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Anaerobic fermentation of whey : acidogenesis 1987

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ANAEROBIC FERMENTATION OF WHEY: ACIDOGENESIS by WILLIAM SSEMPA KISAALITA B.Sc.(Eng.)(Hon.), Makerere Univesity, 1978 M.A.Sc., University of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Chemical Engineering) We accept the thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1987 © William Ssempa K i s a a l i t a , 1987 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 C t f B M l C f l - L - B - M G~( N &£rlQ-. | N (r- The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date <&( f /gT- i i ABSTRACT Based on the i n i t i a l e x p l o r a t o r y r e s u l t s of single-phase (acidogenesis and methanogenesis takes place i n one ve s s e l ) whey biomethanation s t u d i e s , a two-phase (acidogenesis and methanogenesis takes place i n two separated s e r i a l v e s s e l s ) biomethanation process was found to be more s u i t a b l e f o r d e a l i n g w i t h the c u r r e n t whey u t i l i s a t i o n and/or d i s p o s a l problem. Acidogenesis was found to be l e s s understood i n comparison to methanogenesis and t h e r e f o r e acidogenesis became the c e n t r a l problem of t h i s t h e s i s . Given that 90% of the f i v e - d a y biochemical oxygen demand i n whey i s due to l a c t o s e , continuous c u l t u r e (Chemostat) experiments were undertaken to examine the general mechanism of l a c t o s e acidogenesis by a mixed 14 u n d e f i n e d c u l t u r e u s i n g C - l a b e l e d t r a c e r s . A l s o the i n f l u e n c e of whey p r o t e i n (mainly p - l a c t o g l o b u l i n ) on the general fermentation scheme was addressed. Experimental f a c t o r s i n c l u d e d a pH range of 4.0 to 6.5, a m e s o p h i l i c temperature of 35°C and a d i l u t i o n r a t e (D) range of 0.05 to 0.65 h " 1 . At a f i x e d pH l e v e l , the observed v a r i a b i l i t y i n the main acidogenic end products ( a c e t a t e , propionate, butyrate and l a c t a t e ) w i t h respect to D were found to be a consequence of the systematic s e p a r a t i o n of the various m i c r o b i a l g r o u p s i n v o l v e d i n a c i d o g e n e s i s . B a t c h i n c u b a t i o n of a 14 [ C ( U ) ] - l a c t a t e t r a c e r w i t h chemostat e f f l u e n t samples and p r e p a r a t i v e s e p a r a t i o n of the end products f o l l o w e d by a l i q u i d s c i n t i l l a t i o n assay of the l o c a t i o n of the r a d i o a c t i v i t y demonstrated that a m i c r o b i a l p o p u l a t i o n s h i f t w i t h i n c r e a s i n g D was r e s p o n s i b l e f or d i s a b l i n g the conversion of i i i l a c t a t e to other end products and hence the observed increase i n l a c t a t e concentrations at high D values. 14 14 F u r t h e r use of [ C(U)]-butyrate and [ C(2)]-propionate revealed the predominant carbon flow routes from pyruvate to the various end products. A q u a l i t a t i v e lactose acidogenic fermentation model was proposed, i n which lac t o s e i s converted to pyruvate v i a the Embden-Meyerhof-Parnas pathway. Pyruvate i n a p a r a l l e l r e a c t i o n i s then converted to l a c t a t e and butyrate. In the presence of hydrogen reducing methanogens l a c t a t e i s converted to acetate i n a very fast r e a c t i o n and not propionate as previously believed-. The implications of these findings with regard to optimising the acidogenic phase reactor are discussed. Acidogenic fermentation of protein together with lactose did not a f f e c t the carbon flow scheme. In the D range of 0.05 to 0.15 h \ low pH (pH < 5.0) was found to favour the butyrate route at the expense of the l a c t a t e route and at high pH (pH > 5.5) the l a c t a t e route was favoured at the expense of the butyrate route, the pH region of 5.0 to 5.5 being the t r a n s i t i o n range. In order to describe the m i c r o b i a l growth, the Monod chemostat model was chosen among the various a l t e r n a t i v e s , because of i t s s i m p l i c i t y and i t s physico-chemical b a s i s . The estimated model parameters are reported. i v TABLE OF CONTENTS Pages ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF FIGURES x NOMENCLATURE AND ABBREVIATIONS xiv ACKNOWLEDGEMENTS x v i I. INTRODUCTION 1 1.1 Whey 1 1.2 Whey Disposal and/or U t i l i z a t i o n 4 1.3 Biomethanation Process 4 1.4 Whey Biomethanation 7 1.5 Two-Phase Process 11 1.6 Research Objectives 13 1.7 Scope of the Study 15 II. PREVIOUS WORK AND THEORETICAL ASPECTS 17 2.1 Energetics and Metabolic Stages i n Anaerobiosis 17 2.1.1 Energetics 17 2.1.2 Metabolic Stages 19 2.1.2.1 Hy d r o l y t i c B a c t e r i a 22 2.1.2.2 The ^ - p r o d u c i n g Bact e r i a 23 2.1.2.3 Homoacetogenic B a c t e r i a 25 2.1.2.4 The Methanogenic B a c t e r i a 25 2.1.2.5 S p e c i f i c B a c t e r i a l Species Associated with Whey Biomethanation 27 2.1.2.6 Important Process Parameters 28 V Pages 2.2 Mathematical Modeling of M i c r o b i a l Growth 28 2.2.1 Model C l a s s i f i c a t i o n 28 2.2.2 Models for Single Substrate Limited Growth 31 2.2.2.1 Monod Equation 33 2.2.2.2 Other Equations 33 2.2.3 Models with S p e c i f i c Growth Substrate and/or Biomass Growth Dependence 36 2.2.4 M i c r o b i a l Growth Modeling Recommendation 39 2.2.5 Miscellaneous Models 39 2.2.5.1 Models of Growth i n Presence of I n h i b i t i n g Substrate/Product 39 2.2.5.2 Models for Growth Limited by More Than One Substrate 41 2.2.5.3 Models for Product Formation 42 2.3 Mesophilic M i c r o b i a l K i n e t i c Models: L i t e r a t u r e Review .. 43 2.3.1 Whey and/or Lactose Substrates 43 2.3.2 Other Substrates 46 2.4 Mathematical Analysis of Continuous Culture 46 2.5 Experimental Plan 51 2.5.1 Assumptions 51 2.5.2 Experimental Factors 51 I I I . MATERIALS AND METHODS 53 3.1 Inoculum 53 3.2 Media 53 3.2.1 Lactose Limited Growth Sythetic Medium 53 3.2.2 Lactose/Protein Growth Medium 55 v i Pages 3.3 Fermentor Set-up 55 3.4 Fermentor Start-up and Operation Procedure 58 3.5 Set-up f o r R a d i o a c t i v e Tracer I n c o p o r a t i o n 59 3.5.1 Apparatus w i t h pH C o n t r o l 59 3.5.2 Apparatus without pH C o n t r o l 61 3.6 P r e p a r a t i v e Separation of Organic Acids 61 3.6.1 P r i n c i p l e 61 3.6.2 Apparatus 64 3.6.3 M a t e r i a l s and Reagents 67 3.6.4 Procedure 67 3.7 Determination of R a d i o a c t i v i t y 69 3.7.1 P r i n c i p l e 69 3.7.2 Channels R a t i o Method 72 3.7.3 Procedure 72 IV. RESULTS AND DISCUSSION 75 4.1 I n f l u e n c e of D i l u t i o n Rate (D) on Organic Acids (OA) D i s t r i b u t i o n 76 4.2 I n f l u e n c e of D on Gaseous Products D i s t r i b u t i o n •••••••••• 82 4.3 Radio Tracer Studies 90 4.3.1 R e s u l t s of the Radio Tracer Experiments at High D . 91 4.3.1 R e s u l t s of the Radio Tracer Experiments at Low D .. 95 4.4 Proposed Lactose Acidogenic Fermentation Model 102 4.5 S i g n i f i c a n c y of F i n d i n g s 108 4.6 I n f l u e n c e of P r o t e i n on the Fermentation Model I l l 4.7 I n f l u e n c e of pH on Carbon Flow from Pyruvate 115 4.8 M i c r o b i a l Growth Model 117 v i i Pages V. CONCLUSIONS AND RECOMMENDATIONS 132 5.1 C o n c l u s i o n s 132 5.2 Recommendations 135 LITERATURE CITED 136 APPENDIX A 155a APPENDIX B 173 APPENDIX C 183 APPENDIX D 188 v i i i LIST OF TABLES Pages Table 1.1 T y p i c a l Composition Whey S o l i d s 2 Table 1.2 Estimated Q u a n t i t i e s of F l u i d Whey Produced i n the USA 3 Table 1.3 Estimated Q u a n t i t i e s of F l u i d Whey Produced i n Canada 3 Table 1.4 Two-Phase P i l o t and F u l l Scale P l a n t s 12 Table 2.1 O v e r a l l Reaction Schemes of Some Important Acidogenic Fermentation 20 Table 2.2 Gen e r a l i s e d Growth Constants f o r Anaerobiosis 47 Table 2.3 Fact o r s I n v e s t i g a t e d 52 Table 3.1 Lactose L i m i t e d Growth Medium 54 Table 3.2 Lactose and P r o t e i n Growth Medium A n a l y s i s 56 Table 4.1 Maximum S p e c i f i c Growth Rate Values f o r Methanogenic B a c t e r i a 89 Table 4.2 R a d i o a c t i v i t y D i s t r i b u t i o n f o r High D Radio Tracer Experiments 94 Table 4.3 R a d i o a c t i v i t y D i s t r i b u t i o n f o r Low D Radio Tracer Experiments 99 Table 4.4 D i s t r i b u t i o n of T o t a l Free Energy Change f o r Growth of the Two-Phase Anaerobic Process of Glucose to Methane Over D i f f e r e n t M i c r o b i a l Groups 110 Table 4.5 R a d i o a c t i v e D i s t r i b u t i o n f o r Samples from Experiments With L a c t o s e / P r o t e i n Substrate 116 ix Pages Table 4.6 Monod Chemostat Model P r e d i c t i o n s for D i f f e r e n t K s Values Based on u and Y Approximat ions fo r a pH of 6.0 129 Table 4.7 Comparisons of Monod Chemostat Model Constants for Ac idogenes i s 130 Table B l T o t a l Carbon Mass Balance (Lac tose Growth L i m i t e d Subs t r a t e ) at a pH of 6.0 and a Temperature of 35°C 174 Table B2 T o t a l Carbon Mass Balance ( L a c t o s e / P r o t e i n Growth L i m i t e d Subs t r a t e ) at a pH of 6.0 and a Temperature of 35°C 176 Tab le B3 T o t a l Carbon Mass Balance (Lac tose Growth L i m i t e d Subs t r a t e ) at a pH o f 4 .5 and a Temperature of 35°C 177 Tab le B4 T o t a l Carbon Mass Balance (Lac tose Growth L i m i t e d Subs t ra t e ) at a D i l u t i o n Rate of 0.05 h ^ and a Temperature of 35°C 178 Table B5 T o t a l Gas P r o d u c t i o n as a F u n c t i o n of Cummulative E x p e r i m e n t a l Time at a Temperature of 25°C and a P ressu re of One Atmosphere 179 X LIST OP FIGURES Pages F i g u r e 1.1 Three D i f f e r e n t Biomethanat ion Processes 6 F i g u r e 1.2 F i v e B a s i c Anaerobic F i x e d F i l m Reac tor Types 8 F i g u r e 1.3 Three B a s i c Anaerob ic F l o c s / N o n - A t t a c h e d F i l m Reac tor Types 9 F i g u r e 2.1 O v e r a l l R e a c t i o n Schemes of Some Important A c i d o g e n i c Fermenta t ion Types 21 F i g u r e 2.2 E f f e c t of P a r t i a l P ressure of Hydrogen on the Free Energy Change for the Degrada t ion of E t h a n o l , P rop iona t e and Bu ty ra t e w i t h Methane Format ion from H 2 / C 0 2 24 F i g u r e 2.3 P o s s i b l e P e r s p e c t i v e I n t e r a c t i o n s f o r C e l l P o p u l a t i o n K i n e t i c R e p r e s e n t a t i o n 32 F i g u r e 2.4 A Combinat ion of Equat ions fo r the Subs t ra te and/or Biomass C o n c e n t r a t i o n Dependence of the S p e c i f i c Growth Rate 40 F i g u r e 2 .5 The I d e a l Con t inuous -F low S t i r r e d Tank Reac tor (CSTR) . . . 48 F i g u r e 3.1 Schematic Diagram of the Fermentor and A u x i l l i a r y Apparatus 57 F i g u r e 3.2 Schematic Diagram of the R a d i o t r a c e r Experiment Apparatus w i t h pH C o n t r o l 60 F i g u r e 3.3 M o d i f i e d v i a l fo r R a d i o t r a c e r Exper iments Without pH C o n t r o l 62 F i g u r e 3.4 T y p i c a l Chromatogram of a Complex M i x t u r e Separated by a Combinat ion of Simple and Stepwise E l u t i o n 65 x i Pages F i g u r e 3.5 Schematic Diagram of the L i q u i d Chromatography Equipment Assembly 66 F i g u r e 3.6 T y p i c a l Organic A c i d s Chromatogram 70 14 F i g u r e 3.7 C Spec t r a 73 F i g u r e 3.8 E f f i c i e n c y Curve Prepared From Commercial Quenched Standards 73 F i g u r e 4 .1 Products D i s t r i b u t i o n as a F u n c t i o n of D i l u t i o n Rate at a pH of 6.0 78 F i g u r e 4 .2 P o s s i b l e Lac tose Fermenta t ion Models to V a r i o u s End Produc t s 83 F i g u r e 4 .3 Three R e p r e s e n t a t i v e P l o t s of Gas P r o d u c t i o n 85 F i g u r e 4 .4 Three R e p r e s e n t a t i v e P l o t s of Gas P r o d u c t i o n Where the pH was M a i n t a i n e d at the D e s i r e d V a l u e of 6.0 Throughout the E x p e r i m e n t a l P e r i o d 86 F i g u r e 4.5 Fermentor Head Space Gas A n a l y s i s 88 F i g u r e 4.6 A d a p t a t i o n Mechanism i n Organisms and T h e i r Orders of Magnitude of T h e i r R e l a x a t i o n Times 92 F i g u r e 4.7 Radiochromatogram f o r Run Number C4 93 F i g u r e 4 .8 pH-Tirae R e l a t i o n s h i p fo r a Batch R a d i o t r a c e r I n c o r p o r a t i o n Experiment Without pH C o n t r o l 96 F i g u r e 4.9 Radiochromatogram f o r Run Number C l l 97 F i g u r e 4.10 Recovered R a d i o a c t i v i t y D i s t r i b u t i o n for 14 [ C ( U ) ] - B u t y r a t e Trace r a t V a r i o u s Batch Experiment Times 100 x i i Pages Figure 4.11 Recovered R a d i o a c t i v i t y D i s t r i b u t i o n for 14 [ C(U)]-Lactate Tracer at Various Batch Experiment Times 101 14 Figure 4.12 Fermentation Time Course for [ C(U)]-Lactate Degradation i n Single-Phase Lactose Fermentor Sample 104 Figure 4.13 The .Microbial Lactose Fermentation Model i n Three D i s t i n c t But Simultaneous Trophic Phases 105 Figure 4.14 The M i c r o b i a l Acidogenic Fermentation Model 106 Figure 4.15 Comparison of the Main Products D i s t r i b u t i o n f o r Lactose/Protein and Lactose Substrates Experiments 114 Figure 4.16 Products D i s t r i b u t i o n as a Function of pH at a D i l u t i o n Rate of 0.05 h _ 1 118 Figure 4.17 Products D i s t r i b u t i o n as a Function of D i l u t i o n Rate at a pH of 4.5 119 Figure 4.18 Experimental Continuous Culture Data Q u a n t i t a t i v e l y Consistent with the O r i g i n a l Monod Chemostat Model in a Pure Culture of Aerobacter Aerogenes 121 Figure 4.19 Experimental Continuous Culture Data With a Trend Contrary to the O r i g i n a l Monod Chemostat Model i n a Pure Culture of Aerobacter Aerogenes i n a G l y c e r o l Medium 121 Figure 4.20 Dry Biomass Concentration as a Function of D i l u t i o n Rate at a pH of 6.0 122 Figure 4.21 Dry Biomass Concentration as a Function of D i l u t i o n Rate at a pH of 4.5 123 x i i i Pages F i g u r e 4.22 In f luence of the Maintenance C o e f f i c i e n t on the Shape of the Biomass P r e d i c t i o n Curve 126 F i g u r e 4.23 Monod Chemostat Model P r e d i c t i o n s i n Comparison With Expe r imen ta l Data for a pH of 4 .5 128 F i g u r e A l C a l i b r a t i o n Curve fo r Lac tose Us ing the P h e n o l - S u l f u r i c A c i d Method 158 F i g u r e A2 C a l i b r a t i o n Curve fo r P r o t e i n Us ing the B i u r e t - R e a c t i o n Method 160 F i g u r e A3 C a l i b r a t i o n Curve for Formate Using the Method of Lang and Lang (1972) 162 F i g u r e A4 C a l i b r a t i o n Curve for L a c t a t e Us ing a M o d i f i e d Method of Markus (1950) 164 F i g u r e A5 A T y p i c a l V o l a l i t e F a t t y A c i d s Chromatogram 167 F i g u r e A6 V o l a t i l e F a t t y A c i d s C a l i b r a t i o n Curves 167 F i g u r e A7 Fermentor Head Space Gas Chromatograms 169 F i g u r e A8 Fermentor Head Space C a l i b r a t i o n Curves 169 F i g u r e A9 Schematic Diagram of the Automat ic Carbon A n a l y s e r 171 F i g u r e C l Break-down of Glucose to Two Pyruva te v i a Embden- Meyerhof-Parnas Pathway 184 F i g u r e C2 Path of Bu ty ra t e Format ion From Glucose 185 F i g u r e C3 Format ion of P r o p i o n a t e , Ace ta te and Carbond iox ide From D L - L a c t a t e by Megasphaera e l s d e n i i and C l o s t r i d i u m prop ion icum 186 F i g u r e C4 Format ion of L a c t a t e v i a the S u c c i n a t e - P r o p i o n a t e Pathway by P r o p i o n i b a c t e r i a 187 x i v NOMENCLATURE AND ABBREVIATIONS ATP Adenosine triphosphate BOD Biochemical oxygen demand (mg/L) COD Chemical oxygen demand (mg/L) CSTR Continuous flow s t i r r e d tank reactor D D i l u t i o n rate (h 1 ) DNA Deoxyribonucleic acid EMP Embden-Meyerhof-Parnas pathway F Fermentor feed stream flowrate (mL/h) k Constant i n Konak's m i c r o b i a l growth model; see equation 2.6 K' K i n e t i c constant i n Chen and Hashimoto's m i c r o b i a l growth model; see equation 2.19 K^ Constant i n Dabe's m i c r o b i a l growth model; see equation 2.11 K„ Constant i n Dabe's m i c r o b i a l growth model; see equation 2.10 D K^ M i c r o b i a l decay rate (h K^ Substrate i n h i b i t i o n constant (ug/mL) K Product i n h i b i t i o n constant (ug/mL) P K Monod s a t u r a t i o n constant (ug/mL) s L Constant i n Powell's m i c r o b i a l growth model; see equation 2.12 M Parameter i n Rogues and co-workers m i c r o b i a l growth model; see equation (ug/mL) or maintenance c o e f f i c i e n t NAD E l e c t r o n c a r r i e r co-enzyme, nicotinamide adenine d i n u c l e o t i d e NADH Reduced form of NAD OA Organic acids p Constant i n Konak's m i c r o b i a l growth model; see equation 2.6 XV RT Retention time (h) RNA Ribonucleic acid S Concentration of l i m i t i n g nutrient (u-g/mL) s o S value i n feed stream (p,g/mL) fcR Relaxation time (sec) fcHR Hydraulic r e t e n t i o n time (h) V CSTR working volume (mL) VFA V o l a t i l e f a t t y acids VSS V o l a t i l e suspended s o l i d s (g) WPC Whey pro t e i n concentrate X Organism concentration i n the fermentor (ug/mL) X m Maximum value of X that may be reached Y Y i e l d c o e f f i c i e n t (g of c e l l / g of l i m i t i n g nutrient) Y i e l d c o e f f i c i e n t for compound j on compound i Greek Symbols AG° Free energy of rea c t i o n at a temperature of 25°C and pressure of one atmosphere u S p e c i f i c m i c r o b i a l growth rate (h ^) u Maximum u valve (h *) m X Constant i n Moser's m i c r o b i a l growth model; see equation 2.12 xvi ACKNOWLEDGEMENTS The author wishes to express h i s s i n c e r e a p p r e c i a t i o n to Dr. K.L. F i n d e r and Dr. K.V. Lo f o r t h e i r a d v i c e , d i r e c t i o n and encouragement throughout the course of this research p r o j e c t . He i s also thankful to Dr. R.M.R. Branion, a member of h i s research committee, for his constructive c r i t i c i s m s and review of the t h e s i s . He i s indebted for f i n a n c i a l support of t h i s research to the Natural Sciences and Engineering Research Council of Canada and A g r i c u l t u r e Canada. S p e c i a l thanks go to his family members, Rose, Ntumwa and Nkaaku, without whose support and "demands", th i s research would probably never be completed. Last but not l e a s t , s p e c i a l thanks go to Miss Helsa Wong for the exc e l l e n t job of typing the thesis at such a short n o t i c e . - 1 - I. INTRODUCTION 1.1 Whey Whey i s an opaque, greenish-yellow f l u i d that remains i n a cheese vat foll o w i n g the removal of the curd, i n the process of converting milk into cheese. D i f f e r e n t cheese v a r i e t i e s produce whey with somewhat d i f f e r e n t c h a r a c t e r i s t i c s , t y p i c a l of the cheese process. As an example, the whey obtained from rennet-coagulated milk i s re f e r r e d to as "sweet" to d i f f e r e n t i a t e i t from the "acid" whey of cottage cheese. Acid whey forms a very small percentage of the t o t a l whey production i n North America. For a given quantity of milk used i n cheese processing, approximately 10% of the weight ends up as cheese, the balance as f l u i d whey (Harper and H a l l , 1976). As i n d i c a t e d i n Table 1.1, whey contains about 5% lact o s e , 1% pro t e i n (predominantly p - l a c t o g l o b u l i n ) , 0.3% f a t and 0.6% ash (Loehr, 1974). D e s p i t e the f a v o u r a b l e n u t r i e n t c o n t e n t , which o f f e r s i n t e r e s t i n g p o s s i b i l i t i e s for by-product recovery, whey i s an expensive and often a f r u s t r a t i n g d i s p o s a l problem for the cheese manufacturer. The five-day biochemical oxygen demand (BOD) f o r whey ranges between 30,000 and 60,000 mg/L, depending on the cheese process, making d i s p o s a l to streams unacceptable. Generally 90% of the BOD i s due to the lactose component (Green and Kramer, 1979), so the question of whey di s p o s a l and/or u t i l i z a t i o n i s e f f e c t i v e l y a q u e s t i o n of l a c t o s e d i s p o s a l and/or u t i l i z a t i o n . F l u i d whey production i n the USA and Canada was approximately 27 b i l l i o n tonnes and 2 m i l l i o n tonnes r e s p e c t i v e l y during 1985 (Tables 1.2 and 1.3). With the increasing cheese demand i n North America, f l u i d whey production i s expected to increase. - 2 - Table 1.1 T y p i c a l Compos i t ion of Whey S o l i d s Component Compos i t ion (g/100 mL) Carbohydrates Lac tose 5.00 P r o t e i n s p - L a c t o g l o b u l i n .66 ot -Lac toa lbumin .22 Immunoglobul ins .10 Fa t T r i g l y c e r i d e s .30 Ash .6 T o t a l % S o l i d s 6.88 - 3 - Table 1.2 Es t ima ted Q u a n t i t i e s of F l u i d Whey Produced i n the USA. Cheese Type 1980 1981 B i l l i o n 1982 Ki log rams 1983 1984 1985 Amer i can (Cheddar ) 9,700 10,786 11,235 11,953 10,810 11,651 Cottage 6,091 5,801 5,626 5,552 6,388 6,365 Other 6,193 6,250 6,773 7,201 8,271 8,859 T o t a l 21,984 22,837 23,634 24,706 25,470 26,875 Values c a l c u l a t e d u s i n g annual cheese p r o d u c t i o n f i g u r e s from, " D a i r y P r o d u c t s " , p u b l i s h e d by the US Department of A g r i c u l t u r e , S t a t i s t i c s R e p o r t i n g S e r v i c e . I t was assumed tha t fo r every kg of m i l k used, 0.1 kg end up as cheese and 0.9 kg as f l u i d whey. Table 1.3 Es t ima ted Q u a n t i t i e s of F l u i d Whey Produced i n Canada. Cheese Type 1980 1981 M i l l i o n 1982 Ki log rams 1983 1984 1985 Cheddar 956 907 802 895 912 976 Cot tage 260 277 286 284 276 -Other 640 684 727 752 816 882 T o t a l 1,856 1,868 1,815 1,931 2,004 - Values c a l c u l a t e d u s i n g annual cheese p r o d u c t i o n f i g u r e s from, " D a i r y Rev iew" , p u b l i s h e d by S t a t i s t i c s Canada. I t was assumed tha t f o r every kg of m i l k used, 0.1 kg end up as cheese and 0.9 kg as f l u i d whey. - 4 - 1.2 Whey D i s p o s a l and /o r U t i l i z a t i o n Due to the favourable n u t r i e n t content of whey, numerous I n v e s t i g a t i o n s i n t o developing new schemes of whey treatment, w i t h emphasis on product recovery and new product development, have been c a r r i e d out. Among these are: fermentation to p r o t e i n or n i t r o g e n - r i c h feeds (Reddy e t . a l . , 1976; Gerhardt e t . a l . , 1978); fermentation to ethanol f o r beverage or gasohol p r o d u c t i o n (Yang e t . a l . , 1976; Palmer, 1978 & 1979; B e r s t e i n and Tzeng, 1977; F r i e n d and Shahani, 1979; Everson, 1979); n o n - a l c o h o l i c beverage pr o d u c t i o n ; d r y i n g to powder which may be used as animal feed or a supplement i n human food; feeding d i r e c t l y to l i v e s t o c k ( M u l l e r , 1979; Modler e t . a l . , 1980); s e p a r a t i o n of components by membrane technology ( T e i x e i r a e t . a l . , 1982; and land a p p l i c a t i o n (Watson e t . a l . , 1977). The permeate from membrane s e p a r a t i o n schemes i s u s u a l l y high i n BOD and t h e r e f o r e n e c e s s i t a t e s f u r t h e r treatment (Delany, 1981). Most f e r m e n t a t i o n , membrane s e p a r a t i o n and n o n - a l c o h o l i c p r o d u c t i o n schemes are l i m i t e d to l a r g e d a i r y establishments (> 41 m i l l i o n kg f l u i d whey/year) and, whey being a low value bulky product, t r a n s p o r t a t i o n to a c e n t r a l p r o c e s s i n g u n i t i s o f t e n uneconomical (Modler e t . a l . , 1980). Drying r e q u i r e s a l o t of energy. A l s o the m e r i t s of land a p p l i c a t i o n are yet to be e s t a b l i s h e d . D i s p o s a l schemes have taken one or a combination of the f o l l o w i n g forms: dumping d i r e c t l y i n t o sewers or water systems and aerob i c treatment ( M u l l e r , 1979). The author i s not aware of any f u l l s c a l e anaerobic whey treatment f a c i l i t y i n o p e r a t i o n at the present time, w i t h the exception of the M i l l b a n k Cheese and B u t t e r p l a n t (Bellman, 1986). 1.3 B iomethana t ion P r o c e s s e s The b i o c o n v e r s i o n of organic m a t e r i a l to methane and carbon d i o x i d e i n the absence of molecular oxygen i s r e f e r r e d to as the biomethanation process - 5 - or anaerobic methane fermentation. B a s i c a l l y t h i s anaerobic degradation of o r g a n i c compounds i s performed by two groups of b a c t e r i a , the a c i d forming and methane forming. F u r t h e r s u b d i v i s i o n s of the two groups i s considered i n Chapter I I . Biomethanation processes f o r s t a b i l i s i n g o rganic wastes o f f e r s e v e r a l advantages over the c o n v e n t i o n a l a e r o b i c processes (McCarty, 1966), namely: a higher degree of waste s t a b i l i s a t i o n ; lower m i c r o b i a l y i e l d ; lower n u t r i e n t requirements; no oxygen requirement; and methane p r o d u c t i o n . With regard to whey, these advantages are pronounced s i n c e whey c o n s t i t u t e s a high s t r e n g t h organic waste and i n a d d i t i o n , the methane generated can be consumed by the cheese p r o c e s s i n g f a c i l i t y i t s e l f f o r v a r i o u s cooking and h e a t i n g o p e r a t i o n s . A l s o the process may be s u i t a b l e f o r the medium (14-41 m i l l i o n kg f l u i d whey/year) and small s c a l e (< 14 m i l l i o n kg f l u i d whey/year) p l a n t s f o r which the economics of a l t e r n a t i v e whey u t i l i z a t i o n processes are not f a v o u r a b l e . The three most common process l a y o u t s are, the p a r a l l e l , staged and phased processes ( F i g u r e 1.1). G e n e r a l l y , the l a c k of a p p l i c a t i o n of biomethanation f o r t h i s purpose i s probably due to the u n r e l i a b l e o p e r a t i o n t h a t has o f t e n been a s s o c i a t e d w i t h the process. This u n r e l i a b i l i t y has been a t t r i b u t e d to the l a c k of understanding of the fundamental concepts i n v o l v e d i n the process. Other disadvantages h i s t o r i c a l l y have been poor process s t a b i l i t y , a temperature requirement of 35°C, i n a b i l i t y to degrade v a r i o u s substances and l a r g e volume requirements because of slow r e a c t i o n r a t e s (Switzenbaum, 1983). More r e c e n t l y , advances i n b a s i c m i c r o b i o l o g y and b i o c h e m i s t r y along w i t h advances i n b i o - r e a c t o r technology, i n p a r t i c u l a r w i t h immobilised b a c t e r i a systems, sometimes r e f e r r e d to as f i x e d f i l m r e a c t o r s (Young and McCarty, 1969; L e t t i n g a e t . a l . , 1980; Switzenbaum and J e w e l l , 1980; Switzenbaum and Danskin, 1982; Boeing and Larsen, - 6 - Figure 1.1 Three d i f f e r e n t biomethanation processes: (1) i n f l u e n t stream, (2) s o l i d / l i q u i d separator, (3) a c i d (only) producing reactor, (4) methane producing reactor, (5) gas, (6) e f f l u e n t stream. - 7 - 1982; B u l l e t . a l . , 1984; Burgress and M o r r i s , 1984) have helped overcome most of the problems a s s o c i a t e d w i t h a n a e r o b i o s i s . I t i s important to d i s t i n g u i s h between r e a c t o r types and process l a y o u t . In the l a t t e r case one or s e v e r a l r e a c t o r s are in c l u d e d i n a t o t a l process scheme. Henze and Harremoes (1983) have c l a s s f i e d a number of r e a c t o r s , which have been i n v e s t i g a t e d or marketed during the l a s t 5-10 years, i n t o e i g h t b a s i c types, shown i n Fi g u r e s 1.2 and 1.3. 1.4 Whey Biomethanation From Bushwell and M u e l l e r ' s (1952) e m p i r i c a l formula that p r e d i c t s methane p r o d u c t i o n from a knowledge of the chemical composition of the degraded m a t e r i a l , the f o l l o w i n g formula can be w r i t t e n f o r the breakdown of the l a c t o s e i n whey: C12 H22°11 + H 2 ° * 6 C 0 2 + 6 C H 4 Therefore one gram of l a c t o s e (.002775 moles) would y i e l d (6 x .002775 x 22.412) = 0.3722 l i t r e s of CH^ at one atmosphere. For a l i t r e of whey, methane p r o d u c t i o n i s approximately 19 l i t r e s . Most of the l a b o r a t o r y e x p e r i m e n t s c o n d u c t e d to da t e on the biomethanation of whey or l a c t o s e have been designed to evaluate the performance of immobilised b a c t e r i a r e a c t o r systems (Hickey and Owens, 1981; Yang e t . a l . , 1984; Switzenbaum and Danskin, 1982; Boeing and Larsen, 1982; Dehaast e t . a l . , 1983; C a l l a n d e r and B a r f o r d , 1983; Dehaast e t . a l . , 1985). M i c r o b i a l and bio c h e m i c a l k i n e t i c i n f o r m a t i o n on which rudimentary process designs can be based i s v i r t u a l l y n o n - e x i s t e n t . E f f o r t s were made by the present author to e x p e r i m e n t a l l y generate the m i c r o b i a l k i n e t i c s and - 8 - •e- Fixed b e d M o v i n g bed E x p a n d e d b e d F l u i d i s e d bed R e c y c l e d b e d F i g u r e 1.2 F i v e b a s i c anaerobic f i x e d f i l m r e a c t o r types. - 9 - Oo o OQ J i L Recycled floes (contact reactor) O 0 - o I - I I I I Sludge blanket reactor (Clangester type reactor) o O 0 0 0 oo Digester F i g u r e 1.3 Three b a s i c anaerobic f l o c s / n o n - a t t a c h e d f i l m reactor types. - 10 - biochemical data f o r l a c t o s e i n fed-batch, s i n g l e - p h a s e , one l i t r e r e a c t o r s , f o r v a r i o u s d i l u t i o n r a t e s ( D ) and i n f l u e n t l a c t o s e c o n c e n t r a t i o n (unpublished work). At the s t a r t of the experiments the s u b s t r a t e was added to the r e a c t o r s and w i t h i n a few days the pH f e l l r a p i d l y and gas production ceased. This was repeated s e v e r a l times, but each experiment r e s u l t e d i n a "sour" r e a c t o r . S i m i l a r u n s u c c e s s f u l l experiments employing whey or l a c t o s e have been reported by a number of i n v e s t i g a t o r s ( M a r s h a l l and Timbers, 1982; Dehaast e t . a l . , 1983; K e l l y and Switzenbaum, 1984). The r a p i d drop i n pH was a s c r i b e d to the r a p i d formation of organic acids (OA) from l a c t o s e . I n order to solv e t h i s problem, Dehaast and co-workers prefermented the whey, n e u t r a l i s e d the a c i d , d i l u t e d the preferment and used the d i l u t e d product as the s u b s t r a t e f o r biomethanation. Follmann and Maerkl (1979) used a p H - s t a t i c process i n which the c o n t r o l s i g n a l f o r the a d d i t i o n of f r e s h whey was the pH value . When the pH increased beyond 7.0, a pump was t r i g g e r e d to add s u b s t r a t e a u t o m a t i c a l l y u n t i l the pH f e l l to 6.95, at which l e v e l the su b s t r a t e pump was stopped, u n t i l the pH again passed the pH = 7.0 l e v e l , and t h i s t r i g g e r e d a repeat of the sequence. With t h i s k i n d of c o n t r o l they were able to achieve 98% chemical oxygen demand (COD) r e d u c t i o n at a residence time (RT) of 12.5 days. The RT of 12.5 days i s very long and would n e c e s s i t a t e l a r g e r e a c t o r volumes. However Follmann and Maerkl pointed out that t h e i r observed long RT could be reduced approximately i n h a l f i f immobilised b a c t e r i a r e a c t o r s were employed. The above observations suggested that perhaps a two-phase process as p r e v i o u s l y proposed by Babbit and Baumann (1958), Andrews and Pearson (1965) and Pohland and Ghosh (1971) could be more s u c c e s s f u l l i n s t a b i l i s i n g whey or whey permeate. - 11 - 1 .5 T w o - P h a s e P r o c e s s As a l r e a d y I n d i c a t e d , i n a two-phase process, whey or l a c t o s e i s converted i n the f i r s t r e a c t o r to OA by the a c i d forming b a c t e r i a ( a c i d o g e n e s i s ) . The OA are then reduced i n a second s e r i a l phase by the methane forming b a c t e r i a (methanogenesis). Separation of the d i f f e r e n t groups of micro-organisms makes i t p o s s i b l e to m a i n t a i n o p t i m a l c o n d i t i o n s f o r the d i f f e r e n t groups of b a c t e r i a i n v o l v e d i n the process. A l s o i n a two-phase process there i s more o p p o r t u n i t y to c o n t r o l s u b s t r a t e flow between d i f f e r e n t groups of b a c t e r i a than i n one-phase processes, where there i s no p h y s i c a l s e p a r a t i o n of the b a c t e r i a l group p o p u l a t i o n s . Given these advantages, the two-phase process has been a p p l i e d at p i l o t and f u l l - s c a l e l e v e l s f o r s e v e r a l types of high BOD and low suspended s o l i d s (SS) i n d u s t r i a l wastes (Table 1.4). These two-phase p l a n t s c o n s i s t of a completely mixed acid-phase r e a c t o r and an upflow anaerobic sludge-blanket methane r e a c t o r . A l l p l a n t s are operated i n the m e s o p h i l i c temperature range and the carbohydrate content of the wastes i s predominantly a - g l y c o s i d i c types such as s t a r c h and sucrose. As i n d i c a t e d i n the next chapter, there i s a l a r g e volume of l i t e r a t u r e concerning the m i c r o b i a l k i n e t i c s and mechanisms f o r the r e d u c t i o n of OA or methanogenesis and h a r d l y any that i s a p p l i c a b l e to acidogenesis i n a two-phase process. I n the few previous l a b o r a t o r y s t u d i e s on acidogenesis of carbohydrates (Ghosh e t . a l . , 1975; Massey and Pohland, 1979; Cohen e t . a l . 1979; Zeotemeyer e t . a l . , 1982; B u l l e t . a l . 1984) glucose and s t a r c h were employed as the main s u b s t r a t e s . The r e s u l t s obtained by the above authors w i t h regard to optimum pH, a c i d o g e n i c r e a c t o r e f f l u e n t OA d i s t r i b u t i o n , m i c r o b i a l k i n e t i c s and o v e r a l l degradation scheme are v a l i d - 12 - Table 1.4 Two-Phase P i l o t and F u l l - S c a l e P l a n t s Year In d u s t r y L o c a t i o n P l a n t Capacity Reference Type (kg COD/day) 1977 D i s t i l l e r y (enzyme-alcohol) 1980 Beet Sugar D i s t i l l e r y ( y e a s t - a l c o h o l ) 1981 Beet Sugar C i t r i c A c i d Beet Sugar 1980 F l a x R e t t i n g Belgium West Germany Belgium Belgium West Germany West Germany Belgium 1982 Starch to Glucose West Germany 1983 Y e a s t - A l c o h o l Netherlands 1984 Yeast France P i l o t P i l o t P i l o t P i l o t P i l o t P i l o t F u l l - Scale F u l l - Scale F u l l - Scale F u l l - Scale Ghosh et. a l . (1985) 180 45 135 170 120 45 350 20,000 20,000 B e r k o v i t c h (1986) 7,000 - 13 - f o r carbohydrates w i t h cx-1, 4 g l u c o s i d i c bond and may not be v a l i d f o r l a c t o s e (8-1, 4 l i n k a g e ) . 1.6 Research Objectives The o v e r a l l o b j e c t i v e s of t h i s study were two f o l d : 1. To determine the m i c r o b i a l k i n e t i c s and the general mechanism f o r the degradation of l a c t o s e by a mixed, undefined acidogenic 14 b a c t e r i a , u s i n g C-labeled t r a c e r s . 2. To determine the i n f l u e n c e of whey p r o t e i n ( 8 - l a c t o g l o b u l i n ) on the degradation mechanism of l a c t o s e i f degraded together w i t h l a c t o s e . 14 To a l i m i t e d e x t e n t , C - l a b e l e d t r a c e r s have been used to determine the path of carbon and e l e c t r o n flow during anaerobosis of s p e c i f i c matter i n d i v e r s e ecosystems ( J e r i s and McCarty, 1965; Weng and J e r i s , 1976; Cohen, .1982; Lovley and Klug, 1982; Koch e t . a l , 1983). With regard to whey biomethanation, C h a r t r a i n and Zeikus (1986a and b) have used the technique to propose a carbon and e l e c t r o n flow route f o r single-phase biomethanation. P h y s i c a l s e p a r a t i o n of acidogenic and methanogenic b a c t e r i a i n a two-phase system may l e a d to important changes i n the composition of b a c t e r i a l p o p u l a t i o n s as w e l l as i n the i n t e r m e d i a r y routes of s u b s t r a t e degradation (Cohen e t . a l . , 1980). This has been considered a strong enough 14 j u s t i f i c a t i o n f o r s t u d y i n g a c i d o g e n e s i s of l a c t o s e w i t h C t r a c e r s . In a d d i t i o n , s e p a r a t i o n of the two m e t a b o l i c a l l y r e l a t e d groups give s the o p p o r t u n i t y to study the p a r t i c u l a r sub-population i n the r e a c t o r . To the 14 knowledge of t h i s author, no study using C-labeled s u b s t r a t e s to e l u c i d a t e an acidogenic degradation scheme of l a c t o s e , has been conducted p r e v i o u s l y . - 14 - A l l the present experimental work, described i n l a t e r chapters, was accomplished with mixed, undefined c u l t u r e s . A m i c r o b i o l o g i s t might have r e s e r v a t i o n s about the use of taxonomically undefined b a c t e r i a l p o p u l a t i o n s , but the complex nature of the m i c r o b i a l i n t e r a c t i o n s i n v o l v e d i n p r a c t i c e , r e q u i r e s an approach which i m p l i e s a not too d r a s t i c s i m p l i f i c a t i o n of r e a l i t y . T herefore, when the i n t e n t i o n i s to gather i n f o r m a t i o n w i t h a c e r t a i n degree of p r a c t i c a l a p p l i c a b i l i t y , methods which a l l o w a b e t t e r m i c r o b i o l o g i c a l d e s c r i p t i o n such as that obtained w i t h pure c u l t u r e s t u d i e s or s t u d i e s of mixed and w e l l defined populations might be considered l e s s r e l e v a n t . On the other hand, as a consequence of wanting to s o l v e e n g i n e e r i n g problems w i t h s p e c i f i c types of wastes, a n a e r o b i o s i s has o f t e n been t r e a t e d as a form of heterogeneous c a t a l y s i s , c o n s i s t i n g of r e a c t a n t s ( o r g a n i c matter and n u t r i e n t s ) and a c a t a l y s t (the b a c t e r i a ) . This approach has y i e l d e d r e s u l t s under proper process c o n d i t i o n s , but has ignored b i o l o g i c a l l y important f e a t u r e s such as adap t a t i o n at the r e g u l a t o r y or p o p u l a t i o n l e v e l , e c o - p h y s i o l o g i c a l i n t e r a c t i o n s and growth c o n d i t i o n s . For these reasons i t was found u s e f u l to d i r e c t t h i s study towards an in t e r m e d i a t e path between the two extremes mentioned, i n order to connect the f i e l d s of pure and a p p l i e d r e s e a r c h . The choice of l a c t o s e as the sol e carbon source was made to avoid the occurrence of s i d e r e a c t i o n s (Elsden and H i l t o n , 1978; Hirose and S h i b a i , 1980), so that the l a c t o s e degradation scheme could be determined more p r e c i s e l y . The s i g n i f i c a n c e of studying the i n f l u e n c e of 8 - l a c t o g l o b u l i n on l a c t o s e degradation pathway was two-fold: F i r s t organic wastes are f r e q u e n t l y composed of carbohydrates, p r o t e i n s and l i p i d s i n v a r i o u s combinations. No study was found i n the l i t e r a t u r e which addressed the anaerobic degradation mechanism of "complex" organic wastes. - 15 - Previous work has mainly i n v o l v e d complex organic waste c o n s t i t u e n t s i n d i v i d u a l l y and t h e i r i n t e rmediary m e t a b o l i t i e s (eg. amino a c i d s , f a t t y a c i d s , e t c . ) degraded s e p a r a t e l y . The importance of s e p a r a t e l y studying a degradative pathway of an int e r m e d i a r y m e t a b o l i t e l i k e l e u c i n e i s to h i g h l i g h t the p o s s i b i l i t y of determining s p e c i f i c metabolic routes which can be used as models f o r the breakdown of a number of amino a c i d s . However the r e s u l t s of such a study are only u s e f u l f o r a complex waste that contains l e u c i n e , i f the other c o n s t i t u e n t s and t h e i r d e r i v e d i n t e r m e d i a r y m e t a b o l i t e s do not a l t e r the p a r t i c u l a r m i c r o b i a l p o p u l a t i o n . Secondly, w h i l e a s y n t h e t i c waste that contains l a c t o s e may only serve as an analog of whey permeate from whey membrane processes f o r p r o t e i n recovery, a s y n t h e t i c waste that c o n t a i n s l a c t o s e and 8 - l a c t o g l o b u l i n may serve as a whole f l u i d whey waste analog. 1.7 Scope of Study The important f a c t o r s i n the study were i d e n t i f i e d as substate composition, pH and d i l u t i o n r a t e . S p e c i f i c a l l y , the scope of t h i s study c o n s i s t e d of the f o l l o w i n g major t a s k s . 1. With l a c t o s e as the growth l i m i t i n g n u t r i e n t , e s t a b l i s h the organic a c i d s d i s t r i b u t i o n and the biomass w i t h respect to the d i l u t i o n r a t e and pH. The l a t t e r independent v a r i a b l e was r e q u i r e d f o r m i c r o b i a l growth modeling and the former was r e q u i r e d f o r i n i t i a l s p e c u l a t i o n as to the p o t e n t i a l l a c t o s e fermentation model. 2. U t i l i s e a r a d i o t r a c e r methodology to confir m the l a c t o s e degradation scheme proposed i n task 1. - 16 - 3 . With l a c t o s e and p r o t e i n as the growth l i m i t i n g n u t r i e n t s u t i l i s e a r a d i o t r a c e r methodology to e s t a b l i s h the e f f e c t on the degradation scheme f o r l a c t o s e e s t a b l i s h e d i n task 2 of degrading a p r o t e i n w i t h the l a c t o s e . 4 . Estimate k i n e t i c parameters f o r the m i c r o b i a l growth model. I I . PREVIOUS WORK AND THEORETICAL ASPECTS In t h i s chapter, the t h e o r e t i c a l aspects as w e l l as the previous work on energy c o n s e r v a t i o n i n m i c r o b i a l systems and m i c r o b i a l growth modeling f o r a n a e r o b i o s i s i s explored. 2.1 E n e r g e t i c s and Metabo l ic Stages i n Anaerob ios is 2.1.1 E n e r g e t i c s The mechanism of energy gain which supports growth of a h e t e r o t r o p h i c m i c r o b i a l c e l l r e l i e s upon b i o l o g i c a l o x i d a t i o n processes. Chemically bound p o t e n t i a l energy i s rele a s e d by a reduced organic compound w i t h a l e s s reduced compound which ac t s as an o x i d i s e r . By t h i s r e a c t i o n e l e c t r o n s are t r a n s f e r r e d from the reduced compound ( e l e c t r o n donor) to the more o x i d i s e d compound ( e l e c t r o n a c c e p t o r ) . Since fermentation i s a s t r i c t l y anaerobic process, o x i d a t i v e r e a c t i o n s must be anaerobic. U s u a l l y i n these o x i d a t i v e r e a c t i o n s two e l e c t r o n s are removed from the su b s t r a t e molecule (Equations 2.1 and 2.2). 2e x y (2.1) (2.2) Compound x i s o x i d i s e d to compound y, and two e l e c t r o n s are t r a n s f e r r e d to NAD, a common e l e c t r o n c a r r i e r coenzyme. Coenzymes are present In c e l l s i n l i m i t i n g amounts. Wolfe (1983) has l i k e n e d these e l e c t r o n c a r r i e r s to - 18 - t r u c k i n g systems. Unless there i s a point where the cargo ( e l e c t r o n s ) are unloaded, the t r u c k i n g system soon becomes s a t u r a t e d . In a n a e r o b i o s i s , the f i n a l e l e c t r o n acceptor i s formed i n amounts p r o p o r t i o n a l to the s u b s t r a t e being o x i d i s e d so that the reduced e l e c t r o n c a r r i e r can be unloaded ( o x i d i s e d ) and r e t u r n to accept another load ( p a i r s of e l e c t r o n s ) . This energy which i s re l e a s e d by the r e a c t i o n i s stored by the c e l l i n the form of energy r i c h phosphate e s t e r s (ATP) which can be used by the c e l l f o r a l l r e a c t i o n s which support growth. The amount of energy which i s re l e a s e d by such an o x i d a t i o n process (AG°) depends on the d i f f e r e n c e i n s t a t e of r e d u c t i o n of the compound which a c t s as the reductant i n the r e a c t i o n . The amount of ATP which i s gained by the c e l l i n t u r n , depends on the e n e r g e t i c e f f i c i e n c y of i t s metabolism. The m a j o r i t y of anaerobes work a t an e f f i c i e n c y of 25-50% (Thauer e t . a l . , 1977). The e l e c t r o n acceptor can be e i t h e r an Inorganic compound ( r e s p i r a t i o n ) or an organic compound ( f e r m e n t a t i o n ) . When an organic compound ac t s as an e l e c t r o n acceptor, the s u b s t r a t e molecule i s normally s p l i t i n t o two molecules, of which one act s as the e l e c t r o n donor and the other as an e l e c t r o n acceptor. Some ferme n t a t i v e b a c t e r i a possess the a b i l i t y to use protons as e l e c t r o n acceptors i n a d d i t i o n to organic molecules. The r e d u c t i o n of protons i n e l e c t r o n t r a n s f e r r i n g r e a c t i o n s gives r i s e to the prod u c t i o n of molecular hydrogen and i s of importance f o r the c o n t r o l of a n a e r o b i o s i s . Cohen (1982) has pointed out t h a t , i n g e n e r a l , fermentative r e a c t i o n s conform to the f o l l o w i n g p r i n c i p l e s : (1) From the s u b s t r a t e a proper e l e c t r o n donor and e l e c t r o n acceptor must be formed and the amount of e l e c t r o n s s u p p l i e d by the donor must equal the amount accepted by the acceptor; (2) Fermentative r e a c t i o n s occur i n such a way that an opt i m a l ATP ga i n i s accomplished. - 19 - The most important acidogenic fermentation r e a c t i o n s which may occur d u r i n g a n a e r o b i o s i s of carbohydrates are shown i n Table 2.1, a f t e r Thauer e t . a l . (1977). Although the AG° of the d i f f e r e n t r e a c t i o n s are comparable, the ATP gain v a r i e s between 2 and 4. For a more d e t a i l e d t r e a t i s e of the e n e r g e t i c a spects, the reader i s r e f e r r e d to papers by Thauer e t . a l . (1977) and G o t t s c h a l k and Andreesen (1979) . 2.1.2 Metabolic Stages E f f e c t i v e d i g e s t i o n of organic matter i n t o methane r e q u i r e s the combined and c o o r d i n a t e d m e t a b o l i s m of d i f f e r e n t k i n d s of c a r b o n c a t a b o l i s i n g anaerobic b a c t e r i a . At l e a s t four d i f f e r e n t t r o p h i c types of b a c t e r i a have been i s o l a t e d from e i t h e r man-made r e a c t o r s or i n nature (eg. g a s t r o i n t e s t i n a l t r a c t s , lake sediments or thermal w e l l s ) . These b a c t e r i a can be d i s t i n c t l y recognised on the b a s i s of s u b s t r a t e fermented and me t a b o l i c end products formed ( Z e i k u s , 1980). The four m etabolic groups which f u n c t i o n i n a n a e r o b i o s i s i n c l u d e ( F i g u r e 2.1): (1) The h y d r o l y t i c b a c t e r i a which ferment a v a r i e t y of complex organic molecules ( p o l y s a c c h a r i d e s , l i p i d s and p r o t e i n s ) i n t o a b r o a d s p e c t r u m of end p r o d u c t s ( a c e t a t e , H2/CO2, one carbon compounds and o r g a n i c a c i d s longer than a c e t a t e , and n e u t r a l compounds l a r g e r than e t h a n o l ) ; (2) The hydrogen producing acetogenic b a c t e r i a which i n c l u d e both o b l i g a t e and f a c u l t a t i v e species that can ferment organic aci d s l a r g e r than a c e t a t e and n e u t r a l compounds l a r g e r than methanol ( e t h a n o l , propanol) to hydrogen and a c e t a t e ; (3) The homoacetogenic b a c t e r i a which can ferment a very wide spectrum of m u l t i or one carbon compounds to a c e t i c a c i d ; and (4) The m e t h a n o g e n i c b a c t e r i a w h i c h ferment H 2 / C O 2 , one c a r b o n compounds (methanol, CO, methylamine) and acetate to methane. Table 2.1 O v e r a l l Reaction Schemes Of Some Important Acidogenic Fermentation Types AG° ATP Reaction Type ( k J / r e a c t i o n ) ( m o l / r e a c t i o n ) A c e t i c a c i d fermentation C6 H12°6 + 4 H 2 ° 2 C H 3 C H O O ~ + 4 H + + 2 H C 0 3 + 4 I I 2 ~ 2 0 6 4 P r o p i o n i c a c i d fermentation C,-H100, •» — CH oCH oC00~ + J L CH.COO" + J L H + + — HCO" -220 3 - 4 6 12 6 3 3 2 3 3 3 3 3 B u t y r i c a c i d fermentation C-H.-O, + 2H„0 •»• CH-CH.CH-COO" + 3H + + 2H„ + 2HC0~ -255 3 0 1/ O Z J Z Z Z J Ethanol fermentation C6 H12°6 + 2 H 2 ° 2 C H 3 C H 2 O H + 2 H + + 2 C H 0 3 _ 2 2 6 2 L a c t i c a c i d fermentation C.H.,.0. •* 2CH.CH0HC00" + 2H + -198 2 - 2 1 - A c e t a t e Organic M a t t e r Carbohydrates P r o t e i n s L i p i d s y 1 H y d r o l y t i c b a c t e r i a Organic Acid s 2 a c e t o g e n i c b a c t e r i a ( ^ - p r o d u c i n g ) -3- H 2 / C 0 2 homoacetogenie b a c t e r i a \7 4 B methano genie b a c t e r i a ( a c e t a t e d e c a r b o x y l a t i o n ) \7 methanogenic b a c t e r i a ( r e d u c t i v e methane formation) F i g u r e 2 . 1 M e t a b o l i c d i s t i n c t i o n of m i c r o b i a l p o p u l a t i o n s i n v o l v e d i n a n a e r o b i o s i s . - 22 - The raethanogenic b a c t e r i a perform a p i v o t a l r o l e because t h e i r unique metabolism c o n t r o l s the r a t e of organic degradation and d i r e c t s the flow of carbon and e l e c t r o n s by removing t o x i c i n t e rmediary metabolites and enhances the thermodynamic e f f i c i e n c y of i n t e r s p e c i e s intermediary metabolism ( Z e i k u s , 1980). A b r i e f review of the r o l e played by each b a c t e r i a l group i s presented below. For more i n f o r m a t i o n the reader i s r e f e r r e d to a number of recent d e t a i l e d reviews ( Z e i k u s , 1977; Mah e t . a l . , 1977; and Batch e t . a l . 1979). 2.1.2.1 H y d r o l y t i c B a c t e r i a I t has been e s t a b l i s h e d that hexoses are mainly fermented v i a the Erabden-Meyerhof-Parnas (EMP) pathway i n the rumen (Baldwin e t . a l . , 1963; W a l l n o f e r e t . a l . , 1966) and that other metabolic routes are much l e s s i m p o r t a n t . A l s o Wood (1961) showed t h a t the EMP and the hexose monophosphate (HMP) routes are the most important pathways f o r hexose degradation i n anaerobic b a c t e r i a up to pyruvate. Pyruvate occupies the c o n t r o l p o s i t i o n , from which a l c o h o l i c , l a c t i c a c i d or v o l a t i l e f a t t y a c i d fermentations (VFA) s t a r t (Wood, 1961). The l i p i d s are broken down to long c h a i n f a t t y a c i d s (Novak and C a r l s o n , 1970) . The long chain f a t t y a c i d s are f u r t h e r o x i d i s e d by b e t a - o x i d a t i o n to propionate and a c e t a t e . Amino acids from the degradation of n u c l e i c acids and p r o t e i n s are deaminated and the corresponding organic a c i d s are formed. 8 9 P o p u l a t i o n s of 10 -10 h y d r o l y t i c b a c t e r i a per mL of raesophilic sewage sludge have been documented ( Z e i k u s , 1980). The m a j o r i t y of these were u n i d e n t i f i e d gram-negative rods. To date very few d e t a i l e d s t u d i e s or g e n e r i c i d e n t i f i c a t i o n s of the predominant m i c r o b i a l species have been - 23 - re p o r t e d . C l o s t r i d i u m propionicum, C l o s t r i d i u m acetobutyricum and Eubacterium limosum are examples of organisms whose known metabolic pathways l e a d to the formation of end products c h i e f l y formed i n man-made r e a c t o r f e r m e n t a t i o n s , namely, b u t y r a t e s , propionates and a c e t a t e s . 2.1.2.2 The H^-producing Acetogenic Bacteria The ^ - p r o d u c i n g a c e t o g e n i c b a c t e r i a c a t a b o l i s e the products of the f i r s t stage of fermentation (propionate, b u t y r a t e , long chain f a t t y a c i d s (VFA) , a l c o h o l s , aromatics and other organic a c i d s ) to acetate and a I^/CC^ mixture ( F i g u r e 2.1). However these organisms cannot c a t a b o l i s e these s u b s t r a t e s to a c e t a t e when i n the environment i s not at extremely low l e v e l s . F i g u r e 2.2 i l l u s t r a t e s the r e l a t i o n s h i p t h a t e x i s t between p a r t i a l p r e s s u r e and f r e e e n e r g y a v a i l a b l e to the ^ - p r o d u c i n g and H 2 ~ c o n s u m i n g s p e c i e s . For example, i n order f o r energy to be a v a i l a b l e to organisms o x i d i s i n g propionate to acetate and , the p a r t i a l pressure of B.^ can not exceed about 10 ^ atmospheres (Thauer, e t . a l . , 1977). P o p u l a t i o n s of 4.2 x 10^ ̂ - p r o d u c i n g acetogens per mL of sewage sludge have been reporte d (Mclnerney e t . a l . , 1978). These organisms have not been e i t h e r g e n e r i c a l l y i d e n t i f i e d or p h y s i o l o g i c a l l y w e l l c h a r a c t e r i s e d , d e s p i t e the b e l i e f that VFA's are e c o l o g i c a l l y much more important as intermediates than l a c t a t e and ethanol (Mah, 1981). The i s o l a t i o n of the "S" organism from Methanobacillus o m e l i a n s k i i was the f i r s t documentation of a specie i n t h i s g r o up. E t h a n o l was o x i d i s e d to ace t a t e and CO2 reduced to CH^ by the s y n t r o p h i c growth of the S organism and Methanobacillus o m e l i a n s k i i . Mclnerney and Bryant (1981) have r e c e n t l y reported an organism i n t h i s group, Syntrophonas w o l f e i i , that b e t a - o x i d i s e s even-numbered-carbon f a t t y - 24 - Butyrate I 8 0 4 0 0 - 4 0 - B O - 1 2 0 A G at pH 7-0, 25*C (KJ) Figure 2.2 E f f e c t of the p a r t i a l pressure of hydrogen (PH^) on the f r e e energy charge (AG ) f o r the degradation of et h a n o l , propionate and b u t y r a t e w i t h methane formation w i t h U and CO^. The assumption i s that s u b s t r a t e c o n c e n t r a t i o n s of f a t t y a c i d s are 1 mM each and bicarbonate i s 50 mM w i t h the p a r t i a l pressure of CH 4 at 0.5 atm (AG 1 = A G 0 1 +1.36 l o g ( [ p r o d u c t s ] / [ r e a c t a n t s ] ) . Source i s Mclnerney and Bryant (1979). - 25 - a c i d s ( b u t y r a t e , c a p r o a t e ) to a c e t a t e and H 2 ; odd-numbered-carbon f a t t y a c i d s ( v a l e r a t e ) a r e c o n v e r t e d to a c e t a t e , propionate and H 2 . S. W o l f e i i must grow w i t h an ^ - u t i l i s i n g m i c r o b e . Boone and B r y a n t (1980) a l s o r e p o r t e d that Syntrophobacter w o l i n i i o x i d i s e s propionate to a c e t a t e , H 2 and co 2 . 2.2.1.3 Homoacetogenic Bacteria Homoacetogenic b a c t e r i a possess a high thermodynamic e f f i c i e n c y of m e t a b o l i s m as a consequence of not f o r m i n g H 2 and C 0 2 d u r i n g growth on mu l t i - c a r b o n compounds. These b a c t e r i a can ferment a very wide spectrum of s u b s t r a t e s ( s u g a r s a c i d s , C 0 2 , CO, H 2, e t c . ) . The u t i l i z a t i o n of H 2 by homoacetogens i n methane producing r e a c t i o n s i s considered to be of l i t t l e consequence to the o v e r a l l carbon degradation because the C-2 of acetate and C 0 2 g e n e r a l l y account f o r 70% and 30% r e s p e c t i v e l y of the methane carbon i n man-made r e a c t o r s ( J e r i s and McCarty, 1965). Methanogens appear to s u c c e s s f u l l y out compete homoacetogens f o r H 2 i n the g a s t r o i n t e s t i n a l environment ( P r i n s and Lankhorst, 1977). So f a r l i t t l e i s known about the f u n c t i o n a l i m p o r t a n c e o f h o m o a c e t o g e n i c m e t a b o l i s m and t h e acetogenic/methanogenic b a c t e r i a l i n t e r a c t i o n s ( Z e i k u s , 1980). 5 6 P o p u l a t i o n s of 10 - 10 per mL of sewage sludge have been reported (Braun e t . a l . 1979). C l o s t r i d i u m and Acetobacterium are the only recognised genera of H 2 o x i d i s i n g homoacetogenic b a c t e r i a . 2.1.2.4 Methanogenic Bacteria The methanogens are the key organisms i n the pro d u c t i o n of methane from waste m a t e r i a l s . They are the only organisms that are able to break down ac e t a t e and hydrogen to gaseous end products. Without the presence of t h i s - 26 - group of microorganisms i n a m e t h a n i f i c a t i o n p r o c e s s , e f f e c t i v e breakdown of the t o t a l o r g a n i c m a t e r i a l would s top due to the accumu la t ion of the p roduc ts of the p r e v i o u s l y d i s c u s s e d g roups . The methanogens are unusua l s i n c e they are composed of many s p e c i e s w i t h d i f f e r e n t c e l l morphology. T h e i r energy y i e l d i n g mechanisms are not yet known ( Z e i k u s , 1977; Wo l f e , 1979) . They r e q u i r e a s t r i c t l y anaerob ic environment f o r t h e i r g rowth. The methanogens u t i l i z e a na r row range of s u b s t r a t e s ( R ^ / C C ^ , HCOOH, C H 3 N H 2 , C H 3 C O O H , C O ) . A l m o s t a l l s p e c i e s use H 2 and C 0 2 f o r g row th (Equa t i on 2 . 3 ) . 4H. + HC0~ + H + CH. + 3H.0 (2 .3 ) 2 3 4 2 (AG° = -135 .6 k J / r e a c t i o n ) Even though a l a r g e amount of hydrogen i s produced du r i ng a n a e r o b i o s i s , the methanogens m a i n t a i n a low c o n c e n t r a t i o n of hydrogen. A number of s p e c i e s can degrade a c e t a t e (Equa t i on 2 . 4 ) . Of these on l y Methanosars ina b a k e r i , Methanococcus maze! and Methanobacter ium soghngen i i have been i s o l a t e d i n pure c u l t u r e . CH 3C00 + H 2 0 C H 4 + HC0 3 (2 .4 ) (AG° = - 3 1 . 0 k J / r e a c t i o n ) The f r e e energy from r e a c t i o n (2 .4 ) i s b a r e l y enough to form one mole of ATP (AG° = - 3 1 . 6 k J / r e a c t i o n ) . Th i s may e x p l a i n the observed s low growth r a t e o f methanogens on a c e t a t e . g M e t h a n o g e n i c p o p u l a t i o n s of 10 p e r mL of sewage s l u d g e have been d e t e c t e d (Sm i th , 1966) . - 27 - 2.1.2.5 S p e c i f i c B a c t e r i a l S p e c i e s A s s o c i a t e d With Whey Biomethanation I n two r e c e n t papers ( C h a r t r a i n and Z e i k u s , 1986a & b) , the o r g a n i s a t i o n and species composition of b a c t e r i a l groups a s s o c i a t e d w i t h l a c t o s e biomethanation were i n v e s t i g a t e d i n a continuous flow, single-phase, whey degrading r e a c t o r (pH 7.1, temperature of 37°C and d i l u t i o n r a t e of - 1 1 4 .01 h ). C t r a c e r s t u d i e s demonstrated that biomethanation occurred i n three d i s t i n c t but simultaneous phases. Lactose was metabolised p r i m a r i l y t o l a c t a t e , e t h a n o l , a c e t a t e , formate and • These m e t a b o l i t e s were transformed i n t o acetate and H^/CO^ i n a second, acetogenic phase. F i n a l l y , the d i r e c t methane precursors were transformed during the methanogenic phase w i t h acetate accounting f o r 81% of the methane formed. A general scheme was pro p o s e d f o r the c a r b o n and e l e c t r o n f l o w r o u t e d u r i n g l a c t o s e biomethanation. Based on the scheme, pr e v a l e n t m i c r o b i a l populations i n the ecosystem were enumerated, i s o l a t e d , and c h a r a c t e r i s e d . The dominant groups were p r e s e n t i n the f o l l o w i n g c o n c e n t r a t i o n s (per mL); 1 0 ^ f o r h y d r o l y t i c b a c t e r i a ; 10^ - 1 0 ^ f o r acetogenic b a c t e r i a ; and 10^ - 10^ f o r methanogenic b a c t e r i a . The predominant h y d r o l y t i c b a c t e r i a were i d e n t i f i e d as Leuconostoc mesenteroides, K l e b s i e l l a oxytoca and C l o s t r i d i u m butyricum. C l o s t r i d i u m p r o p i o n i c u m and D e s u l f o v i b r i o v u l g a r i s w h i ch were the predominant L a c t a t e u t i l i s i n g , J^-producing acetogens, w h i l e Methanosarcina b a r k e r i and Methanothrix soehngenii were found to be the predominant acetate u t i l i s i n g methanogens. Methanobacterium formicicum was the pre v a l e n t T ^ - u t i l i s i n g s p e c i e . While C h a r t r a i n and Zeikus's work i s very u s e f u l w i t h regard to the m i c r o b i a l ecology of a single-phase biomethanation process, i t i s not c l e a r whether t h e i r f i n d i n g w i l l stand f o r a two-phase process acidogenic r e a c t o r - 28 - where a l l the methanogenlc b a c t e r i a would be washed out due to higher d i l u t i o n r a t e s (>> .01 h . A l s o i t i s d i f f i c u l t to assess from t h e i r f i n d i n g s what o p e r a t i n g the acidogenic r e a c t o r at a lower pH (< 7.0) would do to the b a c t e r i a l p o p u l a t i o n composition r e p o r t e d . 2.1.2.6 Important Process Parameters Some environmental f a c t o r s that i n f l u e n c e b a c t e r i a l degradation are pH, a l k a l i n i t y , temperature, n u t r i e n t s , organic a c i d s c o n c e n t r a t i o n and t o x i c m a t e r i a l . For a c i d o g e n i c r e a c t i o n s the most important process parameters are temperature, d i l u t i o n r a t e and pH. These three v a r i a b l e s can be manipulated to favour the predominant pr o d u c t i o n of a p a r t i c u l a r acidogenic end product. However to date the c h o i c e , of the d e s i r a b l e end product i s a s u b j e c t of c o n s i d e r a b l e controversy (Zeotemeyer, e t . a l . , 1982; Pipyn and V e s t r a e t e , 1981; K i s a a l i t a e t . a l . , 1986). 2.2 Mathematical Modeling of Microbial Growth I n t h i s s e c t i o n models seldom used i n m i c r o b i o l o g y are considered i n a g e n e r a l sense. Emphasis i s placed on those that are more r e l e v a n t to the work reported i n t h i s study; w i t h the aim of i d e n t i f y i n g the most a p p r o p r i a t e form which may be used to d e s c r i b e the m i c r o b i a l growth ob s e r v a t i o n s r e p o r t e d . 2.2.1 Model Classification A conceptual framework, c l a s s f y i n g models of m i c r o b i a l p o p u l a t i o n s , was f i r s t suggested by Tsuchia e t . a l . (1966). This framework has been r e t a i n e d although there i s no u n i v e r s a l agreement. Below, a s l i g h t l y m o d i f i e d framework c o n s i s t i n g of three d i s t i n c t p e r s p e c t i v e s f o r c e l l p o p u l a t i o n - 29 - k i n e t i c r e p r e s e n t a t i o n i s examined. A p e r s p e c t i v e s u i t a b l e f o r the s i t u a t i o n met i n t h i s study w i l l a l s o be i d e n t i f i e d . Two broad approaches to modeling b i o l o g i c a l and other systems e x i s t , namely, continuum and c o r p u s c u l a r methods (Harder and Roels, 1982). In the c o r p u s c u l a r method, the d i s t r i b u t i o n of p r o p e r t i e s among the p o p u l a t i o n i s e x p l i c i t l y recognised and t h e r e f o r e the t y p i c a l behaviour of the system i s caused by the concerted a c t i o n of the p o p u l a t i o n . For a b i o l o g i c a l system, the c e l l s are considered d i s c r e t e and heterogeneous. Given our present-day knowledge of matter, the c o r p u s c u l a r approach must be considered the most r e a l i s t i c method of m i c r o b a l growth modeling. This approach has been used to model m i c r o b i a l systems by F r e d r i c k s o n e t . a l . (1967) and Ramkrishna (1979). They used the term "segregated" to d e s c r i b e t h e i r approach. C l a s s i c a l m i c r o b i o l o g y considers the b a s i c u n i t of a l l f u n c t i o n i n g organisms to be the c e l l . Hence the c o r p u s c u l a r approach lends i t s e l f e a s i l y to m o d e l l i n g m i c r o b i a l sytems. Despite t h i s f a c t , the continuum approach, e a r l i e r termed " d i s t r i b u t i v e " by Tsuchia e t . a l . (1966) and sometimes r e f e r r e d to as "unsegregated" i s the most commonly encountered method used i n d e s c r i p t i o n s of m i c r o b i a l systems. In the continuum approach, the m i c r o b i a l p o p u l a t i o n i s viewed as a lumped s o l u t e biomass which i n t e r a c t s as a whole w i t h i t ' s environment. The c o r p u s c u l a r nature of r e a l i t y i s t h e r e f o r e ignored and the system i s considered to be continuous i n space. The preference f o r the continuum approach may be a t t r i b u t e d to the ease w i t h which mathematical treatment can be employed. The second p e r s p e c t i v e d i s t i n g u i s h e s between the d e t e r m i n i s t i c and s t o c h a s t i c ( p r o b a b l i s t i c ) approaches. The d i f f e r e n c e between these approaches r e s t s on the nature of the p r e d i c t i o n s about the f u t u r e behaviour of the m i c r o b i a l system that the model a l l o w s . In a d e t e r m i n i s t i c approach, - 30 - the knowledge of the s t a t e v e c t o r of the system (a v e c t o r composed of a l l v a r i a b l e s necessary to s p e c i f y the s t a t e of the system at a given moment i n time) a l l o w s an exact p r e d i c t i o n of the f u t u r e behaviour during an a r b i t r a r y p e r i o d . With the s t o c h a s t i c approach, i t i s only p o s s i b l e to s p e c i f y a p r o b a b i l i t y that the s t a t e r e a c t o r w i l l be i n a given r e g i o n of the s t a t e space ( s t a t e space being a coordinate system of d i m e n s i o n a l i t y of the s t a t e v e c t o r and each po i n t i n s t a t e space corresponds to a s i n g l e value of the s t a t e v e c t o r ) . The s t o c h a s t i c approach i s o f t e n used i f the observer i s unable to o b t a i n s u f f i c i e n t i n f o r m a t i o n about the s t a t e of the p o p u l a t i o n and i t ' s subsequent behaviour to a l l o w a d e t e r m i n i s t i c p r e d i c t i o n . Shuler (1985) has pointed out that g e n e r a l l y a t o t a l p o p u l a t i o n g r e a t e r than 10,000 i s s u f f i c i e n t to a l l o w a d e t e r m i n i s t i c treatment of any b i o l o g i c a l system. A l s o , F r e d r i c k s o n (1966) has pointed out that the behaviour of s m a l l numbers of organism, as f o r example during the l a s t stages of s t e r i l i z a t i o n , c a l l s f o r a p r o b a b i l i s t i c approach. Since most m i c r o b i a l systems of engineering i n t e r e s t c o n t a i n populations w e l l i n excess of 10,000, d e t e r m i n i s t i c modeling approaches are more popular than s t o c h a s t i c ones. The t h i r d l e v e l d i s t i n g u i s h e s between s t r u c t u r e d and u n s t r u c t u r e d models. An u n s t r u c t u r e d model assumes that a s i n g l e v a r i a b l e i s adequate to d e s c r i b e the p o p u l a t i o n . T y p i c a l l y the s i n g l e v a r i a b l e i s r e l a t e d to the q u a n t i t y of biomass. I m p l i c i t i n such models i s the idea that b i o s y n t h e t i c c a p a b i l i t i e s of the p o p u l a t i o n are i n v a r i a n t . A s t r u c t u r e d model d i v i d e s the p o p u l a t i o n i n t o subcomponents. With pure c u l t u r e s the a d d i t i o n of s t r u c t u r e i s most o f t e n achieved by d i v i d i n g the c e l l s i n t o two or more re c o g n i s a b l e chemical subcomponents (eg. DNA, RNA, p r o t e i n , storage compounds, e t c . ) . One of the e a r l i e s t two component s t r u c t u r e d , d e t e r m i n i s t i c and continuum model was proposed by W i l l i a m s (1967). - 31 - Campbell (1957) de f i n e d growth over a p e r i o d of time as being balanced, i f d u r i n g that time i n t e r v a l every e x t e n s i v e property of the growing system i n c r e a s e s by the same f a c t o r . So i n balanced growth the composition of a t y p i c a l c e l l i s time i n v a r i a n t . Models which ignore the multicomponent nature of c e l l s may be adequate i n t h i s s i t u a t i o n . T y p i c a l balanced growth s i t u a t i o n s are e x p o n e n t i a l growth i n a batch c u l t u r e and s t e a d y - s t a t e c o n d i t i o n s i n a CSTR (or chemostat). I n F i g u r e 2.3 the p o s s i b l e i n t e r a c t i o n s between two l e v e l s ( c o n t i n u u m / c o r p u s c u l a r and s t r u c t u r e d / u n s t r u c t u r e d ) a r e p r e s e n t e d . P r e d i c t i o n s by any model from each r e g i o n could be e i t h e r s t o c h a s t i c or d e t e r m i n i s t i c . In other words a t h i r d c o r d i n a t e can be added p e r p e n d i c u l a r to the plane of the page w i t h two r e g i o n s ; above the page r e p r e s e n t i n g s t o c h a s t i c model p r e d i c t i o n s and below the page r e p r e s e n t i n g d e t e r m i n i s t i c model p r e d i c t i o n s , thus g i v i n g a t o t a l of e i g h t p o s s i b l e r e g i o n s . The b i o l o g i c a l phase i n t h i s study i s made up of a mixture of species r a t h e r than a s i n g l e s p e c i e . Complete s t r u c t u r e on the one hand n e c e s s i t a t e s e x p l i c i t r e c o g n i t i o n of each specie i n v o l v e d and on the other hand, the model f o r each specie must be c h e m i c a l l y s t r u c t u r e d . Given that the number of species i n the p o p u l a t i o n i s unknown a s t r u c t u r e d approach would be an almost impossible task. Therefore i n t h i s study only models i n r e g i o n one of F i g u r e 2.3 were considered. A l s o given that l a r g e m i c r o b i a l counts were i n v o l v e d only d e t e r m i n i s t i c p r e d i c t i o n s were e n t e r t a i n e d . In the next two subsections p r e v i o u s l y p u b l i s h e d m i c r o b i a l growth models that belong to the above i n d i c a t e d category are reviewed. 2.2.2 Models For Single Substrate Limiting Growth I n the present day theory of continuous c u l t u r e there are two opposing s c h o o l s . In the f i r s t s chool i t i s assumed that the s p e c i f i c growth r a t e - 32 - Unstructured Structured Co nt in um  "D is tr ib ut iv e"  "U ns eg re ga te d"  1. MOST IDEALISED CASE c e l l population treated as one component solute 2. multicomponent average c e l l d e s c r i p t i o n Co rp us cu la r "S eg re ga te d"  3. s i n g l e component heterogeneous i n d i v i d u a l c e l l 4. ACTUAL CASE mult icomponent d e s c r i p t i o n of c e l l to c e l l heterogenity Figure 2.3 Possi b l e perspectives of i n t e r a c t i o n s f o r c e l l population k i n e t i c representation: Region 1 i s an "average c e l l " approximation of Region 3 and a balanced growth approximation of Region 2. Region 3 i s an "average c e l l " approximation of Region 2. - 33 - (|J. = (dX/dt)/X) i s dependent only on the l i m i t i n g s u b s t r a t e c o n c e n t r a t i o n ( S ) . In the second i t i s assumed that u i s e i t h e r dependent on the biomass (X) or i s some f u n c t i o n of both S and X. F i r s t l e t ' s review models that belong to the former s c h o o l . 2.2.2.1 Monod Equation An equation f r e q u e n t l y used i n k i n e t i c d e s c r i p t i o n of growth was proposed by Monod (1942 & 1950) as f o l l o w s : u = (dX/dt)/X = u S/(K + S) (2.5) m s i n w h i c h u. i s t h e maximum s p e c i f i c g r o w t h r a t e . S and X are the m c o n c e n t r a t i o n s of s u b s t r a t e and biomass and K i s a Monod s a t u r a t i o n s c o n s t a n t . Equation 2.5 i s analogous to the Briggs-Haldane s o l u t i o n of the Michaelis-Menten model f o r the k i n e t i c s of a s i n g l e enzyme. I f m i c r o b i a l growth i s considered to be the r e s u l t of a sequence of enzymatic r e a c t i o n s i n which one r e a c t i o n i s much slower than a l l the o t h e r s , then the Michealis-Menten equation can be considered as the p h y s i c a l e x p l a n a t i o n f o r the good f i t that the Monod equation o f t e n g i v e s . This reasoning i s however by no means unique, f o r example the Langmuir a d s o r p t i o n isotherm i s of the same form as equation 2.5. 2.2.2.2 Other Equations Monod's equation i s by no means the only equation which has been proposed f o r the su b s t r a t e c o n c e n t r a t i o n dependence of growth. Numerous other proposals have been suggested. Some of these are discussed below. Konak (1974) assumed that substrate dependence on s p e c i f i c growth r a t e - 34 - i s r e l a t e d to the d i f f e r e n c e between du/dS = i n which k and p are constants. The leads to the f o l l o w i n g : u and |J. (Equation 2.6) m k(u - u ) p (2.6) s o l u t i o n of t h i s d i f f e r e n t i a l equation in e ^ ) f o r p = 1 (2.7) i ( 1 _ p ) - (u - u ) 1 p = (1 - P ) k S f o r p M m m r r (2.8) For p = 2, Equation 2.8 s i m p l i f i e s to: u = a S/(l/ku. + S) (2.9) Equation 2.7 i s s i m i l a r to one that was proposed by T e i s s i e r (1936) and Equation 2.9 i s an analogue of the Monod equation. Therefore the r e l a t i o n s h i p p o s t u l a t e d by Konak seems v e r s a t i l e , however, i t has no c l e a r r e l a t i o n s h i p w i t h any m i c r o b i o l o g i c a l mechanism. Dabes e t . a l . (1973) developed an equation to d e s c r i b e the k i n e t i c s of a s e r i e s of enzymatic r e a c t i o n s f o r the case of steady s t a t e . The three parameter equation f o r growth based on t h e i r work i s : S = \x(Kk + K B ) / ( r i m - V) (2.10) Under s p e c i a l c o n d i t i o n s ( d e t a i l s not presented here) Equation 2.10 reduces to: - 35 - * = V S > V m u = S / V S < V m (2.11) Equations 2.11 were f i r s t proposed by Blackman (1905). Dabes (1970) has indi c a t e d that the Monod equation w i l l a r i s e from th e i r general form (not presented here) as a s p e c i a l case. In a paper Dabes and co-workers published in 1973, they analysed a number of published data using Equations 2.5, 2.10 and 2.11, and found that t h e i r Equation (2.10) always gave a bet t e r f i t . This i s not s u r p r i s i n g since Equation 2.10 includes the Monod and Blackman forms. They also showed Blackman's form to be superior to Monod's. Condrey (1982) has pointed out that Blackmans k i n e t i c s have been neglected i n m i c r o bial growth modeling, perhaps because of the f u n c t i o n a l form being discontinuous. Powell (1967) combined mass transf e r into and in s i d e the organism ( d i f f u s i o n and permeation) with the Michealis-Menten enzyme k i n e t i c s and derived the following expression: u = u- (K + L + S)/2L r l m s u„ = 1 - (1 - 4LS/(K + L + S ) 2 ) * 5 (2.12) z s i n w h i c h L = q /A; q b e i n g the maximum s p e c i f i c r a t e of s u b s t r a t e Mm m̂ ° r consumption and A being a constant determined by the trans f e r resistance i n s i d e and outside the c e l l . Since S, K and L are p o s i t i v e , (K + L + S) > 2 2 2 (L + S) = (L - S) + 4SL. (L - S) i s n e c e s s a r i l y p o s i t i v e , therefore 2 4LS/(K + L + S) i s le s s than unity and i t s expansion by binomial theorem - 36 - i s p e r m i s s i b l e . I f only two terms of the expansion are re ta ined the f o l l o w i n g express ion i s obta inable : li = u (K + L + S) (2.13) m s If L i s smal l compared to the Monod form i s r e t a i n e d . Moser (1958) pos tu lated the fo l lowing modified form of Monod's equat ion: u = u SX/(K + SX) (2.14) m s Unfor tunate ly there i s no p h y s i c a l model to support Equat ion 2.14. There are a v a r i e t y of other suggest ions , mainly of modif ied f i r s t o r d e r f o r m s . These are s p e c i a l cases of the Monod e q u a t i o n (Kg « S). Examples of these are those given by Elmaleh and Aim (1976) and Grady and Wi l l i ams (1975). These types do not p r e d i c t cond i t ions for maximum b i o l o g i c a l a c t i v i t y and system f a i l u r e . In the next s e c t i o n , a t t e n t i o n i s turned to the assumption of the second school mentioned at the beginning of t h i s s ec t ion (2.2.2). 2 . 2 . 3 Models Wi th S p e c i f i c Growth S u b s t r a t e And/Or Biomass Growth Dependence In t h i s type of m i c r o b i a l growth mode l l ing , i t i s assumed that i n a d d i t i o n to substrate c o n c e n t r a t i o n , p. i s a l so dependent on the b i o l o g i c a l p o p u l a t i o n d e n s i t y . The f i r s t example i s the l o g i s t i c law; w r i t t e n as: (2.15) - 37 - i n w hich X i s the maximum biomass c o n c e n t r a t i o n which can be reached, m Equation 2.15 has been used s u c c e s s f u l l y by Co n s t a n t i n i d e s e t . a l . (1970a & b) and Rai and Co n s t a n t i n i d e s (1974). I t s success has been a t t r i b u t e d by Roels and Kossen (1978) to the f a c t that growth curves acc o r d i n g to the l o g i s t i c law bear some resemblence to curves which r e s u l t from m i c r o b i a l modeling e x e r c i s e s . In a l l the approaches considered so f a r the r a t e of growth of biomass i s to some extent dependent upon the growth l i m i t i n g s u b s t r a t e . This c l e a r l y I n d i c a t e s that the l o g i s t i c law can not be s u c c e s s f u l l y a p p l i e d i n s i t u a t i o n s where the r a t e of s u b s t r a t e a d d i t i o n l i m i t s the growth r a t e . This renders i t of l i t t l e value f o r most m i c r o b i a l processes. Fujimoto (1963) considered u t i l i z a t i o n of su b s t r a t e i n three steps; i n t e r a c t i o n at the surface of the c e l l , t r a n s p o r t i n t o the i n t e r i o r and enzymatic r e a c t i o n w i t h i n the c e l l . For the case where the a d s o r p t i o n r a t e i s p r o p o r t i o n a l to the r a t e of t r a n s p o r t i n t o the c e l l i n t e r i o r and subsequent enzymatic conversion there, the o v e r a l l r e a c t i o n was considered as a r e a c t i o n between s u b s t r a t e and enzyme k+1 k+2 X + S "—* (X.S) >[X.S] + P (2.16) k-1 For growth and s u b s t r a t e consumption where the conversion r a t e from the s u b s t r a t e i n t o the c e l l m a t e r i a l i s constant, Fujimoto solved the k i n e t i c s of equation 2.16 and obtained the f o l l o w i n g s o l u t i o n : - 38 - u = au^ (S/X) / (K + (S /X)) (2.17) = \i S/(KX + S) m where a(0 < a < 1) i s the a c t i v i t y of the enzyme. Equation 2.17 was f i r s t proposed by Contois (1959) without a mathematical j u s t i f i c a t i o n . For a CSTR at steady s t a t e X = Y(S - S) (2.18) o where Y i s the y i e l d c o e f f i c i e n t . S u b s t i t u t i n g equation 2.18 i n t o equation 2.17 g i v e s , u = |X mS/(YK(S o - S) + S) ( 2 # 1 9 ) = u S / ( K ' ( S n - S) + S m o Equation 2.19 was f i r s t proposed by Chen and Hashimoto (1978) and has been used s u c c e s s f u l l y i n modeling methane pro d u c t i o n from a g r i c u l t u r a l residues (Chen e t . a l . 1980). Roques e t . a l . (1982) has r e c e n t l y proposed the f o l l o w i n g equation: u = U m S / ( K ( S Q - S) + M + S) (2.20) i n which a t h i r d parameter M was in t r o d u c e d . Equations 2.19 and 2.20 are e q u i v a l e n t s to Monod's equation w i t h K g v a r y i n g as: K = K(S - S) s o and (2.21) K s = K ( S Q - S) + M - 39 - I f M < K ( S Q - S), equation 2.20 becomes analogous to equation 2.19. 2.2.4 Microbial Growth Modelling Recommendation In order to provide an impression of the v a r i e t y of p o s s i b i l i t i e s f o r the modeling of s u b s t r a t e c o n c e n t r a t i o n dependence of s p e c i f i c growth r a t e , some of the equations, considered above are g r a p h i c a l l y represented f o r some values of the parameters ( F i g u r e 2.4). The equations were s c a l e d i n such a way t h a t a l l curves c o i n c i d e at u/u = 1/2 and a r e l a t i v e S value of u n i t y . m I t i s c l e a r , as pointed out by Roels (1983), that d e t a i l e d k i n e t i c s play a minor r o l e i n the f i x i n g of biomass co n c e n t r a t i o n - t i m e r e l a t i o n s h i p s . Therefore there i s l i t t l e j u s t i f i c a t i o n f o r f a v o u r i n g a p a r t i c u l a r equation among those d i s c u s s e d above. In t h i s study the Monod equation was favoured f o r i t s s i m p l i c i t y and the f a c t that i t i s a mathematical homologue of the Michealis-Menten equation, which g i v e s i t a p h y s i c a l m i c r o b i o l o g i c a l i n t e r p r e t a t i o n . There are models that have been proposed that do not f i t the above two considered c l a s s i f i c a t i o n s . This i s u s u a l l y a r e s u l t of the s p e c i a l circumstances surrounding the m i c r o b i o l o g i c a l process i n c o n s i d e r a t i o n . A few of these are b r i e f l y covered next. 2.2.5 Miscellaneous Special Models 2.2.5.1 Models Of Growth In Presence Of Inhibiting Substrate/Product Substrate i n h i b i t i o n of growth i s a subject of i n c r e a s i n g concern. A l a r g e number of p u b l i c a t i o n s have appeared on t h i s s u b j e c t . An o f t e n used model was proposed by Andrews (1968). - 40 - F i g u r e 2.4 A c o m p i l a t i o n o f equations f o r the s u b s t r a t e and/or biomass c o n c e n t r a t i o n dependence of the s p e c i f i c growth r a t e : (1) Monod; (2) Moser, X = 2; (3) Moser, X = 10; (4) T e i s s i e r ; (5) Dabes e t . a l . , = 1; (6) Konak, p = 3; (7) Konak, p = 10; (8) P o w e l l , L/K g = 2; (9) P o w e l l , L/K = °°; (10) Blackman. - 41 - \x - u S/(K + S + S 2/K.) (2.22) m s 1 i n which i s an i n h i b i t i o n constant. L i k e the Monod equation, equation 2.22 i s der i v e d from the theory of i n h i b i t i o n of a s i n g l e enzyme. A number of a l t e r n a t i v e s to equation 2.22 have been proposed, but d i s t i n c t i o n between v a r i o u s proposals i s impossible due to the l i m i t e d e xperimental data (Yano e t . a l . , 1966; Edwards, 1970). The i n t e r e s t e d reader i s r e f e r r e d to Webb (1963), Yamashita e t . a l . (1969), Wayman and Tseng (1975 & 1976), Yang and Humphry (1975) and D i B i a s i o e t . a l . (1981), f o r the v a r i o u s a l t e r n a t i v e s to equation 2.22. Growth can be i n h i b i t e d by me t a b o l i t e s which are excreted as a d i r e c t or i n d i r e c t consequence of growth. One of the models o f t e n used to d e s c r i b e t h i s type of i n h i b i t i o n was proposed by I e r u s a l i m s k y (1967): u- - (S/(K g + S)) (K /(K + P)) (2.23) where P i s the c o n c e n t r a t i o n of the product, i s an i n h i b i t i o n constant. An example of i n h i b i t i o n that i s w e l l known i s that of high ethanol c o n c e n t r a t i o n I n h i b i t i o n of yeast growth (Hinshelwood, 1946; Aiba and Shoda, 1969). For a l t e r n a t i v e s to equation 2.23, the reader i s r e f e r r e d to Ramkrishna e t . a l . (1967) and L e v e n s p i e l (1980). 2 . 2 . 5 . 2 Models For Growth L i m i t e d By More Than One S u b s t r a t e A treatment f o r growth i n f l u e n c e d by the c o n c e n t r a t i o n of more than one su b s t r a t e was given by Tsao and Hanson (1975) : - 42 - H - { l + s } { 2 p. s . til 1 + s. 1 (2.24) i n which Ŝ  are the concentrations of the essential substrates. are the concentrations of the growth rate enhancing substrates, whose presence is not essential for growth, but growth is enhanced when they are supplied. A special case of equation 2.24 is the double substrate kinetics described by Apart from the work of Megee, successful applications of equation 2.25 have been reported by Nagai et. a l . (1973) and Ryder and S inc la ir (1972). A different approach has also been suggested by Bloomfield et. a l . (1973). A number of different groups of product formation processes have been described. The treatment introduced by Gaden (1959), that subdivides these processes into three categories is considered below. In the f i r s t category, the product is a direct result of the primary energy metabolism and is thus strongly associated with growth (eg. ethanol and anaerobic processes without external electron acceptors) . For this class modeling is re lat ive ly simple. The rate of substrate consumption can be assumed to be represented well by the l inear law: Megee (1970); ^ - + S l » + S 2 » (2.25) 2.2.5.3 Models For Product Formation dS/dt = ( d X / d t ) / Y g x + MgX (2.26) - 43 - The r a t e of product formation i s then given by: dP/dt = (dX/dt)/Y + M X (2.27) px p In the second category there i s no obvious d i r e c t or i n d i r e c t c onnection between primary metabolism and product formation (examples of these are p e n i c i l l i n and streptomycin p r o d u c t i o n ) . Modeling i s f a i r l y d i f f i c u l t i n t h i s category. The t h i r d category i n c l u d e s a l l those cases that are intermediate between c a t e g o r i e s one and two. For these the product i s i n d i r e c t l y connected to the energy production pathway (examples are pro d u c t i o n of amino a c i d s and c i t r i c a c i d ) . 2.3 M e s o p h i l i c M i c r o b i a l K i n e t i c Modes; L i t e r a t u r e Review 2 .3 .1 Whey and /o r Lac tose S u b s t r a t e s Despite a comprehensive search only a handful of papers (Rogers e t . a l . , 1978; Yang, 1984) were uncovered concerning acidogenic m i c r o b i a l k i n e t i c modeling of whey/lactose s u b s t r a t e s f e r m e n t a t i o n . However a number of i n v e s t i g a t o r s ( S c h l o t t f e l d t , 1979; Boening and Larsen, 1982; and Yang, 1984) have reporte d v a r i o u s m i c r o b i a l k i n e t i c models f o r defined and undefined single-phase fermentations of l a c t o s e l i m i t e d s u b s t r a t e s . Below, the r e s u l t s of each of the above c i t e d works i s reviewed. Rogers e t . a l . (1978) used a s e m i - s y n t h e t i c l a c t o s e l i m i t e d medium to grow Streptococcus cremoris i n batch c u l t u r e at 30°C and a pH of 6 .0 . S. cremoris i s a l a c t i c a c i d producing micro-organism that has a t t r a c t e d c o n s i d e r a b l e i n t e r e s t as a r e s u l t of the s i g n i f i c a n t r o l e that i t plays as a - 44 - s t a r t e r c u l t u r e i n the d a i r y i n d u s t r y . Various growth models were t e s t e d f o r Sj_ cremoris and the f o l l o w i n g growth i n h i b i t i o n and a product formation r e l a t i o n s were recommended: u = K^S/CK, + S)) ( K p / ( K p + P)) (2.29) dP/dt = K.(dX/dt) + K C(S/K' + S)) <4 J S (2.30) -dS/dt = (dP/dt)/Y (2.31) sp Estimates of the constants were r e p o r t e d . However on t e s t i n g the above model w i t h a continuous c u l t u r e , no washout was observed as p r e d i c t e d by the model. This was a t t r i b u t e d to w a l l growth. S c h l o t t f e l d t (1979) employed an undefined c u l t u r e (obtained from a methane generating r e a c t o r ) i n fed-batch 4L c a p a c i t y l a b o r a t o r y single-phase r e a c t o r s , to t r e a t f r e s h and r e c o n s i t i t u t e d whey. The o v e r a l l f i r s t order COD removal r a t e constant at 35° was determined to be equal to .047 h ̂ . Boening and Larsen (1982) a l s o employed an undefined c u l t u r e (source of inoculum not i n d i c a t e d ) i n a single-phase f l u i d i s e d bed r e a c t o r w i t h a packing medium of crushed c o a l p a r t i c l e s to degrade whey permeate. Three temperature l e v e l s (15, 25 and 35°C) were considered i n the study. A model p r e v i o u s l y proposed by Chen and Hashmoto (1978) of the f o l l o w i n g form was found to be the best among a number t r i e d . S / S o = K ' ( t H R ^ m - 1 + K , ) (2.32) - 45 - where i s the r e t e n t i o n time ( = 1/D) . No values of the constants were r e p o r t e d . Yang (1984) i n an ingenious e f f o r t proposed the use of a defined m i c r o b i a l system comprising of a homo-lactic b a c t e r i a (Streptococcus l a c t i s ) , a homoacetogenic b a c t e r i a ( C l o s t r i d i u m formicoaceticum) and a methanogenic b a c t e r i a (Methanococcus mazei). The f o l l o w i n g growth k i n e t i c modes f o r batch fermentation were reported f o r a temperature of 35°C and pH of 7.0. For Sj_ l a c t i s and C. formicoaceticum, a model s i m i l a r to the one employed by Rogers e t . a l . (1978) (Equations 2.29 - 2.31) was used, w i t h the exception that a decay r a t e (K^ sometimes r e f e r r e d to as the maintenance c o e f f i c i e n t ) term was in c o r p o r a t e d to modify u (Equation 2.33). dX/dt = (u - K.) X (2.33) a For M_̂_ mazei, a s u b s t r a t e i n h i b i t i o n model proposed by Andrews (1968) (Equation 2.22) was u t i l i s e d . Yang's r e s u l t s seems to suggest that there were s e r i o u s a n t a g o n i s t i c e f f e c t s between S_̂  l a c t i s and C^ formicoaceticum, i f the c o c u l t u r e experienced s l i g h t pH v a r i a t i o n s from the optimum value of 7.0. However Yang assumed that each b a c t e r i a i n the c o c u l t u r e would e x h i b i t the same fermentation k i n e t i c s as i n the pure c u l t u r e form. A t h e o r e t i c a l model was d e v e l o p e d f o r the t h r e e b a c t e r i a u s i n g the pure c u l t u r e models. U n f o r t u n a t e l y no experimental v e r i f i c a t i o n of the model was made. However, i t was suggested that a two-phase process w i t h l a c t i c a c i d as the int e r m e d i a t e was the best f o r the def i n e d methane fermentation s t u d i e d . - 46 - 2 . 3 . 2 O t h e r S u b s t r a t e s The two dominating b a c t e r i a l steps i n ana e r o b i o s i s (acidogenesis and methanogenesis) have d i f f e r e n t m i c r o b i a l k i n e t i c c o n s t a n t s . Henze and Herremoes (1983) have c o m p i l e d f o r these steps the reported values of n^, Y, and K g. The d a t a i n c l u d e s e x p e r i m e n t a l l y d e t e r m i n e d c o n s t a n t s and constants used i n modeling, the l a t t e r based on more or l e s s e xtensive l i t e r a t u r e searches. The g e n e r a l i s a t i o n s of Henze and Herremoes are presented i n Table 2.2. 2 . 4 M a t h e m a t i c a l A n a l y s i s o f C o n t i n u o u s C u l t u r e s I n t h i s study continuous c u l t u r e methods were favoured over batch c u l t u r e methods, because pH was an important v a r i a b l e that had to be maintained at a constant l e v e l throughout the experimental p e r i o d . A d d i t i o n of a pH c o r r e c t i n g s o l u t i o n (eg. NaOH) was evaluated f o r batch c u l t u r e , but dismissed due to fear of t o x i c e f f e c t s caused by an accumulation of Na i n the r e a c t o r . Although modeling of continuous c u l t u r e s i s very w e l l documented (Malek and F e n c l , 1966; B a i l e y and O l l i s , 1986), the d e r i v a t i o n of the so c a l l e d Monod chemostat model (CSTR) i s presented below to i l l u s t r a t e the assumptions made i n i t s development. The a n a l y s i s of continuous c u l t i v a t i o n of microorganisms s t a r t s w i t h the b a c t e r i a l mass balance equation ( r e f e r to Fi g u r e 2.5). Table 2.2 Ge n e r a l i s e d Growth Constants For Anaerobic C u l t u r e s \ Parameter C u l t u r e m̂ Maximum s p e c i f i c growth r a t e at 35°C ( h _ 1 ) Y Maximum y i e l d c o e f f i c i e n t Maximum r a t e at g subst 35°C COD/g :rate removal (Kto/Y) ; VSS.h K s Monod S a t u r a t i o n constant g COD/inL g VSS 1/g COD 100% a c t i v e VSS 50% a c t i v e VSS A c e t i c a c i d producing b a c t e r i a .0833 .15 .5417 .2917 .2 x 10~ 3 Methane producing b a c t e r i a .0333 .03 .5417 .2917 .05 x 10~ 3 Combined .0333 .18 .0833 .0417 — 1 VSS - v o l a t i l e suspended s o l i d s - a term used to represent biomass i n waste water technology - 48 - F i g u r e 2.5 The i d e a l c o n t i n u o u s - f l o w s t i r r e d tank r e a c t o r (CSTR). - 49 - r a t e of change = input - output + r e a c t i o n (2.34) In case of a CSTR, there i s a constant r a t e of feeding (F) of a medium to organisms (X) i n the v e s s e l of constant volume ( V ) . This means, of course, that the feed r a t e equals the overflow r a t e . The mass balance f o r the organisms i s : I n most continuous c u l t u r e systems the feed stream i s e i t h e r f r e s h l y made up o r i s s t e r i l e , so X = 0 . A term c a l l e d d i l u t i o n r a t e D which i s equal to o F/V, i s introduced i n t o equation 2.35 to g i v e , The concept of a l i m i t i n g n u t r i e n t i s e s s e n t i a l to the theory of continuous c u l t u r e . The i n g r e d i e n t i n short supply r e l a t i v e to the other i n g r e d i e n t s w i l l be exhausted f i r s t and w i l l thus l i m i t c e l l u l a r or product s y n t h e s i s . The other i n g r e d i e n t s p l a y v a r i o u s r o l e s , such as promoting c e l l u l a r a c t i v i t i e s , but they w i l l not be i n acute short supply as i s the l i m i t i n g n u t r i e n t . A mass balance on the growth l i m i t i n g n u t r i e n t g i v e s , (dX/dt) V = F X q - FX + uX V (2.35) (dX/dt) = uX - DX (2.36) (dS/dt) V = FS - FS - uXV/Y - MXV (2.37) where Y i s the y i e l d c o e f f i c i e n t (g of c e l l per g of l i m i t i n g n u t r i e n t ) . M i s the maintenance c o e f f i c i e n t to keep c e l l s a l i v e . D i v i d i n g by V y i e l d s , - 50 - dS/dt = D S q - DS - uX/Y - MX (2.38) A r e l a t i o n s h i p i s needed between u and S. This i s where the expressions t h a t have been considered i n s e c t i o n 2.2 come i n . As p r e v i o u s l y mentioned, the use of the Monod equation (Equation 2.5) was favoured i n t h i s study. At steady s t a t e , there i s no change, thus the d e r i v a t i v e s i n the d i f f e r e n t i a l equations 2.36 and 2.38 disappear to g i v e , u = D (2.39) and D S Q - DS = uX/Y + MX (2.40) s u b s t i t u t i n g D f o r \i i n equation 2.5 and s o l v i n g f o r S g i v e s , S = DK /(u - D) (2.41) s m S o l v i n g equation 2.40 f o r X a f t e r s u b s t i t u t i n g D f o r u g i v e s , X - D Y ( S Q - S ) / ( D + MY) (2.42) Equations 2.41 and 2.42 are o f t e n c a l l e d the "Monod Chemostat Model". The above a n a l y s i s a p p l i e s to any continuous c u l t u r e that meets the assumption of p e r f e c t mixing and constant volume. The equations are fundamental except f o r the Monod equation which has no time dependence and should be a p p l i e d w i t h c a u t i o n to t r a n s i e n t s t a t e s where there may be a l a g time as \i responds to the changs S. - 51 - 2.5 Experimental Plan Based on the m a t e r i a l presented i n t h i s chapter i t can be concluded t h a t : (1) E f f e c t i v e d i g e s t i o n of organic matter to methane r e q u i r e s the combination of carbon c a t a b o l i s i n g anaerobic b a c t e r i a l groups, of which the methanogens play a key r o l e . Therefore, s e p a r a t i n g the methanogens from the non-methanogens may r e s u l t i n a l t e r a t i o n s i n the int e r m e d i a r y m e t a b o l i t e r o u t e s ; (2) Due to the complexity of the biochemical processes i n a n a e r o b i o s i s , modeling m i c r o b i a l growth w i t h s t r u c t u r e i s an almost Impossible task. Therefore u n s t r u c t u r e d models are found to be most s u i t a b l e ; (3) K i n e t i c data on the acidog e n i c conversion of l a c t o s e i s almost n o n e x i s t e n t . 2.5.1 Assumptions As pointed out i n s e c t i o n 2.1.2.1, i t i s b e l i e v e d that hexoses are mainly fermented v i a the Embden-Meyerhof-Parnas (EMP) pathway. So i t was assumed that l a c t o s e i s mainly fermented v i a the EMP pathway to pyruvate. 14 Thus the t a s k of e l u c i d a t i n g the degradation pathway usi n g C t r a c e r s was reduced to the det e r m i n a t i o n of the f a t e of pyruvate during a c i d o g e n e s i s . I t was a l s o assumed that l a c t o s e i s f i r s t broken down to glucose and ga l a c t o s e v i a 8 - g a l a c t o s i d a s e . Glucose enters the EMP pathway and ga l a c t o s e i s converted to glucose 6-phosphate before i t enters the EMP pathway, a scheme that i s w e l l e s t a b l i s h e d i n Ej_ c o l i • 2.5.2 Experimental Factors The experimental f a c t o r s and l e v e l s s t u d i e d are presented i n Table 2.3. - 52 - Table 2.3 Factors I n v e s t i g a t e d F a c t o r L e v e l s Temperature L i m i t i n g s u b s t r a t e pH D i l u t i o n r a t e R a d i o t r a c e r s 35°C l a c t o s e and l a c t o s e + 8 - l a c t o g l o b u l i n 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 .05, .1, .2, .3, .4, .5 and .6 h L a c t a t e , propionate and b u t y r a t e -1 - 53 - III. MATERIALS AND METHODS 3.1 Inoculum The mixed, undefined c u l t u r e inoculum was sewage sludge. I t was obtained from a l o c a l two-step anaerobic m u n i c i p a l waste treatment f a c i l i t y at Iona I s l a n d . The inoculum samples were drawn from the f i r s t stage, at ten or f i f t e e n f e e t from the s u r f a c e . 3.2 Media Two types of growth medium were employed i n t h i s study: the f i r s t was a c h e m i c a l l y d e f i n e d l a c t o s e l i m i t e d n u t r i e n t s o l u t i o n the second was r e c o n s t i t u t e d whey powder, (comprising of l a c t o s e and p r o t e i n , mainly 8 - l a c t o g l o b u l i n ) . The d e t a i l s of these growth medium are given below. 3.2.1 Lactose Limited Growth Sythetic Medium Since l a c t o s e i s the major s i n g l e component i n whey, the n u t r i e n t medium (shown i n Table 3.1) was composed such that l a c t o s e i s the main carbon source ( s u b s t r a t e ) . The r a t i o s of C:N:P were maintained at approximately 150:5:1, f o r i t has been observed (Speece and McCarty, 1964; H i l l , 1979; Pos e t . a l . , 1981; and Pipyn and V e r s t r a e t e , 1981) that n u t r i t i o n a l requirements f o r s i n g l e phase a n a e r o b i o s i s are d i r e c t l y p r o p o r t i o n a l to the s y t h e s i s of m i c r o b i a l c e l l s and the i n d i c a t e d r a t i o s are necessary to mainta i n n u t r i t i o n a l l y balanced growth. No separate m i c r o b i a l n u t r i t i o n a l i n f o r m a t i o n f o r acidogenic b a c t e r i a were found. Cations of macro elements, calcium, sodium, potassium and magnesium were kept at 100-200 mg/L f o r the former two and 200-240 mg/L f o r the l a t t e r two. These - 54 - Table 3.1 Lactose L i m i t e d Growth Medium Concentration Component (g/L) S u p p l i e r Substrate C12 H22°11 H2° N u t r i e n t s (or macro elements) NH.C1 4 (NH 4) 2HP0 4 MgS0 4 .H20 KC1 CaCl 2.2H 20 NaHC0 3 Trace (or micro) elements F e ( N H 4 ) 2 S 0 4 MnCl 2.4H 20 ZnS0 4 -7H20 CuSO,.5H_0 4 2 NaB.0..10H-0 4 2 2 NaMoO,.2H.0 4 2 C h e l a t i n g agent C i t r a t e 10.53 0.800 0.180 0.150 0.740 0.730 0.300 0.100 0.005 0.005 0.005 0.005 0.005 0.150 BDH BDH FISHER MCB BDH MCB AMACHEM MALLINCRODT AMACHEM MCB AMACHEM BAKER BDH - 55 - c o n c e n t r a t i o n l e v e l s were observed by McCarty (1964) to be s t i m u l a t o r y . Micro elements c o n c e n t r a t i o n s were kept at l e v e l s s i m i l a r to those used by Speece e t . a l . (1983). No growth f a c t o r s were added. A l l the chemicals were reagent grade unless otherwise i n d i c a t e d . 3.2.2 Lactose/Protein Growth Medium This medium was made by adding 15.4 g of sweet whey powder (SIGMA) to 500 mL of d i s t i l l e d water. A f t e r mixing thoroughly the mixture was made up to one l i t r e . An a n a l y s i s of the medium i s shown i n Table 3.2. 3.3 Fermentor Set-Up A modular bench top fermentor (NEW BRUNSWICK) was modified to a l l o w continuous pumping of i n f l u e n t and e f f l u e n t . As shown i n Fi g u r e 3.1, the pH, a g i t a t i o n (rpm) and temperature could be set at d e s i r e d values and monitored. For pH adjustment a 2N, 4N or 6N NaOH s o l u t i o n was used, depending on the r a t e of i n f l u e n t flow. With these c o n c e n t r a t i o n s of c a u s t i c , the pH value could be c o n t r o l l e d to b e t t e r than ±0.1 u n i t s of the d e s i r e d v a l u e , w h i l e causing only a minimal percentage change i n d i l u t i o n r a t e (D). The fermentor had a paddle wheel a g i t a t o r which was run at 400 rpm. A high rpm value was s e l e c t e d to ensure complete mixing ( C h o l e t t e and C l o u t i e r , 1959) and to minimise attachment of micro-organisms on the fermentor v e s s e l components. The d e s i r e d temperature was maintained by hot water c i r c u l a t i o n through s t a i n l e s s s t e e l heat exchanger tubing. The temperature was c o n t r o l l e d at 35 ±0.5°C. The fermentor had a working volume from 1.25 to 5.00 L. - 56 - Table 3.2 Sweet Whey Growth Medium A n a l y s i s Component Concentration (mg/L) Method of A n a l y s i s Substrate C12 H22°11 N u t r i e n t s (macro elements) Ammonium n i t r o g e n T o t a l K j e l d a h l n i t r o g e n T o t a l p r o t e i n as 8 - L a c t o g l o b u l i n Phosphorous S u l f u r Magnesium Potassium Calcium Sodium Trace (micro) elements I r o n Copper 10,000.0 9.5 242.5 2,600.0 18.0 334.0 83.0 134.0 C o l o u r i m e t r i c B i u r e t r e a c t i o n Atomic a b s o r p t i o n 0.7207 0.2495 - 57 - - K J 3 CXD F i g u r e 3.1 The fermentor and a u x i l l i a r y apparatus: (1) ferme- n t o r v e s s e l , (2) heat exchanger t u b i n g , (3) l e v e l s w i t c h , (4) pH probe, (5) pH c o n t r o l l e r , (6) 2N NaOH r e s e r v o i r , (7) s u b s t r a t e r e s e r v o i r , (8) s u b s t r a t e pump, (9) NaOH pump, (10) e f f l u e n t pump, (11) l e v e l c o n t r o l l e r , (12) e f f l u e n t to waste, (13) NaOH to v e s s e l , (14) hot water out, (15) hot water i n , (16) s t i r r e r , (17) wet gas meter. - 58 - 3.4 Fermentor S tar t -Up And Operat ion Procedure For each run at a d e s i r e d f i x e d pH value the fermentor, c o n t a i n i n g 1.5 L of growth medium ( d i l u t e d three times) was f r e s h l y I n o c c u l a t e d w i t h 50 mL of screened inoculum. The fermentor was then purged with at l e a s t twenty fermentor v e s s e l head space volumes of helium to expel a l l the oxygen. I t was operated i n batch mode u n t i l a l l the l a c t o s e was used up, a f t e r which time the pumps were switched on and set f o r d i l u t i o n r a t e (D) of a p p r o x i m a t e l y .05 h 1 . T h i s v a l u e of D was s e l e c t e d to m a i n t a i n the s p e c i f i c growth r a t e (u-) high enough f o r the methanogenic b a c t e r i a to be washed out. This method of acidogenic and methanogenic phase s e p a r a t i o n has been r e f e r r e d to by Pohland and Ghosh (1971) as phase k i n e t i c c o n t r o l . For experiments conducted at higher d i l u t i o n r a t e s , D was stepped up i n steps of .025 h 1 and m a i n t a i n e d a t each s t e p f o r a minimum of two d i l u t i o n s . A c cording to C o l i n e t . a l . (1983) i t i s commonly accepted that a steady s t a t e i s not reached before at l e a s t two or three d i l u t i o n s under the same running c o n d i t i o n s . The fermentor was t h e r e f o r e operated f o r at l e a s t three d i l u t i o n s , once steady s t a t e c o n d i t i o n s had been achieved. On average a p e r i o d ranging between two to three weeks was needed to complete an experiment. Steady s t a t e was defined as when fermentor performance, i n terms of the r a t e of consumption of the pH c o r r e c t i n g s o l u t i o n of NaOH, showed no s i g n i f i c a n t a l t e r a t i o n w i t h i n a pe r i o d of at l e a s t three d i l u t i o n s . Analyses of l a c t o s e , p r o t e i n , formate, l a c t a t e v o l a t i l e f a t t y a c i d s , biomass carbon and dry biomass were made a f t e r steady s t a t e c o n d i t i o n s had been achieved. For most of the runs, these measurements were r e p l i c a t e d every two d i l u t i o n s up to a maximum of four times. The d e t a i l s of the methods used f o r the above analyses are o u t l i n e d i n Appendix A. - 59 - T o t a l gas p r o d u c t i o n was recorded by a wet gas meter (ALEXANDER - 0.25 L per r e v o l u t i o n ) , shown i n F i g u r e 3.1. Procedures used f o r the fermentor head space gas a n a l y s i s and the c a l c u l a t i o n of the a c t u a l gas volume pro d u c t i o n are a l s o o u t l i n e d i n Appendix A. 3.5 Set -Up For R a d i o a c t i v e T r a c e r I n c o r p o r a t i o n In a s e r i e s of experiments, samples from the fermentor were incubated 14 14 under a n a e r o b i c c o n d i t i o n s w i t h [ C(U)] - b u t y r a t e , [ C(2)] - propionate o r [ 1 4 C ( U ) ] - l a c t a t e (NEW ENGLAND NUCLEAR) f o r d e g r a d a t i o n mechanism s t u d i e s . Two types of apparatus were needed f o r these t e s t s . In the f i r s t one i t was necessary to monitor and c o n t r o l the pH at a f i x e d value throughout the p e r i o d of the experiment. In the second case, pH was n e i t h e r monitored nor c o n t r o l l e d . 3 .5 .1 Appara tus Wi th pH C o n t r o l A s m a l l m a g n e t i c a l l y s t i r r e d g l a s s r e a c t o r , w i t h a water j a c k e t f o r temperature c o n t r o l was modified as shown i n Figure 3.2 to accommodate a pH probe and a thermometer. The temperature was c o n t r o l l e d at 35 ±0.5°C. At the beginning of the experiment, the temperature c o n t r o l , water pump and heater were turned on. Enough time was allowed f o r steady s t a t e to be 14 a c h i e v e d then the r e a c t o r was f l u s h e d w i t h U • The s p e c i f i c C - l a b e l e d t r a c e r was i n j e c t e d together w i t h 10 mL of f r e s h s u b s t r a t e . The f r e s h s u b s t r a t e was needed f o r the p r o d u c t i o n of a c i d s that would maint a i n an i n i t i a l downward movement of the pH l e v e l . Otherwise, the pH would i n c r e a s e n e c e s s i t a t i n g a d d i t i o n of some a c i d to the r e a c t o r to m a i n t a i n a f i x e d d e s i r e d l e v e l . A d d i t i o n of a c i d could p o t e n t i a l l y a f f e c t the r e a c t i o n - 60 - Figure 3.2 Schematic diagram f o r r a d i o t r a c e r experiments w i t h pH c o n t r o l : (1) temperature c o n t r o l water i n , (2) n i t r o g e n , (3) pH probe, (4) thermometer, (5) 2N NaOH sy r i n g e f o r pH c o r r e c t i o n s , ( 6 ) sample p o r t , (7) o - r i n g s e a l , (8) clamps, (9) mechanical s t i r r i n g . - 61 - mechanism. A f t e r steady s t a t e was achieved 80 mL of sample from the fermentor were i n j e c t e d i n t o the r e a c t o r . The experiment was run f o r 30 minutes and during t h i s p e r i o d the pH was monitored and maintained at 6. ±0.1 manually. At the end of the experiment, 10 mL of sample were removed from the r e a c t o r by sy r i n g e and immediately introduced i n t o a 24 mL v i a l and f r o z e n at -40°C. P r i o r to a n a l y s i s the samples were d e f r o s t e d and c e n t r i f u g e d ( l h , 4450xg). 1 mL of the supernatant was a c i d i f i e d by adding 1 mL normal s u l p h u r i c a c i d and loaded d i r e c t l y on the l i q u i d chromatography column d e s c r i b e d i n s e c t i o n 3.6. 3 . 5 . 2 Appara tus Wi thout pH C o n t r o l The cap of a 20 mL v i a l was modified to a l l o w continuous flow of i n and out (F i g u r e 3.3). The v i a l was i n i t i a l l y f l u s h e d w i t h N 2. The s p e c i f i c 14 C - l a b e l e d t r a c e r was then introduced i n t o the v i a l by s y r i n g e . A 10 mL sample from the fermentor was then immediately i n j e c t e d i n t o the v i a l . The v i a l was then maintained at 35 ±.5°C f o r a s p e c i f i c p e r i o d . During t h i s p e r i o d a s m a l l amount of N 2 was allowed to flow through the v i a l to a f f e c t complete mixing. At the end of the experiment, the contents of the v i a l were t r e a t e d i n a manner s i m i l a r that d e s c r i b e d i n s e c t i o n 3.5.1. 3.6 P r e p a r a t i v e S e p a r a t i o n Of The Organ ic A c i d s 3 .6 .1 P r i n c i p l e S i n g l e components of a m i x t u r e d i s s o l v e d i n one phase show c o n c e n t r a t i o n changes at the boundary w i t h a second phase. Often a c o n c e n t r a t i o n of components on the sur f a c e of the other phase takes p l a c e . This phenomenon i s r e f e r r e d to as " a d s o r p t i o n " and f o r s i n g l e components i t - 62 - 4 ( CXM—f> 1 2 F i g u r e 3.3 M o d i f i e d v i a l f o r r a d i o t r a c e r experiments wi t h o u t pH c o n t r o l : (1) n i t r o g e n s o u r c e , (2) v i a l , (3) m o d i f i e d v i a l cap, (A) n i t r o g e n to fume hood. - 63 - i s p r o p o r t i o n a l to t h e i r a d s o r p t i o n c o e f f i c i e n t . The d i f f e r e n c e s i n a d s o r p t i o n c o e f f i c i e n t s determine the d i f f e r e n c e s i n c o n c e n t r a t i o n s on the phase boundaries. I f one phase i s moved r e l a t i v e to the other there i s a s e p a r a t i o n of the components which i s the b a s i s of chromatographic s e p a r a t i o n (Mikes, 1979). There are other chromatographic s e p a r a t i o n methods based on d i f f e r e n t p r i n c i p l e s (eg. p a r t i t i o n , i o n exchange, g e l and b i o a f f i n i t y chromatography) that w i l l not be discussed here. The o l d e s t known and commonest a d s o r p t i o n chromatography i s that which takes place between l i q u i d and s o l i d phases. The p a r t i c l e s of a s o l i d ( i n t h i s study c e l i t e coated w i t h sucrose) are placed i n a g l a s s tube, a s u i t a b l e s o l v e n t flows around them c a r r y i n g the components w i t h i t and the s e p a r a t i o n of the components takes place on the adsorbent s u r f a c e . A d s o r p t i o n chromatographic methods may be c a r r i e d out by three d i f f e r e n t procedures namely, f r o n t a l a n a l y s i s , displacement and e l u t i o n chromatography. F r o n t a l a n a l y s i s i s not s u i t a b l e f o r p r e p a r a t i v e purposes. Displacment chromatography i s p r i m a r i l y important as a p r e p a r a t i v e or even a p i l o t - p l a n t method. However i t ' s requirement of the use of a s u i t a b l e a u x i l i a r y substances w i t h a f f i n i t i e s l y i n g between p a i r s of components being separated, makes i t u n s u i t a b l e f o r a n a l y t i c a l purposes. I n e l u t i o n chromatography, a small part of the sample s o l u t i o n i s i n t r o d u c e d i n t o the column, and i s then e l u t e d w i t h a s o l v e n t whose a f f i n i t y f o r the s t a t i o n a r y phase Is s m a l l e r than that of any component. As a r e s u l t of repeated a d s o r p t i o n the components move sl o w l y down the column. Each component i s e l u t e d independent of the o t h e r s , i n order of the components a f f i n i t i e s f o r the s o l i d phase. The component zones ar e v e r y - 64 - o f t e n separated by a zone of pure solvent during t h e i r movement through the column. E l u t i o n of a l l components with the same solvent i s p o s s i b l e i f the separated substances do not d i f f e r too much i n t h e i r a f f i n i t y towards the s t a t i o n a r y phase, so that t h e i r zones are e l u t e d without long time i n t e r v a l s . In s i t u a t i o n s where t h i s i s not the case "stepwise e l u t i o n " may be more s u i t a b l e . Stepwise e l u t i o n i s c a r r i e d out by gradual e l u t i o n of the column by s e v e r a l eluents arranged i n order of i n c r e a s i n g e l u t i n g power. These s o l v e n t s g r a d u a l l y r e l e a s e i n d i v i d u a l components of the mixture from the s t a t i o n a r y phase and e l u t e them ( F i g u r e 3.4). "Gradient e l u t i o n " uses gradual i n s t e a d of abrupt changes i n composition of s o l v e n t s . I n t h i s study an a d s o r p t i o n chromatographic techniques that employs both simple and step e l u t i o n , ( f i r s t reported by Wiseman and I r v i n (1957)) was mo d i f i e d to p r e p a r a t i v e l y separate b u t y r a t e , propionate, a c e t a t e , formate and l a c t a t e . The method employs c e l i t e coated w i t h sucrose and hexane-acetone mixtures as e l u e n t s . I t s d i s t i n c t i v e f e a t u r e i s that the a c i d s i n aqueous s o l u t i o n s are loaded d i r e c t l y to the column. 3.6.2 Apparatus A. Chromatographic tube and a c c e s s o r i e s ( F i g u r e 3.5). 3. Tamping rod, c o n s i s t i n g of a s t a i n l e s s s t e e l rod 3.2 mm i n diameter, 60 cm long, s i l v e r - s o l d e r e d to the centre of a 16 mm i n diameter, 24 - gauge wire punched from a 16 - mesh sc r e e n i n g . C. T i t r a t i o n assembly. - 65 - F i g u r e 3 . 4 T y p i c a l chromatogram of a complex mixture s e p a r a t e d by a combination o f simple and stepwise e l u t i o n : C^-Cg - c o n c e n t r a t i o n s of components, E-^-Ey - e l u t i n g power of the e l u t i n g s o l v e n t s . - 66 - F i g u r e 3.5 Schemat ic d iagram o f the l i q u i d chromatography assembly : (1) 20 mL f r a c t i o n v i a l , (2) s t a i n l e s s f r i t , 10-50 um, (3) QVF g l a s s tube - 1" x 6" , (4) a d s o r b e n t , (5) cap m a t e r i a l , (6) 1/4" swagelook f i t t i n g w i t h septum, (7) e l u e n t t ank , (8) spout f o r r e f i l l i n g the t ank , (9) n i t r o g e n c y l i n d e r . - 67 - 3.6.3 Materials and Reagents A. C e l i t e a n a l y t i c a l f i l t e r a i d (SIGMA), f i n e granulated sugar, anhydrous sodium s u l f a t e and ammonium s u l f a t e . B. C r e s o l red i n d i c a t o r : 1.3 mL of 0.1 N NaOH was added to 50 mg of 0 - c r e s o l s u l f o n p h t h a l e i n i n 20 mL of a l c o h o l and made to 50 mL wi t h d i s t i l l e d water. C Alphamine red-R i n d i c a t o r : 0.4 g were added to 100 mL of d i s t i l l e d water. D. 0.1 N s u l f u r i c a c i d . E. n-Hexane g l a s s d i s t i l l e d (BDH), Acetone, reagent grade (BDH). Var i o u s percentages by volume of acetone i n n-hexane were made up as f o l l o w s : 1, 15, 20, 30 and 50%. These were r e f e r r e d to as BA^, BA^ 5 e t c . To prevent gradual removal of water from the column by a dry e l u e n t , BA^ was e q u i l i b r a t e d a g a i n s t the s t a t i o n a r y phase as f o l l o w s : Two l i t r e s of BA^ were s t i r r e d v i g o r o u s l y w i t h 50 mL of 50% sugar s o l u t i o n to which had been added one mL of sat u r a t e d barium hydroxide s o l u t i o n and a few drops of c r e s o l red i n d i c a t o r to f r e e the solvent of carbon d i o x i d e and any tra c e s of a c i d s . A f t e r s e t t l i n g , the so l v e n t was freed of suspended d r o p l e t s by passing i t through a f i l t e r paper. 3.6.4 Procedure E i g h t mL of alphamine red-R i n d i c a t o r s o l u t i o n were mixed w i t h 20 mL of sugar s o l u t i o n (2 sugar to 1 water by volume) and 0.1 mL of normal s u l f u r i c a c i d , r e s u l t i n g i n a s t a t i o n a r y phase of approximately 50% sugar s o l u t i o n . T h i s mixture was added s l o w l y to a s w i r l i n g suspension of 50 g of c e l i t e i n 500 mL of BA,.n i n a b l e n d e r . S t i r r i n g was v i g o r o u s l y c o n t i n u e d f o r 3 - 63 - minutes. Adsorbent thus prepared was st o r e d i n a glass-stoppered f l a s k i n a r e f r i g e r a t o r u n t i l needed. The column was prepared by f l o w i n g t h e adsorbent s l u r r y from a separatory funnel i n t o two 1" x 6" QVF g l a s s tubes (connected i n s e r i e s , w i t h the bottom end f i t t e d w i t h a s t a i n l e s s s t e e l f r i t and valve as shown i n F i g u r e 3.5) u n t i l they were n e a r l y f u l l . A tamping rod was passed l i g h t l y through t h e s l u r r y t o d i s l o d g e a i r bubbles. With the bottom valve open a pressure of 10 p s i g was a p p l i e d to the chromatographic column, compressing t h e adsorbent to a f i x e d volume i n a r a p i d l y moving s o l v e n t stream. The bottom valve was cl o s e d when the solvent had been expressed to the top of the adsorbent. The top QVF gl a s s tube was then removed. BA^ was added as a f i n e stream down the si d e of the tube, i s o l a t i n g the top surface from f u r t h e r t u r b u l e n t e f f e c t s . E i g h t grams of sodium s u l f a t e , c e l i t e and ammonium s u l f a t e i n weight p r o p o r t i ons of 12:8:1, r e f e r r e d to as cap m a t e r i a l were added as a s l u r r y i n about 25 mL of BA^. The top flange was b o l t e d on the tube and pressure a p p l i e d to compress the cap m a t e r i a l . A p p r o x i m a t e l y 75 mL of 3A^ were f o r c e d t h r o u g h the column to remove the B A ^ g S o l v e n t i n i t i a l l y present. For p r e p a r a t i v e s e p a r a t i o n s , a 2 mL sample from the r a d i o t r a c e r experiments was loaded on the column by a syringe through the septum ( F i g u r e 3.5). The bottom valve was adjusted to give a d r i p r a t e between 1-2 mL/min. BA^ was used to e l u t e b u t y r a t e , BA.^ was used to remove p r o p i o n a t e and acetate i n that order. w a s u s e c * t o e ^ u t e f o l a t e ahead of l a c t a t e . The f r a c t i o n s were c o l l e c t e d by an automatic sample c o l l e c t o r (SUPERRAC, KLB) i n 20 mL u n i t s . I n o r d e r to t e s t the column a n o n r a d i o a c t i v e standard sample was i n i t i a l l y a p p l i e d to the column and the - 69 - progress of the a c i d s down the column was monitored by t i t r a t i o n w i t h 0.005 N barium hydroxide. 10 mL of the 20 mL f r a c t i o n were p i p e t t e d i n t o a t i t r a t i o n f l a s k , approximately 30 mL of carbon d i o x i d e f r e e water were added and the s o l u t i o n was s t i r r e d m a g n e t i c a l l y f o r 3 minutes w h i l e a stream of carbon d i o x i d e f r e e n i t r o g e n was bubbled through the s o l u t i o n to remove t r a c e s of carbon d i o x i d e . Then the s o l u t i o n was t i t r a t e d w i t h 0.005 N barium hydroxide to the c r e s o l red end p o i n t . A t y p i c a l chromatogram i s shown i n F i g u r e 3.6. 3.7 De te rm ina t i on o f R a d i o a c t i v i t y Sample r a d i o a c t i v i t i e s were determined by l i q u i d s c i n t i l l a t i o n spectrometry. 3 .7 .1 P r i n c i p l e Organic compounds c a l l e d " s c i n t i l l a t o r s " have the property of absorbing r a d i a n t energy e i t h e r i n the s o l i d s t a t e or i n s o l u t i o n . The a b s o r p t i o n of t h i s energy by the s c i n t i l l a t o r r e s u l t s i n the formation of e x c i t e d atoms or molecules that then r e t u r n r a p i d l y to the normal or ground s t a t e , r e l e a s i n g energy as photons ( l i g h t energy) and heat. These s c i n t i l l a t o r s are transparent to t h e i r emitted l i g h t , which i s i n the u l t r a v i o l e t or v i s i b l e range. The number of photons emitted i s approximately l i n e a r l y r e l a t e d to the r a d i a n t energy absorbed. A s e n s t i v e p h o t o m u l t i p l i e r , a vacuum tube that converts photons i n t o e l e c t r i c a l energy, can be used as the d e t e c t o r of the photons. The term " l i q u i d s c i n t i l l a t i o n " counting i s used because these s c i n t i l l a t o r s are u s u a l l y d i s s o l v e d i n a s u i t a b l e s o l v e n t c o n t a i n i n g the ^4 O r ao 40 60 ao FRACTION NUMBER (20ml advent/fraction). - r 60 "1 70 Figure 3.6 Organic ac i d s chromatogram: (1) b u t y r a t e , (2) p r o p i o n a t e , (3) a c e t a t e , (4) l a c t a t e . - 71 - r a d i o a c t i v e m a t e r i a l to be assayed. The photon production from t h i s s o l u t i o n r e s u l t s from the f o l l o w i n g sequence of events. The energy of the 8 - p a r t i c l e emitted from a r a d i o a c t i v e source i s f i r s t absorbed by the primary solvent molecules (eg. Toluene), causing them to become e x c i t e d . This e x c i t a t i o n , high frequency energy, i s propagated w i t h i n the solvent and t r a n s f e r r e d to the primary s c i n t i l l a t o r (eg. 2, 5 - D i p h e n y l o x a z o l e ) , causing the s c i n t i l l a t o r molecules to become e x c i t e d . When they r e t u r n to t h e i r ground s t a t e they emit l i g h t at frequencies lower than that at which energy i s t r a n s f e r r e d to them. This lower frequency l i g h t i s propagated by a secondary s o l v e n t to a secondary s c i n t i l l a t o r (eg. 1, 1', 4, 4' - tet r a - p h e n y l b u t a d i e n e ) which emits even lower frequency l i g h t that i s d e t e c t a b l e by a p h o t o m u l t i p l i e r tube. A number of commerical ' s c i n t i l l a t i o n c o c k t a i l s ' (eg. B i o f l u o r (NEW ENGLAND), I n s t a g e l (PACKARD)) c o n t a i n i n g a s o l v e n t , a primary and secondary s c i n t i l l a t o r have been a v a i l a b l e f o r some time. For more d e t a i l s the reader i s r e f e r r e d to an e x c e l l e n t t r e a t i s e on the subject by Kobayashi and Maudsley (1974) . Quenching i s a term a p p l i e d to any f a c t o r that reduces the l i g h t output (photon production) i n the system. Quenching can occur i n s e v e r a l ways: the sample i t s e l f may absorb l i g h t given o f f by the s c i n t i l l a t o r or some of i t s r a d i a t i o n ; the solvent may not t r a n s f e r the energy of the 8 - p a r t i c l e e f f i c i e n t l y to the s c i n t i l l a t o r ; the s c i n t i l l a t o r i t s e l f may absorb some of i t s f l u o r e s c e n c e ; or chemical i n t e r a c t i o n of the components contained i n the counting s o l u t i o n may r e s u l t i n reduced photon output. The determination of quenching i s synonymous w i t h the d e t e r m i n a t i o n of the sample counting e f f i c i e n c y . The most common methods used to determine sample counting e f f i c i e n c y or the methods used to c o r r e c t f o r the l o s s of d e t e c t a b l e - 72 - a c t i v i t y by quenching are: the i n t e r n a l standard method; the e x t e r n a l standard method; and the channels r a t i o method. The channels r a t i o method was employed i n t h i s study and i t s t h e o r e t i c a l b a s i s i s given below. 3.7.2 Channels Ratio Method This method i s based on the f a c t that the pulse height spectrum i s always d i s p l a c e d when quenching occurs. In a two channel instrument 14 a s s a y i n g C, i t i s p o s s i b l e to s e t c h a n n e l A to i n c l u d e a l l 8 - p a r t i c l e s having energies from 0 - 156 KeV, which would be e q u i v a l e n t to a counting e f f i c i e n c y of 100% (see Figure 3.7). Channel 8 can be set to count a l l 8 - p a r t i c l e s having energies from 0 - 5 0 KeV, which would be e q u i v a l e n t to counting e f f i c i e n c y of 50%. I f the channel r a t i o i s a r b t r a r i l y d e fined as the narrow-window e f f i c i e n c y d i v i d e d by the wide window e f f i c i e n c y , the r a t i o i s always a number l e s s than one. Non-quenched samples c o n t a i n i n g 14 v a r i o u s amounts of C m a t e r i a l would have a channels r a t i o n A/B of 0.5. The absolute a c t i v i t y , d i s i n t e g r a t i o n s pec minute (dpm), of each sample i s then obtained by d i v i d i n g the net counts, counts per minute (cpm) appearing i n the narrow window by the counting e f f i c i e n c y determined from the r a t i o - e f f i c i e n c y curve. An e f f i c i e n c y or quenching curve i s u s u a l l y prepared by counting a s e r i e s of d i f f e r e n t l y quenched standards whose a c t u a l a c t i v i t i e s are known and then p l o t t i n g the e f f i c i e n c y a g ainst the channels r a t i o . 3.7.3 Procedure A P h i l i p s PW 4700 l i q u i d s c i n t i l l a t i o n counter (PHILIPS) was used throughout t h i s study. Commercially a v a i l a b l e quenched standards were used - 73 - keV Figure 3.7 s p e c t r a : the s o l i d curve represents unquenched 14 , , 14„ . , C, the dashed curve represents quenched C, A and B represent v a r i o u s window widths. 100 5 z IU i IU go- t o 7 0 - oo- s o - - r 0.4 0.0 0.0 CHANNELS RATIO (A/B). Figure 3.8 E f f i c i e n c y curve prepared from commercial quenched standards. - 74 - to generate the e f f i c i e n c y curve shown i n Figure 3 .8 . One mL from each l i q u i d chromatography f r a c t i o n was p i p e t t e d i n t o a s c i n t i l l a t i o n v i a l . F i v e mL of i n s t a - g e l (PACKARD) c o c k t a i l were added and shaken w e l l . A f t e r accumulating a number of v i a l s , counting was done ov e r n i g h t . Each v i a l was counted f o r f i v e minutes. Peaks of r a d i o a c t i v i t y were detected and i d e n t i f i e d by comparison with the e l u t i o n of standards. IV. RESULTS AND DISCUSSION A l l the experiments conducted i n t h i s study were d i v i d e d i n t o f i v e c a t e g o r i e s : (1) A - s e r i e s experiments were run at an approximately f i x e d D of 0.05 h and temperature, of 35°C. The pH was v a r i e d from 4.0 and 6.5 at i n t e r v a l s of 0.5. The r e s u l t s of these experiments were employed to determine two p o t e n t i a l l y o p t i m a l , pH l e v e l s (4.5 & 6.0) at which f u r t h e r e xperimentation was conducted. A paper based on these experiments has been accepted f o r p u b l i c a t i o n i n "Biotechnology and B i o e n g i n e e r i n g " ( K i s a a l i t a , e t . a l . 1986); (2) B - s e r i e s experiments were run at a pH of 6.0, but t h i s time D was v a r i e d from 0.05 to 0.6 h 1 at approximate i n t e r v a l s of 0.05 h 1 . The r e s u l t s from these experiments were employed to speculate on various p o s s i b l e paths f o r carbon flow from pyruvate to the var i o u s acidogenic end products; (3) As a r e s u l t of organic aci d s d i s t r i b u t i o n found w i t h respect to D; from B - s e r i e s r e s u l t s , three d i l u t i o n r a t e ranges (D < 0.15, 0.15 < D < 0.4, D > 0.4) r e p r e s e n t i n g d i f f e r e n t organic a c i d combinations were s t u d i e d . C - s e r i e s experiments, i n v o l v i n g the use of a number of r a d i o t r a c e r s were conducted i n the f i r s t two regions to d i s c r i m i n a t e between the r i v a l degradation patt e r n s proposed with the help of the r e s u l t s of the B- s e r i e s experiments; (4) D-series experiments were run i n a s i m i l a r f a s h i o n to B - s e r i e s w i t h the exception that the pH was lower (pH 4.5). The r e s u l t s i n t h i s experimental phase together w i t h those i n A were u s e f u l i n determining the i n f l u e n c e of pH on the l a c t o s e degradation model; (5) The s u b s t r a t e used i n a l l the above experimental s e r i e s was only l a c t o s e growth l i m i t e d ( d e s c r i b e d e a r l i e r ) . In E - s e r i e s experiments a l a c t o s e plus 8 - l a c t o g l o b u l i n b a s e d s u b s t r a t e w a s e m p l o y e d . - 76 - The r e s u l t s from the E - s e r i e s have helped i n determining that apparently degrading p r o t e i n w i t h l a c t o s e had no i n f l u e n c e on the l a c t o s e fermentation model. A l l the t a b u l a t e d r e s u l t s are presented i n Appendix B. The f i r s t l e t t e r i n each run number i d e n t i f i e s the experimental phase to which the run belongs. Tables B l , B2, B3 and B4 show the carbon mass balance and Table B5 shows the gas p r o d u c t i o n f o r a l l experiments conducted i n t h i s study, as a f u n c t i o n of experimental cumulative time. The r e s u l t s are presented i n the chapter In an order s l i g h t l y d i f f e r e n t from t h a t i n which they were conducted. F i r s t the i n f l u e n c e of D on organic a c i d and gaseous product d i s t r i b u t i o n s i s considered. Then the r e s u l t s of the r a d i o t r a c e r s t u d i e s are employed to determine the o p e r a t i v e degradation model f o r l a c t o s e . Next the i n f l u e n c e of p r o t e i n and pH on the fermentation model are considered and f i n a l l y the a c i d o g e n i c m i c r o b i a l growth model i s presented. 4.1 I n f l u e n c e Of D i l u t i o n Rate On Organ ic A c i d s (OA) D i s t r i b u t i o n The r e s u l t s of the OA analyses f o r l a c t o s e growth l i m i t e d experiments at a temperature of 35°C and pH of 6.0 are presented i n Table B l (Appendix B ) . For the seven b a s i c experiments and t h e i r r e p l i c a t e s , an e f f o r t was made to mai n t a i n the same temperature and pH, so that the only v a r i a b l e was D. Only one n e u t r a l product, e t h a n o l , was found. The organic a c i d s i n c l u d e d a c e t a t e , propionate, i s o - and normal-butyrate, i s o - and n o r m a l - v a l e r a t e , caproate, l a c t a t e and formate. According to Hobson e t . a l . ( 1 9 7 4 ) , t h e s e can be c o n s i d e r e d normal p r o d u c t s of c a r b o h y d r a t e f e r m e n t a t i o n . Complete a c i d i f i c a t i o n of l a c t o s e was achieved up to D of - 77 - .46 h , beyond w h i c h , d e t e c t a b l e amounts of l a c t o s e were observed. A c a r b o n r e c o v e r y l e s s t h a n 50% was o b s e r v e d at D = .64 h ^ w h i c h was a t t r i b u t e d to b a c t e r i a l washout. Carbon r e c o v e r i e s f o r complete l a c t o s e c o n v e r s i o n were equal to or b e t t e r than 86%. In two analyses, the recovery was found to be l a r g e r than 105%. In the f i r s t case the unreasonably l a r g e recovery f i g u r e was a t t r i b u t e d to an e r r o r i n the measurement of biomass carbon and i n the second case a sample d i l u t i o n e r r o r was suspect. In a l l experiments, formate, i - b u t y r a t e and i - v a l e r a t e were detected, i f at a l l , i n t r a c e amounts. A l s o e t h a n o l , n - v a l e r a t e and caproate were found to be minor products. The major products whose c o n c e n t r a t i o n s s t r o n g l y depended on D were a c e t a t e , propionate, n-butyrate and l a c t a t e . In F i g u r e 4.1, the averaged c o n c e n t r a t i o n s of the major OA are presented as f u n c t i o n s of D. Three D ranges can be employed to c h a r a c t e r i s e the r e l a t i o n s h i p between OA c o n c e n t r a t i o n s and D. The f i r s t D range (D < 0.15) i s c h a r a c t e r i s e d by high acetate and no d e t e c t a b l e l a c t a t e . The second D range (0.15 < D < 0.4) i s c h a r a c t e r i s e d by the f a l l and r i s e of acetate and l a c t a t e c o n c e n t r a t i o n s r e s p e c t i v e l y . The t h i r d D range (D > 0.4) i s c h a r a c t e r i s e d by l e s s than 100% l a c t o s e acidogenesis and a p a r t i a l b a c t e r i a l washout, hence the drop i n a l l OA c o n c e n t r a t i o n s . The appearance and subsequent i n c r e a s e of l a c t a t e c o n c e n t r a t i o n w i t h i n c r e a s i n g D i n t h i s study i s i n agreement w i t h the f i n d i n g s of a number of i n v e s t i g a t i o n s , where pure c u l t u r e s i s o l a t e d from g a s t r o i n t e s t i n a l ecosystems were the agents of carbohydrate a c i d o g e n e s i s . For example at low growth r a t e s ( e q u i v a l e n t to low D i n continuous c u l t u r e ) the rumen bacterium Selenomas ruminatium produces almost e n t i r e l y a c e t a t e and propionate, whereas at high growth r a t e s the fermentation products of glucose are about c re • h-1 T3 '-3 i-i i-i O O T3 Cu H- c O n 3 rr 03 rr a . rs » rr M n r* *—* a* c rr 01 H- o O rr 3 0i rr CD ># 01 a t-h N—^ C 3 tr o c rr rr H- V! O M 3 OJ rr O M l * Cu H> M* C rr o 3 M, ul rr ro 01 rr 01 SC of • o 01 o CD rr 01 rr fD 03 ORGANIC ACID CONCENTRATION (ugC/mL). — -» M I ORGANIC ACIO CONCENTRATION (tigC/mL). ORGANIC ACID CONCENTRATION (ugC/mL). ORGANIC ACID CONCENTRATION (ugC/mL). - 8L - - 79 - 50% l a c t a t e , and 50% acetate and propionate (Hobson, 1965). A l s o Hishinuma e t . a l . (1968) showed that batch c u l t u r e s of Sj_ ruminantium w i t h high i n i t i a l glucose c o n c e n t r a t i o n s ( e q u i v a l e n t to high D i n continuous c u l t u r e ) produced a high p r o p o r t i o n of l a c t a t e and at low glucose c o n c e n t r a t i o n s gave low l a c t a t e w i t h high acetate and propionate c o n c e n t r a t i o n s . L a c t o b a c i l l u s c a s e i i n continuous c u l t u r e produced a high p r o p o r t i o n of l a c t a t e only at high growth r a t e s (De V r i e s e t . a l . , 1970). A number of i n v e s t i g a t i o n s employing mixed c u l t u r e s i n man-made s i n g l e or two-phase fermentors have reported f i n d i n g s i n q u a n t i t a t i v e agreement w i t h the r e s u l t s from t h i s study. For example, i n m u n i c i p a l and animal waste a n a e r o b i o s i s , appearance of l a c t a t e has been a s s o c i a t e d w i t h shock l o a d i n g (Mahr, 1969). A l s o i n a p i l o t i n s t a l l a t i o n (126L) a c i d i f y i n g waste water from a sugar r e f i n e r y , Zeotemeyer e t . a l . (1982a) found that at the s h o r t e s t residence time (high D) the r a t e of l a c t a t e p r oduction exceeded th a t of u t i l i z a t i o n . In e x p l a i n i n g t h e i r f i n d i n g s , Zeotemeyer and h i s c o l l a b o r a t o r s a s s e r t e d that s i n c e the formation of l a c t a t e i s l e s s f a v o u r a b l e from the p o i n t of view of energy (2 mol.ATP/mol.glucose), and a l s o s i n c e the conver s i o n of pyruvate to l a c t a t e i s known to proceed v i a one enzyme ( l a c t a t e dehydrogenase) and t h e r e f o r e p o s s i b l y occurs at a higher r a t e (or i t r e q u i r e s a lower b i o s y n t h e t i c a c t i v i t y of the c e l l ) , l a c t a t e f o r m a t i o n o f f e r s a s o l u t i o n f o r removal of excess r e d u c t i o n e q u i v a l e n t s . In other words micro-organisms r e q u i r e f o r growth as much energy as p o s s i b l e per u n i t time. The highest p o s s i b l e energy conversion i s necessary when there i s a low supply of s u b s t r a t e , which would t r a n s l a t e i n t o formation of VFA. But when there i s a l a r g e supply of s u b s t r a t e , formation of l a c t a t e i s - 80 - not the best route from the energy view po i n t but the q u i c k e s t route w i t h a low e f f i c i e n c y . The production of l a c t a t e at high growth r a t e s i n pure c u l t u r e s l e d Hobson e t . a l . (1974) to conclude that the growth r a t e of b a c t e r i a can a f f e c t fermentation products and s p e c i f i c c o n d i t i o n s of low s u b s t r a t e c o n c e n t r a t i o n or low growth r a t e are not conducive to the formation of l a c t a t e . However Hobson and h i s c o l l a b o r a t o r s were cauti o u s i n equating the mixed substrate/mixed c u l t u r e i n an anaerobic fermentor, where b a c t e r i a may be growing on d i f f e r e n t s u b s t r a t e s at d i f f e r e n t growth r a t e s and where some b a c t e r i a may be dependent on others f o r s u b s t r a t e s or growth f a c t o r s , w i t h pure c u l t u r e s under de f i n e d l a b o r a t o r y c o n d i t i o n s . L a c t a t e i n b a c t e r i a l fermentations i s g e n e r a l l y b e l i e v e d to be mainly broken down to propionate ( G o t t s c h a l k , 1979), f o r l a c t a t e i s known to be the p r e f e r r e d s u b s t r a t e f o r propionate-forming b a c t e r i a . I f t h i s were the case, a high c o n c e n t r a t i o n of propionate under low growth c o n d i t i o n s would suggest that l a c t a t e i s produced under a l l growth c o n d i t i o n s , and at low growth c o n d i t i o n s i t i s converted to propionate. On the c o n t r a r y ( F i g u r e 4.IB) propionate was o b s e r v e d to i n c r e a s e w i t h i n c r e a s i n g D i n the D range up to 0 - 0.15 h \ which i m p l i e s that at low growth c o n d i t i o n s carbon flow from pyruvate may be d i r e c t e d towards i n t e r m e d i a r y m e t a b o l i t e s other than l a c t a t e as suggested by Zeotemeyer and co-workers (1982a). This a s s e r t i o n could e a s i l y be confirmed by the use of a u n i f o r m l y r a d i o a c t i v e l a c t a t e t r a c e r . Under low growth r a t e c o n d i t i o n s , s i n c e no l a c t a t e i s produced, the b a c t e r i a would not be expected to have the c a p a b i l i t y to degrade l a c t a t e i n t o any other m e t a b o l i t e . I f a - 81 - l a c t a t e t r a c e r i s administered under batch c o n d i t i o n s f o r a reasonably short p e r i o d of time, the b a c t e r i a would be expected to convert a very small amount of the t r a c e r to propionate. On the other hand, based on the r e s u l t s p l o t t e d i n F i g u r e s 4.1A and 4.1C, i t was considered a p o s s i b i l i t y that l a c t a t e i s produced under a l l growth c o n d i t i o n s , but at low b a c t e r i a l growth r a t e s i t i s converted mainly to a c e t a t e . Independent evidence to support t h i s p o s s i b i l i t y was published by Nakamura and Takahashi (1971) f o r a n a e r o b i o s i s i n a g a s t r o i n t e s t i n a l ecosystem. Fresh rumen contents of sheep taken at v a r i o u s periods a f t e r 1 4 f e e d i n g , w e r e i n c u b a t e d i_n v i t r o w i t h ( C ( 2 ) ) - l a c t a t e . The r a d i o - a c t i v i t i e s of the consumed l a c t a t e were predominately found i n a c e t a t e , i n most cases, but a l s o i n propionate when the pool s i z e of l a c t a t e was l a r g e . I t was found that the percentage of l a c t a t e r a d i o a c t i v i t y that was found i n acetate depended s t r o n g l y on the type of feed. A l s o Counotte (1981) i n d i c a t e d that once l a c t a t e i s formed i n mixed rumen contents i t can be fermented mainly to acetate i f methanogenesis occurs. The evidence s u p p o r t i n g the l a c t a t e / a c e t a t e conversion c i t e d above was generated from g a s t r o i n t e s t i n a l a n a e r o b i o s i s . No evidence was found from fermentor m i c r o b i a l p o p u l a t i o n s w i t h the e x c e p t i o n of the two recent papers by C h a t r a i n and Zeikus (1986a & b) c i t e d i n the previous chapter. The q u e s t i o n of the source of b u t y r a t e needed a l s o to be examined before running r a d i o t r a c e r experiments to d i s c r i m i n a t e between the two r i v a l hypotheses. G e n e r a l l y only o b l i g a t e anaerobes form b u t y r a t e as a main fermentation product. They belong to the four genera, C l o s t r i d i u m , B u t y r i u b r i o , Subacterium and Fusobacterium. The mechanism of b u t y r a t e f o r m a t i o n was not w e l l understood u n t i l Baker and h i s c o l l a b o r a t o r s i n 1956 - 82 - d i d t h e i r p i o n e e r i n g s t u d i e s on Cj_ k l u y v e r i . The C l o s t r i d i a are known to employ the EMP pathway f o r the degradation of hexoses to pyruvate ( G o t t s c h a l k , 1979). This might suggest that the conversion of pyruvate to b u t y r a t e would go on i n p a r a l l e l w i t h that of other organic a c i d s w i t h of course the e x c e p t i o n of a c e t a t e . On the other hand fermentation of l a c t a t e to b u tyrate has been observed (Zeotemeyer e t . a l . , 1982a; Cohen and de Wit, unpublished r e s u l t s ) , r a i s i n g the p o s s i b i l i t y of l a c t a t e being the p r e c u r sor f o r b u t yrate as w e l l . From the f o r e g o i n g , under low growth r a t e c o n d i t i o n s (D < .15 h *) two p o s s i b l e general degradation schemes were p o s t u l a t e d ( F i g u r e 4.2). The main d i f f e r e n c e between model A and model B i s t h a t , w h i l e i n B l a c t a t e goes to a c e t a t e and propionate i n A l a c t a t e only goes to propionate. A back b u t y r a t e to l a c t a t e o x i d a t i v e r e a c t i o n i s i n c l u d e d because Zeotemeyer and co-workers (1982a) o f f e r e d i t as an e x p l a n a t i o n f o r t h e i r observed c o n v e r s i o n of b u t y r a t e from l a c t a t e . 4 . 2 I n f l u e n c e Of D i l u t i o n R a t e On G a s e o u s P r o d u c t s D i s t r i b u t i o n I n i t i a l l y , fermentor steady s t a t e was d e f i n e d as when fermentor performance i n terms of gas p r o d u c t i o n r a t e showed no s i g n i f i c a n t a l t e r a t i o n (±10% w i t h i n a p e r i o d of at l e a s t three d i l u t i o n s ) . As w i t h previous g l u c o s e a c i d o g e n i c experiments (Zeotemeyer e t . a l . , 1982), CO2 and H 2 were expected to be the main gaseous products f o r acidogenic r e a c t i o n s completely separated from methanogenesis. To the s u r p r i s e of the author, i t was found th a t the manner of pH c o n t r o l during fermentor s t a r t - u p was a s i g n i f i c a n t f a c t o r i n d e t e r m i n i n g whether t h e r e would be p r o d u c t i o n of H„ and C0„. - S3 - Acetate Acetate Figure 4.2 Two p o s s i b l e fermentation models of l a c t o s e to pyruvate and pyruvate to various end products: (1) EPM pathway, (2) s u c c i n a t e - propionate pathway, (3) a c r y l a t e pathway, (4) butyrate fermentation. Known enzyme systems that may be operative i n the model are presented i n Appedix C. - 84 - Fo r a s e r i e s of e x p e r i m e n t s run at D = 0.05 h ( A p p e n d i x B, Table B5, A - s e r i e s ) i t was found that i f the pH f e l l below the value of 4.5, during s t a r t - u p , CO2 and would be d e f i n i t e l y produced even at higher o p e r a t i n g pH l e v e l s . In F i g u r e 4.3, a few r e p r e s e n t a t i v e p l o t s of t o t a l gas produced (25°C and one atmosphere) a g a i n s t cumulative run time are presented. Graph 1 a r e the r e s u l t s (D = 0.05 h 1 and pH = 4.5) f o r the case when the pH was allowed to drop below the 4.5 l e v e l during s t a r t - u p . Graph 2 i s f o r an i d e n t i c a l run i n which the pH l e v e l was maintained above or equal to 4.5. Graph 3 i l l u s t r a t e s the gas pro d u c t i o n r e s u l t s f o r a pH of 4.5 and at a hi g h e r D of 0.357 h 1 . At a higher pH l e v e l of 6.0, w i t h c o n t r o l maintained a t the d e s i r e d pH l e v e l throughout the experimental p e r i o d , i t was i n t e r e s t i n g to note that i n almost a l l the experiments at D > .05 h \ gas pr o d u c t i o n e x h i b i t e d an i r r e g u l a r c y c l i c behaviour. One couldn't help being reminded of the c l a s s i c a l Lotka and V o l t e r r a ( L o t k a , 1920) mathematical model f o r predator-prey i n t e r a c t i o n s that produce c y c l e s on p o p u l a t i o n s i z e s . F i g u r e 4.4 i l l u s t r a t e s such gas pro d u c t i o n c y c l e s . I t was n o t i c e d that f o r some experiments the c y c l e s would d i m i n i s h at an e a r l y stage and f o r others they would p e r s i s t f o r longer p e r i o d s . No s i n g l e experimental f a c t o r was i d e n t i f i e d as being r e s p o n s i b l e f o r the time i t took f o r the c y c l e s to d i m i n i s h . C o i n c i d e n t a l l y , Bungay (1985) has i n d i c a t e d that the fre q u e n c i e s and amplitudes of r e a l prey-predator system are e r r a t i c . The e f f e c t of the gas production p a t t e r n on fermentor steady s t a t e was n e g l i g i b l e , s i n c e the carbon f r a c t i o n i n gaseous form i n comparison to the t o t a l recovered carbon mass was almost always very s m a l l (< 3%). As shown above, gas p r o d u c t i o n was found to be an u n r e l i a b l e measure of fermentor steady s t a t e . I n s t e a d , v a r i a t i o n i n the consumption r a t e of the pH Figure 4.3 Three r e p r e s e n t a t i v e p l o t s of gas p r o d u c t i o n : (1) pH dropped below 4.5 d u r i n g s t a r t - u p , run // A 2 a , ( 2 ) pH was maintained at or above a l e v e l of 4.5, run // A 2 b , (3) pH was maintained at or above a l e v e l of 4.5, run # D4. 2 C 600 TIME (h) F i g u r e 4.4 Three r e p r e s e n t a t i v e p l o t s f o r which the pH was maintained at the d e s i r e d value ( PH6.0): (1) D=0.2109 h " 1 , (2) D = 0.2953 h _ 1 , (3) D = 0.1246 h " 1 . - 87 - c o r r e c t i n g NaOH s o l u t i o n was employed f o r the purpose. I t should be pointed out that some authors such as G i r a r d e t . a l . (1986) have i n d i c a t e d that anaerobic r e a c t o r s are seldom at steady s t a t e , because l o c a l i s e d and short i n h i b i t o r y or s t i m u l a t i n g f a c t o r s cause the m i c r o b i a l p o p u l a t i o n to remain i n a "dynamic" steady s t a t e . Therefore, the steady s t a t e s r e f e r r e d to i n continuous a n a e r o b i o s i s have been termed "pseudo-steady s t a t e s " by many. For those experiments (at a pH of 6.0) where there was gas production at steady s t a t e , F i g u r e 4.5 presents the fermentor head space gas a n a l y s i s . I t c a n be s e e n t h a t below D = 0.15 h \ methane was d e t e c t e d . I t i s b e l i e v e d that the methane must have been formed v i a the r e d u c t i v e methane form a t i o n route ( F i g u r e 2.1) f o r a number of reasons: (1) Table 4.1 summarises the i n f o r m a t i o n p r e s e n t l y a v a i l a b l e on the maximum s p e c i f i c growth r a t e (H ) f o r the formation of methane from hydrogen and a c e t a t e . I t i s c l e a r t h a t u f o r a l l o r g a n i s m s t h a t f o r m methane v i a a c e t a t e m d e c a r b o x y l a t i o n i s w e l l below 0.05 h However t h o s e t h a t reduce e x h i b i t u values w e l l i n the D r e g i o n of i n t e r e s t (remember f o r continuous m c u l t u r e D = i i ) ; (2) Above D = 0.15 h 1 ( F i g u r e 4.5) where no methane was d e t e c t e d r e l a t i v e l y h i g h e r c o n c e n t r a t i o n s of H^ and were observed, o b v i a t i n g the f a c t that below D = 0.15 h 1 they were being converted to CH^; (3) pH 6.0 i s known to be the lowest l i m i t f o r acetate d e c a r b o x y l a t i o n i n most known methanogens, making i t u n l i k e l y that the observed methane was formed v i a the a c e t a t e route. The q u e s t i o n that comes to mind i s what r e l a t i o n s h i p might there be, between the changing gas composition and OA d i s t r i b u t i o n both w i t h respect t o D. By comparing F i g u r e s 4.1 and 4.5, i t was found that the t r a n s i t i o n s  Table 4.1 Maximum S p e c i f i c Growth Rate Values For Methanogenic B a c t e r i a S u b s t r a t e Organism u m ( h 1 ) Temperature Reference (°C) C0 2/H 2 A c e t a t e Methanobacterium thermoaceticum 0.69 65 Schonheit e t . a l . (1980) 0.23 65 Zeikus and Wolfe (1972) Methanobrevibacter a r b o r i p h i l u s AZ 0.058 33 Zehnder and Wuhrman (1977) Methanothrix soehngenii 0.0032 33 Zehnder e t . a l . (1980) Methanosarcina b a r k e r ! (227) 0.02-0.03 36 Smith and Mah (1978) (TM-1) 0.06 50 Zin d e r and Mah (1979) (227) 0.0192 35-37 Yang (1984) (MS) 0.0208 <• Yang (1984) Methanosarcina maze! 0.042 35 Mah (1980) •• 0.0224 35-37 Yang (1984) CO - 90 - i n both OA and gas d i s t r i b u t i o n occurred i n the same D range. I t i s t h e r e f o r e s u g g e s t e d t h a t D's i n the n e i g h b o u r h o o d of 0.15 h * represent washout c o n d i t i o n s f o r the u t i l i s i n g b a c t e r i a l group and consequently the absence of t h i s group r e s u l t s i n a t o t a l l y new b a c t e r i a l p o p u l a t i o n , hence the s h i f t i n OA. However t h i s does not answer the q u e s t i o n that arose i n the f i r s t s e c t i o n of t h i s chapter, whether the s h i f t i n p o p u l a t i o n represents the beginning of pyruvate being converted to l a c t a t e or j u s t d i s a b l e s the conversion of l a c t a t e to other i n t e r m e d i a r y m e t a b o l i t e s , r e s u l t i n g i n the higher observed l a c t a t e c o n c e n t r a t i o n . The answer to t h i s e quation i s d e a l t w i t h i n the next two s e c t i o n s . 4.3 Radiotracer Studies The methods employed to i n c o r p o r a t e the r a d i o t r a c e r s were d e s c r i b e d i n Chapter I I . The value of the technique was f i r s t a p p reciated by Zuntz e t . a l . (1913) and h i s student, Markoff (1913). They employed i t to study rumen m i c r o b i a l f u n c t i o n s . M a r k o f f s experiments are p a r t i c u l a r l y noteworthy because of the e f f o r t made to conduct in v i t r o fermentations i n such a f a s h i o n that q u a n t i t a t i v e r e s u l t s a p p l i c a b l e to the rumen were obtained. The theory behind the technique i s that when a sample of the fermentor contents i s removed, i t continues to f u n c t i o n as i t d i d i n the fermentor u n t i l accumulation of fermentation products, exhaustion of s u b s t r a t e , a v a i l a b i l i t y of new s u b s t r a t e or any other f a c t o r causes i t to change. The onset of the change can be delayed f u r t h e r i f the major fermentor c o n d i t i o n s such as pH and temperature are maintained at comparable l e v e l s i n the new environment. The batch r a d i o t r a c e r experiments, the r e s u l t s from which are r e p o r t e d below, were run, at l o n g e s t , f o r 1,800 seconds. For b i o l o g i c a l - 91 - s y s t e m s , R o e l s (1983) has d e f i n e d r e l a x a t i o n time, t , as the time, which R elapses before the d i f f e r e n c e between a s t a t e value of the system and the i n i t i a l value reaches a f r a c t i o n (1 - 1/e) of the d i f f e r e n c e between the o l d and new steady s t a t e v a l u e s . I n Figure 4.6 a d a p t a t i o n mechanisms and the o r d e r s of magnitude of t h e i r t a r e shown. Since the batch experimental t i m e of 1,800 seconds l i e s at the lower end of the t r e g i o n f o r changes i n enzymic c o n c e n t r a t i o n s , i t was considered to be too short a p e r i o d to have any e f f e c t on the fermentation model d e r i v e d from the r a d i o t r a c e r r e s u l t s . 4.3.1 Results Of The Radiotracer Experiments At High D A procedure f o r r a d i o t r a c e r i n c o r p o r a t i o n d e s c r i b e d i n Chapter I I I was f o l l o w e d to produce a two mL sample that was a p p l i e d to the OA s e p a r a t i o n column a l s o d e s c r i b e d i n Chapter I I I . F i g u r e 4.7 ( f o r Run # C4) i s a t y p i c a l radiochromatogram. B u t y r a t e was e l u t e d w i t h i n the f i r s t ten f r a c t i o n s by BA^, s o l v e n t . A s m a l l amount of propionate was e l u t e d by BA^, as shown by the peak between the 11th and 20th f r a c t i o n . Complete e l u t i o n o f p r o p i o n a t e and a c e t a t e were achieved w i t h BÂ ,. up to the 40th f r a c t i o n , a f t e r which BA^Q was administered to e l u t e l a c t a t e . The second l a c t a t e peak i s a r e s u l t of changing from BA^Q to BA^Q, a stronger s o l v e n t , at the 80th f r a c t i o n so that a l l the l a c t a t e remaining on the column, that would otherwise t a i l badly, was speeded down the column. The r a d i o a c t i v i t y f o r each product f o r each of the four experiments done was summed up to c a l c u l a t e the percentage a c t i v i t y recovery and the recovered a c t i v i t y d i s t r i b u t i o n among the v a r i o u s products. The r e s u l t s f o r the four experiments conducted at high D (D > 0.15) are summarised i n Table 4.2 A l l but one of the experiments were c a r r i e d out i n the small (20 mL) batch - 92 - 10"* TO" 5 1CT4 X T 3 10" 2T0"' O * 1 X 3 ' J IO* 3 X3' 4 X7 5 T O * 6 1 1 1 1 1 1 1 1 1 1 1 1 1 RELAX TON TIME Moss action law 10° (SECONDS) _ Ailosteric controls _ Changes m enzymic Concentrations m-RNA Selection within A control Population ol ore or more species Evolutionary Changes Figure 4 .6 Adaptation mechanisms i n organisms and the orders of magnitude of t h e i r r e l a x a t i o n times. Figure 4.7 Radiochromatogram f o r run number C4: (1) b u t y r a t e , (2) pro- pionate, (3) a c e t a t e , (4) l a c t a t e . Tracer was [14c(U)]-lactate. Table 4.2 Radio Act ivi ty Distribution for High D Radio Tracer Experiments RUN It TRACER RUN TIME (Min) REACTOR TYPE TOTAL TRACER ACTIVITY (dpm) TOTAL RECOVERED TRACER ACTIVITY (dpm) REC0\ /ERED ACTIVIT) f DISTRIBU1 riON PARENT RUN It PARENT RUN D ( h _ 1 ) Butyrate Propionate Acetate Lactate C7c ( U C(U)]-Butyrate 30 S 1 394,130 386,576 98.1% 381,420 98.7% — 5,156 1.3% — B3c 0.2075 C2 L-[ 1 4 C(U)]-Lactate 30 S 359,028 244,048 68% 1,120 0.4% 21,200 8.7% 16,992 7.0% 204,736 83.9% B3c 0.2075 C3 L-[ 1 4 C(U)]-Lactate 30 s 319,810 257,880 80.6% 1,960 0.8% 18,640 7.2% 16,520 6.4% 220.760 85.6% B3c 0.2131 C4 L-[ 1 4 C(U)]-Lactate 30 L 2 455,808 369,520 81.1% 1,620 0.4% 23,520 6.4% 33,200 9.0% 311,180 84.2% B3c 0.2131 S m a l l b a t c h r e a c t o r . L a r g e b a t c h r e a c t o r w i t h p H c o n t r o l . - 95 - r e a c t o r , d e s c r i b e d i n Chapter I I I , S e c t i o n 3.5.2. Since pH was known to have an i n f l u e n c e on product d i s t r i b u t i o n , i t was feared that running a batch r e a c t o r without pH c o n t r o l f o r 30 minutes might a f f e c t the r e s u l t s . To i n v e s t i g a t e the i n f l u e n c e of not c o n t r o l l i n g the pH a l a r g e r r e a c t o r (60 mL) described i n Chapter I I I , S e c t i o n 3.5.1 was run i n a manner s i m i l a r to that used w i t h the 20 mL r e a c t o r and the pH was monitored but not c o n t r o l l e d . F i g u r e 4.8 shows the r e s u l t a n t pH-time r e l a t i o n s h i p . In 30 minutes the pH increased from an i n i t i a l value of 6.06 to 6.54. A comparison of r a d i o a c t i v i t y d i s t r i b u t i o n f o r a run w i t h and without pH c o n t r o l was found necessary to d i s p e l l any m i s g i v i n g s about the e f f e c t s of pH change i n the sm a l l r e a c t o r . Runs number C2 w i t h pH c o n t r o l and C4 without pH c o n t r o l gave r e s u l t s that were considered c l o s e enough to make pH c o n t r o l unnecessary. The r e s t of the r a d i o t r a c e r experiments were c a r r i e d out i n the sm a l l batch r e a c t o r without pH c o n t r o l . Run number C3 was made to demonstrate the r e p l i c a b i l i t y of the experiments. As shown by the combined r e s u l t s of runs number C2, C3 and C4, approximately 15% of the r a d i o a c t i v e l a c t a t e was converted w i t h a r a d i o - a c t i v e product d i s t r i b u t i o n of 0.5% b u t y r a t e , 7.3% propionate and 7.3% a c e t a t e . The low conversion of l a c t a t e Is not s u p r i s i n g s i n c e at high D l a r g e c o n c e n t r a t i o n s of l a c t a t e i n the fermentor were observed, i n d i c a t i n g the i n a b i l i t y of the p o p u l a t i o n to c a t a b o l i s e i t . The bu t y r a t e t r a c e r was recovered w i t h a n e g l i g i b l y s m a l l c o n v e r s i o n to acetate ( 1 . 3 % ) . 4.3.2 Results Of The Radiotracer Experiments At Low D F i g u r e 4.9 presents a t y p i c a l radiochromatogram. Almost a l l the r a d i o a c t i v e l a c t a t e ended up as acetate w i t h minimal q u a n t i t i e s ending up as bu t y r a t e and propionate. The r e s u l t s f o r a l l the runs are summarised TIME (min). Figure 4.8 pH - time r e l a t i o n s h i p f o r the batch r a d i o t r a c e r i n c o p o r a t i o n r e a c t o r experiment, without pH c o n t r o l . 3500 0 50 100 150 200 FRACTION NUMBER (20ml solvent/fraction). Figure 4.9 Radiochromatogram f o r run number C l l : (1) b u t y r a t e , (2) propionate, (3) acetate. Tracer was [ ^ 4 C ( U ) ] - l a c t a t e . - 98 - i n Table 4.3. For the three t r a c e r s employed the batch experimental time was v a r i e d . In Figure 4.10 the conversion of butyrate to acetate i s shown. I t was found to be l i n e a r over the range of time considered. In a p e r i o d of 15 minutes only approximately 15% of the butyrate had been converted to a c e t a t e . The m i c r o b i a l p o p u l a t i o n at low D was found to possess no c a p a b i l i t y f o r c o n v e r t i n g propionate. C o i n c i d e n t a l l y , of a l l the four major OA; propionate was the one found i n l e a s t amounts. This suggest that both the s u c c i n a t e - p r o p i o n a t e and the a c r y l a t e pathways ( F i g u r e 4.2) probably p l a y minor r o l e s . The f a c t that nothing happended to the propionate t r a c e r a l s o precluded the p o s s i b i l i t y of propionate being a major i n t e r m e d i a t e product whose c o n c e n t r a t i o n was observed to be low because i t was being c a t a b o l i s e d . Approximately 85% of the l a c t a t e was converted to acetate i n a very r a p i d r e a c t i o n , almost too f a s t f o r the r a t e to be measurable by the t e c h n i q u e employed i n t h i s s t u d y (see F i g u r e 4.11). N e g l i g i b l e r a d i o a c t i v i t y amounts ended up as b u t y r a t e ( S J3%) and small amounts as propionate (a>12%) . Th i s f i n d i n g not only d i s c r i m i n a t e s between the r i v a l f e rmentation models proposed ( F i g u r e 4.2) i n favour of some form of model B, but a l s o puts the q u e s t i o n that arose i n S e c t i o n 4.1 to r e s t . Indeed the s h i f t i n OA observed In the neighbourhood of D = .15 h 1 , i s not a s h i f t i n carbon flow v i a pyruvate, from VFA p r o d u c t i o n to l a c t a t e p r o d u c t i o n , but an i n a b i l i t y of the b a c t e r i a l p o p u l a t i o n to c a t a b o l i s e l a c t a t e w i t h the concomintant production of a c e t a t e . A l s o , the i n a b i l i t y of the b a c t e r i a l p o p u l a t i o n to c a t a b o l i s e l a c t a t e i s probably a consequence of a b a c t e r i a l Table 4.3 Radio Act ivi ty Distribution for Low D Radio Tracer Experiments RUN II TRACER RUN TIME (Min) REACTOR TYPE TOTAL TRACER ACTIVITY (dpm) TOTAL RECOVERED TRACER ACTIVITY (dpm) REC0\ /ERED ACTIVITl t DISTRIBU' riON PARENT RUN II PARENT RUN , D ( h - 1 ) Butyrate Propionate Acetate Lactate C14a [ U C(U)]-Butyrate 5 S 1 452,240 473,300 104.72 453,080 95.72 — 20,220 4.3% — B2e 0.0947 C15a [ 1 AC(U)]-Butyrate 15 S 447,134 419,060 93.72 357,500 85.3% — 61,560 14.7% — B2e 0.0947 C16 [ 1 AC(2)]-Propionate 5 s 575,880 520,460 90.42 — 520,460 1002 — — B2e 0.1026 C17 [ 1 4C(2)]-Propionate 15 s 560,662 561,240 100.12 — 561,240 1002 — — B2e 0.1026 C19 L-[ 1 A C(U)]-Lactate 1 s 256,438 202,914 79.12 20 02 14,354 7.12 172,260 84.92 16,280 8.02 B2e 0.0947 C l l L-[ 1 A C(U)]-Lactate 5 s 348,336 224,640 64.72 10,660 4.72 17,120 7.62 194,900 86.8% 1,960 0.92 B2d 0.1083 C12 L-[ 1 A C(U)]-Lactate 15 s 320,796 274,580 85.62 8,360 3.02 46,100 16.82 220,120 80.2% — B2d 0.1083 C13 L-[ 1 A C(U)]-Lactate 30 s 311,740 246,436 79.12 7,920 3.22 29,100 11.82 209,416 85.02 — B2d 0.1083 Small batch reactor. Figure 4.10 Recovered r a d i o a c t i v i t y d i s t r i b u t i o n from [ l ^ C ( U ) ] - b u t y r a t e t r a c e r at v a r i o u s batch experiment times: (A) a c e t a t e , (x) b u t y r a t e . 120 100 >- o < o 5 < CC Q U J OC U J > O O 111 tr 80 60 40 20- r • Q - —r- 10 I I-1 o M I 15 T I M E (min) . 20 25 30 Figure 4 .11 Recovered r a d i o a c t i v i t y d i s t r i b u t i o n from [^ 4C(U)]-lactate t r a c e r at various batch experimet times: ([g) l a c t a t e , (•) acetate, (x) propionate, (A) butyrate. - 102 - p o p u l a t i o n a d j u s t m e n t due to the wash out of the u t i l i s i n g methanogens ( F i g u r e 4.5, CH^ c o n c e n t r a t i o n dropped to z e r o between D of 0.1 and .2 h " 1 ) . 4 . 4 Proposed L a c t o s e A c i d o g e n i c Fe rmen ta t i on Model In a l l r a d i o t r a c e r experiments, where l a c t a t e was the r a d i o t r a c e r , a s m a l l amount of r a d i o a c t i v e butyrate (< 4.7% of recovered a c t i v i t y ) was found. As i n t i m a t e d by Cohen (1981), t h i s meagre b u t y r a t e c o n c e n t r a t i o n , whose o r i g i n must have been l a c t a t e , was a t t r i b u t e d to an extremely slow back pyruvate o x i d a t i v e r e a c t i o n as shown i n F i g u r e 4.2A & B. This precludes the p o s s i b i l i t y of l a c t a t e being the precursor f o r b u t y r a t e and t h e r e f o r e confirms the p a r a l l e l nature of carbon flow between bu t y r a t e and the other organic a c i d s , w i t h of course, the e x c e p t i o n of acetate and propionate that i s s y n t h e s i s e d v i a the a c r y l a t e pathway (see F i g u r e 4.2). Before a l a c t o s e fermentation model i s proposed, based on the r e s u l t s i n t h i s study i t i s f i t t i n g to put i n p e r s p e c t i v e the r e l a t i o n s h i p between the f i n d i n g s i n t h i s study and those of some previous authors and the m i c r o b i a l p o p u l a t i o n s group i n t e r a c t i o n s mentioned i n Chapter I I . For t h i s task i n a d d i t i o n to the r e s u l t s i n t h i s study, only the work of C h a r t r a i n and Zeikus (1986a & b) was found to be s p e c i f i c a l l y r e l e v a n t . Using a methodology s i m i l a r to the one d e s c r i b e d i n t h i s study ( w i t h minor v a r i a t i o n s ) f o r r a d i o l a b e l e d l a c t a t e i n c o r p o r a t i o n , C h a r t r a i n and Zeikus proposed models f o r the f a t e of carbon i n samples from a single-phase fermentor run at D = .01 h \ a c o n t r o l l e d pH of 7.1 ± .01 and a temperature - 103 - of 37 ± 1°C. Some of t h e i r r e s u l t s are presented i n Figure 4.12. On the b a s i s of these r e s u l t s and t h e i r m i c r o b i a l enumeration, i s o l a t i o n and gen e r a l c h a r a c t e r i z a t i o n s t u d i e s a l a c t o s e fermentation model ( F i g u r e 4.13) was proposed. C h a r t r a i n and Zeikus's work has been considered to be v a l i d f o r a s i n g l e phase process, and t h i s d i c t a t e s the range of D over which t h e i r f i n d i n g s c o u l d be a p p l i e d ( i . e . D < 0.0333 h _ 1 ) . The v a l u e of .0333 h " 1 (see S e c t i o n 2.3.2, Table 2.2) i s b e l i e v e d to be the l i m i t , below which acidogenesis and methanogenesis can take place s u c c e s s f u l l y i n the same v e s s e l . In t h i s study, three other D ranges of importance w i t h regard to m i c r o b i a l group i n t e r a c t i o n s were d i s c l o s e d : (1) .0333 < D < .150, where b a c t e r i a l group 4B ( S e c t i o n 2.1.2, Figure 2.1) i s washed out; (2) 0.150 < D < .40, where b a c t e r i a l group 4A i s washed out; and (3) D > .40 where general or s e l e c t i v e wash out of the remaining b a c t e r i a l groups commences. The l a c t o s e a c i d o g e n i c fermentation model, proposed i n t h i s study i s presented i n Fi g u r e 4.14. The a t t e n t i o n of the reader i s drawn to the arrow f r o m CO^/H^ t o a c e t a t e b e i n g d o t t e d , to s i g n i f y l a c k of e x p e r i m e n t a l evidence to confirm or d i s p e l t h i s p o s s i b i l i t y . As shown i n Appendix C, F i g u r e C2, butyrate formation from glucose can be summarized as: glucose + 3ADP + 3?± -> but y r a t e + 2C0 2 + 2H 2 + 3ATP (4.1)- where P^ i s i n o r g a n i c phosphorous. Therefore as long as but y r a t e was being detected i n the fermentor e f f l u e n t , concomitant gas pro d u c t i o n was expected. The i r r e g u l a r gas pro d u c t i o n , w h i c h was o b s e r v e d , has a l r e a d y been r e p o r t e d . Assuming that C0„/H_ was - 104 - F i g u r e 4.12 Fermentation time course f o r [ i ^ C ( U ) ] - l a c t a t e degradation i n a single-phase l a c t o s e fermentation sample. - 105 - L A C T O S E C H 4 Figure 4.13 The m i c o b i a l l a c t o s e fermentation nodel i n d i s t i n c t but simultaneous t r o p h i c phases. three - 106 - Lactose 7 • Pyruvate 7 L a c t a t e Propionat Acetate F i g u r e 4.14 The M i c r o b i a l acidogenic l a c t o s e fermentation model - 107 - being produced a l l through the experimental p e r i o d s , but being u t i l i s e d i n some way f o r b i o s y n t h e t i c a c t i v i t i e s , one would be l e d to conclude that the most l i k e l y p r o d u c t ( a p a r t from CH^) would be a c e t a t e and the b a c t e r i a l group i n v o l v e d would be the hydrogen consuming acetogens otherwise r e f e r r e d to as the homoacetogenic b a c t e r i a l (Group 3 i n Figure 2.1). In comparing the r e s u l t s of C h a r t r a i n and Zeikus w i t h the r e s u l t s i n t h i s study, a number of d i f f e r e n c e s are e v i d e n t . For example, w h i l e formate was one of t h e i r main int e r m e d i a t e products, h a r d l y any was detected i n t h i s study. A l s o they detected ethanol i n s u b s t a n t i a l q u a n t i t i e s . In t h i s study there was no production of ethanol i n the fermentor, the ethanol detected under steady s t a t e c o n d i t i o n s was e s t a b l i s h e d to have been produced i n the s u b s t r a t e feed l i n e . The most important short f a l l of t h e i r r e s u l t s , that i s a l s o r e f l e c t e d i n t h e i r proposed model i s the absence of the r o l e played by b u t y r i c a c i d b a c t e r i a . They d i d not use any butyrate t r a c e r s , because they d i d not detect any b u t y r a t e i n the fermentor e f f l u e n t . The reason they d i d n o t d e t e c t any i s b e c ause a t D * .01 h \ b o t h a c i d o g e n e s i s and methanogenesis were s u c c e s s f u l l y t a k i n g place i n the same v e s s e l . The p r o d u c t i o n of methane v i a r e d u c t i o n kept the p a r t i a l pressure of hydrogen low enough, to enable butyrate degradation by the acetogens to be energy y i e l d i n g and thus a l l o w the r e a c t i o n to go to completion (see Figure 2.2). I f they had probed the r e a c t i o n mechanism w i t h a butyrate r a d i o t r a c e r , complete conve r s i o n to acetate would not have been a s u r p r i s i n g r e s u l t . In f a c t one of the h y d r o l y t i c b a c t e r i a that they i d e n t i f i e d was C_i butyricum, a w e l l known b u t y r i c a c i d b a c t e r i a . - 108 - 4 . 5 S i g n i f i c a n c e of Findings So f a r the f i n d i n g w i t h most s i g n i f i c a n t p r a c t i c a l i m p l i c a t i o n s i n t h i s study i s the d i s c r i m i n a t i o n between r i v a l hypotheses; whether l a c t a t e was sy n t h e s i s e d at a l l growth r a t e s or, whether i t s s y n t h e s i s was switched on at a p a r t i c u l a r d i l u t i o n r a t e below which the flow of carbon would be d i r e c t e d away from the l a c t a t e r o u t e . The l a t t e r has been confirmed beyond any doubt u s i n g a l a c t a t e r a d i o t r a c e r . F i r s t t h i s f i n d i n g i s considered not to be l i m i t e d only to the m i c r o b i a l ecology i n t h i s study, but to be v a l i d f o r a l l anaerobic m i c r o b i a l h a b i t a t s were l a c t a t e has been detected under c o n d i t i o n s of high m i c r o b i a l growth r a t e s . Some of these h a b i t a t s were c i t e d e a r l i e r i n t h i s chapter to i n c l u d e , anaerobic m u n i c i p a l waste treatment systems; i n d u s t r i a l food p r o c e s s i n g waste systems and g a s t r o i n t e s t i n a l systems. In other words, a l l systems that c o n t a i n carbohydrates as carbon sources. Secondly, t h i s f i n d i n g may be u s e f u l i n r e s o l v i n g the pre v a l e n t disagreement about the most s u i t a b l e organic a c i d to be used as a marker i n the o p t i m i z a t i o n of pro d u c t i o n by the aci d o g e n i c phase i n a two-phase process. P i p y n and V e r s t r a e t e (1981) were the f i r s t i n v e s t i g a t o r s to address t h i s problem. They devised a framework by which v a r i o u s a l t e r n a t i v e s could be compared, using the f r e e energy of the r e a c t i o n s both In acidogenesis and methanogenesis. For each of the p o t e n t i a l acidogenic end products ( a c e t a t e , propionate, b u t y r a t e , ethanol and l a c t a t e ) , they assumed an acidog e n i c glucose homofermentative process ( i e . a l l the glucose was assumed to be c o n v e r t i b l e to only one p a r t i c u l a r end pr o d u c t ) . Of course t h i s assumption i s u n r e a l i s t i c f o r mixed c u l t u r e systems and f o r many organisms that are known to possess branched bi o c h e m i c a l mechanisms. - 109 - However i t was a necessary approximation to enable them to c a l c u l a t e the f r e e energy f o r each p o s s i b i l i t y . The r e s u l t s of t h e i r c a l c u l a t i o n are presented i n Table 4.4, i n the form of energy quanta a v a i l a b l e f o r acidogenesis on the one hand and methanogenesis on the other. Based on the r e s u l t s of t h e i r c a l c u l a t i o n , they concluded that the p r e f e r r e d acidogenic product should be e i t h e r l a c t a t e or e t h a n o l , because the most d e l i c a t e and r a t e determining b a c t e r i a l group (methanogens) would be a l l o c a t e d a maximum of a v a i l a b l e p o t e n t i a l energy. But from what i s known from the present study l a c t a t e , b u t yrate and propionate are precursors of acetate at d i f f e r e n t l e v e l s of r e d u c t i o n . Therefore i t does not matter whether, f o r example, l a c t a t e i s converted to a c e t a t e i n the acidogenic r e a c t o r ( i m p l y i n g that acetate i s the predominant ac i d o g e n i c r e a c t o r product) or whether l a c t a t e i s converted to acetate i n the methanogenic r e a c t o r ( i m p l y i n g that l a c t a t e i s the predominant a c i d o g e n i c r e a c t o r p r o d u c t ) , f o r that conversion i n e i t h e r case w i l l be c a r r i e d out by the same acidogenic b a c t e r i a w i t h the only d i f f e r e n c e being that i n the l a t t e r case the acetogenic b a c t e r i a w i l l be i n the acidogenic r e a c t o r , w h ile i n the former case they w i l l be i n the methanogenic r e a c t o r . I n both cases the energy a v a i l a b l e f o r the methanogenic r e s p o n s i b l e f o r d e c a r b o x y l a t i o n of acetate w i l l be the same. A second group of authors (Zeotermeyer, e t . a l . , 1982; K i s a a l i t a , e t . a l . 1986) have i n d i c a t e d that the most d e s i r a b l e product d i s t r i b u t i o n from the f i r s t phase of the two-phase process would be one i n which the c o n c e n t r a t i o n of propionate was a minimum. This was based on the w e l l known f a c t that the methanogenesis of propionate i s the slowest of a l l the r e a c t i o n s i n the methanogenic phase (McCarty, 1963; Andrews and Pearson, - 110 - Table 4.4 Distribution of Total Free Energy Change (for Growth) of the Two-Phase Anaerobic Process of Glucose to Methane Over Different Microbial Groups. Acidogenic end product Free energy change for the acidogenic phase (%) Free energy change available for methanogenesis phase H 2gas 1 (%) Otherwise (%) Acetate 51.1 33.6 15.4 Propionate 88.7 0.0 11.3 Butyrate 63.0 16.8 20.2 Ethanol 55.9 0.0 44.2 Lactate 49.0 0.0 51.1 Potentially subject to loss i f head space gases are not reintroduced into the methanogenic phase. - I l l - 1965; Mahr, 1969). Since l a c t a t e had been b e l i e v e d to be the most common prec u r s o r f o r propionate p r o d u c t i o n , i t was determined not to be a d e s i r a b l e end product of the acidogenic phase. The present r e s u l t s have demonstrated t h a t c o n d i t i o n s can be created where l a c t a t e i s converted to acetate and not propionate, and at a very f a s t r a t e ( F i g u r e 4.11). A l s o s i n c e l a c t a t e p r e d o m i n a t e s a t h i g h D ( h i g h D's - reduced t - are d e s i r a b l e because of the b e n e f i t of sm a l l e r r e a c t o r volumes), t h i s makes i t the optimum product f o r the ac i d o g e n i c r e a c t o r . 4 . 6 Influence Of Protein On The Fermentation Model I n i t i a l l y i n t h i s part of the study ( E - s e r i e s experiments) i t was intended to use a pure p r o t e i n , S - l a c t o g l o b u l i n , but the cost was found to be p r o h i b i t i v e . E f f o r t s were made to o b t a i n whey p r o t e i n concentrates (WPC) from v a r i o u s s u p p l i e r s , but e l e c t r o p h o r e t i c analyses of some of these WPC revealed that none of them contained 8 - l a c t o g l o b u l i n o n l y . I n f a c t some of them had s u b s t a n t i a l q u a n t i t i e s of l a c t o s e . So i t was decided to use r e c o n s t i t u t e d whey powder, the a n a l y s i s of which i s presented i n Chapter I I , Table. 3.2. A t o t a l of three experiments were conducted ( E l , E2 and E3). Runs numbered E l and E2 were started-up i n a manner s i m i l a r to the p r e v i o u s l y d i s c u s s e d experiments w i t h the l a c t o s e - only s u b s t r a t e . Run number E3 was i n i t i a l l y started-up w i t h a p u r e l y l a c t o s e growth l i m i t e d n u t r i e n t medium and once steady s t a t e was achieved, the s u b s t r a t e was switched to a l a c t o s e / p r o t e i n growth l i m i t e d s u b s t r a t e . The purpose of t h i s was to i n v e s t i g a t e whether b a c t e r i a that had been adapted to a l a c t o s e - o n l y s u b s t r a t e , i f intr o d u c e d to a l a c t o s e / p r o t e i n s u b s t r a t e would respond d i f f e r e n t l y i n any way. The carbon balances f o r the three experiments are - 112 - presented i n Appendix B, Table B2. As shown, carbon r e c o v e r i e s f o r the three experiments were b e t t e r or equal to 71.5%, with a p r o t e i n conversions ranging between 65 and 70%, probably being p a r t l y r e s p o n s i b l e f o r the comparatively lower carbon r e c o v e r i e s . For the three experiments, only t r a c e amounts of l a c t o s e were d e t e c t a b l e . The p a r t i a l conversion of p r o t e i n and complete conversion of l a c t o s e i m p l i e d that l a c t o s e was a b e t t e r s u b s t r a t e than p r o t e i n . A s i m i l a r o b s e r v a t i o n has been r e c e n t l y reported by Breure e t . a l . (1986a). Breure and co-workers i n v e s t i g a t e d the i n f l u e n c e of ad a p t a t i o n procedure on the simultaneous a c i d o g e n i c fermentation of glucose and g e l a t i n . In one s e r i e s of experiments glucose d i s s o l v e d i n a mi n e r a l s a l t s s o l u t i o n was fed to a mixed p o p u l a t i o n of b a c t e r i a i n a glucose growth l i m i t e d chemostat (or CSTR) at 30°C and d i f f e r e n t pH l e v e l s (5.3, 6.3 and 7.0). At steady s t a t e , when the glucose s u b s t r a t e was switched to g e l a t i n , growth ceased. However when g e l a t i n was added to a medium as a second carbon s u b s t r a t e (a s i t u a t i o n s i m i l a r to one i n t h i s study) the breakdown of the g e l a t i n proceeded to a l i m i t e d extent (< 30%) and the glucose continued to be completely metabolised. In a second s e r i e s of experiments, Breure and co-workers, adapted b a c t e r i a l p opulations to g e l a t i n under comparative c o n d i t i o n s to the ones described i n t h e i r f i r s t s e r i e s of experiments. A f t e r reaching steady s t a t e , glucose was added to the medium as a second carbon s u b s t r a t e . F o l l o w i n g establishment of a new steady s t a t e , they found that g e l a t i n was hyd r o l y s e d but not degraded any f u r t h e r . A l l these r e s u l t s demonstrate the preference f o r carbohydrates over p r o t e i n s by acidogenic b a c t e r i a . In f a c t Breure e t . a l . (1986b) i n a second paper i n which they reported the r e s u l t s of the i n f l u e n c e of VFA and carbohydrates on the h y d r o l y s i s and acidogenic - 113 - fermentation of g e l a t i n , concluded t h a t , " f o r optimal performance of an a n a e r o b i c d i g e s t i o n s y s t e m p u r i f y i n g w a s t e w a t e r s c o n t a i n i n g c a r b o h y d r a t e / p r o t e i n mixtures, fermentation of carbohydrates should be p a r t i a l l y separated from the h y d r o l y s i s and fermentation of p r o t e i n " . The i m p l i c a t i o n of t h i s c o n c l u s i o n w i t h regard to acidogenesis of whey i s that i t would be more s u i t a b l e to use whey permeate (dep r o t e i n a t e d whey) than whole whey ( l a c t o s e / p r o t e i n ) . However given the high p r o t e i n conversion observed i n t h i s study, i t would appear that the preference of l a c t o s e over p r o t e i n r e s u l t e d i n only a slower p r o t e i n conversion. So the slower t o t a l a c i d o g e n i c conversion of l a c t o s e / p r o t e i n (whole whey) would only r e s u l t i n comparatively l a r g e r r e a c t o r s . The e f f e c t of t h i s s l i g h t l y l a r g e r a c i d o g e n i c r e a c t o r volume, to enable t o t a l acidogenesis of whey, on the economics of the process i s something that deserves f u r t h e r c o n s i d e r a t i o n . The q u e s t i o n that t h i s study attempted to address was whether the degradation of l a c t o s e together w i t h p r o t e i n had any i n f l u e n c e on the l a c t o s e fermentation model. In a p r e l i m i n a r y e f f o r t to f i n d an answer, the c o n c e n t r a t i o n of the two main OA products (acetate and l a c t a t e ) f o r l a c t o s e / p r o t e i n acidogenesis were compared to those f o r l a c t o s e acidogenesis at comparable experimental c o n d i t i o n s . In Fi g u r e 4.15 the comparison i s d e p i c t e d by p l o t t i n g the l a c t o s e / p r o t e i n OA c o n c e n t r a t i o n s on the same graph as the l a c t o s e acidogenic products. As shown, acetate c o n c e n t r a t i o n s were found to be comparable but l a c t a t e was found to be on the high s i d e , p o s s i b l y a consequence of the s p e c i f i c growth r a t e being higher f o r l a c t o s e / p r o t e i n a c i d o g e n e s i s ( S q = 5475 .2 p.g C/mL) compared to that of l a c t o s e a c i d o g e n e s i s (S = 4212.5 ug C/mL). The comparison of OA's f o r the - 114 - 2800-1 E — o 3, 1008- z O c z 1000 1000 y 8 0 0 - 0 .8 0.4 O .S o.a 0 .7 2 0 0 0 0 .2 0 . 3 0.4 0 .8 DILUTION RATE(1/h). 0.8 0 .7 Figure 4 . 1 5 Comparison of the main products concentrat ions for l a c t o s e / p r o t e i n and l a c t o s e - o n l y experiments: (A) a c e t a t e , (B) l a c t a t e . (A) l a c t o s e - o n l y , (x) l a c t o s e / p r o t e i n . - 115 - two s u b s t r a t e type processes revealed no evidence to suggest that the p r o t e i n had any i n f l u e n c e on the l a c t o s e breakdown scheme. At the next i n v e s t i g a t i v e l e v e l r a d i o t r a c e r experiments were conducted i n the same manner as p r e v i o u s l y d e s c r i b e d w i t h samples from E - s e r i e s experiments. The summary of the r e s u l t s are shown i n Table 4.5. In general the recovered a c t i v i t y d i s t r i b u t i o n s are comparable to those p r e v i o u s l y presented f o r high D r a d i o t r a c e r experiments. The conversions here were found to be s l i g h t l y lower probably due to the higher s p e c i f i c growth r a t e . P r o t e i n apparently does not a f f e c t i n anyway the pathway f o r l a c t o s e d e g r a d a t i o n . The growth of microorganisms on more than one carbon source, u s u a l l y r e f e r r e d to as d i a u x i c growth i s at the moment a sub j e c t of i n t e r e s t to a number of r e s e a r c h groups around the world. One of these groups (Ramkrishna, 1982; D h u r j a t i , 1982; and Kompala, 1982) have come up w i t h an i n n o v a t i v e method f o r d e s c r i b i n g m i c r o b i a l i n t e r a t i o n termed, " C y b e r n e t i c s " . The c y b e r n e t i c p e r s p e c t i v e of m i c r o b i a l growth contends that the c e l l ' s i n t e r n a l machinery has the a b i l i t y to make r a t i o n a l o ptimal d e c i s i o n s i n response to i t s environment. This has been found to mainfest i t s e l f w e l l i n s i t u a t i o n s of d i a u x i c growth (Demain, 1971) so the p r e f e r e n t i a l u t i l i z a t i o n of a c e r t a i n s u b s t r a t e over another, although each s u b s t r a t e by i t s e l f would h a v e b e e n a c c e p t a b l e t o t h e o r g a n i s m , f i t s t h e c y b e r n e t i c approach s i n c e the preference of a p a r t i c u l a r s u b s t r a t e could w e l l be i n t e r p r e t e d as a r e s u l t of an o p t i m a l s t r a t e g y . 4.7 Influence Of pH On Carbon Flow From Pyruvate At a d i l u t i o n r a t e of approximately 0.05 h ^, the pH was v a r i e d from a l e v e l of 4.0 to a l e v e l of 6.5, at i n t e r v a l s of 0.5 (Run numbers A l to A6) . Table A.5 Radio Act ivi ty Distribution for Samples from Experiments with Lactose Protein Substrate RUN II TRACER RUN TIME (Min) REACTOR TYPE TOTAL TRACER ACTIVITY (dpm) TOTAL RECOVERED TRACER ACTIVITY (dpm) REC0\ /ERED ACTIVITl I DISTRIBU' riON PARENT RUN II PARENT RUN D ( h _ 1 ) Butyrate Propionate Acetate Lactate C6 [ 1 4 C(U)]-Butyrate 30 S 1 411,434 400,180 97.3% 375,900 93.92 — 24,280 6.1% — E2 0.2018 C5b L-[ 1 4 C(U)]-Lactate 30 S 249,145 235,980 94.82 — — 7,220 3.0% 228,760 97.0% E2 0.2018 Cl L-[ 1 A C(U)]-Lactate 30 s 337,414 322,620 95. ex 1,386 .4* 1,224 .4% 12,040 3.7% 307,970 95.52 E2 0.2007 C9b l 1 A C(U)]-Butyrate 30 s 372,218 403,000 108.3% 393,800 97.7% — 9,200 2.32 — E2 . 0.2065 CIO [ 1 A C(U)]-Butyrate 30 s 428,836 439,688 102.5Z 431,700 98.22 — 7,988 1.8% — E2 0.2065 C8 L- [ U C(U) ] -Lac ta te 30 s 233,678 157,018 67.22 — 1,040 .7% 4,146 2.6% 151,832 96.72 E2 0.2124 Small (10 ml) batch reactor. - 117 - The t a b u l a t e d r e s u l t s f o r OA d i s t r i b u t i o n as w e l l as carbon balance are presented i n Appendix B, Table B4. In F i g u r e 4.16 are presented the v a r i a t i o n s of the major OAs a g a i n s t pH. Butyrate predominated below a pH v a l u e of 5.0 and acetate predominated above a pH value of 5.5. The r e g i o n between pH 5.0 and 5.5, being the t r a n s i t i o n range. I t has already been demonstrated, using l a c t a t e , butyrate and propionate r a d i o t r a c e r s that at low D or low s p e c i f i c growth r a t e c o n d i t i o n s , the m i c r o b i a l p o p u l a t i o n i n v o l v e d i n acidogenesis of l a c t o s e does not possess the a b i l i t y to degrade propionate to acetate and that the conversion of b u t y r a t e to acetate i s slow i n comparison to that of l a c t a t e . Since l a c t a t e was only detected i n t r a c e amounts ( F i g u r e 4.16), i t can be concluded that the source of the observed a c e t a t e was l a c t a t e . So the I n f l u e n c e of the pH on the carbon f l o w would be to favour the b u t y r a t e route at low pH (pH < 5.0) and to favour the l a c t a t e route at high pH (pH > 5.5). A s i m i l a r r e l a t i v e change i n OA d i s t r i b u t i o n has been reporte d by Zeotemeyer e t . a l . (1982) and T i k k a (quoted by Thimann (1963) f o r glucose a c i d o g e n e s i s . The most p l a u s i b l e e x p l a n a t i o n f o r t h i s OA s h i f t i s a change i n m i c r o b i a l species composition. By v a r y i n g D at a pH of 4.5 ( F i g u r e 4.17), an OA v a r i a t i o n s i m i l a r to one e x t e n s i v e l y discussed i n S e c t i o n 4.1 ( F i g u r e 4.1) was q u a l i t a t i v e l y reproduced, c o n f i r m i n g the v a l i d i t y of the l a c t o s e fermentation model that has been proposed i n t h i s s tudy. 4 . 8 Microbial Growth Modeling M i c r o b i a l growth modeling approaches were reviewed i n Chapter I I of t h i s study and the use of the Monod equation was p r e f e r r e d f o r reasons of 2600 E 4 4.5 5 5.5 6 6.5 7 pH. Figure 4.16 Products d i s t r i b u t i o n as a function of pH: (•) butyrate, (A) acetate, (x) propionate, (0) l a c t a t e . The d i l u t i o n rate was set at a f i x e d value of 0.05 h ~ l . 0.7 DILUTION RATE (1/h) Figure 4.17 Products d i s t r i b u t i o n as a function of d i l u t i o n rate at a pH of 4.5: (A) acetate, (x) propionate, (•) butyrate, (H) l a c t a t e . - 120 - s i m p l i c i t y and inherent physico-chemical meaning. Two experimental examples of continuous c u l t u r e behaviour from B a i l e y and O l l i s (1986) are shown i n F i g u r e 4.18 and 4.19. The data i n F i g u r e 4.18 i s c o n s i s t e n t w i t h the o r i g i n a l Monod chemostat model. That i s , the observed c e l l mass and s u b s t r a t e c o n c e n t r a t i o n s remain approximately constant over a wider range of c o n d i t i o n s than i n Figure 4.19. The trend shown i n F i g u r e 4.19 which i s c o n t r a r y to the o r i g i n a l Monod Chemostat model was f i r s t e x p l a i n e d by Herbert (1958a & b) who introduced the concept of "endogenous metabolism" i n a continuous c u l t u r e . Endogenous metabolism was I n t e r p r e t e d to mean the e x i s t e n c e of r e a c t i o n s i n c e l l s which consume c e l l substance. Hence he ) i n t r o d u c e d the maintenance c o e f f i c i e n t , M i n the l i m i t i n g n u t r i e n t mass balance (Equation 2.37) when developing the now w i d e l y accepted more general Monod Chemostat model. Since some m i c r o b i a l systems do not e x h i b i t any d e t e c t a b l e endogenous metabolism requirements, i t was found necessary to determine whether the experimental r e s u l t s i n t h i s study i n d i c a t e d any endogenous metabolism requirements before attempts were made to estimate the model parameters. I n Figures 4.20 and 4.21 are shown the dry biomass c o n c e n t r a t i o n data f o r pH 6.0 and pH 4.5 r e s p e c t i v e l y . With r e f e r e n c e to F i g u r e 4.20, between D v a l u e s of 0.0 and 0.1 h *, evidence of endogenous metabolism i s obvious and i s emphasized by the dotted l i n e . However between D v a l u e s of 0.1 and 0.2, there i s a c o l l a p s e i n the biomass c o n c e n t r a t i o n that p i c k s up suddenly and does not show any signs of washout from there t i l l a d i l u t i o n r a t e of 0.65 h * i s reached. I t should be pointed out that f o r a l l the experiments r u n at D v a l u e s h i g h e r than .45 h * and a pH of 6.0, i n s p e c t i o n of the - 121 - o.: 0.4 0.6 0.8 Dilution rate. D. h" 1 1.0 F i g u r e 4.18 E x p e r i m e n t a l continous c u l t u r e data q u a l i t a t i v e l y c o n s i s t e n t w i t h the o r i g i n a l Monod chenostat model i n a pure culture»Aerobacter aerogenes. s 1" 5 B 3 1 - Calculated from batch growth data 3 4 S 6 7 Reciprocal dilution rate. D~x. h F i g u r e 4.19 E x p e r i m e n t a l continous c u l t u r e data w i t h a t r e n d c o n t r a r y to the o r i g i n a l Monod chemostat model i n a continous c u l t u r e of Aerobacter aerogenes i n a g l y c e r o l medium. 2500 2000 1600 1000 500 0.0 0.2 0.3 0.4 0.6 DILUTION RATE(1/h). 0.6 0.7 Figure 4.20 Dry biomass c o n c e n t r a t i o n at a pH of 6.0. DRY BIOMASS CONCENTRATION (ug/mL). oo c a rl O 3 CB cn cn o o 3 n re 3 rt H CB O 3 - ZZT - - 124 - fermentor v e s s e l at the end of each experiment revealed copious m i c r o b i a l growth adhering to the w a l l s the fermentor v e s s e l ( i e . w a l l growth). So the a c t u a l c r i t i c a l d i l u t i o n r a t e at which washout should have occurred could not be determined e a s i l y . As already pointed out i n Chapter I I , Rogers e t . a l . (1978) observed a s i m i l a r problem w i t h S t r e p t o c o c i u s c r e m o r i s , a l a c t i c a c i d b a c t e r i a , f o r experiments run at D values above the c r i t i c a l v a l u e . The sudden drop i n l a c t a t e c o n c e n t r a t i o n ( F i g u r e 4.1) at D = 0.4 h \ however i s evidence of b a c t e r i a l washout, f o r during washout c o n c e n t r a t i o n s of biomass and other products are known to decrease r a p i d l y i f the c o n d i t i o n s of complete mixing are met. But the slow biomass decrease l i k e one observed here f o r D > .4 h ^ i s c h a r a c t e r i s t i c of m i c r o b i a l systems w i t h problems of w a l l growth ( F i e c h t e r , 1984). I t i s i n t e r e s t i n g to note that the o b s e r v e d drop i n biomass c o n c e n t r a t i o n a f t e r D = 0.1 h ^ c o i n c i d e s w i t h the main OA d i s t r i b u t i o n s h i f t that was considered to be a consequence of m i c r o b i a l p o p u l a t i o n s h i f t . The m i c r o b i a l p o p u l a t i o n s h i f t at a pH of 4.5 i s not as obvious as i n the previous case ( F i g u r e 4.21). Given that l a c t a t e was determined to be the p r e f e r r e d product f o r the o p e r a t i o n of an acidogenic r e a c t o r i n a two-phase process, the ope r a t i n g D range of i n t e r e s t would have to be above a value of 0.2 h ^ ( i n p r a c t i c e 80 - 90% c r i t i c a l d i l u t i o n r a t e v a l u e ) . Therefore the m i c r o b i a l group of i n t e r e s t i s that formed a f t e r the s h i f t and s i n c e , f o r t h i s group, the i n i t i a l r i s e i n m i c r o b i a l biomass c o n c e n t r a t i o n i s sudden ( i m p l y i n g a n e g l i g i b l e endogenous metabolism requirement), i t was decided to drop the M parameter i n the Monod chemostat model so equations 2.41 and 2.42 i n Chapter I I reduce to: - 125 - S = D K / ( u -s in D) (4.2) X = Y ( S Q - S ) (4.3) To i l l u s t r a t e the i n f l u e n c e of M on the biomass c o n c e n t r a t i o n curve, h y p o t h e t i c a l parameters i n p r a c t i c a l ranges were employed to produce Figure 4.22. For curves with shapes s i m i l a r to curve numbers 4, 5 & 6, M i s n e g l i g i b l e . To o b t a i n the parameter estimates i n the Monod chemostat model, a s u b r o u t i ne NL2S0L from the U n i v e r s i t y of B r i t i s h Columbia (Moore, 1984) was used. The F o r t r a n program w r i t t e n to d e f i n e the problem and then c a l l NL2S0L i s presented i n Appendix D. For the biomass data generated at a pH l e v e l of" 4.5, the f o l l o w i n g estimates were obtained: u = 0.3596 ± 0.0026 h" 1 m K g = 8.3258 ± 3.5216 ug C/mL Y = 0.2053 ± 0.0114 In F i g u r e 4.23 a good agreement between the model p r e d i c t i o n s and the e x p e r i m e n t a l d a t a i s shown. A h i g h e r r o r i n K g i s due to the unsteady nature of biomass c o n c e n t r a t i o n at or near the c r i t i c a l d i l u t i o n r a t e . U n f o r t u n a t e l y , f o r the biomass data generated at a pH l e v e l of 6.0, no s e n s i b l e s o l u t i o n could be found, because of l a c k of data of f r a c t i o n a l s u b s t r a t e a c i d o g e n e s i s , caused by the w a l l growth problem, already mentioned. For w i t h w a l l growth the assumption of complete mixing broke down. However the biomass c o n c e n t r a t i o n data i s not completely u s e l e s s . F o r r u d i m e n t a r y process e v a l u a t i o n purposes, the a may be considered to be Figure 4.22 Influence of the maintenance c o e f f i c i e n t (M) on the shape of the biomass p r e d i c t i o n curve: (1) M = 0.5 h - ^ , (2) M = 0.2 h" 1, (3) M = 0.1 h _ 1 (4) M = 0.01 h - 1 , (5) M = 0.005 h _ 1 , (6) M = 0.0001 h - 1 . TJ H-0Q C i-i fD J> to fD > X o a fD o ft) i-l a. X H-T3 3 o fD fD r r i-i 3 fD H- rr 3 s Co o fD (—1 CD a rr rr O . Co Co Cu rr t—1 rt CO 3 Cu o tu i-h a. rt O fD ce t-t M Co •o rj X) fD SC a. o n i-h rr H* O • 3 Ln H- 3 >—s 1 o 1 o <•—' 3 X! 3 Co o i i a H- n> CD i — • o • 3 € rr r r th ro DRY BIOMASS CONCENTRATION (ug/mL). O O o p La. Ol IO - C O H io 3 — L O CA Ol CD O O © O O i I ( - m - - 128 - e q u a l to .41 h ( b a c t e r i a l washout commenced at 0 = .4 h , based on the v a r i a t i o n of l a c t a t e w i t h respect to D-Figure 4.1C). Al s o i n the D range of i n t e r e s t , an average dry biomass c o n c e n t r a t i o n of 1500 ug/mL, gives a y i e l d c o e f f i c i e n t M, of .3561 (S = 4212.5 ug C/mL). A f t e r f i x i n g a and Y, model o m p r e d i c t i o n s at v a r i o u s p r a c t i c a l values of K g were performed and the r e s u l t s a r e shown i n T a b l e 4.6. For K g v a l u e s above 3.0 |ig C/mL at the c r i t i c a l d i l u t i o n r a t e (D = u ), the model p r e d i c t s a negative biomass c o n c e n t r a t i o n . m A t K g = 2.0 [ig C/mL and below, the model p r e d i c t s a p o s i t i v e biomass c o n c e n t r a t i o n . So i t can be c o n c l u d e d t h a t an e s t i m a t e of a K v a l u e s between 2.0 and 3.0 \ig C/mL i s reasonable. A more accurate d e t e r m i n a t i o n of K g i s not j u s t i f i a b l e given that both Y and values were approximations. Comparison of m i c r o b i a l growth constants to those p r e v i o u s l y p u b l i s h e d (Table 4.7) revealed that the growth of an undefined mixed m i c r o b i a l p o p u l a t i o n predominantly producing l a c t a t e on l a c t o s e was f a s t e r than on g l u c o s e . This i s shown by the r e l a t i v e l y high v a l v e of u. . However the u m m values obtained i n t h i s study are not unreasonable, given that they are l o w e r than the \i v a l u e r e p o r t e d f o r a pure l a c t a t e c u l t u r e by Rogers e t . a l . ( 1 9 7 8 ) . The u. v a l u e r e p o r t e d by Ghosh and P o l a n d (1974) i s m u n r e a s o n a b l y h i g h . The h i g h u and e x t r e m e l y low K values suggest that m s probably there were w a l l growth problems, f o r the m i c r o b i a l mass curve form p r e d i c t e d by the Monod Chemostat model w i t h the parameters of Ghosh and Poland (1974) i s c h a r a c t e r i s t i c of those w i t h w a l l growth. In order to compare K g v a l u e s generated i n t h i s study to others a c o n v e r s i o n f a c t o r of 32/12 was used to put them on the same b a s i s as the o t h e r s . I t i s c l e a r t h a t the values from t h i s study are an order of magnitude higher. The only e x p l a n a t i o n that can be o f f e r e d at t h i s time i s that t h i s may be - 129 - Table 4.6 Monod Chemostat model p r e d i c t i o n s f o r d i f f e r e n t K s values based on u m and Y approximations f o r a pH of 6.0 D ( h _ 1 ) P r e d i c a t e d dry biomass c o n c e n t r a t i o n K s ( t i g C/mL) 1.8 2.0 3.0 5.0 10.0 0.2400 1499.1 1499.1 1498.6 1497.6 1495.0 0.2600 1499.0 1498.8 1498.2 1497.0 1493.9 0.2800 1498.7 1498.5 1497.8 1496.2 1492.4 0.3000 1498.3 1498.1 1497.2 1495.2 1490.4 0.3200 1497.8 1497.5 1496.3 1493.7 1487.4 0.3400 1497.0 1496.6 1494.9 1491.4 1482.8 0.3600 1495.5 1494.9 1492.4 1487.3 1474.4 0.3900 1487.6 1486.2 1479.2 1465.4 1430.6 0.4000 1474.4 1471.6 1457.3 1428.9 1357.6 0.4040 1456.9 1452.1 1428.1 1380.2 1260.3 0.4060 1435.0 1427.8 1391.6 1319.4 1138.6 0.4080 1369.3 1357.8 1282.1 1136.8 773.6 0.4090 1237.9 1208.8 1063.1 771.8 43.6 0.4095 975.1 916.8 625.1 41.8 N 0.4098 186.7 40.8 N 1 N N Negative biomass c o n c e n t r a t i o n . Table 4.7 Monod Chemostat Model constants f o r acidogenesis Maximum S p e c i f i c Growth rat e pH Y i e l d C o e f f i c i e n t Monod S a t u r a t i o n Constant Temp- erature (°C) S u b s t r a t e / c u l t u r e References HmC""1) Y Basis K g Basis 0.36 4.5 0.21 kg/kg C 22.2 kg COD/m3 35 l a c t o s e / mixed & undefined This study 0.41 6.0 0.36 kg/kg C 5.3-8.0 kg COD/m3 35 l a c t o s e / mixed & undefined This study 0.33 6.0 0.13 NR1 NR 30 glucose/ mixed & undefined Zeotemeyer e t . a l . (1982) 0.30 NR 0.14 kg VSS/kg COD 0.37 kg COD/m3 35 glucose/ mixed & undefined Ghosh and Kl a s s (1978) 1.25 NR 0.17 kg VSS/kg COD 0.023 kg COD/m3 37 glucose/ mixed & undefined Ghosh and Pohland (1974) 0.56 6.0 NA 2 NA 30 l a c t o s e / S. cremoris Rogers e t . a l . (1978) Not reported. Not a p p r e c i a b l e . - 131 - c h a r a c t e r i s t i c of the b a c t e r i a l p o p u l a t i o n that predominated as a consequence of the s u b s t r a t e being l a c t o s e . L a s t l y a word about the i n f l u e n c e of the nature of the carbon source chemical bond i s c a l l e d f o r . Zeoteraeyer e t . a l . (1982b) pointed out that the h y d r o l y s i s of a - 1, 4 - g l y c o s i d i c bonds (as i n s t a r c h and sucrose) i s f a s t e r than that of B - 1 , 4 - g l y c o s i d i c bond s u b s t r a t e s (as i n c e l l u l o s e ) . Since l a c t o s e i s a d i s a c c h a r i d e of g a l a c t o s e and glucose l i n k e d by a 8-1, 4 l i n k a g e , the acidogenesis of l a c t o s e was p r e d i c t a b l y expected to be slower t h a n t h a t of g l u c o s e . The higher values reported i n t h i s study suggest t h a t the o v e r a l l acidogenesis of l a c t o s e i s f a s t e r than that of glucose. Again the only p l a u s i b l e e x p l a n a t i o n f o r t h i s at the present time i s the type of m i c r o b i a l p o p u l a t i o n that predominates the acidogenic process induced by the presence of l a c t o s e as opposed to the one that would predominate i n a process i n the presence of glucose. - 132 - V. CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions V a r y i n g the d i l u t i o n r a t e (D) of an acidoge n i c chemostat or CSTR i n the me s o p h i l i c temperature range at a pH of 6.0, up to the poin t of b a c t e r i a l washout proved to be a very e f f e c t i v e t o o l f o r s e p a r a t i n g the microorganisms i n v o l v e d i n a n a e r o b i o s i s i n t o v a r i o u s groups w i t h s i m i l a r or over l a p p i n g s p e c i f i c growth r a t e ranges. Above a D value or s p e c i f i c growth r a t e of 0.05 h , the methanogens that form methane by the acetate d e c a r b o x y l a t i o n route were s u c c e s s f u l l y e l i m i n a t e d from the ecosystem. Above a D value of 0.15 h , the methanogens t h a t form methane v i a the r o u t e of hydrogen r e d u c t i o n were a l s o s u c c e s s f u l l y e l i m i n a t e d from the ecosystem. Above a D value of 0.4 h \ s e l e c t i v e or general m i c r o b i a l washout commenced. At a pH of 4.5, a s i m i l a r trend was q u a l i t a t i v e l y reproduced w i t h the acetate d e c a r b o x y l a t i n g and hydrogen reducing methanogens being e l i m i n a t e d above 0.05 and 0.1 h ^ r e s p e c t i v e l y . M i c r o b i a l washout at t h i s low pH commenced at a D value of between 0.3 and 0.35 h Based on the r e l a t i v e c o n c e n t r a t i o n s of the main acidogenic end products ( a c e t a t e , propionate, b u t y r a t e and l a c t a t e ) , produced over the s p e c i f i c d i l u t i o n r a t e ranges corresponding to the d i f f e r e n t m i c r o b i a l groups, two p o s s i b l e hypotheses emerged, namely: (1) The s h i f t i n the m i c r o b i a l p o p u l a t i o n i n the neighbourhood of a D value of 0.15 and 0.1 h ̂ f o r pH l e v e l s of 6.0 and 4.5 r e s p e c t i v e l y represented the beginning of pyruvate c o n v e r s i o n to l a c t a t e ; (2) or the m i c r o b i a l s h i f t d i s a b l e d the conv e r s i o n of l a c t a t e to other i n t e r m e d i a r y m e t a b o l i t e s . Batch i n c u b a t i o n 14 of [ C ( U ) ] - l a c t a t e w i t h the a c i d o g e n i c chemostat e f f l u e n t samples and - 133 - p r e p a r a t i v e s e p a r a t i o n of the predominant organic acids followed by a l i q u i d s c i n t i l l a t i o n assay of the l o c a t i o n of the r a d i o a c t i v i t y , d i s c r i m i n a t e d beyond any doubt between the two r i v a l hypotheses i n favour of the l a t t e r h y p o t h e s i s . This f i n d i n g i s b e l i e v e d not only to be v a l i d f o r l a c t o s e fermentation but f o r a l l mixed undefined anaerobic fermentations of carbohydrates. 14 14 Fu r t h e r use of [ C(U)]-butyrate and [ C(2)]-propionate at a pH of 6.0 and i n the me s o p h i l i c temperature range revealed the predominant carbon flow r o u t e s . I t was assumed that l a c t o s e i s broken down to pyruvate v i a the Embden-Meyerhof-Parnas pathway. So the flow routes of concern were from pyruvate to the v a r i o u s observed organic a c i d s . I t was found that the flow of carbon from pyruvate to butyrate and l a c t a t e was p a r a l l e l . A l s o i t was found, that i n the presence of hydrogen reducing methanogens, l a c t a t e was almost completely converted to acetate and not propionate. Butyrate was found to be converted to acetate at a slow r a t e as long as hydrogen reducing methanogens were present. F u r t h e r conversion of propionate was not p o s s i b l e under a l l D ranges considered. This together w i t h the r e l a t i v e l y low propionate c o n c e n t r a t i o n s observed suggested that the r o l e played by p r o p i o n i b a c t e r i a i n t h i s l a c t o s e acidogenic ecosystem was minor. This knowledge together w i t h the knowledge of the m i c r o b i a l i n t e r a c t i o n s i n r e l a t i o n to D, were used to propose a q u a l i t a t i v e l a c t o s e fermentation model. I n t h i s model l a c t o s e I s c o n v e r t e d to p y r u v a t e v i a the Embden-Meyerhof-Parnas pathway. Pyruvate i n a p a r a l l e l r e a c t i o n i s converted to l a c t a t e and b u t y r a t e . In the presence of hydrogen reducing methanogens, l a c t a t e i s converted i n a very f a s t r e a c t i o n to a c e t a t e . Also b u t y r a t e i s converted to acetate but at a much slower r a t e . In the l i g h t of - 134 - this model i t was concluded that lactate is the most suitable marker for optimising an acidogenic reactor in a two-phase biomethanation process. For that reason only biomass concentration data in the D range where the production of lactate predominated were used to model microbial growth. The Monod chemostat model was favoured among various alternatives because of i t s s impl ic i ty and the physico-chemical basis. The maintenance coefficient was not included in the model as i t was found to be negligible by inspection. At a pH of 4.5 the model parameter estimates were found to be: = 0.3596 ± 0.0026 h " 1 ; K g = 8.3258 ± 3.5216 |J.g C/mL; and Y = 0.2053 ± 0.0114 ug dry biomass/ug C. The large error in K g was a t t r i b u t e d to the d i f f i c u l t y encountered in maintaining a steady biomass concentration as D approached the c r i t i c a l value at which complete bacterial washout occurs. At a pH of 6 .0 , wa l l growth was observed at D values above 0.4 l i ^ and therefore a decrease in biomass as D approached the c r i t i c a l value was not observed. The presence of wall growth invalidated the assumption of complete mixing on which the Monod chemostat model is based. However the c r i t i c a l value of D could be estimated based on the observed sudden drop in the major organic acid concentrations. Degradation of protein (mainly B-lactoglobulin) together with lactose did not in anyway affect the carbon flow scheme. In the D range of 0.05 to 0.15 h * low pH (pH < 5.0) was found to favour the butyrate route at the expense of the lactate route and at high pH (pH > 5.5) the lactate route was favoured at the expense of the butyrate pathway, the pH region of 5.0 to 5.5 being the transi t ion range. - 135 - 5.2 Recommendations Recommendations f o r f u t u r e s t u d i e s i n c l u d e the f o l l o w i n g : (1) The problem of e r r a t i c gas production needs to be s t u d i e d f u r t h e r so that the proposed l a c t o s e fermentation model can be made more complete. A 2x2x2 experimental p r o t o c o l (pH = 4.5, 6.0; D = 0.1, 0.3 h ^; s t a r t - u p procedure = promote gas p r o d u c t i o n , discourage gas production) i s hereby proposed. For each of the e i g h t experiments, samples should be withdrawn and incubated 14 w i t h C-labeled sodium bicarbonate. Then usi n g the procedures d e s c r i b e d i n t h i s study to assay f o r the l o c a t i o n of the r a d i o a c t i v i t y among the major o r g a n i c a c i d s one c o u l d c o n f i r m or d i s p e l l the proposed f a t e of H2 /CO2 p r e c i s e l y . (2) L a c t a t e has been proposed as the most d e s i r a b l e a c i d o g e n i c end product. I t can be p r e d i c t e d from the r e s u l t s of t h i s study that i n the methanogenic s e r i a l r e a c t o r of the two-phase process, l a c t a t e would be converted to a c e t a t e . However, t h i s p r e d i c t i o n needs to be e x p e r i m e n t a l l y v e r i f i e d together w i t h the methanogenic m i c r o b i a l growth model, s i n c e i t i s necessary f o r process design. (3) I t i s a l s o recommended t h a t , m i c r o b i a l p o p u l a t i o n enumeration, c h a r a c t e r i s a t i o n and i s o l a t i o n of the p r e v a l e n t s p e c i e s f o r the two D ranges of i n t e r e s t (.05 < D < 0.15 and 0.15 < D < 0.4) should be attempted. Then by studying the behaviour of the i s o l a t e s i n continuous c u l t u r e f o r the i n f l u e n c e they have on each other a robust mixed and d e f i n e d c u l t u r e f o r a two-phase process could be developed. (4) F i n a l l y t h e economics of the two-phase f e r m e n t a t i o n p r o c e s s f o r whey and d e p r o t e i n a t e d whey (whey permeate) should be e s t a b l i s h e d . - 136 - LITERATURE CITED A i b a , S. and Shoda, M. 1969. Reassessment of the product i n h i b i t i o n i n a l c o h o l f e r m e n t a t i o n . J o u r n a l of Fermentation Technology, 47:790-797. Andrews, J.F. 1968. 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The method could not d i s c r i m i n a t e between l a c t o s e and i t s monomers, glucose and g a l a c t o s e . A l . l Principle T h i s procedure sometimes r e f e r r e d to as the p h e n o l - s u l f u r i c a c i d method was f i r s t p r o posed by Doubis e t . a l . ( 1 9 5 6 ) . S i m p l e s u g a r s , o l i g o s a c c h a r i d e s , p o l y s a c c h a r i d e s and t h e i r d e r i v e r t i v e s I n c l u d i n g the methyl e s t e r s w i t h f r e e or p o t e n t i a l l y f r e e reducing groups, g i v e an orange-yellow c o l o u r when tr e a t e d w i t h phenol and concentrated s u l f u r i c a c i d . The r e a c t i o n i s s e n s i t i v e and the c o l o r i s s t a b l e . Al.2 Reagents and Chemicals A. To ten mL reagent grade phenol (MCB), 180 mL of d i s t i l l e d water were added. B. Concentrated s u l f u r i c a c i d (BDH). A1.3 Procedure One mL of c e n t r i f uged (1 h, 4450 x g) sample, d i l u t e d such that i t contained between 10 - 70 [ig of l a c t o s e , was p i p e t t e d i n t o a c o l o r i m e t e r tube. One mL of reagent A was added and f i v e mL of concentrated s u l f u r i c a c i d were added r a p i d l y . The tubes were allowed to stand f o r ten minutes i n a i r , shaken and placed f o r ten to twenty minutes i n a water bath at twenty to t h i r t y degrees c e n t i g r a d e , before readings were taken at 480 nm using a spectrophotometer, S p e c t r o n i c 70 (BAUSH & L0MB). Blanks were prepared by - 157 - s u b s t i t u t i n g d i s t i l l e d water f o r the sample. A t y p i c a l c a l i b r a t i o n curve i s shown i n Figure A l . A.2 Determination of Whey Protein A2.1 Principle P r o t e i n r e a c t s w i t h c u p r i c i o n i n an a l k a l i n e sodium potassium t a r t r a t e s o l u t i o n to form a complex c o l o r e d compound. Any compound that has i n i t s molecular s t r u c t u r e p a i r s of carbamyl groups l i n k e d through n i t r o g e n or carbon (or peptide l i n k a g e s ) w i l l show a p o s i t i v e b i u r e n t r e a c t i o n . The name of the t e s t i s der i v e d from the simpl e s t of such compounds, b i u r e t . When b i u r e t i s t r e a t e d w i t h an a l k a l i n e potassium copper t a r t r a t e s o l u t i o n , two b i u r e t molecules are jo i n e d to form the f o l l o w i n g complex v i o l e t - c o l o r e d compound. CONH, 2 NH OH OH CONH--K / 1 OH 0.8 0.6 O CO O OL OC O CO cn < 0.4 0.2 Ln 00 0.0-ip 0 —r 10 —r— 20 30 40 50 60 LACTOSE CONCENTRATION (ug/mL). 70 Figure A l C a l i b r a t i o n Curve f o r Lactose Using the P h e n o l - S u l f u r i c Acid Method. - 159 - Since p r o t e i n s c o n t a i n these l i n k a g e s , they react to give the c h a r a c t e r i s t i c c o l o r which i s d i r e c t l y p r o p o r t i o n a l to the amount of p r o t e i n present. A2.2 Reagents and Chemicals B i u r e t reagent (BDH). A2.3 Procedure One mL of f i l t e r e d (.45 fim MILLIPORE membrane) sample was p i p e t t e d i n t o a c o l o r i m e t e r tube. Four mL of b i u r e t reagent were added. The s o l u t i o n was throughly mixed and incubated at room temperature f o r 30 minutes. The absorbance was then determined at a wavelength of 540 nm. Blanks were prepared by s u b s t i t u t i n g d i s t i l l e d water f o r the sample. A t y p i c a l c a l i b r a t i o n curve i s shown i n Figure A2. A3 Determination of Formate A3.1 Principle This procedure was f i r s t d e s cribed by Lang and Lang (1972). B r i g h t y e l l o w and g r e e n - y e l l o w f l u o r e s c e n t r e a c t i o n p r o d u c t s from the f o r m a t e - c i t r i c a c i d r e a c t i o n , change to raspberry red i n the same medium, at room temperature. The i n t e n s i t y of the c o l o r i s p r o p o r t i o n a l to the c o n c e n t r a t i o n of formate and/or formic a c i d . I t ' s main l i g h t a b s o r p t i o n band i s at 515 nm. A3.2 Reagents and Chemicals A. 0.5 g c i t r i c a c i d and ten g acetamide were d i s s o l v e d i n 100 mL i s o p r o p a n o l . E c o -* O H Q. OC O CO 03 < 0.0 1 2 3 4 6 PROTEIN CONCENTRATION (mg/mL). Figure A2 C a l i b r a t i o n Curve f o r Protein Using the Biuret-Reaction Method. - 161 - B. A sodium acetate s o l u t i o n , aqueous, 30 g i n 100 mL was made up. C . A c e t i c a n h y d r i d e . A3.3 Procedure 0.5 mL of c e n t r i f u g e d (1 h, 4450 x g) sample, d i l u t e d such that i t contained l e s s than 200 mg/mL formate was p i p e t t e d i n t o a c o l o r i m e t e r tube. 50 uL reagent B to which 3.5 mL a c e t i c a n h y d r i d e had been added and one mL of reagents A were added to the tube. The mixture was shaken w e l l and incubated at 50°C f o r t h i r t y minutes to speed up the r e a c t i o n . The absorbance was then taken at 515 nm. Blanks were prepared by s u b s t i t u t i n g d i s t i l l e d water f o r the sample. A t y p i c a l c a l i b r a t i o n curve i s shown i n Fi g u r e A3. A4 Determinat ion of L a c t a t e A4.1 P r i n c i p l e When a very d i l u t e s o l u t i o n of l a c t i c a c i d i s heated i n the presence of a high c o n c e n t r a t i o n of s u l f u r i c a c i d i t i s coverted to acetaldehyde. The acetaldehyde may then be determined by the s e n s i t i v e c o l o r t e s t employing phydroxydiphenyl. The procedure was taken from a paper by Markus (1950). In the m o d i f i c a t i o n of t h i s procedure given below, s u f f i c i e n t heat to convert l a c t a t e to acetaldehyde i s generated by r a p i d mixing of s u l f u r i c a c i d and water. A4.2 Reagents and Chemicals A. F o u r g of CuSO^.SH^O were d i s s o l v e d i n water and the volume adjusted to 100 mL. 1.2 E c m z o I- Q. tr o co 03 < 0.8 0.4 0.2 O N 100 200 300 400 600 FORMATE CONCENTRATION (ug/mL). 600 Figure A3 C a l i b r a t i o n Curve f o r Formate Using the Method of Lang and Lang (1972). - 163 - B. One g of p-hydroxydiphenyl (SIGMA) was dissolved i n 100 mL of 0.08N NaOH and store i n a brown b o t t l e i n the r e f r i g e r a t o r u n t i l needed. C. Concentrated s u l f u r i c a c i d . A4.3 P rocedure One mL of centrifuged (1 h, 4450 x g) sample d i l u t e d such that i t contained l e s s than 10 ug/mL l a c t a t e was pipetted into a colorimeter tube. 50 \iL of reagent A were added. Six mL of concentrated s u l f u r i c a c i d was syringed i n t o the tubes, and allowed to stand for f i v e minutes i n a i r , then cooled below 20°C i n cold water. 50 \iL of reagent B were added without touching the wall of the tube, mixed thoroughly and the tubes allowed to stand for 6 h at room temperature (or overnight). The absorbance was then measured at a wavelength of 570 nm. A blank, prepared by s u b s t i t u t i n g d i s t i l l e d water for the sample, were run with each set of determinations. A t y p i c a l c a l i b r a t i o n curve i s shown i n Figure A4. A5 D e t e r m i n a t i o n o f V o l a t i l e F a t t y A c i d s (VFA) and E t h a n o l VFA concentrations were determined using a gas chromatograph. A5.1 Appara tus A. A n a l y t i c a l gas chromatography (GC) - Model 311 (CARLE), equipped with both a Thermal Conductivity Detector (TCD) and a Flame I o n i z a t i n Detector (FID). B. Gas c y l i n d e r s of A i r , H„ and Helium (UNION CARBIDE). 0.30 0.26 0.20 A g 0.16 Q. CC O o.10 m < 0.06 1.00 ^ f ^ - 2 4 6 8 10 LACTATE CONCENTRATION (ug/mL). 12 Figure A4 C a l i b r a t i o n Curve f o r Lact a t e Using a M o d i f i e d Method of Markus (1950). - 165 - C. A 60/80 Carbopack C/0.3 Carbowax 20 M/0.1% H-PO, column, 30" xc 2 4 1/8" s t a i n l e s s s t e e l (SUPELCO). D. A computing i n t e g r a t o r - SP4100 (SPECTRA-PHYSICS). A5.2 Sample P r e p a r a t i o n The VFA can only be detected i n free form. I t was th e r e f o r e necessary to a c i d i f y the samples. In general: HA H + + A ( A l ) ( f r e e a c i d ) At e q u i l i b r i u m , K a = [A~] [H +]/[HA] (A2) - + where [A ], [H ] and [HA] are the molar co n c e n t r a t i o n s of the anion, c a t i o n and a c i d r e s p e c t i v e l y . I f , P K a = - l o g 1 Q K a (A3) and '10 pH = - l o g 1 f J H + ] (A4) then - ( P K -pH) [A ]/[HA] =10 a (A5) For most of the a c i d s (>99%) to be i n f r e e form: [A~]/[HA] < 0.01 (A6) or - 166 - 10 -( PK a-pH) < 10 -2 (A7) Thus pK - pH > 2 (A8) S i n c e the l o w e s t pK a , of a l l the VFA's i s that of a c e t i c a c i d which equals 4.76 (Lehninger, 1970), the pH of the sample had to be l e s s than 2.73. The low pH was achieved by adding a combination of formic a c i d and phosphoric a c i d s . The samples were c e n t r i f u g e d (1 h, 4450 x g) before a d j u s t i n g pH. A5.2 Procedure Once the GC had been i n s t a l l e d , the Helium, A i r and H^ r e g u l a t o r s were set at 12, 20 and 23 p s i g r e s p e c t i v e l y . The oven was set at 120°C. The system was allowed to s t a b i l i s e , a f t e r which the FID was turned on and 5-10 minutes was allowed f o r b a s e l i n e s t a b i l i z a t i o n . One uL sample was i n j e c t e d and the run s t a r t e d . The run was stopped a f t e r 9 minutes and the i n t e g r a t e d chromatogram was obtained from the computing I n t e g r a t o r . Each sample was analysed at l e a s t four times and the mean values r e p o r t e d . A t y p i c a l chromatogram and some of the standard c a l i b r a t i o n curves are shown i n Fi g u r e s A5 and A6 r e s p e c t i v e l y . A6 Gas Volume and Composit ion Determinat ion A6.1 Gas Volume Gas volumes produced over a known pe r i o d of time were measured by wet gas metres (ALEXANDER - 0.25 L per r e v o l u t i o n ) . The t o t a l volume of gas - 167 - Figure A 5 A t y p i c a l v o l a t i l e f a t t y a c i d chronatogran: (Retention Time (RT) = .39 min) ethanol, (RT - 1.94) acetate, (RT = 2.57) propionate, (RT = 3.80) butyrate, (RT 5.64) v a l e r a t e , (RT = 8.39) caproate. 38000 30000 - CO 28000 20000- 18000 10000 8000 2000 4000 8000 8000 10000 ORGANIC ACID CONCENTRATION (ug/mL). 12000 "igure A6 Gome v o l a t i l e f a t t y acids t y p i c a l c a l i b r a t i o n curves: (x) butyrate (A) acetate. - 168 - recorded (V ) was l a r g e r than the a c t u a l volume of gas produced (V ) due f S C 3.C t to moisture. V was c a l c u l a t e d from the equation: act 1 V = (p ̂  " P*) V /p t (A9) act ratm c rec ratm * * where p i s the vapour p r e s s u r e of water at 25°C and one atmosphere (p 23.76 mm Hg) and p i s the atmospheric pressure. ratm r * A6.2 Gas Composition The GC desc r i b e d i n s e c t i o n A5 was used f o r gas a n a l y s i s . A 10% carbowax 20 M on chromosorb W-HF 80/100 mesh column, 8' x 1/8" s t a i n l e s s s t e e l (CHROMATOGRAPHIC SPECIALTIES) w i t h Helium (12 psig) as c a r r i e r gas was used f o r the gas s e p a r a t i o n (^; CH^ and C02)- D e t e c t i o n was by a thermal c o n d u c t i v i t y d e t e c t o r (TCD). For the measurement of the same column and TCD were used w i t h ^ (15 p s i g ) as the c a r r i e r gas. Helium could not be used f o r because the thermal c o n d u c t i v i t i e s of both gases are very c l o s e r e s u l t i n g i n a very poor TCD s e n s i t i v i t y . Once the c a r r i e r gas flow rate and oven temperature (35°C) were s e t , the TCD was turned on and 30 - 45 minutes were allowed f o r b a s e l i n e s t a b i l i z a t i o n . A 500 p.L sample was taken d i r e c t l y from the fermentor head space us i n g a gas t i g h t s y r i nge and i n j e c t e d i n t o the GC In f i g u r e A7 and A8 are shown t y p i c a l chromatograms and c a l i b r a t i o n curves. - 169 - F i g u r e A7 T y p i c a l f e r m e n t o r h e a d s p a c e gas c h r o m a t o g r a m s : ( R e t e n t i o n T ime = . 1 1 m i n ) n i t r o g e n , (RT = . 2 0 ) m e t h a n e , (RT = . 5 1 ) c a r b o n . d i o x i d e , (RT = . 2 8 ) h y d r o g e n . 600 800- CO t 400- < UJ 300-1 200- 100- a . / / / / : / ,' / : I • / / i i i i 1000 2000 3000 4000 QAS SAMPLE SIZE (uL). 8000 6000 r i g u r e A8 Fementor head space gas c a l i b r a t i o n curves: (x) carbon d i o x i d e , (•) methane, (A) hydrogen. - 1 7 0 - A7 Determinat ion of Dry Biomass and Biomass Carbon Dry biomass was determined by g r a v i m e t r i c means. Biomass and sub s t r a t e carbon were determined by a t o t a l carbon a n a l y s e r . A schematic diagram of a the carbon a n a l y s e r used i n t h i s study i s shown i n Figure A9. A7.1 P r i n c i p l e By examining the s p e c t r a of a pure substance a wave len g t h may be found at which a b s o r p t i o n i s c o n s i d e r a b l y g r e a t e r than f o r other compounds present i n the mixture. For a n a l y s i s i t i s simply necessary to measure absorbance at the s e l e c t e d wavelength. Use i s made of t h i s p r i n c i p l e i n the t o t a l carbon a n a l y s e r . A l i q u i d sample i s i n j e c t e d i n t o the r e a c t o r with a strong o x i d i z i n g agent l i k e sodium p e r s u l f a t e . A l l carbon c o n t a i n i n g m a t e r i a l s are o x i d i z e d to carbon d i o x i d e . The carbon d i o x i d e i s c a r r i e d by a stream of oxygen through an i n f r a r e d a n a l y s e r which i s s p e c i f i c a l l y designed to measure and record the c o n c e n t r a t i o n of carbon d i o x i d e present. T o t a l carbon i s measured by t h i s procedure, but I f the sample i s f i r s t prepared by a c i d i f i c a t i o n and a e r a t i o n to remove a l l the i n o r g a n i c carbon, then a s e l e c t i v e measure of the organic carbon present may be obtained. For more d e t a i l s the reader i s r e f e r r e d to Sawyer and McCarty (1978) . A7.2 Apparatus and Reagents A. T o t a l carbon a n a l y s e r (ASTRO). B. 238 g of u l t r a - p u r e reagent grade sodium p e r s u l f a t e (ASTRO) were d i s s o l v e d i n d i s t i l l e d water to make one l i t r e of s o l u t i o n . 6 9 1 0 Figure A9 Schematic diagram of the carbon a n a l y s e r : (1) i n j e c t i o n p o r t , (2) p e r s u l f a t e reagent, (3) oxygen supply c y l i n d e r , (4) auto i n j e c t i o n v a l v e , (5) sample overflow d r a i n , (6) r e a c t o r , (7) g a s / l i q u i d s e p a r a t i o n , (8) l i q u i d d r a i n , (9) CC^ d e t e c t o r , (10) s i g n a l p r o c e s s o r / p r i n t e r . - 172 - A7.3 Procedure The t o t a l carbon analyser was operated i n the t o t a l carbon (TC) node. A f t e r f o l l o w i n g the s t a r t u p i n s t r u c t i o n s , a syringe w i t h 20 mL of sample d i r e c t from the fermenter was loaded i n t o the i n j e c t i o n p o r t , l e a v i n g the s y r i n g e connected to t h i s p o r t . The onboard microprocessor was turned on which assumed the a n a l y s i s task i n c l u d i n g the i n j e c t i o n of the proper sample volume at the a p p r o p r i a t e time and p r i n t i n g out of the sample a n a l y s i s r e s u l t s . A s i m i l a r procedure was repeated f o r a sample from the fermentor, which had f i r s t been f i l t e r e d through a 0.45 urn MILLIPORE membrane. The biomass carbon was assumed to be the d i f f e r e n c e i n carbon c o n c e n t r a t i o n between the f i l t e r e d and u n f i l t e r e d samples. The biomass that remained on the f i l t e r was washed, d r i e d overnight at 105 ± 1°C and weighed. -173- APPENDIX B TABULATED RESULTS TABLE Bl - TOTAL CARBON MASS BALANCES (LACTOSE GROMTH LIMITED SUBSTRATE) AT pH = 6.0 AND TEMFERATl'REOs'c RUN I 81 EXPERIMENTAL FACTORS SOLUTE PRODUCTS CARBON (ugC/aLl GASEOUS PRODUCTS CARBON (ugC/uLI TEMP OIL 'N RATE CARBON BIOMASS CARBON CARBON RECOVERY CARBON RECOVERY (1 OF INFLUENT I'CI pH ETHANOL ACETATE PROPIONATE BUTYRATE BUTYRATE VALERATE VALERATE CAPROATE LACTATE FORMATE DIOXIDE METHANE (ugC/aL) (ugC/oLI CARBON) 35.0 6.05 0.0405* 197.8 2430.5 478.4 27.9 268.8 T 2 38.5 ND NA 4 NA ND . ND NA 3905.3 92.7 s 34.5 6.05 0.0408 266.1 2125.0 446.0 71.6 534.1 T 105.1 ND 5.6 ND t in ND 496 4049.5 96.1 34.5 6.OB 0.0420 322.5 2002.0 310.8 58.8 496.8 ND3 86.5 ND NA " NA ND ND NA 3741.0 88.8 34.5 6.10 0.0401 221.4 2230.0 210.6 21.3 368.6 26.9 50.1 NO NA NA ND ND 420 3634.5 86.3 B2a 34.5 6.00 0.1035 ND 2390.0 340.6 ND 281.9 ND 33.9 ND NA NA 6.9 3.6 948 4005.3 95.1 ND 6.00 0.1091 ND 2325.0 261.9 ND 386.3 ND 41.0 142.9' NA NA 24.6 12.9 867 4061.6 96.4 ND 6.00 0.1029 ND 2105.0 166.3 ND 420.4 ND T 253.3 NA NA 78.S 41.5 934 3999.3 94.9 ND 6.00 0.1105 ND 1861.3 156.5 ND 480.6 ND T 215.0 T ND 104.4 55.0 NA 3788.8 B9.9 B2b 36.5 6.00 0.1108 ND 2533.0 152.4 T 261.3 ND ND ND NA NA 24.9 59. i 1139 4169.7 99.0 36.0 6.00 0.1080 ND 2536.1 199.0 I 379.5 NO NO ND NA NA 42.4 100.6 1634 4791.2 113.7 36.5 6.00 0.1097 ND 2396.3 194.9 T 382.3 ND ND ND NA NA 29.2 69.1 NA 4)11.0 97.6 - 'z^: ' 36.0 6.00 0.1088 NA NA NA NA NA NA NA NA NA NA 30,0 71.2 939.4 - - B2c 34.0 5.95 0.I2B7 T 1839.5 235.0 ND 100.3 ND T T ND NA 94.8 ,163.2 662 3894.8 92.5 34.5 6.00 0.1249 T 1178.3 139.0 NO 1262.5 NO T 176.0 NA NA V7l.~6 181.1 603 3811.5 90.5 34.5 6.00 0.1216 T 1370.0 136.9 ND 1006.9 ND T 240.3 ND "NA 220.1 146.8 550 3671.0 87.1 34.0 6.00 0.1233 ND 2232.3 IBI.6 T 961.8 NO T 131.6 NA NA 119.5 79.7 481 4187.4 99.4 B?d 35.0 5.90 0.1145 T 2181.9 213.1 ND 362.4 NA NA NA 4.4 NA 116.2 205.3 664.5 3747.8 B9.0 34.0 6.00 0.1054 T 2239.3 220.9 ND 391.5 NA NA NA T NA 91.8 162.1 636.0 3741.3 BB.B B2<T 35.0 6.10 0.1026 T 2154.4 211.9 ND 755.6 NA NA NA 2.5 NA 171,1 264.4 769.2 4329.6 102.8 35.0 6.00 0.0947 T 1858.6 268.5 ND 693.8 NA NA NA T NA "120.9 185.2 B72.5 4019.5 95.4 Bib 35.0 6.00 0.2386 13S.7 - B79.4 235.7 ND 695.7 167.7 T I NA NA 'NA NA 798.8 3879.1 92.1 35.5 6.00 0.2423 130.3 923.5 252.4 NO 876.0 107.9 27.4 T NA NA NA NA 901.2 4320.5 102.6 35.5 6.00 0.2459 150.1 1019.3 260.3 NO 939.3 T T NO 1101.8 NA NA NA 490.4 3961.2 94.0 35.5 6.00 0.2430 153.4 1008.3 295.3 ND 929.2 T T ND NA NA NA NA B42.8 4177.4 99.2 1. V - 1470 itL; 2. Trace asountsj 3. Not detected) 4. Not analysed) 5. Average influent carbon; S g = 4212.5 ugC/il CON'T TABLE Bl - TOTAL CARBON MASS BALANCES (LACTOSE 6REWTH LIMITED SUBSTRATE) AT pH = 6.0 AND TEMPERATURE«35*C SOLUTE PRODUCTS CARBON 6ASE0US PRODUCTS RUN EXPERIMENTAL FACTORS (ugC/iLI CARBON (ugC/iLI CARBON I RECOVE DIL'N BIOMASS CARBON (1 OF TEMP RATE i- n- i- n- CARBON CARBON RECOVERY INFLUE (°CI pH (h"'l ETHANOL ACETATE PROPIONATE BUTYRATE BUTYRATE VALERATE VALERATE CAPROATE LACTATE FORMATE DIOXIDE METHANE (ugC/sL) (ugC/uLI CARBON 63c 35.0 4.00 0.2075 89.2 585.3 684.6 ND 979.6 T 387.2 ND 637.0 NA 40.8 ND 906.6 4310.3 102.3 35.0 5.90 0.2131 93.9 843.0 663.1 T 1159.6 T 370.0 NO 497.2 NA 26.6 ND 489.3 4142.1 98.3 34.5 5.95 0.2121 T 1023.3 727.1 T 1742.6 T 294.8 ND NA NA 159.9 ND 796.5 4744.2 112.6 B4a 35.0 6.00 0.2896 112.0 1451.5 342.6 ND 1390.1 ND T ND NA NA 157.9 T 790.7 4297.8 102.0 35.5 6.05 0.2840 125.6 1314.4 320.0 ND 1218.0 ND T ND NA NA 121.3 T 932.4 4084.7 97.0 35.5 6.00 0.3005 90.9 1334.1 277.1 ND 1265.8 ND T ND NA NA 150.4 T 856.8 4028.4 95.4 34.5 6.00 0.2891 101.5 1574.3 335.4 ND 1395.9 ND T ND 53.0 ND 112.9 T 705.6 4278.6 101.6 84b 35.0 6.00 0.2907 142.1 1416.7 508.4 ND 542.B T 36.9 ND 413.5 NA ND ND 834.8 3975.2 94.4 35.0 6.00 0.3035 196.2 1510.8 629.5 ND 362.0 T 39.4 ND NA NA ND ND 783.1 4250.1 1G0.9 35.5 6.00 0.2971 93.2 1190.5 557.3 ND 615.1 T 104.9 ND 807.5 NA ND ND 965.2 4333.7 102.9 35.5 6.00 0.2898 95.4 1158.7 438.0 ND 699.1 T 122.7 ND 966.3 NA ND ND 794.0 4274.2 101.5 B5 34.5 6.00 0.3520 T 659.2 223.9 ND 750.0 T T ND NA NA 86.6 T 590.6 3651.8 86.7 35.0 6.00 0.3401 NA NA NA ND NA NA NA ND NA NA 79.3 T 667.2 - - 34.0 6.00 0.3537 T 692.1 223.0 ND 667.2 ND 38.0 ND NA NA 72.9 T 583.7 3618.4 85.9 34.0 6.00 0.3560 59.4 983.0 243.6 ND 963.5 T 58.8 ND 1341.5 ND 113.9 T 654.4 4438.1 105.4 Ei 35.0 5.93 0.4784 T 592.2 189.6 ND 669.5 T T ND NA NA 157.3 T 584.4 2975.5 70.6 35.5 5.98 0.4624 NA HA NA ND NA NA NA NA NA NA 151.1 T 571.8 - - 35.0 6.00 0.4716 T 645.5 263.4 ND 597.5 T 27.7 ND NA NA 93.1 T 695.2 3015.7 71.6 35.0 6.00 0.4762 T 910.8 322.0 ND 690.0 T 48.3 ND 782.5 T 83.8 T NA 2B37.4 67.4 B7 35.0 6.00 0.640 T 403.9 238.4 ND ; 597.1 ND 73.2 ND 702.0 NA ND ND 656.4 2014.6 47.8 TABLE B2 - TOTAL CARBON MASS BALANCES (LACTOSE/PROTEIN 6RQNTH LIMITED SUBSTRATE) OF pH = 6.0 AND TEMPERATURE = 35°C RUN EXPERIMENTAL FACTORS SOLUTE PRODUCTS CARBON t i - n- l- n- TEMP pH DIL'N ETHANOL ACETATE PROPIONATE BUTYRATE BUTYRATE VALERATE VALERATE CAPROATE LACTATE l°C) • RATE liT1) El 34.5 4.10 (h ) 0.3061 B1.8 1021.4 274.2 2 207.5 ND3 131.7 152. B NA4 34.5 4.10 0.3005 98.1 1088.1 283.8 T 235.8 ND 113.8 124.0 1940.0 35.0 6.10 0.3061 84.6 1126.2 408.0 T 290.1 NO 103.9 141.6 1281.5 34.0 6.10 0.3061 71.4 1107.5 486.8 T 204.2 ND 141.0 181.3 1118.6 35.0 6.10 0.3061 B3.9 861.5 310.4 T 242.4 ND 127.6 1B7.5 1501.0 E2 34.0 6.00 0.2029 69.6 725.3 188.5 T 233.9 NO 89.9 NO 1551.9 34.0 6.00 0.2029 69.7 1689.7 212.5 T 284.5 ND 143.5 ND 1720.7 35.0 6.00 0.2017 101.0 1386.7 172.3 T 208.5 ND 160.8 ND 1728.9 E3 34.5 6.00 0.2029 ND 1699.1 592.7 T . 481.8 76.6 65.7 T 962.8 33.7 5.95 0.2097 7B.9 1263.1 563.4 T 416.2 76.1 50.8 T 909.4 34.5 5.85 0.2124 100.8 1307.4 656.2 T 461.7 131.6 36.7 T 1IB2.0 34.5 5.97 0.2071 99.1 1339.8 526.8 T 346.9 154.3 34.9 T NA V = 1470 aL; 2. Trace aiounts; 3. Not detected; 4, Nat analysed; 5. Influent carbon concentration i s 0 = 5475.2 ugC/iL EASEOUS PRODUCTS CARBON CARBON lugC/iL) RECOVERY BIOMASS CARBON (1 OF t PROTEIN FORMATE CARBON METHANE CARBON RECOVERY INFLUENT FERMENTED DIOXIDE lugC/iL) lugC/sL) CARBON) NA 22.9 ND 958.4 4311.0 78.7s NA 21.9 ND 617.0 4522.5 B2.6 NA 30.9 ND 876.0 4342.8 79.3 65.1 NA 17.2 ND 719.2 4047.2 73.9 NA 21.2 ND NA 4128.2 75.4 NA 19.2 ND 1053.2 3912.3 71.5 NA 3.1 ND 1356.6 5477.2 100.0 68.7 NA 10.0 ND NA 4963.1 90.6 NA ND ND 561.0 4439.7 Bl . l NA ND ND 662.9 4020.B 73.4 69.9 NA ND ND 643.1 4519.5 82.5 NA ND ND 682.8 4202.7 76.8 TABLE B3 - TOTAL CARBON MASS BALANCE (LACTOSE 6R0NTH LIMITED SUBSTRATE) AT pH = 4.5 AND TEMPERATURE = 35°t RUN I EXPERIMENTAL FACTORS SOLUTE PRODUCTS CARBON (ugC/iL) 6ASE0US PRODUCTS CARBON (ugC/iL) CARBON RECOVERY DIL'N1 BIOMASS CARBON (t OF TEMP RATE i- n- i - n- CARBON CARBON RECOVERY INFLUENT l°CI pH (ffl ETHANOL ACETATE PROPIONATE BUTYRATE BUTYRATE VALERATE VALERATE CAPROATE LACTATE FORMATE DIOXIDE METHANE (ugC/iL) (ugC/iL) CARBON) 35.0 4.6 0.0495 261.9 1175.9 711.1 75.0 1391.5 42.0 118.5 361.4 NA4 NA ND ND NA 4450.4 105.6 35.5 4.6 0.0505 257.5 1260.9 641.9 75.3 1382.4 T 2 112.3 326.3 NA NA ND ND 15B 4214.6 100.0 35.5 4.6 0.0496 250.0 1141.1 434.8 66.9 1503.6 T 90.6 308.4 4.8 ND3 ND ND 396 4196.2 99.6 36.0 4.5 0.0505 373.1 1554.0 4BB.0 96.9 1640.0 T 84.1 407.1 NA NA ND ND 371 5019.0 119.5 D2 36.0 4.5 0.1036 ND 1044.1 520.9 T 1369.1 ND 109.3 ND NA NA ND ND 348 3407.3 B0.9 35.0 4.5 0.1014 ND 960.1 671.3 T 1232.5 ND 73.3 ND NA NA ND ND NA 3347.7 79.5 36.0 4.5 o'.ion ND 990.0 622.6 I 1346.3 ND T ND 15.9 ND ND ND 410 3385.6 BO. 4 35.0 4.5 0.1006 ND 1097.3 594.9 T 1297.4 ND T NO NA NA NO NO 426 3431.5 B1.5 03 35.0 4.5 0.2071 T 824.0 506.5 ND 473.1 ND 44.4 ND 1347.9 NA ND ND 692 3887.8 92.3 35.0 4.5 0.1959 NA NA NA NA NA NA NA NA NA NA NA NA NA - - 35.U 4.5 0.20BO NA NA NA NA NA NA NA NA NA NA NA NA 65B - - D4 36.0 4.a 0.3571 53.3 268.1 B5.B ND 2B4.0 ND T ND NA NA ND ND 366 2110.4 50.1 35.0 4.6 NA 42.1 279.1 105.4 ND 226.6 ND T NO NA NA ND ND 348 2054.4 4B.8 36.5 4.6 0.3560 51.9 379.1 105.4 ND 1 133.0 ND T ND NA NA ND ND 292 2014.6 47.8 36.5 4.6 0.3578 42.5 473.9 135.9 ND 162.5 ND T ND 1053.4 NA ND ND 406 2274.2 54.0 1. V- 1470 EL; 2. Trace amounts; 3. Not detected; 4. Not analysed; 5. Average influent carbon, S 0 = 4212.5 ug/iL TABLE 64 - TOTAL CARBON MASS BALANCE (LACTOSE 6RQUTH LIMITED SUBSTRATE) AT D = O.OStT'ftND TEMPERATURE = 35°C . RUN EXPERIMENTAL FACTORS SOLUTE PRODUCTS CARBON BASEOUS PRODUCTS CARBON t , (ugC/iL) CARBON (ugC/iL) RECOVERY DIL'N BIOMASS CARBON (I OF TEMP RATE i- n- i - n- CARBON CARBON RECOVERY INFLUENT <°C) pH (If1) ETHANOL ACETATE PROPIONATE BUTYRATE BUTYRATE VALERATE VALERATE CAPROATE LACTATE FORMATE DIOXIDE METHANE (ugC/iLI (ugC/iL) CARBON) Al 34.9 4.0 0.0520 241.9 351.9 ND3 ND 1315.0 ND NO T* 41.6 ND 513.4 ND 308.0 2258.4 53. t 4 A2a 35.4 4.6 0.0500 222.2 1014.3 386.5 59.4 1336.5 ND 80.5 274.0 4.8 ND ND ND 372.0 3750.2 89.0 I A3a 35.0 5.0 0.0430 305.6 1078.5 190.4 ND 1418.4 ND 121.4 200.1 12.8 NO ' ND ND 770.0 4097.2 97.3 M ^4 A4a 34.8 5.6 0.0530 172.4 1635.0 473.B 9.0 456.0 NO 43.5 166.3 2.0 ND ND ND 646.0 3938.2 93.5 0 0 I A5 34.0 6. 1 0.0400 265.9 1969.3 446.3 71.3 534.4 ND 105.1 ND 7.0 ND NO ND 496.0 4050.5 96.2 fit 34.2 6.5 0.0480 105.6 1993.0 196.4 4.1 564.4 ND 79.4 393.1 14.0 ND ND ND 767.0 4117.0 97.7 A2D 35.4 4.6 0.0501 103.9 850.6 110.9 54.8. 1863.3 NO ND NO ND ND 721.8 NO 374.0 4079.3 96.8 Alb 35.0 5.0 0.0430 104.5 622.8 538.1 46.B 1502.2 ND 153.3 ND 5.2 T 415.6 ND 340.6 3729.1 B8.5 A4b 35.0 5.5 0.0536 243.6 1635.0 2B0.9 16.0 604.0 78.4 85.8 120.0 7.2 ND 330.1 177.4 589.0 4172.0 99.0 I . V - 1470 iL; 2. Trace amounts; 3. Not detected; 4. Average influent carbon, S 0 = 4212.5 ugC/iL T ' i .E ES - TOTAL GAS PRODUCTION AS A FRACTION OF CUMULATIVE EXPERIMENT TIME AT 25°C AND ONE ATMOSPHERE A-SERIES EXPERIMENTS Al A2a,0l A3 A4 A5a TIKE GAS' TIME GAS TIME GAS TIME 6AS TIME (h) (L/LI (hi IL/LI (hi IL/LI Ihl (L/LI (hi 0 _ 0 - 0 - 0 - 0 23. - 23. - 44. - 20. 0 24. 46. - 48. - 68. 0 43. 0 34. 54. 0 56. 0 92. 0 66. 0 48. 89. 0.539 89. 0.2124 116. 0 102. 0 72. 106. 0.5301 106. 0.0 140.5 0 115. 0 99. 128. 1.036 128. 0.0 174.0 0 139. 0 120. 153. 0.253 153. 0.9115 199. 0 164. 0 143. ;?fc. 0,'t59 17b. 2.163 2S3. 0 188. 0 167. 200. 0.1587 200. 2.756 23B. 0 212. 0 191. 224. 0.74 224. 2.326 267. 0 215. 249.5 1.284 249.5 1.716 285. 0 239. 274.0 1.435 274.0 0.2778 308. 0 264.5 296. 1.495 296. 0. 332. 0 321. 2.164 321. 0 353. 0 344. 2.075 344. 0 348. 2.225 366. 0 372. 2.121 372. 0 1. In litres nf gas ptr litre effluent. A6 A26 A36 A46 GAS TIME 6AS TIME 6AS TIME GAS TIME GAS (L/LI Ihl (L/L) (hi (L/L) Ih) (L/LI <h) (L/LI _ 0. - 0 - 0 - 0 - 20. 0 24. - 24. - 44. - 0 43. 0 48. - 48. - 68. - 0 66. 0.030 72. 0 72. 0 92. 0 0 102. 0.086 96. 0 96. 5.912 116. 0.639 0 115. 0.94 128. 0 128. 4.733 140.5 0.286 0 139. 0.35 144. 0 144. 4.697 174. 0.708 0 179. 0 179. 4.485 199. 0.200 0 192. 0.602 192. 3.960 213. 0.368 0 216. 2.287 216. 2.813 23B. 0.490 0 240. 2.965 240. 2.079 267. 0.174 0 264. 3.601 264. 0.611 285. 0.219 0 288. 3.301 288. 0.656 308. 0.290 312. 3.924 312. 1.495 332. 0.451 333. 3.895 333. 1.812 353. 0.7S2 358. 3.790 358. 2.805 371. 0.828 384. 3.349 384. 2.706 395. 0.897 407.5 2.969 407.5 1.377 419. 1.315 431.5 2.973 431.5 1.313 443. 1.285 455.5 2.830 455.5 1.793 467. 1.313 480.5 3.167 480.5 1.824 491. 1.116 505.0 2.953 505.0 2.125 515. 1.116 527.5 2.615 527.5 1.402 -539. 1.199 553.0 2.638 1.188 TABLE B5 - TOTAL 6AS PRODUCTION AS A FRACTION OF CUMULATIVE EXPERIMENT TINE BAS TINE In) IL/L) (h) 0 - 0 24 - 18 34 0 42 48 0 68.5 72 0 92.0 99 0 116.0 120 0 140.0 143 0 164.0 147 0 168.0 191 0 212.0 215 0 236.0 239 0 260.0 244.5 0 284.0 308.0 i B2b BAS TINE GAS (L/LI In) (L/L) 0 40 0.170 0 64 0.305 0.293 89 0.15a 0.00 113 0.418 0.061 135 0.316 0.218 162 0.609 0.366 184 0.299 0.219 20B 0.205 0.207 232 0.349 0.027 256 0.240 0.096 2B0 0.247 0.308 0.408 B2c TIME GAS Ih) (L/LI 0 - B 0 50 0.001 74 0.323 107.5 0.034 124 0.004 149 0.038 173 0.131 19B 0.073 234 0.113 256 0.3B6 2B7 0.634 312 0.712 325 0.529 349 0.484 373 0.452 400 0.279 423 0.337 453 0.966 493 0.783 517 0.425 526 0.230 TINE AT 25°C AND ONE ATMOSPHERE (CQN'TI B-SERIES EXPERIMENTS B2d B2e B3b B3c TIME 6AS TIME 6AS TINE BAS TIME BAS (hi (L/L) (h) (L/L) (hi (L/L) (h) (L/L) 0.0 - 0 - 0 - 0 - 20 0 25 0 18. - 24. 0 36 0 37. 0.397 26. 0 44. 0.272 47 0 61. 0 43. 0 55. 0.424 71 0 85. 0 55. 0 68. 0.18 95 0 109. 0 67. 0.660 80. 0.048 119 0 133. 0 79. 1.498 92. 0.238 143 0 157. 0 91. 2.877 104. 0.214 167 0.113 181. 0 102. 3.154 116. 0.016 191 0.325 211. 0 114. 0.562 128. 0.958 221 0.473 229. 0.236 126. 0.371 140. 1.644 239 0.257 254. 1.147 138. 0.464 152. 1.070 264 0.244 27B. 0.825 150. 0.235 164. 0.092 288 0.464 302. 0.859 163. 0 176. 0.11B 312 0.114 326. 1.043 175. 0 188. 0.126 336 0.703 350. 1.069 186. 0.14 200. 0.204 360 0.759 374. 0.749 196. 0 212. 0.160 384 0.600 398. 0.545 211. 0.153 224. 0.231 408 0.636 422. 0.582 222. 0.169 236. 0.181 432 0.633 448. 0.768 234. 0.436 24B. 0.286 456 0.769 472. 0.109 246. 0.481 260. 0.16B 480 0.725 496. 0.061 271. 0.250 504 0.71? 283. 0.116 295. 0.185 307. 0.075 319. 0.152 338. 0.341 355. 0.451 CON'I TABLE B5 - TOTAL 6A5 PRODUCTION AS A FRACTION OF CUMULATIVE EXPERIMENT TINE AT 25°C AND ONE ATMOSPHERE 643 TIME GAS (h) (L/LI 0 - 36. 0 60. 1.60 72. 0 84. 0 96. 0.171 106. 0.158 120. 0.070 132. 0.156 144. 0.174 156. 0.441 168. 0.599 180. 0.652 192. 0.501 204. 0.621 214. 0.466 B4b TIME 6AS Ihl (L/L) 18. _ 20. 0 43. 0.789 55. 0.841 67. 0.293 79. 0.424 91. 0.211 102. 0.020 114. 0 126. 0.020 138. 0.269 150. 0.136 163. 0.073 175. 0 186. 0 196. 0 211. 0 222. 0 234. 0.053 246. 0 259. 0 271. 0.042 B-SER1ES B5 TIME GAS (hi (L/LI 0 18. 0 32. 0.195 42. 0.067 55. 0.031 67. 0.088 79. 0.336 91. 0.592 102. 0.455 114. 0.419 126. 0.349 132. 0.320 139. 0.294 151. 0.368 157. 0.459 163. 0.526 B6 TIME GAS (hi (L/L) 0 18. 0 32. 0.211 42. 0.213 55. 0.582 67. 0.446 79. . 0.196 91. 0.495 102. 0.767 114. 0.922 126. 1.013 132. 0.949 139. 0.667 151. 0.585 157. 0.526 163. 0.498 B7 TIME 6AS (h) IL/L) 173. 0.376 179. 0.037 187. 0.036 199. 0 CON'T TABLE B5 - TOTAL BAS PRODUCTION AS A FRACTION OF CUMULATIVE EXPERIMENT TINE AT 25°C AND ONE ATMOSPHERE D-SER1ES EXPERIMENTS D2 03 D4 TIME BAS TIME BAS TIME 6AS (h) (L/LI th) (L/L) (hi (L/L) 0 - 0 - 0 - 21. 0 36. 0 B. 0 45. 1.993 60. 0.100 50. 1.447 67. 1.066 72. 1.653 74. 1.214 89. 0 84. 0.390 107.5 1.27 113. 0 96. 0 124. 1.527 138. 0.072 108. 0.169 149. 1.672 162. 0 120. 0.409 173. 1.155 168. 0.056 132. 0.531 198. 1.194 210. 0.063 144. 0.580 234. 0.926 235. 0.201 156. 0.633 256. 0.161 259. 0 168. 0.418 2B7. 0 180. 0 312. 0.041 192. 0 325. 0.197 204. 0 349. 0.06 214. 0 373. 0 400. 0 426. 0 453.5 0 493. 0.211 517. 0.104 526. 0.050 E-SERIES EXPERIMENTS El E2 E3 TIME 6AS TIME GAS TIME 6AS (hi (L/LI (h) (L/L) (hi (L/L) 0 -• 0 - 383. 0.616 21. 0 24. 0 395. 0.467 32. 0.500 44. 0.043 408. 0.391 44. 0.745 55. 0.061 419. 0.643 57. 0.214 68. q. 125 431. 0.526 69. 0.121 80. 0.139 443. 0.295 BO. 0.016 92. 0.091 455. 0.042 92. 0.119 104. 0.103 467. 0 105. 0.126 116. 0.045 479. 0 116. 0.147 128. 0.022 494. 0 128. 0.183 140. 0.028 503. 0 141. 0.249 152. 0.0B7 515. 0 152. 0.213 164. 0.1098 527. 0 164. 0.243 176. 0.105 538. 0 176. 0.283 188. 0.086 IBB. 0.232 200. 0.078 200. 0.253 212. 0.113 212.1 0.238 224. 0.085 224. 0.259 236. 0.201 236.; 0.183 248. 0.263 248. 0.185 260. 0 260. 0.225 271. 0 283. 0.344 295. 0.137 307. 0.056 319. 0.053 338. 0.108 355. 0.179 - 183 - APPENDIX C ENZYME SYSTEMS - 184 - C H , O H c=o C H j O ® IIO-C I H C H - C - O H I H OH C H i O © Iructose-fi-phosphate H-C =0 H -C OH HO -C -H I H - C - O H I H C -OH C ' H ; 0 ® glucuse-6-phosphate ruetose-1. b- hisphosphate di hydroxy ace lone phosphate' dyceraldehyde-3- phusphate NAD| + Pi C O O ® H C - O H C H 2 0 © -<ADP) COOH H C - O H C ' H i O ® C O O H H C - O ® C H , O H 4-* COOH - c - o ® II w C H , C O O ® H C - O H C H - . 0 ® • | K (ATP) C O O H H C - O H C H , 0 ® coo» c=o ^ CH-i.J 1. 3-bispliospho- glycerate 3-phospho- glycerate 2-phospho- tdveerate phosphoenol- pyruvate Figure C l Breakdown o f glucose to 2 pyruvate v i a the Embden-Meyerhof- Parnas pathway: (1) PEP: glucose phosphotransferase; (2) glucose phosphate isomerase; (3) phosphofructokinase; (4) f r u c t o s e bisphosphate a l d o l a s e ; (5) t r i o s e phosphate isomerase; (6) glyceraldehyde-3-phosphate dehydrogenase; (7) 3-phosphoglycerate k i n a s e ; (8) phosphoglycerate mutase; (9) enolase; (10) pyruvate k i n a s e . Source i s Gottschalk (1979). - 185 - ( . ' l ( 3 - O l ; C l l 2 COOH p. butyrate CoA acetyl-CoA ^ acetyl-P acetate C H j - C H 2 - C H 2 - C O - C o A butyryl-CoA N A D - ^ f N A D H j — * | C H j - C H = C H - C O - C o A crotonyl-CoA H 2 0 C H 3 - C H - C H 2 - C O - C o A O H L (+ H3-hydroxybutyry 1-CoA t C H 3 - C O - - C H ! - C O - C o A acetoacetyl-CoA NAD N A D H i sum: glucose + 3 ADP + 3 P, • butyrate + : C O j + 2 H2 +3 ATP Figure C2 Path of b u t y r a t e formation from glucose: (1) phosphotransferase system and Embden-Meyerhof pathway; (2) p y r u v a t e - f e r r o d o x i n oxidoreductase; (3) hydrogenase; (4) a c e t y l - C o A - a c e t y l t r a n s f e r a s e ( t h i o l a s e ) ; (5) L(+)-8-hydroxybutyryl-CoA dehydrogenase; (6) L-3-hydroxyacyl-CoA hydrolyase ( c r o t o n a s e ) ; (7) butyryl-CoA dehydrogenase; (8) CoA-transferase; (9) phosphotransacetylase; (10) acetate k i n a s e . Source i s Gotschalk (1979). - 186 - OH (L)2CH3 - C - C O O H H til Ir H (D) CH 3 -C-COOH OH OH I : C H 3 - C - C O - C O A :CH,=CH-CO-CoA - ETF - ETF • H 2 • CHj-CO-COOH CoA -vL Fd CoA - ^ L , fd ~-*Y~ [ C O T I V ^ - Fd • H 2 — ETF • H 2 ' — ETF — C H 3 - C O - C 0 A 2 C H 3 - C H 2 - C O - C o A 2CH3-CH3-COOH sum: 3 lactate • 2 propionate + acetate + C 0 2 Figure C3 Formation of propi o n a t e , a c e t a t e , and CC^ from D L - l a c t a t e by Megasphaera e l s d e n i i and C l o s t r i d i u m propionicum: (1) l a c t a t e racemase; (2) CoA t r a n s f e r a s e ; (3) r e a c t i o n not e s t a b l i s h e d ; (4) dehydrogenase, which employs reduced e l e c t r o n - t r a n s f e r r i n g f l a v o p r o t e i n (ETF'R^) as H-donor; (5) D-lactafce dehydrogenase; (6) p y r u v a t e - f e r r e d o x i n oxidoreductase; (7) transhydrogenase; (8) phosphotransacetylase + acetate k i n a s e . Source i s Got t s c h a l k (1979). - 187 - HOOC-CH,-CO-COOH NADH; NAD HOOC-CH:-CHOH-COOH HOOC-CH=CH-COOH HOOC-CH 2-CH 2-COOH (ATP) QM3P + sum: lactate + NADH 2 + ADP 4- P, » propionate + NAD + ATP F i g u r e C4 Fermentation of l a c t a t e v i a the su c c i n a t e - p r o p i o n a t e pathway by p r o p i o n i b a c t e r i a : (1) l a c t a t e dehydrogenase (the H-acceptor i s probably a f l a v o p r o t e i n ) ; (2) (S)-methyImalonyl-CoA-pyruvate t r a n s c a r b o x y l a s e ; (3) malate dehydrogenase; (4) fumarase; (5) fumarate reductase; (6) CoA t r a n s f e r a s e ; (7) (R)-methyImalonyl-CoA mutase; (8) methyImalonyl-CoA racemase. Source i s Got t s c h a l k (1979). - 1 8 8 - APPENDIX D A COMPUTER PROGRAM FOR ESTIMATING THE PARAMETERS OF THE MICROBIAL GROWTH MODEL - 189 - C THIS PROGRAM ESTIMATES THE PARAMETERS OF A NONLINEAR C MICROBIAL K I N E T I C MODEL EITHER USING A L E A S T SQUARES C OR A CHERBTCHEV CURVE F I T T I N G METHOD. C I M P L I C I T REAL*8 ( A - H , 0 - Z ) DIMENSION P ( 3 ) , I V ( 6 3 ) , V(282 ) EXTERNAL C A L C R , C A L C J COMMON X ( 2 0 ) , 7 ( 2 0 ) , SO, EPS C SETTING I N I T I A L VALUES AND READING IN DATA EPS = 0.001 DO SO= 4212.5D0 N=5 M=3 P( 1 ) = .200D0 P(2)=60 .D0 P(3)= .36D0 DO I I » 1,11 CALL FREAD ( 5 , ' 2R*8 : ' , X ( I ) , 7 ( D ) 1 CONTINUE C SETTING DEFAULT VALUES IN I V AND V CALL D F A L T ( I V . V ) CC REQUESTING FOR THE PRINTING OF THE COVARIANCE MATRIX CC AFTER CONVERGENCE HAS OCCURED. IV( 14) = 1 IV(21)=6 C C A L L I N G THE MAIN SUBROUTINE NL2SOL C A L L N L 2 S O L ( N , M , P , C A L C R , C A L C J , I V , V , I P A R M , R P A R M , F P A R M ) C WRITING THE RETURN CODE AND SOLUTION . W R I T E ( 6 , 1 1 0 ) I V ( 1 ) 110 FORMAT('RETURN CODE=' , 12) W R I T E ( 6 , 1 2 0 ) ( P ( I ) , 1=1 ,M) , V(10 ) 120 F O R M A T U F 1 6 . 8 ) STOP END C SUBROUTINE DEFINING THE MODEL SUBROUTINE C A L C R ( N , M , P , N F , R , I P A R M , R P A R M , F P A R M ) I M P L I C I T REAL*8 ( A - H , O - Z ) DIMENSION P ( M ) , R(N) COMMON X ( 2 0 ) , 7 ( 2 0 ) , SO, EPS C C IMPOSING A CONSTRAINT ON X ( I ) I F (P(3 ) . L T . (X(N)+EPS)) GO TO 131 C C PLACING RESIDUALS IN R DO 130 1=1,N DUMP = P ( 2 ) * X ( D / ( P ( 3 ) - X ( D ) R(I )=P(1 )* (SO-DUMP) - 7 ( 1 ) 130 CONTINUE RETURN - 1 9 0 - 131 NF=0 RETURN END C SUBROUTINE DEFINING THE PARTIAL D E L I V E R T I V E S SUBROUTINE C A L C J ( N , M , P , N F , D , I P A R M , R P A R M , F P A R M ) I M P L I C I T REAL*8 ( A - H , O - Z ) DIMENSION P ( M ) , D ( N , M ) COMMON X ( 2 0 ) , Y ( 2 0 ) , SO, EPS C C IMPOSING A CONSTRAINT ON X ( I ) I F ( P ( 3 ) . L T . ( X ( N ) + E P S ) ) GO TO 131 C PUTTING D E R V I A T I V E S I N D DO 130 1=1,N D ( I , 1 ) = S O - ( P ( 2 ) * X ( I ) / ( P ( 3 ) - X ( I ) ) ) D ( I , 2 ) = - P ( 1 ) * X ( I ) / ( P ( 3 ) - X ( I ) ) DUMP = P ( 3 ) - X ( I ) D ( I , 3 ) = P ( 1 ) * P ( 2 ) * X ( I ) / ( D U M P * D U M P ) 130 CONTINUE RETURN 131 NF=0 RETURN END

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