey ultrafiltrate\", Biotechnology and Bioengineering, 22:1277-81 (1980) E.R. Hall and G.P. Adams, \"Anaerobic treatment of cheese whey\", Proc. Seminar on Anaerobic Fixed-Film Digestion, Ontario Ministry of Environment/Pollution Control Association of Ontario, Toronto, pp65-76 (1986) 213 Bibliography 214 J . C . Young and P.L. McCarty, \"Anaerobic filter for waste treatment\", Journal of Water Pollution Control Federation, 41:R160 (1969) M.S. Switzenbaum, \" A comparison of the anaerobic filter and the anaerobic ex-panded/fluidized bed processes\", Water Science and Technology, 15:345-358 (1983) G . Lettinga, A . F . M . van Velsen, S.W. Hobna, W . de Zeeuw and A . Klapwijk, \"Use of upflow sludge blanket (USB) reactor concept for biological wastewater treatment, especially for anaerobic treatment\", Biotechnology and Bioengineering, 22:699-734 (1980) M.S. Switzenbaum and S .G. Danskin, \"Anaerobic expanded bed treatment of whey\", Proceedings (36th) of Industrial Waste Conference, Purdue University, A n n Arbor Science, Michigan, 1982 P .H. Boeing and V . F . Larsen, \"Anaerobic fluidized bed whey treatment\", Biotech-nology and Bioengineering, 24:2539-56 (1982) J . Dehaast, T . J . Britz, J . C . Novello and P . M . Lategan, \"Anaerobic digestion of cheese whey using a stationary fixed-bed reactor\", New Zeland Journal of Dairy Science and Technology, 18:261-71 (1983) L. van den Berg and K . J . Kennedy, \"Dairy waste treatment with anaerobic sta-tionary fixed-bed film reactors\", Water Science and Technology, 15:359(1983) F . X . Wildenauer and J . Winter, \"Anaerobic degestion of high-strength acidic whey in pH-controlled upflow fixed film loop reactor\", Applied Microbiology and Biotech-nology, 22:367 (1985) P.J. Reynolds and E . Colleran, \"Comparison of start-up and operation of anaerobic fixed bed and hybrid sludge-bed/fixed-bed reactors treating whey wastewater\", Proceedings of E W P C A Conference Anaerobic Treatment, Amsterdam, p515 1986 F . G . Pohland and S. Ghosh, \"Developments in anaerobic stabilization of organic wastes\u00E2\u0080\u0094the two-phase concept\", Environmental Letters, 1(14):255 (1971) F . E . Mosey, \"Methamatical modeling of the anaerobic digestion process: regulatory mechanisms for the formation of short-chain volatile acids from glucose\", Water Science and Technology, 15:209-32 (1983) M . J . Hammer et al, \"Dialysis separation of sewage sludge digestion\", Journal of the American Society of TJivil Engineers, SA5, 907(1969) S. Ghosh, J .R. Conrad and D . L . Klass, \"Anaerobic acidogenesis of waste water sludge\" Journal of Water Pollution Control Federation, 47:31-45 (1975) Bibliography 215 [27] S. Ghosh and M . D . Henry, \"Stabihzation and gasification of soft drink manufac-turing waste by conventional and two-phase anaerobic digestion\", Proceedings of the 36th Purdue Industrial Wasts Conference, Lafayette, IN. p292, 1981 [28] S. Ghosh et al, \"Two-phase upflow anaerobic digestion of concentrated sludge\", Proceedings of the 5th Symposium on Biotechnology for Fuels and Chemicals\", Gattinburg, T N , 1983 [29] J . D . Keenan, \"Multiple staged methane recovery from sohd wastes\", Journal of Environmental Science and Health, All(8/9) :525 (1976) [30] S. Ghosh, J.P. Ombregt and P. Pipyn, \"Methane production from industrial wastes by two-phase anaerobic digestion\", Water Research 19(9):1083-88 (1985) [31] I. Berkovitch, \" T h e treatment plant that gives more than just clean water\", Process Engineering, April p77 (1986) [32] R .F . Hickey and R . W . Owens, \"Methane generation from high- strength industrial wastes with the anaerobic biological fluidized bed\", Biotechnology and Bioengi-neering Symposium, No.11 1981 [33] S.-T. Yang, I.-C. Tang and M . R . Okos, \"Defined bacterial culture development for methane generation from lactose\" Biotechnology and Bioengineering, 32:28-37 (1988) [34] I.J. Callander and J.P. Barford, \"Cheese whey anaerobic digestion \u00E2\u0080\u0094 effect of chemical flocculant addition\" Biotechnology Letters, 5:153-58 (1983) [35] J . Dehaast, T . J . Britz, J . C . Novello, and E . W . Veywey, \"Anaerobic digestion of deproteined cheese whey\", Journal of Dairy Research, 52:457-67 (1985) [36] S.R. Harper and F . G . Pohland, \"Recent developments in hydrogen manage-ment during anaerobic biological waste water\", Biotechnology and Bioengineering, 28:558-602 (1985) [37] F. Ehhnger, J . M . Audic, D . Verrier and G . M . Faup, \" T h e influence of the car-bon source on microbiological clogging in an anaerobic filter\", Water Science and Technology, 19:261-273 (1987) [38] J . C . Young and M . F . Dahob, \"Effect of media design on the performence of fixed bed anaerobic reactors\", Water Science and Technology, 15(8/9):369 (1983) [39] E .R. Hall, \"Improving hydraulic efficiency in high rate anaerobic systems\", Proc. of Seminar Bridging the Gap between Research and Full Scale Operation in Wastew-ater Treatment, Ontario Ministry of Environment/Pollution Control Association of Ontario, pp55-57 (1984) Bibliography 216 [40] Kuroda Masao et al, \"Methanogenic bacteria attached to solid supports\", Water Research, 25(5):653-656 (1988) [41] A . Bhadra, J . M . Scharer and M . Moo-Young, \"Methanogenesis from volatic fatty acids in down flow stationary fixed-bed reactor\", Biotechnology and Bioengineering, 30:314-319 (1986) [42] R. Wollersheim, \"Adhesion and biofilm development of acetate- and butyrate-degrading microorganisms on glass surfaces\", Biotechnology Letters, 11(10):749-752 (1989) [43] W . D . Murray and L. van den Berg, \"Effects of support material on the development of microbial fixed films converting acetic acid to methane\", Journal of Applied Bacteriology, 51:257-265 (1981) [44] W.S. Kisaalita, K . V . Lo and K . L . Pinder, \"Acidogenic fermentation of lactose\", Biotechnology and Bioengineering, 30:88-95 (1987) [45] W.S . Kisaalita, K . V . Lo and K . L . Pinder, \"Influence of dilution rate on the acido-genic phase products distribution during two-phase lactose anaerobiosis\", Biotech-nology and Bioengineering, 34:1235-1250 (1989) [46] W.S . Kisaalita, \"Anaerobic fermentation of whey: acidogenises \" , P h D thesis, Uni-versity of British Columbia, British Columbia, Canada, 1987 [47] P. Harremoes, \"Biofilm kinetics\" in \"Water pollution microbiology\", ed. by R. Mitchell, John Wiley & Sons, New York, N . Y . Vol.2, P P71-109 (1978) [48] L . G . Scheve, \"Elements of biochemistry\", Allyn and Bacon, Inc. Newton, Mas-sachusetts, p355 (1984) [49] D . F . Toerien and W.H.Journal Hattingh, \"Anaerobic digestion I. the microbiology of anaerobic digestion\", Water Research, 3:385-416 (1969) [50] R . E . McKinney, \"Microbiology for sanitary engineers\", McGraw-Hil l , New York, (196.2) [51] D . F . Toerien, M . L . Siebert and W.H.Journal Hattingh, \" T h e bacterial nature of the acid-forming phase of anaerobic digestion\", Water Research, 1:497 (1967) [52] F.Journal Post, A . D . Allen and T . C . Reid, \"Simple medium for the selective isola-tion of Bacteroids and related organisms and their occurence in sludge\", Applied Microbiology, 15:213 (1967) Bibliography 217 [53] H . W . Doelle, \"Basic metabolic processes\" in \"Biotechnology, a comprehensive trea-tise in 8 volumes: vol.1 microbial fundamentals\", ed. by H.-Journal Rehm and G . Reed, P113 (1981) [54] P.O. McCarty, J.S. Jeris, R . E . McKinney, K . Reed and C . A . Vath, \"Microbiology of anaerobic digestion\" Report No. R62-29 Sedgwick Lab. of Sanitary Science, M I T Cambridge, (1962) [55] G . Gottschalk \"Bacterial metabolism\", Springer Verlag (1986) [56] R . E . Hungate, \"Hydrogen as an intermediate in the rumen fermentation\", Archives of Microbiology, 59:158-164 (1967) [57] J . G . Zeikus, \" T h e biology of methanogenic bacteria\", Bacteriological Reviews, 41(1):514-541 (1977) [58] M . C . Siebert, D . F . Toerien and W.H.Journal Hattingh, \"Bacterial nature of the acid-forming phase of anaerobic digestion\", Water Research, 2:545-554 (1968) [59] A . Kiener and T . Leisinger, \"Oxygen sensitivity of methanogenic bacteria\", Sys-tematic and Apphed Microbiology, 4:305-312 (1983) [60] B . C . McBride and R.S. Wolfe, \" A new coenzyme of methyl transfer, coenzyme M \" , Biochemistry, 10:2317-2324 (1971) [61] C D . Taylor and R.S. Wolfe, \"Structure and Methylation of coenzyme M (HSCH2CH2S03)\", Journal Biological Chemistry, 249:4879-4885 (1974) [62] S.F. Tzeng, R.S. Wolfe and M . P . Bryant, \"Factor 420- dependent pyridine nucleotide-hnked formate metabohsm of Methanobacterium ruminantium\", Jour-nal of Bacteriology, 121:184-191 (1975) [63] J.S. Jeris and P .C . McCarty, \"Biochemistry of C H 4 fermentation using 1 4 C tracers\", Journal Water Pollution Control Federation, 37:178 (1966) [64] P .H. Smith and R . A . Mah, \"Kinetics of Acetate Metabohsm During Sludge Diges-tion\", Apphed Microbiology, 14:368-371 (1966) [65] M.Journal Pine and H . A . Barker, \"Studies on the methane fermentation XII The pathway of hydrogen in acetate fermentation\", Journal of Bacteriology, 71:644-648 (1956) [66] M.Journal Pine and W . Vishniae, \" T h e methane fermentation of acetate and methanol\", Journal of Bacteriology, 73:736-742 (1957) Bibliography 218 [67] K . Decker, K . Jtmgermann and R . K . Thauer, \"Energy production in anaerobic or-ganisms\", Angewandte Chemie, Internationa] Edition in English, 9:138-158 (1970) [68] T . C . Stadtman and H . A . Barker, \"Studies on methane fermentation IX The origen of methane in the acetate and methanol fermentations by Methanosarcina\", Journal of Bacteriology, 61:81-86 (1951) [69] J . G . Zeikus, P.Journal Weimez, D . R . Nelson and L . Paniels, \"Bacterial methano-genesis: acetate as a methane prosursor in pure culture\", Archives of Microbiology, 104:129-134 (1975) [70] T . E . Cappenberg, \" A study of mixed continus culture of sulfate-reducing and methane-producing bacteria\", Microbial Ecology, 2:60-72 (1975) [71] L. van den Berg, G . B . Patel, P.S. Clack and C P . Lentz, \"Factors affecting rate of methane fermentation from acetic acid by enriched methanogenic cultures\", Cana-dian Journal of Microbiology, 22:1312-1319 (1976) [72] M.Journal Wolin, \"Hydrogen transfer in microbial communities\" in \"Microbial in-teractions and communities\", ed. by A . T . Bull and J . H . Slater Academic Press, London (1982) [73] M . P . Bryant, E . A . Wolin, M.Journal Wolin and R.S. Wolfe, \"Methanobacillus omelianskii, a symbiotic association of species of bacteria\", Archives of Microbiol-ogy, 59:20-31 (1967) [74] Journal Dolnng, \"Acetogenesis\" in \"Biology of anaerobic microorganisms\", ed. by A . J . B . Zehnder, New York, Wiley, pp417-468 (1988) [75] M.Journal Mclnerney, M.P . Bryant, R .B . Hespell and J . W . Costertar, \"Syn-trophomonas wolfei gen. nov. sp. nov., an anaerobic, syntrophic fatty acid-oxidizing bacterium\", Applied and Environmental Microbiology, 41:1029-1039 (1981) [76] P.R. Boone and M.P . Bryant, \"Propionate-degrading bacterium, Syntrophobacter wolinii sp. nov. gen. nov., from methaogenic ecosystems\", Applied and Environ-mental Microbiology, 40:626-632 (1981) [77] D . O . Mountfort, W.Journal Brulla, L .R . Krumholz and M.P . Bryant, \"Isolation and Characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge\", Archives of Microbiology, 133:249-256 (1982) [78] N . G . Wofford, P.S. Beaty and M.Journal Mclnerney, \"Prepartion of cell-free ex-tracts and enzymes involved in fatty acid metabolism in Syntrophomonas wolfei\", Journal of Bacteriology, 167:179-185 (1986) Bibliography 219 M . Koch, Journal Dolfing, K . Wuhrman and A . J . B . Zehnder, \"Pathways of propi-onate degradation by enriched methanogenic cultures\", Applied and Environmental Microbiology, 45:1411-1414 (1983) M.S. Salkinoja-Salonen, E.-Journal Nyns, P . M . Sutton, L. van den Berg and A . D . Wheatley, \"Start-up of an anaerobic fixed-film reactor\", Water Science and Tech-nology, 15:305-308 (1983) J . D . Bryers, \"Biologically active surfaces: processes governing the formation and persistence of biofilms\", Biotechnology Progress, 3(2):57-68 (1987) M.S. Powell and N . K . H . Slater, \" T h e deposition of bacterial cells from laminar flows onto sohd surfaces\", Biotechnology and Bioengineering, 25:891 (1983) J .E . Duddridge, C . A . Kent and J .F . Laws, \"Effects of surface shear stress on the attachment of Pseudomonas fluoresceins to stainless steel under defined flow con-ditions\", Biotechnology and Bioengineering 24:153 (1982) C E . Zobell, \"Influence of bacterial activity on source sediment\", Journal of Bac-teriology, 46:39-56 (1943) K . C Marshall, R. Stout and R. Mitchell, \"Mechanism of the initial enents in the sorption of marine bacteria to surfaces\", Journal of General Microbiology, 68:337-348 (1971) J . D . Bryers and W . G . Characklis, \"Processes governing biofilm formation\", Biotechnology and Bioengineering, 24:2451 (1982) Y . K . Park and B . C . Rivera \" T h e effect of shear stress on biofilm loss rate\", Biotech-nology and Bioengineering, 24:501-506 (1982) C M . Brown, D . C . Ellwood and J.R. Hunter, \"Growth of bacteria at surfaces: influence of nutrient limitation\", F E M S Microbiology Letters, 1:163-166 (1977) D . C . Ellwood, J .R. Hunter and V . M . C . Longyear, \"Growth of Stretococcus mutants in a chemostat\", Archives of Oral Biology, 19:659-664 (1974) M . Fletcher, \" T h e effects of culture concentration and age, time and temperature on bacterial attachment to polystyrene\", Canadian Journal of Microbiology, 23:1-6 (1977) R . E . Baier, \"Influence of the initial surface condition of materials on bioadhession\" in Proceedings of 3rd Inter. Congress, on Marine Corrosion and Fouling, ed. by R.F . Acker, B . F . Brown, J.R. Depalma and W.P . Iverson, Northwestern Univ. Press., pp 633-639 (1973) Bibliography 220 [92] S. Takakuwa, T . Fujimori and H . Iwasaki, \"Some properties of cell-sulfur adhesion in ThiobaciUus thioxidans\", Journal of General and Apphed Microbiology, 25:21-29 (1979) [93] S.C. Dexter, \"Influence of substratum critical surface tension on bacterial adhesion-in situ studies\", Journal of CoUoid and Interface Science, 70:346-453 (1979) [94] M . Fletcher and G.I. Loeb, \" T h e influence of substratum characteristics on the at-tachment of a marine pseudomonad to sohd surfaces\", Apphed and Environmental Microbiology, 37:67-72 (1979) [95] M . C . M . van Loosdrencht, Journal Lyklema, W . Worde, G . Schraa and A . J . B . Zehnde, \" T h e role of bacterial cell wall hydrophobicity in adhesion\", Apphed and Environmental Microbiology, 53:1893-1897 (1987) [96] M . C . M . van Loosdrencht, Journal Lyklema, W . Worde, G . Schraa and A . J . B . Zehnde \"Elecrophoretic mobihty and hydrophobicity in adhesion\", Apphed and Environmental Microbiology, 53:1898-1901 (1987) [97] T . A . Stenstrom, \"Bacterial hydrophobicity, an overall parameter for the measure-ment of adhesion potential to soil particles\", Apphed and Environmental Microbi-ology, 55(1):142-147 (1989) [98] M . Fletcher, \"Adherence of marine micro-organisms to soomth surfaces\" in \"Bac-terial adherence\", ed. by E . H . Beachey, London and New York, Chapman and Hall, pp 347-371 (1980) [99] P.R. Rutter, \" T h e physical chemistry of the adhesion of of bacteria and other cells\" in \" Cell adhesion and motility-3rd symposium of the British Socity for cell biology\", ed. by A . S . G . Curtis and J . D . Pitts, Cambridge University Press., ppl03-135 (1980) [100] T h . F . Tadros, \"Particle-surface adhesion\" in \"Microbioal adhesion to surfaces\", ed. by R . C . W . Berkiley et al, Ellis Horwood, Chichester, England, pp93-116 (1980) [101] K . C . Marshall, \"Interfaces in Microbial Ecology\", Haward University Press. Cam-bridge Mass, (1976) [102] S.C. Dexter, J . D . Sullivan Jr., Journal Wilhans III and S.W. Watson, \"Influence of substrate wetability on the attachment of marine bacteria to various surfaces\", Apphed and Environmental Microbiology, 30(2):298-308 (1975) [103] M . Fletcher and G . D . Floodgate, \" A n electron microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to sohd sur-face\", Journal of General Microbiology, 74:325-334 (1973) Bibliography 221 [104] W . A . Corpe and H . Winters, \" T h e biology of microfouling of sohd surfaces with special reference to power plant heat exchangers\", Condenser Biofouhng Control Symposium Proceedings, Electric Power Research Institute, ed. by J .F . Garey et al, A n n Arbor, Mich. pp29-42 (1980) [105] I.W. Sutherland, \"Surface carbohydrates of the prokaryote cell\", Academic Press. London and New York, p472 (1977) [106] W . F . Dudman and J .F . Wilkinson, \"The composition of extracellular polysaccha-rides of Aerobacter-Klebsiell strains\", Biochemical Journal, 62:289-295 (1959) [107] M . Shapiro and M.S. Switzenbaum, \"Initial anaerobic biofilm development\", Pro-ceedings of 2nd Inter. Conference on Fixed-film Biological Processes, ed. by J . T . Bandy et al, Vol. 1, Arhngton, Virginia, July 10-12 (1984) [108] W . D . Murray and L. van den Berg, \"Effects of support material on the development of microbial fixed films converting acetic acid to methane\", Journal of Apphed Bacteriology, 51:257-265 (1981) [109] L. van den Berg and K.Journal Kennedy, \"Support materials for stationary fixed film reactors for high-rate methanogenic fermentations\", Biotechnology Letters, 3(4):165-170 (1981) [110] P. Huysman, P. van Meenen, P. van Assche and W . Verstraete, \"Fators affecting the colonization of non porous and porous packing materials in model upflow methane reactors\", Biotechnology Letters, 5(9):643- 648 (1983) [111] D . Verrier and G . Albagnac, \"Adhesion of anaerobic bacteria from methanogenic sludge onto inert sohd surfaces\", E U R report # 10024 pp537-541 (1985) [112] D . Verrier, B. Mortier, H . C . Dubourguier and G . Albagnac, \"Adhesion of anaerobic bacteria to inert supports and development of methanogenic biofilms\", Proc. 5th Inter. Symposium on Anaerobic Digestion, ed. by E .R. Hall and P.N. Hobson, Pergamou Press, Oxford, Bologna, Italy, pp61-79 (1988) [113] M.S. Switzenbaum, K . C . Scheuer and K . E . Kallmeyer \"Influence of materials and precoating on initial anaerobic biofilm development\", Biotechnology Letters, 7(8):585-588 (1985) [114] J . M . Scharer, A . Bhadra and M . Moo-Young, \"Methane production in immmobi-lized cell bioreactor\" in \"Bioreator immobihzed enzymes and cells: fundamentals and application\", ed. by M . Moo-Young, Elsevier Apphed Science Publications Ltd. , (1988) [115] G . Astarita, \"Mass transfer with chemical reaction\", Elsevier, Amsterdam, (1967) Bibliography 222 V . G . Levich, \"Physicochemical hydrodynamicas\", New York, Prentice Hall, (1962) M . Moo-Young and H . W . Blanch, \"Design of biochemical reactors, mass transfer criteria for simple and complex systems\", Advances in Biochemical Engineering, 19:1-57 (1981) J . C . Kissel, \"Modeling mass transfer in biological wastewater treatment processes\", Water Science and Technology, 18:35-45 (1986) E.Journal LaMotta, \"External mass transfer in a biological film reactor\", Biotech-nology and Bioengineering, 18:1359-1370 (1976) R . W . Robinson, D . E . Akin, R . A . Nordstedt, M . V . Thomas and H . C . Aldrich, \"Lingt and electron microscopic examinations of methae- producing biofilms from anaerobic fixed-bed reactors\", Apphed and Environmental Microbiology, 48(1):127-136 (1984) T . T . Eighmy, D . Maratea and P.L. Bishop, \"Electron microscopic examination of wastewater biofilm formation and structural components\", Apphed and Environ-mental Microbiology, 45(6):1921-1931 (1983) M . Harvey, C . W . Forsberg, T.Journal Beveridge, Journal Pos and J.R. Ogilvie, \"Methanogenic activity and strustural characteristics of the microbial biofilm on a needle-punched polyester support\", Apphed and Environmental Microbiology, 48(3):633-638 (1984) S.B. Libicki, P . M . Salmon and C R . Robertson, \" T h e effective diffusive perme-ability of a nonreacting solute in microbial cell aggregates\", Biotechnology and Bioengineering, 32:68-85 (1988) E.Journal LaMotta, \"Internal diffusion and reaction in biological films\", Environ-mental Science and Technology, 10(8):765-769 (1976) H.R. Bungay and D . M . Harold, \"Simulation of oxygen transfer in mcirobial slimes\", Biotechnology and Bioengineering, 13:569-579 (1971) Y.S . Chen and H.R. Bungay, \"Microelectorde studies of oxygen transfer in trickling filter slimes\", Biotechnology and Bioengineering, 23:781-792 (1981) K. Williamson and P.L. McCarty, \"Verification studies of the biofilm model for bacterial substrate utihzation\", Journal of Water Pollution Control Federation, 48(2):281-296 (1976) J . V . Matson and W . G . Characklis, \"Diffusion into microbial aggregates\", Water Research, 10:877-885 (1976) Bibliography 223 [129] M . Onuma and T . Omura, \"Mass-transfer characteristics within microbial sys-tems\", Water Science and Technology, 14:553-568 (1982) [130] H . H . Siegrist and W . Gujer, \"Mass transfer mechanisms in a heterotrophic biofilm\", Water Research, 19(11): 1369-1378 (1985) [131] P . G . Smith and P. Coackley, \"Diffusivity, tortuousity and pore structrue of acti-vated sludge\", Water Research, 18(1): 117-122 (1984) [132] A . D . Meunier and K.Journal Williamson, \"Packed bed biofilm reactors: design\", Journal of Environmental Engineering Division, A S C E , 107:319-337 (1981) [133] B . E . Rittmann and P.L. McCarty, \"Variable-order Model of bacterial-film kinetics\", Journal of Environmental Engineering Division, A S C E , 104:889-900 (1978) [134] Y . - T Wang, M . T . Suidan and B . E . Rittmann, \"Kinetics of methanogens in an expanded-bed reactor\", Journal of Environmental Engineering Division, A S C E , 112:155-170 (1986) [135] N.P. Harris and G.S. Hansford, \" A study of substrate removal in a microbial film reactor\", Water Research, 10:935-943 (1976) [136] B. Atkinson and I.S. Daoud, \"Diffusion effects with microbial films\", Transactions, Institute of Chemical Engineers, 48:245-254 (1970) [137] V . H . Edwards, \" T h e influence of high substrate concentraions on microbial kinet-ics\", Biotechnology and Bioengineering, 12:679-712 (1970) [138] C .P .L . Grady, Jr, \"Modeling of biological fixed films - A state-of-the-art review\" in \"Fixed-film, biological processes for wastewater treatment\", ed. by Y . C . Wu and E . D . Smith, Noyes Data Corporation pp75-134 (1983) [139] W . K . Shieh and L . T . Mulcahy, \"Experimental determination of intrinsic kinetic coefficients for biological wastewater treatment systems\", Water Science and Tech-nology, 18:1-10 (1986) [140] I.Journal Callander and'J.P. Barford, \"Cheese whey anaerobic digestion-effect of chemical flowclant addition\", Biotechnology Letters, 5:153-158 (1983) [141] R . F . Hickey, \"Methane generation from high-strengh industrial wastes with the anaerobic biological fluidised bed\", Biotechnology and Bioengineering Symposium, Number 11, John Wiley and Sons, New York, N Y , (1981) [142] A . W . Lawrence and P.L. McCarty, \"Kinetics of methane fermentation in anaerobic treatment\", Journal of Water Pollution Control Federation, 41(2):R1-R17 (1969) Bibliography 224 [143] Chiu-Yue Lin , Kazuaki Sato, Tatsuya Noike and Junichiro Matsumoto, \"Methanogenic digestion using mixed substrate of acetate, propionic and butyric acids\", Water Research, 20(3):385-394 (1986) [144] M . Henze and P. Harremoes, \"Anaerobic treatment of wastewater in fixed film reactors \u00E2\u0080\u0094 a literature review\", Water Science and Technology, 15:1-101 (1983) [145] R.Journal Zoetemyer, J . C . Heuvel, and A. Cohen, \" p H influence on acidogenic dissimilation of glucose in an anaerobic digester\", Water Research, 16(3):303-311 (1982) [146] R.S. Oremland, L. Marsh, and D.Journal DesMarais, \"Methanogenesis in big soda lake nevada: an alkaline, moderately hypersaline desert lake\", Apphed and Envi-ronmental Microbiology, 43:462-468 (1982) [147] R . C . Harriss, E . Gorham, D.I. Sebacher, K . B . Bartlett and P.A. Flebbe, \"Methane flux from northern peatlands\", Nature, 315:652-654 (1985) [148] A . J . B . Zehnder, K . Ingvorsen and T . Marti, \"Microbiology of methane bacteria\", The Second International Symposium on Anaerobic Digestion, 6-11 Sept. Trave-munde, Germany, (1981) [149] R . A . M a h and M . R . Smith, \" T h e Methanogenic bacteria\" in \" T h e prokaryotes, vol. 1\", ed. by M.P . Starr, H . Stolp, H . G . Truper, A . Balows and H . G . Schlegel, Springer, Berlin, pp948-977 (1981) [150] L. Bhatnagar, M . K . Jain, J.P. Aubert and J . G . Zeikus, \"Comparison of assimila-tory organic nitrogen, sulfur, and carbon sources for growth of Methanobacterium species\", Apphed and Environmental Microbiology, 48:785-790 (1984) [151] L. van den Berg and C P . Lentz, \"Food processing waste treatment by anaerobic digestion\", Proceedings of the 32nd Industrial Waste Conference, Purdue Univer-sity, Lafayette, Indiana, A n n Arbor Science, A n n Arbor, Mich. , 1978, pp252-258 (1977) [152] R . E . Speece and P.L. McCarty, \"Nutrient requirements and biological accumulation in anaerobic digestion\" in \" Advances in Water Pollution Research\", ed. by W . W . Eckenfelder, Perganon Press, Oxford, U K , (1964) [153] P. Scherer and H . Sahm, \"Influence of sulphur-containing compounds on the growth of Methanosarcina barkeri in a defined medium\", European Journal of Applied Microbiology and Biotechnology, 12:28-35 (1981) Bibliography 225 [154] L. Daniels, N . Belay and B.S. Rajagopal, \" Assimilatory reduction of sulfate and sul-fite by methanogenic bacteria\", Applied and Environmental Microbiology, 51:703-709 (1986) [155] D.Journal Hill , \"Effects of carbon: nitrogen ratio on anaerobic digestion of dairy manure\", Agricultural Wastes, 1:267-278 (1979) [156] P. Pipyn and W . Verstraete, \"Lactate and Ethanol as Intermediates in Two-Phase Anaerobic Digestion\", Biotechnology and Bioengineering, 23:1145-1154 (1981) [157] P.L. McCarty, \" T h e methane fermentation\" in \" Principles and apphcations in aquatic microbiology\", ed. by H . Heukelekian and N . C . Dondero, John Wiley and Sons, New York, N Y , (1964) [158] R . E . Speece, G . F . Parkin and D . Gallagher, \"Nickel stimulation of anaerobic di-gestion\", Water Research, 17:677-683 (1983) [159] K.Journal Kennedy and R.L . Droste, \"Startup of anaerobic downflow station-ary fixed film (DSFF) reactors\", Biotechnoloty and Bioengineering, 27:1152-1165 (1985) [160] J . A . HoweU and B. Atkinson, \"Sloughing of microbial film in trickling filters\", Water Research, 10:307-315 (1976) [161] M . H . Turakhia and W . G . Charackhs, \"Activity of Pseudomonas aeraginosa in biofilms: effect of calcium\", Biotechnology and Bioengineering, 33:406-414 (1989) [162] S. Ghosh and D . L . Klass, \"Two-phase anaerobic digestion\", Process Biochemistry, 13(4):15-24 (1978) [163] P.L. McCarty, \"Energetics and kinetics of anaerobic treatment\" in \"Anaerobic bio-logical treatment processes\", Advances in biochemistry series 105 A C S , Washington D . C , pP91-107 (1971) [164] J .F . Andrews, \"Kinetics and characteristics of volatile acid production in anaerobic fermentation processes\", International Journal of Air and Water Pollution Controll, 9:439-461 (1965) [165] J . A . Mueller and J .L . Mancini, \"Anaerobic filter kinetics and apphcations\", Pro-ceedings of the 30th industrial waste conference 1975 Purdue University, Lafayette, Indiana, A n n Arbor Science, A n n Arbor, Mich 1977 pp423-447 (1975) [166] M . Lindgren, \"Mathematical modeling of the anaerobic filter process\", Water Sci-ence and Technology, 15(8-9):197-207 (1983) Bibliography 226 [167] R . W . Harvey and L . Y . Young, \"Enumeration of particle- bound and unattached respiring bactria in th salt mash enviroment\", Apphed and Environmental Micro-biology, 40:156-160 (1980) [168] C . W . Hendricks, \"Sorpotion of hetreotrophic and euteric bactria to glass surfaces in the continuous culture of river water\", Applied Microbiology, 28:572-578 (1974) [169] W . H . Jeffrey and J . H . Paul, \"Activity measurements of planktomic microbial and microfouling communities in an eutrophic estnary\", Apphed and Environmental Microbiology, 46:157-162 (1986) [170] M . C . M . van Loosdrecht, Journal Kyklema, Journal Morde and A . J . B . Zehnder, \"Influence of interfaces on microbial activity\", Microbiological Reviews, 54(l):5-87 (1990) [171] W . H . Jeffrey and J . H . Paul, \"Activity of an attached and free-living Vibrio sp. as measured by thrymidine incorporation, p-indonitrotetrazolium reduction, and A T P / A D P Ratios\", Apphed and Environmental Microbiology, 51:150-156 (1986) [172] T . C . Kieft and D . E . Caldwell, \"Chemostat and in-situ colonization kinetics of Thermotrix Thiopara on caldite and pyrite surfaces\", Geomicrobiology Journal, 3:217-229 (1984) [173] D . E . Caldwell and J.R. Lawrence, \"Growth kinetics of Pseudomonas fluorescens microcolonies within the hydrodynamic boundary layers of surface microenviron-ments\", Microbial Ecology, 12:299-312 (1986) [174] A.S . Gordon ans F.Journal Millero, \"Adsorption-mediated decrease in the biodegra-dation rate of organic compounds\", Microbial Ecology, 11:289-298 (1985) [175] G . Stucki and M . Alexander, \"Role of dissolution rate and solubihty in biodegrada-tion of aromatic compounds\", Apphed and Environmental Microbiology, 53:292-297 (1987) [176] J.Journal Bright and M . Fletcher, \"Amino assimilation and electron transport sys-tem activity in attached and free-living marine bacteria\", Apphed and Environ-mental Microbiology, 45:818-825 (1983) [177] R. Hattori and T . Hattori, \"Growth rate and molar growth yield of E. coli adsorbed on an anion exchange resin\", Journal of General and Apphed Microbiology, 27:287-298 (1981) [178] A . Cohen, J . M . van Germert, R.Journal Zoetemeyer, A . M . Breure, \" M a i n charac-teristics and stoichiometric aspects of acidogenesis of soluble carbohydrate contain-ing wastewaters\", Process biochemistry, 19(6):228-232 (1984) Bibliography 227 [179] R . K . Thauer, K . Jungermann and K . Decker, \"Energy conservation in chemotrophic anaerobic bacteria\", Bacteriological Reviews, 41(1)100-180 (1977) [180] G . Gottschalk, \"Bacterial Metabolism\" Springer-Verlag New York Inc. New York, P P167-121 (1979) [181] J . C . van den Heuvel, H . H . Beeftink and P . G . Verschuren, \"Inhibition of the aci-dogenic dissimilation of glucose in anaerobic continuous cultures by free butyric acid\", Applied Microbiology and Biotechnology, 29:89-24 (1988) [182] P.J.F. Henderson, \"Iron transport by energy-conserving biological membranes\", Annual Review of Microbiology, 25:393-428 (1971) [183] Journal Domingue, \"Probing the chemistry of the solid/liquid interface\", American Laboratory, pp50-55, Oct. (1990) [184] J.Journal Bikerman, \"Surface chemistry - theory and applications\", Academic Prss Inc. New York, (1958) [185] D . C . Ellwood, C . W . Keevil, P .D. Marsh, C M . Brown and J . N . Wardell, \"Surface-associated growth\", Philosophical Transations of the Royal Society of London, Ser. B, 297:517-532 (1982) [186] A . C Bajpaj, L .R. Mustoe, and D . Walker, \"Advanced engineering mathematics\", John Wiley & Sons, Ltd. , p l l O (1977) [187] M . C . M . van Loosdrecht, Journal Lyklema, N . Norde, G . Schraa and A . J . B . Zehn-der, \" T h e role of bacterial cell wall hydrophobicity in adhesion\", Apphed and E n -vironmental Microbiology, 53(8):1893-1897 (1987) [188] J . H . Pringle and M . Fletcher, \"Influence of substratum wettability on attachment of freshwater bacteria to sohd surfaces\", Apphed and Environmental Microbiology, 45:811-817 (1983) [189] M . Fletcher and G . D . Floodgate, \" T h e adhesion of bacteria to sohd surfaces\" in \"Microbial ultrastructure: the use of the electron microscope\", ed. by R. Fuller and D . W . Lovelock, Academic Press, London, pplOl-107 (1976) [190] D . R . Absolom, \" T h e role of bacterial hydrophobicity in infection\", Canadian Jour-nal of Microbiology 34(3):287-298 (1988) [191] N . Mozes, D . E . Amorg, A.Journal Leonard and P . G . Rouxhet, \"Surface properties of microbial cells and their role in adhesion and flocculation\", Colloids and Surfaces, 42(3-4):313 (1989) Bibliography 228 B. Dahneke, \" T h e influence of flatting on the adhesion of particles\", Journal of CoUoid and Interface Science, 40:1-13 (1972) R.Journal Shimp and F. Pfaender, \"Effects of surface area and flow rate on ma-rine bacterial growth in activated carbon columns\", Apphed and Environmental Microbiology 44(2):471-477 (1982) F . A . L . Dulhen, \"New relation between viscocity and the diffusion coefficients based on Lamm's theory of diffusion\", Transactions of the Fraraday Society, 59:856-68 (1963) R . K . Ghai, H . Ertt and F . A . L . Dulhen, \"Liquid diffusion of nonelectrolytes I\", A I C h E Journal, 19:881-900 (1973) T . Lofin and E . McLaughlin, \"Diffusion in binary hquid mixtures\", Journal of Physical Chemistry, 73:186(1969) R . C . Reid, J . M . Prausnitz and B . E . Pohng, \" T h e Properties of Gases ans Liquids\", McGraw-Hill Book Company, New York, pp577-624 (1987) C R . Wilke and P. Chang, \"Correlation of diffusion coefficients in diluted solutions\", A I C h E Journal, 1:264 (1955) D . R. Lide, \"Handbook of chemistry and physics\", 71th Edition, C R S Press 1990/1991 M . E . Davis \"Numerical methods and modehng for chemical engineers\", John Wiley and Sons, Inc., p l8 (1984) L . E . Muller, E . Hindin, J . V . Lunsford and G . H . Dunstan, \"Some characteristics of anaerobic sludge digestion I. effects of loading\", Journal of Water Pollution Control Federation, 31:669-676 (1959) E . Hindin and G . H . Dunstan, \"Effect of detention time on anaerobic digestion\" Journal of Water Pollution Control Federation, 32:930-938 (1960) J . A . Eastman and J .F . Ferguson, \"Solubihzation of organic carbon during the acid phase of anaerobic digestion\", Journal of Water Pollution Control Federation, 53:352-366 (1981) M . L . Massey and F . G . Pohland, \"Phase separation of anaerobic stabihzation by ki-netic controls\", Journal of Water Pollution Control Federation, 50:2204-2222 (1978) C . P.L. Grady Jr. and H . C Lim, \"Biological wastewater treatment: theory and apphcations\", Marcel Dekher, New York, N Y (1980) Bibliography 229 J . M . Scharer and M . Moo-Young, \"Methane generation by anaerobic digestion of cellulose-containing waste\", Advances in Biochemical Engineering, 11:85 (1979) Journal Neter, W . Wasserman and G . A . Whitmore, \"Apphed Statistics\", Allyn and Bacon, Inc. Boston, pp518-521 (1982) L . van den Berg, \"Effect of temperature on growth and activity of a methanogenic culture utihzing acetate\", Canadian Journal of Microbiology, 23:898-902 (1977) W . Gujer and A . J . B . Zehnder, \"Conversion processes in anaerobic digestion\", Wa-ter Science and Technologh, 15:127-167 (1983) C L . Cooney, \"Growth of microorganisms\" in \"Biotechnology: a conprehensive treaties in 8 vol. Volume 1. microbial fundamentals\", ed. by H.Journal Rehm and G . Reed, Verlag Chemie Gmbh, D-6940 Weinheim (1981) L . G . M . Gorris, J . M . A . van Deursen, C . van der Drift and G . D Vogels, \"Biofilm development in laboratory methanogenic fluidized bed reactors\", Biotechnology and Bioengineering, 33:687-693 (1989) W . L . Bolle, Journal van Breugel, G . C . van Eybergen, N . W . F . Kossen and W . van Gils, \"Kinetics of anaerobic purification of industrial wastewater\", Biotechnology and Bioengineering, 28:542-548 (1986) R . L . Droste and K.Journal Kennedy, \"Steady state kinetics of anaerobic downflow stationary fixed film reactors\", Water Science and Technology, 19:275-285 (1987) M . Dohanyos, B. Dosova, Journal Zabranska and P. Grau, \"Production and utihza-tion of volatile fatty acids in various types of anaerobic reactors\", Water Science and Technology, 17:191-205 (1985) M . Dubios, K . A . Gilles, J . K . Hamilton, P .A. Rebers and F. Smith \"Colorimetric method for determination of surgars and related substrances\", Analytical Chem-istry, 28(3):350-356 R . L . Markus, \"Colorimetric determination of lactic acid in body fluids utihsing cation exchange of deproteinization\", Archives of Biochemistry, 29:159-165 Appendix A Fermentation Pathways A . l Four Fermentation Pathways of Glucose to Pyruvate 1. Embden-Meyerhof-Parnas ( E M P ) pathway of glucose utihzation is considered to be widely used among fungi, yeasts and bacteria and has the overall reaction Glucose + 2 A T P + 2NAD+ 2Pyruvate + 4 A T P + 2 ( N A D H + H+). Figure A.81 shows the reaction intermediates and the enzymes which catalyses the formation of these intermediates. 2. Hexose monophosphate (HMP) pathway is largely concerned with biosynthetic metabohsm because of its provision of pentoses which are needed for the synthesis of nucleic acids and nucleotide-containing prosthetic groups. The H M P pathway can be operated in two different variations. The first variation is referred to as the em pentose shunt or pentose cycle which does not lead directly to pyruvate as shown in Figure A.82 [53]. The overall reaction is Glucose + 12NADP+ + 7 H 2 0 + A T P \u00E2\u0080\u0094 \u00E2\u0080\u00A2 6CO2 + 1 2 ( N A D P H + H + ) + H 3 P 0 4 + A D P . Another variation is used by oxidative microorganisms, the partly complete cycle to produce pyruvate from glyceraldehyde 3-phosphate, catalyzed by the same enzymes as the E M P pathway. The sum of reactions for this second variation is 230 Appendix A. Fermentation Pathways 231 3Glucose + 6 N A D P + + A T P \u00E2\u0080\u0094> 2Ffructose 6-P + Glyceraldehyde 3-P + 3 C 0 2 + A D P + 6 ( N A D P H + H+) + H 3 P 0 4 . and the pathway is shown in Figure A.83. 3. The Entner-Doudoroff (ED) pathway was discovered by Entner and Doudoroff during metabohc studies of Pseudomonas saccharophila as shown in Figure A.84. 4. In addition to the well known E M P , H M P and E D pathways, there is another pathway, phosphoketolase (PK) pathway as shown in Figure A.85. It is, however, possessed by only a small group of bacteria, the heterofermentative Lactobacilli. A.2 Formation of Organic Acids from Pyruvate The following figures show the mechanisms of the formation of acetate (Figure A.86), butyrate (Figure A.87), mixed products (Figure A.88) and propionate (Figure A.89) from glucose via the common intermediate, pyruvate. Appendix A. Fermentation Pathways 232 G L U C O S E EC 2. 7. 1. 1. glucose 6-phosphate EC 5. 3. 1. 9. fructose 6-phosphate ' \" A T ? * \u00E2\u0080\u0094 / S-ADP fructose 1,6-bisphosphate dihydroxyacetone-phosphate methylglyoxal D-lactate - glyceraldehyde 3-phosphate EC 1.2.1.12. \u00E2\u0080\u00A2^NADH+H* 1,3-bisphosphoglycerate E C 2. 7. 2. 3. \u00E2\u0080\u00A2ADP ^ A T P -NAD* \u00E2\u0080\u00A2\u00E2\u0080\u00A2NADH + H* 3-phosphoglycerate EC 2.7.5. 3. ? t 2-phosphoglycerate EC 4. 2.1.11. phosphoenolpyruvate L-Al EC 2.7.1.40 I 2 ATP \u00E2\u0080\u00A2ATP P Y R U V A T E Figure A.81: EMP pathway of glucose conversion to pyruvate. Key to the enzymes: EC 2.7.1.1: hexokinase; EC 5.3.1.9: glucosephosphate isomerase; EC 2.7.1.11: phosphofruc-tokinase; EC 4.2.1.13: fructosebisphosphate aldolase; EC 5.3.1.1: triosephosphate iso-merase; EC 1.2.1.12: glyceraldehyde 3-phosphate dehydrogenase; EC 2.7.2.3: phospho-glycerate kinase; EC 2.7.5.3: phosphoglyceromutase; EC 4.2.1.11: Enolase; EC 2.7.1.40: pyruvate kinase. Source is [52]. Appendix A. Fermentation Pathways 233 g l u c o s e i glucose 6-phosphate \u00E2\u0080\u00A2* T \" ribulose 5-phosphate I Pentose phosphates i \u00E2\u0080\u00A2 glyceraldehyde 3-P \u00E2\u0080\u009E dihydroxyacetone-P fructose 1,6-bisphosphate fructose 6 -P \u00E2\u0080\u00A2 E M P pathway phosphoenolpyruvate I pyruvate Figure A.82: Schematic representation of the cychc (pentose shunt) and non-cychc nature of the HMP pathway. Source is [52]. Appendix A. Fermentation Pathways 234 GLUCOSE ,-ATP \"ADP glucose 6-phosphate NADP* 'NADPH + H+ EC 1. 1.1.49 glucono-lactone 6-phosphate EC 3.1.1.17 EC 5.1.3.1 xylulose 5-phosphate 6-phosphogluconate U-NADP* 111\" L.NADPH+H* ribulose 5-phosphate / \ E C 5. 3. 1. 6 |ribose 5-phosphate| glyceraldehyde fructose 6-P \ fructose 6-P X \ I E C S . 3 . 1.9 NAD*^ L-ADP NADH+H*\u00C2\u00AB7>\u00C2\u00BB-A'IT , glucose 6-phosphate-P Y R U V A T E Figure A.83: HMP pathway of glucose utihzation. S 7-P: sedoheptulose 7-phosphate; GA 3- P: glyceraldehyde 3-phosphate; DHAP: dihydroxy-acetonephosphate; E 4-P: erythrose 4- phosphate; F 6-P: fructose 6-phosphate; FDP: fructose 1,6-bisphosphate. Key to the enzymes: EC2.7.1.1: hexkoinase; EC 1.1.1.49: glucose 6-phosphate dehydrogenase; EC 3.1.1.17: gluconolactonaise; EC 1.1.1.44: 6-phosphogluconate dehydrogenase; EC 5.1.3.1: ribulosephosphate 3-epimerase: EC 5.3.1.6: ribose 5-phosphate isomerase; EC 2.2.1.1: transketolase; EC 4.1.2.13: fructose bisphosphate aldolase; EC 3.1.3.11: hexose diphos-phatase; EC 5.3.1.9: glucose 6-phosphate isomerase. Source is [52]. Appendix A. Fermentation Pathways 235 G L U C O S E g l u c o s e 6 - p h o s p h a t e g l u c o n a t e L\u00E2\u0080\u0094NADP* EC 1.1. 1.49 f K^NADPH + H* g l u c o n o - l a c t o n e 6 - p h o s p h a t e EC 3.1.1.17 6 - p h o s p h o g l u c o n a t e EC 4. 2. 1. 12 2 - k e t o - 3 - d e x y - 6 - p h o s p h o g l u c o n a t e EC4.1.2.14 f r u c t o s e 6 - P P Y R U V A T E g l y c e r a l d e h y d e / * 3 - p h o s p h a t e ~V A T P ' . P O O L / 2 ATP 2 ADP f r u c t o s e 6 - P EC 2. 2.1.1 e r y t h r o s e 4-p h o s p h a t e s e d o h e p t u l o s e g l y c e r a l d e h y d e 7 - p h o s p h a t e 3 - p h o s p h a t e EC2.2.1.1 | L r i b o s e 5 - p h o s p h a t e x y l u l o s e 5 -p h o s p h a t e |ECJ. 1.3. 1 r i b u l o s e 5 -p h o s p h a t e Figure A.84: ED pathway of glucose utilization. The abbreviations as in HMP pathway. Key to the enzymes: EC 2.7.1.1: hexokinase; EC 1.1.1.49: glucose 6-phosphate dehy-drogenase; EC 3.1.1.17: gluconolactonase; EC 4.2.1.12: 'phosphogluconate dehydratase; EC 4.1.2.14: phospho-2- keto-3-deoxygluconate aldolase; EC 2.2.1.1: transketolase; EC 2.2.1.2: transaldolase; EC 5.1.3.1: ribulosephosphate 5- phosphate isomerase. Source is [52]. Appendix A. Fermentation Pathways 236 glucose L - A T P k\u00C2\u00BB-ADP D-ribose ^ - A T P EC 2 . 7 . 1 . 1 5 ^ -\u00C2\u00BB -ADP glucose 6 - P U - N A D P * E C 1 1 1 \" ' p > N A D P H + H * glucono-lactone 6 - P EC 3 . 1 . 1 . 1 7 6-phosphogluconate U-NADP+ EC 1. 1 .1 . 44 L NADPH-H* ribulose 5-phosphate L-arabinose EC 5. 3 . 1 . 3 D-ribose 5 - P EC 5 . 3 . 1 . 6 L-ribulose A T P \" \u00E2\u0080\u00A2 A D F xylulose 5-phosphate IEC4. 1 .2 . 9 D-xylose EC 5. 3. 1.5 D- xylulose A T P >-*-ADP A D P A T P pyruvate glyceraldehyde 3 - P 1 \u00C2\u00AB t E M P acetyl-phosphate \u00E2\u0080\u0094 ^ ^ \u00C2\u00BB acetate Figure A.85: PK pathway of hexose and pentose utihzation. Key to the enzymes: EC 2.7.1.1: hexokinase; EC 1.1.1.49: glucose 6-phosphate dehydrogenase; EC 3.1.1.17: glu-conolactonase; EC 1.1.1.44: phosphogluconate dehydrogenase; EC 5.1.3.1: ribulose phos-phate 3-epimerase; EC 2.7.1.15: ribokinase; EC 5.3.16: ribosephosphate isomerase; EC 5.3.1.3: arabinose isomerase; EC 2.7^ 1.47: ribulokinase; EC 5.3.1.5: xylose isomerase; EC 2.7.1.17: xylulokinase; EC 4.1.2.9: phosphokeitolase; EC 2.7.2.1: acetokinase. Source is [52]. Appendix A. Fermentation Pathways 237 fructose 2ADP + 2P;-2ATP-2NAD \u00E2\u0080\u00A2 2NADH + 2H* r pyruvate pyruvate CoA- \u00E2\u0080\u00A2Fd \u00E2\u0080\u00A2 FdH 2 CoA-acetyl-CoA 1 C 0 2 -Fd \u00E2\u0080\u00A2FdH, acetyl-CoA CoA-acetate ADP + Pj ATP r X H 2 ' 3 CoA formate ATP\u00E2\u0080\u0094-X H 4 F -ADP + Pj -^1 5 10-formyl-H4F 5, 10-methenyl-H4F NADH + rT J NAD* -*-A 5, 10-methylene-H\u00E2\u0080\u00A2 NAD* C H 3 - C H - C H 2 - C O - S C o A \u00E2\u0080\u0094 O H ( J - h y d r o x y b u t y r y l - C o A C H 3 \u00E2\u0080\u0094 C O - S C o A > c o 3 C H 3 - C O - C O O H p y r u v a t e HjO C H 3 - C H 2 - C H 2 - C O - S C o A b u t y r y l - C o A N A D * - * N N A D H + H * \u00E2\u0080\u00A2 C H 3 - C H = C H - G O - S C o A c r o t o n y l - C o A Figure A.87: Butyrate formation from pyruvate by Clostridia. Key to the enzymes: 1, pyruvate-ferrodoxin oxidoreductase; 2, acetyl-Co A-acetyl transferase; 3, 3-hydroxybu-tyryl-CoA dehydrogenase; 4, 3-hydroxyacyl-CoA hydrolyase; 5, butyryl-CoA dehydroge-nase; 6, fatty acid CoA transferase. Source is [52]. Appendix A. Fermentation Pathways 239 glucose succinate\"] \u00E2\u0080\u00A2 ^ - \u00E2\u0080\u00A2 ^ oxaloacetate - 4\u00E2\u0080\u0094^\u00E2\u0080\u0094 P E P LADP XS*-ATP (H) CO, -(H) ADP 'ATP lactate (H) 5 (H) acetaldehyde \u00E2\u0080\u0094 ^ acetyl-CoA pyruvate X (H) ethanol \u00E2\u0080\u00A2I: :etyl-formate CoA acetyl- (?) ADP ATP X CO, Figure A.88: Formation of mixed products (ethanol, lactate, etc.). 1, enzymes for the EMP pathways; 2, lactate dehydrogenase; 3, pyruvate- formate lyase; 4, for-mate-hydrogen lyase; 5, acetaldehyde dehydrogenase; 6, alcohol dehydrogenase; 7, phos-photransacetylase; 8, acetate kinase; 9, PEP carboxylase; 10, malate dehydrogenase, fumarase and fumarate reductase. Source is [54]. Appendix A. Fermentation Pathways 240 H O O C - C - C O - S C o A I H D s - m e t h y l m a J o n y l - C o A H O O C - C - C O - S C o A I C H 3 L n - m e t h y l m a l o n y l - C o A H O O C - C H , - C H 2 - C O - S C o A s u c c i n y l - C o A C H j C H , C O O H p r o p i o n a t e \u00E2\u0080\u00A2 C H j - C H j - C O S C o A p r o p i o n y l - C o A C H j - C O - P O j 1 \" -C H 3 - C O ~ S C o A a c e t y l - C o A - C H j - C O - C O O H p y r u v a t e / - A T P ^ A M P \u00E2\u0080\u00A2 Pj ADP ATP H O O C - C H 2 - C O - C O O H o x a l a c e t a t e fi L\u00E2\u0080\u0094NADH \u00E2\u0080\u00A2 H* ' r* NAD* H O O C - C H j - C H O H - C O O H m a l a t e r H , 0 C H j = C - C O O \" O pof-p h o s p h o e n o l p y r u v a t e H O O C - C H = C H - C O O H f u m a r a t e i^-ADP \u00E2\u0080\u00A2 pt - H O O C ^ C H , - C H , - C O O H C H 3 - C O O H a c e t a t e - C H , - C H O H - C O O H l a c t a t e Figure A.89: Formation of propionate from pyruvate or lactate. 1, lactate dehydro-genase; 2, pyruvate-ferredoxin oxidorecuctase; 3, phosphoacetyl transferase; 4, acetate kinase; 5, Ds-methylmalonyl- CoA-pyruvate transcarboxylase; 6, malate dehydroge-nase; 7, fumarase; 8, fumarate reductase; 9, succinyl-CoA transferase; 10, L R - methyl-malonyl-CoA mutase; 11, methylmalonyl-CoA racemase; 12, pyruvate-phosphate diki-nase; 13, PEP-carboxytransphosphorylase. Source is [52]. Appendix B Analysis Methods The first two sections describe the analysis of a liquid sample for lactose, lactate, acetate, propionate, butyrate, and ethanol. The third section covers the measurement of the production rate and compositions of the gaseous products. The last one explains the analysis of sohd samples including total organic carbon ( T O C ) , dry weight and ash content of a biomass, etc. B . l Determination of Lactose The lactose concentration in the feed mixtures or the fermenter effluents was assayed with a colorimetric method. Simple sugars, oligosaccharides, polysaccharides, and their derivatives, including the methyl ethers with free or potentially free reducing groups, give an orange-yellow color when treated with phenol and concentrated sulfuric acid. The method is simple, rapid, and sensitive, and gives reproducible results because the color produced is permanent. Therefore, it is unnecessary to pay special attention to the control of the analytical conditions [215]. B . l . l Reagents and Apparatus \u00E2\u0080\u00A2 Phenol solution, 5 % by weight, prepared by adding 20 grams of reagent grade phenol (BDH) to 380 grams of distilled water, which was used throughout the experiments. 241 Appendix B. Analysis Methods 242 \u00E2\u0080\u00A2 Sulfuric acid, reagent grade 95.5 % (BDH), specific gravity 1.84. B . l .2 P r o c e d u r e 1. Five mL of filtrate of the hquid sample filtrated with a membrane filter (d = 0.45 /x, Sartorius, W . Germany) were diluted to such that it contained between 10-100 mg lactose/1. 2. After one mL of the phenol solution and one mL of the diluted solution was pipetted into a test tube (15 mL), five m L of sulfuric acid were added rapidly, the stream of acid being directed against the hquid surface to obtain good mixing. 3. The tube was allowed to stand 10 minutes in air, and then, was shaken in a water bath to be maintained at 20-30 \u00C2\u00B0 C for 10 minutes. 4. In parallel, two such samples were prepared for one sugar solution. A blank was prepared by substituting distiUed water for the sugar solution. 5. Put the samples and the blank in three colorimeter tubes and read the absorbance of the samples at a wave length of 480 nm after the meter's zero point was adjusted on the blank. 6. The concentration of lactose was then determined from the readings by reference to the standard curve in Figure B.90 which had been obtained by analyzing a few lactose solutions with known concentration. B.2 D e t e r m i n a t i o n of Lactate When a very dilute solute solution of lactic acid is heated in the presence of a high concentration of sulfuric acid, it is converted to acetaldehyde. The acetaldehyde can Appendix B. Analysis Methods 243 Absorbance Figure B.90: Standard curve of lactose concentration vs absorbance at 480 nm, The hne is calculated from the equation: S ^ ^ e = 120.3369 'absorbance' - 0.07461. then be assayed by a sensitive color test employing p-hydroxydiphenyl which gives the sample a purple color [216]. This method was modified by adding of sulfuric acid to the aqueous samples so rapidly that a large amount of reaction heat released could be used to supply sufficient heat for the conversion of lactate to acetaldehyde. B.2.1 Reagents and Apparatus \u00E2\u0080\u00A2 CuS0 4 solution; 4 grams of CuS0 4.5H 20 (BDH) were dissolved in 100 mL of distilled water. \u00E2\u0080\u00A2 Hydroxydiphenyl solution; 1 gram of p-hydroxydiphenyl (BDH) was dissolved in 100 mL of 0.08N NaOH solution and stored in a brown bottle in a refrigerator till needed. \u00E2\u0080\u00A2 Sulfuric acid; reagent grade 95.5 % (BDH). Appendix B. Analysis Methods 244 \u00E2\u0080\u00A2 Spetronic 70 ( B A U S H & L O M B ) spectrophotometer. B.2.2 procedure 1. Five mL of filtrate of reactor effluent filtered through a membrane filter (d = 0.45 p) were diluted to give 1-9 mg lactate pre mL. 2. After one m L of the diluted solution and 50 pL CUSCM were added into a test tube (15mL), 5 m L of sulfuric acid was added rapidly into the sample by directing the stream of the acid onto the liquid surface. 3. The sample was allowed to stand in air for 20 minutes and then cooled below 20 \u00C2\u00B0C in tap water. 4. 50 pL of hydroxydiphenyl solution were added without touching the tube's wall, mixed thoroughly, and the sample was allowed to stand for 6 hours. 5. In paraUel, two such samples were prepared for each effluent solution. A blank was also prepared by replacing the effluent solution with distilled water. 6. Put the two samples and the blank in three colorimetric tubes of the spectropho-tometer. The lactate concentration was then determined from the readings at a wave length of 570 nm, after the meter's zero point had been adjusted using the blank, by reference to the standard hne of lactate concentration vs absorbance as shown in Figure B.91. B.3 Determination of Volatile Fatty Acids and Ethanol Acetic acid, propionic acid, butyric acid and ethanol are easily vaporized. Therefore, they can be assayed with a gas chromatograph. Appendix B. Analysis Methods 245 lO-, Absorbance Figure B.91: Standard curve of lactate concentration vs absorbance. The hne is calcu-lated from the equation: Sioctate \u00E2\u0080\u0094 12.0048 'absorbance + 0.005522. B.3.1 Reagents and Apparatus \u00E2\u0080\u00A2 0.2N Phosphoric acid solution. \u00E2\u0080\u00A2 9 standard samples having known concentrations of ethanol (8-600 mg/l), acetic acid (500-7200 mg/l), propionic acid (200-1300 mg/l), butyric acid (600-1800 mg/l), iso-butyric acid (7-20 mg/l) and valeric acid (0-2000 mg/l). \u00E2\u0080\u00A2 Analytical gas chromatograph (GC)-Model 311 (CARLE), equipped with both a TCD and a FID. \u00E2\u0080\u00A2 Gas cylinders of air, hydrogen and He (UNION CARBIDE). \u00E2\u0080\u00A2 A 60/80 Carbopack C/0.3 Carbowax 20 M/0.1 % H 3 P0 4 column, 30\" x 1/8\" stainless steel (SUPELCO). 0 Appendix B. Analysis Methods 246 \u00E2\u0080\u00A2 A computing intergrator-SP 4100 ( S P E C T R A - P H Y S I C S ) . B.3.2 Procedure 1. Five mL of filtrate of the reactor effluent, filtered through a membrane filter (d = 0.45 p), was titrated with 0.2N phosphoric acid solution until the p H value of the sample was below 2. It was calculated by Kisaalita [46] that the p H value of a sample must be acidified below 2.73 if the strongest acid in the sample, acetic acid, could be in free form. The amount of added acid solution was recorded to calculated the dilution factor. 2. The air, hydrogen and He regulators were set at 12, 20, and 23 psig respectively since it is advised by the G C manufacture that these pressures would give an optimal ratio of air to hydrogen and a suitable residence time of the components. The oven temperature was set at 120 \u00C2\u00B0 C . 3. After the G C was maintained at the desired state, the FID was turned on and 5-10 minutes were allowed for stabihzing the basehne of the computing integrator. 4. 0.5 pL of the acidified sample was injected for each run. Each sample was analyzed at least three times and then an average was reported. 5. For each sample, 0.5 pL of a standard sample which had the nearest composition to the unknown sample, was also injected. Therefore, the composition of the unknown sample was determined from the concentration factors calculated from the standard sample, /,-, (the concentrations, Sstandardti devided by the peak areas, Astandard Substituting Equation F.122 for the fraction of active enzyme centers in Equation F.119 gives ^ - w r r a From reactions (1) and (2), the consumption rate of the substrate can be written as follows: r = ~ = KIE^S] - k^[ES} + 8k2[ES}[S] (F.124) Put Equation F.121 into Equation F.124 to give r = k2[ES)(l + 8[S]) (F.125) Finally, replacing the fraction of the substrate-enzyme complex in Equation F.125 by its expression,Equation F.123, the substrate (propionate) utihzation rate can be ex-pressed as a function of substrate concentration, S, k2S(l+8S) r = , T/, o F.126 (fc_x + k2)/ki + S v ' F .2 U t i l i z a t i o n R a t e M o d e l of B u t y r a t e The utihzation of butyrate by acetogens in the methanogenic biofilms is affected by the presence of propionate, especially at the high concentrations of the latter. The reaction mechanism is assumed as follows: E + Sb ^ E S b - ^ P + E (1) Appendix F. Derivation of Utilization Rate Models of Propionate and Butyrate 263 ESb + Sp ^ SpESb (deactivation) (2) With the assumptions that the sum of the fractions of the three types of active centers (E, ESb a n d SpESb) should equals one, and that the fraction of deactivated centers is a constant at steady state, [E] + [ESb] + [SpESb] = 1 (F.127) and, d[SpESb] dt k3[ESb][Sp] - k_3[SpESb] = 0 (F.128) or, [SpESb] = p-[ESb][Sp] (F.129) \" - 3 The Michaelis-Menten assumption that the substrate-enzyme complex is a constant at steady state gives d[ES] dt k^Sb] - k^ESb] ~ k2[ESb] - k3[ESb][Sp] + k_3[SpESb] = 0 k1[E][Sb] = (k-1 + ki)[ESb] (F.130) The fraction of active empty enzyme centers, [E], can be expressed by l\u00C2\u00A3l = W 1 \u00C2\u00A3 S t l ( F 1 3 1 ) Put the expressions for [E] and [SpESb], Equations F.129 and F.131 into Equa-tion F.127 to give the expression of [ESb] a s the foUowing, Appendix F. Derivation of Utilization Rate Models of Propionate and Butyrate 264 [ESb] [sb + (k3/k_3)[s[b][sp] + (*,. + fca)/fcx ( F ' 1 3 2 ) The substrate b is consumed by the reaction (1) only, and its utihzation rate is n = ~lt = fcl^5fc] _ k-i\ESh] (F.133) By using Equation F.130, the rate expression becomes rb = k2[ESb] (F.134) Substituting Equation F.132 for [ESb] in Equation F.134, the digestion rate of butyrate is expressed as a function of the concentrations of butyrate and propionate, Sb and Sp, T b + k2)/kt + Sb + (k3/k_3)SpSb ( F ' 1 3 5 ) A p p e n d i x G N u m e r i c a l M e t h o d s The numerical methods used in this study include a direct search method for the esti-mation of parameters in a non-linear function and the Runge-Kutta-Fehlberg ( R K F ) method for the numerical solution of a differential equation. G.l D i r e c t Search M e t h o d The estimation of several parameters in a non-hnear function is generally converted to an optimization problem, minimizing the difference between the experimental results and the data predicted by the model (a non-hnear function) by changing the values of the parameters. For a non-hnear function of several variables, it is common that a direct search method is used because of the complexity of the differentiations of the function. There have been several methods devised which comprise two major stages in a direct search, an exploratory stage and a pattern stage. One of the simplest methods is due to Hooke and Jeeves [186] which has been used in this study. The algorithm is designed to seek the best direction of search and move fast in that direction as shown in Figure G.97. First of all, a base camp is estabhshed at x \u00C2\u00B0 . For this first stage of the expedition, this point is also the advanced camp x 1 , from which explorations are made. A step of a given length, d, is taken in each of the coordinate directions in turn. If a success is encountered in a direction insofar as a lower objective function value is achieved, progress has been made. If it is not, the step in this direction is decreased by d from the start point and tested again; if it still produces no decrease, stay at the start point. When all 265 Appendix G. Numerical Methods 266 variables are thus dealt with, the exploratory state is complete. It is deemed a success if the value of the function at the latest point, x', is less than f ( x \u00C2\u00B0 ) . Otherwise it is a failure. If successful, the base camp is moved to x' and the advanced camp is moved to x1 = x' + (x' - x0); A new search is carried out from the new advanced camp and the process repeated. A computer program in Pascal language is attached here which has been successfuUy used in a personal computer with Turbo Parscal. G.2 Runge-Kutta-Fehlberg Method Since there are relatively few differential equations arising from practical problems like the reaction-diffusion model for which analytical solutions are known, one must resort to numerical methods. In general, an mth-order differential equation, y{m) = f(x,y,y',y\",---,y{rn~1)) with initial conditions y(xo) = yo y'M = y'0 y^ixo) = can be rewritten as an equivalent system of m first-order equations. To do so, a new set of dependent variables yi(x), y2(x), . . . , y m (x) are defined by yi=y 2/2 = y' endix G. Numerical Methods 267 (START) I N P U T B A S E P O I N T X ( 0 > S T E P S I Z E D A N D D M I N | E V A L U A T E F U N C T I O N A T X<\u00C2\u00B0> | \u00E2\u0080\u00A2 1 P U T X < 0 = X ^ ' F O R S T A R T P H A S E r [PUT I = I I I OUTPUT RESULTS | f STOP) Figure G.97: Flow chart of the direct search method Appendix G. Numerical Methods 268 2/3=2/ Therefore, a mth-order differential equation (e.g. the reaction-diffusion model is a 2nd-order differential equation) can be numerically solved like m first-order differential equations. R u n g e - K u t t a methods are expbcit algorithms that involve evaluation of the function f at points in a small interval, between x,- and x ; + i . In order to estimate the errors occurring during computation, a local error would be calculated from the difference between u * t + 1 and U i + i where u J + i is calculated using a step-size of h and u * 1 + 1 using a step-size of h/2 . Since the accuracy of the numerical method depends upon the step-size to a certain power, u * 2 + 1 wi l l be a better estimate for y ( x I + 1 ) than U i + 1 . Therefore, \zi+i - U | < \zi+i - ui+1\ and so, e t + l = z i + l ~ u i + l \u00E2\u0080\u0094 u + l \u00E2\u0080\u0094 u i + l R u n g e - K u t t a formulas, if two half-step procedure is used, would need many com-putations. A better procedure is Runge-Kutta-Fehlberg's method [200], which uses a Runge-Kut ta formula of higher-order accuracy than used for u l + i to compute u * l + 1 . In this study, the Runge-Kutta-Fehlberg fourth-order method was used and the pari of formulas is . 25 , 1408, 2197, 1, , n , . . U; + i = li; -f K i H \u00C2\u00AB 3 H KA K 5 e, + i = 0(/l ) 1 + 1 L216 2565 4104 5 J + V ' . 16 , 6656 , 28561, 9 , 2 , . n . f 6 . u*i+i = u{ + + \u00E2\u0080\u0094 k3 + \u00E2\u0080\u0094 k4 - - k 5 + - k 6 ] ei+1 = 0(h ) Appendix G. Numerical Methods 269 where fcj = hf(xi,Ui) k2 = hf(xi + \h,Ui -k3 = hf(xi + lh,Ui -kA - hf(xi + ^h,u{ k5 = hf(xi + h,Ui + k6 = hf(x{ + \h,Ui - n 40 Notice that the formula for U{ +i is fourth-order accurate but requires five function evaluations as compared with the four of the Rung-Kut ta -GiU method, which is of the same order accuracy. However, if e j + 1 is to be estimated, the half-step method using the Runge-Kutta-Gil l method requires eleven function evaluations while the R K F method requires only six. The following computer program , written in Pascal language, was used to solve numerically the reaction-diffusion model which has been described in Chapter 7 on mass transfer in active biofilms. G.3 The Computer Programs in Pascal Language The direct search program (Procedure M U L T I V A R ) and the R K F program (Procedure RungeKutta) are called by a main program (Program S E A R C H ) . The main program reads the experimental data from a D O S file 'Data' (Procedure G E T D A T A ) , calculates the value of the objective function at a set of estimated parameters (Procedure S U M ) , determines if a search is successful by comparing the values of the objective function at two search points (Procedure T E S T A R R Y ) and completes the search for a set of param-eters which give the minimum value of the objective function (Procedure M U L T I V A R ) . Appendix G. Numerical Methods 270 The objective function used in this study was a sum of square errors between the ex-perimental data and the values predicted by a model at a set of estimated parameters. For the model of build-up of symbiotic methanogenic biofilms, the predicted values could be directly calculated from the model. However, for the reaction-diffusion model, the program (Procedure RungeKutta) of Runge-Kutta-Fehlber's method must be called in by the procedure S U M to calculate the substrate concentrations on the biofilm surface with the model, and then, by comparing the calculated values with the experimentally measured values, a set of optimal parameters (diffusion coefficients) were determined. PROGRAM SEARCH; {The main program} CONST dimen = 1 ; {dimensions of parameter set } nequatn = 1;{number of e q u a t i o n s } ntime = 1;{number of e x p e r i m e n t a l data of} {independent v a r i a b l e x} nmax = 2; {number of dependent v a r i a b l e s y p l u s 1} TYPE {type of v a r i a b l e s ; r e a l , l n t e g r , etc} a r r y 2 = A R R A Y [ 1 . . n t ime, 1 . .nmax] OF r e a l ; paramet = A R R A Y [ 1 . . d i m e n ] OF r e a l ; a r r y l = A R R A Y [ 1 . . n e q u a t n ] OF r e a l ; -'VAR {names of the v a r i a b l e s } d a t a : arry2; paraO : paramet; s t e p , s tepm: r e a l ; i : i n t e g e r ; f i l v a r : t e x t ; PROCEDURE GETDATA(VAR d a t a : a r r y 2 ) ; {read data from a d i s k f i l e } VAR i , J : i n t e g e r ; Appendix G. Numerical Methods 271 BEGIN { read data from the DOS f i l e ' D a t a ' } a s s I g n ( f i 1 v a r , ' D a t a ' ) ; {$!-} {turn o f f e r r o r checking} r e s e t ( f i l v a r ) ; {$!+} {turn i t back on} FOR i := 1 TO ntlme DO BEGIN {Data f i l e } FOR j := 1 TO nmax DO {x, y} r e a d ( f i 1 v a r , d a t a [ i , j ] ) ; {x, y} r e a d l n ( f i l v a r ) {....} END {x, y} END; PROCEDURE SUM(x:paramet; VAR y v a l u e : REAL) ; { c a l c u l a t e the v a l u e s of the o b j e c t i v e f u n c t i o n } {x g i v e s the parameters} {yvalue sends out the c a l c u l a t i o n r e s u l t } VAR equatn : a r r y l ; i , j : i n t e g e r ; y : r e a l ; {$i RungKuta.pas} {Runge-Kutta method to s o l v e a d i f f e r e n t i a l equation} {It s h o u l d be removed i f d i f f e r e n t i a l e q u a t i o n s not i n v o l v e d } BEGIN RungeKutta ( y ) ; { s o l u t i o n of a d i f f e r e n t i a l equation} y v a l u e := s q r ( y - d a t a [ l , l ] ) {sum of e r r o r } END; PROCEDURE TESTARRY(xlary ,x2ary :paramet ;VAR r 1 , r 2 : I N T E G E R ) ; {compare the v a l u e s of the o b j e c t i v e f u n c t i o n at two p o i n t s } {and determine i f a s e a r c h i s success} VAR i : INTEGER; BEGIN r l := 1; r2 := 1; FOR i := 1 TO dimen DO BEGIN IF x l a r y C i ] = x 2 a r y [ i ] THEN r e s u l t 1 := i ELSE r e s u l t 2 := 0 END END; Appendix G. Numerical Methods 272 {$i m u l t i v a r . p a s } {the d i r e c t s e a r c h program} BEGIN {main program} w r i t e l n ( ' i n p u t your i n i t i a l guess of p a r a m e t e r s ' ) ; FOR I := 1 TO dimen DO BEGIN W R I T E C p ' . l , ' = ' ) ; r e a d l n ( p a r a O [ i ] ) END; w r i t e ( ' s t e p l e n g t h =') ; r e a d l n ( s t e p ) ; wr i t e ( ' m i n i m u m s t e p l e n g t h =') ; r e a d l n ( s t e p m ) ; g e t d a t a ( d a t a ) ; m u l t i v a r ; w r i t e l n ( ' a p e r f e c t e x i t ' ) END. Appendix G. Numerical Methods 273 PROCEDURE MULTIVAR; {the d i r e c t s e a r c h method} VAR p a r a , p a r a l , p a r a 2 : paramet; i , x l e x O , x l n e x O : i n t e g e r ; f v a l u e , f v a l u e O , f v a l u e l , f v a l u e 2 , \u00C2\u00A3 : r e a l ; BEGIN{procedure m u l t i v a r } FOR i := 1 TO dimen DO begi n p a r a [ i ] := p a r a O [ i ] ; p a r a l [ i ] := p a r a O t i ] end; REPEAT {reduce s t e p l e n g t h loop} for i := 1 to dimen do p a r a [ i ] := p a r a O C i ] ; FOR i := 1 TO dimen DO BEGIN {N p a r a m e t e r s ' e x p l o r a t i o n } p a r a C i ] := p a r a l [ i ] + s t e p ; S U M ( p a r a , f ) ; f v a l u e := f ; S U M ( p a r a l , f ) ; f v a l u e l := f ; IF f v a l u e >= f v a l u e l THEN BEGIN {backward one step} p a r a [ i ] := p a r a l [ i ] - s t e p ; IF p a r a C i 3 < 0 THEN para [ i ] : = 0; S U M ( p a r a , f ) ; f v a l u e := f ; S U M ( p a r a l , f ) ; f v a l u e l := f ; IF f v a l u e >= f v a l u e l THEN p a r a 2 [ i ] := p a r a l [ i ] ELSE p a r a 2 [ i ] := p a r a [ i ] END {end badkward one step} ELSE p a r a 2 [ i ] : = p a r a [ i ] END; {end n p r a m e t e r s ' e x p l o r a t i o n } SUM(para2, f ) ; f v a l u e 2 := f ; SUM(paraO, f ) ; f v a l u e O := f ; wr 1 1 e l n ( 1 f u n c t i o n v a l u e f v a l u e O = ' , f v a l u e O ) ; IF f v a l u e 2 >= f v a l u e O THEN BEGIN Appendix G. Numerical Methods 274 T E S T A R R Y ( p a r a l , p a r a O , x l e x O , x l n e x O ) ; I F x l e x O = x l n e x O THEN s t e p := s t e p / 2 E L S E FOR i := 1 TO d i m e n DO p a r a l [ i ] := p a r a O [ i ] END E L S E FOR i := 1 TO d i m e n DO B E G I N p a r a l C i ] := p a r a 2 [ i ] + p a r a 2 [ i ] - p a r a O [ i ] ; p a r a O [ i ] : - p a r a 2 [ i ] E N D ; I F ( 2 * s t e p ) < s t e p m THEN B E G I N W R I T E L N ( ' T H E MINIMUM F U N C T I O N = ' , f v a l u e O ) ; w r i t e l n ( ' N p a r a m e t e r s ' ) ; FOR i := 1 TO d i m e n DO wr i t e l n ( ' p a r a [ ' , ! , ' ] = ' , p a r a O [ i ] ) E N D ; FOR i := 1 TO d i m e n DO W R I T E C ' , p a r a O [ i ] ) ; wr i t e1n U N T I L < 2 * s t e p ) < s t e p m E N D ; Appendix G. Numerical Methods PROCEDURE RungeKutta (VAR S i n i t i a l : r e a l ) ; { R u n g e - K u t t a - F e h l b e r g ' s method} l a b e l s t a r t ; const a l l = 25/216; a l 3 = 1408/2565; a l 4 = 2197/4104; a21 = 16/135; a23 = 6656/12825; a24 = 28561/56430; a25 = 9/50; a26 = 2/55; b31 = 3/32; b32 = 9/32; b41 = 1932/2197; b42 = 7200/2197; b43 = 7296/2197; b51 = 439/216; b53 = 3680/513; b54 = 845/4104; b61 = 8/27; b63 = 3544/2565; b64 = 1859/4104; b65 = 11/40; m = 4; 12 t imes the number of d i f f e r e n t i a l e q u a t i o n s } e l t = 0.00001; {error of l a c a l t r u n c t i o n } ex = 0 .001; {error of f i n a l length} d i v i s i o n = 1000; { d i v i s i o n of a b i o f i l m t h i c k n e s s } t h i c k n e s s = 0.078; { b i o f i l m t h i c k n e s s (cm)} Var yO, y, z , u : a r r a y [ l . . m ] of r e a l ; s t e p , l m a r k : r e a l ; k : a r r a y [ 1 . . 6 , 1 . . m ] of r e a l ; i : i n t e g e r ; {y0 ,u : HBu, dHBu/dx, HPr, dHPr/dx} {Four f u n c t i o n s are d e f i n e d as f o l l o w i n g } FUNCTION F l ( a , b , c , d : r e a l ) : r e a l ; { a , b , c , d are f a l s e v a r i a b l e s } b e g i n F l := b ; end; Appendix G. Numerical Methods 276 FUNCTION F 2 ( a , b , c , d : r e a l ) : r e a l ; { a , b , c , d are f a l s e v a r i a b l e s } begl n F2 :=x[ l ]*a / (0 .027+a+0 .296*a*c) ; {the u t i l i z a t i o n r a t e of butyra te } end; FUNCTION F 3 ( a , b , c , d : r e a l ) : r e a l ; begi n F3 := d ; end; FUNCTION F 4 < a , b , c , d : r e a l ) : r e a l ; b e g i n F4 : = 2 7 . l * c * ( l + 0 . 2 * c ) / ( 0 . 0 5 + c ) ; {the u t i l i z a t i o n r a t e of propionate } end; Begin s t e p := t h i c k n e s s / d i v i s i o n ; ltnark := 0; y 0 [ l ] := 0.326; {HBu c o n c e n . (mg/ml) i n s i d e the c e l l } y0 [2 ] := 0; {ds/dx of HBu i n s i d e the c e l l } y 0 [ 3 ] := 1.715; {HPr c o n c e n . (mg/ml) i n s i d e the c e l l } y0[4 ] := 0; {ds/dx of HPr i n s i d e the c e l l } {Four i n i t i a l c o n d i t i o n s } s t a r t : f o r i := 1 to m do u [ i ] := y 0 [ i ] ; k [ l , l ] := s t e p * F l ( u [ l ] , u C 2 ] , u [ 3 ] , u [ 4 ] ) ; k [ l , 2 ] := s t e p * F 2 ( u [ l ] , u [ 2 ] , u t 3 ] , u [ 4 ] ) ; k [ l , 3 ] := s t e p * F 3 ( u [ l ], u [ 2 ] , uC3] , u [ 4 ] ) ; k [ l , 4 ] := s t e p * F 4 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; f o r I := 1 to u do u [ i ] . : = y 0 [ i J + k [ l , i ] /4; k [ 2 , l ] := s t e p * F l ( u [ l ] , u [ 2 ] , u [ 3 ] J u t 4 ] ) ; k [ 2 , 2 ] := s t e p * F 2 < u [ l ] , u t 2 ] , u [ 3 ] , u [ 4 ] ) ; k [ 2 , 3 ] := s t e p * F 3 ( u [ l ] , u [ 2 ] , u [ 3 ] J u [ 4 ] ) ; k [ 2 , 4 ] := s t e P * F 4 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; f o r i := 1 to m do u [ l ] := y 0 [ i ]+b31*k[l , i ]+b32*k[2, i ] ; k [ 3 , l ] := s t e p * F l ( u [ l ] > u [ 2 ] , u [ 3 ] , u t 4 ] ) ; k [ 3 , 2 ] := s t e p * F 2 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; k [ 3 , 3 ] := s t e p * F 3 ( u [ l ], u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; k [ 3 , 4 ] := s t e p * F 4 ( u [ l ] , u [ 2 ] , u [ 3 ] , u t 4 ] ) ; for i : = 1 to m do Appendix G. Numerical Methods u [ i ] := y O [ i ] + b 4 1 * k [ l , i ] - b 4 2 * k [ 2 , i ] + b 4 3 * k [ 3 , i ] ; k [ 4 , l ] := s t e p * F l ( u [ l ] , u [ 2 ] , u [ 3 ] , u t 4 ] ) ; k [ 4 , 2 ] := s t e p * F 2 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; k [ 4 , 3 ] := s t e p * F 3 ( u [ l ] , u [ 2 ] , u [ 3 ] , u t 4 ] ) ; k [ 4 , 4 ] := s t e p * F 4 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; for i := 1 to m do u [ i ] := y O [ i ] + b 5 1 * k [ l , 1 ] - 8 * k [ 2 , i ] + b 5 3 * k[3 , i ] -b54*k[4, 1 ]; k [ 5 , l ] := s t e p * F l ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; k [ 5 , 2 ] := s t e p * F 2 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 3 ) ; k [ 5 , 3 ] := s t e p * F 3 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; k [ 5 , 4 ] := s t e p * F 4 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; for i : = l t o m d o u [ i ] := y O [ i ] - b 6 1 * k [ l , i ] + 2 * k [ 2 , i ] - b 6 3 * k [ 3 , i ]+b64*k[4, i ] -b65*k[5 , 1 ]; k [ 6 , l ] := s t e p * F l ( u [ l ] , u [ 2 ] , u C 3 ] , u [ 4 ] ) ; k [ 6 , 2 ] := s t e p * F 2 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; k [ 6 , 3 ] := s t e p * F 3 ( u t l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; k [ 6 , 4 ] := s t e p * F 4 ( u [ l ] , u [ 2 ] , u [ 3 ] , u [ 4 ] ) ; f o r i := 1 to m do begin y [ i ] : = y O [ i ] + a l l * k [ l , i ] + a l 3 * k [ 3 , i ] + a 1 4 * k[4 , i ] -k [5 , i ] /5 ; z [ i ] : = y O [ i ] + a 2 1 * k [ l , i ] + a 2 3 * k [ 3 , i ] + a 2 4 *k[4 , i ] -a25*k[5, i ]+a26*k[6, i ] ; end; i f a b s ( y [ l ] - z [ l ] ) > e l t then b e g i n s t e p := s t e p / 2 ; goto s t a r t ; end; i f a b s ( y [ 2 ] - z [ 2 ] ) > e l t then b e g i n s t e p := s t e p / 2 ; goto s t a r t ; end; lmark := lmark + s t e p ; i f a b s ( t h i c k n e s s - lmark) > ex then b e g i n f o r i := 1 to m do y O [ i ] := z [ i ] ; i f ( lmark+2*step) <= t h i c k n e s s then b e g i n s t e p := 2*s tep ; goto s t a r t ; end; s t e p := t h i c k n e s s - lmark; goto s t a r t ; end; wr i t e l n ( ' c o n c n = z [ l ] ) ; w r i t e l n ( ' d s / d x =\u00E2\u0080\u00A2',- z [ 2 ] ) ; S i n i t i a l := z [ l ] ; end; "@en . "Thesis/Dissertation"@en . "10.14288/1.0059041"@en . "eng"@en . "Chemical and Biological Engineering"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Two-phase methanation of lactose in biofilm reactors"@en . "Text"@en . "http://hdl.handle.net/2429/31900"@en .