10 Liquid Fuels from Carbonates by a Microbial System M O R R I S W A Y M A N and M A R Y W H I T E L E Y
Chemistry for Energy Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/12/19. For personal use only.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 1A4
I n h i s r e v i e w o f t e c h n i q u e s f o r t h e e n r i c h m e n t , i s o l a t i o n and m a i n t e n a n c e o f t h e p h o t o s y n t h e t i c b a c t e r i a , v a n N i e l (1) c o n t r a s t s two k i n d s o f c y c l e s o f m a t t e r , a " p r i m i t i v e c y c l e " i n w h i c h s u l f i d e i s o x i d i z e d t o s u l f a t e by p h o t o s y n t h e t i c b a c t e r i a , t h e r e s u l t i n g s u l f a t e b e i n g r e d u c e d t o s u l f i d e a g a i n by o t h e r , n o n p h o t o s y n t h e t i c m i c r o b e s ; and a " t e r r e s t r i a l c y c l e " i n w h i c h g r e e n p l a n t p h o t o s y n t h e s i s p r o v i d e s an o r g a n i c b a s e f o r b i o l o g i c a l r e g e n e r a t i o n o f c a r b o n d i o x i d e . He i l l u s t r a t e d t h e two c y c l e s as shown i n F i g u r e 1. Carbon d i o x i d e i s e s s e n t i a l i n b o t h c y c l e s , b e i n g f i x e d i n p h o t o s y n t h e s i s and r e l e a s e d i n f e r m e n t a t i o n . To t h e e x t e n t t h a t t h e two c y c l e s i n t e r c h a n g e C 0 , t h e y a r e i n t e r active. However, t h e two c y c l e s d i f f e r w i t h r e s p e c t t o some p o r t i o n s o f t h e s u n ' s r a d i a t i o n u t i l i z e d by t h e r e d b a c t e r i a and t h e g r e e n p l a n t s , w h i c h may be s i g n i f i c a n t i n t h e f u n c t i o n i n g o f t h e e c o s y s t e m as a w h o l e . The p h o t o s y n t h e t i c b a c t e r i a a r e a u t o t r o p h s , w h e r e a s t h e s u l f a t e r e d u c e r s l i v e on t h e o r g a n i c m a t t e r f o r m e d by them. P l a n t , a n i m a l and most m i c r o b i a l o x i d a t i o n s r e q u i r e m o l e c u l a r oxygen, whereas m i c r o b i a l s u l f a t e f o r m a t i o n from s u l f i d e t a k e s p l a c e a n a e r o b i c a l l y , w i t h o u t the use o f m o l e c u l a r oxygen. P h o t o s y n t h e t i c b a c t e r i a and s u l f a t e r e d u c i n g b a c t e r i a a r e found i n n a t u r e i n c l o s e a s s o c i a t i o n ( 1 , 2 ) , both groups b e i n g engaged i n t h e s u l f u r c y c l e . Upon i s o l a t i o n o f t h e p u r e c u l t u r e s , t h e r e c y c l i n g o f s u l f u r compounds b e t w e e n them i s u p s e t . Membranes e p a r a t e d c u l t u r e o f f e r s an o p p o r t u n i t y t o s t u d y t h e g r o w t h and m e t a b o l i s m o f e a c h o f t h e two k i n d s o f o r g a n i s m s , and a t t h e same t i m e t h e i n t e r a c t i o n b e t w e e n them (3) . T h i s p a p e r r e p o r t s on such i n t e r a c t i o n s i n membrane-separated a n a e r o b i c c u l t u r e of a r e d p h o t o s y n t h e t i c b a c t e r i u m and a c o l o u r l e s s n o n p h o t o s y n t h e t i c s u l fate reducing bacterium. I n t h i s m i c r o b i a l s y s t e m , c a r b o n a t e was the o n l y source of carbon f o r growth. The g r o w t h o f t h e s e p a r a t e s p e c i e s has b e e n f o l l o w e d , as w e l l as t h e f o r m a t i o n and d i s a p p e a r a n c e o f s u l f i d e . The h a r v e s t e d m i c r o b e s were a n a l y s e d f o r p r o t e i n c o n t e n t . 2
This chapter not subject to U.S. Copyright. Published 1979 American Chemical Society.
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O r g a n i s m s « B o t h new i s o l a t e s w e r e o b t a i n e d f r o m a r e d - p u r p l e b l o o m w h i c h o c c u r s i n Durum L a k e , S a s k a t c h e w a n . Durum L a k e i s r i c h i n sodium s u l f a t e , the h e a v i e r l a y e r s c o n t a i n i n g about 7 t o 8 percent of t h i s s a l t . The b l o o m o c c u r s a t a l o w e r c o n c e n t r a t i o n , a b o u t 0.35 p e r c e n t , 1 t o 3 cm b e l o w t h e s u r f a c e . I t c o n t a i n s many d i f f e r e n t m i c r o o r g a n i s m s , i n c l u d i n g a l g a e and p r o t o z o a . A C h l o r e l l a i s o l a t e d f r o m t h e b l o o m has b e e n s t u d i e d s e p a r a t e l y ( 4 ) . The s e p a r a t i o n o f m i c r o o r g a n i s m s i n t o u s e f u l g r o u p s was begun u s i n g a W i n o g r a d s k y c o l u m n and l o w i n c a n d e s c e n t i l l u m i n a t i o n . The u p p e r l a y e r s w e r e g r e e n and t h e l o w e r l a y e r s r e d - p u r p l e . The i n t e r d e p e n d e n c e o f t h e p h o t o s y n t h e t i c b a c t e r i a and t h e s u l f a t e r e d u c i n g b a c t e r i a was o b s e r v e d d u r i n g t h e e n r i c h m e n t and i s o l a t i o n process. As t h e pH o f P f e n n i g ' s s t a n d a r d medium (5) was r a i s e d t h e r e was an i n c r e a s e i n t h e r e d p h o t o s y n t h e t i c b a c t e r i a i n t h e crude enrichment c u l t u r e s . R i c h l y c o l o u r e d r e d c u l t u r e s were o b t a i n e d a t t h e h i g h e r pH v a l u e s , and t h i s r e a c h e d a maximum a t i n i t i a l pH 9.0. U s i n g s o - m o d i f i e d P f e n n i g ' s medium, c u l t u r e s o f t h e r e d p h o t o s y n t h e t i c b a c t e r i a c o u l d be d i r e c t l y i s o l a t e d f r o m the bloom samples w i t h o u t p r e - e n r i c h m e n t i n a Winogradsky column. G r e e n p h o t o s y n t h e t i c b a c t e r i a c o u l d a l s o be i s o l a t e d i n t h i s m o d i f i e d medium, b u t o n l y f r o m a W i n o g r a d s k y c o l u m n . F o l l o w i n g g r o w t h o f t h e r e d b a c t e r i a i n t h i s medium, t h e c r u d e c u l t u r e s w e r e f o u n d t o h a v e pH 7.5 - 7.8, as t h e medium was n o t b u f f e r e d t o pH 9. In o r d e r t o o b t a i n pure c u l t u r e s , the crude c u l t u r e which cont a i n e d Chromâtium s p e c i e s was s e r i a l l y d i l u t e d i n t h i s m o d i f i e d medium as s u g g e s t e d by v a n N i e l ( 1 ) . T h i s was r e p e a t e d s e v e r a l times to ensure the p u r i t y of the c u l t u r e . The a g a r s h a k e method was n o t u s e d as t h e l a r g e p h o t o s y n t h e t i c b a c t e r i a f a i l t o grow i n a g a r s h a k e t u b e s (1) . I n p u r e c u l t u r e t h e Chromâtium s p e c i e s d i d n o t grow as w e l l as i n t h e m i x e d c u l t u r e s i t u a t i o n . The c u l t u r e was n o t as d e n s e o r as r i c h i n c o l o u r as t h e c r u d e c u l t u r e s . The r e d p h o t o s y n t h e t i c b a c t e r i u m a p p e a r s t o be i d e n t i c a l morp h o l o g i c a l l y and i n i t s b e h a v i o u r w i t h a s t o c k c u l t u r e o f Chromâtium w a r m i n g i i o b t a i n e d f r o m t h e A m e r i c a n Type C u l t u r e C o l l e c t i o n , No. 14 959. I t w i l l be r e f e r r e d t o as Chromâtium w a r m i n g i i NI (NI = new i s o l a t e ) . The s u l f a t e r e d u c i n g b a c t e r i u m was i s o l a t e d f r o m t h e c r u d e c u l t u r e by p r e - e n r i c h m e n t i n a l a c t a t e - s u l f a t e b r o t h (1) . S e v e r a l anomalous p r o p e r t i e s o f t h i s s u l f a t e r e d u c e r w e r e n o t e d . I t grew w e l l on Medium C o f B u t l i n ( 6 ) , p l u s a g a r , and f o r m e d b l a c k c o l o n i e s , b u t f a i l e d t o grow on s u b c u l t u r e . The a g a r a r o u n d t h e c o l o n i e s showed b l a c k e n i n g . T h i s b l a c k e n i n g , and t h e b l a c k c o l o n i e s , i m p l y s u l f a t e r e d u c t i o n . The c u l t u r e was i n c u b a t e d i n an anaerobe j a r w i t h a H2-CO2 atmosphere. C o t t o n wool soaked w i t h l e a d a c e t a t e was p l a c e d i n t h e j a r t o p r e v e n t H2S p o i s o n i n g o f t h e Pd c a t a l y s t ( 7 ) . B l a c k l e a d s u l f i d e became e v i d e n t . S t r u c t u r a l l y , the s u l f a t e r e d u c i n g b a c t e r i a a r e p o i n t e d , nonmotile rods, often associated i n p a i r s . They a r e g r a m - n e g a t i v e and n o n - s p o r u l a t i n g . T h e s e p r o p e r t i e s do n o t c o r r e s p o n d w i t h t h e
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d e s c r i p t i o n s of the s u l f a t e reducing b a c t e r i a belonging to the D e s u l f o v i b r i o or DesuIfomaculum groups. However, N. H v i d - H a n s e n has d e s c r i b e d (8) a s u l f a t e r e d u c i n g b a c t e r i u m w h i c h he i s o l a t e d f r o m s u l f i d e c o n t a i n i n g w a t e r s c h a r a c t e r i z e d by a s l i g h t l y a l k a l i n e r e a c t i o n , h i g h t o t a l s o l i d s and h i g h b i c a r b o n a t e , l o w s u l f a t e and a l o w c o n t e n t o f o t h e r d i - and t r i - v a l e n t i o n s . T h i s d e s c r i p t i o n m a t c h e s r a t h e r c l o s e l y t o t h e m o d i f i e d P f e n n i g medium we u s e d , and o u r s u l f a t e r e d u c e r s were a b l e t o grow i n t h i s medium a l o n e . H v i d - H a n s e n r e p o r t e d a f a i l u r e o f h i s o r g a n i s m s t o grow on s u b c u l t u r e i n m e d i a s u i t a b l e f o r t h e g r o w t h o f D e s u l f o v i b r i o . He named his organism D e s u l f o r i s t e l l a h y d r o c a r b o n o b l a s t i c a , the s p e c i f i c name b e i n g c h o s e n b e c a u s e t h e o r g a n i s m was f o u n d t o c o n t a i n b i t u minous s u b s t a n c e s . Our s u l f a t e r e d u c i n g b a c t e r i u m was f o u n d t o grow, and was m a i n t a i n e d , on t h e medium o f v a n N i e l f o r p h o t o s y n t h e t i c microorganisms, p l u s agar. Whether o r n o t o u r s u l f a t e reducer i s D e s u l f o r i s t e l l a hydrocarbonoblastica i s questionable, and we c a n n o t p r o v e i t s i n c e t h e o r i g i n a l c u l t u r e has b e e n l o s t . However, due t o t h e many s i m i l a r i t i e s we h a v e t e n t a t i v e l y c a l l e d i t D e s u l f o r i s t e l l a sp. M e d i a . The m e d i a e m p l o y e d w e r e b a s e d on P f e n n i g ' s s t a n d a r d medium (5) f o r t h e g r o w t h o f p h o t o s y n t h e t i c s a t pH 6.8. I t was m o d i f i e d f o r o t h e r e x p e r i m e n t s t o pH 9.0. A t t h i s pH a p r e c i p i t a t e f o r m e d w h i c h was f i l t e r e d o f f w i t h Whatman No. 1, f o l l o w e d by passage through a M i l l i p o r e f i l t e r f o r s t e r i l i z a t i o n . A l l c u l t u r e s w e r e grown a t room t e m p e r a t u r e (21 ± 1°C). Illumination. Chromâtium c u l t u r e s w e r e i l l u m i n a t e d c o n t i n u o u s l y by t h r e e 24 w a t t i n c a n d e s c e n t b u l b s a t a d i s t a n c e o f 25 t o 30 cm. Membrane-Separated C u l t u r e Apparatus. One o f t h e a d v a n t a g e s of membrane-separated c u l t u r e s i s t h a t the e x t e n t of c e l l growth o f e a c h o f two ( o r more) c u l t u r e s c a n be c o n v e n i e n t l y m e a s u r e d , and i n t e r a c t i o n can be o b s e r v e d a t t h e same t i m e ( 3 ) . I t was t h e r e f o r e d e c i d e d t o use t h i s t y p e of apparatus t o study the i n t e r a c t i o n o f Chromâtium w a r m i n g i i and D e s u l f o r i s t e l l a s p . A d i a g r a m o f t h e a p p a r a t u s i s shown i n F i g u r e 2. A c o m m e r c i a l membranes e p a r a t e d c u l t u r e a p p a r a t u s ( B e l c o G l a s s Co.) was a d a p t e d t o a c c o modate f o r a n a e r o b i c c o n d i t i o n s and e a s y s a m p l i n g . The domed l i d s o f t h e o r i g i n a l a p p a r a t u s w e r e r e p l a c e d by f l a t a l u m i n u m l i d s so t h a t t h e f e r m e n t e r s c o u l d be c o m p l e t e l y f i l l e d . In a d d i t i o n , ext e n s i o n t u b e s w e r e a t t a c h e d w h i c h r e a c h e d a l e v e l above t h e l i d s f o r f i l l i n g and s a m p l i n g . The a p p a r a t u s was f i l l e d t h r o u g h t h e s e e x t e n s i o n tubes through M i l l i p o r e f i l t e r s . An o v e r f l o w t u b e i n t h e t o p o f each l i d a l l o w e d t h e a p p a r a t u s t o be f i l l e d t o t h e t o p . E a c h f e r m e n t e r - e a c h s i d e - h e l d a b o u t 1700 m l . A f t e r t h e a p p a r a t u s was f i l l e d and i n o c u l a t e d , s t e r i l e M i l l i p o r e f i l t e r s w e r e attached to the e x t e n s i o n tubes. These were used t o f e e d s m a l l amounts o f m e d i a i n t o t h e u n i t v i a a s y r i n g e . T h i s a d d i t i o n c a u s e d
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Primitive Cycle of Matter Microbial fermentations and sulfate reduction
Light, photosynthetic bacteria
~ Organic matter + H SO4" 2
Terrestrial Cycle of Matter -C0 H 0 +
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Plant, animal and bacterial fermentations and oxidations
Light, green plant photosynthesis
Organic matter +Ο2* Figure
1.
Primitive
cycle of matter
MEMBRANE - SEPARATED CULTURE APPARATUS M - Nuclepore Membrane 0 - Overflow S - Magnetic Stirrers
Ε - Extension Tubes F - Millipore Filters
Figure 2. Membrane-separated culture apparatus, modified for anaerobic opera tion. M, nucleopore membrane; O, overflow; S, magnetic stirrers; E, extension tubes; F, millipore filters.
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e q u a l v o l u m e s o f t h e c u l t u r e s t o o v e r f l o w , and t h e s e were c o l l e c t e d f r o m t h e o v e r f l o w t u b e s . A p p r o x i m a t e l y 10 m l s a m p l e s w e r e t a k e n f o r e a c h r e a d i n g . The membrane u s e d t o s e p a r a t e t h e c u l t u r e s was a N u c l e p o r e membrane, p o r e s i z e 0.2 ym. G r o w t h R a t e s . The f o l l o w i n g m e m b r a n e - s e p a r a t e d c u l t u r e s y s tems w e r e s e t u p , and d u r i n g t h e c o u r s e o f g r o w t h , d r y w e i g h t o f m i c r o o r g a n i s m and t h e pH o f t h e c u l t u r e s w e r e f o l l o w e d : Side 1 Fermenter C. w a r m i n g i i (ATCC) π ( ) " " " (NI) Η A X C C
(
" π
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Ι
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(ATCC)* ·· (ATCC)*
S i d e 2 Fermenter D e s u l f o r i s t e l l a sp. , » . , . , h h π „ I. „ C. w a r m i n g i i ( N I ) " " " (NI)
I n i t i a l pH 6.8 9,o 6 9
#
8
0
6.8 9.0
* C o n t r o l s i n w h i c h t h e c u l t u r e s were n o t membrane-separated, b u t s e p a r a t e d by a p i e c e o f i m p e r v i o u s rubber s h e e t i n g . Dry Weight. The g r o w t h o f t h e c u l t u r e s was f o l l o w e d b y d r y weight: o p t i c a l d e n s i t y c o u l d n o t be used s i n c e s u l f u r g l o b u l e s which accumulate w i t h i n the c e l l s i n t e r f e r e ( 9 ) . D r y w e i g h t was m e a s u r e d by f i l t e r i n g 10 m l s a m p l e s a n d d r y i n g t h e f i l t e r membrane plus culture i n a desiccator. The f i l t e r s u s e d w e r e M i l l i p o r e t y p e GSWP 025 0 0 , p o r e s i z e 0.2 ym. Inocula. Chromâtium: 5 m l f r e s h l y p r e p a r e d o v e r n i g h t c u l ture. Desulforistella: 1 ml f r e s h l y prepared overnight c u l t u r e . C u l t u r e p u r i t y was c h e c k e d f r e q u e n t l y d u r i n g g r o w t h , b o t h m i c r o s c o p i c a l l y and by p l a t i n g o u t . P r o t e i n D e t e r m i n a t i o n . P r o t e i n was d e t e r m i n e d b y t h e b i u r e t method (10) a n d e x p r e s s e d a s % o f d r y w e i g h t . The s t a n d a r d was b o v i n e serum a l b u m i n (BDH). Sulfur. These e x p e r i m e n t s were performed i n 4 o z . Brockway b o t t l e s c o n t a i n i n g P f e n n i g ' s medium a t pH 9.0, w i t h e i t h e r s u l f i d e o r s u l f a t e a s t h e s u l f u r s o u r c e s . P a r a l l e l c u l t u r e s w e r e s e t up as f o l l o w s : 5 b o t t l e s C. w a r m i n g i i ( N I ) ; 5 b o t t l e s D e s u l f o r i s t e l l a sp.; 5 b o t t l e s mixed c u l t u r e . The i n o c u l a t o t a l l e d 1 m l i n e a c h c a s e , t h e m i x e d c u l t u r e h a v i n g 0.5 m l i n o c u l a o f e a c h o f t h e two o r g a n i s m s . F o r each e x p e r i m e n t , a b o t t l e was t a k e n o f e a c h c u l t u r e a n d t h e s u l f i d e a n d d r y w e i g h t m e a s u r e d . S u l f i d e was d e t e r m i n e d b y t h e method o f Pachmayr a s d e s c r i b e d b y T r u p e r and S c h l e g e l ( 1 1 ) . The c o n c e n t r a t i o n o f s u l f i d e was measured b y a b s o r b a n c e u s i n g a Beckman DB s p e c t r o p h o t o m e t e r a t 670 nm, a n d a s t a n d a r d i z e d c a l i b r a t i o n c u r v e .
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Results The r e s u l t s a r e g i v e n i n T a b l e I and F i g u r e s 3 t o 9. Table I p r e s e n t s the r e s u l t s of the p r o t e i n d e t e r m i n a t i o n . Table
I.
P r o t e i n Determination
Results Protein % of dry weight
Membrane-separated c u l t u r e s Chromât ium w a r m i n g i i pH
9.0
65 -
70 68
"
pH
6.8
62 -
D e s u l f o r i s t e l l a sp.
pH
9.0
38
pH
6.8
32
"
"
"
"
"
F i g u r e 3 shows t h e r a t e o f g r o w t h o f e a c h o r g a n i s m i n membranes e p a r a t e d c u l t u r e s a t i n i t i a l pH 9.0, and F i g u r e 4 shows t h e c h a n g e s i n pH d u r i n g g r o w t h o f e a c h o r g a n i s m . F i g u r e 5 shows t h e r a t e o f g r o w t h o f t h e C. w a r m i n g i i i n p u r e c u l t u r e , a t two pH l e v e l s . Figu r e s 6 and 7 p r e s e n t t h e r e s u l t s o f g r o w t h measurement and s u l f i d e d e t e r m i n a t i o n i n t h e b o t t l e e x p e r i m e n t s u s i n g s u l f i d e medium, and t h e f i n a l two f i g u r e s , 8 and 9, g i v e g r o w t h and s u l f i d e d u r i n g c u l t u r e i n s u l f a t e medium. Discussion As i s e v i d e n t i n F i g u r e 3, t h e C. w a r m i n g i i b e g a n t o grow f i r s t , f o l l o w e d by v e r y r a p i d g r o w t h o f t h e D e s u l f o r i s t e l l a . The f i n a l p o p u l a t i o n o f t h e C. w a r m i n g i i was h i g h e r . The d e l a y i n ons e t o f r a p i d g r o w t h by t h e s u l p h a t e r e d u c e r may be due t o t h e n e e d t o a c c u m u l a t e s u l f a t e b e i n g p r o d u c e d by t h e p h o t o s y n t h e t i c bact e r i u m as i t o x i d i z e d s u l f i d e . I t i s probable t h a t the sodium s u l f i d e s o l u t i o n a l r e a d y c o n t a i n s a v e r y s m a l l amount o f s u l f a t e , as P o s t g a t e (7) has s u g g e s t e d . P o s t g a t e a l s o has r e p o r t e d t h a t a t l e a s t i n D e s u l f o v i b r i o an i n i t i a l pH o f 8.6 c a u s e d a more r a p i d growth of the organisms, but a reduced s t a t i o n a r y p o p u l a t i o n . I t s h o u l d be n o t e d t h a t t h e i n o c u l u m o f D e s u l f o r i s t e l l a was o n l y onef i f t h t h a t o f t h e C. w a r m i n g i i , so t h e v e r y r a p i d i n i t i a l g r o w t h c a n h a r d l y be a t t r i b u t e d t o i n o c u l u m s i z e . A t pH 9, a t a b o u t 20 30 h o u r s a f t e r t h e D e s u l f o r i s t e l l a e n t e r e d e x p o n e n t i a l p h a s e t h e Chromâtium c u l t u r e s began t h e i r e x p o n e n t i a l p h a s e . Lower g r o w t h y i e l d s i n t h i s m i c r o b i a l s y s t e m were o b s e r v e d a t pH 6.8, and t h i s may be a t t r i b u t a b l e t o t h e D e s u l f o r i s t a l l a , w h i c h d i d n o t grow w e l l a t t h e l o w e r pH, and h e n c e d i d n o t p r o v i d e t h e n e e d e d s u l f i d e f o r t h e Chromâtium. The change i n pH o f t h e m e m b r a n e - s e p a r a t e d c u l t u r e s i s shown i n F i g u r e 4. The medium as m o d i f i e d t o pH 9.0 was n o t b u f f e r e d , and t h e p r o d u c t i o n o f end p r o d u c t s o f t h e r e s p i r a t o r y c h a i n s u c h as
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l a c t a t e c a u s e d t h e pH t o f a l l t o 7.4. The pH o f t h e D e s u l f o r i s t e l l a f e l l f i r s t a t a t i m e c o r r e s p o n d i n g t o v e r y r a p i d g r o w t h (30 or 40 t o 60 h o u r s ) , and t h e l a t e r f a l l i n pH o f t h e Chromâtium a l s o c o r r e s p o n d e d w i t h t h e p e r i o d o f i t s most r a p i d g r o w t h (50 t o 80 o r 70 t o 100 h o u r s ) . A t t h e end o f g r o w t h , when b o t h c u l t u r e s w e r e i n s t a t i o n a r y p h a s e , t h e pH was t h e same on b o t h s i d e s o f t h e membrane. The g r o w t h o f p u r e c u l t u r e s o f C. w a r m i n g i i i n t h i s medium was s l o w and t h e y i e l d s a t s t a t i o n a r y p h a s e w e r e much l o w e r t h a n i n t h e membrane-separated c u l t u r e s , as s e e n i n F i g u r e 5. Their g r o w t h was l i m i t e d by t h e a v a i l a b i l i t y o f s u l f i d e . T r u p e r and S c h l e g e l (11) r e p o r t e d t h a t t h e b i o m a s s y i e l d o f Chromâtium c o u l d be i n c r e a s e d by t h e a d d i t i o n o f s o d i u m s u l f i d e a f t e r t h e i n i t i a l s u p p l y was e x h a u s t e d . A t e x h a u s t i o n o f i n i t i a l s u l f i d e , t h e i r y i e l d s were a b o u t 150 mg/1 a t pH 6.8, w h i l e i n o u r c a s e , a t t h a t pH o u r y i e l d s w e r e o n l y 60 t o 100 mg/1. T h e i r s t u d y u s e d C. o k e n i i , a s p e c i e s o f l a r g e r b a c t e r i a t h a n C. w a r m i n g i i . They i n c r e a s e d t h e i r y i e l d s t o 250 mg/1 upon a d d i t i o n o f a s e c o n d s u p p l y o f s u l fide. I n t h e p r e s e n t s t u d y , y i e l d s o f C. w a r m i n g i i w e r e somewhat h i g h e r a t pH 9.0 t h a n a t t h e l o w e r pH, b e i n g 100 t o 150 mg/1. In t h e s e e x p e r i m e n t s , pH changes w e r e much s m a l l e r i n p u r e c u l t u r e s of C. w a r m i n g i i t h a n i n t h e m e m b r a n e - s e p a r a t e d c u l t u r e s , a r e s u l t a s s o c i a t e d w i t h the lower y i e l d s . The e x p e r i m e n t s w i t h s u l f i d e and s u l f a t e m e d i a w e r e d e s i g n e d t o r e l a t e changes o f t h e s u l f u r compounds t o b a c t e r i a l g r o w t h , and a l s o t o t r y t o e x p l a i n t h e i n c r e a s e d y i e l d s a t t h e h i g h e r pH. The r e s u l t s a r e shown i n F i g u r e s 6-9. I n s u l f i d e medium, s u l f i d e c o n c e n t r a t i o n i n c r e a s e d d u r i n g D e s u l f o r i s t e l l a growth, showing the r e d u c t i o n of s u l f a t e , a p r o bable contaminant of the s u l f i d e . When t h i s m i c r o o r g a n i s m r e a c h e d s t a t i o n a r y p h a s e , no s u l f a t e c o u l d be d e t e c t e d by b a r i u m p r e c i p i tation. D u r i n g Chromâtium g r o w t h , s u l f i d e d e c r e a s e d t o a l m o s t complete d i s a p p e a r a n c e , a t which p o i n t growth ceased. I n the m i x e d c u l t u r e , h o w e v e r , t h e r e was an i n i t i a l i n c r e a s e i n s u l f i d e b e f o r e i t s c o n c e n t r a t i o n began t o f a l l . M i x e d c u l t u r e g r o w t h was much more r a p i d . However, by a b o u t 90 h o u r s , s u l f i d e c o n c e n t r a t i o n had f a l l e n t o a l o w l e v e l , and t h e m i x e d c u l t u r e e n t e r e d t h e s t a t i o n a r y phase. The y i e l d a t s t a t i o n a r y p h a s e was a b o u t 400 mg/1, w h e r e a s t h e p u r e c u l t u r e s r e a c h e d o n l y 70 mg/1 (C. w a r m i n g i i ) o r 100 mg/1 ( D e s u l f o r i s t e l l a ) . These p u r e c u l t u r e y i e l d s o b t a i n e d i n b o t t l e s a r e q u i t e comparable t o those o b t a i n e d i n pure c u l t u r e growth i n the f e r m e n t e r s . The r e s u l t s o f g r o w t h i n t h e s u l f a t e m e d i a a r e shown i n F i g u r e 8, and t h e c o r r e s p o n d i n g s u l f i d e c o n c e n t r a t i o n s a r e shown i n F i g u r e 9. C. w a r m i n g i i c a n n o t u t i l i z e s u l f a t e f o r g r o w t h (12, p. 3 7 ) , a s f o u n d h e r e . The C. w a r m i n g i i d i d n o t grow. However, t h e D e s u l f o r i s t e l l a d i d grow s l o w l y t o a b o u t 70 mg/1, w h i l e i n t h e m i x e d c u l t u r e g r o w t h r e a c h e d 110 mg/1. The m i x e d c u l t u r e became p i n k i n c o l o u r , and t h e m i c r o s c o p e r e v e a l e d t h e p r e s e n c e o f b o t h t h e
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Chromâtium and t h e D e s u l f o r i s t e l l a . Sulfide production closely p a r a l l e l the growth r e s u l t s . No s u l f i d e was p r o d u c e d by t h e Chromâtium c u l t u r e , w h i l e s m a l l amounts w e r e p r o d u c e d by t h e D e s u l f o r i s t e l l a and i n t h e m i x e d c u l t u r e s . From a l l t h e s e r e s u l t s i t c a n be c o n c l u d e d t h a t t h e i n t e r a c t i o n o f C. w a r m i n g i i and t h e D e s u l f o r i s t e l l a s p . i n c r e a s e d t h e g r o w t h y i e l d s o v e r p u r e c u l t u r e s i n b o t h m e m b r a n e - s e p a r a t e d and m i x e d c u l t u r e s , and t h a t t h i s i n c r e a s e i n y i e l d c a n be e x p l a i n e d by t h e c y c l i n g o f t h e s u l f u r b e t w e e n t h e two o r g a n i s m s , i n t h e manner d e s i g n a t e d by v a n N i e l a s a p r i m i t i v e c y c l e o f m a t t e r . The b a c t e r i a o f t h i s s t u d y w e r e grown i n m i n e r a l m e d i a . The a u t o t r o p h y o f s u l f a t e r e d u c e r s h a s been q u e s t i o n e d ( 7 ) . G r o w t h o f the D e s u l f o r i s t e l l a on t h e m i n e r a l medium a l o n e was n o t g r e a t . However, i n m i x e d c u l t u r e o r i n m e m b r a n e - s e p a r a t e d c u l t u r e , t h e s u l f a t e r e d u c e r grew v e r y w e l l , a t t i m e s i n c r e a s i n g 1 0 - f o l d i n a 24 h o u r p e r i o d , a d o u b l i n g t i m e o f a b o u t 7 h o u r s . I t i s not c l e a r l y shown i n t h i s w o r k w h e t h e r t h e c a r b o n f o r t h e g r o w t h came f r o m the c a r b o n a t e o r f r o m o r g a n i c compounds p r o d u c e d by t h e Chromât ium. S t i l l i t i s beyond q u e s t i o n t h a t the m i c r o b i a l system i s a u t o trophic. As c a n be s e e n f r o m t h e p r o t e i n a n a l y s i s d a t a i n T a b l e I , C. warmingii i s r i c h i n protein. T r u p e r and S c h l e g e l (11) r e p o r t a p r o t e i n c o n t e n t o f 82.6% i n t h e i r p a p e r on C. o k e n i i . They a s s o c i a t e the h i g h p r o t e i n c o n t e n t w i t h the chromatophore f r a c t i o n of the c e l l s . Chromâtium c o u l d be c o n s i d e r e d as a s o u r c e o f s i n g l e c e l l p r o t e i n (13) and s i n c e i t was grown on c a r b o n a t e as a s o l e s o u r c e o f c a r b o n , i t s c o s t o f p r o d u c t i o n s h o u l d be l o w compared w i t h s i n g l e c e l l p r o t e i n b a s e d on m i c r o b e s grown on more e x p e n s i v e carbon s u b s t r a t e s . One c a n f u r t h e r c o n s i d e r a more c o m p l e x s y s t e m c o n t a i n i n g a n i t r o g e n f i x e r t o f u r t h e r reduce s u b s t r a t e c o s t s . The l o w p r o t e i n c o n t e n t o f t h e D e s u l f o r i s t e l l a r a i s e s t h e question of the composition of the c e l l s . Hvid-Hansen found a " b i t u m i n o u s o i l - l i k e s u b s t a n c e " i n h i s c e l l s , and he r e f l e c t e d t h a t f i n d i n g i n h i s s p e c i f i c name h y d r o c a r b o n o b l a s t i c a . If Desulforist e l l a i s indeed capable of forming hydrocarbons, then the m i c r o b i a l s y s t e m we h a v e b e e n d i s c u s s i n g may be a m o d e l f o r t h e f o r m a t i o n o f p e t r o l e u m u n d e r p r i m i t i v e c y c l e c o n d i t i o n s , i n w h i c h C O 2 , as c a r bonate, i s reduced t o hydrocarbons. Z o B e l l (14) h a s d i s c u s s e d s e v e r a l p o s s i b l e r o l e s f o r microorganisms i n petroleum formation, b u t t h e r e i s as y e t l i t t l e a g r e e m e n t on how m i c r o b e s h a v e p a r t i c i p a t e d i n t h e p r o c e s s . The p r e s e n t w o r k s u g g e s t s one p o s s i b i l i t y , n a m e l y t h a t m i c r o b i a l p h o t o s y n t h e s i s i s t h e p r i m a r y p r o c e s s , and t h a t s u l f a t e r e d u c e r s s u c h as we have b e e n d i s c u s s i n g , c o n t i n u e t h e r e d u c t i o n o f CO2 t o h y d r o c a r b o n s . The f o r m a t i o n o f h i g h e n e r g y s u b s t a n c e s by f e r m e n t a t i o n i s w e l l e s t a b l i s h e d i n p r o c e s s e s s u c h as t h o s e w h i c h p r o d u c e m e t h a n e , a l c o h o l , o r a c e t o n e and b u t a n o l . T h e s e p r o c e s s e s r e q u i r e o r g a n i c s u b s t r a t e s . A u t o t r o p h i c m i c r o b i a l p r o c e s s e s s u c h as a l g a l g r o w t h have h i g h l y e f f i c i e n t p h o t o s y s t e r n s and u s e l o w e n e r g y r a d i a t i o n , that i s the longer wavelengths. P a r t of t h e i r energy f o r C0 2
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f i x a t i o n a n d m e t a b o l i s m comes f r o m t h e o x i d a t i o n o f s u l f i d e s . The energy b a l a n c e i n t h i s m i c r o b i a l system i s worthy o f f u r t h e r study. The above s p e c u l a t i o n a b o u t h y d r o c a r b o n f o r m a t i o n s u g g e s t s a n o t h e r a d v a n t a g e o f membrane-separated c u l t u r e . We c a n v i s u a l i z e p r o d u c t i o n o f h i g h p r o t e i n b i o m a s s i n one f e r m e n t e r , w h i l e h i g h energy biomass i s produced by i n t e r a c t i o n i n a n o t h e r fermenter o f such a m i c r o b i a l system. Acknowledgements T h i s w o r k was s u p p o r t e d i n p a r t b y t h e N a t i o n a l R e s e a r c h C o u n c i l o f Canada. We a r e a l s o g r a t e f u l t o D r . P h i l l i p R u e f f e l , T e c h n i c a l Manager, S a s k a t c h e w a n M i n e r a l s Company, f o r s u p p l i e s o f m i c r o b i a l b l o o m f r o m Durum L a k e .
Abstract This is a study of the anaerobic growth and interaction in membrane-separated cultures of a red photosynthetic sulfide oxi dizer identified as Chromatium warmingii and a colourless, nonphotosynthetic sulfate reducer, tentatively placed with Desulforistella. The microorganisms were obtained from purple bloom in a sodium sulfate lake, Durum Lake, Saskatchewan. Each organism grew much better with interaction than in pure culture. The micro bial system was autotrophic, carbonate being the sole source of carbon. Sulfide was utilized by the Chromatium and formed by the Desulforistella. The system fits van Niel's (1) description of a primitive cycle of matter. The Chromatium contained 62 to 70% protein, while the Desulforistella contained only 32 to 38% pro tein. These results suggest an autotrophic source of single c e l l protein, and also may have some bearing on the early formation of petroleum. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.
van Niel, C.B., Methods in Enzymology (1971) 23, 3-28. Pfennig, Ν., Annual Review of Microbiology (1967) 21, 285-324. Smith, B.S., M.A.Sc. Thesis (1977), Department of Chemical Eng ineering and Applied Chemistry, University of Toronto, Toronto. Arciero, G., Thesis (1977), Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada. Lapage, S.P., Shelton, J.E. and Mitchell, T.G., Methods in Microbiology, 3A, 119-120, Media Tables No. 76. Butlin, K.R., Adams, M.E. and Thomas, Μ., J . Gen. Microbiol. (1948) 3, 46-59. Postgate, J.R., J . Gen. Microbiol. (1950) 5, 714-724; Bacteriol. Rev. (1965) 29, 425-441. Hvid-Hansen, Ν., Acta Pathologica Microbiol. Scand. (1951) 29, 314-334. van Gemerden, Η., Arch. Mikobiol. (1968) 64, 103-110.
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10. Herbert, D., Phipps, P . J . and Strange, R.E., Methods in Microbiology (1971) 56, 244-248. 11. Trüper, H.G. and Schlegel, H.G., Antonie van Leeuwenhoek (1964) 30, 225-238. 12. Bergey's Manual of Determinative Microbiology, Eighth Edition, 1974. 13. Protein-calorie Advisory Group of the United Nationls Organization, PAG Bulletin (1976) 6(3), September. 14. ZoBell C.E., Science (1945) 102, 364-369. RECEIVED July 25, 1978.
