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Anaerobic fermentation of whey : acidogenesis Kisaalita, William Ssempa 1987-12-31

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ANAEROBIC FERMENTATION OF WHEY:  ACIDOGENESIS  by  WILLIAM SSEMPA KISAALITA  B.Sc.(Eng.)(Hon.),  Makerere  U n i v e s i t y , 1978  M.A.Sc., U n i v e r s i t y 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  in  THE  FACULTY OF GRADUATE STUDIES  (Department  of Chemical E n g i n e e r i n g )  We accept the t h e s i s as conforming to the r e q u i r e d standard  THE  UNIVERSITY OF BRITISH COLUMBIA January, 1987  © W i l l i a m Ssempa K i s a a l i t a ,  1987  In  presenting  degree  this  at the  thesis  in  University of  freely available for reference copying  of  department publication  this or of  thesis for by  this  his  or  partial fulfilment  of  British  I agree  Columbia,  and study.  requirements that the  I further agree  scholarly purposes her  the  representatives.  may be It  thesis for financial gain shall not  CtfBMlCfl-L-  Date  <&(  f  /gT-  advanced  Library shall make it for extensive  granted  head  is  by the  understood be  B - M G~( N &£rlQ-.| N (r-  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  an  that permission  permission.  Department of  for  that  allowed without  of  my  copying  or  my written  ii  ABSTRACT  Based on the i n i t i a l and a  methanogenesis two-phase  serial  with  Acidogenesis and  and methanogenesis  biomethanation the  current  process whey  was  takes  found  utilisation  was found t o be l e s s u n d e r s t o o d  due  that  place  examine  the g e n e r a l  studies,  i n two  t o be more  and/or  separated  suitable for  disposal  problem.  i n c o m p a r i s o n t o methanogenesis  90% o f the f i v e - d a y b i o c h e m i c a l  to l a c t o s e , continuous  (acidogenesis  i n one v e s s e l ) whey b i o m e t h a n a t i o n  t h e r e f o r e a c i d o g e n e s i s became t h e c e n t r a l problem o f t h i s Given  to  place  (acidogenesis  vessels)  dealing  takes  e x p l o r a t o r y r e s u l t s of s i n g l e - p h a s e  thesis.  oxygen demand i n whey i s  c u l t u r e (Chemostat) e x p e r i m e n t s were mechanism  of  lactose  acidogenesis  undertaken  by  a  mixed  14 undefined protein  culture using  (mainly  addressed. mesophilic  C-labeled  p - l a c t o g l o b u l i n ) on  Experimental temperature  factors  of 35°C  tracers.  A l s o t h e i n f l u e n c e of whey  the g e n e r a l  included  a  and a d i l u t i o n  pH  fermentation range  rate  o f 4.0  (D) range  scheme  was  t o 6.5, a of 0.05 t o  0.65 h " . 1  At end  a fixed  products  pH l e v e l ,  the observed  (acetate, propionate,  variability  butyrate  acidogenic  and l a c t a t e ) w i t h r e s p e c t  were found  t o be a consequence of the s y s t e m a t i c  microbial  groups  involved  i n the main  to D  s e p a r a t i o n of the v a r i o u s  i n acidogenesis.  Batch  i n c u b a t i o n of a  14 [  C(U)]-lactate tracer with  s e p a r a t i o n of the end p r o d u c t s  chemostat e f f l u e n t  samples and p r e p a r a t i v e  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 o f t h e r a d i o a c t i v i t y shift  with  i n c r e a s i n g D was  demonstrated t h a t a m i c r o b i a l p o p u l a t i o n  responsible  for disabling  the c o n v e r s i o n  of  iii  lactate  to  other  end  products  and  hence  the  observed  increase  in  lactate  c o n c e n t r a t i o n s at high D v a l u e s . Further  use  of  predominant carbon qualitative lactose  is  Pyruvate In  the  The  C ( U ) ] - b u t y r a t e and  acidogenic  converted  to  in a parallel of  i n a very  implications  14  [  f l o w r o u t e s from p y r u v a t e  lactose  presence  acetate  [  hydrogen  fast  of  reaction  the  i s then  reducing  reaction  these  via  and  C ( 2 ) ] - p r o p i o n a t e r e v e a l e d the  to the v a r i o u s end  fermentation  pyruvate  14  model  was  f i n d i n g s with  proposed,  in  Embden-Meyerhof-Parnas  converted  to  methanogens  not  products.  lactate  propionate regard  lactate  and  A  which  pathway. butyrate.  i s converted  to  as p r e v i o u s l y believed-.  to o p t i m i s i n g the a c i d o g e n i c  phase r e a c t o r are d i s c u s s e d . Acidogenic the  carbon  5.0)  was  route  of  transition In was  flow  found  and  expense  f e r m e n t a t i o n of p r o t e i n t o g e t h e r w i t h l a c t o s e d i d not  at  scheme.  to  favour  high  the  pH  In the  (pH  butyrate  >  t h e D range of 0.05 butyrate 5.5)  route,  route  the the  at  lactate pH  region  to 0.15 the  expense  route of  h \  was  5.0  affect  low pH  of  the  lactate  favoured to  5.5  (pH <  at  the  being  the  range.  order  to  describe  the  m i c r o b i a l growth,  the  Monod  chemostat  chosen among the v a r i o u s 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  physico-chemical basis.  The  e s t i m a t e d model parameters are r e p o r t e d .  model  and i t s  iv  TABLE OF CONTENTS Pages ABSTRACT  i i  TABLE OF CONTENTS  iv  LIST OF TABLES  viii  LIST OF FIGURES  x  NOMENCLATURE AND ABBREVIATIONS  xiv  ACKNOWLEDGEMENTS  xvi  I.  II.  INTRODUCTION  1  1.1  Whey  1  1.2  Whey D i s p o s a l and/or U t i l i z a t i o n  4  1.3  Biomethanation Process  4  1.4  Whey B i o m e t h a n a t i o n  7  1.5  Two-Phase P r o c e s s  11  1.6  Research O b j e c t i v e s  13  1.7  Scope of the Study  15  PREVIOUS WORK AND THEORETICAL ASPECTS  17  2.1  E n e r g e t i c s and M e t a b o l i c Stages i n A n a e r o b i o s i s  17  2.1.1  Energetics  17  2.1.2  M e t a b o l i c Stages  19  2.1.2.1  Hydrolytic Bacteria  22  2.1.2.2  The ^ - p r o d u c i n g B a c t e r i a  23  2.1.2.3  Homoacetogenic  25  2.1.2.4  The Methanogenic  2.1.2.5  S p e c i f i c B a c t e r i a l Species Associated  2.1.2.6  Bacteria Bacteria  25  w i t h Whey B i o m e t h a n a t i o n  27  Important  28  P r o c e s s Parameters  V  Pages 2.2  M a t h e m a t i c a l M o d e l i n g 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 f o r S i n g l e S u b s t r a t e L i m i t e d Growth  31  2.2.2.1  Monod E q u a t i o n  33  2.2.2.2  Other E q u a t i o n s  33  2.2.3  Models w i t h S p e c i f i c Growth S u b s t r a t e and/or Biomass Growth Dependence  36  2.2.4  M i c r o b i a l Growth M o d e l i n g Recommendation  39  2.2.5  M i s c e l l a n e o u s Models  39  2.2.5.1  Models of Growth i n P r e s e n c e of Inhibiting  2.2.5.2  2.2.5.3 2.3  III.  Substrate/Product  39  Models f o r Growth L i m i t e d by More Than One S u b s t r a t e  41  Models f o r P r o d u c t F o r m a t i o n  42  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 Models:  L i t e r a t u r e Review  ..  43  2.3.1  Whey and/or L a c t o s e S u b s t r a t e s  43  2.3.2  Other S u b s t r a t e s  46  2.4  M a t h e m a t i c a l A n a l y s i s of C o n t i n u o u s C u l t u r e  46  2.5  Experimental Plan  51  2.5.1  Assumptions  51  2.5.2  Experimental Factors  51  MATERIALS AND METHODS  53  3.1  Inoculum  53  3.2  Media  53  3.2.1  Lactose Limited  Growth S y t h e t i c Medium  3.2.2  L a c t o s e / P r o t e i n Growth Medium  53 55  vi  Pages 3.3  Fermentor Set-up  55  3.4  Fermentor S t a r t - u p and O p e r a t i o n P r o c e d u r e  58  3.5  Set-up f o r R a d i o a c t i v e T r a c e r I n c o p o r a t i o n  59  3.5.1  A p p a r a t u s w i t h pH C o n t r o l  59  3.5.2  A p p a r a t u s w i t h o u t pH C o n t r o l  61  3.6  3.7  IV.  P r e p a r a t i v e S e p a r a t i o n of O r g a n i c A c i d s  61  3.6.1  Principle  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  D e t e r m i n a t i o n of R a d i o a c t i v i t y  69  3.7.1  Principle  69  3.7.2  Channels R a t i o Method  72  3.7.3  Procedure  72  RESULTS AND DISCUSSION 4.1  75  I n f l u e n c e of D i l u t i o n Rate (D) on O r g a n i c A c i d s (OA) Distribution  4.2  I n f l u e n c e of D on Gaseous P r o d u c t s D i s t r i b u t i o n  4.3  Radio Tracer S t u d i e s  76 ••••••••••  4.3.1  R e s u l t s of the Radio T r a c e r E x p e r i m e n t s a t H i g h D  4.3.1  R e s u l t s o f the Radio T r a c e r E x p e r i m e n t s a t Low D  82 90  .  91  ..  95  4.4  Proposed L a c t o s e A c i d o g e n i c F e r m e n t a t i o n 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 F e r m e n t a t i o n Model  4.7  I n f l u e n c e o f pH on Carbon Flow from P y r u v a t e  115  4.8  M i c r o b i a l Growth Model  117  I l l  vii  Pages  V.  CONCLUSIONS AND RECOMMENDATIONS  132  5.1  Conclusions  132  5.2  Recommendations  135  LITERATURE CITED  136  APPENDIX A  155a  APPENDIX B  173  APPENDIX C  183  APPENDIX D  188  viii  LIST OF TABLES Pages T a b l e 1.1  T y p i c a l C o m p o s i t i o n Whey S o l i d s  T a b l e 1.2  E s t i m a t e d Q u a n t i t i e s o f F l u i d Whey  2  Produced i n t h e USA T a b l e 1.3  3  E s t i m a t e d Q u a n t i t i e s o f F l u i d Whey Produced i n Canada  T a b l e 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  T a b l e 2.1  O v e r a l l R e a c t i o n Schemes of Some I m p o r t a n t  3 12  Acidogenic Fermentation  20  T a b l e 2.2  G e n e r a l i s e d Growth C o n s t a n t s f o r A n a e r o b i o s i s  47  T a b l e 2.3  Factors Investigated  52  T a b l e 3.1  L a c t o s e L i m i t e d Growth Medium  54  T a b l e 3.2  L a c t o s e and P r o t e i n Growth Medium A n a l y s i s  56  T a b l e 4.1  Maximum S p e c i f i c Growth Rate V a l u e s f o r Methanogenic B a c t e r i a  T a b l e 4.2  89  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 H i g h D Radio Tracer Experiments  T a b l e 4.3  94  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  T a b l e 4.4  99  D i s t r i b u t i o n of T o t a l F r e e Energy Change f o r Growth of the Two-Phase A n a e r o b i c P r o c e s s o f G l u c o s e t o Methane Over D i f f e r e n t M i c r o b i a l Groups  T a b l e 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 With L a c t o s e / P r o t e i n Substrate  110 Experiments 116  ix  Pages Table 4.6  Monod Chemostat  Model P r e d i c t i o n s  V a l u e s Based on u  for D i f f e r e n t K  s  and Y A p p r o x i m a t i o n s f o r a pH  o f 6.0 Table 4.7  129  Comparisons of Monod Chemostat  Model C o n s t a n t s  for  Acidogenesis Table B l  T o t a l Carbon Mass B a l a n c e ( L a c t o s e S u b s t r a t e ) at  T a b l e B2  130 Growth L i m i t e d  a pH of 6.0 and a Temperature of 35°C  T o t a l Carbon Mass B a l a n c e ( L a c t o s e / P r o t e i n L i m i t e d S u b s t r a t e ) at  Growth  a pH of 6.0 and a Temperature  of 35°C T a b l e B3  176  T o t a l Carbon Mass B a l a n c e ( L a c t o s e Growth L i m i t e d S u b s t r a t e ) at a pH o f 4 . 5 and a Temperature o f 35°C  T a b l e B4  174  177  T o t a l Carbon Mass B a l a n c e ( L a c t o s e Growth L i m i t e d S u b s t r a t e ) at a D i l u t i o n Rate of 0.05 h ^ and a Temperature o f 35°C  T a b l e 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 o f  178 Cummulative  E x p e r i m e n t a l Time at a Temperature o f 25°C and a P r e s s u r e 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  Biomethanation  Processes  F i g u r e 1.2  Five Basic Anaerobic Fixed F i l m Reactor  F i g u r e 1.3  Three B a s i c A n a e r o b i c F l o c s / N o n - A t t a c h e d  6 Types  8  Film  R e a c t o r Types F i g u r e 2.1  9  O v e r a l l R e a c t i o n Schemes of Some I m p o r t a n t Acidogenic Fermentation  F i g u r e 2.2  Effect  21  of P a r t i a l P r e s s u r e of Hydrogen on the  Energy Change Propionate from H / C 0 2  F i g u r e 2.3  Types  for  the D e g r a d a t i o n  and B u t y r a t e  Free  of E t h a n o l ,  w i t h Methane  Formation 24  2  Possible Perspective  Interactions for  Cell  Population K i n e t i c Representation F i g u r e 2.4  A C o m b i n a t i o n of E q u a t i o n s Biomass C o n c e n t r a t i o n  f o r the  32 Substrate and/or  Dependence of the  Specific  Growth Rate  40  Figure 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  F i g u r e 3.1  Schematic  Diagram of the Fermentor  and  (CSTR)  F i g u r e 3.3  57  Schematic  Diagram o f the R a d i o t r a c e r  Apparatus  w i t h pH C o n t r o l  Modified v i a l  for Radiotracer  Experiment 60  Experiments  W i t h o u t pH C o n t r o l F i g u r e 3.4  48  Auxilliary  Apparatus F i g u r e 3.2  ...  T y p i c a l Chromatogram  62 of a Complex M i x t u r e S e p a r a t e d  by a C o m b i n a t i o n of S i m p l e and S t e p w i s e  Elution  65  xi Pages F i g u r e 3.5  F i g u r e 3.6  Schematic  Diagram of the L i q u i d  Equipment  Assembly  Chromatography 66  T y p i c a l O r g a n i c A c i d s Chromatogram  70  14 F i g u r e 3.7 F i g u r e 3.8  F i g u r e 4.1  C Spectra Efficiency  73 Curve P r e p a r e d  From Commercial  Quenched  Standards  73  Products  D i s t r i b u t i o n as a F u n c t i o n of  Dilution  Rate at a pH of 6.0 Figure 4.2  78  P o s s i b l e Lactose Fermentation  Models to V a r i o u s  End P r o d u c t s  83  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  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 Where the pH was M a i n t a i n e d at of 6.0 Throughout  Production  the D e s i r e d V a l u e  the E x p e r i m e n t a l P e r i o d  F i g u r e 4.5  Fermentor  F i g u r e 4.6  A d a p t a t i o n Mechanism i n Organisms  86  Head Space Gas A n a l y s i s  88 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  93  F i g u r e 4.8  pH-Tirae R e l a t i o n s h i p Incorporation  F i g u r e 4.9  f o r Run Number C4 for a Batch  Radiotracer  Experiment W i t h o u t pH C o n t r o l  Radiochromatogram  f o r Run Number C l l  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  96 97  for  14 [ C ( U ) ] - B u t y r a t e Tracer at Various Batch Experiment Times  100  xii Pages F i g u r e 4.11 Recovered R a d i o a c t i v i t y 14 [ C(U)]-Lactate  Distribution for  Tracer at Various  Batch  Experiment Times  101 14  Figure  4.12 F e r m e n t a t i o n Time Course f o r [ Degradation i n Single-Phase  C(U)]-Lactate  Lactose  Fermentor  Sample F i g u r e 4.13 The  104  .Microbial  Lactose  F e r m e n t a t i o n Model i n  Three D i s t i n c t But Simultaneous T r o p h i c F i g u r e 4.14 The M i c r o b i a l  Acidogenic  F e r m e n t a t i o n Model  F i g u r e 4.15 Comparison of the Main P r o d u c t s for Lactose/Protein  Phases  and L a c t o s e  Substrates 114  D i s t r i b u t i o n as a F u n c t i o n  a D i l u t i o n Rate o f 0.05 h F i g u r e 4.17 P r o d u c t s  Distribution  of pH a t 118  _ 1  as a F u n c t i o n  of D i l u t i o n  Rate a t a pH o f 4.5 F i g u r e 4.18 E x p e r i m e n t a l Consistent  119  Continuous C u l t u r e Data  with  F i g u r e 4.19 E x p e r i m e n t a l  Quantitatively  the O r i g i n a l Monod Chemostat Model  i n a Pure C u l t u r e of A e r o b a c t e r  Contrary  106  Distribution  Experiments F i g u r e 4.16 P r o d u c t s  105  Aerogenes  121  Continuous C u l t u r e Data With a Trend  to the O r i g i n a l Monod Chemostat Model i n a  Pure C u l t u r e of A e r o b a c t e r  Aerogenes i n a  Glycerol  Medium F i g u r e 4.20 Dry Biomass C o n c e n t r a t i o n  121 as a F u n c t i o n  of D i l u t i o n  Rate a t a pH of 6.0 F i g u r e 4.21 Dry Biomass C o n c e n t r a t i o n Rate a t a pH of 4.5  122 as a F u n c t i o n  of D i l u t i o n 123  xiii Pages F i g u r e 4.22  Influence of  of the Maintenance  Coefficient  Shape  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 Experimental Figure A l  on the  i n Comparison W i t h  Data f o r a pH of 4 . 5  128  C a l i b r a t i o n Curve f o r L a c t o s e U s i n g the  Phenol-Sulfuric  A c i d Method F i g u r e A2  158  C a l i b r a t i o n Curve f o r P r o t e i n U s i n g the  Biuret-Reaction  Method F i g u r e A3  160  C a l i b r a t i o n Curve f o r Formate U s i n g the Method of Lang and Lang (1972)  F i g u r e A4  162  C a l i b r a t i o n Curve f o r L a c t a t e  U s i n g a M o d i f i e d Method  o f 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 A u t o m a t i c  Carbon  Analyser Figure C l  171  Break-down of G l u c o s e to Two P y r u v a t e Meyerhof-Parnas  v i a Embden-  Pathway  F i g u r e C2  P a t h of B u t y r a t e  F i g u r e C3  F o r m a t i o n of P r o p i o n a t e ,  184  F o r m a t i o n From G l u c o s e Acetate  From D L - L a c t a t e by Megasphaera  and  Carbondioxide  e l s d e n i i and C l o s t r i d i u m  propionicum F i g u r e C4  F o r m a t i o n of L a c t a t e  185  186 v i a the  Pathway by P r o p i o n i b a c t e r i a  Succinate-Propionate 187  xiv  NOMENCLATURE AND ABBREVIATIONS  ATP  Adenosine t r i p h o s p h a t e  BOD  B i o c h e m i c a l oxygen demand (mg/L)  COD  Chemical  CSTR  Continuous  D  Dilution  DNA  Deoxyribonucleic  EMP  Embden-Meyerhof-Parnas  F  Fermentor f e e d stream  k  Constant  K'  K i n e t i c constant  oxygen demand (mg/L) flow s t i r r e d  tank r e a c t o r  r a t e (h ) 1  acid pathway f l o w r a t e (mL/h)  i n Konak's m i c r o b i a l growth model; see e q u a t i o n 2.6 i n Chen and Hashimoto's m i c r o b i a l growth model;  see e q u a t i o n 2.19 K^  Constant  i n Dabe's m i c r o b i a l growth model;  see e q u a t i o n 2.11  K„  Constant  i n Dabe's m i c r o b i a l growth model; see e q u a t i o n 2.10  D  K^  M i c r o b i a l decay r a t e (h  K^  Substrate i n h i b i t i o n constant  K  Product  K  P s  i n h i b i t i o n constant  Monod s a t u r a t i o n c o n s t a n t  L  Constant  M  Parameter i n Rogues equation  (ug/mL)  (ug/mL)  (ug/mL)  i n P o w e l l ' s m i c r o b i a l growth model; see e q u a t i o n 2.12 and co-workers m i c r o b i a l growth model; see  (ug/mL) o r maintenance  NAD  E l e c t r o n c a r r i e r co-enzyme,  NADH  Reduced  form  OA  Organic  acids  p  Constant  coefficient  n i c o t i n a m i d e adenine d i n u c l e o t i d e  of NAD  i n Konak's m i c r o b i a l growth model;  see e q u a t i o n 2.6  XV  RT  Retention  RNA  Ribonucleic  S  C o n c e n t r a t i o n of l i m i t i n g n u t r i e n t (u-g/mL)  s  o  R  fc  time (h) acid  S v a l u e i n feed stream  (p,g/mL)  R e l a x a t i o n time ( s e c )  fc  H y d r a u l i c r e t e n t i o n time (h)  V  CSTR working volume (mL)  VFA  Volatile  fatty acids  VSS  Volatile  suspended s o l i d s (g)  WPC  Whey p r o t e i n c o n c e n t r a t e  X  Organism c o n c e n t r a t i o n i n the fermentor  X  Maximum v a l u e of X t h a t may be  HR  m Y  (ug/mL)  reached  Yield  coefficient  (g o f c e l l / g o f l i m i t i n g n u t r i e n t )  Yield  coefficient  f o r compound j on compound i  Greek Symbols AG°  F r e e energy of r e a c t i o n a t a temperature o f 25°C and p r e s s u r e o f one atmosphere  u u X  Specific m  m i c r o b i a l growth r a t e (h ^)  Maximum u v a l v e (h *) Constant  i n Moser's m i c r o b i a l growth model; see e q u a t i o n  2.12  xvi  ACKNOWLEDGEMENTS  The Dr.  author  K.L.  Finder  encouragement thankful  wishes and  throughout  i s indebted  Sciences  without  thanks  whose  completed. excellent  support  his  sincere  for their  of t h i s  appreciation  advice,  research  direction  project.  a member o f h i s r e s e a r c h  for financial  go  Lo  the course  and review  and E n g i n e e r i n g  Special  express  D r . K.V.  to Dr. R.M.R. B r a n i o n ,  constructive criticisms He  to  to and  He i s a l s o  committee,  for h i s  o f the t h e s i s . support  of t h i s  research  to the N a t u r a l  Research C o u n c i l of Canada and A g r i c u l t u r e Canada. to h i s f a m i l y  and "demands",  L a s t but not l e a s t ,  members,  this  special  Rose,  research  Ntumwa  would  and Nkaaku,  probably  never be  thanks go to Miss H e l s a Wong f o r the  j o b o f t y p i n g the t h e s i s a t such a s h o r t n o t i c e .  - 1 -  I.  INTRODUCTION  1.1 Whey Whey  i s an opaque,  following cheese.  the removal Different  characteristics, obtained  very  of the c u r d ,  cheese  typical  from  differentiate  greenish-yellow f l u i d  i t from  quantity  of m i l k used  whey  process.  milk  is  indicated  i n Table  (predominantly  i n cheese  Despite  the  possibilities  favourable  processing,  disposal  biochemical  oxygen  mg/L,  and  utilization  is  f a t and  content,  (BOD)  1979),  so  effectively  a  the  5%  ash  1%  offers  manufacturer. ranges  of  of  The  1974).  a  five-day  30,000  disposal  lactose  protein  and o f t e n  between  whey  1976).  interesting  and  to streams  i s due to the l a c t o s e  question  For a  (Loehr,  i s an e x p e n s i v e  question  to  A c i d whey forms a  lactose,  0.6%  p r o c e s s , making  90% of the BOD  "sweet"  a p p r o x i m a t e l y 10% of the  which  f o r whey  on the cheese  Generally Kramer,  0.3%  as  the whey  i n N o r t h America.  about  f o r the cheese  demand  to  into  different  whey (Harper and H a l l ,  r e c o v e r y , whey  problem  depending  unacceptable. (Green  contains  nutrient  f o r by-product  frustrating  60,000  1.1, whey  milk  As an example,  whey p r o d u c t i o n  p-lactoglobulin),  i n a cheese v a t  somewhat  referred  weight ends up as cheese, t h e b a l a n c e as f l u i d As  with  whey of c o t t a g e cheese.  s m a l l p e r c e n t a g e o f the t o t a l  given  produce  o f the cheese  the " a c i d "  remains  i n the p r o c e s s of c o n v e r t i n g  varieties  rennet-coagulated  that  component  disposal  and/or  disposal  and/or  utilization. Fluid  whey  production  billion  tonnes and 2 m i l l i o n  1.3).  With  the i n c r e a s i n g  i n the USA  and  Canada  was  approximately  27  tonnes r e s p e c t i v e l y d u r i n g 1985 ( T a b l e s 1.2 and cheese  p r o d u c t i o n i s expected to i n c r e a s e .  demand  i n North  America,  fluid  whey  -  T a b l e 1.1  2  -  T y p i c a l C o m p o s i t i o n of Whey S o l i d s  Component  Carbohydrates Lactose Proteins p-Lactoglobulin ot-Lactoalbumin Immunoglobulins  Composition ( g / 1 0 0 mL)  5.00  .66 .22 .10  Fat Triglycerides Ash Total % Solids  .30 .6 6.88  - 3 -  T a b l e 1.2  E s t i m a t e d Q u a n t i t i e s of F l u i d Whey Produced i n the USA.  Cheese Type  American(Cheddar)  Cottage Other Total  1981  9,700 6,091 6,193  10,786 5,801 6,250  11,235 5,626 6,773  21,984  22,837  23,634  Values c a l c u l a t e d using annual " D a i r y P r o d u c t s " , p u b l i s h e d by S t a t i s t i c s Reporting Service. of m i l k u s e d , 0.1 kg end up as  T a b l e 1.3  B i l l i o n Kilograms 1982 1983  1980  cheese the US I t was cheese  Total  1985  11,953 5,552 7,201  10,810 6,388 8,271  11,651 6,365 8,859  24,706  25,470  26,875  p r o d u c t i o n f i g u r e s from, Department of A g r i c u l t u r e , assumed t h a t f o r e v e r y kg and 0.9 kg as f l u i d whey.  E s t i m a t e d Q u a n t i t i e s of F l u i d Whey Produced i n Canada.  Cheese Type  Cheddar Cottage Other  1984  M i l l i o n Kilograms 1982 1983  1980  1981  956 260 640  907 277 684  802 286 727  1,856  1,868  1,815  1984  1985  895 284 752  912 276 816  976  1,931  2,004  V a l u e s c a l c u l a t e d u s i n g a n n u a l cheese p r o d u c t i o n f i g u r e s from, " D a i r y R e v i e w " , 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 t h a t f o r e v e r y kg o f m i l k u s e d , 0.1 kg end up as cheese and 0.9 kg as f l u i d whey.  -  882  -  - 4 1.2  Whey D i s p o s a l a n d / o r  Utilization  Due t o t h e 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 into  developing  recovery are:  new  and new p r o d u c t  fermentation  Gerhardt  (Yang  Friend  treatment,  fermentation  to ethanol  e t . a l . , 1976; Palmer,  and  Shahani,  drying  supplement  i n human  to  food;  which  feeding  separation  may  be  permeate  from  separation  schemes  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 and n o n - a l c o h o l i c  large dairy establishments a  low v a l u e  bulky  or  1976;  gasohol  as  animal  beverage  feed  or  ( M u l l e r , 1979;  by membrane  technology  (Watson e t . a l . , 1977). i s usually  (Delany,  1981).  production  high Most  schemes  a  i n BOD  The and  fermentation,  are l i m i t e d  to  (> 41 m i l l i o n kg f l u i d whey/year) and, whey b e i n g  product,  o f t e n uneconomical (Modler  these  (Reddy e t . a l . ,  to l i v e s t o c k  components  e t . a l . , 1982; and l a n d a p p l i c a t i o n membrane  product  Among  non-alcoholic  used  directly of  out.  on  f o r beverage  1979);  (Teixeira  separation  emphasis  1978 & 1979; B e r s t e i n and Tzeng,  1979; E v e r s o n ,  powder  e t . a l . , 1980);  membrane  with  t o p r o t e i n o r n i t r o g e n - r i c h feeds  production;  Modler  o f whey  development, have been c a r r i e d  e t . a l . , 1978);  production 1977;  schemes  o f whey, numerous I n v e s t i g a t i o n s  t r a n s p o r t a t i o n to a c e n t r a l processing u n i t i s e t . a l . , 1980).  D r y i n g r e q u i r e s a l o t o f energy.  A l s o t h e m e r i t s o f l a n d a p p l i c a t i o n a r e y e t t o be e s t a b l i s h e d . Disposal forms:  schemes  have  dumping d i r e c t l y  (Muller, treatment  1979). facility  taken  one o r a  o f the f o l l o w i n g  i n t o sewers o r w a t e r systems and a e r o b i c  The a u t h o r  i s not aware o f any f u l l  i n o p e r a t i o n a t the present  the M i l l b a n k Cheese and B u t t e r p l a n t ( B e l l m a n ,  1.3  combination  treatment  scale anaerobic  whey  time, w i t h the e x c e p t i o n of 1986).  Biomethanation Processes The  b i o c o n v e r s i o n o f o r g a n i c m a t e r i a l t o methane and c a r b o n d i o x i d e i n  the absence o f m o l e c u l a r  oxygen i s r e f e r r e d t o as t h e b i o m e t h a n a t i o n  process  - 5 -  or  a n a e r o b i c methane f e r m e n t a t i o n .  o r g a n i c compounds i s performed and in  methane f o r m i n g . Chapter  offer  II.  several namely:  yield;  lower  two  Biomethanation  a  this  groups  over  higher  nutrient  of b a c t e r i a ,  processes  degree  of  waste  requirements;  no  stabilisation;  oxygen  a  various for  can  be  c o o k i n g and  the  medium  million  kg  s t r e n g t h o r g a n i c waste  consumed  by  the  cheese  heating operations.  (14-41  organic  million  kg  microbial  and  a r e pronounced i n addition,  methane  s i n c e whey the  methane  processing f a c i l i t y i t s e l f A l s o the p r o c e s s may  fluid  f l u i d whey/year) p l a n t s  and  wastes  (McCarty,  lower  requirement;  constitutes  forming  i s considered  the c o n v e n t i o n a l a e r o b i c p r o c e s s e s  W i t h r e g a r d t o whey, t h e s e advantages  generated  the a c i d  groups  for stabilising  production.  high  a n a e r o b i c d e g r a d a t i o n of  F u r t h e r s u b d i v i s i o n s of the two  advantages  1966),  by  Basically  whey/year) and  f o r which  be  suitable  small scale  the economics of  for  (<  14  alternative  whey u t i l i z a t i o n p r o c e s s e s a r e not f a v o u r a b l e . The phased  t h r e e most common p r o c e s s processes  (Figure  biomethanation f o r t h i s that  has  often  been a t t r i b u t e d involved  1.1).  purpose  to  the  lack  i s p r o b a b l y due  i n the p r o c e s s .  Other  a temperature  various  substances  and  (Switzenbaum, biochemistry  large  1983). along  the  parallel,  lack  of  More r e c e n t l y , advances  of  application  the  fundamental  and  Jewell,  1980;  Switzenbaum  1969; and  reaction  i n basic microbiology  bio-reactor  technology,  L e t t i n g a e t . a l . , 1980; Danskin,  1982;  poor  to degrade  because of s l o w  advances  concepts  p a r t i c u l a r w i t h i m m o b i l i s e d b a c t e r i a systems, sometimes r e f e r r e d t o as f i l m r e a c t o r s (Young and M c C a r t y ,  of  has  have been  of 35°C, i n a b i l i t y  in  and  This u n r e l i a b i l i t y  disadvantages h i s t o r i c a l l y requirement  staged  to the u n r e l i a b l e o p e r a t i o n  volume r e q u i r e m e n t s  with  the  process.  of u n d e r s t a n d i n g  stability,  and  G e n e r a l l y , the  been a s s o c i a t e d w i t h  process  rates  layouts are,  Boeing  in  fixed  Switzenbaum and  Larsen,  -  F i g u r e 1.1  6  -  Three d i f f e r e n t b i o m e t h a n a t i o n p r o c e s s e s : (1) i n f l u e n t stream, (2) s o l i d / l i q u i d s e p a r a t o r , (3) a c i d ( o n l y ) p r o d u c i n g r e a c t o r , (4) methane p r o d u c i n g r e a c t o r , (5) gas, (6) e f f l u e n t stream.  -  1982; most  Bull  e t . a l . , 1984; B u r g r e s s  o f t h e problems  distinguish one  7 -  and M o r r i s , 1984) have h e l p e d  associated with  between r e a c t o r t y p e s  anaerobiosis.  and p r o c e s s  I t i s important to  layout.  I n the l a t t e r  o r s e v e r a l r e a c t o r s a r e i n c l u d e d i n a t o t a l p r o c e s s scheme.  Harremoes  (1983)  have  classfied  a  number  overcome  of r e a c t o r s ,  which  case  Henze and have  been  i n v e s t i g a t e d o r marketed d u r i n g t h e l a s t 5-10 y e a r s , i n t o e i g h t b a s i c t y p e s , shown i n F i 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 from  (1952)  a knowledge  empirical  formula  of the chemical  that  composition  predicts  methane  production  of the  degraded  m a t e r i a l , t h e f o l l o w i n g f o r m u l a c a n be w r i t t e n f o r t h e breakdown o f  the l a c t o s e i n whey:  C  Therefore  12 22°11 H  one gram o f l a c t o s e  2 2 . 4 1 2 ) = 0.3722 l i t r e s  +  H  2° *  6 C 0  2  +  6 C H  4  (.002775 m o l e s ) would y i e l d  o f CH^ a t one a t m o s p h e r e .  (6 x .002775 x  F o r a l i t r e o f whey,  methane p r o d u c t i o n i s a p p r o x i m a t e l y 19 l i t r e s . Most  of  biomethanation performance  the laboratory o f whey  experiments  or lactose  have  conducted  been  to date  designed  on t h e  to evaluate the  o f i m m o b i l i s e d b a c t e r i a r e a c t o r systems ( H i c k e y and Owens, 1981;  Yang e t . a l . , 1984; Switzenbaum and D a n s k i n ,  1982; B o e i n g and L a r s e n , 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). Microbial designs present  and b i o c h e m i c a l k i n e t i c  c a n be based author  to  i s virtually experimentally  i n f o r m a t i o n on w h i c h non-existent. generate  rudimentary  process  E f f o r t s were made by t h e  the m i c r o b i a l  kinetics  and  -  8  -  Fixed  •e-  Figure  1.2  Five basic anaerobic  bed  Moving  bed  Expanded  bed  Fluidised  bed  Recycled  bed  fixed film reactor  types.  -  Oo o  9  -  J Recycled floes (contact reactor)  OQ i L  I  0 - o O  -I I I I  Sludge blanket reactor (Clangester type reactor)  o O 0  0 0  oo  F i g u r e 1.3  Three b a s i c a n a e r o b i c r e a c t o r types.  Digester  flocs/non-attached  film  - 10 -  b i o c h e m i c a l d a t a f o r l a c t o s e i n f e d - b a t c h , s i n g l e - p h a s e , one l i t r e for  various  dilution  (unpublished work). to  (D)  rates  and  influent  r a p i d l y and gas p r o d u c t i o n  T h i s was r e p e a t e d s e v e r a l t i m e s , but each experiment  "sour" r e a c t o r .  concentration  A t t h e s t a r t o f the e x p e r i m e n t s the s u b s t r a t e was added  the r e a c t o r s and w i t h i n a few days t h e pH f e l l  ceased.  lactose  reactors,  resulted i na  S i m i l a r u n s u c c e s s f u l l e x p e r i m e n t s employing whey o r l a c t o s e  have been r e p o r t e d by a number o f i n v e s t i g a t o r s ( M a r s h a l l and T i m b e r s , 1982; Dehaast  e t . a l . , 1983; K e l l y  was a s c r i b e d order  t o the r a p i d  to solve  this  substrate  The r a p i d drop i n pH  f o r m a t i o n o f o r g a n i c a c i d s (OA) from l a c t o s e .  problem, Dehaast  n e u t r a l i s e d the a c i d , d i l u t e d the  and Switzenbaum, 1984).  and co-workers  prefermented  In  the whey,  the p r e f e r m e n t and used t h e d i l u t e d p r o d u c t as  f o r biomethanation.  Follmann  and M a e r k l  (1979)  used  a  p H - s t a t i c p r o c e s s i n which t h e c o n t r o l s i g n a l f o r t h e a d d i t i o n o f f r e s h whey was the pH v a l u e . add  substrate  substrate and  When the pH i n c r e a s e d beyond 7.0, a pump was t r i g g e r e d to  automatically  until  t h e pH f e l l  pump was s t o p p e d , u n t i l  t h e pH a g a i n passed  t h i s t r i g g e r e d a r e p e a t o f t h e sequence.  were  able  residence would  to achieve time  necessitate  pointed  98% c h e m i c a l  (RT) o f 12.5 days.  out that  large their  reactor observed  t o 6.95, a t w h i c h  oxygen  the pH = 7.0  level,  W i t h t h i s k i n d o f c o n t r o l they demand  (COD) r e d u c t i o n  The RT o f 12.5 days volumes.  l e v e l the  However  l o n g RT c o u l d  i s v e r y l o n g and  Follmann  be reduced  at a  and M a e r k l  approximately i n  h a l f i f i m m o b i l i s e d b a c t e r i a r e a c t o r s were employed. The  above  o b s e r v a t i o n s suggested  that  perhaps  a two-phase p r o c e s s as  p r e v i o u s l y proposed by B a b b i t and Baumann ( 1 9 5 8 ) , Andrews and P e a r s o n and or  Pohland whey  and Ghosh (1971)  permeate.  c o u l d be more s u c c e s s f u l l  i n stabilising  (1965) whey  - 11 -  1.5  Two-Phase  As  Process  already  Indicated,  in  first  converted  the  a  two-phase  reactor are  to  methane  b a c t e r i a (methanogenesis).  for  the  different  two-phase between  different  Separation  of  phase by the  industrial  is  more  opportunity  groups  of  b a c t e r i a than  two-phase  wastes  mixed  process  f o r s e v e r a l types (Table  acid-phase  reactor.  to  process.  control  1.4).  carbohydrate  are  i n one-phase  has high  been BOD  These  r e a c t o r and  A l l plants  the  of  in a  substrate  flow  content  of  low  at  and  hardly  any  two-phase p r o c e s s .  I n the  few  of carbohydrates  were  solids  two-phase p l a n t s c o n s i s t of anaerobic  i n the the  sludge-blanket  mesophilic  wastes  with  distribution,  is  previous  (Ghosh e t . a l . , 1975;  as  the  regard  optimum  microbial kinetics  and  a p p l i c a b l e to  is  temperature predominantly  literature  Bull  pH,  acidogenesis  l a b o r a t o r y s t u d i e s on Massey and  main s u b s t r a t e s . to  a  sucrose.  that  Zeotemeyer e t . a l . , 1982;  employed  authors  and  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 o f OA  methanogenesis  1979;  pilot  suspended  As i n d i c a t e d i n the next c h a p t e r , t h e r e i s a l a r g e volume of  al.  different  Also  applied  and  an u p f l o w  operated  a - g l y c o s i d i c t y p e s such as s t a r c h and  concerning  the  makes i t p o s s i b l e t o 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  there  the  levels  and  serial  bacteria  Given  advantages,  range  forming  is  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 .  these  methane  i n a second  lactose  where  i s no  completely  acid  or  processes,  there  (SS)  reduced  the  whey  groups of b a c t e r i a i n v o l v e d i n the  process  full-scale  process,  by  The  groups of m i c r o - o r g a n i s m s  then  OA  (acidogenesis). forming  OA  in  Pohland,  e t . a l . 1984) The  glucose  obtained  acidogenic  reactor  overall  degradation  a  acidogenesis  1979;  results  in  or  by  Cohen e t . and the  starch above  effluent  scheme a r e  OA  valid  - 12 -  T a b l e 1.4  Two-Phase P i l o t  and F u l l - S c a l e  Year  Industry  Location  Plant Type  1977  Distillery (enzyme-alcohol)  Belgium  Pilot  180  1980  Beet  West Germany  Pilot  45  Distillery (yeast-alcohol)  Belgium  Pilot  135  Beet  Belgium  Pilot  170  West Germany  Pilot  120  West Germany  Pilot  45  Belgium  FullScale  350  1981  Sugar  Sugar  Citric Beet  Acid  Sugar  Plants  Capacity ( k g COD/day)  1980  Flax Retting  1982  S t a r c h to G l u c o s e West Germany  FullScale  20,000  1983  Yeast-Alcohol  Netherlands  FullScale  20,000  1984  Yeast  France  FullScale  7,000  Reference  Ghosh e t . a l . (1985)  Berkovitch  (1986)  - 13 -  for  carbohydrates  with  cx-1,  4  glucosidic  bond  and  may  not  be  valid  for  l a c t o s e (8-1, 4 l i n k a g e ) .  1.6  Research O b j e c t i v e s  The  o v e r a l l o b j e c t i v e s of t h i s s t u d y were two  1.  To  determine  the  the m i c r o b i a l k i n e t i c s  degradation  of  lactose  by  fold:  and  a  the g e n e r a l mechanism f o r  mixed,  undefined  acidogenic  14 bacteria, using 2.  C-labeled  tracers.  To d e t e r m i n e  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  degradation  mechanism  of  lactose  if  degraded  together  the with  lactose. 14 To  a limited  extent,  the p a t h of c a r b o n  and  C - l a b e l e d t r a c e r s have been used t o  e l e c t r o n flow during anaerobosis  i n d i v e r s e ecosystems ( J e r i s and M c C a r t y , 1965; .1982;  Lovley  and  biomethanation,  Klug,  1982;  C h a r t r a i n and  Koch  et.  Zeikus  of s p e c i f i c  matter  Weng and J e r i s , 1976;  Cohen,  a l , 1983).  (1986a and  With  regard  b) have used the  t o propose a c a r b o n and e l e c t r o n f l o w r o u t e f o r s i n g l e - p h a s e Physical system  s e p a r a t i o n of a c i d o g e n i c and may  lead  to  p o p u l a t i o n s as w e l l (Cohen  et.  important as  in  the  composition  of  i n the i n t e r m e d i a r y r o u t e s of s u b s t r a t e  a l . , 1980).  This  has  been  considered  to  whey  technique  biomethanation.  methanogenic b a c t e r i a i n a  changes  determine  a  two-phase bacterial  degradation  strong  enough  14 justification addition, opportunity  for studying acidogenesis  separation to study  of  the  two  the p a r t i c u l a r  of l a c t o s e w i t h  metabolically related sub-population 14  knowledge o f t h i s a u t h o r , no s t u d y u s i n g  C tracers. groups  gives  i n the r e a c t o r .  To  In the the  C - l a b e l e d s u b s t r a t e s to e l u c i d a t e  an a c i d o g e n i c d e g r a d a t i o n scheme of l a c t o s e , has been conducted p r e v i o u s l y .  - 14 -  All  the p r e s e n t  accomplished  with  experimental  mixed,  work, d e s c r i b e d  undefined  cultures.  i n later  the complex n a t u r e  requires  an  reality. certain  degree  microbiological or  of  which when  implies  a  not too d r a s t i c  the i n t e n t i o n i s to g a t h e r  practical  applicability,  methods  d e s c r i p t i o n such as t h a t o b t a i n e d  s t u d i e s of mixed and w e l l d e f i n e d the  bacterial  relevant.  On  engineering  problems w i t h  other  hand,  populations  as  specific  a  biologically  results  under  important  proper  allow  features  such  a  as  of  wanting  to  less solve  has o f t e n  c o n s i s t i n g of reactants  conditions,  adaptation  This  but  has  approach ignored  a t the r e g u l a t o r y o r  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 . these  reasons  intermediate the  i t was  path  found  between  useful  to  direct  this  the two extremes m e n t i o n e d ,  f i e l d s of pure and a p p l i e d r e s e a r c h .  a  better  might be c o n s i d e r e d  of w a s t e s , a n a e r o b i o s i s  process  of  information with  which  consequence  types  simplification  ( o r g a n i c m a t t e r and n u t r i e n t s ) and a c a t a l y s t ( t h e b a c t e r i a ) . yielded  populations,  w i t h pure c u l t u r e s t u d i e s  been t r e a t e d as a form o f heterogeneous c a t a l y s i s ,  has  have  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 ,  approach  Therefore,  was  A m i c r o b i o l o g i s t might  r e s e r v a t i o n s about the use of t a x o n o m i c a l l y u n d e f i n e d but  chapters,  The c h o i c e  study  towards  i n order  For an  t o connect  of l a c t o s e as the s o l e  c a r b o n source was made to a v o i d the o c c u r r e n c e  o f s i d e r e a c t i o n s ( E l s d e n and  Hilton,  that  1978; H i r o s e  and S h i b a i ,  1980),  scheme c o u l d be d e t e r m i n e d more p r e c i s e l y . influence First lipids  so  the l a c t o s e  The s i g n i f i c a n c e of s t u d y i n g the  o f 8 - l a c t o g l o b u l i n on l a c t o s e d e g r a d a t i o n  pathway was  o r g a n i c wastes a r e f r e q u e n t l y composed of c a r b o h y d r a t e s , i n various combinations.  addressed  the a n a e r o b i c  No s t u d y  degradation  degradation  two-fold:  p r o t e i n s and  was found i n t h e l i t e r a t u r e which  mechanism of "complex" o r g a n i c  wastes.  - 15 -  Previous  work has m a i n l y  individually  and t h e i r  i n v o l v e d complex  intermediary  a c i d s , e t c . ) degraded s e p a r a t e l y . degradative  pathway  of  an  organic  metabolities  waste c o n s t i t u e n t s  ( e g . amino  acids,  The i m p o r t a n c e of s e p a r a t e l y s t u d y i n g a  intermediary  h i g h l i g h t the p o s s i b i l i t y o f d e t e r m i n i n g  metabolite  like  leucine  o f such a s t u d y  leucine,  i f  metabolites  the  However t h e  a r e o n l y u s e f u l f o r a complex waste t h a t  other  constituents  do n o t a l t e r  i s to  s p e c i f i c m e t a b o l i c r o u t e s which c a n  be used as models f o r t h e breakdown o f a number o f amino a c i d s . results  fatty  the p a r t i c u l a r  and  their  derived  contains  intermediary  microbial population.  Secondly,  w h i l e a s y n t h e t i c waste t h a t c o n t a i n s l a c t o s e may o n l y s e r v e as an a n a l o g o f whey permeate from whey membrane p r o c e s s e s  l a c t o s e and 8 - l a c t o g l o b u l i n may s e r v e  waste t h a t c o n t a i n s whey waste  1.7  f o r protein recovery, a synthetic as a whole  fluid  analog.  Scope o f Study  The  important  composition,  factors  pH and d i l u t i o n  i n the rate.  study  were  Specifically,  identified  as  substate  t h e scope of t h i s  study  c o n s i s t e d o f t h e f o l l o w i n g major t a s k s . 1.  W i t h l a c t o s e as t h e 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 t h e o r g a n i c acids rate  distribution and pH.  microbial  The  and t h e biomass latter  with  independent  respect  to the d i l u t i o n  v a r i a b l e was  required f o r  growth m o d e l i n g and t h e 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 t o t h e p o t e n t i a l l a c t o s e f e r m e n t a t i o n model. 2.  Utilise degradation  a  radiotracer  methodology  scheme proposed i n t a s k 1.  to  confirm  the  lactose  - 16 -  3.  W i t h 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 r a d i o t r a c e r methodology scheme  for lactose  to e s t a b l i s h the e f f e c t on the d e g r a d a t i o n  established  i n task  2  of d e g r a d i n g a  w i t h the l a c t o s e . 4.  a  E s t i m a t e k i n e t i c parameters f o r the m i c r o b i a l growth  model.  protein  II.  PREVIOUS WORK AND THEORETICAL ASPECTS  In on  this  energy  chapter,  t h e t h e o r e t i c a l a s p e c t s as w e l l as the p r e v i o u s work  conservation  for  anaerobiosis  2.1  Energetics 2.1.1  i n microbial  systems and m i c r o b i a l  and M e t a b o l i c  Stages i n  Anaerobiosis  Energetics  which s u p p o r t s growth o f a  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 p r o c e s s e s . potential  energy  i s released  by a reduced  reduced compound w h i c h a c t s as an o x i d i s e r . transferred  heterotrophic  C h e m i c a l l y bound  organic  compound  By t h i s  reaction electrons are  with  from t h e reduced compound ( e l e c t r o n donor) t o the more  (electron  acceptor).  Since  fermentation  p r o c e s s , o x i d a t i v e r e a c t i o n s must be a n a e r o b i c . reactions  modeling  i s explored.  The mechanism of energy g a i n  compound  growth  two e l e c t r o n s  a r e removed from  is a strictly  Usually  the s u b s t r a t e  i n these molecule  a  less  oxidised anaerobic oxidative  (Equations  2.1 and 2 . 2 ) .  2e x  (2.1)  y  (2.2)  Compound x i s o x i d i s e d  t o compound y, and two e l e c t r o n s  NAD, a common e l e c t r o n c a r r i e r coenzyme. limiting  amounts.  Wolfe  (1983)  a r e t r a n s f e r r e d to  Coenzymes a r e p r e s e n t I n c e l l s i n  has l i k e n e d  these  electron  c a r r i e r s to  - 18 -  trucking  systems.  Unless  there  i s a p o i n t where t h e c a r g o ( e l e c t r o n s ) a r e  u n l o a d e d , t h e t r u c k i n g system soon becomes s a t u r a t e d . final  e l e c t r o n acceptor  being  oxidised  so  I n a n a e r o b i o s i s , the  i s formed i n amounts p r o p o r t i o n a l t o t h e s u b s t r a t e  that  the reduced  electron  carrier  c a n be  unloaded  ( o x i d i s e d ) and r e t u r n t o a c c e p t a n o t h e r l o a d ( p a i r s o f e l e c t r o n s ) . T h i s energy w h i c h i s r e l e a s e d by t h e r e a c t i o n i s s t o r e d by t h e c e l l i n the form o f energy r i c h phosphate e s t e r s (ATP) w h i c h c a n be used by t h e c e l l for  a l l reactions  released state  by such  an e f f i c i e n c y  compound  When an o r g a n i c  substrate molecule i s normally the e l e c t r o n  fermentative acceptors electron  which  acts  donor  bacteria  i n addition  by t h e c e l l i n t u r n , depends on The m a j o r i t y o f anaerobes work The e l e c t r o n  into  two m o l e c u l e s ,  pointed  following  out t h a t ,  principles:  e l e c t r o n acceptor donor  must  equal  compound  as  an  the a b i l i t y  to organic  rise  electron  acceptor.  t o use p r o t o n s  molecules. gives  o f w h i c h one a c t s  The r e d u c t i o n  i n general,  to the production  fermentative  ( 1 ) From t h e s u b s t r a t e  reactions  as  Some electron  of protons i n  h y d r o g e n and i s o f i m p o r t a n c e f o r t h e c o n t r o l o f a n a e r o b i o s i s . has  acceptor  compound a c t s as an e l e c t r o n a c c e p t o r , t h e  and t h e o t h e r  transferring reactions  i n the  ( r e s p i r a t i o n ) o r an o r g a n i c  split  possess  which i s  as t h e r e d u c t a n t  o f 25-50% (Thauer e t . a l . , 1977).  be e i t h e r an I n o r g a n i c  o f energy  ( A G ° ) depends on t h e d i f f e r e n c e i n  process  o f t h e compound  The amount  e f f i c i e n c y of i t s metabolism.  (fermentation).  as  growth.  The amount o f ATP w h i c h i s g a i n e d  the energetic  can  support  an o x i d a t i o n  of reduction  reaction.  at  which  of  molecular  Cohen (1982)  conform  to t h e  a p r o p e r e l e c t r o n donor and  must be formed and t h e amount o f e l e c t r o n s s u p p l i e d by t h e t h e amount  accepted  by t h e a c c e p t o r ;  (2) F e r m e n t a t i v e  r e a c t i o n s o c c u r i n such a way t h a t an o p t i m a l ATP g a i n i s a c c o m p l i s h e d .  - 19 -  The during  most  important acidogenic fermentation reactions  anaerobiosis  et.  a l . (1977).  the  ATP  w h i c h may  of c a r b o h y d r a t e s a r e shown i n T a b l e 2.1,  occur  after  Thauer  A l t h o u g h the AG° of the d i f f e r e n t r e a c t i o n s a r e comparable, 4.  g a i n v a r i e s between 2 and  F o r 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 s p e c t s , the r e a d e r i s r e f e r r e d t o papers by Thauer e t . a l . (1977) and G o t t s c h a l k and Andreesen  2.1.2  Metabolic  Effective combined  and  catabolising  Stages  digestion  of  anaerobic bacteria.  gastrointestinal be  organic  matter  coordinated metabolism  b a c t e r i a have been i s o l a t e d  can  (1979) .  tracts,  distinctly  of  At l e a s t  into  methane  different  requires  kinds  four d i f f e r e n t  of  trophic  the  carbon t y p e s of  from e i t h e r man-made r e a c t o r s o r i n n a t u r e ( e g .  l a k e sediments o r t h e r m a l w e l l s ) .  r e c o g n i s e d on  the  basis  of  These  substrate  bacteria  fermented  and  m e t a b o l i c end p r o d u c t s formed ( Z e i k u s , 1980). The  four  metabolic  (Figure  2.1):  complex  organic molecules  broad  (1)  spectrum  organic  acids  ethanol);  hydrogen  o f end longer  which  hydrolytic  function  bacteria  (polysaccharides,  in  which lipids  anaerobiosis ferment and  a  include  variety  proteins)  into  a  p r o d u c t s ( a c e t a t e , H2/CO2, one c a r b o n compounds and than  acetate,  and  neutral  compounds  larger  and and  f a c u l t a t i v e s p e c i e s t h a t can ferment o r g a n i c a c i d s l a r g e r neutral  compounds l a r g e r  acetate;  (3) The  t h a n methanol  homoacetogenic  (ethanol,  than  bacteria  which  methanogenic  b a c t e r i a which  ( m e t h a n o l , CO, methylamine)  ferment H2/CO2,  and a c e t a t e t o methane.  one  than  propanol) to can ferment  v e r y wide s p e c t r u m of m u l t i o r one c a r b o n compounds t o a c e t i c a c i d ; and The  of  (2) The hydrogen 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 w h i c h i n c l u d e b o t h  o b l i g a t e and acetate  The  groups  carbon  a  (4)  compounds  T a b l 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 F e r m e n t a t i o n  AG°  R e a c t i o n Type  Acetic acid C  6 12°6 H  +  4 H  (kJ/reaction)  2°  10  2  C  H  3  C  H  O  O  ~  +  4  o  H  +  +  2 H C 0  3  +  ~  4 I I 2  o  3  3  Butyric acid  3  +  + — 3  2 0 6  HCO" 3  +  O  C  6 12°6 H  J  Z  Ethanol  4  -220  3 -4  -255  3  fermentation  C-H.-O, + 2H„0 •»• CH-CH.CH-COO" + 3 H + 2H„ + 2HC0~ 1/  ATP (mol/reaction)  fermentation  Propionic acid fermentation C,-H 0, •» — CH CH C00~ + J L CH.COO" + J L H 6 12 6 3 2 3  0  Types  Z  Z  Z  J  fermentation +  2 H  2°  2 C H  3  C H  2  O H  +  2  H  L a c t i c acid fermentation C.H.,.0. •* 2CH.CH0HC00" + 2 H  +  +  +  2 C H 0  3  _  2  2  6  2  -198  2  -  21  -  Carbohydrates Proteins Lipids  Organic Matter  y  Organic  1Hydrolytic  bacteria  Acids  2 acetogenic b a c t e r i a (^-producing)  Acetate  -3-  H /C0 2  2  homoacetogenie b a c t e r i a  \7  Figure  2.1  4 B methano g e n i e bacteria (acetate decarboxylation)  \7  Metabolic d i s t i n c t i o n of microbial involved i n anaerobiosis.  methanogenic bacteria ( r e d u c t i v e methane formation)  populations  -  The  raethanogenic  22  -  b a c t e r i a perform  a pivotal  role  m e t a b o l i s m c o n t r o l s the r a t e of o r g a n i c d e g r a d a t i o n c a r b o n and the  1980).  i s presented of  recent  al.  efficiency  A brief  below.  review  of  interspecies  directs  of  the r o l e p l a y e d  reviews  ( Z e i k u s , 1977;  Mah  unique  the f l o w of  intermediary  enhances  metabolism  by each b a c t e r i a l  F o r more i n f o r m a t i o n the r e a d e r  detailed  group  i s r e f e r r e d to a number  e t . a l . , 1977;  and  Batch  et.  via  the  1979).  2.1.2.1 It  Hydrolytic  has  been  Wallnofer  et.  Also  monophosphate  (HMP)  control  that  that  (1961)  routes  hexoses  pathway  and  Wood  i n anaerobic  position,  fermentations  (EMP)  a l . , 1966)  important.  degradation  Bacteria  established  Erabden-Meyerhof-Parnas  are  i n the other  showed the  b a c t e r i a up  to  start  (Wood, 1961).  f u r t h e r o x i d i s e d by the  degradation  corresponding  of  nucleic acids  routes  that  EMP  important  lactic The  unidentified generic  are  and  lipids  much the  pathways Pyruvate  acid  The  e t . a l . , 1963; less  hexose  for  hexose  occupies  or v o l a t i l e  the  fatty  acid  are b r o k e n down to l o n g  long c h a i n f a t t y a c i d s are and  acetate.  Amino a c i d s  p r o t e i n s a r e deaminated and  the  o r g a n i c a c i d s a r e formed. 9  P o p u l a t i o n s o f 10 -10 have  the  pyruvate.  and  fermented  rumen ( B a l d w i n  b e t a - o x i d a t i o n to propionate  8  sludge  mainly  metabolic  most  from w h i c h a l c o h o l i c ,  (VFA)  are  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) .  from  and  their  e l e c t r o n s by removing t o x i c i n t e r m e d i a r y m e t a b o l i t e s and  thermodynamic  (Zeikus,  because  been  documented  gram-negative  identifications  of  h y d r o l y t i c b a c t e r i a per mL (Zeikus, rods. the  To  1980). date  predominant  The very  of  raesophilic  majority few  of  detailed  microbial  species  these  sewage were  studies have  or  been  - 23 -  reported.  Clostridium  propionicum,  Clostridium  acetobutyricum  E u b a c t e r i u m limosum a r e examples o f organisms whose known m e t a b o l i c lead  to the formation  o f end p r o d u c t s  chiefly  f e r m e n t a t i o n s , namely, b u t y r a t e s , p r o p i o n a t e s  formed  and  pathways  i n man-made r e a c t o r  and a c e t a t e s .  2.1.2.2 The H^-producing Acetogenic Bacteria The first  ^-producing  stage  acetogenic  of fermentation  (VFA) , a l c o h o l s , aromatics mixture  (Figure  substrates  2.1).  to acetate  levels.  Figure  partial  pressure  H2~consuming  (propionate,  butyrate,  long  chain  of the  fatty  acids  and o t h e r o r g a n i c a c i d s ) t o a c e t a t e and a I^/CC^  However when  these  organisms  cannot  catabolise  these  i n t h e environment i s not a t extremely low  2.2 i l l u s t r a t e s and f r e e  species.  b a c t e r i a c a t a b o l i s e the products  the r e l a t i o n s h i p  energy  that  exist  between  a v a i l a b l e to the ^ - p r o d u c i n g  and  F o r example, i n o r d e r f o r energy t o be a v a i l a b l e t o  organisms o x i d i s i n g propionate  t o a c e t a t e and  , the p a r t i a l pressure  o f B.^  can n o t exceed about 10 ^ atmospheres ( T h a u e r , e t . a l . , 1977). P o p u l a t i o n s o f 4.2 x 10^ ^ - p r o d u c i n g have been r e p o r t e d ( M c l n e r n e y  a c e t o g e n s p e r mL o f sewage  e t . a l . , 1978).  sludge  These organisms have n o t 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 characterised, despite the b e l i e f than  t h a t VFA's a r e e c o l o g i c a l l y much more i m p o r t a n t  lactate  and e t h a n o l  from M e t h a n o b a c i l l u s this  group.  syntrophic Mclnerney group,  of  and B r y a n t  Syntrophonas  The i s o l a t i o n  o m e l i a n s k i i was t h e f i r s t  Ethanol growth  (Mah, 1981).  as i n t e r m e d i a t e s  o f t h e "S" o r g a n i s m  documentation of a specie i n  was o x i d i s e d t o a c e t a t e and CO2 r e d u c e d t o CH^ by t h e the S  organism  (1981)  have  wolfeii,  that  and  recently  Methanobacillus reported  omelianskii.  an o r g a n i s m  i n this  b e t a - o x i d i s e s even-numbered-carbon  fatty  - 24 -  I  Butyrate  80  40  AG  F i g u r e 2.2  0  -40  at p H 7-0, 2 5 * C  -BO  -120  (KJ)  E f f e c t of the p a r t i a l pressure  o f hydrogen (PH^)  on  the f r e e energy c h a r g e (AG ) f o r t h e d e g r a d a t i o n o f ethanol, propionate formation with U  and b u t y r a t e w i t h methane  and CO^.  substrate concentrations each and b i c a r b o n a t e  The a s s u m p t i o n i s t h a t  o f f a t t y a c i d s a r e 1 mM  i s 50 mM w i t h t h e p a r t i a l  pressure of CH  a t 0.5 atm ( A G = A G 1  4  0 1  +1.36 log ([products]/  [reactants]). Source i s M c l n e r n e y and B r y a n t  (1979).  - 25 -  acids  (butyrate,  c a p r o a t e ) t o a c e t a t e a n d H ; odd-numbered-carbon f a t t y  acids  ( v a l e r a t e ) a r e c o n v e r t e d t o a c e t a t e , p r o p i o n a t e and H .  2  2  m u s t grow w i t h a n ^ - u t i l i s i n g  microbe.  Boone a n d B r y a n t  S. W o l f e i i (1980)  also  r e p o r t e d t h a t S y n t r o p h o b a c t e r w o l i n i i o x i d i s e s p r o p i o n a t e t o a c e t a t e , H and 2  co . 2  2.2.1.3  Homoacetogenic Bacteria  Homoacetogenic metabolism  bacteria  as a consequence  m u l t i - c a r b o n compounds. substrates  possess  a  high  of not forming H  C0  2  2  and C 0  2  efficiency  (sugars acids,  C 0 , CO, H , e t c . ) . 2  2  of  d u r i n g growth on  These b a c t e r i a c a n ferment a v e r y wide  homoacetogens i n methane p r o d u c i n g r e a c t i o n s consequence  thermodynamic  spectrum o f  The u t i l i z a t i o n o f H  2  by  i s c o n s i d e r e d t o be o f l i t t l e  t o t h e o v e r a l l c a r b o n d e g r a d a t i o n because t h e C-2 o f a c e t a t e and  g e n e r a l l y a c c o u n t f o r 70% and 30% r e s p e c t i v e l y o f the methane c a r b o n i n  man-made  reactors  successfully environment functional  (Jeris  and M c C a r t y ,  1965).  o u t compete homoacetogens f o r H  ( P r i n s and L a n k h o r s t , 1977). importance  of  Methanogens 2  Populations (Braun  o f 10  e t . a l . 1979).  r e c o g n i s e d genera of H 2.1.2.4  2  i s known about t h e  metabolism  acetogenic/methanogenic b a c t e r i a l i n t e r a c t i o n s (Zeikus, 5  to  i n the g a s t r o i n t e s t i n a l  So f a r l i t t l e  homoacetogenic  appear  and t h e  1980).  6 - 10  p e r mL o f sewage s l u d g e have been r e p o r t e d  Clostridium  and A c e t o b a c t e r i u m  o x i d i s i n g homoacetogenic  are the only  bacteria.  Methanogenic Bacteria  The methanogens a r e the k e y organisms i n t h e p r o d u c t i o n o f methane from waste  materials.  They a r e t h e o n l y organisms  a c e t a t e and hydrogen  t o gaseous  end p r o d u c t s .  t h a t a r e a b l e t o break down Without the presence of t h i s  - 26  -  group of m i c r o o r g a n i s m s i n a m e t h a n i f i c a t i o n the  total  products  organic  of  since  they  Their  energy  1979).  the are  material  previously composed  yielding  CH NH , 2  (Equation  CH COOH, 3  stop  of  many  species  mechanisms a r e  utilize  CO).  due  to  discussed groups.  They r e q u i r e a s t r i c t l y  The m e t h a n o g e n s 3  would  process, effective  not  the  breakdown of  accumulation  The methanogens  with yet  different known  cell  (Zeikus,  a n a e r o b i c environment  for  are  of  the  unusual  morphology. 1977;  their  Wolfe,  growth.  a n a r r o w range of s u b s t r a t e s ( R ^ / C C ^ , HCOOH,  Almost  all  s p e c i e s use H  2  and C 0  2  for  growth  2.3).  4H. + HC0~ + H 2 3  CH. + 3H.0 4 2  +  (AG° = - 1 3 5 . 6  (2.3)  kJ/reaction)  Even though a l a r g e amount of hydrogen i s produced d u r i n g a n a e r o b i o s i s , 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  can  Of  these only  Methanosarsina b a k e r i ,  and M e t h a n o b a c t e r i u m  soghngenii  have been i s o l a t e d  degrade a c e t a t e  Methanococcus pure  maze!  (Equation  2.4).  hydrogen.  A number of  the  species  in  culture.  CH C00 3  + H 0  (AG° = - 3 1 . 0  The f r e e energy from r e a c t i o n ( 2 . 4 ) (AG° = - 3 1 . 6  kJ/reaction).  C H + HC0  2  4  3  (2.4)  kJ/reaction)  i s b a r e l y enough t o form one mole of ATP  T h i s may e x p l a i n  the o b s e r v e d s l o w growth  rate  o f methanogens on a c e t a t e . g Methanogenic detected (Smith,  populations  1966).  of  10  p e r mL of  sewage s l u d g e h a v e been  - 27 -  2.1.2.5  Specific  Bacterial  Species  Associated  With  Whey  Biomethanation  In  two  recent  organisation  papers  and s p e c i e s  l a c t o s e biomethanation whey d e g r a d i n g  (Chartrain  composition  and Z e i k u s ,  of b a c t e r i a l  groups  were i n v e s t i g a t e d i n a c o n t i n u o u s  r e a c t o r (pH 7.1,  temperature  1986a  &  b) , t h e  associated  with  flow, single-phase,  o f 37°C and d i l u t i o n r a t e o f  - 1 1 4 .01  h  three to  ).  C t r a c e r s t u d i e s demonstrated that biomethanation  distinct  lactate,  transformed  but simultaneous  phases.  e t h a n o l , a c e t a t e , formate  Lactose and  was m e t a b o l i s e d •  primarily  T h e s e m e t a b o l i t e s were  i n t o a c e t a t e and H^/CO^ i n a second, a c e t o g e n i c phase.  t h e d i r e c t methane p r e c u r s o r s were t r a n s f o r m e d with acetate accounting proposed  occurred i n  biomethanation.  d u r i n g the methanogenic phase  f o r 81% o f the methane formed.  f o r the carbon  and  electron  Finally,  flow  A g e n e r a l scheme was  route  during  lactose  Based on t h e scheme, p r e v a l e n t m i c r o b i a l p o p u l a t i o n s i n t h e  e c o s y s t e m 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  1 0 ^ for hydrolytic  i n t h e f o l l o w i n g c o n c e n t r a t i o n s ( p e r mL);  b a c t e r i a ; 10^ - 1 0 ^ f o r a c e t o g e n i c b a c t e r i a ; and 10^ - 10^ f o r methanogenic bacteria.  The  Leuconostoc  predominant  mesenteroides,  Clostridium  propionicum  hydrolytic  Klebsiella  oxytoca  utilising  methanogens.  T^-utilising While  were  acetogens,  s o e h n g e n i i were found Methanobacterium  identified  and C l o s t r i d i u m  and D e s u l f o v i b r i o v u l g a r i s  predominant Lactate u t i l i s i n g , J^-producing b a r k e r i and M e t h a n o t h r i x  bacteria  which  while  as  butyricum. were t h e  Methanosarcina  to be t h e predominant a c e t a t e  formicicum  was  the p r e v a l e n t  specie. C h a r t r a i n and Z e i k u s ' s  m i c r o b i a l ecology  work  i s very  of a s i n g l e - p h a s e b i o m e t h a n a t i o n  useful with  regard  t o the  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 s t a n d f o r a two-phase p r o c e s s  acidogenic reactor  - 28 -  where  a l l the  dilution  methanogenlc  rates  (>>  .01  b a c t e r i a would  h  f i n d i n g s what o p e r a t i n g  .  Also  the a c i d o g e n i c  are  For  end  of  general  higher  from t h e i r  (< 7.0)  would  reported.  and  acids  concentration  most i m p o r t a n t  pH.  These  process  three  and  toxic  can  (Zeotemeyer, e t . a l . , 1982;  be  acidogenic  the c h o i c e , of the d e s i r a b l e end p r o d u c t  controversy  pH,  parameters  variables  the predominant p r o d u c t i o n of a p a r t i c u l a r  considerable  Pipyn  is a and  K i s a a l i t a e t . a l . , 1986).  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 a r e c o n s i d e r e d sense.  reported  Emphasis in  this  appropriate  form  observations  reported.  2.2.1  suggested  although framework  which  Model  A conceptual first  pH  to  Mathematical Modeling of M i c r o b i a l Growth In  work  rate  However t o date  V e s t r a e t e , 1981;  2.2  r e a c t i o n s the  dilution  to f a v o u r  product.  subject  to a s s e s s  r e a c t o r at a lower  n u t r i e n t s , organic  acidogenic  temperature,  manipulated  due  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  temperature,  material.  out  Important Process Parameters  Some e n v i r o n m e n t a l alkalinity,  washed  i t is difficult  do to the b a c t e r i a l p o p u l a t i o n c o m p o s i t i o n  2.1.2.6  be  there  i s placed study; may  be  on  those  with  the  used  to  t h a t a r e more r e l e v a n t t o aim  of  describe  identifying the  the  microbial  in a the most  growth  Classification framework, c l a s s f y i n g models o f m i c r o b i a l p o p u l a t i o n s ,  by T s u c h i a e t . a l . ( 1 9 6 6 ) . is  no  T h i s framework has been r e t a i n e d  u n i v e r s a l agreement.  c o n s i s t i n g of  three  distinct  was  Below,  perspectives  a  slightly  for  cell  modified population  - 29 -  kinetic  representation  s i t u a t i o n met Two  is  examined.  A  i n t h i s s t u d y w i l l a l s o be  broad  approaches  perspective  suitable  for  identified.  to modeling b i o l o g i c a l  and  o t h e r systems  namely, continuum and c o r p u s c u l a r methods ( H a r d e r and R o e l s , 1982). 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 explicitly  r e c o g n i s e d and  therefore  the t y p i c a l  knowledge of m a t t e r , the c o r p u s c u l a r  to  For a b i o l o g i c a l  (1979).  They  systems  used  the  by  Fredrickson  term  considered  be  the  modelling  cell.  Hence  microbial  the  e t . a l . (1967)  "segregated"  sytems.  corpuscular Despite  to  describe  and  Ramkrishna  their  approach.  fact,  systems.  In  a l . (1966)  the  continuum  and  to  approach,  t o as " u n s e g r e g a t e d " i s the most commonly e n c o u n t e r e d method microbial  et.  continuum  easily  referred  of  Tsuchia  the  itself  termed  descriptions  by  lends  organisms  earlier  in  "distributive"  approach  this  the most  T h i s a p p r o a c h has been used  C l a s s i c a l m i c r o b i o l o g y c o n s i d e r s 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 to  system,  G i v e n our p r e s e n t - d a y  a p p r o a c h must be  method of m i c r o b a l growth m o d e l i n g .  model m i c r o b i a l  I n the  b e h a v i o u r of the system i s  c e l l s a r e c o n s i d e r e d d i s c r e t e and h e t e r o g e n e o u s .  realistic  exist,  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  c a u s e d by the c o n c e r t e d a c t i o n of the p o p u l a t i o n . the  the  sometimes used  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 w h i c h i n t e r a c t s as a  whole  therefore  with  i t ' s environment.  ignored  and  the system  The  corpuscular  i s considered  The p r e f e r e n c e f o r the continuum approach may  nature  of  reality  is  t o be c o n t i n u o u s i n space.  be a t t r i b u t e d t o the ease w i t h  w h i c h m a t h e m a t i c a l t r e a t m e n t can be employed. The stochastic  second  perspective  (probablistic)  distinguishes approaches.  between The  the  deterministic  difference  between  and these  approaches r e s t s on the n a t u r e o f the p r e d i c t i o n s about the f u t u r e b e h a v i o u r of  the m i c r o b i a l system t h a t 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  v a r i a b l e s necessary  s t a t e v e c t o r of  the  system (a v e c t o r  to s p e c i f y the s t a t e of the system a t a g i v e n moment i n  t i m e ) a l l o w s an e x a c t p r e d i c t i o n of the f u t u r e b e h a v i o u r period.  With  probability  the  that  s t o c h a s t i c approach,  the  state  and  unable and  each p o i n t  vector).  a coordinate  be  i n f o r m a t i o n about  the  to a s i n g l e v a l u e  the  a  state  i f the  s t a t e of  of  the  observer  the  is  population  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 .  to a l l o w a d e t e r m i n i s t i c t r e a t m e n t  o f o r g a n i s m , as  f o r example d u r i n g  a probabilistic contain  approach. populations  third  level  of any  biological  t h a t the b e h a v i o u r  the  Shuler  last  stages  well  in  excess  system.  of s m a l l numbers  of s t e r i l i z a t i o n ,  S i n c e most m i c r o b i a l systems of  m o d e l i n g approaches a r e more p o p u l a r The  specify  p o i n t e d out t h a t 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  sufficient  interest  to  r e g i o n of  s t o c h a s t i c approach i s o f t e n used  sufficient  arbitrary  system of d i m e n s i o n a l i t y of the s t a t e  A l s o , F r e d r i c k s o n (1966) has p o i n t e d out  for  d u r i n g an  possible  i n a given  i n s t a t e space c o r r e s p o n d s  i t ' s subsequent b e h a v i o u r  (1985) has is  The  to o b t a i n  i t i s only  state reactor w i l l  space ( s t a t e space b e i n g vector  composed of a l l  of  10,000,  calls  engineering  deterministic  t h a n s t o c h a s t i c ones.  distinguishes  between  structured  and  unstructured  models.  An u n s t r u c t u r e d model assumes t h a t a s i n g l e v a r i a b l e i s adequate to  describe  the  population.  q u a n t i t y of biomass. capabilities the  of  population  structure  the  recognisable compounds, deterministic  Implicit population  into  i s most  often  and  are  achieved  invariant. With  by  of  continuum  the model  (eg.  the  s t r u c t u r e d model d i v i d e s cultures  the  DNA,  earliest was  A  pure  dividing  subcomponents One  s i n g l e v a r i a b l e i s r e l a t e d to  i n such models i s the i d e a t h a t b i o s y n t h e t i c  subcomponents.  chemical etc.).  T y p i c a l l y the  two  proposed  the  addition  cells  into  RNA,  protein,  component by  two  or  of  more  storage  structured,  Williams  (1967).  - 31 -  C a m p b e l l (1957) d e f i n e d during  that  increases typical nature  time  growth o v e r a p e r i o d of  interval  every  by  the  same f a c t o r .  cell  is  time  of c e l l s may  situations  are  extensive So  property  i n balanced  invariant.  time as b e i n g  Models  growth  in  which  a  the  growing  ignore  batch  the  of  a  multicomponent  T y p i c a l balanced culture  if  system  growth the c o m p o s i t i o n  be adequate i n t h i s s i t u a t i o n .  exponential  of  balanced,  and  growth  steady-state  c o n d i t i o n s i n a CSTR ( o r c h e m o s t a t ) . In  Figure  2.3  the  (continuum/corpuscular Predictions  by  any  deterministic. to  the  plane  stochastic  possible and  model  interactions  between  structured/unstructured)  from  each  region  could  be  two  are  the  page w i t h  two  model p r e d i c t i o n s and  regions;  below the  above  presented.  e i t h e r s t o c h a s t i c or  I n o t h e r words a t h i r d c o r d i n a t e can be added of  levels  the  perpendicular  page  representing  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  biological  rather  than  necessitates  a  phase i n t h i s  single  explicit  study  specie.  i s made up of a m i x t u r e  Complete  r e c o g n i t i o n of each s p e c i e  hand, the model f o r each s p e c i e must be the  number of  species  i n the  would be an a l m o s t i m p o s s i b l e r e g i o n one counts the  were  next  belong  of F i g u r e  2.3  involved  two  task.  the  i n v o l v e d and  on  i s unknown a  Therefore  species  one  hand  the  other  Given that  structured  approach  i n t h i s s t u d y o n l y models i n  Also given that large m i c r o b i a l  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 . previously published are  m i c r o b i a l growth models  In that  reviewed.  Models For Single Substrate Limiting Growth  I n the p r e s e n t schools.  on  chemically structured.  were c o n s i d e r e d .  only  subsections  population  t o the above i n d i c a t e d c a t e g o r y  2.2.2  structure  of  In  the  day  theory  of c o n t i n u o u s  c u l t u r e t h e r e a r e two  first  school  i t i s assumed t h a t the  specific  opposing  growth  rate  - 32 -  Continum "Distributive" "Unsegregated"  Unstructured  1. MOST IDEALISED CASE c e l l population t r e a t e d as one component solute  2.  3.  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  Corpuscular "Segregated"  s i n g l e component heterogeneous individual cell  Figure  2.3  Structured  multicomponent average c e l l description  P o s s i 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 " a p p r o x i m a t i o n o f Region 3 and a b a l a n c e d growth a p p r o x i m a t i o n o f Region 2. Region 3 i s an "average c e l l " a p p r o x i m a t i o n o f R e g i o n 2.  -  (|J. = ( d X / d t ) / X ) i s dependent o n l y (S). (X)  33 -  on the l i m i t i n g  concentration  I n the second i t i s assumed that u i s e i t h e r dependent on t h e biomass o r i s some f u n c t i o n o f b o t h  belong  S and X.  First  let's  review  models  that  to the former s c h o o l .  2.2.2.1 An  Monod Equation  equation  frequently  proposed by Monod (1942  used  & 1950)  i n kinetic  description  o f growth  was  as f o l l o w s :  u = (dX/dt)/X  in  substrate  = u S/(K + S) m s  (2.5)  which  u. i s t h e m a x i m u m s p e c i f i c g r o w t h r a t e . S and X a r e t h e m c o n c e n t r a t i o n s o f s u b s t r a t e and b i o m a s s a n d K i s a Monod s a t u r a t i o n s constant.  Equation  Michaelis-Menten  2.5 i s analogous t o the B r i g g s - H a l d a n e s o l u t i o n o f t h e  model f o r t h e k i n e t i c s  growth i s c o n s i d e r e d in  which  one  Michealis-Menten  to be t h e r e s u l t  reaction  i s much  equation  I f microbial  o f a sequence o f e n z y m a t i c r e a c t i o n s  slower  than  can be c o n s i d e r e d  the good f i t t h a t the Monod e q u a t i o n by  o f a s i n g l e enzyme.  a l l the others,  then t h e  as t h e p h y s i c a l e x p l a n a t i o n f o r  often gives.  This reasoning  no means u n i q u e , f o r example t h e Langmuir a d s o r p t i o n  isotherm  i s however i s of the  same form as e q u a t i o n 2.5.  2.2.2.2 Other Equations Monod's proposed  equation  i s by no means  f o r the s u b s t r a t e  other proposals  the only  concentration  have been s u g g e s t e d .  equation  dependence  which  o f growth.  Some o f these a r e d i s c u s s e d  has been Numerous below.  Konak (1974) assumed that s u b s t r a t e dependence on s p e c i f i c growth r a t e  - 34 -  is related  to the d i f f e r e n c e between u and m  du/dS = k(u  i n which  k and  l e a d s to the  p are c o n s t a n t s .  |J. ( E q u a t i o n  - u)  2.6)  (2.6)  p  The 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  following:  e ^)  in  i  ( 1 _ p )  m  -  F o r p = 2, E q u a t i o n 2.8  (u  m  - u)  1  p  for p = 1  (1 - ) k S  =  P  (2.7)  for p  r  M  r  (2.8)  s i m p l i f i e s to:  u = a S / ( l / k u . + S)  Equation  2.7  Equation  2.9  relationship  i s similar is  an  to one  analogue  t h a t was of  the  (2.9)  proposed Monod  by  Teissier  equation.  p o s t u l a t e d by Konak seems v e r s a t i l e ,  (1936)  Therefore  however, i t has  no  and the  clear  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 a  series  of  enzymatic  an e q u a t i o n to d e s c r i b e the k i n e t i c s  r e a c t i o n s f o r the  case  of  steady  state.  The  of  three  parameter e q u a t i o n f o r growth based on t h e i r work i s :  S = \x(K  k  + K )/( i B  r  m  - V)  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 p r e s e n t e d h e r e ) E q u a t i o n 2.10 to:  (2.10)  reduces  - 35  * V =  u =  Equations  2.11  were  indicated  that  the  presented  here)  published 2.5,  better and  Monod  as  i n 1973,  2.10  and  fit.  first  a  This  Blackman  equation  and  forms.  found  by  Condrey  (1982) has  neglected  i n m i c r o b i a l growth  (2.11)  V  m  Blackman  will  arise In  (1905).  from  a  their  paper  Dabes  (1970)  general  form  Dabes  and  their  Equation  since Equation  showed  pointed  Blackman's  out  modeling,  (2.10)  (not  Equations  always  gave  a  2.10  i n c l u d e s the Monod  form  to  be  superior  t h a t Blackmans k i n e t i c s perhaps  has  co-workers  a number of p u b l i s h e d d a t a u s i n g that  also  Monod's.  Vm  S <  V  surprising  They  >  case.  analysed  i s not  /  proposed  special  they  2.11,  S  S  -  because  of  to  have been  the  functional  form b e i n g d i s c o n t i n u o u s . Powell (diffusion derived  (1967) and  combined  mass  permeation)  with  transfer the  into  and  inside  the  Michealis-Menten  enzyme  kinetics  = u- (K + L + m s  r  S)/2L  u„ = 1 - (1 - 4LS/(K + L + S ) ) * z s 2  which  L  =  q /A; m  q ^m  M  and  inside  o u t s i d e the c e l l .  +  S)  4LS/(K  and 2  = +  A  being  being °  consumption  (L  and  the f o l l o w i n g e x p r e s s i o n :  u l  in  organism  (L - S) L +  S)  2  2  +  a  4SL.  i s less  the  constant  maximum determined  S i n c e S, K and (L -  S)  2  specific r  by  the  r a t e of transfer  L are p o s i t i v e ,  is necessarily  than u n i t y and  (2.12)  5  i t s expansion  (K  substrate resistance + L + S) >  positive, therefore by b i n o m i a l  theorem  - 36 -  is  permissible.  following  If  expression  only is  two  terms  of  m  s m a l l compared to  (1958)  Moser  expansion  are  retained  the  obtainable:  li = u (K  If L is  the  + L + S)  the Monod form i s  postulated  (2.13)  s  the  retained.  following  modified  form  of  Monod's  equation:  u = u S /(K  + S)  X  m  Unfortunately There order  are  forms.  Examples  of  Williams  second  a  These these  activity  the  is  no p h y s i c a l model to  variety  (1975).  biological In  there  next  are  are  Models  other  special  those  These  types  suggestions, cases  given  support E q u a t i o n 2.14.  of  the  mainly Monod  of  modified  equation  (K  g  first «  S).  by E l m a l e h and Aim (1976) and Grady and do  not  predict  conditions  for  maximum  and system f a i l u r e . section,  s c h o o l mentioned  2.2.3  of  (2.14)  X  s  at  With  attention  is  turned  the b e g i n n i n g of  Specific  Growth  this  to  the  assumption  the  (2.2.2).  section  Substrate  of  And/Or  Biomass  Growth  Dependence In addition  this to  type  of  substrate  population density.  microbial  growth  concentration, The f i r s t  example  modelling,  p. i s is  also the  it  is  dependent  logistic  assumed on  the  that  in  biological  law; w r i t t e n  as:  (2.15)  -  in  w h i c h X i s t h e maximum b i o m a s s m  Equation (1970a  2.15 &  b)  attributed according  has  been  used  and R a i and  by  Roels  concentration  Constantinides  and K o s s e n  t o the l o g i s t i c  -  successfully  (1978)  by  successfully limits  applied  c a n be r e a c h e d ,  Constantinides  (1974).  I t s success  t o the f a c t  that  has been  growth  curves  i n s i t u a t i o n s where This  so f a r  dependent upon the growth  T h i s c l e a r l y I n d i c a t e s t h a t the l o g i s t i c  the growth r a t e .  et.a l .  I n a l l the approaches c o n s i d e r e d  r a t e o f growth o f biomass i s to some e x t e n t  l i m i t i n g substrate.  which  law bear some resemblence t o c u r v e s which r e s u l t  from m i c r o b i a l m o d e l i n g e x e r c i s e s . the  37  the r a t e  renders i t of l i t t l e  law c a n not be  of s u b s t r a t e  value  addition  f o r most m i c r o b i a l  processes. Fujimoto interaction  (1963)  considered  a t the s u r f a c e  utilization  o f the c e l l ,  e n z y m a t i c r e a c t i o n w i t h i n the c e l l . is  proportional  subsequent  to  the rate  enzymatic conversion  of  of substrate  transport  into  i n three  steps;  the i n t e r i o r  F o r t h e case where the a d s o r p t i o n transport  there,  into  the o v e r a l l  the c e l l  interior  r e a c t i o n was  and rate and  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 X + S "—* (X.S)  k+2 >[X.S] + P  (2.16)  k-1  For  growth  substrate of equation  and s u b s t r a t e i n t o the c e l l  consumption  where  m a t e r i a l i s constant,  2.16 and o b t a i n e d  the conversion  rate  Fujimoto solved  the f o l l o w i n g s o l u t i o n :  from the  the k i n e t i c s  - 38 -  u = a u ^ (S/X)  =  \i  m  / (K + ( S / X ) )  S/(KX +  (2.17)  S)  where a(0 < a < 1) i s the a c t i v i t y of the enzyme.  E q u a t i o n 2.17  proposed by C o n t o i s (1959) w i t h o u t a m a t h e m a t i c a l j u s t i f i c a t i o n . at  was  first  F o r a CSTR  steady s t a t e  X = Y(S  where Y i s t h e y i e l d c o e f f i c i e n t . e q u a t i o n 2.17  -  o  S)  (2.18)  S u b s t i t u t i n g e q u a t i o n 2.18  into  gives, u = |X S/(YK(S m  o  - S) + S)  (  2  #  1  9  )  = u S / ( K ' ( S - S) + S m o n  E q u a t i o n 2.19 used  was  successfully  first  proposed by Chen and Hashimoto  (1978) and has  i n m o d e l i n g methane p r o d u c t i o n from a g r i c u l t u r a l  been  residues  (Chen e t . a l . 1980). Roques e t . a l . (1982) has r e c e n t l y p r o p o s e d t h e f o l l o w i n g e q u a t i o n :  u = U S / ( K ( S - S) + M + S) m  in  which  a  third  (2.20)  Q  parameter M was  introduced.  e q u i v a l e n t s t o Monod's e q u a t i o n w i t h K K  s  g  = K(S  E q u a t i o n s 2.19  and  2.20  are  v a r y i n g as: o  - S)  and  (2.21) K  s  = K(S  Q  - S) + M  -  If M < ( S K  -  S ) , e q u a t i o n 2.20 becomes a n a l o g o u s to e q u a t i o n  Q  2.2.4  39  2.19.  M i c r o b i a l Growth Modelling Recommendation  In order the m o d e l i n g  t o p r o v i d e an i m p r e s s i o n 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  o f 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  rate,  some of the e q u a t i o n s , c o n s i d e r e d above a r e g r a p h i c a l l y r e p r e s e n t e d f o r some v a l u e s o f the p a r a m e t e r s ( F i g u r e 2 . 4 ) . way  t h a t a l l c u r v e s c o i n c i d e a t u/u  The e q u a t i o n s were s c a l e d i n such a  = 1/2 and a r e l a t i v e S v a l u e o f u n i t y . m  It  i s clear,  minor  role  Therefore  as p o i n t e d out by R o e l s in  the  fixing  there i s l i t t l e  concentration-time  I n t h i s study  i t s s i m p l i c i t y and the f a c t  Michealis-Menten  biomass  that d e t a i l e d k i n e t i c s play a relationships.  j u s t i f i c a t i o n f o r favouring a p a r t i c u l a r  among t h o s e d i s c u s s e d above. for  of  (1983),  equation,  t h e Monod e q u a t i o n was  that i t i s a mathematical  which  gives  i t  a  equation favoured  homologue of the  physical microbiological  interpretation. There a r e models t h a t have been proposed t h a t do not f i t the above two considered  classifications.  circumstances  surrounding  This  a  result  the m i c r o b i o l o g i c a l p r o c e s s  few o f these a r e b r i e f l y c o v e r e d  2.2.5  i s usually  of  the  special  i n consideration.  A  next.  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 o f growth i s a s u b j e c t o f i n c r e a s i n g c o n c e r n .  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 model was proposed by Andrews  (1968).  subject.  A  An o f t e n used  - 40 -  F i g u r e 2.4  A compilation of equations  f o r t h e s u b s t r a t e and/or  b i o m a s s c o n c e n t r a t i o n dependence o f t h e s p e c i f i c growth r a t e :  (1) Monod; (2) M o s e r , X = 2; (3) M o s e r ,  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 (9) P o w e l l , L/K  = °°; (10) B l a c k m a n .  g  = 2;  - 41 -  \x - u S/(K + S + S /K.) m s 1  (2.22)  2  i n which equation A  i s an i n h i b i t i o n c o n s t a n t .  2.22 i s d e r i v e d from t h e t h e o r y o f i n h i b i t i o n o f a s i n g l e enzyme.  number  of  distinction experimental reader Tseng  L i k e t h e Monod e q u a t i o n ,  alternatives  between data  i s referred  to  various  (Yano  equation  proposals  2.22  have  i s impossible  e t . a l . , 1966; Edwards,  t o Webb  (1963),  Yamashita  been  proposed,  but  due t o t h e l i m i t e d  1970).  The  e t . a l . (1969),  interested Wayman and  (1975 & 1976), Yang and Humphry (1975) and D i B i a s i o e t . a l . ( 1 9 8 1 ) ,  f o r t h e v a r i o u s a l t e r n a t i v e s t o e q u a t i o n 2.22. Growth c a n be i n h i b i t e d  by m e t a b o l i t e s w h i c h a r e e x c r e t e d as a d i r e c t  o r i n d i r e c t consequence o f g r o w t h .  One o f t h e models o f t e n used t o d e s c r i b e  t h i s type o f 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  u- - ( S / ( K  g  (1967):  + S ) ) (K /(K + P ) )  (2.23)  where P i s t h e 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  An  i s that  example  of i n h i b i t i o n  that  i s well  known  c o n c e n t r a t i o n I n h i b i t i o n o f y e a s t growth ( H i n s h e l w o o d , 1969).  For alternatives  to equation  R a m k r i s h n a e t . a l . (1967) and L e v e n s p i e l  2.2.5.2 A treatment  of high  constant. ethanol  1946; A i b a and Shoda,  2.23, t h e r e a d e r  i s referred  to  (1980).  Models F o r Growth L i m i t e d By More Than One S u b s t r a t e f o r growth i n f l u e n c e d by t h e c o n c e n t r a t i o n o f more than one  s u b s t r a t e was g i v e n by Tsao and Hanson (1975) :  - 42 -  p.  H - {l + s  in  }  til  { 2  s.  1  which S^ are the concentrations of the e s s e n t i a l  concentrations not e s s e n t i a l  of  the  (2.24)  + s1.  substrates.  growth rate enhancing substrates,  are the  whose presence  is  for growth, but growth i s enhanced when they are supplied.  A  s p e c i a l case of equation 2.24  is  the double substrate k i n e t i c s described by  Megee (1970);  ^ -  +  S  l »  +  Apart from the work of Megee, successful been  reported by Nagai et.  al.  (1973)  S  (2.25)  2 »  applications of equation 2.25 have and Ryder and S i n c l a i r  d i f f e r e n t approach has also been suggested by Bloomfield et.  (1972).  A  a l . (1973).  2.2.5.3 Models For Product Formation A number of d i f f e r e n t described.  groups of product formation processes have been  The treatment introduced by Gaden (1959),  processes into three categories In  the  first  category,  is considered below.  the  product i s  a direct result  energy metabolism and i s  thus strongly associated  and  without  anaerobic  processes  that subdivides these  external  c l a s s modeling i s r e l a t i v e l y simple.  of  the primary  with growth (eg.  electron  acceptors) .  ethanol For  this  The rate of substrate consumption can  be assumed to be represented w e l l by the l i n e a r law:  dS/dt = ( d X / d t ) / Y  g x  +M X g  (2.26)  -  The  r a t e of p r o d u c t  43  -  f o r m a t i o n i s then g i v e n by:  dP/dt = ( d X / d t ) / Y  In  the  connection these  second  between  i n this  The between  third  and  there  i s no  metabolism  2.3.1  streptomycin  category one  includes and  direct  formation  production).  a l l those  two.  or  indirect  (examples of  Modeling  is  fairly  For  cases  these  that  are intermediate  the p r o d u c t  is  indirectly  acid).  Whey a n d / o r L a c t o s e  Despite  a  L i t e r a t u r e Review  Substrates  comprehensive  search  only  a l . , 1978; Yang, 1984) were u n c o v e r e d  k i n e t i c modeling  of whey/lactose  investigators have  undefined  obvious  and p r o d u c t  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;  1984)  (2.27)  t o t h e energy p r o d u c t i o n pathway (examples a r e p r o d u c t i o n o f amino  a c i d s and c i t r i c  et.  + M X p  category.  categories  connected  of  primary  are p e n i c i l l i n  difficult  2.3  category  px  (Schlottfeldt,  reported  single-phase  various  a  handful  concerning  of  papers  acidogenic m i c r o b i a l  substrates fermentation.  However a number  1979; Boening and L a r s e n , microbial  fermentations  kinetic  models  of l a c t o s e l i m i t e d  (Rogers  1982; and Yang, f o r defined  substrates.  and  Below,  the r e s u l t s o f each o f the above c i t e d works i s r e v i e w e d . 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 t o grow S t r e p t o c o c c u s cremoris  is a  cremoris  lactic  acid  i n batch  c u l t u r e a t 30°C and a pH o f 6 . 0 .  producing  micro-organism  that  has  S.  attracted  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 t h e s i g n i f i c a n t r o l e t h a t i t p l a y s as a  - 44 -  starter  culture  i n the d a i r y  industry.  Various  growth models were t e s t e d  f o r Sj_ c r e m o r i s and t h e f o l l o w i n g growth i n h i b i t i o n and a p r o d u c t  formation  r e l a t i o n s were recommended:  u = K ^ S / C K , + S)) ( K / ( K  + P))  (2.29)  dP/dt = K . ( d X / d t ) + K ( S / K ' + S ) )  (2.30)  p  p  C  J  <4  S  -dS/dt = ( d P / d t ) / Y  (2.31) sp  Estimates  of the constants  were  reported.  However on t e s t i n g  model w i t h a c o n t i n u o u s c u l t u r e , no washout was observed model.  T h i s was a t t r i b u t e d t o w a l l  Schlottfeldt  (1979)  t h e above  as p r e d i c t e d by t h e  growth.  employed  an u n d e f i n e d  culture  (obtained  from  a  methane g e n e r a t i n g r e a c t o r ) i n f e d - b a t c h 4L c a p a c i t y l a b o r a t o r y s i n g l e - p h a s e reactors,  to treat  COD removal Boening inoculum  f r e s h and r e c o n s i t i t u t e d  whey.  r a t e c o n s t a n t a t 35° was d e t e r m i n e d  The o v e r a l l f i r s t  order  t o be e q u a l t o .047 h ^.  and L a r s e n (1982) a l s o employed an u n d e f i n e d c u l t u r e ( s o u r c e o f  not i n d i c a t e d ) i n a single-phase  p a c k i n g medium o f c r u s h e d  coal  particles  fluidised  bed r e a c t o r w i t h  t o degrade whey permeate.  a  Three  temperature  levels  ( 1 5 , 25 and 35°C) were c o n s i d e r e d i n t h e s t u d y .  A model  previously  proposed  by Chen and Hashmoto (1978) o f t h e f o l l o w i n g  form was  found  t o be t h e b e s t 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 v a l u e s o f t h e c o n s t a n t s were  reported. Yang  (1984)  microbial  system  lactis),  a  i n an  ingenious  comprising  homoacetogenic  of  effort a  bacteria  proposed  homo-lactic  the use of a bacteria  defined  (Streptococcus  ( C l o s t r i d i u m formicoaceticum)  and  a  methanogenic b a c t e r i a (Methanococcus m a z e i ) . The for  a  f o l l o w i n g growth k i n e t i c modes f o r b a t c h f e r m e n t a t i o n were r e p o r t e d temperature  formicoaceticum, (1978) rate  of  35°C  a model  (Equations  and  similar  pH  of  7.0.  i n c o r p o r a t e d to modify  Sj_  t o t h e one employed  2.29 - 2.31) was used, w i t h  (K^ sometimes r e f e r r e d  For  lactis  and  C.  by Rogers  et.  al.  the e x c e p t i o n  t h a t a decay  t o as t h e m a i n t e n a n c e c o e f f i c i e n t ) term was  u ( E q u a t i o n 2.33).  dX/dt = ( u - K.) X a  For  M_^_ m a z e i ,  a  substrate  ( E q u a t i o n 2.22) was Yang's effects  experienced Yang  slight  assumed  that  model  proposed  by  Andrews  (1968)  utilised.  results  between  inhibition  (2.33)  seems  S_^  t o suggest  lactis  and  pH v a r i a t i o n s each  C^ from  t h a t t h e r e were s e r i o u s a n t a g o n i s t i c formicoaceticum, t h e optimum v a l u e  b a c t e r i a i n t h e c o c u l t u r e would  i f the o f 7.0. exhibit  coculture However t h e same  f e r m e n t a t i o n k i n e t i c s as i n t h e pure c u l t u r e form.  A t h e o r e t i c a l model was  developed  pure  f o r the  three  bacteria  using  the  culture  U n f o r t u n a t e l y no e x p e r i m e n t a l v e r i f i c a t i o n o f t h e model was made. it  was  suggested  that  a  two-phase  process  with  lactic  acid  models. However, as t h e  i n t e r m e d i a t e was t h e b e s t f o r t h e d e f i n e d methane f e r m e n t a t i o n s t u d i e d .  - 46 -  2.3.2  The  Other  Substrates  two d o m i n a t i n g  methanogenesis) Herremoes  different  steps  The d a t a  g  used  literature  i n a n a e r o b i o s i s ( a c i d o g e n e s i s and  microbial  kinetic  constants.  i n c l u d e s e x p e r i m e n t a l l y determined  i n modeling,  searches.  The  Henze and  f o r these s t e p s the r e p o r t e d v a l u e s o f n^,  ( 1 9 8 3 ) have c o m p i l e d  Y, and K . constants  have  bacterial  the l a t t e r  based  on more  g e n e r a l i s a t i o n s of Henze  c o n s t a n t s and  or l e s s  extensive  and Herremoes a r e  p r e s e n t e d i n T a b l e 2.2.  2.4  Mathematical A n a l y s i s of  In  this  culture  study  methods,  Continuous  continuous  because  Cultures  culture  pH was  an  methods important  maintained a t a constant l e v e l throughout o f a pH c o r r e c t i n g dismissed  were  favoured  variable  that  over  batch  had t o be  the experimental p e r i o d .  Addition  s o l u t i o n ( e g . NaOH) was e v a l u a t e d f o r b a t c h c u l t u r e , but  due t o f e a r o f t o x i c  effects  caused  by an a c c u m u l a t i o n  o f Na i n  the r e a c t o r . Although modeling and  o f c o n t i n u o u s c u l t u r e s i s v e r y w e l l documented  F e n c l , 1966; B a i l e y  Monod  chemostat  assumptions The  model  and O l l i s , (CSTR)  1986),  (Malek  t h e d e r i v a t i o n o f t h e so c a l l e d  i s presented  below  to  illustrate  the  made i n i t s development.  analysis  of continuous  cultivation  of microorganisms  the b a c t e r i a l mass b a l a n c e e q u a t i o n ( r e f e r t o F i g u r e 2 . 5 ) .  starts  with  T a b l e 2.2  G e n e r a l i s e d Growth C o n s t a n t s F o r A n a e r o b i c C u l t u r e s  ^m \  Parameter  Culture  Maximum specific rate at  growth  Y Maximum yield coefficient  Maximum subst: r a t e r e m o v a l r a t e a t 35°C (Kto/Y)  g VSS /g  100%  1  35°C ( h  _ 1  )  COD  g COD/g; VSS.h active VSS  50%  active VSS  K  s  Monod S a t u r a t i o n constant g COD/inL  Acetic acid producing bacteria  .0833  .15  .5417  .2917  .2 x 1 0 ~  Methane producing bacteria  .0333  .03  .5417  .2917  .05 x 1 0 ~  Combined  .0333  .18  .0833  .0417  —  3  3  1 VSS - v o l a t i l e suspended  s o l i d s - a term used t o r e p r e s e n t biomass  i n waste water t e c h n o l o g y  -  F i g u r e 2.5  The  48  -  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 = i n p u t - o u t p u t + r e a c t i o n  In  case of a CSTR, t h e r e  organisms (X) that  the  i s a constant  r a t e of  i n the v e s s e l of c o n s t a n t  feed  rate  equals  the  feeding  volume ( V ) .  overflow  rate.  (2.34)  (F) of a medium to  T h i s means, of c o u r s e ,  The  mass b a l a n c e  for  the  organisms i s :  (dX/dt) V = F X  q  - FX + uX  I n most c o n t i n u o u s c u l t u r e systems the f e e d or  is sterile,  F/V,  so  i s introduced  s t r e a m i s e i t h e r f r e s h l y made up  =0. A term c a l l e d d i l u t i o n r a t e D w h i c h i s e q u a l to o i n t o e q u a t i o n 2.35 t o g i v e ,  DX  (2.36)  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 t o the  culture.  The  will  be  The  other  ingredient  exhausted  activities, nutrient.  first  ingredients but  i n short and  will  play  they w i l l  not  supply  relative  thus l i m i t  various be  theory  t o the o t h e r  cellular  roles,  such  i n acute short  of c o n t i n u o u s ingredients  or p r o d u c t as  supply  synthesis.  promoting  cellular  as  limiting  i s the  A mass b a l a n c e on the growth l i m i t i n g n u t r i e n t g i v e s ,  ( d S / d t ) V = FS  where Y i s the y i e l d is  (2.35)  X  ( d X / d t ) = uX -  The  V  - FS - uXV/Y -  c o e f f i c i e n t (g of c e l l  the maintenance c o e f f i c i e n t to keep c e l l s  per  MXV  (2.37)  g of l i m i t i n g n u t r i e n t ) .  alive.  D i v i d i n g by V  M  yields,  - 50 -  dS/dt = D S  A r e l a t i o n s h i p i s needed  q  - DS - uX/Y - MX  between u and S.  This  t h a t have been c o n s i d e r e d i n s e c t i o n 2.2 come i n .  (2.38)  i s where t h e e x p r e s s i o n s As p r e v i o u s l y m e n t i o n e d ,  t h e use o f t h e Monod e q u a t i o n ( E q u a t i o n 2.5) was f a v o u r e d i n t h i s s t u d y . steady  s t a t e , t h e r e i s no change, thus  At  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  e q u a t i o n s 2.36 and 2.38 d i s a p p e a r t o g i v e ,  u = D  (2.39)  and  DS  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 e q u a t i o n 2.5 and s o l v i n g f o r S g i v e s , S = DK / ( u - D) s m  (2.41)  S o l v i n g e q u a t i o n 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 -  DY(S  Q  -  S)/(D  +  (2.42)  MY)  E q u a t i o n s 2.41 and 2.42 a r e o f t e n c a l l e d t h e "Monod Chemostat M o d e l " . The  above  assumption  of  analysis perfect  f u n d a m e n t a l except  applies mixing  t o any c o n t i n u o u s and  constant  culture  volume.  The  that  meets t h e  equations  are  f o r t h e Monod e q u a t i o n w h i c h has no time dependence and  s h o u l d be a p p l i e d w i t h c a u t i o n t o t r a n s i e n t s t a t e s where t h e r e may be a l a g t i m e as \i r e s p o n d s t o t h e changs S.  - 51 -  2.5  Experimental Based  that:  Plan  on t h e m a t e r i a l p r e s e n t e d  (1) E f f e c t i v e  combination  d i g e s t i o n of organic  non-methanogens (2)  anaerobiosis, Impossible suitable;  may  Due  result to  the  modeling  task.  Therefore,  chapter  matter  of carbon c a t a b o l i s i n g anaerobic  methanogens p l a y a key r o l e .  routes;  i n this  i t can be  t o methane  r e q u i r e s the  b a c t e r i a l g r o u p s , of w h i c h t h e  s e p a r a t i n g t h e methanogens from t h e  i n alterations  i n the i n t e r m e d i a r y  complexity  of  the  biochemical  growth  with  structure  microbial  Therefore  concluded  unstructured  models  metabolite  processes is  a r e found  an  in  almost  t o be  most  (3) K i n e t i c d a t a on t h e a c i d o g e n i c c o n v e r s i o n of l a c t o s e i s a l m o s t  nonexistent.  2.5.1 As mainly  Assumptions  pointed  o u t i n s e c t i o n 2.1.2.1,  i t i s believed  fermented v i a t h e Embden-Meyerhof-Parnas  assumed  that  l a c t o s e i s mainly  fermented  that  hexoses a r e  (EMP) pathway.  So i t was  v i a the EMP pathway t o p y r u v a t e . 14  Thus  t h e t a s k o f e l u c i d a t i n g the degradation  reduced  to the d e t e r m i n a t i o n  It  also  was  assumed  that  o f t h e f a t e of p y r u v a t e lactose  galactose v i a8-galactosidase. is  converted  to glucose  pathway u s i n g  is first  broken  C t r a c e r s was  during down  acidogenesis.  to glucose  and  G l u c o s e e n t e r s t h e EMP pathway and g a l a c t o s e  6-phosphate  before  i t enters  t h e EMP  pathway, a  scheme t h a t 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  The e x p e r i m e n t a l  Factors  f a c t o r s and l e v e l s s t u d i e d a r e p r e s e n t e d  i n T a b l e 2.3.  - 52 -  T a b l e 2.3  Temperature substrate  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  pH Dilution  Investigated  Levels  Factor  Limiting  Factors  rate  Radiotracers  6.5  .05, .1, .2, .3, .4, .5 and .6 h L a c t a t e , p r o p i o n a t e and b u t y r a t e  -1  - 53 -  III.  MATERIALS AND METHODS  3.1  Inoculum The  mixed,  obtained at ten  Iona  culture  inoculum  was sewage  sludge.  I t was  from a l o c a l two-step a n a e r o b i c m u n i c i p a l waste t r e a t m e n t Island.  The i n o c u l u m  samples were drawn from t h e f i r s t  facility stage, at  o r f i f t e e n f e e t from t h e s u r f a c e .  3.2  Media Two  a  undefined  t y p e s o f growth medium were employed i n t h i s s t u d y :  chemically defined  reconstituted  whey  8-lactoglobulin).  powder,  limited  nutrient  (comprising  solution  of lactose  Lactose Limited Growth Sythetic Medium  Since  lactose  (shown  carbon  source  approximately  i s t h e major  i n Table  single  component  3.1) was composed  and p r o t e i n ,  (substrate).  The  that  of  C:N:P  ratios  150:5:1, f o r i t has been o b s e r v e d  requirements  f o r single  i n whey,  such  1979; Pos e t . a l . , 1981; and P i p y n  nutritional  t h e second  was  mainly  The d e t a i l s o f t h e s e growth medium a r e g i v e n below.  3.2.1  medium  Hill,  lactose  t h e f i r s t was  phase  the nutrient  lactose were  (Speece  i s t h e main  maintained  at  and M c C a r t y , 1964;  and V e r s t r a e t e , anaerobiosis  1981) t h a t  are  directly  p r o p o r t i o n a l t o t h e s y t h e s i s o f m i c r o b i a l c e l l s and t h e i n d i c a t e d r a t i o s a r e n e c e s s a r y t o m a i n t a i n n u t r i t i o n a l l y b a l a n c e d growth. nutritional macro  information f o r acidogenic bacteria  elements,  calcium,  sodium,  potassium  No s e p a r a t e m i c r o b i a l  were  found.  and magnesium  Cations of were  kept a t  100-200 mg/L f o r t h e former two and 200-240 mg/L f o r t h e l a t t e r two.  These  - 54 -  T a b l e 3.1  L a c t o s e L i m i t e d Growth Medium  Concentration (g/L)  Component  Supplier  Substrate 12 22°11 2° N u t r i e n t s ( o r macro e l e m e n t s ) C  H  H  10.53  BDH  0.800  BDH  0.180  FISHER  MgS0 .H 0  0.150  MCB  KC1  0.740  BDH  CaCl .2H 0  0.730  MCB  NaHC0  0.300  AMACHEM  0.100  MALLINCRODT  NH.C1 4 (NH ) HP0 4  2  4  4  2  2  2  3  T r a c e ( o r m i c r o ) elements Fe(NH ) S0 4  2  4  MnCl .4H 0  0.005  Z n S 0 -7H 0 CuSO,.5H_0 4 2 NaB.0..10H-0 4 2 2 NaMoO,.2H.0 4 2  0.005  AMACHEM  0.005  MCB  0.005  AMACHEM  0.005  BAKER  0.150  BDH  2  4  Chelating Citrate  2  2  agent  - 55 -  concentration  levels  M i c r o elements Speece  were  observed  by McCarty  (1964)  c o n c e n t r a t i o n s were k e p t a t l e v e l s  e t . a l . (1983).  No growth  factors  t o be  similar  were added.  stimulatory.  t o those used by A l l the chemicals  were r e a g e n t grade u n l e s s o t h e r w i s e i n d i c a t e d .  3.2.2  Lactose/Protein Growth Medium  T h i s medium was made by a d d i n g 15.4 g o f sweet whey powder (SIGMA) t o 500 mL o f d i s t i l l e d t o one l i t r e .  3.3  water.  A f t e r m i x i n g t h o r o u g h l y t h e m i x t u r e was made up  An a n a l y s i s o f the medium i s shown i n T a b l e 3.2.  Fermentor Set-Up A modular  continuous pH,  monitored.  rate  on  and e f f l u e n t .  the r a t e  a  could  2N, 4N  of i n f l u e n t  o r 6N NaOH With  400  rpm.  and  Cloutier,  had a paddle  A h i g h rpm v a l u e was s e l e c t e d 1959) and t o m i n i m i s e  f e r m e n t o r v e s s e l components. circulation  through  agitator  was  used,  concentrations of  change i n d i l u t i o n which  was r u n a t  t o ensure complete m i x i n g ( C h o l e t t e  attachment  of m i c r o - o r g a n i s m s  on t h e  t e m p e r a t u r e was m a i n t a i n e d by hot  steel  t e m p e r a t u r e was c o n t r o l l e d a t 35 ±0.5°C. from 1.25 t o 5.00 L.  percentage  wheel  The d e s i r e d stainless  these  solution  v a l u e s and  t o b e t t e r than ±0.1 u n i t s o f t h e  value, while causing only a minimal The f e r m e n t o r  As shown i n F i g u r e 3.1, t h e be s e t a t d e s i r e d  flow.  t h e pH v a l u e c o u l d be c o n t r o l l e d  (D).  water  (NEW BRUNSWICK) was m o d i f i e d to a l l o w  (rpm) and t e m p e r a t u r e F o r pH a d j u s t m e n t  depending  desired  top f e r m e n t o r  pumping o f i n f l u e n t  agitation  caustic,  bench  heat  exchanger  tubing.  The  The f e r m e n t o r had a w o r k i n g volume  - 56 -  T a b l e 3.2  Sweet Whey Growth Medium A n a l y s i s  Component  Concentration (mg/L)  Method o f Analysis  10,000.0  Colourimetric  Substrate 12 22°11 N u t r i e n t s (macro e l e m e n t s ) C  H  Ammonium  nitrogen  Total Kjeldahl  nitrogen  9.5 242.5  T o t a l p r o t e i n as 8-Lactoglobulin  2,600.0  Biuret  reaction  Phosphorous Sulfur Magnesium  18.0  Potassium  334.0  Calcium Sodium Trace  83.0 134.0  ( m i c r o ) elements  Iron  0.7207  Copper  0.2495  Atomic  absorption  - 57 -  - K J  3  CXD  F i g u r e 3.1  The f e r m e n t o r and a u x i l l i a r y a p p a r a t u s : (1) fermen t o r v e s s e l , (2) h e a t exchanger t u b i n g , (3) l e v e l s w i t c h , (4) pH p r o b e , (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 t o w a s t e , (13) NaOH t o v e s s e l , (14) h o t w a t e r o u t , (15) h o t w a t e r i n , (16) s t i r r e r , (17) wet gas meter.  - 58 -  3.4  Fermentor  Start-Up  For  run at a desired  fixed  pH v a l u e  1.5 L o f growth medium ( d i l u t e d  three  times)  50  each  mL of s c r e e n e d  twenty  fermentor  oxygen.  And O p e r a t i o n  inoculum. vessel  The f e r m e n t o r  head  I t was o p e r a t e d  Procedure  space  the fermentor,  was f r e s h l y  was then  volumes  i n b a t c h mode u n t i l  containing  Inocculated  purged  of helium  with  with at least  to expel  a l l the  a l l the l a c t o s e was used up,  a f t e r w h i c h time the pumps were s w i t c h e d on and s e t f o r d i l u t i o n r a t e (D) o f approximately  .05 h .  specific  rate  growth  washed o u t .  .025  h  1  According state  t o by Pohland  conducted  to C o l i n  running c o n d i t i o n s .  period  terms showed  no  a t each s t e p  e t . a l . (1983) before  b a c t e r i a t o be  between  Analyses  every  t h e methods  and  a  for at least  weeks  was  needed  steady  within protein,  d r y biomass  a  period  t o complete  formate, were  at  lactate  made  an  performance, i n  solution  of  three  On average a  as when f e r m e n t o r  o f t h e pH c o r r e c t i n g  alteration  two d i l u t i o n s used  was t h e r e f o r e o p e r a t e d  to three  of l a c t o s e ,  carbon  that  two o r t h r e e d i l u t i o n s under t h e same  s t a t e was d e f i n e d  significant  f o r a minimum o f two d i l u t i o n s .  s t a t e c o n d i t i o n s had been a c h i e v e d . two  For  up i n s t e p s o f  i t i s commonly a c c e p t e d  at least  The f e r m e n t o r  steady  Steady  biomass  replicated  f o r t h e methanogenic  and Ghosh (1971) as phase k i n e t i c c o n t r o l .  c o n d i t i o n s had been a c h i e v e d .  of  enough  o f t h e r a t e o f consumption  dilutions. acids,  once  ranging  experiment.  o f D was s e l e c t e d t o m a i n t a i n t h e  a t h i g h e r d i l u t i o n r a t e s , D was stepped  and maintained  i s n o t reached  dilutions,  (u-) h i g h  value  T h i s method o f a c i d o g e n i c and methanogenic phase s e p a r a t i o n has  been r e f e r r e d experiments  This  1  o f NaOH,  least  three  volatile  fatty  steady  state  after  F o r most o f t h e r u n s , these measurements were up t o a maximum o f f o u r t i m e s .  f o r t h e above  analyses  are outlined  The d e t a i l s  i n Appendix  A.  - 59  T o t a l gas  production  revolution), space gas  was  r e c o r d e d by a wet  shown i n F i g u r e  a n a l y s i s and  the  3.1.  S e t - U p For  under anaerobic or  [  1 4  one  Two  i t was  the  f o r the  a c t u a l gas  conditions with  fermentor  volume  to  per head  production  [  14  C(U)]  f e r m e n t o r were  - butyrate,  [  14  C(2)]  incubated  -  propionate  ENGLAND NUCLEAR) f o r d e g r a d a t i o n  t y p e s of a p p a r a t u s were needed f o r t h e s e t e s t s . necessary  L  Incorporation  e x p e r i m e n t s , samples from the  C ( U ) ] - l a c t a t e (NEW  studies.  meter (ALEXANDER - 0.25  A.  Radioactive Tracer  I n a s e r i e s of  gas  P r o c e d u r e s used  c a l c u l a t i o n of  a r e a l s o o u t l i n e d i n Appendix  3.5  -  monitor  and  control  t h r o u g h o u t the p e r i o d of the e x p e r i m e n t .  the  pH  at  mechanism I n the  first  fixed  value  a  I n the second c a s e , pH was  neither  m o n i t o r e d nor c o n t r o l l e d .  3.5.1 A  A p p a r a t u s W i t h pH  small  magnetically  t e m p e r a t u r e c o n t r o l was probe and the  heater  were  of  the  turned  stirred  modified  a thermometer.  beginning  Control  The  glass  with  as shown i n F i g u r e  t e m p e r a t u r e was  experiment,  on.  reactor,  Enough  the time  3.2  jacket  for  to accommodate a  c o n t r o l l e d a t 35 ±0.5°C.  temperature was  a water  c o n t r o l , water  allowed  for  steady  pump  state  to  pH At and be  14 achieved tracer  then  was  substrate  the  injected was  flushed with U •  r e a c t o r was together  needed  for  the  with  desired  level.  addition  of  Addition  some of  mL  production  i n i t i a l downward movement of the pH necessitating  10  level.  acid  acid  to  could  of of  The  specific  fresh acids  C - labeled  substrate. that  would  The  maintain  O t h e r w i s e , the pH would the  reactor  to  maintain  potentially affect  fresh  the  an  increase a  fixed  reaction  -  F i g u r e 3.2  60  -  Schematic d i a g r a m f o r r a d i o t r a c e r e x p e r i m e n t s w i t h pH control: (1) t e m p e r a t u r e c o n t r o l w a t e r i n , (2) n i t r o g e n , (3) pH p r o b e , (4) thermometer, (5) 2N NaOH s y 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) m e c h a n i c a l s t i r r i n g .  - 61 -  mechanism. fermentor minutes  After were  steady  state  injected into  and d u r i n g  ±0.1 m a n u a l l y .  this  was  achieved  the r e a c t o r .  period  at  normal  o f sample  from t h e  The e x p e r i m e n t was r u n f o r 30 and m a i n t a i n e d  a t 6.  A t the end o f the e x p e r i m e n t , 10 mL o f sample were removed  -40°C.  Prior  c e n t r i f u g e d ( l h , 4450xg). mL  mL  the pH was m o n i t o r e d  f r o m the r e a c t o r by s y r i n g e and i m m e d i a t e l y frozen  80  to  analysis  i n t r o d u c e d i n t o a 24 mL v i a l and the  samples  1 mL o f t h e s u p e r n a t a n t  s u l p h u r i c a c i d and l o a d e d  directly  were  defrosted  and  was a c i d i f i e d by a d d i n g 1  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 The and  A p p a r a t u s W i t h o u t pH C o n t r o l c a p o f a 20 mL v i a l was m o d i f i e d  to a l l o w continuous  o u t ( F i g u r e 3 . 3 ) . The v i a l was i n i t i a l l y  flushed with N . 2  flow of  in  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 i n t r o d u c e d i n t o t h e v i a l by s y r i n g e . sample from t h e f e r m e n t o r was then i m m e d i a t e l y vial  was then m a i n t a i n e d  period  complete mixing.  i n j e c t e d i n t o the v i a l .  a t 35 ±.5°C f o r a s p e c i f i c  a s m a l l amount o f N  2  was a l l o w e d  A 10 mL  period.  During  The this  to flow through the v i a l to a f f e c t  A t t h e end o f the e x p e r i m e n t ,  the contents  o f the v i a l  were t r e a t e d i n a manner s i m i l a r t h a t 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 O r g a n i c A c i d s 3.6.1  Principle  Single concentration concentration  components changes  at  of  a  mixture  the boundary  dissolved with  a  i n one  second  o f components on the s u r f a c e o f the o t h e r  phase  phase.  show  Often  phase t a k e s  a  place.  T h i s phenomenon i s r e f e r r e d t o 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 radio t r a c e r experiments w i 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 c a p , (A) n i t r o g e n to fume hood.  -  is  proportional  adsorption  to  their  coefficients  phase b o u n d a r i e s . of  the  separation  (Mikes,  -  adsorption  determine  I f one  separation  63  coefficient.  the  1979).  which  There  is  are  methods based on d i f f e r e n t p r i n c i p l e s  the  to the  basis  other  o l d e s t known and  p l a c e between l i q u i d  and  this  study  with  suitable  solvent  coated  flows  there  the is a  chromatographic  chromatographic  (eg. p a r t i t i o n ,  on  in  separation  i o n exchange, g e l  and  here.  commonest a d s o r p t i o n chromatography i s t h a t w h i c h  takes  celite  other  of  b i o a f f i n i t y chromatography) t h a t w i l l not be d i s c u s s e d The  differences  differences i n concentrations  phase i s moved r e l a t i v e  components  The  around  solid  phases.  sucrose)  The  are  p a r t i c l e s of a s o l i d ( i n  placed  in  a  glass  tube,  them c a r r y i n g the components w i t h i t and  a the  s e p a r a t i o n of the components takes p l a c e on the a d s o r b e n t s u r f a c e . Adsorption different  chromatographic  procedures  chromatography.  namely,  methods  frontal  may  be  F r o n t a l a n a l y s i s i s not  method.  However  out  a n a l y s i s , displacement  by and  suitable for preparative  D i s p l a c m e n t chromatography i s p r i m a r i l y i m p o r t a n t pilot-plant  carried  i t ' s requirement  three elution  purposes.  as a p r e p a r a t i v e or even a of  the  use  of  a  suitable  a u x i l i a r y s u b s t a n c e s 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 b e i n g separated, In  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 p u r p o s e s .  elution  chromatography,  i n t r o d u c e d i n t o the column, and  a  small  part  of  the  sample  i s t h e n 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 I s s m a l l e r t h a n t h a t o f any  component.  of  down the  repeated  component affinities  adsorption  the  components move s l o w l y  i s eluted  independent  for  solid  the  solution is  of  the  phase.  others, The  i n order  component  of  As a r e s u l t  column. the  zones  Each  components are  very  - 64  o f t e n separated  -  by a zone of pure s o l v e n t d u r i n g  t h e i r movement t h r o u g h  the  column. Elution separated  of  a l l components  substances  do so  with  not  differ  that  their  the too  stationary  phase,  intervals.  I n s i t u a t i o n s where t h i s  same s o l v e n t  much i n t h e i r  zones  are  i s not  eluted  i s possible affinity  if  the  towards  the  without  the case " s t e p w i s e  long  time  elution"  may the  be more s u i t a b l e .  S t e p w i s e e l u t i o n i s c a r r i e d out by g r a d u a l  e l u t i o n of  column by  eluents  e l u t i n g power.  several  These s o l v e n t s the  s t a t i o n a r y phase and  gradual  release  i n order  individual  this  both simple  study and  modified  formate  and  an  step to  adsorption  The as  3.6.2  reported  separate  method eluents.  from  elution"  uses  of s o l v e n t s .  by  techniques  that  employs  Wiseman and  Irvin  (1957))  celite  propionate,  coated  Its distinctive  acetate,  with  sucrose  and  feature  i s that  the  d i r e c t l y to the column.  Apparatus  C h r o m a t o g r a p h i c tube and Tamping  d i a m e t e r , 60 cm  rod,  accessories  consisting  of  a  (Figure  T i t r a t i o n assembly.  3.5).  stainless steel  l o n g , s i l v e r - s o l d e r e d to the c e n t r e  24 - gauge w i r e punched from a 16 - mesh C.  the m i x t u r e  "Gradient  butyrate,  employs  a c i d s i n aqueous s o l u t i o n s are l o a d e d  3.  components of  chromatographic  (first  preparatively  lactate.  increasing  e l u t e them ( F i g u r e 3.4).  elution,  hexane-acetone mixtures  A.  of  i n s t e a d of a b r u p t changes i n c o m p o s i t i o n  In  was  gradually  arranged  screening.  rod  of a 16 mm  3.2  mm  in  i n diameter,  -  Figure 3.4  65  -  T y p i c a l chromatogram o f a complex m i x t u r e s e p a r a t e d by a c o m b i n a t i o n o f s i m p l e 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 o f components, E-^-Ey - e l u t i n g power o f t h e eluting solvents.  - 66 -  F i g u r e 3.5  S c h e m a t i c d i a g r a m o f the l i q u i d c h r o m a t o g r a p h y 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" s w a g e l o o k f i t t i n g w i t h s e p t u m , (7) e l u e n t t a n k , (8) s p o u t f o r r e f i l l i n g t h e t a n k , (9) n i t r o g e n cylinder.  - 67  3.6.3 A.  M a t e r i a l s and Reagents Celite  anhydrous B.  -  analytical  filter  a i d (SIGMA),  fine  granulated  sugar,  sodium s u l f a t e and ammonium s u l f a t e . Cresol  red i n d i c a t o r :  0-cresolsulfonphthalein  1.3 mL o f 0.1 N NaOH was added t o 50 mg o f  i n 20 mL of a l c o h o l and made to 50 mL w i t h  distilled  water. C  Alphamine  red-R i n d i c a t o r :  0.4 g were added t o 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  E. Various  n-Hexane  glass  percentages  by  follows:  distilled volume  (BDH),  of acetone  i n n-hexane  were  grade  (BDH).  made  up as  removal o f water from t h e column by a d r y e l u e n t , BA^ was  against  were s t i r r e d  vigorously  one mL  t h e s t a t i o n a r y p h a s e as f o l l o w s : with  of saturated  After  settling,  i t through a f i l t e r  3.6.4  Two l i t r e s o f BA^  50 mL o f 50% sugar s o l u t i o n t o w h i c h had been barium  hydroxide  c r e s o l r e d i n d i c a t o r t o f r e e the s o l v e n t  passing  reagent  5  equilibrated  acids.  Acetone,  1, 1 5 , 2 0 , 30 and 50%. These were r e f e r r e d to as BA^, BA^ e t c .  To p r e v e n t g r a d u a l  added  acid.  the s o l v e n t  solution  and a few drops o f  of c a r b o n d i o x i d e and any t r a c e s o f  was  freed  o f suspended  droplets  by  paper.  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 s u g a r s o l u t i o n (2 sugar t o 1 water by volume) and 0.1 mL o f normal acid,  r e s u l t i n g i n a s t a t i o n a r y phase  This  m i x t u r e was added s l o w l y  500  mL o f BA,. i n a b l e n d e r . n  sulfuric  o f a p p r o x i m a t e l y 50% sugar s o l u t i o n .  t o a s w i r l i n g s u s p e n s i o n o f 50 g o f c e l i t e i n Stirring  was v i g o r o u s l y  continued  for3  -  minutes.  A d s o r b e n t thus prepared  -  63  was s t o r e d i n a g l a s s - s t o p p e r e d  flask i n a  r e f r i g e r a t o r u n t i l needed. The  column  separatory w i t h  funnel  prepared  into  by  t h e adsorbent  flowing  two 1" x 6" QVF g l a s s  tubes  3.5) u n t i l t h e slurry  through pressure  t o dislodge  to a f i x e d  valve  was c l o s e d  the a d s o r b e n t . fine  were n e a r l y  full.  stream  A tamping r o d was passed  a i r bubbles.  With  when the s o l v e n t had been e x p r e s s e d  The t o p QVF g l a s s tube was then removed. the s i d e  turbulent  effects.  ammonium  sulfate  i n weight  material  were a d d e d as a s l u r r y i n about 25 mL o f BA^.  bolted  on  the tube  Approximately B A ^ g S o l v e n t  For  (Figure 1-2  was  3.5).  propionate of  initially  mL/min.  (SUPERRAC,  of of  applied  separations,  loaded  The  t o the top of  BA^ was added as a  the top s u r f a c e  from  sulfate,  celite  12:8:1,  referred  to as cap  t o compress through  and  The t o p f l a n g e was the cap m a t e r i a l .  t h e column t o remove t h e  on  BA^ was u s e d  a  mL  sample  t h e column  by  a  was a d j u s t e d  syringe to give  to e l u t e b u t y r a t e ,  i n that order.  w  a  from  s  u s e c  the r a d i o through  BA.^ was u s e d *  t o  e  ^  u t e  i n 20  standard  mL  units.  In  sample was i n i t i a l l y  order  to  test  tracer  t h e septum  a drip rate  The f r a c t i o n s were c o l l e c t e d by an a u t o m a t i c  nonradioactive  stream.  sodium  2  The bottom v a l v e  KLB)  compressing  present.  and a c e t a t e  lactate.  p r o p o r t i ons  and p r e s s u r e  isolating  grams  75 mL o f 3A^ w e r e f o r c e d  preparative  experiments  Eight  lightly  the bottom v a l v e open a  volume i n a r a p i d l y moving s o l v e n t  o f the tube,  a  and v a l v e as shown i n  down  further  from  i n series,  o f 10 p s i g was a p p l i e d t o the c h r o m a t o g r a p h i c column,  adsorbent  bottom  they  slurry  (connected  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  Figure  the  was  t o remove  folate sample  between  ahead collector  the column  a  a p p l i e d t o the column and the  - 69 -  m o n i t o r e d by t i t r a t i o n w i t h 0.005  p r o g r e s s o f the a c i d s down the column was N  barium  10 mL o f t h e  hydroxide.  20 mL f r a c t i o n were  pipetted  into a  t i t r a t i o n f l a s k , a p p r o x i m a t e l y 30 mL of c a r b o n d i o x i d e f r e e w a t e r were added and  the  s o l u t i o n was s t i r r e d  carbon  dioxide  traces  of carbon  barium  hydroxide  shown i n F i g u r e  3.7  free  nitrogen  dioxide. t o the  magnetically  f o r 3 minutes w h i l e  was b u b b l e d  through  Then  the  c r e s o l red  the  a stream o f  solution  s o l u t i o n was t i t r a t e d  end p o i n t .  A typical  determined  by  t o remove 0.005 N  with  chromatogram i s  3.6.  Determination of R a d i o a c t i v i t y Sample  radioactivities  were  liquid  scintillation  spectrometry.  3.7.1  Principle  O r g a n i c compounds c a l l e d " s c i n t i l l a t o r s " have the p r o p e r t y r a d i a n t energy e i t h e r i n the s o l i d  state or i n solution.  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 f o r m a t i o n  The  of  absorbing  absorption  of  of e x c i t e d atoms o r  m o l e c u l e s t h a t t h e n r e t u r n r a p i d l y t o the normal o r ground s t a t e , r e l e a s i n g energy  as photons  transparent range.  (light  energy)  t o t h e i r emitted  These  s c i n t i l l a t o r s are  l i g h t , w h i c h i s i n the u l t r a v i o l e t o r  The number o f photons e m i t t e d  the r a d i a n t energy a b s o r b e d . converts  and h e a t .  visible  i s approximately l i n e a r l y r e l a t e d to  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 t h a t  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  scintillators  "liquid are  scintillation"  usually  dissolved  counting  in a suitable  i s used solvent  because  these  containing the  ^4  O  ao  FRACTION NUMBER  F i g u r e 3.6  r 40  -r 60 60  "1  ao  70  (20ml advent/fraction).  O r g a n i c a c i d s chromatogram: (3) a c e t a t e , (4) l a c t a t e .  (1) b u t y r a t e ,  (2) p r o p i o n a t e ,  - 71 -  radioactive solution  material  results  8-particle primary  to  from  emitted  solvent  be  assayed.  the f o l l o w i n g  from  a  molecules  The  sequence  radioactive  photon  production  of e v e n t s .  source  is  The  first  ( e g . T o l u e n e ) , c a u s i n g them  from  energy  absorbed to become  this of the  by  the  excited.  T h i s e x c i t a t i o n , h i g h f r e q u e n c y energy, i s p r o p a g a t e d w i t h i n the s o l v e n t transferred causing their  the  primary  the s c i n t i l l a t o r  ground  energy a  to  state  light  to them.  solvent  to  tetra-phenylbutadiene)  a  at  solvent, time. the  (eg.  Biofluor  emits  (NEW  scintillator even  (photon  sample i t s e l f may  its  radiation;  the  system.  absorb l i g h t  solvent  may  (eg.  1,  1', light  4,  4'  that  by is  'scintillation  (PACKARD)) c o n t a i n i n g  to an e x c e l l e n t  a  f o r some  treatise  on  (1974) . t h a t reduces the l i g h t  Quenching  can  occur  i n several  output ways:  g i v e n o f f by the s c i n t i l l a t o r or some of  not  t o the s c i n t i l l a t o r ;  i s propagated  have been a v a i l a b l e  i s a term a p p l i e d t o any f a c t o r  the  When they r e t u r n t o  frequency  Instagel  the r e a d e r i s r e f e r r e d  p r o d u c t i o n ) i n the  efficiently  lower  scintillator  s u b j e c t by K o b a y a s h i and Maudsley Quenching  - Diphenyloxazole),  A number of c o m m e r i c a l  ENGLAND),  secondary  F o r more d e t a i l s  5  T h i s lower frequency l i g h t  secondary  which  a p r i m a r y and  2,  f r e q u e n c i e s l o w e r than t h a t a t which  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 t u b e . cocktails'  (eg.  m o l e c u l e s to become e x c i t e d .  they emit  i s transferred  secondary  scintillator  and  transfer  the energy  the s c i n t i l l a t o r  of  i t s e l f may  the  8-particle  a b s o r b some of  i t s f l u o r e s c e n c e ; or c h e m i c a l i n t e r a c t i o n of the components c o n t a i n e d i n the c o u n t i n g s o l u t i o n may quenching  is  efficiency. efficiency  synonymous The  or  r e s u l t i n reduced photon o u t p u t .  the  most  with  the  d e t e r m i n a t i o n of  common methods  methods  used  to  used  correct  to  The d e t e r m i n a t i o n of the  sample  counting  determine  sample  counting  for  the  loss  of  detectable  -  activity  by  quenching  s t a n d a r d method; and was  are:  the  the c h a n n e l s  72  -  internal ratio  standard  method.  method;  The  below.  Channels Ratio Method  This always  external  c h a n n e l s r a t i o method  employed i n t h i s s t u d y and i t s t h e o r e t i c a l b a s i s i s g i v e n  3.7.2  the  method  i s based  displaced  when  on  the  quenching  fact  that  occurs.  the  pulse height  In  a  two  spectrum  channel  is  instrument  14 assaying having  C,  e n e r g i e s from  efficiency 8-particles counting the  of  100%  various  of 50%.  a  number 14  amounts of  narrow  curve.  be e q u i v a l e n t to a c o u n t i n g  Channel  0-50  divided  KeV,  less  than  by one.  disintegrations  window  ratio-efficiency  would  C m a t e r i a l would  absolute a c t i v i t y ,  the  which  8 can  which  I f the c h a n n e l r a t i o  efficiency  t h e n o b t a i n e d by d i v i d i n g in  KeV,  e n e r g i e s from  efficiency  i s always  0 - 156  t o 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  (see F i g u r e 3.7).  having  narrow-window  ratio  The  i t i s possible  the  be  s e t to count a l l  would  be  i s a r b t r a r i l y d e f i n e d as  wide  window  Non-quenched  the An  efficiency,  samples  pec minute  counting  (dpm), of each  efficiency  efficiency or  (cpm)  curve  of  0.5.  sample i s  determined  quenching  the  containing  have a c h a n n e l s r a t i o n A/B  the net c o u n t s , c o u n t s per minute  by  e q u i v a l e n t to  appearing from  is  the  usually  p r e p a r e d by c o u n t i n g a s e r i e s of d i f f e r e n t l y quenched s t a n d a r d s whose a c t u a l activities  a r e known and  then p l o t t i n g  the e f f i c i e n c y  a g a i n s t the  channels  ratio.  3.7.3 A  Procedure  Philips  throughout  this  PW  4700  study.  liquid  scintillation  counter  (PHILIPS)  was  used  C o m m e r c i a l l y a v a i l a b l e quenched s t a n d a r d s were used  - 73 -  keV  F i g u r e 3.7  spectra:  14  t h e s o l i d curve r e p r e s e n t s unquenched , , 14„ . , C, t h e dashed curve r e p r e s e n t s quenched C, A and  B r e p r e s e n t v a r i o u s window w i d t h s .  100  go-  5z IU  i  to  70-  IU  oo-  so0.4  0.0  -r  0.0  CHANNELS RATIO (A/B).  F i g u r e 3.8  E f f i c i e n c y curve prepared standards.  from c o m m e r c i a l quenched  -  to  g e n e r a t e the e f f i c i e n c y  74  c u r v e 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 . mL  of  insta-gel  (PACKARD)  cocktail  a c c u m u l a t i n g a number of v i a l s , counted  for  five  minutes.  were  added  and  shaken  c o u n t i n g was done o v e r n i g h t . Peaks  of  radioactivity  i d e n t i f i e d by c o m p a r i s o n w i t h the e l u t i o n o f s t a n d a r d s .  were  well.  Five After  Each v i a l  was  detected  and  IV.  RESULTS AND DISCUSSION  All  the experiments  categories:  (1) A-series  o f 0.05 h  conducted  o f 0.5.  determine  two p o t e n t i a l l y  The r e s u l t s  et.  a l . 1986);  results  possible  were  employed to  (4.5 & 6.0) a t which  ( 3 ) As a r e s u l t  0.4, D  >  studied.  (Kisaalita,  e x p e r i m e n t s were r u n a t a pH o f 6.0, but t h i s 1  a t a p p r o x i m a t e i n t e r v a l s o f 0.05 h . 1  of o r g a n i c a c i d s d i s t r i b u t i o n  0.4) r e p r e s e n t i n g C-series  found w i t h r e s p e c t  t h r e e d i l u t i o n r a t e ranges (D < 0.15, 0.15 < D different  experiments,  organic  involving  r a d i o t r a c e r s were conducted i n t h e f i r s t  acid  t h e use  combinations of  a  experimental  determining  phase  the i n f l u e n c e  s u b s t r a t e used limited  of  two r e g i o n s to d i s c r i m i n a t e between  d e g r a d a t i o n p a t t e r n s proposed w i t h the h e l p o f t h e r e s u l t s of t h e  to B-series with the exception that this  were  number  B - s e r i e s e x p e r i m e n t s ; (4) D - s e r i e s e x p e r i m e n t s were r u n i n a s i m i l a r  in  further  t h e s e e x p e r i m e n t s were employed t o s p e c u l a t e on v a r i o u s  t o D; from B - s e r i e s r e s u l t s ,  the r i v a l  five  p a t h s f o r c a r b o n f l o w from p y r u v a t e t o t h e v a r i o u s a c i d o g e n i c end  products;  <  pH l e v e l s  experiments  i n " B i o t e c h n o l o g y and B i o e n g i n e e r i n g "  (2) B - s e r i e s  from  into  A paper based on t h e s e e x p e r i m e n t s has been  t i m e D was v a r i e d from 0.05 t o 0.6 h The  divided  The pH was v a r i e d from 4.0 and 6.5 a t  of these  optimal,  e x p e r i m e n t a t i o n was c o n d u c t e d . forpublication  s t u d y were  e x p e r i m e n t s were r u n a t an a p p r o x i m a t e l y f i x e d D  and t e m p e r a t u r e , o f 35°C.  intervals  accepted  i n this  fashion  the pH was l o w e r (pH 4 . 5 ) . The r e s u l t s  together  with  those  o f pH on t h e l a c t o s e  in A  were  useful  d e g r a d a t i o n model; (5) The  i n a l l t h e above e x p e r i m e n t a l s e r i e s was o n l y l a c t o s e  (described  earlier).  8 - l a c t o g l o b u l i n  based  In E-series  in  experiments  s u b s t r a t e  was  a  lactose  growth plus  e m p l o y e d .  - 76 -  The  results  degrading  from  the  E - s e r i e s have h e l p e d  p r o t e i n w i t h l a c t o s e had  no  i n determining  that  apparently  i n f l u e n c e on the l a c t o s e f e r m e n t a t i o n  model. All  the  tabulated  results  are  presented  i n Appendix  B.  The  first  l e t t e r i n each r u n number i d e n t i f i e s the e x p e r i m e n t a l phase t o w h i c h the r u n belongs.  T a b l e s B l , B2, B3 and B4 show the c a r b o n mass b a l a n c e and T a b l e  shows the gas  p r o d u c t i o n f o r a l l experiments  f u n c t i o n of experimental cumulative The  and  i n t h i s s t u d y , as  they were c o n d u c t e d .  gaseous p r o d u c t  First  distributions  model a r e  different  the i n f l u e n c e o f D on o r g a n i c  i s considered.  t h e r a d i o t r a c e r s t u d i e s a r e employed to d e t e r m i n e model f o r l a c t o s e .  a  time.  r e s u l t s a r e p r e s e n t e d i n the c h a p t e r I n an o r d e r s l i g h t l y  from t h a t i n which acid  conducted  B5  Then the r e s u l t s  of  the o p e r a t i v e d e g r a d a t i o n  Next the i n f l u e n c e of p r o t e i n and pH on the f e r m e n t a t i o n  considered  and  finally  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 R a t e On O r g a n i c A c i d s (OA) The  r e s u l t s of the OA  a t a temperature B).  For  the  analyses  of 35°C and  f o r l a c t o s e growth l i m i t e d  pH of 6.0  seven b a s i c e x p e r i m e n t s  made to m a i n t a i n the same t e m p e r a t u r e D.  Only  one  included  n e u t r a l product,  acetate,  propionate,  are presented and  and  ethanol, iso-  their pH, was and  n o r m a l - v a l e r a t e , c a p r o a t e , l a c t a t e and f o r m a t e . (1974),  these  fermentation.  can  be  Complete  Distribution  considered acidification  normal of  experiments  i n Table B l  replicates,  effort  was  so t h a t the o n l y v a r i a b l e  was  found.  The  an  (Appendix  organic  normal-butyrate,  acids  iso-  and  A c c o r d i n g t o Hobson e t . a l . products  l a c t o s e was  of  achieved  carbohydrate up  to D  of  -  .46  h  , beyond w h i c h ,  carbon  recovery  attributed  to  less  bacterial  77  -  d e t e c t a b l e amounts of  l a c t o s e were observed.  than  at D =  50%  was  washout.  observed  Carbon  found  t o be  recovery  figure  c a r b o n and In if  at  found  I n the f i r s t  was  an  to  all,  formate,  i n t r a c e amounts.  e r r o r i n the measurement of  be  minor  i - b u t y r a t e and  Also  products.  major  large biomass  suspect.  i - v a l e r a t e were d e t e c t e d ,  e t h a n o l , n - v a l e r a t e and  The  was  recovery  case the u n r e a s o n a b l y  i n the second case a sample d i l u t i o n e r r o r was  a l l experiments,  to  I n two a n a l y s e s , the  l a r g e r than 105%. attributed  h ^ which  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 e q u a l to o r b e t t e r t h a n 86%. was  .64  A  products  caproate  whose  were  concentrations  s t r o n g l y depended on D were a c e t a t e , p r o p i o n a t e , n - b u t y r a t e and  lactate.  In  Figure  presented  as  4.1,  functions  the of  averaged  D.  Three  r e l a t i o n s h i p between OA is  c h a r a c t e r i s e d by  r a n g e (0.15 lactate  concentrations  of  the  D  be  employed  ranges  can  c o n c e n t r a t i o n s and D.  high  < D < 0.4)  a c e t a t e and  no  respectively.  c h a r a c t e r i s e d by l e s s t h a n 100%  The  are  to  characterise  f i r s t D range (D <  detectable lactate.  i s c h a r a c t e r i s e d by  concentrations  major OA  The  third  D  range  (D  >  l a c t o s e a c i d o g e n e s i s and a p a r t i a l  0.15)  second  the f a l l and r i s e of a c e t a t e  The  the  0.4)  D  and is  bacterial  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 i n g D i n t h i s study investigations,  where  i n c r e a s e of  pure  cultures  isolated  produces  almost  from  acidogenesis.  g r o w t h r a t e s ( e q u i v a l e n t t o low D i n c o n t i n u o u s ruminatium  concentration with  i s i n agreement w i t h the f i n d i n g s of a number of  ecosystems were t h e a g e n t s of c a r b o h y d r a t e  Selenomas  lactate  gastrointestinal F o r example a t  low  c u l t u r e ) the rumen b a c t e r i u m  entirely  acetate  whereas a t h i g h growth r a t e s the f e r m e n t a t i o n p r o d u c t s  and  propionate,  of g l u c o s e are about  c re  •  h-  ORGANIC ACID CONCENTRATION (ugC/mL). —  03 rr rs  a.  n  01 o rr 0i rr  ORGANIC ACIO CONCENTRATION (tigC/mL).  I  '-3 i-i  O Cu  *—*  M  1  T3 i-i O T3 HO  3  -»  c  n rr  »  rr M  r*  a* c rr HO  3  CD  >#  01  a t-h N—^ C  tr crr  V!  M  OJ  rr *  3  o rr HO  3  O  Ml  Cu H> M*  C  rr  ORGANIC ACID CONCENTRATION (ugC/mL).  o  3  M,  ul  rr ro 01 rr 01  SC of  • o  01  o  CD  rr 01 rr  fD 03  - 8L -  ORGANIC ACID CONCENTRATION (ugC/mL).  - 79 -  50%  l a c t a t e , and 50% a c e t a t e and p r o p i o n a t e  et.  a l . (1968)  initial  glucose  showed  that  batch  concentrations  (Hobson, 1965).  cultures  o f Sj_ r u m i n a n t i u m  l a c t a t e w i t h h i g h a c e t a t e and p r o p i o n a t e  casei  i n continuous  with  ( e q u i v a l e n t to high D i n continuous  p r o d u c e d a h i g h p r o p o r t i o n o f l a c t a t e and a t low g l u c o s e low  A l s o Hishinuma  c u l t u r e produced  culture)  c o n c e n t r a t i o n s gave  concentrations.  a high  high  Lactobacillus  p r o p o r t i o n of l a c t a t e  only at  h i g h growth r a t e s (De V r i e s e t . a l . , 1970). A number o f i n v e s t i g a t i o n s e m p l o y i n g mixed c u l t u r e s i n man-made s i n g l e or  two-phase  with  fermentors  the r e s u l t s  waste  anaerobiosis,  loading water  this  from a sugar residence  favourable  from  study.  refinery, time  of l a c t a t e  that  since  of view  a l s o s i n c e the c o n v e r s i o n of pyruvate enzyme rate  (lactate  installation  per  words m i c r o - o r g a n i s m s  unit  time.  The h i g h e s t  t h e r e i s a low s u p p l y VFA.  (126L) a c i d i f y i n g waste t h a t a t the exceeded  findings,  Zeotemeyer  and h i s  formation  o f energy  of  lactate  is  less  (2 m o l . A T P / m o l . g l u c o s e ) , and  t o l a c t a t e i s known t o proceed v i a one p o s s i b l y occurs  biosynthetic activity  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 o f e x c e s s other  shock  production  dehydrogenase) and t h e r e f o r e  ( o r i t r e q u i r e s a lower  animal  of l a c t a t e  their the  and  has been a s s o c i a t e d w i t h  ( h i g h D) t h e r a t e  the point  i n municipal  Zeotemeyer e t . a l . (1982a) found  In explaining  asserted  f i n d i n g s i n q u a n t i t a t i v e agreement  F o r example,  Also i n a p i l o t  of u t i l i z a t i o n .  collaborators  reported  appearance  (Mahr, 1 9 6 9 ) .  shortest that  from  have  of the c e l l ) ,  higher lactate  reduction equivalents.  r e q u i r e f o r growth as much energy p o s s i b l e energy  at a  conversion  In  as p o s s i b l e  i s necessary  when  o f s u b s t r a t e , w h i c h would t r a n s l a t e i n t o f o r m a t i o n o f  But when t h e r e i s a l a r g e s u p p l y o f s u b s t r a t e , f o r m a t i o n o f l a c t a t e i s  - 80 -  not  the b e s t  low  efficiency. The  route  from the energy v i e w p o i n t but  production  of  Hobson  et.  affect  fermentation  a l . (1974)  concentration lactate. mixed be  l a c t a t e at  or  to  products  low  growth  However Hobson and  substrate/mixed  b a c t e r i a may  be  fermentations  rate  that  the  specific are  rates  growth  conducive  others  defined  bacteria.  propionate  low  converted  to  observed  of  bacteria low  the  for substrates  laboratory  formation  where b a c t e r i a  conditions  would  a l l growth c o n d i t i o n s , and  to i n c r e a s e w i t h  where some  Lactate  in  b r o k e n down to the  bacterial propionate  preferred substrate for  On  the  contrary  at  suggest  low  that  lactate  (Figure  4.IB)  propionate  i n c r e a s i n g D i n the D range up t o 0 - 0.15  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 c o - w o r k e r s (1982a).  T h i s a s s e r t i o n c o u l d e a s i l y be  h  may  to degrade l a c t a t e i n t o any  \ be by  confirmed  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 p r o d u c e d , the b a c t e r i a would not be capability  is  was  o t h e r than l a c t a t e as s u g g e s t e d  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 .  t o have the  of  growth c o n d i t i o n s i t i s  w h i c h i m p l i e s t h a t a t low growth c o n d i t i o n s c a r b o n f l o w from p y r u v a t e  Zeotemeyer and  may  or growth f a c t o r s , w i t h  conditions. mainly  of the  I f t h i s were the c a s e , a h i g h c o n c e n t r a t i o n  growth  propionate.  can  substrate  s u b s t r a t e s a t d i f f e r e n t growth r a t e s and  propionate-forming  under  of  to  fermentor,  ( G o t t s c h a l k , 1979), f o r l a c t a t e i s known t o be  produced  rate  conditions  not  i s g e n e r a l l y b e l i e v e d to be  under  i n pure c u l t u r e s l e d  h i s c o l l a b o r a t o r s were c a u t i o u s i n e q u a t i n g  dependent on  c u l t u r e s under  and  growth  c u l t u r e i n an a n a e r o b i c  growing on d i f f e r e n t  pure  conclude  high  the q u i c k e s t r o u t e w i t h a  expected  other metabolite.  If a  - 81 -  l a c t a t e t r a c e r i s a d m i n i s t e r e d under b a t c h c o n d i t i o n s f o r a r e a s o n a b l y  short  period  small  of  time,  the  b a c t e r i a would  be  expected  to  convert  a  very  amount of the t r a c e r to p r o p i o n a t e . On 4.1C,  the  other  i t was  hand,  based  considered  a  on  the  results  possibility  that  plotted  lactate  i n F i g u r e s 4.1A  i s produced  under a l l  g r o w t h c o n d i t i o n s , but a t low b a c t e r i a l growth r a t e s i t i s c o n v e r t e d to acetate.  Independent e v i d e n c e  by  and  Nakamura  ecosystem.  Takahashi  Fresh  to s u p p o r t  (1971)  t h i s p o s s i b i l i t y was  for anaerobiosis  rumen c o n t e n t s  of  sheep  taken  in a  and  mainly  published  gastrointestinal  at various  periods  after  C (2) ) - l a c t a t e .  The  1 4 feeding,  were  incubated  radio-activities  of  the  i_n v i t r o  consumed  with  lactate  (  were  predominately  found  a c e t a t e , i n most c a s e s , but a l s o i n p r o p i o n a t e when the p o o l s i z e of was  large.  was  found  I t was  fermented  supporting  mainly  the  of l a c t a t e r a d i o a c t i v i t y  the type of f e e d .  to  acetate  i f methanogenesis  before  lactate/acetate conversion anaerobiosis.  populations  question running  that  A l s o Counotte  with  No  the  cited  evidence  exception  of  occurs.  above was was  found  the  two  C h a t r a i n and Z e i k u s (1986a & b) c i t e d i n the p r e v i o u s The  lactate  t h a t once l a c t a t e i s formed i n mixed rumen c o n t e n t s i t can  gastrointestinal microbial  t h a t the p e r c e n t a g e  i n a c e t a t e depended s t r o n g l y on  (1981) i n d i c a t e d be  found  in  of  radio  rival  hypotheses.  main  fermentation  the  source  tracer  Generally product.  of  butyrate  experiments only  Subacterium  and  f o r m a t i o n was  not w e l l u n d e r s t o o d  to  Fusobacterium.  evidence  generated from  recent  needed  fermentor papers  also  to  be  d i s c r i m i n a t e between  the  four The  from  by  chapter.  o b l i g a t e anaerobes  They b e l o n g  Butyriubrio,  to  The  form  examined the  butyrate  genera,  mechanism  two as  a  Clostridium, of  butyrate  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 -  did  their  employ  pioneering  the  EMP  (Gottschalk, butyrate course  pathway  1979).  would  go  Cj_ k l u y v e r i .  s t u d i e s on for  the  unpublished  degradation  Clostridia of  on  i n p a r a l l e l w i t h t h a t of o t h e r  been observed  On  a r e known to  hexoses  T h i s might s u g g e s t t h a t the c o n v e r s i o n  the e x c e p t i o n of a c e t a t e .  t o b u t y r a t e has  The  to  of p y r u v a t e  to  acids with  of  organic  the o t h e r hand f e r m e n t a t i o n o f  From the  lactate  (Zeotemeyer e t . a l . , 1982a; Cohen and de  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 b e i n g the  f o r b u t y r a t e as  pyruvate  Wit,  precursor  well. 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 *)  possible general degradation  schemes were p o s t u l a t e d ( F i g u r e 4.2).  The  two 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  acetate  lactate  and  butyrate  propionate  to  co-workers  lactate (1982a)  in  A  only  goes  to  propionate.  A  back  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 offered  it  as  an  explanation  for  their  and  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  Initially,  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  when  fermentor  p e r f o r m a n c e 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  alteration  (±10%  within  a  fermentor  period  of  steady  at  state  least  three  acidogenic  expected  t o be the main gaseous p r o d u c t s  that  from m e t h a n o g e n e s i s .  the manner of  factor  defined  dilutions).  as  As  with  previous  e x p e r i m e n t s (Zeotemeyer e t . a l . , 1982), CO2 and H  glucose  separated  was  Distribution  pH  i n determining  To  the s u r p r i s e of the a u t h o r ,  control during whether  for acidogenic reactions  fermentor  there would  be  s t a r t - u p was production  2  completely  i t was a  were  found  significant  o f H„  and  C0„.  -  S3  -  Acetate Acetate  F i g u r e 4.2 Two p o s s i b l e f e r m e n t a t i o n models o f l a c t o s e t o p y r u v a t e and p y r u v a t e t o v a r i o u s end p r o d u c t s : (1) EPM pathway, (2) s u c c i n a t e p r o p i o n a t e pathway, (3) a c r y l a t e pathway, (4) b u t y r a t e f e r m e n t a t i o n . Known enzyme systems t h a t may be o p e r a t i v e i n the model a r e p r e s e n t e d i n A p p e d i x C.  - 84 -  For a s e r i e s A-series)  of experiments  i t was found CO2 a n d  start-up, pH l e v e l s .  that  r u n a t D = 0.05 h i f t h e pH f e l l  (Appendix  below  B, T a b l e B5,  t h e v a l u e o f 4.5, d u r i n g  would be d e f i n i t e l y produced even a t h i g h e r o p e r a t i n g  I n 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 o f t o t a l gas produced  (25°C and one atmosphere) a g a i n s t c u m u l a t i v e r u n time a r e p r e s e n t e d . 1 are the r e s u l t s allowed  t o drop  identical Graph  (D = 0.05 h  below  3 illustrates 1  the  desired  interesting  level  during start-up.  pH  to note  f o r a pH o f 4.5 and a t a  A t a h i g h e r pH l e v e l o f 6.0, w i t h c o n t r o l m a i n t a i n e d level  throughout  the  experimental  model  that for  of the c l a s s i c a l  f o r predator-prey  sizes.  Lotka  f o r some e x p e r i m e n t s  factor  was  identified  cycles  to d i m i n i s h .  and V o l t e r r a  interactions  F i g u r e 4.4 i l l u s t r a t e s  o t h e r s they would  persist as b e i n g  total above, steady  produce  Coincidentally,  production  Bungay  cycles  I t was n o t i c e d  on  fermentor  t h e c a r b o n f r a c t i o n i n gaseous  s t a g e and  i t took  has i n d i c a t e d  p r e y - p r e d a t o r system  pattern  on p o p u l a t i o n  No s i n g l e e x p e r i m e n t a l  f o r t h e time (1985)  gas  1920) m a t h e m a t i c a l  d i m i n i s h a t an e a r l y  responsible  the since  (Lotka,  f o r longer periods.  effect  negligible,  that  t h e c y c l e s would  and a m p l i t u d e s o f r e a l gas  i t was  One c o u l d n ' t h e l p b e i n g  such gas p r o d u c t i o n c y c l e s .  frequencies of  period,  t h a t i n a l m o s t a l l t h e e x p e r i m e n t s a t D > .05 h \  p r 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 b e h a v i o u r . reminded  Graph 2 i s f o r an  t h e pH l e v e l was m a i n t a i n e d above o r e q u a l t o 4.5.  t h e gas p r o d u c t i o n r e s u l t s  h i g h e r D o f 0.357 h . at  and pH = 4.5) f o r t h e case when t h e pH was  1  the 4.5  run i n which  Graph  f o r the that the  are e r r a t i c .  The  steady  was  state  form i n c o m p a r i s o n  to the  r e c o v e r e d c a r b o n mass was a l m o s t always v e r y s m a l l (< 3 % ) . As shown gas p r o d u c t i o n was found state.  Instead,  t o be an u n r e l i a b l e  variation  measure o f f e r m e n t o r  i n t h e consumption  rate  of  the pH  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 o f gas p r o d u c t i o n : (1) pH d r o p p e d below 4.5 s t a r t - u p , r u n // A 2 a , ( 2 ) pH was m a i n t a i n e d a t o r above a l e v e l o f 4.5, A 2 b , (3) pH was m a i n t a i n e d a t o r above a l e v e l o f 4.5, r u n # D4.  during r u n //  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 w h i c h t h e pH was m a i n t a i n e d a t t h e d e s i r e d v a l u e ( H 6 . 0 ) : (1) D=0.2109 h " , (2) D = 0.2953 h , (3) D = 0.1246 h " . 1  P  _ 1  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 p u r p o s e .  out  as  that  some a u t h o r s  such  anaerobic  r e a c t o r s are  seldom a t  inhibitory in  a  or  For  steady  those  can  steady  a l . (1986) have  state.  indicated  s t a t e , because l o c a l i s e d  Therefore,  the  steady  e x p e r i m e n t s ( a t a pH  s t a t e , F i g u r e 4.5  be  believed  seen  that  formation  of 6.0)  presents  where t h e r e was  h \  m e t h a n e was  (Figure  2.1)  information  that  u  decarboxylation exhibit  u  culture  D = ii);  detected  for  a  number  presently  of  gas  m  for a l l organisms m i s w e l l b e l o w 0.05 h  that  the  (2)  A b o v e D = 0.15 higher  h  maximum  methane  ( F i g u r e 4.5)  1  concentrations  (3)  limit  i s known t o be  the  most known methanogens, making formed v i a the a c e t a t e question  between the  form  that  lowest  1  o f H^  acetate. via  that  f o r acetate that  the  It  acetate  reduce continuous  where no methane  and  4.1  specific  was  were observed,  they were b e i n g c o n v e r t e d  i t unlikely  It is  Table  v a l u e s w e l l i n the D r e g i o n o f i n t e r e s t (remember f o r  relatively  6.0  production  (1)  However those  h  By  remain  r e d u c t i v e methane  reasons:  a v a i l a b l e on  o b v i a t i n g the f a c t t h a t below D = 0.15  t o D.  to  short  detected.  the methane must have been formed v i a the  the  clear  The  and  s t a t e s r e f e r r e d to i n  growth r a t e (H ) f o r the f o r m a t i o n o f methane from hydrogen and  pH  that  the f e r m e n t o r head space gas a n a l y s i s .  t h a t b e l o w D = 0.15  route  summarises  is  pointed  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.  a t steady It  et.  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  "dynamic"  continuous  Girard  I t s h o u l d be  to  decarboxylation  observed  CH^; in  methane  was  i s what r e l a t i o n s h i p might t h e r e  be,  route. comes to mind  c h a n g i n g gas  composition  comparing F i g u r e s  4.1  and  and 4.5,  OA  d i s t r i b u t i o n both with  i t was  found t h a t the  respect  transitions  T a b l e 4.1  Substrate  C0 /H 2  2  Organism  Temperature (°C)  Reference  u (h )  0.69  65  Schonheit e t . a l . (1980)  0.23  65  Z e i k u s and W o l f e (1972)  0.058  33  Zehnder and Wuhrman ( 1 9 7 7 )  Methanothrix soehngenii  0.0032  33  Zehnder e t . a l . (1980)  Methanosarcina  0.02-0.03  36  S m i t h and Mah (1978)  (TM-1)  0.06  50  Z i n d e r and Mah (1979)  (227)  0.0192  35-37  (MS)  0.0208  <•  1  m  Methanobacterium thermoaceticum  Methanobrevibacter Acetate  Maximum S p e c i f i c Growth R a t e V a l u e s F o r M e t h a n o g e n i c B a c t e r i a  Methanosarcina ••  arboriphilus  b a r k e r ! (227)  maze!  AZ  0.042 0.0224  35 35-37  Yang  (1984)  Yang (1984) Mah  (1980)  Yang (1984)  CO  - 90 -  in  both  OA  therefore  and  gas  distribution  suggested  t h a t D's  washout c o n d i t i o n s f o r the absence of t h i s the  shift  the  first  represents  the  utilising  of  this  does not chapter,  beginning  of  pyruvate  conversion  of  lactate  disables  the  resulting  i n the h i g h e r o b s e r v e d  D  range.  of 0.15  It is  h * represent  new  the  b a c t e r i a l p o p u l a t i o n , hence  answer the q u e s t i o n t h a t a r o s e i n whether  being to  the  shift  in  to  lactate  converted  other  population or  just  intermediary metabolites, The  answer t o t h i s  sections.  Radiotracer Studies The  Chapter al.  same  lactate concentration.  e q u a t i o n i s d e a l t w i t h i n the next two  4.3  the  b a c t e r i a l group and c o n s e q u e n t l y  in a totally  However t h i s  section  in  i n the neighbourhood  group r e s u l t s  i n OA.  occurred  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 II.  The  v a l u e of the t e c h n i q u e was  (1913) and h i s s t u d e n t , M a r k o f f ( 1 9 1 3 ) .  microbial  functions.  because  of  fashion  that  The  theory  contents until  the  Markoffs  effort  made  quantitative  behind  the  to  conduct  results  technique  availability  o f new  of  in  are  vitro  applicable  to  to f u n c t i o n  the  s u b s t r a t e o r any  rumen were  sample  as  products,  particularly fermentations  i s t h a t when a  fermentation  a p p r e c i a t e d by Zuntz e t .  They employed i t to s t u d y rumen  experiments  i s removed, i t c o n t i n u e s  accumulation  first  of  noteworthy i n such  a  obtained.  the  fermentor  i t d i d i n the  fermentor  exhaustion  o t h e r f a c t o r causes  of  substrate,  i t t o change.  The  o n s e t o f the change can be d e l a y e d f u r t h e r i f the major f e r m e n t o r c o n d i t i o n s such  as  pH  environment. reported  and  temperature  The  are maintained  a t comparable l e v e l s  batch r a d i o t r a c e r experiments,  below, were  run,  at  l o n g e s t , f o r 1,800  i n the  new  the r e s u l t s from w h i c h  are  seconds.  For  biological  - 91 -  systems, Roels  ( 1 9 8 3 ) has  d e f i n e d r e l a x a t i o n t i m e , t , as the t i m e , w h i c h R  elapses  before  the  d i f f e r e n c e between a  i n i t i a l v a l u e reaches and  new  steady  orders time  a f r a c t i o n (1 - 1/e)  state values.  of magnitude of  of  1,800  their  t  shown.  S i n c e the b a t c h  seconds l i e s a t the lower end of the t  Results Of The A procedure  followed column  considered  to be  too  typical  experimental  r e g i o n f o r changes i n s h o r t a p e r i o d t o have  by BA^,  shown by  of  propionate  a result so  otherwise  tail  was  for  Figure was  4.7  eluted  from BA^Q  a l l the  each  20th  conducted  but  the  the  the  various  on  experiments recovery  products.  a t h i g h D (D > 0.15) were  carried  separation #  C4)  the  is  and  ten  e l u t e d by  BA^,  Complete  elution  fraction,  second l a c t a t e peak  The  the  column,  The done the  that  was  summed  recovered  results  i n the  80th would  radio a c t i v i t y for  for  small  (20  up  to  activity the  a r e summarised i n T a b l e 4.2 out  a  first  up to the 4 0 t h The  I I I was  a s t r o n g e r s o l v e n t , a t the  remaining  four  activity  experiments  within  fraction.  speeded down the column.  percentage among  t o BA^Q,  lactate  of  ( f o r Run  A s m a l l amount of p r o p i o n a t e was  a d m i n i s t e r e d to e l u t e l a c t a t e .  experiments of  a p p l i e d to the OA  a c e t a t e were a c h i e v e d w i t h BA^,.  b a d l y , was  the  distribution  III.  Butyrate  solvent.  and  that  product  calculate  sample t h a t was  i n Chapter  of changing  fraction  one  mL  the peak between the 1 1 t h and  a f t e r w h i c h BA^Q  each  two  described  as  results.  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  radiochromatogram.  fractions  the  Radiotracer Experiments At High D  t o produce a  also  the  a d a p t a t i o n mechanisms and  e f f e c t on the f e r m e n t a t i o n model d e r i v e d from the r a d i o t r a c e r  4.3.1  is  are  the system and  of the d i f f e r e n c e between the o l d  I n F i g u r e 4.6  enzymic c o n c e n t r a t i o n s , i t was any  s t a t e v a l u e of  mL)  four All batch  - 92 -  10"* TO" 1CT X T 5  1  1  Moss action law  4  1  1  3  1  10" T0"'  O*  2  1  1  1  10° _ Ailosteric controls  Figure 4.6  1  1  X 3 ' IO* X 3 ' J  1  3  1  1  X7  4  1  5  TO*  6  RELAX TON TIME (SECONDS)  _ Changes m enzymic Concentrations m-RNA Selection within A control Population ol ore or more species Evolutionary Changes  A d a p t a t i o n mechanisms i n o r g a n i s m s and t h e o r d e r s o f magnitude o f t h e i r r e l a x a t i o n t i m e s .  F i g u r e 4.7  Radiochromatogram f o r r u n number C4: (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 . T r a c e r was [14c(U)]-lactate.  Table 4.2  RUN  RUN TIME (Min)  TRACER  It  Radio A c t i v i t y D i s t r i b u t i o n  REACTOR TYPE  TOTAL TRACER ACTIVITY (dpm)  for High D Radio Tracer  TOTAL RECOVERED TRACER ACTIVITY (dpm)  Butyrate  Propionate  394,130  386,576 98.1%  381,420 98.7%  —  Experiments  REC0\/ERED ACTIVIT)f DISTRIBU1riON  It  Acetate  C7c  ( C(U)]-Butyrate  30  S  C2  L-[ C(U)]-Lactate  30  S  359,028  244,048 68%  1,120 0.4%  21,200 8.7%  16,992 7.0%  C3  L-[ C(U)]-Lactate  30  s  319,810  257,880 80.6%  1,960 0.8%  18,640 7.2%  C4  L-[ C(U)]-Lactate  30  L  455,808  369,520 81.1%  1,620 0.4%  23,520 6.4%  U  14  14  14  S m a l l  batch  Large  batch  r e a c t o r . r e a c t o r  with  pH c o n t r o l .  1  2  PARENT RUN  5,156 1.3%  PARENT RUN D(h  _ 1  )  Lactate  —  B3c  0.2075  204,736 83.9%  B3c  0.2075  16,520 6.4%  220.760 85.6%  B3c  0.2131  33,200 9.0%  311,180 84.2%  B3c  0.2131  - 95 -  reactor, have  an i n f l u e n c e  batch To  described  i n Chapter  I I I , Section  on p r o d u c t  distribution,  reactor without  that  used  minutes  Figure  4.8  mL  reactor  shows  increased  without  of r a d i o a c t i v i t y  i n the small  to  demonstrate  an  and  t h e pH  initial  distribution to d i s p e l l  reactor.  value  monitored  for a  of  6.06  run with  any m i s g i v i n g s  r e a c t o r without  o f runs  7.3% p r o p i o n a t e  pH c o n t r o l .  and w i t h o u t  pH  about the e f f e c t s o f pH c o n t r o l and C4  c l o s e enough t o make pH  i t .  conversion  to acetate  4.3.2  The b u t y r a t e  As  shown  w i t h a r a d i o - a c t i v e product and 7.3% a c e t a t e .  by t h e  15% o f t h e distribution  The low c o n v e r s i o n o f  the i n a b i l i t y  t r a c e r was r e c o v e r e d  carried  Run number C3 was made  of the experiments.  f e r m e n t o r were o b s e r v e d , i n d i c a t i n g  catabolise  I n 30 A  l a c t a t e I s not s u p r i s i n g s i n c e a t high D l a r g e c o n c e n t r a t i o n s the  but n o t  t o 6.54.  number C2, C3 and C4, a p p r o x i m a t e l y  l a c t a t e was c o n v e r t e d  o f 0.5% b u t y r a t e ,  was  Runs number C2 w i t h  the r e p l i c a b i l i t y  results  radioactive  the r e s u l t s .  The r e s t o f the r a d i o t r a c e r e x p e r i m e n t s were  i n the small batch  combined  a  t h e pH a l a r g e r r e a c t o r (60  pH c o n t r o l gave r e s u l t s t h a t were c o n s i d e r e d  c o n t r o l unnecessary. out  running  the r e s u l t a n t pH-time r e l a t i o n s h i p .  from  c o n t r o l was found n e c e s s a r y change  that  pH c o n t r o l f o r 30 m i n u t e s might a f f e c t  t h e 20  t h e pH  comparison  pH was known t o  i n C h a p t e r I I I , S e c t i o n 3.5.1 was r u n i n a manner s i m i l a r t o  with  controlled.  Since  i t was f e a r e d  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  mL) d e s c r i b e d  pH  3.5.2.  of l a c t a t e i n  of the p o p u l a t i o n to  with a negligibly  small  (1.3%).  Results Of The Radiotracer Experiments At Low D Figure  4.9  presents  a  typical  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 a c e t a t e w i t h m i n i m a l q u a n t i t i e s e n d i n g up as butyrate  and p r o p i o n a t e .  The  results  f o r a l l t h e runs  a r e summarised  TIME (min). F i g u r e 4.8  pH - time r e l a t i o n s h i p f o r t h e b a t c h r a d i o t r a c e r r e a c t o r e x p e r i m e n t , w i t h o u t pH c o n t r o l .  incoporation  3500  0  50  100  150  FRACTION NUMBER (20ml solvent/fraction).  200  F i g u r e 4.9 Radiochromatogram f o r r u n number C l l : (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 . T r a c e r was [ ^ C ( U ) ] - l a c t a t e . 4  - 98 -  in  Table  was  For  varied.  I t was 15  4.3.  the  three  I n F i g u r e 4.10  t r a c e r s employed the b a t c h  only  acetate.  The  the range of time c o n s i d e r e d .  approximately microbial  15%  of  population  propionate  was  the  succinate-propionate  p l a y minor r o l e s . also  precluded  product  whose  catabolised. very  the one  The  the  found  and  fact  concentration Approximately  radioactivity propionate  ( >12%) .  a l s o puts  shift  i n OA  too  ended  of  propionate  observed  of  concomintant population  the  been  found  f o r the  study  (see  up  as  being be  low  not  only  a  possess  no  4.2)  major  r a t e to be  probably  SJ  from VFA  to c a t a b o l i s e l a c t a t e  measurable by  the  Negligible  small  amounts the  as rival  i n f a v o u r of some form of model B,  production  acetate.  being  to a c e t a t e i n a  d i s c r i m i n a t e s between  i n S e c t i o n 4.1  population  tracer  i t was  4.11).  ( 3%) and  both  intermediate  because  to r e s t . 1  of  to  to  (Figure  converted  Figure  butyrate  that arose  bacterial  production  converted  T h i s suggest t h a t  I n the neighbourhood of D = .15 h ,  carbon flow v i a pyruvate, inability  to  fast  finding  the q u e s t i o n  observed  had  C o i n c i d e n t a l l y , of a l l the f o u r major  f e r m e n t a t i o n models proposed ( F i g u r e 4.2) but  was  a c r y l a t e pathways  was  this  This  a  D  85% of the l a c t a t e was  in  amounts  low  i n l e a s t amounts.  the  possibility  employed  butyrate  I n a p e r i o d of  t h a t n o t h i n g happended to the p r o p i o n a t e  r a p i d r e a c t i o n , almost  technique  the at  c a p a b i l i t y for converting propionate. OA;  time  the c o n v e r s i o n of b u t y r a t e to a c e t a t e i s shown.  found to be l i n e a r over  minutes  experimental  to  Also,  i s probably  Indeed  i s not a s h i f t i n  to l a c t a t e p r o d u c t i o n , but catabolise  the  inability  the  lactate of  the  a consequence of  a  with  an the  bacterial bacterial  Table 4.3  RUN  TRACER  II  RUN TIME (Min)  Radio A c t i v i t y D i s t r i b u t i o n for Low D Radio Tracer  REACTOR TYPE  TOTAL TRACER ACTIVITY (dpm)  Experiments  TOTAL RECOVERED TRACER ACTIVITY (dpm)  Butyrate  Propionate  452,240  473,300 104.72  453,080 95.72  —  20,220 4.3% 61,560 14.7%  REC0\/ERED ACTIVITlt DISTRIBU'r i O N  II  PARENT RUN , D(h )  —  B2e  0.0947  —  B2e  0.0947  PARENT RUN  Acetate  - 1  Lactate  C14a  [ C(U)]-Butyrate  5  S  C15a  [ C(U)]-Butyrate  15  S  447,134  419,060 93.72  357,500 85.3%  —  C16  [ C(2)]-Propionate  5  s  575,880  520,460 90.42  —  520,460 1002  —  —  B2e  0.1026  C17  [ C(2)]-Propionate  15  s  560,662  561,240 100.12  —  561,240 1002  —  —  B2e  0.1026  C19  L-[ C(U)]-Lactate  1  s  256,438  202,914 79.12  172,260 84.92  16,280 8.02  B2e  0.0947  Cll  L-[ C(U)]-Lactate  s  348,336  1,960 0.92  B2d  0.1083  U  1A  1A  14  1A  1A  5  1  C12  L-[ C(U)]-Lactate  15  s  320,796  C13  L-[ C(U)]-Lactate  30  s  311,740  1A  1A  Small batch  reactor.  20  14,354  02  7.12  224,640 64.72  10,660  17,120  4.72  7.62  194,900 86.8%  274,580 85.62  8,360  46,100 16.82  220,120 80.2%  —  B2d  0.1083  3.02  246,436 79.12  7,920 3.22  29,100 11.82  209,416 85.02  —  B2d  0.1083  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 f r o m [ 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 b a t c h e x p e r i m e n t t i m e s : (A) a c e t a t e , (x) b u t y r a t e .  120  >-  o < o  100  80  5 <  60  Q UJ OC UJ  40  r  • Q -  I Io  1  CC  >  O O  M I  20-  111 tr  —r10  15  20  25  30  T I M E (min). F i g u r e 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 [ ^ C ( U ) ] - l a c t a t e t r a c e r a t v a r i o u s b a t c h experimet t i m e s : ([g) l a c t a t e , (•) a c e t a t e , (x) p r o p i o n a t e , (A) b u t y r a t e . 4  - 102  p o p u l a t i o n a d j u s t m e n t due (Figure .2  4.5,  CH^  t o the wash out of the  c o n c e n t r a t i o n dropped  u t i l i s i n g methanogens  to zero  between D of  0.1  and  h" ). 1  4.4  P r o p o s e d L a c t o s e A c i d o g e n i c F e r m e n t a t i o n Model In  a l l r a d i o t r a c e r experiments,  small  amount  found.  As  of  back  the  other  the  of  this  the p a r a l l e l acids,  was  as  lactate  of  of  the  radiotracer, a  recovered  activity)  meagre b u t y r a t e attributed  shown being  nature  with  4.7%  was  in the  to an  Figure  extremely  4.2A  &  B.  of c a r b o n f l o w between b u t y r a t e  and  the  exception  of  acetate  this  study  the  results  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  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 C h a p t e r I I .  For  this  i n a d d i t i o n to the Zeikus  (1986a  &  methodology  similar  variations)  for  b) to  and  4.2).  and  and  in  to put  and  authors  task  findings  i t is fitting  This and  a l a c t o s e f e r m e n t a t i o n model i s p r o p o s e d , based on  study  slow  for butyrate  course,  precursor  was  concentration,  t h a t 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  Before this  reaction  possibility  organic  propionate  Cohen ( 1 9 8 1 ) ,  oxidative  therefore confirms the  (<  must have been l a c t a t e ,  pyruvate  precludes  where l a c t a t e  radioactive butyrate  i n t i m a t e d by  whose o r i g i n  in  -  those  results was  i n this  found  the  one  to  models  for  the  fate  fermentor  r u n a t D = .01 h \  of  be  some  study,  previous  o n l y the work of C h a r t r a i n  specifically  described  radiolabeled lactate  proposed  of  in  this  relevant. study  (with  i n c o r p o r a t i o n , C h a r t r a i n and  carbon  in  samples  a c o n t r o l l e d pH of 7.1  from  a  ± .01 and  Using  a  minor Zeikus  single-phase a temperature  - 103 -  o f 37 ± 1°C. basis  of  general  these  proposed.  for  a single  and  their  microbial  C h a r t r a i n and Z e i k u s ' s phase  process,  f i n d i n g s could  .0333 h " which  results  r e s u l t s are presented  i n F i g u r e 4.12.  enumeration,  On t h e  isolation  and  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 f e r m e n t a t i o n model ( F i g u r e 4.13)  was  their  Some o f t h e i r  1  and t h i s  work has been c o n s i d e r e d d i c t a t e s t h e range  be a p p l i e d ( i . e . D < 0.0333 h  _ 1  t o be v a l i d  o f D over  ).  The v a l u e o f  (see S e c t i o n 2.3.2, T a b l e 2.2) i s b e l i e v e d t o be t h e l i m i t ,  acidogenesis  same v e s s e l .  and methanogenesis  c a n take  place  below  s u c c e s s f u l l y i n the  I n t h i s s t u d y , t h r e e o t h e r D ranges o f i m p o r t a n c e w i t h  t o 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 :  which  ( 1 ) .0333 < D < .150,  b a c t e r i a l group 4B ( S e c t i o n 2.1.2, F i g u r e 2.1) i s washed out;  regard where  ( 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 g e n e r a l o r s e l e c t i v e wash o u t o f t h e r e m a i n i n g The  lactose acidogenic  presented from  fermentation  model, proposed  i n F i g u r e 4.14. The a t t e n t i o n o f t h e r e a d e r  CO^/H^  evidence  b a c t e r i a l groups commences.  t o acetate  to confirm  being  or dispel  dotted, this  to s i g n i f y  possibility.  i n this  study i s  i s drawn t o t h e a r r o w lack of  experimental  As shown i n A p p e n d i x C,  F i g u r e C2, b u t y r a t e f o r m a t i o n from g l u c o s e c a n be summarized a s :  g l u c o s e + 3ADP + 3?  ±  -> b u t y r a t e + 2 C 0  2  + 2 H + 3ATP 2  (4.1)-  where P^ i s i n o r g a n i c phosphorous. Therefore concomitant  as l o n g as b u t y r a t e was b e i n g d e t e c t e d gas p r o d u c t i o n  w h i c h was o b s e r v e d ,  was e x p e c t e d .  has a l r e a d y  i n the fermentor  The i r r e g u l a r  been r e p o r t e d .  effluent,  gas p r o d u c t i o n ,  Assuming t h a t C0„/H_ was  - 104  F i g u r e 4.12  -  Fermentation time course f o r [ ^ C ( U ) ] - l a c t a t e degradation i n a single-phase l a c t o s e f e r m e n t a t i o n sample. i  - 105  -  LACTOSE  CH  F i g u r e 4.13  4  The m i c o b i a l l a c t o s e f e r m e n t a t i o n n o d e l i n t h r e e d i s t i n c t but s i m u l t a n e o u s t r o p h i c phases.  - 106  -  Lactose  7 • Pyruvate  7 Lactate  Propionat  Acetate  F i g u r e 4.14  The M i c r o b i a l a c i d o g e n i c l a c t o s e f e r m e n t a t i o n model  - 107  being  produced a l l through  some way  the  experimental  for biosynthetic a c t i v i t i e s ,  most l i k e l y  product  (apart  group i n v o l v e d would be  -  one  f r o m CH^)  p e r i o d s , but  comparing  the  w o u l d be  a c e t a t e and  one  study.  results  of  C h a r t r a i n and  Zeikus  under s t e a d y substrate  production  of  ethanol  s t a t e c o n d i t i o n s was  feed  line.  i s also reflected butyric acid  not  The  i n the  bacteria.  any  were  enough,  to  fermentor,  short f a l l  They d i d not  use  i s because at D * successfully taking  enable  any  yielding  and  thus  If  had  probed  they  a l l o w the  complete c o n v e r s i o n f a c t one  the  .01  detected i n t h i s In t h i s  the e t h a n o l  study  detected  of t h e i r r e s u l t s ,  butyrate  tracers,  effluent.  h \  place  r e d u c t i o n kept  butyrate  both  in  the  The  that  played because  reason  they  acidogenesis same  and  vessel.  The  the p a r t i a l p r e s s u r e of hydrogen  degradation  r e a c t i o n t o go  r e a c t i o n mechanism  by  the  acetogens  to c o m p l e t i o n with  a  to be  (see F i g u r e  butyrate  energy 2.2).  radiotracer,  to a c e t a t e would not have been a s u r p r i s i n g r e s u l t .  of the h y d r o l y t i c b a c t e r i a t h a t they i d e n t i f i e d was  w e l l known b u t y r i c a c i d b a c t e r i a .  in  e s t a b l i s h e d to have been produced i n the  most i m p o r t a n t  p r o d u c t i o n o f methane v i a low  the r e s u l t s  i n t h e i r proposed model i s the absence o f the r o l e  detect  methanogenesis  bacterial  F o r example, w h i l e f o r m a t e  t h e y d i d not d e t e c t any b u t y r a t e i n the f e r m e n t o r did  with  A l s o they d e t e c t e d e t h a n o l i n s u b s t a n t i a l q u a n t i t i e s . no  t h a t the  2.1).  of t h e i r main i n t e r m e d i a t e p r o d u c t s , h a r d l y any was  t h e r e was  by  the  in  the hydrogen consuming a c e t o g e n s o t h e r w i s e r e f e r r e d  t h i s s t u d y , a number of d i f f e r e n c e s a r e e v i d e n t . was  utilised  would be l e d to c o n c l u d e  t o as the homoacetogenic b a c t e r i a l (Group 3 i n F i g u r e In  being  C_i b u t y r i c u m ,  In a  - 108 -  4.5  S i g n i f i c a n c e of Findings  So f a r t h e 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  lactate  was  s y n t h e s i s e d a t a l l growth r a t e s o r , whether i t s s y n t h e s i s was s w i t c h e d on a t a p a r t i c u l a r d i l u t i o n r a t e below w h i c h t h e f l o w o f c a r b o n would be d i r e c t e d away from t h e l a c t a t e r o u t e . using a lactate First  The l a t t e r has been c o n f i r m e d  beyond any doubt  radiotracer.  this  finding  i s considered  n o t t o be  limited  only  to the  m i c r o b i a l e c o l o g y i n t h i s s t u d y , b u t t o be v a l i d f o r a l l a n a e r o b i c 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 d e t e c t e d growth r a t e s . include,  under c o n d i t i o n s o f h i g h m i c r o b i a l  Some o f t h e s e h a b i t a t s were c i t e d e a r l i e r  anaerobic  municipal  waste  treatment  systems;  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. systems t h a t c o n t a i n c a r b o h y d r a t e s Secondly,  this  finding  may  as c a r b o n be  o p t i m i z a t i o n of production  process. address  Pipyn this  and V e r s t r a e t e  problem.  They  by  industrial  food  I n o t h e r words, a l l  sources.  useful  i n resolving  d i s a g r e e m e n t about t h e most s u i t a b l e o r g a n i c a c i d the  i n t h i s chapter to  t o be used as a marker i n  the acidogenic  (1981)  were  devised  a  the prevalent  phase  the f i r s t framework  in a  two-phase  investigators by  which  to  various  a l t e r n a t i v e s c o u l d be compared, u s i n g t h e f r e e energy o f t h e r e a c t i o n s b o t h In acidogenesis end  products  and m e t h a n o g e n e s i s .  (acetate, propionate,  F o r each o f t h e p o t e n t i a l butyrate,  ethanol  assumed an a c i d o g e n i c g l u c o s e h o m o f e r m e n t a t i v e p r o c e s s was  assumed  course  t o be c o n v e r t i b l e t o o n l y  one p a r t i c u l a r  acidogenic  and l a c t a t e ) ,  they  ( i e . a l l the glucose end p r o d u c t ) .  Of  t h i s a s s u m p t i o n 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  a r e known  to  possess  branched  biochemical  mechanisms.  - 109  However free  i t was  energy  presented  a  for  in  necessary each  Table  results  of  product  s h o u l d be  rate  their  approximation  possibility. 4.4,  a c i d o g e n e s i s on the one  in  The  the  to  enable  results  form  of  them  of  to c a l c u l a t e  the  calculation  are  available  for  their  energy  quanta  hand and methanogenesis on the o t h e r .  calculation,  determining  -  either  they  lactate  concluded  t h a t the  o r e t h a n o l , because  Based on  the  preferred acidogenic the most d e l i c a t e  and  b a c t e r i a l group (methanogens) would be a l l o c a t e d a maximum  o f a v a i l a b l e p o t e n t i a l energy. But  from what  propionate Therefore  are  i s known from  precursors  of  i t does not m a t t e r  the  present  acetate  at  study  lactate,  different  levels  whether, f o r example, l a c t a t e  butyrate of  the  r e a c t o r product)  methanogenic  acidogenic carried that  reactor  reactor  out  by  i n the  both  lactate  (implying  that  product),  for  that  i s converted  lactate  conversion  is  in  predominant  to a c e t a t e  the  either  to  in  predominant case  will  be  the same a c i d o g e n i c b a c t e r i a w i t h the o n l y d i f f e r e n c e b e i n g  latter  case  the  r e a c t o r , w h i l e i n the former In  or whether  reduction.  i s converted  a c e t a t e i n the a c i d o g e n i c r e a c t o r ( i m p l y i n g t h a t a c e t a t e i s the acidogenic  and  cases  the  energy  acetogenic case  bacteria w i l l  they w i l l  available  for  be  the  be  i n the  acidogenic  i n the methanogenic r e a c t o r . methanogenic  responsible for  d e c a r b o x y l a t i o n of a c e t a t e w i l l be the same. A al. the  second group of a u t h o r s  1986) first  have i n d i c a t e d phase  of  the  (Zeotermeyer,  t h a t the most d e s i r a b l e p r o d u c t two-phase  process  c o n c e n t r a t i o n of p r o p i o n a t e was  a minimum.  fact  of  that  reactions  the in  methanogenesis  the  e t . a l . , 1982;  methanogenic  T h i s was  propionate  phase  would  (McCarty,  is  be  Kisaalita,  distribution one  in  which  et. from the  based on the w e l l known the  1963;  slowest  of  a l l the  Andrews and  Pearson,  - 110 -  Table 4.4  Acidogenic end product  Distribution of Total Free Energy Change (for Growth) of the Two-Phase Anaerobic Process of Glucose to Methane Over Different Microbial Groups.  Free energy change for the acidogenic phase (%)  Free energy change available for methanogenesis phase H gas (%) 2  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.  - Ill-  1965;  Mahr, 1969).  precursor end  Since  f o r propionate  l a c t a t e had been b e l i e v e d  production,  p r o d u c t o f the a c i d o g e n i c  to be the most common  i t was d e t e r m i n e d n o t t o be a d e s i r a b l e  phase.  The p r e s e n t r e s u l t s have d e m o n s t r a t e d  t h a t c o n d i t i o n s can be c r e a t e d where l a c t a t e i s c o n v e r t e d t o a c e t a t e  and not  propionate,  lactate  and  at a very  predominates at high the  4.6  rate  (Figure  4.11).  D's - reduced t  Also  since  - a r e d e s i r a b l e because o f  r e a c t o r v o l u m e s ) , t h i s makes i t the optimum p r o d u c t  reactor.  Influence Of Protein On The Fermentation Model Initially  intended  i n this  from v a r i o u s revealed  part  of  the s t u d y  (E-series  experiments)  i t was  t o 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 c o s t was found t o  be p r o h i b i t i v e .  them  D (high  b e n e f i t of s m a l l e r  f o r the a c i d o g e n i c  fast  E f f o r t s were made t o o b t a i n whey p r o t e i n c o n c e n t r a t e 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 a n a l y s e s  t h a t none o f them c o n t a i n e d  had  substantial  quantities  (WPC)  o f some of t h e s e  WPC  8-lactoglobulin only.  I n f a c t some o f  lactose.  decided  of  So  i t was  t o 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 o f w h i c h i s p r e s e n t e d i n Chapter I I , T a b l e . 3.2. Runs  A total  numbered  El  previously  discussed  number E3  was  nutrient switched was  medium  of three  and  E2  were  experiments  initially and  e x p e r i m e n t s were conducted started-up with  started-up  once  steady  ( E l , E2 and E 3 ) .  in a  manner  similar  the l a c t o s e  - only  substrate.  with state  a was  purely  lactose  achieved,  t o 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  to  the Run  growth l i m i t e d substrate  was  The purpose o f t h i s  t o i n v e s t i g a t e whether b a c t e r i a t h a t had been adapted t o a l a c t o s e - o n l y  substrate,  i f introduced  differently  i n any way.  to  a  lactose/protein  substrate  The c a r b o n b a l a n c e s f o r t h e t h r e e  would  respond  experiments are  - 112 -  presented  i n Appendix  three experiments ranging  between  comparatively  B, T a b l e  B2.  As shown, carbon  r e c o v e r i e s f o r the  were b e t t e r o r e q u a l t o 71.5%, w i t h a p r o t e i n c o n v e r s i o n s 65  and  lower  70%, p r o b a b l y  carbon  being  recoveries.  partly  responsible  F o r the t h r e e  f o r the  experiments,  only  t r a c e amounts o f l a c t o s e were d e t e c t a b l e . The  partial  implied  that  conversion  lactose  was  o f p r o t e i n and complete c o n v e r s i o n a  better  substrate  o b s e r v a t i o n has been r e c e n t l y r e p o r t e d and  co-workers  simultaneous of  than  by B r e u r e  protein.  A  similar  e t . a l . (1986a).  Breure  i n v e s t i g a t e d the i n f l u e n c e o f a d a p t a t i o n  procedure  a c i d o g e n i c f e r m e n t a t i o n o f g l u c o s e and g e l a t i n .  experiments  glucose  of l a c t o s e  dissolved i n a mineral salts  on the  I n one s e r i e s  s o l u t i o n was f e d to a  mixed p o p u l a t i o n o f b a c t e r i a i n a g l u c o s e growth l i m i t e d chemostat ( o r CSTR) a t 30°C and d i f f e r e n t the g l u c o s e  pH l e v e l s  s u b s t r a t e was s w i t c h e d  gelatin  was  similar  t o one i n t h i s  limited  ( 5 . 3 , 6.3 and 7 . 0 ) . A t s t e a d y  added  extent  metabolised.  (<  t o g e l a t i n , growth ceased.  t o a medium as a second study)  30%)  and  I n a second  carbon  the  series  glucose  continued  of experiments,  Breure  state,  glucose  Following  was  first  added  establishment  s e r i e s of experiments.  t o the medium  o f a new s t e a d y  state,  h y d r o l y s e d b u t n o t degraded any f u r t h e r . preference Breure of  f o r carbohydrates  as  a  they  found  to a  be  completely  and  co-workers,  c o n d i t i o n s t o the  After  second  (a s i t u a t i o n  proceeded  to  adapted b a c t e r i a l p o p u l a t i o n s t o g e l a t i n under c o m p a r a t i v e ones d e s c r i b e d i n t h e i r  However when  substrate  the breakdown o f the g e l a t i n  s t a t e , when  reaching  carbon  steady  substrate.  t h a t g e l a t i n was  A l l these r e s u l t s demonstrate the  over p r o t e i n s by a c i d o g e n i c b a c t e r i a .  In fact  e t . a l . (1986b) i n a second paper i n which they r e p o r t e d the r e s u l t s  the i n f l u e n c e o f VFA and c a r b o h y d r a t e s  on the h y d r o l y s i s and a c i d o g e n i c  - 113 -  fermentation  of g e l a t i n ,  anaerobic  digestion  carbohydrate/protein partially  separated  implication it  would  whole  i n this  protein  resulted  mixtures,  acidogenic  suitable  However  i n only a slower  waters  of an  containing  carbohydrates  should  to acidogenesis  given  The  of whey i s t h a t  ( d e p r o t e i n a t e d whey)  the h i g h  be  protein  than  conversion  question  reactors.  that  to e n a b l e  this  study  together  l a c t o s e f e r m e n t a t i o n model. the  The  effect total  of  So the s l o w e r  two  attempted  with  this  total  OA  o f whey, on the  t o address  p r o t e i n had  products  larger  further consideration.  In a preliminary effort main  slightly  acidogenesis  i s something t h a t d e s e r v e s  lactose  of  protein conversion.  of l a c t o s e / p r o t e i n (whole whey) would o n l y r e s u l t i n  economics of the p r o c e s s  concentration  performance  s t u d y , i t would appear t h a t the p r e f e r e n c e o f l a c t o s e over  larger  of  of  t o use whey permeate  r e a c t o r volume,  degradation  waste  fermentation  conclusion with regard  conversion  comparatively  " f o r optimal  purifying  (lactose/protein).  observed  acidogenic  system  that,  from the h y d r o l y s i s and f e r m e n t a t i o n of p r o t e i n " .  of t h i s  be more  whey  The  concluded  any  was  whether  the  i n f l u e n c e on the  t o f i n d an answer, the  (acetate  and  lactate)  for  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 were compared t o those f o r l a c t o s e a c i d o g e n e s i s at  comparable  experimental  conditions.  In Figure  4.15  the comparison i s  d e p i c t e d by p l o t t i n g t h e 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 t h e same g r a p h as  the l a c t o s e acidogenic  found  t o be  possibly  a  comparable consequence  lactose/protein lactose  products. but l a c t a t e of  the  acidogenesis  a c i d o g e n e s i s (S  As shown, a c e t a t e c o n c e n t r a t i o n s were  (S  was  found  specific q  growth  t o be rate  = 5475 .2 p.g C/mL)  = 4212.5 ug C/mL).  on  the h i g h  being compared  side,  higher f o r t o t h a t of  The c o m p a r i s o n of OA's f o r the  -  114 -  2800-1  E —  o  3,  1008-  z  O  c z  1000  1000  y  800-  0.8  0.4  O.S  o.a  0.7  0.3  0.4  0.8  0.8  0.7  2000  0.2  DILUTION  RATE(1/h).  F i g u r e 4 . 1 5 Comparison o f the main p r o d u c t s c o n c e n t r a t i o n s 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 e x p e r i m e n t s : (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 .  for  - 115  two  substrate  p r o t e i n had  type  any  processes  -  revealed  no  evidence  to  suggest  that  the  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 e x p e r i m e n t s were c o n d u c t e d in  the  same  manner  experiments. the  The  recovered  presented  Protein  previously  described  with  samples  from  summary o f the r e s u l t s a r e shown i n T a b l e 4.5.  activity  for high  f o u n d to be  as  D  slightly  apparently  distributions  radio lower  does  are  comparable  t r a c e r experiments. probably  not  due  affect  The  anyway  In  those  general  previously  conversions  to the h i g h e r in  to  E-series  h e r e were  s p e c i f i c growth r a t e .  the  pathway  for  lactose  carbon source,  usually  degradation. The referred number  growth of m i c r o o r g a n i s m s t o as of  on  more t h a n one  d i a u x i c growth i s a t the moment a s u b j e c t of i n t e r e s t  research  ( R a m k r i s h n a , 1982;  groups  around  D h u r j a t i , 1982;  the  and  world.  One  Kompala, 1982)  of  these  cybernetic  internal  perspective  machinery  has  the  response to i t s environment. situations  of  microbial  ability  approach  to make r a t i o n a l  of d i a u x i c growth (Demain, 1971)  been  acceptable  since  the  contends  optimal  to  preference  the of  cell's in  itself well in utilization  each s u b s t r a t e by i t s e l f would  organism, a  the  decisions  so the p r e f e r e n t i a l  although  an  "Cybernetics".  that  T h i s has been found to m a i n f e s t  of a c e r t a i n s u b s t r a t e over another, have  growth  a  groups  have come up w i t h  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, The  to  particular  fits  the  substrate  cybernetic could  well  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 a p p r o x i m a t e l y  l e v e l o f 4.0  t o a l e v e l of 6.5,  0.05  h ^, the pH was  a t i n t e r v a l s of 0.5  v a r i e d from a  (Run numbers A l to A6) .  Table A.5 Radio A c t i v i t y D i s t r i b u t i o n for Samples from Experiments with Lactose Protein  RUN  TRACER  II  RUN TIME (Min)  REACTOR TYPE  TOTAL TRACER ACTIVITY (dpm)  TOTAL RECOVERED TRACER ACTIVITY (dpm)  Butyrate  Propionate  411,434  400,180 97.3%  375,900 93.92  —  24,280 6.1%  —  —  7,220 3.0% 12,040 3.7%  REC0\/ERED ACTIVITlI DISTRIBU'riON  [ C(U)]-Butyrate  30  S  C5b  L-[ C(U)]-Lactate  30  S  249,145  235,980 94.82  Cl  L-[ C(U)]-Lactate  30  s  337,414  322,620 95. ex  1,386 .4*  C9b  l C(U)]-Butyrate  30  s  372,218  403,000 108.3%  393,800 97.7%  —  CIO  [ C(U)]-Butyrate  30  s  428,836  439,688 102.5Z  431,700 98.22  —  C8  L-[ C(U)]-Lactate  30  s  233,678  157,018 67.22  —  14  1A  1A  1A  U  Small (10 ml) batch reactor.  1  PARENT RUN  II  C6  14  Substrate  1,224 .4%  1,040 .7%  Acetate  9,200 2.32 7,988 1.8% 4,146 2.6%  PARENT RUN D(h ) _ 1  Lactate  —  E2  0.2018  228,760 97.0%  E2  0.2018  307,970 95.52  E2  0.2007  —  E2  . 0.2065  —  E2  0.2065  E2  0.2124  151,832 96.72  - 117  The  tabulated  presented  in  variations value  of  o f 5.0  between pH  D  or  involved  for  Appendix  B,  and 5.0  5.5,  using  distribution  Table  acetate and  low  the  growth  pH.  rate  and  be  propionate the  (pH  > 5.5).  ( F i g u r e 4.17), an OA  validity  of  (Figure the  presented  the  I t has  The  pH  region  already  been  r a d i o t r a c e r s that  at  microbial  population  p o s s e s s the a b i l i t y  to degrade  S i n c e l a c t a t e was concluded  only detected i n trace  t h a t the s o u r c e  (pH  < 5.0)  and  of the  observed  was  distribution  (1982) and T i k k a (quoted  similar  t o one  qualitatively  lactose fermentation  be  lactate  by Thimann  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  variation  4.1)  to f a v o u r the  A s i m i l a r r e l a t i v e change i n OA  s h i f t i s a change i n m i c r o b i a l s p e c i e s c o m p o s i t i o n .  4.1  are  So the I n f l u e n c e of the pH on the c a r b o n f l o w would  (1963) f o r g l u c o s e a c i d o g e n e s i s .  Section  are  balance  predominated below a  range.  conditions,  has been r e p o r t e d by Zeotemeyer e t . a l .  4.5  carbon  4.16  Butyrate  transition  t o f a v o u r the b u t y r a t e r o u t e a t low pH a t h i g h pH  as  t h a t the c o n v e r s i o n of b u t y r a t e t o a c e t a t e i s s l o w  amounts ( F i g u r e 4.16), i t can  route  well  Figure  of l a c t o s e does not  to a c e t a t e and  lactate.  In  butyrate  i n c o m p a r i s o n to t h a t of l a c t a t e .  a c e t a t e was  as  predominated above a pH v a l u e o f 5.5.  lactate,  specific  B4.  against  being  i n acidogenesis  propionate  OA  the major OAs  demonstrated, low  results  -  By v a r y i n g D a t a pH  OA of  extensively discussed i n  reproduced,  model t h a t has  confirming  been proposed  the  in this  s tudy.  4.8  Microbial Growth Modeling Microbial  this  study  and  growth the  use  modeling of  approaches  were r e v i e w e d  the Monod e q u a t i o n  was  i n Chapter  II  of  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. F i g u r e 4.16  Products d i s t r i b u t i o n as a f u n c t i o n of pH: (•) b u t y r a t e , (A) a c e t a t e , (x) p r o p i o n a t e , (0) lactate. The d i l u t i o n r a t e was s e t a t a f i x e d v a l u e of 0.05 h ~ l .  0.7 DILUTION R A T E F i g u r e 4.17  (1/h)  P r o d u c t s 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 r a t e a t a pH of 4.5: (A) a c e t a t e , (x) p r o p i o n a t e , (•) b u t y r a t e , (H) lactate.  - 120  simplicity of  and i n h e r e n t p h y s i c o - c h e m i c a l meaning.  continuous  Figure  4.18  original  -  culture and  Monod  behaviour  4.19.  The  chemostat  from  data  Bailey  and  i n Figure  model.  That  Two  e x p e r i m e n t a l examples  Ollis  4.18  (1986) a r e shown i n  i s consistent with  i s , the  observed  cell  the  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 a p p r o x i m a t e l y c o n s t a n t over a w i d e r range of conditions contrary Herbert a  than to  the  existence introduced  Monod  original  (1958a & b) who  continuous  balance  i n F i g u r e 4.19.  of  culture.  the  maintenance  endogenous  model  F i g u r e s 4.20  pH 6.0  D values and 0.2, and 0.65 run  explained  by  of "endogenous m e t a b o l i s m "  in  model  was  was  M  first  I n t e r p r e t e d t o mean  i n the  substance. limiting  systems  requirements,  i t was  experimental  requirements  results  Hence he  nutrient  mass  w i d e l y a c c e p t e d more g e n e r a l  some m i c r o b i a l  metabolism  the  in  this  do  not  found study  exhibit  any  necessary  to  indicated  b e f o r e a t t e m p t s were made to e s t i m a t e  any the  parameters. In  for  is  consume c e l l  coefficient,  Since  the  endogenous m e t a b o l i s m  which  which  when d e v e l o p i n g the now  model.  whether  Chemostat  Endogenous m e t a b o l i s m  reactions i n cells  Chemostat  determine  Monod  t r e n d shown i n F i g u r e 4.19  i n t r o d u c e d the concept  ( E q u a t i o n 2.37)  detectable  The  and  pH 4.5  o f 0.0  and  i s emphasized by  and  4.21  a r e shown the d r y biomass c o n c e n t r a t i o n d a t a  respectively. 0.1  h *,  W i t h r e f e r e n c e t o F i g u r e 4.20,  e v i d e n c e of endogenous m e t a b o l i s m  the d o t t e d l i n e .  i s obvious  However between D v a l u e s of 0.1  t h e r e 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 t h a t p i c k s up does not  show any  h * i s reached.  s i g n s of washout from  there t i l l  a dilution  I t s h o u l d be p o i n t e d out t h a t f o r a l l the  at D values h i g h e r  than  .45  between  h * and  a pH  o f 6.0,  and  suddenly r a t e of  experiments  i n s p e c t i o n of the  )  - 121 -  o.:  0.4  0.6  0.8  Dilution rate. D. h"  1.0  1  F i g u r e 4.18 E x p e r i m e n t a l c o n t i n o u s c u l t u r e d a t a 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 t h e o r i g i n a l Monod c h e n o s t a t model i n a p u r e culture»Aerobacter a e r o g e n e s .  s  1" 5  B  3  1-  Calculated from batch growth data 3  4  S  6  7  Reciprocal dilution rate. D~ . h x  F i g u r e 4.19 E x p e r i m e n t a l c o n t i n o u s c u l t u r e d a t a w i t h a t r e n d c o n t r a r y t o t h e o r i g i n a l Monod c h e m o s t a t model i n a c o n t i n o u s c u l t u r e o f A e r o b a c t e r 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). F i g u r e 4.20 Dry biomass c o n c e n t r a t i o n a t a pH o f 6.0.  0.6  0.7  DRY BIOMASS CONCENTRATION (ug/mL).  c  oo  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 a t the end of each e x p e r i m e n t  growth adhering actual  revealed  copious  microbial  t o the w a l l s the f e r m e n t o r v e s s e l ( i e . w a l l g r o w t h ) .  critical  dilution  r a t e a t which washout  have o c c u r r e d  not  be d e t e r m i n e d e a s i l y .  al.  (1978) o b s e r v e d 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 ,  acid The  As a l r e a d y p o i n t e d  should  b a c t e r i a , f o r experiments sudden drop  biomass  conditions one  and  other  products  o f complete m i x i n g  value.  ( F i g u r e 4.1) a t D = 0.4  a r e known  a r e met.  a lactic  above the c r i t i c a l  however i s e v i d e n c e of b a c t e r i a l washout, f o r d u r i n g washout of  could  out i n Chapter I I , Rogers e t .  run at D values  i n lactate concentration  So the  to  decrease  h  \  concentrations  rapidly  i f the  But t h e slow biomass d e c r e a s e  like  o b s e r v e d 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 interesting  t o 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  microbial  population  shift  shift.  t h a t was c o n s i d e r e d  t o be a consequence of  The m i c r o b i a l p o p u l a t i o n  shift  a t a pH of 4.5  i s n o t as o b v i o u s as i n the p r e v i o u s case ( F i g u r e 4.21). G i v e n t h a t l a c t a t e was d e t e r m i n e d operation  of an a c i d o g e n i c  range of i n t e r e s t 80 - 90% c r i t i c a l interest initial  i s that rise  r e a c t o r i n a two-phase p r o c e s s ,  would have dilution formed  t o be the p r e f e r r e d product  i n microbial  the s h i f t  biomass  Therefore  II  concentration  for this  i s sudden  i t was d e c i d e d  i n the Monod chemostat model so e q u a t i o n s  reduce t o :  the m i c r o b i a l group o f  and s i n c e ,  n e g l i g i b l e endogenous m e t a b o l i s m r e q u i r e m e n t ) , parameter  the o p e r a t i n g D  t o be a b o v e a v a l u e o f 0.2 h ^ ( i n p r a c t i c e  rate value).  after  f o r the  group, t h e (implying  a  t o drop the M  2.41 and 2.42 i n Chapter  - 125 -  S  =  X  To  illustrate  hypothetical 4.22.  the  influence  D K / ( u - D) s in  =  of  S)  M  the  on  parameters i n p r a c t i c a l  For  curves with  shapes  (4.3)  Y(S Q  (4.2)  biomass  concentration  ranges were employed  similar  to c u r v e  curve,  to produce  numbers  4,  Figure  5 & 6,  M is  negligible. To  obtain  the  parameter  e s t i m a t e s i n the  Monod  chemostat  model,  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) used.  The  Fortran  program  written  to  NL2S0L i s p r e s e n t e d i n Appendix D.  define  the  problem  and  then  a was  call  F o r the biomass d a t a g e n e r a t e d a t a pH  l e v e l of" 4.5, the f o l l o w i n g e s t i m a t e s were o b t a i n e d :  u K  m  = 0.3596 ± 0.0026 h "  = 8.3258 ± 3.5216 ug  g  1  C/mL  Y = 0.2053 ± 0.0114 In  Figure  4.23  experimental  a  good  agreement  d a t a i s shown.  between  A high  the  error  model  in K  g  predictions  i s due  n a t u r e of biomass c o n c e n t r a t i o n a t or near the c r i t i c a l  and  to the unsteady  dilution  rate.  U n f o r t u n a t e l y , f o r the biomass d a t a g e n e r a t e d a t a pH l e v e l of 6.0, sensible  solution  substrate mentioned. down. For  could  be  acidogenesis, For with  However  found,  caused  wall  the biomass  growth  because  by  the  of wall  lack  of d a t a of  growth  the  no  fractional  problem,  already  the a s s u m p t i o n of complete m i x i n g broke  concentration  d a t a i s not  r u d i m e n t a r y p r o c e s s e v a l u a t i o n p u r p o s e s , the a  may  completely useless. be c o n s i d e r e d to be  F i g u r e 4.22 I n f l u e n c e o f 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 " , (3) M = 0.1 h (4) M = 0.01 h , (5) M = 0.005 h , (6) M = 0.0001 h . -  1  _ 1  _ 1  - 1  - 1  TJ H-  DRY BIOMASS CONCENTRATION (ug/mL).  0Q  C i-i fD  fD  X  O © O  I  La.  Ol  o  a fD o ft) i-l a . X HT3 3 o fD fD r r fD i-i 3 H- rr 3 Co o fD (— CD rr a rr O . Co Co Cu rr t— rt CO 3 Cu o tu i-h a . rt O fD ce t-t M  s  CD O O  i  p  to  >  o  O O  J>  IO -  1  1  Co •o rj X) fD SC a .  o n i-h rr •  Ln >—s 1 1  <•—'  3  o a n > i—• •  H* O 3  C O H io 3  —L  O  H3  o o  3 X! Co ii HCD  ( CA Ol  o 3 €  rr rr th  ro  - m  -  - 128  equal  to  .41  h  (bacterial  -  washout commenced a t 0 = .4 h  v a r i a t i o n of l a c t a t e w i t h r e s p e c t to D - F i g u r e 4.1C).  (S  ug/mL, g i v e s a y i e l d  After fixing a  = 4212.5 ug C/mL).  and Y, model  o  m  p r e d i c t i o n s a t v a r i o u s p r a c t i c a l v a l u e s of K are  shown i n T a b l e  4.6.  the  A l s o i n the D range of  i n t e r e s t , an average d r y biomass c o n c e n t r a t i o n of 1500 c o e f f i c i e n t M, of .3561  , based on  For  K  values  g  were performed and  g  above 3.0  |ig C/mL  the  a t the  results critical  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 n e g a t i v e biomass c o n c e n t r a t i o n . m At K = 2.0 [ig C/mL and b e l o w , t h e m o d e l p r e d i c t s a p o s i t i v e b i o m a s s concentration. So i t c a n be c o n c l u d e d t h a t an e s t i m a t e o f a K value s g  between 2.0 K  g  and 3.0  i s not  of  4.7)  population  lower  revealed  obtained than  that  constants  the  growth  producing  v a l u e s were to  of  those  an  lactate  on  approximations.  previously  undefined  mixed  l a c t o s e was  study  value  are  not  unreasonable,  published microbial  faster  than  However the  given  that  they  are  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 .  u. v a l u e r e p o r t e d by G h o s h a n d P o l a n d ( 1 9 7 4 ) i s m high. The h i g h u and e x t r e m e l y low K v a l u e s suggest t h a t m s  by  the  was  used  Monod  Chemostat  is characteristic  values  g  generated  t o put  model of  with  those  the  parameters  with wall  growth.  of In  that  can  them on  be  form  Ghosh  and  order  to  i n t h i s s t u d y to o t h e r s a c o n v e r s i o n f a c t o r o f the  same b a s i s as  the o t h e r s .  I t i s clear  t h a t the v a l u e s from t h i s s t u d y a r e an o r d e r of magnitude h i g h e r . explanation  u  The  (1974)  compare K  on  m  t h e r e were w a l l growth p r o b l e m s , f o r the m i c r o b i a l mass c u r v e  predicted  32/12  growth  i n this  t h e \i  unreasonably probably  A more a c c u r a t e d e t e r m i n a t i o n o f  T h i s i s shown by the r e l a t i v e l y h i g h v a l v e o f u. . m  al . (1978).  Poland  microbial  predominantly  glucose. values  i s reasonable.  j u s t i f i a b l e g i v e n t h a t b o t h Y and  Comparison (Table  \ig C/mL  offered  at  this  time  is  that  this  The may  only be  - 129  T a b l e 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 v a l u e s based on u and Y a p p r o x i m a t i o n s f o r a pH of 6.0 s  m  Predicated  d r y biomass c o n c e n t r a t i o n  D(h ) _ 1  K (tig  C/mL)  s  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  N  N e g a t i v e biomass  N  1  concentration.  Table 4.7  Maximum Specific Growth r a t e  Monod Chemostat Model c o n s t a n t s f o r a c i d o g e n e s i s  Yield Coefficient  Monod S a t u r a t i o n Constant  Temperature (°C)  pH Y  HmC"" ) 1  Basis  K  Substrate/culture  References  Basis  g  0.36  4.5  0.21  kg/kg C  22.2  kg COD/m  35  lactose/ mixed & u n d e f i n e d  This study  0.41  6.0  0.36  kg/kg C  5.3-8.0  kg COD/m  35  lactose/ mixed & u n d e f i n e d  This study  0.33  6.0  0.13  NR  30  glucose/ mixed & u n d e f i n e d  Zeotemeyer e t . a l . (1982)  0.30  NR  0.14  kg VSS/kg COD  0.37  kg COD/m  35  glucose/ mixed & u n d e f i n e d  Ghosh and K l a s s (1978)  1.25  NR  0.17  kg VSS/kg COD  0.023  kg COD/m  37  glucose/ mixed & u n d e f i n e d  Ghosh and P o h l a n d (1974)  0.56  6.0  30  lactose/ S. c r e m o r i s  Rogers e t . a l . (1978)  Not r e p o r t e d . Not a p p r e c i a b l e .  NA  2  3  3  NR  1  3  3  NA  - 131  characteristic  of  the  bacterial  consequence of the s u b s t r a t e b e i n g Lastly chemical the  a word about  bond  i s called  -  population  predominated  than  that  of  the  i n f l u e n c e of  for.  Zeoteraeyer  the n a t u r e  of  the c a r b o n  B - 1 , 4 - g l y c o s i d i c bond  the a c i d o g e n e s i s  than  t h a t of g l u c o s e .  that  the  Again  overall  the of  induced  only  the  higher  acidogenesis  plausible  microbial by  of l a c t o s e was  The  substrates  predominate i n a process  of  (as  in  sucrose)  lactose i s faster at  predominates  lactose  i n the presence  as  opposed  of g l u c o s e .  than  the  to be  the to  t h a t of  present  one  4  slower suggest  glucose.  time  acidogenic the  is  cellulose).  values r e p o r t e d i n t h i s study  that  that  g l u c o s e l i n k e d by a 8-1,  p r e d i c t a b l y expected  explanation for this  population  presence  of  a  source  e t . a l . (1982b) p o i n t e d out  S i n c e 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 linkage,  as  lactose.  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  faster  type  that  that  is  the  process would  - 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) o f an a c i d o g e n i c chemostat o r CSTR i n t h e mesophilic  temperature  range a t a pH o f 6.0, up t o t h e p o i n t o f b a c t e r i a l  washout proved t o be a v e r y 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 m i c r o o r g a n i s m s involved  i n anaerobiosis  specific  growth r a t e r a n g e s .  0.05  h  route 0.15  into  various  groups w i t h  similar  or overlapping  or specific  growth r a t e o f  , t h e methanogens t h a t form methane by t h e a c e t a t e  decarboxylation  Above a D v a l u e  were s u c c e s s f u l l y e l i m i n a t e d from t h e ecosystem. h  , t h e methanogens  that  form methane  Above a D v a l u e o f  v i a the route  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 t h e e c o s y s t e m . v a l u e o f 0.4 h \ of  s e l e c t i v e o r g e n e r a l m i c r o b i a l washout commenced.  4.5, a s i m i l a r  decarboxylating 0.05  Above a D  trend  was q u a l i t a t i v e l y  and hydrogen  reducing  reproduced  methanogens  with  being  A t a pH  the acetate  eliminated  above  a n d 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 a t t h i s l o w pH commenced  a t a D v a l u e o f between 0.3 and 0.35 h Based  on t h e r e l a t i v e  products  (acetate,  specific  dilution  groups,  for  rate  conversion  butyrate  ranges  and l a c t a t e ) ,  corresponding  hypotheses  emerged,  o f t h e main  a c i d o g e n i c end  produced  to the d i f f e r e n t  namely:  over t h e microbial  ( 1 ) The s h i f t  in  the  p o p u l a t i o n i n the n e i g h b o u r h o o d o f a D v a l u e o f 0.15 and 0.1 h ^  pH l e v e l s  pyruvate  of  propionate,  two p o s s i b l e  microbial  concentrations  o f 6.0 and 4.5 r e s p e c t i v e l y r e p r e s e n t e d  conversion  to lactate;  of lactate  to other  14 [ C(U)]-lactate with  the beginning of  (2) or the m i c r o b i a l s h i f t intermediary metabolites.  the acidogenic  d i s a b l e d the  Batch  chemostat e f f l u e n t  incubation samples and  - 133 -  p r e p a r a t i v e s e p a r a t i o n o f the predominant o r g a n i c a c i d s f o l l o w e d by a l i q u i d scintillation  assay  o f the l o c a t i o n  beyond any doubt between  o f the r a d i o a c t i v i t y ,  the two r i v a l  hypothesis.  This  finding  i s believed  fermentation  but  f o r a l l mixed  discriminated  hypotheses i n favour not o n l y  undefined  of the l a t t e r  t o be v a l i d  anaerobic  f o r lactose  fermentations  of  carbohydrates. 14 F u r t h e r use o f [ C ( U ) ] - b u t y r a t e and  a t a pH o f 6.0  i n t h e m e s o p h i l i c t e m p e r a t u r e range r e v e a l e d the predominant carbon f l o w  routes.  I t was assumed  Embden-Meyerhof-Parnas pyruvate of  14 and [ C ( 2 ) ] - p r o p i o n a t e  that  pathway.  t o the v a r i o u s observed  carbon from p y r u v a t e  found,  that  almost  completely  a l lD  converted  relation model.  t o D, In  i n this with  were  this  to l a c t a t e  methanogens, l a c t a t e butyrate i s converted  via  were  the  from  I t was found t h a t the f l o w A l s o i t was  methanogens, l a c t a t e  and n o t p r o p i o n a t e .  Butyrate  was was  F u r t h e r c o n v e r s i o n o f p r o p i o n a t e was not p o s s i b l e This  observed lactose  together  suggested acidogenic  the knowledge  used  model  Embden-Meyerhof-Parnas converted  of concern  and l a c t a t e was p a r a l l e l .  to acetate  considered.  concentrations  together  to p y r u v a t e  t o a c e t a t e a t a slow r a t e as l o n g as hydrogen r e d u c i n g  ranges  propionibacteria knowledge  routes  organic acids.  to butyrate  methanogens were p r e s e n t .  propionate  So the f l o w  i n t h e presence o f hydrogen r e d u c i n g  found to be c o n v e r t e d  under  l a c t o s e i s b r o k e n down  t o propose lactose  pathway.  a  Is  that  the r e l a t i v e l y the  ecosystem  converted in  I n the presence  played  minor.  by This  fermentation  pyruvate  parallel  v i a the  reaction i s  o f hydrogen  reducing  i n a very f a s t r e a c t i o n to a c e t a t e .  t o a c e t a t e b u t a t a much s l o w e r r a t e .  low  interactions i n  lactose  to a  role  was  the m i c r o b i a l  qualitative  Pyruvate  and b u t y r a t e . i s converted  of  with  Also  I n the l i g h t o f  - 134 -  this  model  it  was  concluded that  lactate  is  the most  suitable  marker for  optimising an acidogenic reactor i n a two-phase biomethanation process. that  reason  only  biomass  production of lactate  concentration  data  in  the  D range  where  the  predominated were used to model microbial growth.  The  Monod chemostat model was favoured among various a l t e r n a t i v e s s i m p l i c i t y and the physico-chemical b a s i s . not  included i n the model as i t  because of  The maintenance c o e f f i c i e n t  was found to be n e g l i g i b l e  by  At a pH of 4.5 the model parameter estimates were found to be: ± 0.0026 h " ; K 1  g  C.  encountered  i n maintaining a steady biomass  6.0,  The l a r g e  error  in K  value at which complete  w a l l growth was observed  decrease  its was  inspection. = 0.3596  = 8.3258 ± 3.5216 |J.g C/mL; and Y = 0.2053 ± 0.0114 ug dry  biomass/ug  the c r i t i c a l  For  i n biomass  as  g  was a t t r i b u t e d  to  the  concentration as D approached  b a c t e r i a l washout occurs.  at D v a l u e s  D approached the  difficulty  At a pH of  above 0.4 l i ^ and therefore a  critical  value was not  observed.  The presence of wall growth invalidated the assumption of complete mixing on which the Monod chemostat could be estimated acid  based on the  based.  However the c r i t i c a l value of D  observed sudden drop i n the major organic  concentrations. Degradation of  protein (mainly B - l a c t o g l o b u l i n )  did not i n anyway affect 0.15  model i s  the carbon flow scheme.  h * low pH (pH < 5.0)  expense of the lactate  was  found to  together with lactose  In the D range of 0.05  favour the butyrate route at the  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 being the t r a n s i t i o n range.  to  5.5  - 135  5.2  Recommendations  Recommendations p r o b l e m of proposed  erratic  for gas  future  lactose fermentation  promote gas of  studies  p r o d u c t i o n needs model  e x p e r i m e n t a l p r o t o c o l (pH = 4.5,  each  -  6.0;  production, discourage  the  eight  experiments,  can  include to be be  following:  s t u d i e d f u r t h e r so  made more c o m p l e t e .  D = 0.1,  gas  the  0.3  (1)  The  that  the  A  2x2x2  h ^; s t a r t - u p p r o c e d u r e  p r o d u c t i o n ) i s hereby p r o p o s e d .  samples  should  be  withdrawn  and  = For  incubated  14 with this  C - l a b e l e d sodium b i c a r b o n a t e . study  organic  to a s s a y  a c i d s one  precisely.  f o r the  Then u s i n g the p r o c e d u r e s  l o c a t i o n of  the r a d i o a c t i v i t y  c o u l d c o n f i r m or d i s p e l l  (2) L a c t a t e has  been proposed as  the  described i n  among the major  proposed  f a t e of  H2/CO2  the most d e s i r a b l e a c i d o g e n i c  end p r o d u c t .  I t can be p r e d i c t e d from the r e s u l t s o f t h i s s t u d y t h a t i n the  methanogenic  serial  converted verified  reactor  to a c e t a t e .  of  the  However, t h i s  two-phase  process,  lactate  p r e d i c t i o n needs to be  be  experimentally  t o g e t h e r 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 population  f o r process  design.  enumeration,  (3)  I t i s a l s o recommended t h a t , m i c r o b i a l  characterisation  and  isolation  of  the  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  should  behaviour  of  be  continuous  attempted.  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Al.l  Principle T h i s procedure  was  first  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  proposed  oligosaccharides, methyl  esters  orange-yellow acid.  Al.2  by  Doubis  polysaccharides  with colour  free  or  when  et. and  a l . (1956). their  potentially  treated  with  Simple  derivertives  free  reducing  phenol  and  sugars,  Including  groups,  concentrated  the  give  an  sulfuric  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 .  Reagents and A.  To  Chemicals  t e n mL  reagent  grade  phenol  (MCB), 180 mL  of d i s t i l l e d  water  were added. B.  A1.3  Concentrated  s u l f u r i c a c i d (BDH).  Procedure One  contained tube.  mL  of c e n t r i f uged  between 10 - 70 [ig of l a c t o s e ,  One mL  of reagent  a c i d were added r a p i d l y . air, to  (1 h, 4450 x g) was  sample,  pipetted  A was added and f i v e mL  degrees  spectrophotometer,  into  such  that i t  a colorimeter  of c o n c e n t r a t e d  sulfuric  The tubes were a l l o w e d t o s t a n d f o r t e n minutes i n  shaken and p l a c e d f o r t e n to twenty minutes  thirty  diluted  i n a water b a t h a t twenty  c e n t i g r a d e , b e f o r e r e a d i n g s were taken a t 480 nm u s i n g a 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 F i g u r e A l .  A.2  Determination of Whey Protein  A2.1  Principle Protein  solution  with cupric  i o n i n an a l k a l i n e sodium p o t a s s i u m t a r t r a t e  t o form a complex c o l o r e d  molecular carbon  reacts  structure  pairs  compound.  of carbamyl  (or peptide linkages)  will  Any compound t h a t  groups  show  linked  a positive  through  biurent  has i n i t s n i t r o g e n or  reaction.  The  name o f t h e t e s t i s d e r i v e d from t h e s i m p l e s t o f such compounds, b i u r e t .  CONH,  2  NH  When solution,  biuret  i s treated  two b i u r e t  violet-colored  with  molecules  an a l k a l i n e  are joined  t o form  compound.  OH  OH  CONH--K  /  OH  1  potassium  copper  the f o l l o w i n g  tartrate complex  0.8  0.6 O CO  O 0.4 Ln  OL  00  OC  O  CO  cn 0.2  <  0.0-ip  0  —r  10  —r— 20  30  40  50  60  70  LACTOSE CONCENTRATION (ug/mL). F i g u r e A l C a l i b r a t i o n Curve f o r L a c t o s e U s i n g t h e P h e n o l - S u l f u r i c A c i d Method.  - 159 -  S i n c e 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 r e a c t to g i v e the c h a r a c t e r i s t i c c o l o r w h i c h 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 p r e s e n t .  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  a c o l o r i m e t e r tube. throughly  mixed  absorbance  was  prepared  by  (.45  fim MILLIPORE membrane) sample was  Four mL  of b i u r e t reagent were added.  and  incubated  then  determined  substituting  at  room  at  temperature  a wavelength  distilled  water  of  The  f o r 30 540  pipetted  into  solution  was  minutes.  nm.  for  the  sample.  Lang  and  Lang  The  Blanks  were  A  typical  (1972).  Bright  c a l i b r a t i o n c u r v e i s shown i n F i g u r e A2.  A3  Determination of Formate  A3.1  Principle This  yellow  procedure and  was  first  green-yellow  d e s c r i b e d by fluorescent  reaction  products  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 r a s p b e r r y r e d i n the same medium, a t room  temperature.  concentration  of  band i s a t 515  A3.2  0.5  intensity  formate  and/or  of  the  color  formic a c i d .  is  proportional  I t ' s main  to  the  light absorption  nm.  Reagents and A.  The  g  Chemicals  citric  isopropanol.  acid  and  t e n g acetamide  were d i s s o l v e d  i n 100  mL  E c o -* O H Q. OC O  CO 03 <  0.0  1  2  3  4  6  PROTEIN CONCENTRATION (mg/mL). F i g u r e A2  C a l i b r a t i o n Curve f o r P r o t e i n U s i n g Method.  the B i u r e t - R e a c t i o n  - 161 -  A3.3  B.  A sodium a c e t a t e s o l u t i o n , aqueous,  C.  Aceticanhydride.  30 g i n 100 mL was made up.  Procedure  0.5 mL o f c e n t r i f u g e d contained less  (1 h, 4450 x g) sample,  than 200 mg/mL formate was p i p e t t e d  diluted  such  that i t  i n t o a c o l o r i m e t e r tube.  50 uL r e a g e n t 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  incubated  at  absorbance  was then t a k e n a t 515 nm.  distilled  50°C  to the tube.  water  for thirty  The m i x t u r e  minutes  f o r t h e sample.  to speed  was  up  shaken  w e l l and  the r e a c t i o n .  The  B l a n k s were p r e p a r e d by s u b s t i t u t i n g  A typical  calibration  c u r v e i s shown i n  F i g u r e A3.  A4  A4.1  Determination  of  Lactate  Principle  When a v e r y d i l u t e s o l u t i o n o f l a c t i c a c i d i s heated i n t h e presence o f a high c o n c e n t r a t i o n of s u l f u r i c  acid  i t i s c o v e r t e d to a c e t a l d e h y d e .  a c e t a l d e h y d e may then be d e t e r m i n e d by the s e n s i t i v e phydroxydiphenyl. In  lactate  test  employing  The p r o c e d u r e was t a k e n from a paper by Markus ( 1 9 5 0 ) .  the m o d i f i c a t i o n  convert  color  The  of t h i s  procedure  given  to a c e t a l d e h y d e i s g e n e r a t e d  below, by r a p i d  sufficient  heat  to  mixing of s u l f u r i c  a c i d and w a t e r .  A4.2  Reagents and C h e m i c a l s  A.  Four  g o f CuSO^.SH^O were d i s s o l v e d  a d j u s t e d to 100 mL.  i n water  and t h e v o l u m e  1.2  E c  0.8  m z  o I-  ON  Q.  0.4  03  0.2  tr o co <  100  200  300  400  600  600  FORMATE CONCENTRATION (ug/mL). F i g u r e A3  C a l i b r a t i o n Curve f o r Formate U s i n g t h e Method o f Lang and Lang (1972).  - 163  B.  One  g of p - h y d r o x y d i p h e n y l  NaOH and C.  -  (SIGMA) was  s t o r e i n a brown b o t t l e  Concentrated  sulfuric  dissolved  i n 100 mL  i n the r e f r i g e r a t o r u n t i l  of 0.08N needed.  acid.  Procedure  A4.3  One  mL  of  centrifuged  contained  less  50  reagent  \iL of  syringed cooled  into  stand  10  A  the  were  added.  20°C  i n cold  wall  of  f o r 6 h at  4450  the  water.  of  diluted  into  such  50  \iL of  concentrated  reagent  thoroughly  sulfuric  (or o v e r n i g h t ) . blank,  The  i n a i r , then added  tubes  by  without  allowed  absorbance  prepared  was  was  to  then  water f o r the sample, were run w i t h each s e t of d e t e r m i n a t i o n s .  A5.1  A  the  acid  distilled  substituting A  c a l i b r a t i o n c u r v e i s shown i n F i g u r e A4.  Determination of V o l a t i l e VFA  nm.  B were  and  that i t  a c o l o r i m e t e r tube.  to s t a n d f o r f i v e minutes  mixed  570  sample  at  A5  of  g)  pipetted  S i x mL  room temperature  wavelength  was  allowed  tube,  x  measured  typical  a  h,  ug/mL l a c t a t e  the tubes, and  below  touching  than  (1  F a t t y A c i d s (VFA) and  c o n c e n t r a t i o n s were determined  u s i n g a gas  Ethanol chromatograph.  Apparatus A.  Analytical with  both  gas a  chromatography Thermal  (GC)  - Model  311  Conductivity Detector  (CARLE),  (TCD)  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).  and  equipped a  Flame  0.30 A  0.26  0.20  g  0.16  Q.  CC O  o.10  m < 0.06  1.00  ^f^-  2  4  6  8  10  12  LACTATE CONCENTRATION (ug/mL). F i g u r e A4  C a l i b r a t i o n Curve f o r L a c t a t e U s i n g a M o d i f i e d Method o f Markus (1950).  - 165 -  A5.2  C.  A 60/80  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  Sample  Carbopack  C/0.3  Preparation  The VFA can o n l y  be d e t e c t e d  to a c i d i f y the samples.  i n free  form.  I t was t h e r e f o r e  necessary  In general:  HA  H  (free At  (SPECTRA-PHYSICS).  + A  +  (Al)  acid)  equilibrium, K  where  = [A~] [ H ] / [ H A ] +  a  +  [A ] , [H ] and [HA] a r e the molar c o n c e n t r a t i o n s  and a c i d  (A2)  of the a n i o n ,  cation  respectively.  If, P  K  a  = - log  K  1 Q  (A3)  a  and pH = - l o g J H ] '10 +  1 f  (A4)  then -( K P  [A ]/[HA] = 1 0  F o r most of the a c i d s  -pH)  a  (>99%) t o be i n f r e e form:  [A~]/[HA] < 0.01 or  (A5)  (A6)  - 166  -( K -pH) P  10  -  -2 10  a  <  (A7)  Thus pK  Since 4.76  the  lowest  pK  , of a l l the VFA's i s t h a t of a c e t i c a c i d which  a  ( L e h n i n g e r , 1970),  low  (A8)  - pH > 2  t h e pH of the sample had adding  a combination  to be l e s s than 2.73.  pH  was  achieved  acids.  The  samples were c e n t r i f u g e d (1 h, 4450 x g) b e f o r e a d j u s t i n g pH.  A5.2  by  equals  of f o r m i c a c i d  and  The  phosphoric  Procedure  Once t h e GC had been i n s t a l l e d , the H e l i u m , A i r and H^ r e g u l a t o r s were s e t a t 12, 20 and 23 p s i g r e s p e c t i v e l y . The and  system  5-10  uL  9  and  computing I n t e g r a t o r . mean  values  injected the  and  the  A6.1  Gas  i n t e g r a t e d chromatogram  Each sample was  reported.  Gas  Gas gas  Volume and  the FID was  A  run s t a r t e d .  typical  The  was  chromatogram  and  run was  obtained  analysed at l e a s t  c a l i b r a t i o n c u r v e s a r e shown i n F i g u r e s A5 and A6  A6  s e t a t 120°C. turned  on  allowed for baseline s t a b i l i z a t i o n .  sample was  minutes  oven was  a l l o w e d to s t a b i l i s e , a f t e r which  m i n u t e s was  One after  was  The  stopped from  f o u r times and  some of  the  the the  standard  respectively.  Composition Determination  Volume  volumes produced  metres  over  (ALEXANDER - 0.25  a known p e r i o d of time were measured by L  per  revolution).  The  total  volume of  wet gas  - 167 -  Figure A 5  A typical v o l a t i l e fatty acid chronatogran: (Retention Time (RT) = .39 min) e t h a n o l , (RT - 1.94) a c e t a t e , (RT = 2.57) p r o p i o n a t e , (RT = 3.80) b u t y r a t e , (RT 5 . 6 4 ) 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).  " i g u r e A6  Gome v o l a t i l e f a t t y a c i d s t y p i c a l (x) b u t y r a t e (A) a c e t a t e .  12000  calibration  curves:  - 168 -  recorded  (V  ) was l a r g e r than the a c t u a l volume o f gas produced (V  f SC  to m o i s t u r e .  V was c a l c u l a t e d from the e q u a t i o n : act 1  V  *  where p  ) due  3.C t  i s the vapour  act  = (p ^ atm r  " P*) V /p rec atm c  (A9)  t  r  p r e s s u r e o f water a t 25°C and one atmosphere (p  *  23.76 mm Hg) and p i s the a t m o s p h e r i c p r e s s u r e . atm * r  A6.2  r  Gas Composition The  carbowax  GC d e s c r i b e d  i n s e c t i o n A5 was used  (12 p s i g ) as c a r r i e r gas was  f o r t h e g a s 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 t h e r m a l  c o n d u c t i v i t y d e t e c t o r (TCD). TCD w e r e u s e d w i t h ^ used  A 10%  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 H e l i u m used  f o r gas a n a l y s i s .  for  F o r the measurement o f  t h e same column and  ( 1 5 p s i g ) as t h e c a r r i e r g a s . H e l i u m  c o u l d n o t be  because t h e t h e r m a l c o n d u c t i v i t i e s o f both gases a r e v e r y c l o s e  r e s u l t i n g i n a v e r y poor TCD s e n s i t i v i t y . Once t h e c a r r i e r the  TCD was t u r n e d  stabilization.  gas f l o w r a t e and oven  temperature  on and 30 - 45 m i n u t e s  A 500 p.L sample was t a k e n  were  directly  space u s i n g a gas t i g h t s y r i n g e and i n j e c t e d  allowed  f o r baseline  from t h e f e r m e n t o r  i n t o the G C  A8 a r e shown t y p i c a l chromatograms and c a l i b r a t i o n  (35°C) were s e t ,  curves.  head  I n f i g u r e A7 and  - 169 -  F i g u r e A7  T y p i c a l fermentor head space gas chromatograms: ( R e t e n t i o n T i m e = . 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 ) hydrogen.  600  800-  CO t  400-  < UJ 300-1 ,'  :  / /  / /  /  /  200: I • / / 100-  a . i  1000  i  2000  i  3000  i  4000  8000  6000  QAS SAMPLE SIZE (uL).  r  igure  A8  Fementor head space gas c a l i b r a t i o n c u r v e s : c a r b o n d i o x i d e , ( • ) methane, (A) h y d r o g e n .  (x)  -  A7  Determination  Dry biomass  170  -  of Dry Biomass and Biomass Carbon  was d e t e r m i n e d by g r a v i m e t r i c means.  c a r b o n were d e t e r m i n e d by a t o t a l c a r b o n a n a l y s e r . the  Biomass and s u b s t r a t e  A s c h e m a t i c diagram of a  c a r b o n a n a l y s e r used i n t h i s s t u d y i s shown i n F i g u r e A9.  A7.1  Principle  By e x a m i n i n g the s p e c t r a o f a pure s u b s t a n c e a wave l e n 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 o t h e r compounds p r e s e n t  in  the m i x t u r e .  at  the s e l e c t e d  For analysis wavelength.  carbon a n a l y s e r .  i t i s s i m p l y n e c e s s a r y t o measure Use i s made o f t h i s  oxygen  to c a r b o n through  measure  an  and r e c o r d  dioxide. infrared  A l l carbon c o n t a i n i n g m a t e r i a l s are  The c a r b o n d i o x i d e a n a l y s e r which  i s carried  by a stream o f  i s specifically  the c o n c e n t r a t i o n o f c a r b o n  dioxide  and a e r a t i o n  t o remove  a l l the i n o r g a n i c  s e l e c t i v e measure o f t h e o r g a n i c c a r b o n  to  Total  p r e p a r e d by  carbon,  p r e s e n t may be o b t a i n e d .  d e t a i l s t h e r e a d e r i s r e f e r r e d to Sawyer and McCarty  A7.2  designed  present.  c a r b o n i s measured by t h i s p r o c e d u r e , but I f t h e sample i s f i r s t acidification  i n the t o t a l  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 w i t h a s t r o n g  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 . oxidized  principle  absorbance  then  a  F o r more  (1978) .  A p p a r a t u s and Reagents  A. B.  T o t a l c a r b o n a n a l y s e r (ASTRO). 238 g o f u l t r a - p u r e dissolved  reagent  grade  sodium  persulfate  (ASTRO) were  i n d i s t i l l e d water t o make one l i t r e o f s o l u t i o n .  6  9  F i g u r e A9  1  0  Schematic diagram o f the c a r b o n 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 r e a g e n t , (3) oxygen s u p p l y 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 o v e r f l o w 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 processor/printer.  -  172  -  Procedure  A7.3  The After  total  following  direct  from  syringe  c a r b o n a n a l y s e r was the  startup  instructions,  the f e r m e n t e r was  connected  to t h i s  o p e r a t e d i n the t o t a l  loaded i n t o  port.  The  a  syringe  carbon (TC) node.  with  the i n j e c t i o n  onboard  20 mL port,  of  leaving  m i c r o p r o c e s s o r was  at  results. which biomass  the  appropriate  and  A s i m i l a r p r o c e d u r e was  had  first  carbon  been was  between the f i l t e r e d the  time  filtered  assumed and  to  printing  out  of  r e p e a t e d f o r a sample  through a 0.45 be  unfiltered  the  sample  sample  analysis  from the f e r m e n t o r ,  urn MILLIPORE membrane.  difference  samples.  the  The  i n carbon biomass  f i l t e r was washed, d r i e d o v e r n i g h t a t 105 ± 1°C and  the  t u r n e d on  w h i c h assumed the a n a l y s i s t a s k i n c l u d i n g the i n j e c t i o n of the p r o p e r volume  sample  The  concentration  t h a t remained  weighed.  on  -173-  APPENDIX B TABULATED RESULTS  TABLE Bl - TOTAL CARBON MASS BALANCES (LACTOSE GROMTH LIMITED SUBSTRATE) AT pH = 6.0 AND TEMFERATl'REOs'c  SOLUTE PRODUCTS CARBON EXPERIMENTAL FACTORS  RUN  GASEOUS PRODUCTS CARBON (ugC/uLI  (ugC/aLl  I OIL 'N RATE  TEMP  81  B2a  pH  35.0 34.5 34.5 34.5  6.05 6.05 6.OB 6.10  0.0405* 0.0408 0.0420 0.0401  34.5  6.00 6.00  ND ND ND B2b  - 'z^:  B2c  '  Bib  BUTYRATE  BUTYRATE VALERATE  VALERATE CAPROATE LACTATE  197.8 266.1 322.5 221.4  2430.5 2125.0 2002.0 2230.0  478.4 446.0 310.8 210.6  27.9 71.6 58.8 21.3  268.8 534.1 496.8 368.6  T T ND 26.9  0.1035 0.1091 0.1029 0.1105  ND ND ND  2390.0 2325.0 2105.0 1861.3  340.6 261.9 166.3 156.5  ND ND ND ND  281.9 386.3 420.4 480.6  ND ND ND ND  41.0 T T  ND ND  152.4 199.0 194.9 NA  T  I T NA  261.3 379.5 382.3 NA  ND NO ND NA  ND  2  3  38.5 105.1 86.5 50.1  ND ND ND NO  NA 5.6 NA NA  33.9  ND 142.9' 253.3 215.0  NA NA NA T  NO ND NA  ND ND ND NA  NA NA NA NA  4  NA ND " NA NA  NA NA NA ND  6.00  0.1108 0.1080  0.1097  ND  0.1088  NA  2533.0 2536.1 2396.3 NA  5.95 6.00 6.00 6.00  0.I2B7 0.1249 0.1216 0.1233  T T T ND  1839.5 1178.3 1370.0 2232.3  235.0 139.0 136.9 IBI.6  ND NO ND T  100.3 1262.5 1006.9 961.8  ND NO ND NO  T T T T  T 176.0 240.3 131.6  ND NA ND NA  NA NA "NA NA  5.90 6.00  0.1145 0.1054  2181.9 2239.3  213.1 220.9  ND ND  362.4 391.5  NA NA  NA NA  NA NA  4.4 T  35.0  6.10 6.00  0.1026 0.0947  2154.4 1858.6  211.9 268.5  ND ND  755.6 693.8  NA NA  NA NA  NA NA  35.0 35.5 35.5 35.5  6.00 6.00 6.00 6.00  0.2386  B79.4 923.5 1019.3 1008.3  235.7 252.4 260.3 295.3  ND NO NO ND  695.7 876.0 939.3 929.2  167.7 107.9 T T  T  I T NO ND  34.0  35.0 34.0  B2<T  6.00 6.00  ACETATE PROPIONATE  6.00 6.00 6.00  36.5 36.0 36.5 36.0  34.5 34.5 34.0 B?d  ETHANOL  I'CI  CARBON FORMATE DIOXIDE  35.0  0.2423  0.2459 0.2430  T T  T T  13S.7 130.3 150.1 153.4  -  1. V - 1470 itL; 2. Trace asountsj 3. Not detected) 4. Not analysed) 5. Average influent carbon;  ND  27.4  T T  S = 4212.5 ugC/il g  NA  ND tin  ND ND  BIOMASS CARBON CARBON RECOVERY METHANE (ugC/aL) (ugC/oLI .  ND ND ND ND  6.9  3.6  24.6  12.9  78.S  41.5 55.0  104.4  NA  24.9 42.4  NA NA  30,0  29.2  94.8  59. i 100.6 69.1 71.2  CARBON RECOVERY (1 OF INFLUENT CARBON)  NA 496 NA 420  3905.3 4049.5 3741.0 3634.5  92.7 96.1 88.8 86.3  948 867 934  4005.3 4061.6 3999.3 3788.8  95.1 96.4 94.9 B9.9  4169.7 4791.2 4)11.0  99.0 113.7 97.6  NA 1139  1634 NA 939.4  -  550  3894.8 3811.5 3671.0  481  4187.4  92.5 90.5 87.1 99.4  162.1  664.5 636.0  3747.8 3741.3  B9.0 BB.B  171,1 "120.9  264.4 185.2  769.2 B72.5  4329.6 4019.5  102.8  'NA NA NA NA  NA NA NA NA  798.8 901.2 490.4 B42.8  3879.1 4320.5 3961.2 4177.4  92.1 102.6 94.0 99.2  V7l.~6  ,163.2 181.1  220.1 119.5  146.8  NA NA  116.2 91.8  205.3  2.5 T  NA NA  NA NA 1101.8 NA  NA NA NA NA  79.7  662  -  603  95.4  s  CON'T TABLE Bl - TOTAL CARBON MASS BALANCES (LACTOSE 6REWTH LIMITED SUBSTRATE) AT pH = 6.0 AND TEMPERATURE«35*C  RUN I  SOLUTE PRODUCTS CARBON (ugC/iLI  EXPERIMENTAL FACTORS  TEMP (°CI  pH  DIL'N RATE (h"'l  63c  35.0 35.0 34.5  4.00 5.90 5.95  0.2075 0.2131 0.2121  89.2 93.9 T  585.3 843.0 1023.3  684.6 663.1 727.1  ND T T  979.6 1159.6 1742.6  T T T  B4a  35.0 35.5 35.5 34.5  6.00 6.05 6.00 6.00  0.2896 0.2840 0.3005 0.2891  112.0 125.6 90.9 101.5  1451.5 1314.4 1334.1 1574.3  342.6 320.0 277.1 335.4  ND ND ND ND  1390.1 1218.0 1265.8 1395.9  ND ND ND ND  84b  35.0 35.0 35.5 35.5  6.00 6.00 6.00 6.00  0.2907 0.3035 0.2971 0.2898  142.1 196.2 93.2 95.4  1416.7 1510.8 1190.5 1158.7  508.4 629.5 557.3 438.0  ND ND ND ND  542.B 362.0 615.1 699.1  T T T T  B5  34.5 35.0 34.0 34.0  6.00 6.00 6.00 6.00  0.3520 0.3401 0.3537 0.3560  T NA T 59.4  659.2 NA 692.1 983.0  223.9 NA 223.0 243.6  ND ND ND ND  750.0 NA 667.2 963.5  35.0 35.5 35.0 35.0  5.93 5.98 6.00 6.00  0.4784 0.4624 0.4716 0.4762  T NA T T  592.2 HA 645.5 910.8  189.6 NA 263.4 322.0  ND ND ND ND  35.0  6.00  0.640  T  403.9  238.4  ND ;  Ei  B7  iniETHANOL ACETATE PROPIONATE BUTYRATE BUTYRATE VALERATE  6ASE0US PRODUCTS CARBON (ugC/iLI  nVALERATE  CAPROATE LACTATE  CARBON FORMATE DIOXIDE  BIOMASS CARBON CARBON RECOVERY METHANE (ugC/sL) (ugC/uLI  CARBON RECOVE (1 OF INFLUE CARBON  ND NO ND  637.0 497.2 NA  NA NA NA  40.8 26.6 159.9  ND ND ND  906.6 489.3 796.5  4310.3 4142.1 4744.2  102.3 98.3 112.6  ND ND ND ND  NA NA NA 53.0  NA NA NA ND  157.9 121.3 150.4 112.9  T T T T  790.7 932.4 856.8 705.6  4297.8 4084.7 4028.4 4278.6  102.0 97.0 95.4 101.6  36.9 39.4 104.9 122.7  ND ND ND ND  413.5 NA 807.5 966.3  NA NA NA NA  ND ND ND ND  ND ND ND ND  834.8 783.1 965.2 794.0  3975.2 4250.1 4333.7 4274.2  94.4 1G0.9 102.9 101.5  T NA ND T  T NA 38.0 58.8  ND ND ND ND  NA NA NA 1341.5  NA NA NA ND  86.6 79.3 72.9 113.9  590.6 667.2 583.7 654.4  3651.8  86.7  T T  3618.4 4438.1  85.9 105.4  669.5 NA 597.5 690.0  T NA T T  T NA 27.7 48.3  ND NA ND ND  NA NA NA 782.5  NA NA NA T  157.3 151.1 93.1 83.8  T T  597.1  ND  73.2  ND  702.0  NA  ND  387.2 370.0 294.8 T T T T  T  T  T  T  ND  -  -  584.4 571.8 695.2 NA  2975.5  70.6  3015.7 2B37.4  71.6 67.4  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  EASEOUS PRODUCTS CARBON lugC/iL)  t TEMP  pH  l°C)  DIL'N • RATE  ETHANOL  innlACETATE PROPIONATE BUTYRATE BUTYRATE VALERATE VALERATE CAPROATE LACTATE  liT )  FORMATE  CARBON DIOXIDE  METHANE  BIOMASS CARBON lugC/iL)  CARBON RECOVERY CARBON (1 OF t PROTEIN RECOVERY INFLUENT FERMENTED lugC/sL) CARBON)  1  El  E2  E3  34.5 34.5 35.0 34.0 35.0  4.10 4.10 6.10 6.10 6.10  (h ) 0.3061 0.3005 0.3061 0.3061 0.3061  B1.8 98.1 84.6 71.4 B3.9  1021.4 1088.1 1126.2 1107.5 861.5  274.2 283.8 408.0 486.8 310.4  34.0 34.0 35.0  6.00 6.00 6.00  0.2029 0.2029 0.2017  69.6 69.7 101.0  725.3 1689.7 1386.7  188.5 212.5 172.3  34.5 33.7 34.5 34.5  6.00 5.95 5.85 5.97  0.2029 0.2097 0.2124 0.2071  ND 7B.9 100.8 99.1  1699.1 1263.1 1307.4 1339.8  592.7 563.4 656.2 526.8  2 T T T T  T  T T  T  T T T  .  207.5 235.8 290.1 204.2 242.4  ND ND NO ND ND  131.7 113.8 103.9 141.0 127.6  152. B 124.0 141.6 181.3 1B7.5  NA 1940.0 1281.5 1118.6 1501.0  NA NA NA NA NA  22.9 21.9 30.9 17.2 21.2  ND ND ND ND ND  958.4 617.0 876.0 719.2 NA  4311.0 4522.5 4342.8 4047.2 4128.2  78.7 B2.6 79.3 73.9 75.4  233.9 284.5 208.5  NO ND ND  89.9 143.5 160.8  NO ND ND  1551.9 1720.7 1728.9  NA NA NA  19.2 3.1 10.0  ND ND ND  1053.2 1356.6 NA  3912.3 5477.2 4963.1  71.5 100.0 90.6  481.8 416.2 461.7 346.9  76.6 76.1 131.6 154.3  65.7 50.8 36.7 34.9  T T T T  962.8 909.4 1IB2.0 NA  NA NA NA NA  ND ND ND ND  ND ND ND ND  561.0 662.9 643.1 682.8  4439.7 4020.B 4519.5 4202.7  Bl.l 73.4 82.5 76.8  3  V = 1470 aL; 2. Trace aiounts; 3. Not detected; 4, Nat analysed; 5. Influent carbon concentration i  s = 0  4  5475.2 ugC/iL  s  65.1  68.7  69.9  TABLE B3 - TOTAL CARBON MASS BALANCE (LACTOSE 6R0NTH LIMITED SUBSTRATE) AT pH = 4.5 AND TEMPERATURE = 35°t  RUN  EXPERIMENTAL FACTORS  SOLUTE PRODUCTS CARBON (ugC/iL)  I  DIL'N RATE  6ASE0US PRODUCTS CARBON (ugC/iL)  1  D2  03  D4  TEMP l°CI  pH  (ffl  35.0 35.5 35.5 36.0  4.6 4.6 4.6 4.5  0.0495 0.0505 0.0496 0.0505  36.0 35.0 36.0 35.0  4.5 4.5 4.5 4.5  0.1036 0.1014  35.0 35.0 35.U 36.0 35.0 36.5 36.5  innCARBON iETHANOL ACETATE PROPIONATE BUTYRATE BUTYRATE VALERATE VALERATE CAPROATE LACTATE FORMATE DIOXIDE  CARBON RECOVERY BIOMASS CARBON (t OF CARBON RECOVERY INFLUENT METHANE (ugC/iL) (ugC/iL) CARBON)  1175.9 1260.9 1141.1 1554.0  711.1 641.9 434.8 4BB.0  75.0 75.3 66.9 96.9  1391.5 1382.4 1503.6 1640.0  42.0 T T T  118.5 112.3 90.6 84.1  361.4 326.3 308.4 407.1  NA NA 4.8 NA  NA NA ND NA  ND ND ND ND  ND ND ND ND  NA 15B 396 371  4450.4 4214.6 4196.2 5019.0  105.6 100.0 99.6 119.5  0.1006  ND ND ND ND  1044.1 960.1 990.0 1097.3  520.9 671.3 622.6 594.9  T T I T  1369.1 1232.5 1346.3 1297.4  ND ND ND ND  109.3 73.3 T T  ND ND ND NO  NA NA 15.9 NA  NA NA ND NA  ND ND ND NO  ND ND ND NO  348 NA 410 426  3407.3 3347.7 3385.6 3431.5  B0.9 79.5 BO. 4 B1.5  4.5 4.5 4.5  0.2071 0.1959 0.20BO  T NA NA  824.0 NA NA  506.5 NA NA  ND NA NA  473.1 NA NA  ND NA NA  44.4 NA NA  ND NA NA  1347.9 NA NA  NA NA NA  ND NA NA  ND NA NA  692 NA 65B  3887.8  92.3  4.a  0.3571 NA 0.3560 0.3578  53.3 42.1 51.9 42.5  B5.B 105.4 105.4 135.9  ND ND ND ND  2B4.0 226.6 133.0 162.5  ND ND ND ND  T T T T  ND NO ND ND  NA NA NA 1053.4  NA NA NA NA  ND ND ND ND  ND ND ND ND  366 348 292 406  2110.4 2054.4 2014.6 2274.2  4.6 4.6 4.6  o'.ion  261.9 257.5 250.0 373.1  268.1 279.1 379.1 473.9  1  2  4  1. V- 1470 EL; 2. Trace amounts; 3. Not detected; 4. Not analysed; 5. Average influent carbon, S = 4212.5 ug/iL 0  3  -  -  -  -  50.1 4B.8 47.8 54.0  TABLE 64 - TOTAL CARBON MASS BALANCE (LACTOSE 6RQUTH LIMITED SUBSTRATE) AT D = O.OStT'ftND TEMPERATURE = 35°C . RUN t  EXPERIMENTAL FACTORS  SOLUTE PRODUCTS CARBON (ugC/iL)  ,  BASEOUS PRODUCTS CARBON (ugC/iL)  CARBON RECOVERY BIOMASS CARBON (I OF ini nCARBON CARBON RECOVERY INFLUENT ACETATE PROPIONATE BUTYRATE BUTYRATE VALERATE VALERATE CAPROATE LACTATE FORMATE DIOXIDE METHANE (ugC/iLI (ugC/iL) CARBON)  TEMP <°C)  pH  DIL'N RATE (If )  Al  34.9  4.0  0.0520  241.9  351.9  ND  ND  1315.0  ND  NO  T*  41.6  ND  513.4  ND  308.0  2258.4  53. t  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  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  1  ETHANOL  3  4  I M  ^4  A4a  34.8  5.6  0.0530  172.4  1635.0  473.B  9.0  456.0  NO  43.5  A5  34.0  6. 1  0.0400  265.9  1969.3  446.3  71.3  534.4  ND  105.1  fit  34.2  6.5  0.0480  105.6  1993.0  196.4  4.1  564.4  ND  A2D  35.4  4.6  0.0501  103.9  850.6  110.9  54.8.  1863.3  Alb  35.0  5.0  0.0430  104.5  622.8  538.1  46.B  A4b  35.0  5.5  0.0536  243.6  1635.0  2B0.9  16.0  I.V  166.3  2.0  ND  ND  ND  646.0  3938.2  93.5  ND  7.0  ND  NO  ND  496.0  4050.5  96.2  79.4  393.1  14.0  ND  ND  ND  767.0  4117.0  97.7  NO  ND  NO  ND  ND  721.8  NO  374.0  4079.3  96.8  1502.2  ND  153.3  ND  5.2  T  415.6  ND  340.6  3729.1  B8.5  604.0  78.4  85.8  120.0  7.2  ND  330.1  177.4  589.0  4172.0  99.0  - 1470 iL; 2. Trace amounts; 3. Not detected; 4. Average influent carbon, S = 4212.5 ugC/iL 0  0  0  I  T'i.E ES - TOTAL GAS PRODUCTION AS A FRACTION OF CUMULATIVE EXPERIMENT TIME AT 25°C AND ONE ATMOSPHERE  A-SERIES EXPERIMENTS  A2a,0l  Al TIKE (h) 0 23. 46. 54. 89. 106. 128. 153. ;?fc. 200. 224. 249.5 274.0 296. 321. 344. 348. 372.  GAS'  (L/LI _  0 0.539 0.5301 1.036 0.253 0,'t59 0.1587 0.74 1.284 1.435 1.495 2.164 2.075 2.225 2.121  TIME (hi 0 23. 48. 56. 89. 106. 128. 153. 17b. 200. 224. 249.5 274.0 296. 321. 344. 366. 372.  A4  A3  GAS  IL/LI  0 0.2124 0.0 0.0 0.9115 2.163 2.756 2.326 1.716 0.2778 0. 0 0 0 0  TIME (hi 0 44. 68. 92. 116. 140.5 174.0 199. 2S3. 23B. 267. 285. 308. 332. 353.  1. In litres nf gas ptr litre effluent.  GAS  IL/LI  0 0 0 0 0 0 0 0 0 0 0 0 0  A5a  TIME Ihl  6AS (L/LI  TIME (hi  0 20. 43. 66. 102. 115. 139. 164. 188. 212.  -  0 24. 34. 48. 72. 99. 120. 143. 167. 191. 215. 239. 264.5  0 0 0 0 0 0 0 0 0  A6  (L/LI  TIME Ihl  (L/L)  _  0.  -  -  20.  GAS  0 0 0 0 0  43.  6AS  0 0  TIME (hi  (L/L)  0  -  24. 48.  A46  A36  A26  6AS  -  TIME Ih)  (L/LI  0  -  24. 48.  GAS  -  66.  0.030  72.  0  72.  0  102.  0.086  96.  0  96.  5.912  TIME <h)  GAS  (L/LI  0 44. 68. 92. 116.  0 0.639  115.  0.94  128.  0  128.  4.733  140.5  0.286  139.  0.35  144.  0  144.  4.697  174.  0.708  0  179.  0  179.  4.485  199.  0.200  0 0  192.  0.602  192.  3.960  213.  0.368  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.  527.5  2.615  527.5  1.402  -539.  553.0  2.638  1.116 1.199 1.188  TABLE B5 - TOTAL 6AS PRODUCTION AS A FRACTION OF CUMULATIVE EXPERIMENT TINE AT 25°C AND ONE ATMOSPHERE (CQN'TI B-SERIES EXPERIMENTS  i TINE In)  BAS IL/L)  TINE (h)  0 24 34 48 72 99 120 143 147 191 215 239 244.5  -  0 18 42 68.5 92.0 116.0 140.0 164.0 168.0 212.0 236.0 260.0 284.0 308.0  0 0 0 0 0 0 0 0 0 0 0  B2c  B2b BAS (L/LI  0 0.293 0.00 0.061 0.218 0.366 0.219 0.207 0.027 0.096 0.308 0.408  TINE In)  GAS (L/L)  0 40 64 89 113 135 162 184 20B 232 256 2B0  0.170 0.305 0.15a 0.418 0.316 0.609 0.299 0.205 0.349 0.240 0.247  B2d  TIME Ih)  GAS (L/LI  0 B 50 74 107.5 124 149 173 19B 234 256 2B7 312 325 349 373 400 423 453 493 517 526  0 0.001 0.323 0.034 0.004 0.038 0.131 0.073 0.113 0.3B6 0.634 0.712 0.529 0.484 0.452 0.279 0.337 0.966 0.783 0.425 0.230  -  B2e  TIME (hi  6AS (L/L)  TIME (h)  0.0 20 36 47 71 95 119 143 167 191 221 239 264 288 312 336 360 384 408 432 456 480 504  0 0 0 0 0 0 0 0.113 0.325 0.473 0.257 0.244 0.464 0.114 0.703 0.759 0.600 0.636 0.633 0.769 0.725 0.71?  0 25 37. 61. 85. 109. 133. 157. 181. 211. 229. 254. 27B. 302. 326. 350. 374. 398. 422. 448. 472. 496.  B3b  6AS (L/L)  TINE  -  0 18. 26. 43. 55. 67. 79. 91. 102. 114. 126. 138. 150. 163. 175. 186. 196. 211. 222. 234. 246.  0 0.397 0 0 0 0 0 0 0 0.236 1.147 0.825 0.859 1.043 1.069 0.749 0.545 0.582 0.768 0.109 0.061  (hi  B3c  BAS (L/L)  TIME  -  0 24. 44. 55. 68. 80. 92. 104. 116. 128. 140. 152. 164. 176. 188. 200. 212. 224. 236. 24B. 260. 271. 283. 295. 307. 319. 338. 355.  0 0 0 0.660 1.498 2.877 3.154 0.562 0.371 0.464 0.235 0 0 0.14 0 0.153 0.169 0.436 0.481  (h)  BAS (L/L)  0 0.272 0.424 0.18 0.048 0.238 0.214 0.016 0.958 1.644 1.070 0.092 0.11B 0.126 0.204 0.160 0.231 0.181 0.286 0.16B 0.250 0.116 0.185 0.075 0.152 0.341 0.451  CON'I TABLE B5 - TOTAL 6A5 PRODUCTION AS A FRACTION OF CUMULATIVE EXPERIMENT TINE AT 25°C AND ONE ATMOSPHERE B-SER1ES B4b  643  TIME (h)  GAS (L/LI  0 36. 60. 72. 84. 96. 106. 120. 132. 144. 156. 168. 180. 192. 204. 214.  0 1.60 0 0 0.171 0.158 0.070 0.156 0.174 0.441 0.599 0.652 0.501 0.621 0.466  B6  B5  TIME Ihl  6AS (L/L)  18. 20. 43. 55. 67. 79. 91. 102. 114. 126. 138. 150. 163. 175. 186. 196. 211. 222. 234. 246. 259. 271.  _  0 0.789 0.841 0.293 0.424 0.211 0.020 0 0.020 0.269 0.136 0.073 0 0 0 0 0 0.053 0 0 0.042  B7  TIME (hi  GAS (L/LI  TIME (hi  GAS (L/L)  0 18. 32. 42. 55. 67. 79. 91. 102. 114. 126. 132. 139. 151. 157. 163.  0 0.195 0.067 0.031 0.088 0.336 0.592 0.455 0.419 0.349 0.320 0.294 0.368 0.459 0.526  0 18. 32. 42. 55. 67. 79. . 91. 102. 114. 126. 132. 139. 151. 157. 163.  0 0.211 0.213 0.582 0.446 0.196 0.495 0.767 0.922 1.013 0.949 0.667 0.585 0.526 0.498  TIME (h)  6AS IL/L)  173. 179. 187. 199.  0.376 0.037 0.036 0  CON'T TABLE B5 - TOTAL BAS PRODUCTION AS A FRACTION OF CUMULATIVE EXPERIMENT TINE AT 25°C AND ONE ATMOSPHERE  D-SER1ES EXPERIMENTS  E-SERIES EXPERIMENTS  TIME (h)  BAS (L/LI  0 B. 50. 74. 107.5 124. 149. 173. 198. 234. 256. 2B7. 312. 325. 349. 373. 400. 426. 453.5 493. 517. 526.  0 1.447 1.214 1.27 1.527 1.672 1.155 1.194 0.926 0.161 0 0.041 0.197 0.06 0 0 0 0 0.211 0.104 0.050  TIME th) 0 21. 45. 67. 89. 113. 138. 162. 168. 210. 235. 259.  El  D4  03  D2  BAS (L/L)  TIME (hi  -  0 36. 60. 72. 84. 96. 108. 120. 132. 144. 156. 168. 180. 192. 204. 214.  0 1.993 1.066 0 0 0.072 0 0.056 0.063 0.201 0  6AS (L/L)  0 0.100 1.653 0.390 0 0.169 0.409 0.531 0.580 0.633 0.418 0 0 0 0  E3  E2  TIME (hi  6AS (L/LI  TIME (h)  GAS (L/L)  TIME (hi  6AS (L/L)  0 21. 32. 44. 57. 69. BO. 92. 105. 116. 128. 141. 152. 164. 176. IBB. 200. 212.1 224. 236.; 248. 260.  -•  0 24. 44. 55. 68. 80. 92. 104. 116. 128. 140. 152. 164. 176. 188. 200. 212. 224. 236. 248. 260. 271. 283. 295. 307. 319. 338. 355.  -  383. 395. 408. 419. 431. 443. 455. 467. 479. 494. 503. 515. 527. 538.  0.616 0.467 0.391 0.643 0.526 0.295 0.042 0 0 0 0 0 0 0  0 0.500 0.745 0.214 0.121 0.016 0.119 0.126 0.147 0.183 0.249 0.213 0.243 0.283 0.232 0.253 0.238 0.259 0.183 0.185 0.225  0 0.043 0.061 q. 125 0.139 0.091 0.103 0.045 0.022 0.028 0.0B7 0.1098 0.105 0.086 0.078 0.113 0.085 0.201 0.263 0 0 0.344 0.137 0.056 0.053 0.108 0.179  - 183 -  APPENDIX C ENZYME SYSTEMS  - 184 -  CHjO®  CH,OH  c=o IIO-C H  H  ruetose-1. bhisphosphate  I  C OH  H - C -OH I  CHiO© Iructose-fi-phosphate  H -C  dyceraldehyde-3phusphate  di hydroxy ace lone phosphate'  H - C =0 OH  NAD| + Pi  HO -C -H  I  H-C-OH I  H  C -OH C'H 0® ;  glucuse-6-phosphate  COO®  C H  2  COO® HC-OH  HC-OH 0 ©  1. 3-bispliosphoglycerate  CH-.0®  -<ADP)  •|K(ATP) COOH  COOH  HC-OH  HC-OH  C'HiO®  3-phosphoglycerate  C H , 0 ®  COOH HC-O®  2-phosphotdveerate  CH,OH  4-* COOH  -c-o® II  phosphoenol-  w  CH,  coo» c=o ^  pyruvate  CH-i.J  F i g u r e C l Breakdown o f g l u c o s e t o 2 p y r u v a t e v i a t h e Embden-MeyerhofParnas pathway: (1) PEP: g l u c o s e p h o s p h o t r a n s f e r a s e ; (2) g l u c o s e phosphate i s o m e r a s e ; (3) p h o s p h o f r u c t o k i n a s e ; (4) f r u c t o s e b i s p h o s p h a t e a l d o l a s e ; (5) t r i o s e phosphate i s o m e r a s e ; (6) g l y c e r a l d e h y d e - 3 - p h o s p h a t e dehydrogenase; (7) 3 - p h o s p h o g l y c e r a t e k i n a s e ; (8) p h o s p h o g l y c e r a t e mutase; (9) e n o l a s e ; (10) p y r u v a t e kinase. Source i s G o t t s c h a l k (1979).  - 185 -  (.'l( -Ol;  Cll  3  p. CoA  COOH  2  butyrate acetyl-CoA  ^  acetyl-P acetate  CHj-CH -CH -CO-CoA 2  2  butyryl-CoA  NAD-^f NADHj—*| CHj - C H = C H - C O - C o A crotonyl-CoA H 0 2  CH -CH-CH -CO-CoA 3  CH -CO--CH!-CO-CoA acetoacetyl-CoA  2  3  OH L (+ H3-hydroxybutyry 1-CoA  t NAD sum: glucose + 3 A D P + 3 P,  F i g u r e C2  NADHi • butyrate + : C O j + 2 H  2  +3 A T P  P a t h o f b u t y r a t e f o r m a t i o n from g l u c o s e : (1) p h o s p h o t r a n s f e r a s e s y s t e m and Embden-Meyerhof pathway; (2) p y r u v a t e - f e r r o d o x i n o x i d o r e d u c t a s e ; (3) h y d r o g e n a s e ; (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 - h y d r o x y b u t y r y l - C o A dehydrogenase; (6) L - 3 - h y d r o x y a c y l - C o A h y d r o l y a s e ( c r o t o n a s e ) ; (7) b u t y r y l - C o A dehydrogenase; (8) C o A - t r a n s f e r a s e ; (9) p h o s p h o t r a n s a c e t y l a s e ; (10) a c e t a t e k i n a s e . Source i s G o t s c h a l k (1979).  - 186 -  OH  OH  I  (L)2CH - C - C O O H  :CH -C-CO-COA  3  3  H  til Ir H (D) C H - C - C O O H 3  :CH,=CH-CO-CoA  OH  ETF -  -  ETF • H • 2  CHj-CO-COOH CoA CoA -vL -^L, [ C O T I V ^ -  Fd fd ~-*Y~ Fd • H  CH3-CO-C0A  2  —  ETF • H ' 2  — ETF — 2CH -CH -CO-CoA 3  2  2CH3-CH3-COOH sum: 3 lactate •  2 propionate + acetate + C 0  2  F i g u r e C3 F o r m a t i o n o f p r o p i 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 p r o p i o n i c u m : (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 n o t e s t a b l i s h e d ; (4) dehydrogenase, w h i c h employs r e d u c e d 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 o x i d o r e d u c t a s e ; (7) t r a n s h y d r o g e n a s e ; (8) p h o s p h o t r a n s a c e t y l a s e + a c e t a t e k i n a s e . Source i s G o t t s c h a l k (1979).  - 187 -  HOOC-CH,-CO-COOH NADH; NAD  HOOC-CH -CHOH-COOH :  HOOC-CH -CH -COOH 2  HOOC-CH=CH-COOH  2  (ATP) QM3P + sum: lactate + NADH + ADP 4- P, 2  F i g u r e C4  » propionate + NAD + ATP  F e r m e n t a t i o n o f l a c t a t e v i a t h e 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 : (1) l a c t a t e dehydrogenase ( t h e H - a c c e p t o r i s p r o b a b l y a f l a v o p r o t e i n ) ; (2) ( S ) - m e t h y I m a l o n y l - C o A - p y r u v a t e t r a n s c a r b o x y l a s e ; (3) m a l a t e dehydrogenase; (4) fumarase; (5) fumarate r e d u c t a s e ; (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 G o t t s c h a l k (1979).  -  188  -  APPENDIX D A COMPUTER PROGRAM FOR ESTIMATING THE PARAMETERS OF THE MICROBIAL GROWTH MODEL  - 189 -  C T H I S PROGRAM E S T I M A T E S T H E PARAMETERS OF A NONLINEAR C MICROBIAL K I N E T I C MODEL E I T H E R USING A L E A S T SQUARES C OR A CHERBTCHEV CURVE F I T T I N G METHOD. C IMPLICIT REAL*8 ( A - H , 0 - Z ) DIMENSION P ( 3 ) , I V ( 6 3 ) , V ( 2 8 2 ) EXTERNAL C A L C R , C A L C J COMMON X ( 2 0 ) , 7 ( 2 0 ) , S O , EPS C S E T T I N G I N I T I A L VALUES AND READING  IN DATA  EPS = 0.001 DO SO= 4 2 1 2 . 5 D 0 N=5 M=3 P( 1 ) = . 2 0 0 D 0 P(2)=60.D0 P(3)=.36D0  DO I I » 1,11  C A L L FREAD ( 5 , ' 2R*8 1 CONTINUE  :',  X(I),  7(D)  C S E T T I N G D E F A U L T VALUES I N I V AND V CALL  DFALT(IV.V)  CC REQUESTING FOR T H E PRINTING OF T H E COVARIANCE MATRIX CC A F T E R CONVERGENCE HAS OCCURED. IV( 14) = 1 IV(21)=6 C C A L L I N G T H E MAIN SUBROUTINE NL2SOL CALL 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 T H E RETURN CODE AND SOLUTION . WRITE(6,110) IV(1) 110 FORMAT('RETURN C O D E = ' , 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 D E F I N I N G T H E 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 ) IMPLICIT REAL*8 ( A - H , O - Z ) DIMENSION P ( M ) , R ( N ) COMMON X ( 2 0 ) , 7 ( 2 0 ) , S O , EPS C C IMPOSING A CONSTRAINT ON X ( I ) I F ( P ( 3 ) . L T . ( X ( N ) + E P S ) ) 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  -  131  190  -  NF=0 RETURN END  C SUBROUTINE D E F I N I N G THE P A R T I A L 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 ) IMPLICIT REAL*8 ( A - H , O-Z) DIMENSION P ( M ) , D ( N , M ) COMMON X ( 2 0 ) , Y ( 2 0 ) , S O , EPS C C IMPOSING IF  A CONSTRAINT ON X ( I )  (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) = SO-(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)/(DUMP*DUMP) 130 CONTINUE RETURN 131 NF=0 RETURN END  

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