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Possible methods of decreasing cell yields in waste treatment systems Nix, Peter G. 1972

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POSSIBLE METHODS OF DECREASING CELL YIELDS IN WASTE TREATMENT SYSTEMS by PETER G. NIX B.Sc, University of Guelph, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Microbiology We accept this thesis as conforming to the required standard August, 1972 In present ing th i s thes is in pa r t i a l f u l f i lmen t of the requirements for an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ib ra ry sha l l make it f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th i s thes i s for s cho l a r l y purposes may be granted by the Head of my Department or by h is representa t i ves . It is understood that copying or pub l i c a t i on o f th i s thes i s fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Depa rtment The Un ivers i t y of B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT It has previously been shown that low pD^ stimulated the respira-tion of facultative bacteria suggesting that precise control of dissolved oxygen in waste treatment systems might be an effective method of increasing carbon dioxide production at the expense of c e l l yield. In continuous cultures of glucose limited Escherichia c o l i B, controlled at less than 8mmHg, we have demonstrated a 57% increase i n carbon dioxide production at the expense of both c e l l yield and supernatant carbon. Batch cultures of Pseudomonas aeruginosa 9027 showed an even greater loss of efficiency at low oxygen tensions. Studies with mixed populations (batch culture) showed some evidence of yield reduction (in spite of inherent d i f f i c u l t i e s with floculation and natural selection). However, the reduction was considered to be too small to warrant the practical application of dissolved oxygen control in waste treatment systems. While completely anaerobic E_. c o l i B_ cells appear to be uncoupled by aeration, alternating a continuous culture between anaerobic and aerobic growth did not similarily affect the cells - evidently E_. c o l i B requires a substantial amount of time (over 4 hours) to f u l l y adapt to anaerobic conditions. In any case, unlike E_. c o l i B_, the growth of stable anaerobic mixed populations did not become "uncoupled" when aerated - indicating that this technique would not be suitable in waste systems. S t u d i e s w i t h an u l t r a - v i o l e t i r r a d i a t e d c o n t i n u o u s c u l t u r e o f E. c o l i B showed c o n s i d e r a b l e i n c r e a s e s i n c a r b o n d i o x i d e p r o d u c t i o n a t t h e expense o f c e l l y i e l d . I n a d d i t i o n , the p r o c e s s o f f l o c u l a t i o n was g r e a t l y enhanced. The p r a c t i c a l p o s s i b i l i t i e s o f i m p l e m e n t a t i o n i n waste t r e a t m e n t systems, w a r r e n t f u r t h e r i n v e s t i g a t i o n o f t h e e f f e c t s o f u.v. i r r a d i a t i o n on b a c t e r i a l growth. i i i TABLE OP CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 7 CULTURE TECHNIQUE FOR OXYGEN CONTROLLED EXPERIMENTS 7 DISSOLVED OXYGEN PARTIAL PRESSURE 8 OXYGEN TENSION CONTROL SYSTEM 8 ASSAY FOR CYTOCHROME B 9 CELL SIZE 9 GROWTH OF MIXED CULTURES AT LOW OXYGEN TENSIONS 10 USE OF WARBURG APPARATUS 10 ALTERNATIONS BETWEEN ANAEROBIC AND AEROBIC GROWTH 11 CULTURE TECHNIQUE FOR ULTRA-VIOLET IRRADIATION 11 CALCULATION OF U.V. IRRADIATION DOSE LEVELS: 12 CALCULATION OF CARBON BALANCES 13 C e l l Carbon 13 S u p e r n a t a n t Carbon 14 Carbon D i o x i d e 14 RESULTS 15 RE S PIROMETER 15 OSCILLATIONS 15 TABLE OF CONTENTS (Continued) i v Page GROWTH OF E . COLI B AT LOW OXYGEN TENSIONS 18 GROWTH OF PSEUDOMQNAS AERUGINOSA 9027 AT LOW OXYGEN TENSIONS.. 21 EFFECT OF LOW OXYGEN TENSIONS ON CYTOCHROME B 21 EFFECT OF LOW OXYGEN TENSIONS ON CELL SIZE 25 EFFECT OF LOW OXYGEN TENSIONS ON THE GROWTH OF MIXED POPULATIONS 25 EFFECT OF ALTERNATING BETWEEN AEROBIC AND ANAEROBIC GROWTH 29 EFFECT OF U.V. IRRADIATION ON THE GROWTH OF E . COLI B 37 DISCUSSION 43 OSCILLATIONS 43 GROWTH OF E . COLI B AT LOW OXYGEN TENSIONS 44 EFFECT OF LOW OXYGEN TENSIONS ON LEVELS OF CYTOCHROME B 45 EFFECT OF LOW OXYGEN TENSION ON CELL SIZE 46 GROWTH OF PSEUDOMQNAS AERUGINOSA 9027 AT LOW OXYGEN TENSIONS. . 46 BIBLIOGRAPHY 51 V LIST OF TABLES Page T a b l e I . E f f e c t o f oxygen t e n s i o n on carbon d i o x i d e p r o d u c t i o n i n E . c o l i B i n c o n t i n u o u s c u l t u r e 19 T a b l e I I . E f f e c t o f oxygen t e n s i o n on ca r b o n d i o x i d e p r o d u c t i o n o f E_. c o l i B i n c o n t i n u o u s c u l t u r e s . . . . 20 T a b l e I I I . E f f e c t o f oxygen t e n s i o n on ca r b o n d i o x i d e p r o d u c t i o n i n Pseudomonas a e r u g i n o s a 9027 i n b a t c h c u l t u r e 24 T a b l e IV. E f f e c t o f oxygen t e n s i o n on c e l l s i z e 27 T a b l e V. E f f e c t o f low oxygen t e n s i o n s on the growth o f mixed p o p u l a t i o n s i n b a t c h c u l t u r e 30 T a b l e V I . Endogenous r e s p i r a t i o n o f f l o c u l a n t c u l t u r e s showing t h e d e c r e a s e i n c e l l mass o v e r time a t pO o f 8mmHg and 144mmHg 31 T a b l e V I I . R e s p i r a t i o n r a t e and ca r b o n uptake r a t e o f fJ.oces m a i n t a i n e d p r e v i o u s l y f o r 5.5 hours a t (a) 8mmHg and (b) 144mmHg 32 T a b l e V I I I . The e f f e c t o f a e r a t i n g an a n a e r o b i c c o n t i n u o u s c u l t u r e o f E . c o l i B_ 33 T a b l e IX. Carbon d i o x i d e p r o d u c t i o n from a c o n t i n u o u s c u l t u r e o f E_. c o l i B a l t e r n a t e d between a e r o b i c and anae-r o b i c c o n d i t i o n s 35 v i L IST OF TABLES (Continued) Page T a b l e X. The e f f e c t o f a e r a t i n g an a n a e r o b i c c o n t i n u o u s c u l t u r e o f mixed p o p u l a t i o n s 38 T a b l e X I . E f f e c t o f i n c r e a s i n g dose l e v e l s o f u l t r a - v i o l e t l i g h t on t h e growth o f E_. c o l i B_ i n c o n t i n u o u s c u l t u r e 40 T a b l e X I I . E f f e c t o f i r r a d i a t i n g a normal a e r o b i c c o n t i n u o u s c u l t u r e o f E. c o l i B w i t h u l t r a - v i o l e t l i g h t 42 v i i LIST OF FIGURES Page F i g u r e 1. The R e s p i r o m e t e r 16 F i g u r e 2. Data from r e s p i r o m e t e r showing o s c i l l a t i o n s i n r e s p i r a t i o n o f a c o n t i n u o u s c u l t u r e o f E . c o l i B i n g l u c o s e m i n i m a l medium 17 F i g u r e 3. E f f e c t o f oxygen t e n s i o n s on growth l a g o f Pseudomonas a e r u g i n o s a 9027 i n b a t c h c u l t u r e s 22 F i g u r e 4. E f f e c t o f oxygen t e n s i o n on growth r a t e o f Pseudomonas a e r u g i n o s a 9027. 23 F i g u r e 5. E f f e c t o f oxygen t e n s i o n on l e v e l o f Cytochrome B. 26 F i g u r e 6. E f f e c t o f oxygen t e n s i o n on t h e r e l a t i o n s h i p between O.D. and c e i l mass 28 F i g u r e 7. E f f e c t o f changing from a e r o b i c t o a n a e r o b i c c o n d i t i o n s i n a c o n t i n u o u s c u l t u r e o f E . c o l i B... 36 1 INTRODUCTION B i o l o g i c a l waste t r e a t m e n t systems c o n v e r t o r g a n i c c a r b o n t o c a r b o n d i o x i d e and c e l l c a r b o n ( s l u d g e ) . E f f i c i e n c y , i n b i o l o g i c a l terms, r e f l e c t s t h e a b i l i t y o f b a c t e r i a l c e l l s t o form c e l l c a r b o n , however, i n terms o f waste d i s p o s a l , the p r o d u c t i o n o f c e l l c a r b o n i s n o t d e s i r a b l e - eg. c o n v e r t i n g one form o f waste i n t o a n o t h e r . The o b j e c t i v e o f t h e p r e s e n t work was t o maximize the p r o d u c t i o n o f c a r b o n d i o x i d e a t t h e expense o f c e l l c a r b o n and thus l i m i t the q u a n t i t y o f s l u d g e p r o d u c e d by waste t r e a t m e n t systems. One might d e c r e a s e t h e amount o f c e l l c a r b o n by i n c r e a s i n g t h e maintenance energy r e q u i r e m e n t s o f t h e b a c t e r i a - eg. by growing them under a d v e r s e c o n d i t i o n s ; o r by e f f e c t i n g a p r o c e s s o f energy u n c o u p l i n g -eg. by a c t i v a t i o n o f an ATPase o r by d e c r e a s i n g t h e e f f i c i e n c y o f ATP p r o d u c t i o n . Uncoupled growth, i n the c o n t e x t o f t h i s work, r e f e r s t o a d e c r e a s e i n t h e e f f i c i e n c y o f growth and might n o t d i r e c t l y i n v o l v e t h e p r o c e s s o f o x i d a t i v e p h o s p h o r y l a t i o n . D e c r e a s i n g t h e e f f i c i e n c y o f energy g e n e r a t i o n by i n t r o d u c i n g c h e m i c a l s u n c o u p l e r s i s " p o s s i b l e b u t i m p r a c t i c a l a t t h i s time; t h e use o f 2 , 4 , d i n i t r o p h e n o l (1) p r e s e n t s the d i f f i c u l t y o f c o n t a m i n a t i o n o f t h e e f f l u e n t w i t h a t o x i c c h e m i c a l w h i l e t h e use o f methylene b l u e o r o t h e r a l t e r n a t e e l e c t r o n a c c e p t o r s compounds t h i s d i f f i c u l t y by r e q u i r i n g s t o i c h i o m e t t r i c (thus v a s t ) q u a n t i t i e s . However, w h i l e 2 perhaps n o t p r o v i d i n g a p r a c t i c a l s o l u t i o n , the a b i l i t y o f t h e s e c h e m i c a l s t o d e c r e a s e energy p r o d u c t i o n does emphasize t h a t an im-p o r t a n t c h a r a c t e r i s t i c o f o x i d a t i v e p h o s p h o r y l a t i o n i n b a c t e r i a i s i t s s u s c e p t i b i l i t y t o wide f l u c t u a t i o n s i n e f f i c i e n c y ( 2 , 3 ) . P a s t workers have demonstrated t h i s c h a r a c t e r i s t i c by a v a r i e t y o f t e c h n i q u e s . The p r e s e n c e o f a growth f a c t o r i n l i m i t i n g amounts may cause u n c o u p l e d growth ( 4 ) . Temperatures below and above the optimum f o r growth may r e s u l t i n d e c r e a s e d y i e l d s ( 5 ) . V e r y h i g h o r low growth r a t e s i n c o n t i n u o u s c u l t u r e e f f e c t y i e l d v a l u e s ( 6 ) , low d i l u t i o n r a t e s i n c r e a s e the r e l a t i v e amount o f energy used f o r maintenance, w h i l e h i g h r a t e s may cause "uncoupled" growth perhaps due t o t h e l i m i t i n g c o n c e n t r a t i o n o f some e s s e n t i a l growth f a c t o r ( 4 ) . The p r i n c i p a l weakness o f a d a p t i n g t e c h n i q u e s such as t h e s e t o a waste t r e a t m e n t system i s the f a c t t h a t one i s now d e a l i n g w i t h a d i v e r s i t y o f b a c t e r i a l s p e c i e s . Thus n a t u r a l s e l e c t i o n s h o u l d f a v o u r more e f f i c i e n t s p e c i e s . F o r t h i s r e a s o n , we hoped t o employ a method t h a t would e f f e c t mixed p o p u l a t i o n s o f b a c t e r i a . By v a r y i n g t h e c o n c e n t r a t i o n o f d i s s o l v e d oxygen, marked changes o c c u r i n r e s p i r a t i o n r a t e , c e l l s t r u c t u r e , e n z y m e - c o n s t i t u t i o n , as w e l l as c e l l and p r o d u c t y i e l d ( 7 ) . The most s t r i k i n g e f f e c t s o c c u r i n f a c u l t a t i v e o rganisms. S i n c e our c o n c e r n was p r i n c i p a l l y w i t h a e r o b i c waste t r e a t m e n t systems, we d e c i d e d t o i n v e s t i g a t e t h e a b i l i t y o f o b l i g a t e l y and f a c u l t a t i v e l y a e r o b i c b a c t e r i a t o produce c e l l c a r b o n 3 a t v a r y i n g l e v e l s o f oxygen t e n s i o n . P a s t workers (8) have i n d i c a t e d t h a t t h e r e s p i r a t i o n r a t e i s i ndependent o f d i s s o l v e d oxygen u n t i l a c r i t i c a l l e v e l under 15 mmHg was r e a c h e d . As the oxygen t e n s i o n d e c r e a s e d , i n c r e a s e s i n cytochrome c o n t e n t became a p p a r e n t (9, 1 0 ) . H a r r i s o n and P i r t (5) found i n s t u d i e s w i t h c o n t i n u o u s c u l t u r e s o f A. aerogenes, t h a t a t low oxygen t e n s i o n s o s c i l l a t i o n s i n t h e r e s p i r a t i o n r a t e o c c u r r e d - eg. p e r i o d s o f s t i m u l a t e d r e s p i r a t i o n . D u r i n g t h i s p e r i o d , c e l l y i e l d d e c r e a s e d w h i l e g l u c o s e f e r m e n t a t i o n p r o d u c t s and c a r b o n d i o x i d e i n c r e a s e d . A l t h o u g h the i n d u c t i o n o f r e s p i r a t o r y enzymes might be i n v o l v e d , t h e speed o f r e s p o n s e ( l e s s t h a n 5 minutes) s u g g e s t e d o t h e r w i s e (5, 8 ) . H a r r i s o n and M a i t r a (3) demonstrated t h a t t h i s s t i m u l a t i o n i n t h e r e s p i r a t i o n r a t e was n o t accompanied by l a r g e v a r i a t i o n s i n ATP c o n t e n t and c o n c l u d e d t h a t e i t h e r t h e t u r n o v e r r a t e had i n c r e a s e d o r t h a t t h e P/0 r a t i o had d e c r e a s e d . Degn e t al_. (11) have su g g e s t e d t h a t r e s p i r a t i o n i s b o t h d e r e p r e s s e d and d i f f u s i o n l i m i t e d a t low l e v e l s o f oxygen and t h a t i t i s t h e o p p o s i n g n a t u r e o f t h e s e two f a c t o r s t h a t r e s u l t s i n o s c i l l a t i o n s o f t h e r e s p i r a -t i o n r a t e . Our o b j e c t i v e , t h e r e f o r e , was t o m a i n t a i n a l e v e l o f oxygen low enough t o i n d u c e r e s p i r a t i o n y e t s u f f i c i e n t l y h i g h t o e x c l u d e l i m i t a t i o n by d i f f u s i o n . We hoped t h a t t h i s would a c h i e v e a h i g h l e v e l o f c a r b o n d i o x i d e p r o d u c t i o n a t the expense o f c e l l c a r b o n w i t h o u t c o n c u r r e n t p r o d u c t i o n o f f e r m e n t a t i o n p r o d u c t s . 4 S i n c e oxygen l e v e l s i n t h e o s c i l l a t i n g c u l t u r e s o f H a r r i s o n and P i r t (8) c l o s e l y approached a n a e r o b i c v a l u e s , i t seemed p r o b a b l e t h a t - due t o d e c r e a s i n g amounts o f e l e c t r o n a c c e p t o r s (oxygen) -the c e l l would d e v e l o p a n a e r o b i c pathways. The i n c r e a s e i n fe r m e n t -a t i o n p r o d u c t s s u g g e s t e d t h a t t h i s was, i n f a c t , t h e c a s e . Thus t h e a l t e r n a t i n g m e t a b olism ( a n a e r o b i c v s a e r o b i c ) m i g h t have been a f a c t o r i n the d e c r e a s e d e f f i c i e n c y o f growth. C l a r k and B u r k i (23) ob s e r v e d t h a t growth o f two s t r a i n s o f Pseudomonas and Achromobacter a t oxygen l e v e l s below 4mmHg r e s u l t e d i n a d e c l i n e i n growth r a t e and lower y i e l d s - t h e l a t t e r b e i n g a s s o c i a t e d w i t h t h e i n h i b i t o r y e f f e c t s o f low oxygen t e n s i o n s . Maclennan e_t a l . (24) r e c e n t l y showed t h a t oxygen v a l u e s below 28mmHg produ c e d i n c r e a s e s i n c a r b o n d i o x i d e p r o d u c t i o n d u r i n g t h e growth o f Pseudomonas AMI a t t h e expense o f s u p e r n a t a n t c a r b o n r a t h e r t h a n c e l l c a r b o n - t h e maximum e f f e c t was o n l y a 7% i n c r e a s e i n ca r b o n d i o x i d e p r o d u c t i o n a t 7mmHg. They s u g g e s t e d t h a t t h e e f f i c i e n c y o f o x i d a t i v e p h o s p h o r y l a t i o n was d e c r e a s e d by low l e v e l s o f oxygen. A more p r a c t i c a l t e c h n i q u e i n v o l v i n g oxygen c o n t r o l i n waste t r e a t m e n t systems might be t o s i m p l y a l t e r n a t e between a e r o b i c and a n a e r o b i c p e r i o d s o f growth. There appeared t o be a p o s s i b i l i t y t h a t such a l t e r n a t i o n s m i g h t r e s u l t i n "uncoupled" growth. C a v a r i e t a l . (25) i n d i c a t e d t h a t a n a e r o b i c c e l l s o f E. c o l i , when s w i t c h e d t o a e r o b i c c o n d i t i o n s i n a chemostat, r e a c h t h e i r maximum r e s p i r a t i o n r a t e i n t h e 5 f i r s t hour; while "the effectiveness of energy transformation" 32 (expressed as the amount of P incorporated per atom of oxygen) only reached i t s peak after two to three hours. Harrison and P i r t (8) also noted that i t required ten or more generations for anaerobically grown A. aerogenes to adapt to aerobic conditions. An i n i t i a l aim of this investigation was to construct a respiro-meter which would continuously record respiration rates while the oxygen tension i n the chemostat was maintained at precise levels. A respirometer of this type would also have practical applications in waste treatment systems. Patterson et al_. (17) have summarized the inadequacies of present methods i n obtaining biological data - such as Chemical Oxygen Demand (COD) and Biological Oxygen Demand (BOD). The major problem i s the time lag between collection and completion of these tests - 2 hours for COD's and 5 days for BOD's. Because of this time lag, these parameters are only useful for monitoring the results of a treatment operation and cannot be applied to automatically control the biolo-gical activity of the system. In contrast, a continuous measure of respiration rate with a lag of only a minute or two provides a direct indication of the rate of substrate removal and could be coupled to a system to regulate sub-strate inflow or dissolved oxygen concentration so that a maximal rate i s maintained. In addition, the measurement could be used to 6 a c t i v a t e an a l a r m i f a c t i v i t y dropped because o f wash-out o r t h e i n t r o d u c t i o n o f a t o x i c c h e m i c a l w i t h t h e waste. We a l s o p r o p o s e d t o st u d y t h e e f f e c t s o f u l t r a - v i o l e t i r r a d i a t i o n on growth i n a chemostat i n a f u r t h e r attempt t o d e v e l o p a p r a c t i c a l method o f d e c r e a s i n g y i e l d s i n waste t r e a t m e n t systems. Exposure o f b a c t e r i a t o h a r m f u l agents (such as u.v.) might e f f e c t i v e l y c o u n t e r -a c t t h e d i f f i c u l t i e s o f n a t u r a l s e l e c t i o n by i n c r e a s i n g t h e maintenance energy o f t h e c e l l s . I t has been assumed (12) t h a t t h e major e f f e c t s o f u.v. l i g h t a r e t o pro d u c e p y r i m i d i n e d i m e r s i n t h e DNA m o l e c u l e ; however, o t h e r l e s s w e l l known phenomena - such as u r a c i l dimers o r DNA and RNA c r o s s -l i n k a g e s w i t h p r o t e i n - may be o f s i g n i f i c a n c e . P a s t r e s e a r c h has i n d i c a t e d t h a t , i n c e l l f r e e e x t r a c t s , u.v. i r r a d i a t i o n d e s t r o y s p h o s p h o r y l a t i o n (13) and s p e c i f i c a l l y , p a r t i a l l y d e s t r o y s an i s o l a t e d c o u p l i n g f a c t o r ( 14). To our knowledge, t h e r e has been no p r e v i o u s attempt t o r e l a t e t h e e f f e c t o f u.v. l i g h t on t h e e f f i c i e n c y o f growth i n a chemostat. 7 MATERIALS AND METHODS A s t r a i n o f E . c o l i B_ was o b t a i n e d from t h e l a b o r a t o r y o f Dr. R.A.J. Warren (Department o f M i c r o b i o l o g y , U n i v e r s i t y o f B.C.); Pseudomonas a e r u g i n o s a 9027 was o b t a i n e d from t h e l a b o r a t o r y o f Dr. J.J.R. Campbell (Department o f M i c r o b i o l o g y , U n i v e r s i t y o f B.C.). Both s t r a i n s were m a i n t a i n e d on g l u c o s e m i n i m a l media (see media) a t r e f r i g e r a t i o n t e m p e r a t u r e s . CULTURE TECHNIQUE FOR OXYGEN CONTROLLED EXPERIMENTS: A 100 ml w a t e r - j a c k e t e d f l a s k was employed t o r b o t h c o n t i n u o u s and b a t c h c u l t u r e s . A temperature r e g u l a t e d water pump m a i n t a i n e d the temperature a t 37 C ±1 C. F o r t h e c o n t i n u o u s c u l t u r e , a p e r i s t a l t i c pump f o r c e d s t e r i l e media i n a t a r a t e o f 1.0 ml/min - d i l u t i o n r a t e o f .6. E f f l u e n t was f o r c e d o u t by t h e p r e s e n c e o f t h e gases p a s s e d i n t h r o u g h a s i n t e r e d g l a s s s p a r g e r (oxygen and n i t r o g e n o r a i r ) . Samples were c o l l e c t e d by p u s h i n g t h e g l a s s o u t l e t s l i g h t l y f a r t h e r i n t o t h e v e s s e l t o q u i c k l y f o r c e o u t a 5 - 10 ml volume. Pseudomonas a e r u g i n o s a 9027 was grown i n b a t c h c u l t u r e s s i n c e i t had a tendency t o l y s e i n c o n t i n u o u s c u l t u r e s ; E s c h e r i c h i a c o l i B_ was always grown i n c o n t i n u o u s c u l t u r e . The g l u c o s e l i m i t e d medium c o n s i s t e d o f : 8 . 5 g / 1 2 . 0 g / 1 -.2 g / 1 1 . 5 mg/1 3 . 6 g / 1 1 . 4 g / 1 The oxygen e l e c t r o d e and c u l t u r e v e s s e l were a u t o c l a v e d f o r 1 5 minutes a t 1 5 p s i g . The a p p a r a t u s was c o o l e d , and medium was added a s e p t i c a l l y and t h e v e s s e l was m a i n t a i n e d a t 3 7 C f o r a t l e a s t 2 4 hours t o a l l o w t h e e l e c t r o d e t o s t a b i l i z e i n a i r s a t u r a t e d media. A magnetic s t i r r e r , r o t a t e d a t a c o n s t a n t speed, was used f o r m i x i n g . DISSOLVED OXYGEN PARTIAL PRESSURE: (pC>2) The oxygen e l e c t r o d e responds t o t h e d i s s o l v e d oxygen p a r t i a l p r e s s u r e (pC^ oxygen t e n s i o n ) r a t h e r t h a n c o n c e n t r a t i o n . The oxygen t e n s i o n (T) and t h e c o n c e n t r a t i o n (C) a r e r e l a t e d by t h e e x p r e s s i o n T = FC where F i s an a c t i v i t y c o e f f i c i e n t which i s c o n s t a n t f o r a g i v e n s e t o f e x p e r i m e n t a l c o n d i t i o n s . Changes i n oxygen t e n s i o n ( e x p r e s s e d as mmHg) can be r e l a t e d d i r e c t l y t o changes i n oxygen c o n c e n t r a t i o n . g l u c o s e ammonium s u l p h a t e - ( N H 4 ) 2 S 0 4 Magnesium s u l p h a t e - MgSO^^I^O F e r r o u s s u l p h a t e - FeSO^ Pot a s s i u m phosphate ( D i b a s i c ) K^HPO^ Po t a s s i u m phosphate (Monobasic) KH P0 2 ' P H 7 . 0 ± . 1 OXYGEN TENSION CONTROL SYSTEM: The system f o r c o n t r o l l i n g t h e p0 used steam s t e r i l i z a b l e oxygen 9 e l e c t o d e (15). D i s s o l v e d oxygen p a r t i a l p r e s s u r e (pC^ i n mmHg) i s c o n t i n u o u s l y m o n i t e r e d by an oxygen e l e c t r o d e c o u p l e d t o a 10 mv p r o p -o r t i o n a l c o n t r o l l e r . The oxygen e l e c t r o d e s i g n a l i s a p p l i e d t h r o u g h a v a r i a b l e g a i n de e m p l i f i e r which a l l o w s s c a l e e x p a n s i o n f o r o p e r a t i o n s a t low pO^. When the e l e c t r o d e senses an oxygen d e f i c i t i n t h e c u l t u r e v e s s e l , t h e c o n t r o l l e r a c t i v a t e s a s o l e n o i d v a l v e i n t h e oxygen.supply l i n e . Oxygen i s the n r e l e a s e d i n t o t h e c o n t i n u o u s f l o w o f n i t r o g e n gas e n t e r i n g i n t o t h e c u l t u r e t h r o u g h a s i n t e r e d g l a s s s p a r g e r . T o t a l f l o w approximated 100 - 150 mls/min. ASSAY FOR CYTOCHROME B: Cytochrome B was a s s a y e d on whole c e l l s u s p e n s i o n s o f E_. c o l i B_ (O.D. between 5 and 18) s p e c t r o p h o t o m e t r i c a l l y i n open c u v e t t e s u s i n g a C a r y 14 r e c o r d i n g s p e c t r o p h o t o m e t e r . A d i f f e r e n c e spectrum between r e d u c e d (.05 mis o f 25% g l u c o s e ) and o x i d i z e d (.05 mis o f 3% hydrogen p e r o x i d e ) was o b t a i n e d u s i n g 2.3 ml samples. G e n e r a l l y , t h e b e s t r e -s u l t s were o b t a i n e d f i f t e e n minutes a f t e r t h e a d d i t i o n o f t h e g l u c o s e and hydrogen p e r o x i d e . CELL SIZE: Samples from t h e chemostat (E_. c o l i B_ c e l l s ) were p l a c e d i n a 1% f o r m a l i n s o l u t i o n and a n a l y z e d i n a m o d i f i e d c o u l t e r c o u n t e r ( 1 6 ) . 10 GROWTH OF MIXED CULTURES AT LOW OXYGEN TENSIONS; A stock culture of bacteria from an inoculum of s o i l and sewage samples was used for a l l experiments with mixed populations. The medium was the same as the glucose limited medium used previously except that the glucose was replaced by skim milk (.5 g/1) and bactotryptone (.5 g/1). The total organic carbon concentration was 400 ppm/ml. Temperature was maintained at 37 C ± 1 C; pH 7.0 ±.1. In order to study the effect of low oxygen tension on the decrease in c e l l weight of endogenously respiring floculent cultures, floculent cells were centrifuged and washed; tnen resuspended i n tap water and maintained at a p0 2 of either 8mmHg or 144mmHg. Five ml samples were taken every hour and the decrease i n weight recorded. After 5 hours, 5 one ml samples from both flasks were placed in Warburg flasks. The oxygen uptake rate was recorded over one hour and at various inter-vals, the contents of individual flasks were f i l t e r e d for analysis of supernatant carbon. USE OF WARBURG APPARATUS: Two mis of glucose limited medium were placed i n each flask and one ml of the floculant culture placed in the side-arm. Standard mono-metric methods were used. Due to the d i f f i c u l t y i n obtaining precise 11 d a t a w i t h f l o c u l a n t c u l t u r e s , t h e oxygen uptake r a t e was c a l c u l a t e d o v e r a p e r i o d o f one hour. ALTERNATIONS BETWEEN ANAEROBIC AND AEROBIC GROWTH: A c o n t i n u o u s c u l t u r e was spa r g e d w i t h n i t r o g e n and i n t e r m i t t e n t l y s p a r g e d w i t h a i r from an e l e c t r i c pump c o n t r o l l e d by a s w i t c h i n g p r o -grammer. The same g l u c o s e medium was used as i n p r e v i o u s experiments and t h e d i l u t i o n r a t e was m a i n t a i n e d a t .6 h r \ An oxygen e l e c t r o d e r e c o r d e d t h e oxygen t e n s i o n t o ensure t h a t a n a e r o b i c l e v e l s were r e a c h e d . The l e n g t h o f time t h a t t h e c u l t u r e was k e p t e i t h e r a e r o b i c o r anae-r o b i c v a r i e d from 1 - 1 5 m i n u t e s . CULTURE TECHNIQUE FOR ULTRA-VIOLET IRRADIATION: The work w i t h an u.v. i r r a d i a n t c u l t u r e o f E_. c o l i B_ was done i n a c i r c u l a r p l e x i - g l a s s v e s s e l (40 mm by 70 mm) h a v i n g a t o t a l volume o f 60 m i s . A f l a t p i e c e o f q u a r t z (15 mm x 5 mm) from a c u v e t t e was f i t t e d i n t o a "window" on t h e s i d e o f t h e v e s s e l t o a l l o w u.v. l i g h t t o e n t e r u n h i n d e r e d . Media e n t e r e d v i a a p e r i s t a l t i c pump (.4 ml/min) and was f o r c e d o u t a t t h e 40 mis l e v e l by t h e a i r p r e s s u r e p r o d u c e d by s p a r g i n g s t e r i l e a i r t h r o u g h a s i l i c o n e t u b i n g i n t o the v e s s e l . The d i l u t i o n r a t e was m a i n t a i n e d a t .6 h r 1 w i t h g l u c o s e m i n i m a l medium used p r e v i o u s l y (pH 7.0 ± . 1 ) . The e n t i r e a p p a r a t u s was l o c a t e d i n a c l o s e d c o n t a i n e r and temperature was c o n t r o l l e d a t 30 C ± 1 C w i t h a 12 p r o p o r t i o n a l temperature c o n t r o l l e r . A Beckman q u a r t z (model DU) s p e c t r o p h o t o m e t e r was used as a u.v. s o u r c e . F o r one s e r i e s o f ex p e r i m e n t s , the q u a r t z window was p o s i t i o n e d a d j a c e n t t o t h e sp e c t r o p h o t o m e t e r and i n t h i s way the c u l t u r e c o u l d be exposed t o l i g h t o f a s p e c i f i c wave l e n g t h . I n subsequent experiments the u.v. lamp from t h e s p e c t r o p h o t o m e t e r was p l a c e d i n f r o n t o f t h e q u a r t z window and t h e dose l e v e l was v a r i e d by v a r y i n g t h e d i s t a n c e between t h e u.v. lamp and t h e window. CALCULATION OF U.V. IRRADIATION DOSE LEVELS: A photometer c a l i b r a t e d i n f o o t - c a n d l e s was used t o measure t h e i n t e n s i t y o f l i g h t r e c e i v e d by t h e s u r f a c e o f t h e q u a r t z window (ar e a = 75 mm). A f i l t e r a l l o w e d o n l y l i g h t below a wave l e n g t h o f 3240°A t o be measured. 2 C o n v e r s i o n o f f o o t - c a n d l e s ( f t - c ) t o ergs/mm / s e c . 1 f t - c = 10.76 lumens/sq. meter 1 lumen = .0015 watt s Thus 1 f t - c = 10.75 x .0015 = .016 w a t t s / s q . meter 5 = 1.6 x 10 e r g s / s q . meter/sec 2 = .16 ergs/mm / s e c I n t e n s i t y o f u.v. l i g h t below 3240°A a t a d i s t a n c e o f 18 mm. I n t e n s i t y w i t h f i l t e r = 24 f t - c I n t e n s i t y w i t h u.v. lamp o f f = 14 f t - c (eg. background) I n t e n s i t y o f u.v. below 3240 = 10 f t - c 2 10 x .16 ergs'/mm / s e c 2 1.6 ergs/mm / s e c C a l c u l a t i o n o f dose l e v e l . Dose l e v e l s o f u.v. l i g h t were o b t a i n e d by v a r y i n g t h e d i s -t a n c e between t h e u.v. lamp and t h e "window" and u s i n g t h e f o r m u l a h = ( d 2 ) 2 2 2 where I i s the i n t e n s i t y i n ergs/mm / s e c , i 2 «v and d i s t h e d i s t a n c e t o mm. 2 D i s t a n c e (mm) I n t e n s i t y (ergs/mm /sec) 40 .3 25 .8 18 1.6 12 3.7 3 60.0 NOTE; a r e a o f q u a r t z window = 75 mm CALCULATION OF CARBON BALANCES: C e l l Carbon: F i v e ml samples were f i l t e r e d t h r o u g h two m i l l i p o r e f i l t e r s i n s e r i e s . The second f i l t e r s e r v e d as a c o n t r o l f o r w e i g h t g a i n due t o salts from the medium. Both f i l t e r s were incubated at 110 C for at least two hours and then cooled for one half hour. The weight of the bottom f i l t e r was subtracted from the weight of the top f i l t e r to give the dry weight of the cells per 5 ml. Normal values for an aerobic continuous culture of E_. c o l i B were: top f i l t e r (14 mg) minus bottom f i l t e r (2 mg) = 12 mg/5 mis = .24 mg/ml. Cell carbon was calculated with the assumption that 50% of the cell ' s dry weight i s carbon. Thus .24 mg/ml =120 ppm/ml carbon. Supernatant Carbon: The f i l t r a t e from the dry weight samples was analyzed by a Beckman Total Carbon Analyzer. A 20 Ul sample was oxidized at a high tempera-ture (950 C) for total carbon analysis. The carbon dioxide evolved was analyzed i n a spectrophotometer using infra-red light. Inorganic carbon was oxidized at a lower temperature (150 C). The figure for inorganic carbon (4 - 6 ppm/ml) was subtracted from the total carbon to give total organic carbon (supernatant carbon). Standardization was done with glucose carbon in dilutions of 25, 50 and 100 ppm/ml. Carbon Dioxide: Values for c e l l carbon and supernatant carbon (ppm/ml) were sub-tracted from the i n i t i a l substrate carbon to calculate the amount of carbon given off as carbon dioxide (ppm/ml). RESULTS RESPIROMETER: T h i s a p p a r a t u s ( F i g u r e 1) was c o n c e i v e d w i t h a view towards p r a c t i c a l a p p l i c a t i o n i n m o n i t o r i n g t h e o p e r a t i o n o f a c t i v a t e d s l u d g e p l a n t s ; b u t p r o v e d t o be r a t h e r complex and d i f f i c u l t t o o p e r a t e . A major problem w i t h t h e a p p a r a t u s was t h e a c c u m u l a t i o n o f b a c t e r i a l c e l l s on t h e membrane o f t h e oxygen e l e c t r o d e , p r o h i b i t i n g p r o l o n g e d usage. The n e c e s s i t y o f c o m p l e t e l y s a t u r a t i n g t h e c u l t u r e i n t h e r e - a e r a t i o n chamber i n t r o d u c e d f u r t h e r c o m p l i c a t i o n s . T h i s problem c o u l d be e l i m i n a t e d by u s i n g two e l e c t r o d e s i n s e r i e s so t h a t a change i n pO^ c o u l d be r e c o r d e d a t any i n i t i a l l e v e l o f oxygen t e n s i o n . F u r t h e r m o r e , w i t h mixed c u l t u r e s , w i t h o u t the need f o r a s t e r i l e system, i t s h o u l d be p o s s i b l e t o s i m p l y p l a c e t h e e l e c t r o d e i n an open r e c e p t a c l e t o p r e v e n t the b u i l d - u p o f d e p o s i t s . OSCILLATIONS: When t h e p 0 2 i n t h e chemostat f e l l below 15mmHg, non-uniform o s c i l l a t i o n s i n r e s p i r a t i o n were o b s e r v e d ( F i g u r e 2 ) . S i m i l a r t o H a r r i s o n ' s (8) e s t i m a t e o f a 33% i n c r e a s e i n t h e r e s p i r a t i o n r a t e , d i f f i c u l t i e s i n h e r e n t i n t h e a p p a r a t u s l i m i t e d i t s u s e f u l n e s s i n d e t e r m i n i n g i f , a t a s p e c i f i c pC^f a s t a b l e s t i m u l a t e d r a t e o f r e -s p i r a t i o n c o u l d be a c h i e v e d and t h i s q u e s t i o n remains u n r e s o l v e d . C U L T U R E R E T U R N E D TO C H E M 0 8 T A T © X Y G E N E L E C T R O D E 6T D I S T A N C E O U T - F L O W F i g u r e 1. The R e s p i r o m e t e r . SAMPLE P U M g £ 0 CONT5& UOUS.LY F R O M CHE&IOSTAT IN S T I A L R E - A E R A T I Z7 A I R V5T ^ t i j ^ - D E F E C T OR ^-RE~&E§*AT1 ^ ^ . S P A R G E R 1 M A G N E T & C ST IHRE R OXYGEN TENS 8 ON ,. % o f 1 ramHg SAT 860 100 144 90 • 128. 8 0 -1 112 70 -96 8 0 60 • 5 0 -64 40 " 30 20 • JO • OXYGEN TENSION IN CHEMOSTAT OXYGEW TENSJOft IN RES PIR 0JV3ETE R F i g u r e 2. u " u—— i r 2 3 4 5 T r s 1 II 1 0 n i a 8 9 10 Bi 12 13 !4 15 16 6 7 T I M E ( H r . ) Data from r e s p i r o m e t e r showing o s c i l l a t i o n s i n r e s p i r a t i o n o f a c o n t i n u o u s c u l t u r e o f E_. c o l i B i n g l u c o s e m i n i m a l medium. Temperature 37 C, pH 7.0. ? oxygen t e n s i o n i n c u l t u r e v e s s e l ; oxygen t e n s i o n i n t h e r e s p i r o m e t e r 18 The n o n - u n i f o r m i t y o f t h e o s c i l l a t i o n s a g r e e s w i t h H a r r i s o n ' s (5) r e s u l t s on a g l u c o s e l i m i t e d c u l t u r e . GROWTH OF E. COLI B AT LOW OXYGEN TENSIONS: H a r r i s o n and P i r t (8) demonstrated an a p p a r e n t i n c r e a s e i n c a r b o n d i o x i d e p r o d u c t i o n and d e c r e a s e i n c e l l c a r b o n when A. aerogenes was grown a t low oxygen t e n s i o n s ; however, s i n c e t h e c e l l s were grown i n exc e s s g l u c o s e (amount u n s p e c i f i e d ) and t h e amount o f s u p e r n a t a n t c a r b o n was not r e c o r d e d , i t was n o t p o s s i b l e t o determine and q u a n t i t a t e t h e a p p a r e n t i n c r e a s e i n s u b s t r a t e c a r b o n t h a t was c o n v e r t e d t o c a r b o n d i o x i d e a t t h e s e low l e v e l s o f oxygen. Hence, t h e i r s u g g e s t i o n t h a t c u l t i v a t i o n a t low p 0 2 caused u n c o u p l e d growth was n o t r e a l l y s u b s t a n -t i a t e d . The p r e s e n t work, un d e r t a k e n t o i n v e s t i g a t e t h i s phenomenon, i s summarized i n T a b l e I . I n c o n t i n u o u s c u l t u r e s w i t h g l u c o s e as t h e l i m i t i n g s u b s t r a t e , c o n t r o l l i n g t h e oxygen t e n s i o n between 3 - 8mmHg r e s u l t e d i n a 20% d e c r e a s e i n c e l l d r y weight from a e r o b i c l e v e l s w h i l e t h e amount o f s u b s t r a t e c a r b o n c o n v e r t e d t o c a r b o n d i o x i d e i n c r e a s e d by 57%, eg. from 21% t o 33%. C o n c o m i t a n t l y , t h e s u p e r n a t a n t c a r b o n d e c r e a s e d from an average o f 43 t o 33 ppm/ml. When t h e oxygen t e n s i o n was c o n t r o l l e d below 3mmHg, a n o t i c e a b l e i n c r e a s e i n s u p e r n a t a n t c a r b o n , eg. f e r m e n t a t i o n p r o d u c t s , was e v i d e n t . When g l y c e r o l was t h e l i m i t i n g s u b s t r a t e , t h e r e was no change i n ca r b o n d i o x i d e p r o d u c t i o n and c e l l c a r b o n from t h o s e v a l u e s o b t a i n e d d u r i n g growth i n ex c e s s oxygen (Ta b l e I I ) . 19 T a b l e I E f f e c t o f oxygen i t e n s i o n on c a r b o n d i o x i d e p r o d u c t i o n i n E . c o l i B_ i n c o n t i n u o u s c u l t u r e ; l i m i t i n g s u b s t r a t e - g l u c o s e - 2 0 0 ppm/ml carbon. . Temperature 37 C :, pH 7.0. No. o f oxygen d r y wgt. c e l l c a r b o n s u p e r n a t a n t c a r b o n d i o x i d e e v o l v e d e x p t s . t e n s i o n mg/ml ppm/ml ca r b o n ppm/ml ppm/ml % o f s u b s t r a t e mmHg .24 120 42 .24 120 52 6 144 .22 110 38 42 ± 7.7 21 .22 110 44 .23 115 38 .23 115 45 2 0 .08 40 132 25 ± 3.0 13 .06 30 148 2 3 .16 80 60 59 ± 1.0 30 .14 70 72 .20 100 38 .22 110 39 5 3-8 .22 110 38 65 ± 1 2 . 1 33 .16 80 42 .18 90 28 20 Table II. Effect of oxygen tension on carbon dioxide production of E_. c o l i B_ i n continuous cultures; limiting substrate-glycerol-200 ppm/ml carbon. Temperature 37 C, pH 7.0. No. of oxygen dry wgt. c e l l carbon supernatant carbon dioxide evolved expts. tension mg/ml ppm/ml carbon ppm/ml ppm/ml % of substrate mmHg 148 .28 .28 .27 .27 140 140 135 135 28 20 20 20 42 ± 5.4 22 3-8 .27 .26 .26 .25 135 130 130 125 24 44 31 31 43 ± 4.8 22 GROWTH OF PSEUDOMONAS AERUGINOSA 9027 AT LOW OXYGEN TENSIONS: Oxygen t e n s i o n s below 120 mmHg (75% o f s a t u r a t i o n ) r e s u l t e d i n a l a g o f g r e a t e r t h a n 6 hours i n the growth o f Pseudomonas a e r u g i n o s a 9027 i n b a t c h c u l t u r e s when the s t a r t i n g c u l t u r e O.D. was l e s s t h a n .04 ( F i g u r e 3 ) . Because o f t h i s l a g , the e x t e n t o f which was n o t det e r m i n e d , a l l b a t c h c u l t u r e s o f P_. a e r u g i n o s a 9027 were grown up t o an O.D. o f .07 i n f u l l y a e r a t e d medium b e f o r e the oxygen t e n s i o n was c o n t r o l l e d a t lower l e v e l s . The s p e c i f i c growth r a t e i n c u l t u r e s i n which oxygen t e n s i o n s were between 144 and 160mmHg was .86 h r ^; however, i n c u l t u r e s grown a t l e s s t h a n 16mmHg, i t d e c l i n e d t o .46 h r ^ ( F i g u r e 4 ) . The d a t a f o r c a r b o n b a l a n c e s r e v e a l e d t h a t , a t a low p 0 2 , t h e amount o f c a r b o n d i o x i d e e v o l v e d was i n c r e a s e d a t t h e expense o f c e l l c a r b o n ( T a b l e I I I ) . The v a r i a b l e f i g u r e s f o r s u p e r n a t a n t c a r b o n a r e the r e s u l t o f h a r v e s t i n g a t s l i g h t l y d i f f e r e n t s t a g e s i n t h e growth c u r v e , and a r e t h e r e f o r e , s i g n i f i c a n t o n l y when c o n s i d e r e d w i t h t h e v a l u e s f o r c e l l c a r b o n . When the oxygen t e n s i o n was g r e a t e r t h a n 144mmHg, 14% o f s u b s t r a t e c a r b o n was c o n v e r t e d t o c a r b o n d i o x i d e . T h i s was i n c r e a s e d t o 27% f o r v a l u e s between 8 and 16mmHg and t o 37% between 3 and 8mmHg. EFFECT OF LOW OXYGEN TENSIONS ON CYTOCHROME B: I n a c o n t i n u o u s c u l t u r e o f E_. c o l i B_, t h e c o n c e n t r a t i o n o f c y t o -chrome B i n c r e a s e d a t l e v e l s o f oxygen t e n s i o n below a p p r o x i m a t e l y 22 F i g u r e 3. E f f e c t o f oxygen t e n s i o n s on growtn l a g o f Pseudomonas a e r u g i n o s a 9027 i n b a t c h c u l t u r e ; g l u c o s e m i n i m a l medium; Temperature 37 C, pH 7.0. Note: O.D. o f s t a r t i n g c u l t u r e i s l e s s t h a n .04. 23 OXYGEN IN E X C E S S ^ s X ^ P E R I ©.B O F C O N T R O L L E D Q X Y G E f c S <©0.3 H <0 025^ -0.2 - 1 to QO.15-1 <OJ A o a. GREATER THAN ) L E S S T H A N 3 6 • m SR M q 2 3 T I M E ( Hr.) F i g u r e 4. E f f e c t o f oxygen t e n s i o n on growth r a t e o f Pseudomonas  a e r u g i n o s a 9027. 24 T a b l e I I I . E f f e c t o f oxygen t e n s i o n on c a r b o n d i o x i d e p r o d u c t i o n i n Pseudomonas a e r u g i n o s a 9027 i n b a t c h c u l t u r e ; l i m i t i n g sub-s t r a t e - g l u c o s e - 2 0 0 ppm/ml c a r b o n . Temperature 37 C, pH 7.0. No. o f e x p t s . oxygen t e n s i o n mmHg d r y wgt. c e l l c a r b o n s u p e r n a t a n t c a r b o n d i o x i d e e v o l v e d mg/ml ppm/ml ca r b o n ppm/ml ppm/ml % o f s u b s t r a t e 144 .24 .18 120 120 90 58 48 80 28 ± 3.9 14 8-16 .17 .14 85 70 66 74 63 ± 4.0 27 .18 90 .17 85 31 44 75 ± 4.0 37 Oxygen t e n s i o n was c o n t r o l l e d w i t h oxygen and n i t r o g e n r a t h e r than s p a r g i n g w i t h a i r as were t h e o t h e r c u l t u r e s a t a p 0 2 o f 144 mmHg. 25 llmitiHg ( F i g u r e 5) . I t appeared t h a t cytochrome B d i d n o t l i m i t r e -s p i r a t i o n , eg. d i d n o t i n c r e a s e p r o p o r t i o n a l l y w i t h an i n c r e a s e i n r e s p i r a t i o n , t h u s t h e r e was l i t t l e j u s t i f i c a t i o n f o r c o n t i n u e d s t u d y . EFFECT OF LOW OXYGEN TENSIONS ON CELL SIZE; When E_. c o l i B_ c e l l s were i n j e c t e d i n t o t h e c o u l t e r c o u n t e r , i t became a p p a r e n t t h a t an approximate 20% r e d u c t i o n i n s i z e o c c u r r e d when t h e oxygen t e n s i o n was r e d u c e d i n a c o n t i n u o u s c u l t u r e from 144mmHg t o between 3 and 8mmHg (Tabl e I V ) . T h i s d a t a was c o n f i r m e d by t h e changing r e l a t i o n s h i p between O.D. and d r y w e i g h t s (mg/ml) under t h e s e d i f f e r e n t l e v e l s o f oxygen t e n s i o n ( F i g u r e 6 ) . F o r an e q u i v a l e n t O.D. the d r y w e i g h t o f c e l l s grown a t low p 0 2 was l e s s t h a n t h a t o f c e l l s grown w i t h e x c e s s oxygen, i n d i c a t i n g t h a t , a t low oxygen t e n s i o n , t h e r a t i o o f s u r f a c e a r e a t o c e l l w e i g h t had i n c r e a s e d , eg. c e l l s i z e had d e c r e a s e d . EFFECT OF LOW OXYGEN TENSIONS ON THE GROWTH OF MIXED POPULATIONS: When c o n t i n u o u s c u l t u r e s were used, c o n s t a n t d r y wei g h t s o f b a c t e r i a were d i f f i c u l t t o a t t a i n , s i n c e f l o c u l a t i o n p r o d u c e d an o s c i l l a t i n g p a t t e r n o f growth as t h e c e l l s formed i n t o f l o e s , adhered t o t h e g l a s s s u r f a c e o f t h e v e s s e l and t h e n s l o u g h e d o f f . I t i s p r o b a b l e t h a t many v i a b l e c e l l s a r e l o c a t e d i n s i d e t h e f l o e s and t h e r e f o r e a r e p r o b a b l y n ot a f f e c t e d i n a d i r e c t way by c o n t r o l o f 26 «= (A! ^ 8 - 11 mm H q 44 mm H< 580 §70 960 530 540 W A V E L E N 6 T H (mu ) F i g u r e 5. E f f e c t o f oxygen t e n s i o n on l e v e l o f cytochrome B. E_. c o l i B i n c o n t i n u o u s c u l t u r e ; g l u c o s e l i m i t e d - 2 0 0 ppm ca r b o n ; Temperatue, 37 C; pH 7.0; D i l u t i o n r a t e , .6 h r " 1 . 27 Table IV. Effect of oxygen tension on c e l l size; E_. c o l i B i n continuous culture; glucose limited-200 ppm/ml carbon; temperature 37 C, pH 7.0; dilution rate .6 hr ^. * Relative c e l l size Experiment 144mmHg 8mmHg 162 130 130 100 * See methods. 28 o o M D R Y W E I G H T (M.G. / 2 0 M.L.) F i g u r e 6. K f f e c t o f oxygen t e n s i o n on t h e r e l a t i o n s h i p between O.D. and c e l l mass (mg/ml); Temperature 37 C, pH 7.0; g l u c o s e m i n i m a l medium (.1%). E . c o l i B i n c o n t i n u o u s c u l t u r e ; d i l u t i o n r a t e , .6 h r \ 29 t h e oxygen t e n s i o n . B a t c h c u l t u r e s r e v e a l e d t h a t i n c r e a s e s i n c a r b o n d i o x i d e p r o d u c t i o n d u r i n g growth a t low oxygen t e n s i o n s (8 - 16mmHg) c o u l d be a c h i e v e d , however, t h e s e i n c r e a s e s were n e i t h e r l a r g e n o r c o n s i s t e n t ( T a b l e V ). S i n c e c o n t r o l l i n g t h e l e v e l o f oxygen i n waste t r e a t m e n t systems c o n t a i n i n g huge volumes would be r a t h e r e x p e n s i v e , i t was hoped t h a t by c o n t r o l l i n g t h e oxygen t e n s i o n s o f r e c y c l i n g s l u d g e , one might l e s s e n t h i s problem s i n c e t h e c e l l s i n a f l o c u l a n t s t a t e a r e much more c o n c e n t r a t e d . To s t u d y t h i s p o s s i b i l i t y , f l o c u l a n t endogenously r e s p i r i n g c u l t u r e s were c o n t r o l l e d a t low oxygen t e n s i o n s ( T a b l e VI ) . However, t h e f l o e s c o n t r o l l e d a t 8mmHg showed o n l y a s l i g h t l y g r e a t e r d e c r e a s e i n d r y we i g h t o v e r a 5 t o 9 hour p e r i o d , as opposed t o f l o e s c o n t r o l l e d a t 144mmHg. A l s o , when r e - a e r a t e d , t h e f l o e s c o n t r o l l e d a t 8mmHg d i d n o t show any i n c r e a s e i n r e s p i r a t i o n o r c a r b o n u t i l i z a -t i o n from t h o s e c o n t r o l l e d a t h i g h l e v e l s o f oxygen ( T a b l e V I I ) . Thus t h e r e i s no j u s t i f i c a t i o n f o r c o n t r o l l i n g s l u d g e a t low oxygen t e n s i o n s and r e c y c l i n g i t back i n t o a waste t r e a t m e n t system. EFFECT OF ALTERNATING BETWEEN AEROBIC AND ANAEROBIC GROWTH; A s i m p l e t e c h n i q u e o f a l t e r n a t i n g between a e r o b i c and a n a e r o b i c growth m i g h t l e a d t o d e c r e a s e d y i e l d s i n waste t r e a t m e n t systems. When a s t a b l e a n a e r o b i c c o n t i n u o u s c u l t u r e o f E. c o l i B_ was su d d e n l y s p a r g e d w i t h a i r , "uncoupled" growth r e s u l t e d f o r a p p r o x i m a t e l y 90 minutes a f t e r the i n t r o d u c t i o n o f t h e a i r ( T a b l e VIII),. A f t e r 30 30 T a b l e V. E f f e c t o f low oxygen t e n s i o n s on t h e growth o f mixed p o p u l a t i o n s i n b a t c h c u l t u r e . No. o f oxygen d r y wgt. c e l l c a r b o n s u p e r n a t a n t c a r b o n d i o x i d e e v o l v e d e x p t . t e n s i o n mg/ml ppm/ml c a r b o n ppm/ml ppm/ml % o f s u b s t r a t e mmHg 1 ex c e s s .4 200 97 103 52 8 - 1 5 -4 200 78 122 61 2 e x c e s s .25 125 195 80 40 8 - 15 .27 135 180 85 43 3 excess .36 180 115 105 53 8 - 1 5 .3 150 98 152 76 4 e x c e s s .32 160 136 104 52 8 - 15 .31 155 136 107 54 N.B. A s t o c k c u l t u r e from an inoculum o f s o i l b a c t e r i a was used i n a l l e x p e r i m e n t s . The c e l l s were h a r v e s t e d i n l o g phase. The medium was the same as i n o t h e r experiments (see Methods) e x c e p t t h a t g l u c o s e was r e p l a c e d by skim m i l k (.5 g/1) and B a c t o Tr-yptone (.5 g / 1 ) -c a r b o n (400 ppm/ml). Temperature 37 C, pH 7.0. 31 T a b l e V I . Endogenous r e s p i r a t i o n o f f l o c u l a n t c u l t u r e s showing t h e d e c r e a s e i n c e l l mass o v e r time a t pO o f 8mmHg and 144mmHg. Dry Weights mg/ml Experiment #1 Expe r i m e n t #2 8mmHg l44mmHg 8mmHg 144mmHg 0 3.05 3.05 3.05 3.05 1 2.9 3.05 2 - 2.95 3.5 2.85 - 2.9 2.9 5.5 2.55 ± .092 .83 ± .22 2.4 2.6 9 - - 2.2 ± .03 2.5 ± .17 Time (hrs) 32 T a b l e V I I . R e s p i r a t i o n r a t e and c a r b o n uptake r a t e o f f l o c e s m a i n t a i n e d p r e v i o u s l y f o r 5.5 hours a t (a) 8mmHg and (b) 144mmHg. (a) (b) Q0 2 (Ul/mg/hr) 1860 1980 c a r b o n uptake (ppm/mg/hr) 125 123 33 T a b l e V I I I . The e f f e c t o f a e r a t i n g an a n a e r o b i c c o n t i n u o u s c u l t u r e o f E. c o l i B. Oxygen t e n s i o n time (min) c e l l c a r b o n s u p e r n a t a n t c ar bo n d i o x i d e e v o l v e d c a r bo n ppm/ml % o f s u b s t r a t e a n a e r o b i c 0 40 132 28 14 s t e a d y s t a t e a e r o b i c 15 50 63 87 44 30 50 51 99 50 45 55 48 97 49 60 80 .35 85 43 75 110 26 64 32 90 110 22 68 34 a e r o b i c s t e a d y s t a t e 115 38 47 23 N.B. A c o n t i n u o u s c u l t u r e o f E_. c o l i B was grown a n a e r o b i c a l l y t o s t e a d y s t a t e and a t time 0 s w i t c h e d t o a e r o b i c c o n d i t i o n s ; g l u c o s e m i n i m a l medium; Temperature 37 C, pH 7.0, d i l u t i o n r a t e .6 hr--'-. 34 m i n u t e s , s u b s t r a t e c a r b o n c o n v e r t e d t o ca r b o n d i o x i d e r o s e from 14% ( a n a e r o b i c l e v e l s ) t o a maximum o f 50%. However, when c u l t u r e s o f E . c o l i B were a l t e r n a t e d between a e r o b i c and a n a e r o b i c c o n d i t i o n s a t i n t e r v a l s from 1 t o 15 mi n u t e s , t h e r e was no i n c r e a s e i n ca r b o n d i o x i d e p r o d u c t i o n above normal v a l u e s f o r a e r o b i c growth ( T a b l e IX ) . I t seems l i k e l y t h a t t h i s f a i l u r e was due t o t h e l o n g p e r i o d o f time ( g r e a t e r t h a n 4 hours) needed f o r a e r o b i c E_. c o l i t o c o m p l e t e l y adapt t o a n a e r o b i c growth ( F i g u r e 7 ) . The most s t r i k i n g a s p e c t o f t h i s " l a g " i s the a p p a r e n t washout o f c e l l s . G l u c o s e remained below 10 ppm/ml w h i l e s u p e r n a t a n t c a r b o n i n c r e a s e d a t a l o g r a t e . D u r i n g t h i s " l a g " , t h e c e l l s c o n t i n u e d t o m e t a b o l i z e g l u c o s e , however, no growth o c c u r r e d and the p r o d u c t s were e j e c t e d i n t o t h e s u p e r n a t a n t ( F i g u r e 7 ) . T h i s s u g g e s t e d t h a t a b l o c k i n t h e c a t a b o l i s m o f g l u c o s e o c c u r r e d , p r e v e n t i n g t h e c e l l from o b t a i n i n g energy f o r growth. Indeed, s i n c e t h e l o g d e c r e a s e i n c e l l w e i g h t exceeded t h a t of washout, i t appeared t h a t t h e c e l l s were u s i n g up energy, and t h e r e -f o r e , s t o r a g e p r o d u c t s , i n t h e i n i t i a l s t e p s o f g l u c o s e c a t a b o l i s m . Whatever t h e mechanism, t h i s " u n c o u p l i n g " p r o c e s s r e v e a l s a weakness i n t h e c o n t r o l system o f t h e c e l l . However, s i n c e tne o b j e c t i v e o f t h i s work was t o i n c r e a s e t h e l e v e l o f ca r b o n d i o x i d e p r o d u c t i o n , no f u r t h e r s t u d y was u n d e r t a k e n r e g a r d i n g t h i s phenomena. 35 T a b l e IX. Carbon d i o x i d e p r o d u c t i o n from a c o n t i n u o u s c u l t u r e o f E_. c o l i B a l t e r n a t e d between a e r o b i c and a n a e r o b i c c o n d i t i o n s . Temperature 37 C, pH 7.0; medium-glucose l i m i t e d - 200 ppm/ml c a r b o n ; d i l u t i o n r a t e , .6 h r A n a e r o b i c v s a e r o b i c Time i n minute s c e l l c a r b o n s u p e r n a t a n t c a r b o n d i o x i d e e v o l v e d A n a e r o b i c a e r o b i c ppm/ml c a r b o n ppm/ml ppm/mi % o f s u b s t r a t e 3 3 70 6 4 110 7 3 90 3 1 55 4 1 50 15 15 90 normal a e r o b i c c u l t u r e 100 30 15 68 22 11 79 31 16 91 54 27 136 14 7 66 44 22 40 45 23 3b 30 60 9 0 120 !50 18 0 210 2 40 T I M E ( M in.) Figure 7. Effect of changing from aerobic to anaerobic conditions in a continuous culture of E. c o l i B; glucose limited medium; carbon, 200 ppm/ml; Temperature 37 C, pH 7.0. At time = 0. the culture was changed from aerobic to anaerobic conditions. 0 = O.D.; • = log of supernatant carbon; A = glucose carbon; w r= log of dry weights. 37 Gray e_t a l . (27) indicated that anaerobically grown E_. c o l i has many of the structural and enzymatic properties of obligate aerobes. They concluded that this may enable these cells to rapidly adjust to changes in oxygen tension. Our results indicate that this i s not the case. Harrison (26) states that the time required for E. c o l i to adapt to aerobic or anaerobic conditions i s 8 and 14 hours respectively. Further evidence that alternating between aerobic and anaerobic growth would not "uncouple" cells i n waste treatment systems, was pro-vided in Table X . Unlike E_. c o l i , a stable anaerooic mixed culture did not become "uncoupled" when sparged with a i r . EFFECT OF U.V. IRRADIATION ON THE GROWTH OF E. COLI B: With the hope of selectively inactivating a coupling factor without destroying c e l l DNA, a continuous culture of E_. c o l i B_ was irradiated with light of specific wave lengths. No effects were ob-served except at wave lengths around 2600°A. Since proteins are approximately 100 times less sensitive to incident light than i s DNA, i t i s possible that the intensity of light at these specific wave lengths was insufficient to affect proteins and this approach, therefore, was abandoned. Thereafter, we u t i l i z e d the whole spectrum of light emitted from the u.v. lamp (see Methods), varying the intensity by positioning i t at various distances from the quartz window. 38 T a b l e X. The e f f e c t o f a e r a t i n g an a n a e r o b i c c o n t i n u o u s c u l t u r e o f mixed p o p u l a t i o n s . oxygen t e n s i o n time (min) c e l l c a r b o n s u p e r n a t a n t carbon c a r b o n ppm/ml d i o x i d e e v o l v e d % o f s u b s t r a t e a n a e r o b i c 0; 65 120 15 8 a e r o b i c 15 70 112 18 9 30 80 114 6 3 45 85 110 5 3 60 85 100 15 8 75 90 95 15 8 90 95 84 21 11 120 100 80 20 10 N.B. A c o n t i n u o u s c u l t u r e o f mixed p o p u l a t i o n s , d i l u t i o n r a t e = .6, was s w i t c h e d from a n a e r o b i c t o a e r o b i c c o n d i t i o n s ; g l u c o s e m i n i m a l medium; 200 ppm/ml carbon; Temperature 37 C; pH 7.0. A t time = 0 the n i t r o g e n s p a r g e d c u l t u r e was made a e r o b i c by s p a r g i n g w i t h a i r . 39 T a b l e XI shows t h e r e s u l t o f a c o n t i n u o u s c u l t u r e i n which t h e dose o f u.v. i r r a d i a t i o n was i n c r e a s e d g r a d u a l l y . Below a dose or 1.6 2 ergs/mm / s e c , t h e r e was l i t t l e change i n t h e c a r b o n b a l a n c e between c e l l c a r b o n and c a r b o n d i o x i d e . As t h e i n t e n s i t y i n c r e a s e d , however, a g r e a t e r p e r c e n t a g e o f s u b s t r a t e c a r b o n was c o n v e r t e d t o c a r b o n 2 d i o x i d e r a t h e r t h a n t o c e l l s , a t an i n t e n s i t y o f 60 ergs/mm / s e c . Normal a e r o b i c l e v e l s o f c a r b o n d i o x i d e were i n c r e a s e d from 20% t o 50% o f s u b s t r a t e c a r b o n . A t t h i s l e v e l , c e l l y i e l d was o n l y s l i g h t l y h i g h e r t h a n would be e x p e c t e d from an a n a e r o b i c c u l t u r e w h i l e s u p e r -n a t a n t c a r b o n approached a e r o b i c l e v e l s ; d r a m a t i c e v i d e n c e o f i n -e f f i c i e n t (uncoupled) growth. D u r i n g t h i s experiment ( T a b l e XI ) , f i l a m e n t o u s c e l l s d e v e l o p e d soon a f t e r t h e i n i t i a l exposure t o u.v. and t h e y became l o n g e r w i t h i n c r e a s i n g l e v e l s o f u.v. i r r a d i a t i o n . F l o c u i a t i o n , eg. v i s i b l e clumps o f f i l a m e n t o u s c e l l s , became e v i d e n t a t t h e l e v e l o f 1.6 e r g s / 2 mm / s e c , and t h e f i o c s i n c r e a s e d i n s i z e from t h a t t i m e . As a conse-quence o f t h e s e f l o e s , t h e r e was a d i s r u p t i o n i n our d a t a s i n c e 5 ml samples p r o d u c e d wide f l u c t u a t i o n s i n d r y w e i g h t s . To e l i m i n a t e t h i s problem, samples were o b t a i n e d from t h e e f f l u e n t c o l l e c t e d o v e r n i g h t i n an i c e water b a t h . I t was assumed t h a t t h i s l a r g e sample i n d i c a t e d t h e r e a l c a r b o n b a l a n c e o f t h e system. The e f f l u e n t c o n s i s t e d o f d i s -p e r s e d c e l l s and s m a l l f l o e s . The degree o f f l o c u i a t i o n appeared t o remain c o n s t a n t i n t h e c u l t u r e v e s s e l and i t was assumed t h a t t h e f l o e s T a b l e XI. E f f e c t o f i n c r e a s i n g dose l e v e l s o f u l t r a - v i o l e t l i g h t on the growth o f E_. c o l i B_ i n c o n t i n u o u s c u l t u r e . Dose l e v e l Time Dry weight C e l l carbon S u p e r n a t a n t c a r b o n d i o x i d e e v o l v e d ergs/mm^/sec (Hrs) mg/ml ppm/ml carbon ppm/ml ppm/ml % o f s u b s t r a t e 0 1 .24 120 40 40 20 .3 2 .24 120 44 36 18 19 .24 120 34 46 23 22 .24 129 40 40 20 25 .22 110 43 47 . 24 .8 27 .24 120 40 40 20 30 .23 115 45 40 20 1.6 40 mm 45 - - 32 - -50 — **" 40 — — 3.7 7 2 * 35 _ _ 96 .15 75 47 78 39 60.0 1 0 ° * „ _ mm mm 120 ; .10 50 44 106 53 D i l u t i o n r a t e = .6; Temperature, 37 C; pH 7.0; g l u c o s e m i n i m a l medium, 200 ppm/ml c a r b o n . * Samples c o l l e c t e d from a volume o f e f f l u e n t o b t a i n e d i n an i c e b a t h o v e r a 12 hour p e r i o d (see Methods). 41 broke up as t h e y were v i g o r o u s l y e j e c t e d from t h e v e s s e l by t h e o u t -f l o w i n g a i r . To ensure t h a t f l o e s were n o t l a r g e enough t o cause d i s c r e p a n c i e s w i t h t h e r e s u l t s , s e v e r a l s h o r t term experiments were c a r r i e d o u t t o f u r t h e r i n v e s t i g a t e t h e e f f e c t o f u.v. on t h e growth o f E_. c o l i B 2 (Tabl e X I I ) . Repeated experiments a t a dose or 1.6 ergs/mm / s e c showed an i n c r e a s e i n t h e amount o f s u b s t r a t e c a r b o n c o n v e r t e d t o c a r b o n d i o x i d e (from 20% t o 34%) w i t h a c o r r e s p o n d i n g d e c r e a s e i n c e l l d r y w e i g h t (Ta b l e X I I ) . When t h e u.v. lamp was s h u t o f f , t h e c h a r a c t e r -i s t i c h i g h l e v e l o f c a r b o n d i o x i d e p r o d u c t i o n c o n t i n u e d f o r a t l e a s t 24 h o u r s . Table XII. Effect of irradiating a normal aerobic continuous culture of E_. c o l i B_ with ultra-violet light. Cell Experiment Dose level Time Dry wgt. c a r j - , o n Supernatant Carbon dioxide evolved No. ergs/mm2/sec (Hrs.) mg/ml . , carbon ppm/ml ppm/ml % of substrate ppm/ml *•* :— —: : 1 0 22 .24 120 43 23 .24 120 40 24 .23 115 38 40 ± 1.7 20 25 .23 115 40 0 .24 120 42 38 .5 - - . 212 -2 .24 120 94 -5 .21 105 41 54 8 .14 70 78 52 10 .12 60 84 56 22 .16 80 54 23 .16 80 62 24 .14 70 64 66 ± i 25 .14 70 54 26 .18 90 47 27 — — — 46 .18 90 38 47 .16 80 40 48 .16 80 40 78 ± . 49 .18 90 32 3 20 .15 21 .14 1.6 22 .14 75 55 70 65 67 ± 2.0 34 70 65 Dose, 1.6 ergs/mm2/sec; glucose minimal medium; 200 ppm/ml, carbon; Temperature, 37 C; pH 7.0. N.B. Experiment #2 shows that the characteristic of inefficient growth i s maintained 24 hours after teh ultra-violet lamp i s shut off. 43 DISCUSSION The p r i m a r y o b j e c t i v e i n c o n t r o l l i n g oxygen l e v e l s was t o i n d u c e a c o n s t a n t s t a t e o f s t i m u l a t e d r e s p i r a t i o n as opposed t o t h e o s c i l -l a t i n g r a t e s o b s e r v e d by H a r r i s o n and P i r t ( 8 ) , and t o e l i m i n a t e t h e p r o d u c t i o n o f f e r m e n t a t i o n p r o d u c t s . H a r r i s o n and P i r t s u g g e s t e d t h a t t h i s p e r i o d o f s t i m u l a t e d r e s p i r a t i o n r e s u l t e d i n a re d u c e d ATP y i e l d / m o l e o f g l u c o s e o x i d i z e d - eg. u n c o u p l e d growth - and we f e l t t h a t , i f t r u e , t h e consequent d e c r e a s e i n c e l l c a r b o n would be advan-tageous i n waste t r e a t m e n t systems. OSCILLATIONS: I n t h e o s c i l l a t i n g c u l t u r e a t low oxygen l e v e l s , H a r r i s o n and P i r t (8) found an i n c r e a s e d p r o d u c t i o n o f ca r b o n d i o x i d e and a d e c r e a s e i n c e l l c a r b o n - i n d i c a t i n g u n c o u p l e d growth - a l t h o u g h t h e concen-t r a t i o n o f f e r m e n t a t i o n p r o d u c t s i n c r e a s e d . I t i s i m p o r t a n t t o d i s t i n g u i s h between t h e o s c i l l a t i o n s f o u nd by H a r r i s o n and P i r t , w hich o c c u r r e d i n t h e chemostat between oxygen l e v e l s o f .2 and 15mmHg, and the o s c i l l a t i o n s i n our system, which o c c u r r e d i n a c o n t i n u o u s f l o w o f c u l t u r e t h r o u g h t h e r e s p i r o m e t e r w h i l e t h e oxygen l e v e l i n t h e chemostat was m a i n t a i n e d c o n s t a n t ( F i g u r e 2 ) . In our system, t h e o s c i l l a t i o n s o c c u r r e d w e l l above a p 0 2 o f 15mmHg and t h e r e was no i n c r e a s e i n s u p e r n a t a n t c a r b o n ( f e r m e n t a t i o n 44 p r o d u c t s ) , s u g g e s t i n g t h a t a n a e r o b i c metabolism was n o t a f a c t o r i n the phenomena o f "uncoupled" growth a t low l e v e l s o f oxygen t e n s i o n . GROWTH OF E. COLI B AT LOW OXYGEN TENSIONS: The c o n t r o l l e r r e g u l a t e d oxygen t e n s i o n i n t h e c u l t u r e s t o w i t h i n 2.5mmHg, hence t h e r e s u l t s a r e e x p r e s s e d f o r l e v e l s below 3mmHg and be-tween 3-8mmHg ( T a b l e I ) . A n a e r o b i c p r o d u c t s accumulated below 3mmHg -a c c o u n t i n g f o r t h e p r e s e n c e o f f e r m e n t a t i o n p r o d u c t s i n t h e r e s u l t s o f H a r r i s o n and P i r t ( 8 ) . I n a pa p e r t h a t p a r a l l e l e d t h i s work, H a r r i s o n and L o v e l e s s (4) o b t a i n e d low y i e l d s w i t h E . c o l i when grown under " l i m i t e d " oxygen; however, t h e c o n c e n t r a t i o n o f f e r m e n t a t i o n p r o d u c t s i n c r e a s e d s i g n i f i c a n t l y . T h i s demonstrates the need t o c o n t r o l t h e oxygen t e n s i o n above 3mmHg i n o r d e r t o a v o i d i n c r e a s e s o f s u p e r n a t a n t c a r b o n i n t h e e f f l u e n t . A t l e v e l s between 3 and 8mmHg i n g l u c o s e l i m i t e d c u l t u r e s , t h e y i e l d o f c e l l c a r b o n was re d u c e d by about 20% w i t h no i n c r e a s e i n s u p e r n a t a n t c a r b o n . S i n c e t h e s e r e s u l t s d i d n o t s i g n i f i c a n t l y d i f f e r from t h o s e o f H a r r i s o n ( 8 ) , we d i d n o t a c h i e v e a n o n - o s c i l l a t i n g s t i m u l a t i o n o f t h e r e s p i r a t i o n r a t e o r t h a t such a c o n d i t i o n d i d n o t r e s u l t i n f u r t h e r d e c r e a s e d i n c e l l c a r b o n , eg. t h e degree o f "uncoup-l i n g " was n o t i n c r e a s e d . A d e c r e a s e i n c e l l c a r b o n would be a l o g i c a l r e s u l t o f a d e c r e a s e i n energy a v a i l a b l e t o t h e c e l l . H a r r i s o n and M a i t r a (18) s u p p o r t 45 this concept by observing that ATP levels remain nearly constant during periods of stimulated respiration suggesting an increased turnover rate of ATP or a f a l l i n the P/0 ratio. However, when glycerol was the sole carbon source (Table II), no "uncoupled" growth resulted at low oxygen tensions. This suggests that, although the oscillations i n pO^ were well above 3mmHg, anaerobic metabolism, or at least the potential to metabolize a substrate anaerobically, might be a factor influencing the efficiency of growth at low oxygen tensions. (Glycerol cannot be metabolized anaerobically without the addition of an electron acceptor.)(19). EFFECT OF LOW OXYGEN TENSIONS ON LEVELS OF CYTOCHROME B: We did not find any relationship between the increased content of cytochrome B (Figure 5) and the increase in the respiration rate -eg. cytochrome B does not limit respiration. Clark-Walter et a l . (20) have suggested that low oxygen levels de-repress an ALA synthase enzyme involved i n the synthesis of heme. Since low oxygen levels changes the balance of induction and repression i n the synthesis of cytochromes, other components of oxidative phosphorylation may be similarily affected - resulting perhaps i n a decrease i n the e f f i c i -ency of energy production. 46 EFFECT OF LOW OXYGEN TENSION ON CELL SIZE: A t oxygen l e v e l s below 8mmHg, c e l l s i z e d e c r e a s e d by a p p r o x i -m a t e l y 20% (Tab l e I V ) . I t i s p o s s i b l e t h a t "uncoupled" growth r e -s t r i c t s t h e c e l l ' s a b i l i t y t o s y n t h e s i z e normal amounts o f macromolecular compounds due t o a d e f i c i e n c y i n a v a i l a b l e energy, and th u s t h e c e l l s i z e would d i m i n i s h . A l t e r n a t i v e l y , assuming t h a t because the s p e c i f i c growth r a t e i s c o n s t a n t , t h e q u a n t i t y o f macromolecules - eg. r i b o -somes - i s t h e r e f o r e a l s o c o n s t a n t , t h e n t h e d e c r e a s e i n s i z e c o u l d i n d i c a t e a d e c r e a s e i n s t o r a g e p r o d u c t s . GROWTH OF PSEUDOMONAS AERUGINOSA 9027 AT LOW OXYGEN TENSIONS: I n h i s r e v i e w o f oxygen and m i c r o b i a l m e t a b o l i s m ( 7 ) , Wimpenny n o t e d t h a t s t r i c t a erobes as w e l l as f a c u l t a t i v e b a c t e r i a were a f f e c t e d by low v a l u e s o f oxygen t e n s i o n . However, a p a r t from n o t i n g i n c r e a s e d l e v e l s o f t h e components o f t h e r e s p i r a t o r y system, l i t t l e p r e c i s e i n f o r m a t i o n was a v a i l a b l e . R e c e n t l y , C l a r k and B u r k i (23) o b s e r v e d w i t h s t r a i n s o f Pseudomonas and Achromobacter, t h a t growth a t oxygen l e v e l s below 15mmHg e x h i b i t e d l a g p e r i o d s . Our work w i t h Pseudomonas a e r u g i n o s a 9027 c o n s i s t a n t l y showed l a g p e r i o d s e x c e e d i n g 6 hours w i t h s m a l l i n o c u l a a t any l e v e l o f oxygen t e n s i o n below 120mmHg ( s t a r t i n g c u l t u r e a t an O.D. o f l e s s t h a n . 0 4 ) ( F i g u r e 3 ) . The c o n t r o l c u l t u r e s (excess oxygen) were sp a r g e d w i t h a i r - c o n t a i n i n g .03% c a r b o n d i o x i d e , as opposed t o t h e complete 47 l a c k o f c a r b o n d i o x i d e i n t h e gases (oxygen and n i t r o g e n ) u s ed f o r c o n t r o l a t oxygen t e n s i o n s l e s s t h a n 120mmHg. I t seemed p o s s i b l e , t h e r e f o r e , t h a t the l a g was a r e s u l t o f c a r b o n d i o x i d e s t r i p p i n g from t h e c u l t u r e s . However, when t h e c e l l s were grown above 120mmHg, u s i n g oxygen and n i t r o g e n t o c o n t r o l t h e oxygen t e n s i o n , no l a g d e v e l o p e d . Thus t h e r e i s n o t r e a d i l y a v a i l a b l e e x p l a n a t i o n s f o r t h i s l a g phenomena. Our r e s u l t s showed a d r a m a t i c e f f e c t o f low oxygen l e v e l s on t h e growth o f P. a e r u g i n o s a 9027. The c a r b o n b a l a n c e ( T a b l e I I I ) i n d i c a t e d t h a t c a r b o n d i o x i d e p r o d u c t i o n i n c r e a s e d from 14% t o 37% (of s u b s t r a t e carbon) when oxygen l e v e l s were r e d u c e d from 144mmHg t o 8mmHg. I t was a p p a r e n t t h a t "uncoupled" growth a t low oxygen t e n s i o n s i s even more s i g n i f i c a n t i n t h i s s t r a t i n o f Pseudomonas - a s t r i c t aerobe -t h a n i s t h e ca s e w i t h E . c o l i B_ - a f a c u l t a t i v e b a c t e r i a . F u r t h e r m o r e , the " u n c o u p l i n g " e f f e c t s o f low oxygen l e v e l s on s t r i c t aerobes may be much g r e a t e r t h a n what has been p r e v i o u s l y r e p o r t e d . The d a t a s u g g e s t s t h a t a r e l a t i o n s h i p e x i s t s between t h e e f f i c i e n c y o f o x i d a t i v e p h o s p h o r y l a t i o n and oxygen l e v e l s . I f one assumes t h a t Y , m „ i s c o n s t a n t , i t i s p r o b a b l e t h a t a d e c r e a s e ATP i n y i e l d r e l e c t s a d e c r e a s e d p r o d u c t i o n o f ATP. The changes i n c y t o -chrome c o n t e n t s a t low oxygen t e n s i o n may r e f l e c t a s w i t c h t o an a l t e r -n a t e e l e c t r o n pathway p o s s i b l y h a v i n g fewer s i t e s f o r t h e p r o d u c t i o n o f ATP. I t might be t h a t an i n c r e a s e i n a s p e c i f i c cytochrome - eg. 48 cytochrome B i n E . c o l i - i n d i c a t e s t h e p o i n t a t which t h i s a l t e r n a t e pathway branched from t h e o r i g i n a l To summarize: l e v e l s o f oxygen t e n s i o n between 3 and 16mmHg, t h e a b i l i t y o f a e r o b i c and f a c u l t a t i v e b a c t e r i a t o produce c e l l c a r b o n from a g i v e n amount o f s u b s t r a t e may be d e c r e a s e d . T h i s m ight r e f l e c t a d e c r e a s e i n t h e e f f i c i e n c y o f o x i d a t i v e p h o s p h o r y l a t i o n a t t h e s e low oxygen c o n c e n t r a t i o n s . The mechanism r e s p o n s i b l e f o r t h i s "un-c o u p l i n g " i s n o t u n d e r s t o o d b u t p r o b a b l y does n o t i n v o l v e t h e " i n i -t i a t i o n " o f a n a e r o b i c m e t a b o l i s m . Mixed p o p u l a t i o n s were l e s s n o t i c e a b l y a f f e c t e d ( a n d i t appeared t h a t f l o c u i a t i o n and n a t u r a l s e l e c t i o n would n u l l i f y t h e " u n c o u p l i n g " e f f e c t s o f low l e v e l s o f oxygen i n waste t r e a t m e n t s y s t e m s ) . A n a e r o b i c E_. c o l i B c e l l s a r e u n c o u p l e d when a e r a t e d . However, mixed p o p u l a t i o n s d i d n o t d u p l i c a t e t h i s e f f e c t , i n d i c a t i n g t h a t t h i s t e c h n i q u e would n o t be s u i t a b l e i n waste t r e a t m e n t systems. Work on an u l t r a - v i o l e t i r r a d i a t e d c o n t i n u o u s c u l t u r e o f E . c o l i B was u n d e r t a k e n w i t h the same o b j e c t i v e : t h a t i s , t o o x i d i z e more o f t h e s u b s t r a t e t o c a r b o n d i o x i d e and t h e r e f o r e t o p r o d u c e l e s s c a r b o n . I t was a n t i c i p a t e d t h a t t h e r e s u l t o f u.v. i r r a d i a t i o n would be t o s e l e c t a r e s i s t a n t p o p u l a t i o n w i t h i n c r e a s e d maintenance energy r e -q u i r e m e n t s - eg. due t o dimer r e p a i r a c t i v i t y . A n o t h e r p o s s i b l e e f f e c t m i g h t be t o produce u n c o u p l e d growth due t o t h e damage o f s p e c i f i c p r o t e i n s . I n any c a s e , i t was hoped t h a t t h e e f f e c t ( s ) o f u.v. 49 i r r a d i a t i o n would a p p l y t o a l l b a c t e r i a l s p e c i e s - eg. n a t u r a l s e l e c t i o n would n o t be a f a c t o r . By g r a d u a l l y i n c r e a s i n g t h e i n t e n s i t y o f u.v. i r r a d i a t i o n ( T a b l e X I ) , no l y s i s o c c u r r e d . F i l a m e n t o u s c e l l s appeared i m m e d i a t e l y and i n c r e a s e d i n s i z e as t h e dosage i n c r e a s e d . T h i s s u g g e s t e d t h a t t h e s e c e l l s had t h e a b i l i t y t o d i v i d e a l t h o u g h w i t h i n c r e a s i n g d i f f i c u l t y a t h i g h e r dose l e v e l s . F l o c u l a t i n g f i l a m e n t o u s c e l l s were dominant i n the c u l t u r e beyond 2 a dose l e v e l o f 1.6 ergs/mm / s e c . a l t h o u g h t h e r e were always a s m a l l e r p o p u l a t i o n o f normal s i z e d d i s p e r s e d c e l l s . I t i s i m p o r t a n t t o note t h a t E ^ c o l i c e l l s r e s i s t a n t t o u.v. may o r may n o t be f i l a m e n t o u s (28). S i n c e t h e development o f f l o c u l a t i n g c u l t u r e s i s a v e r y i m p o r t a n t a s p e c t o f waste t r e a t m e n t systems (31), t h i s a b i l i t y o f f i l a m e n t o u s c e l l s t o f l o c u l a t e i s r e g a r d e d as e x t r e m e l y s i g n i f i c a n t . I n c r e a s i n g t h e dose l e v e l o f u.v. p r o d u c e d c o r r e s p o n d i n g de-c r e a s e s i n c e l l c a r b o n and i n c r e a s e s i n t h e p r o d u c t i o n o f c a r b o n d i o -x i d e . T h i s s u g g e s t e d t h a t i n c r e a s i n g r e q u i r e m e n t s f o r maintenance energy were i n v o l v e d . However, when t h e u.v. lamp was s h u t o f f , t h i s c h a r a c t e r i s t i c o f i n e f f i c i e n t growth remained s t a b l e f o r a t l e a s t 24 h ours a l t h o u g h t h e c e l l s r e t u r n e d t o a normal s i z e . I t now appeared t h a t a change i n p o p u l a t i o n (E_. c o l i s t r a i n s ) had o c c u r r e d , and t h a t t h e "uncoupled" growth was n o t an e x c l u s i v e p r o p e r t y o f f i l a m e n t o u s c e l l s . E v i d e n t l y a s t a b l e (over a 24 hour p e r i o d ) p o p u l a t i o n w i t h a de-c r e a s e d e f f i c i e n c y o f s u b s t r a t e u t i l i z a t i o n . 50 With a d i l u t i o n r a t e o f 6 h r , i t i s d i f f i c u l t t o u n d e r s t a n d why a h i g h r a t e o f washout d i d n o t o c c u r i n i t i a l l y u n t i l t h e new popu-l a t i o n became dominant. The experiments i n which t h e u.v. was a p p l i e d ( T a b l e X I I ) , a t an i n i t i a l l y h i g h e r l e v e l t h a n i n t h e o r i g i n a l e x p e r i -ment, o c c a s i o n a l l y d i d r e s u l t i n immediate l y s i s , b u t n o t washout. T h i s may have been caused by t h e i n d u c t i o n o f phage. These experiments have p r o v i d e d i m p o r t a n t i n s i g h t s r e g a r d i n g t h e i n f l u e n c e o f u.v. l i g h t on t h e growth o f E_. c o l i . Many q u e s t i o n s r e g a r d i n g i t s b i o l o g i c a l e f f e c t s r e m a i n unanswered; y e t i t i s c l e a r t h a t t h i s t e c h n i q u e has an i m p o r t a n t p o t e n t i a l t o r a d i c a l l y improve waste t r e a t m e n t o p e r a t i o n s w i t h E . c o l i . I t has e f f e c t i v e l y i n c r e a s e d the c o n v e r s i o n o f s u b s t r a t e c a r b o n t o c a r b o n d i o x i d e from 20% t o o v e r 50% and f u r t h e r i n c r e a s e s may be p o s s i b l e . Moreover, i t en s u r e s t h a t r e s i d u a l i n s o l u b l e c a r b o n ( c e l l s ) can be s e p a r a t e d . I t i s n o t known i f t h i s e f f e c t can be d u p l i c a t e d w i t h c u l t u r e s i n v o l v i n g mixed p o p u l a t i o n s . 51 BIBLIOGRAPHY 1. P i n c h o t , G.B. 1967. The mechanism o f u n c o u p l i n g o f o x i d a t i v e p h o s p h o r y l a t i o n by 2 - 4 - d i n i t r o p h e n o l . J . B i o l . Chem. 242:4577-4583. 2. Deamer, D.W. 1969. ATP s y n t h e s i s : The c u r r e n t c o n t r o v e r s y . J . o f Chem. Ed. 46:198-206. 3. M i t c h e l l , P. 1966. Chemiosmotic c o u p l i n g i n o x i d a t i v e and p h o t o -s y n t h e t i c p h o s p h o r y l a t i o n . B i o l . Rev. 41:455-502. 4. H a r r i s o n , D.E.F., J . E . L o v e l e s s . 1971. The e f f e c t o f growth c o n d i -t i o n s on r e s p i r a t o r y a c t i v i t y and growth e f f i c i e n c y i n f a c u l t a t i v e anaerobes grown i n chemostat c u l t u r e . J . Gen. M i c r o b i o l . 68:35-43. 5. Ng, H. 1965. E f f e c t o f d e c r e a s i n g growth temperature on c e l l y i e l d o f E s c h e r i c h i a c o l i . J . B a c t . 89:232-237. 6. Uden, N.V. 1968. Y i e l d and maintenance a n a l y s i s i n the chemostat: A t o o l f o r m e t a b o l i c s t u d i e s o f growing c e l l s . A r c h , f u r M i k r o . 62:34-40. 7. Wimpenny, J.W.T. 1969. Oxygen and M i c r o b i a l M e t a b o l i s m . P r o c e s s Biochem. V o l . IV: 19. 8. H a r r i s o n , D.E.F., S . J . P i r t . 1967. t h e i n f l u e n c e o f d i s s o l v e d oxygen c o n c e n t r a t i o n on t h e r e s p i r a t i o n and g l u c o s e m e t a b o l i s m o f K l e b s i e l l a  aerogenes d u r i n g growth. J . Gen. M i c r o b i o l . 46:193-211. 9. Moss, F. 1952. The i n f l u e n c e o f oxygen t e n s i o n on r e s p i r a t i o n and cytochrome a 2 f o r m a t i o n o f E s c h e r i c h i a c o l i . A u s t . J . exp. B i o l , med. S c i . 30:531-540. 10. Moss, F. 1954. A d a p t a t i o n s o f t h e cytochromes o f A e r o b a c t e r aerogenes i n r e s p o n s e t o e n v i r o n m e n t a l oxygen t e n s i o n . A u s t . J . exp. 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The r e g u l a t i o n o f DNA r e p l i c a t i o n and c e l l d i v i s i o n i n E . c o l i B / r . C o l d S p r i n g Harbour Symp. Quant. B i o l . 33:823-838. 17. P a t t e r s o n , J.W., B r e z o n i k , P.L. and H.P. Putnam. 1970. Sludge a c t i v i t y parameters and t h e i r a p p l i c a t i o n t o t o x i c i t y measurements i n a c t i v a t e d s l u d g e . Dept. o f E n v i r o n m e n t a l Eng., U n i v e r s i t y o f F l o r i d a , G a i n s v i l l e , U.S.A. 18. H a r r i s o n , D.E.F. and P.K. M a i t r a . 1969. C o n t r o l o f r e s p i r a t i o n and m e t a b o l i s m i n growing K l e b s i e l l a a erogenes. Biochem. J . 112: 647-656. 19. Chused, T.M. 1962. BA Honours t h e s i s , H a r v a r d U n i v e r s i t y . 20. C l a r k - W a l t e r , C D . , B. R i t t e n b e r g and J . L a s l e l l e s . 1967. Cytochrome s y n t h e s i s and i t s r e g u l a t i o n i n S p i r i l l u m i t e r s o n i i . J . B a c t . 94:1648-1655. 21. Holme, T. 1957. Cont i n u o u s c u l t u r e s t u d i e s on g l y c o g e n s y n t h e s i s i n E s c h e r i c h i a c o l i B. A c t a . Chem. Scand. 11:763-780. 22. W ilkonson, J . F . and A.L.S. Munro. 1967. M i c r o b i a l P h y s i o l o g y and Con t i n u o u s C u l t u r e . P r o c . o f t h e T h i r d I n t e r n a t i o n a l Symposium, p.183. 23. C l a r k , D.S. and T. B u r k i . 1971. Oxygen r e q u i r e m e n t s o f s t r a i n s o f Pseudomonas and Achromobacter. Can. J . M i c r o b i o l . 18:321-^326. 24. Maclennan, D.G., Ousby, J.C., R.B. Vasey and N.T. C o t t o n . 1971. The i n f l u e n c e o f d i s s o l v e d oxygen i n Pseudomonas AMI grown on methanol i n c o n t i n u o u s c u l t u r e . J . Gen. M i c r o b i o l . 69:395-404. 25. C a v a r i , B.Z., Y. A v i - d o r and N. G r o s s o w i c z . 1968. 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