Polysaccharide Formation by a

5 Polysaccharide Formation by a Methylomonas

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 6, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch005

ΚΑΙ T. ΤΑΜ and R. K. FINN School of Chemical Engineering, Cornell University, Ithaca, NY 14853

Extracellular microbial polysaccharides show great diversity as well as novelty in their structures and properties ( 1 ) . The applications of some of these biopolymers as stabilizers, emulsifiers, or thickeners in foods; as additives for recovery of pe troleum by water flooding; as plasma extenders or as selective adsorbents in laboratory research, are well documented (2,3,4). A new polysaccharide-producing bacterium called Methylomonas mucosa NRRL B-5696, was isolated from s o i l as an obligate methylotroph and the batch production of polymer and some of its prop erties have been described (5,6). Kinetics for growth of the cells and for polymer production in shake flasks and chemostats are reported here. Materials and Methods The bacteria were maintained on agar plates with a 3% (v/v) methanol basal medium which contained 3.0 g ΚΗ ΡO , 3.7 g Na HPO , 2.5 g NaNO , 0.4 g MgSO ·7 H O, 0.07 g Fe (NH SO ) , 0.025 g Ca (NO ) ·4 H O, 0.001 g ZnSO ·H O, in one liter of d i s t i l l e d water. Methanol concentration was determined by a gas chromatograph with a flame ionization detector using ethanol as the internal standard. Cell dry weight was calibrated against a modified Lowry's protein assay (7), and the latter was used for routine measurements. Polysaccharide concentration was expressed as glu cose equivalent by the phenol-sulfuric acid method of Dubios et. al. (8) with D-glucose as standard. Effluent gas composition was analyzed by a Fisher-Hamilton gas partitioner, model 29, using helium as a carrier gas. Dissolved oxygen measurements were made with membrane probes constructed as described by Johnson and Borkowski (9, 10). Polymer was recovered by acetone precipita tion (11). Viscosity was measured in a Brookfield SynchroLectric Viscometer, model LVT with U. L. adaptor. Fermenter broths diluted in the range 1:5 to 1:10, were f i r s t degassed in vacuum, and then viscosity measurements for each dilution were made at various shearing rates. In some cases, viscosity of the 2

3

3 2

2

4

2

4

4

4

2

4 2

2

58

Sandford and Laskin; Extracellular Microbial Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

4

5.

ΤΑΜ

Polysaccharide

A N D FINN

Formation

by a Methylomonas

59

f i n a l b r o t h was d e t e r m i n e d a t a s h e a r i n g r a t e o f 30 RPM w i t h a No. 3 s p i n d l e u s i n g 150 m l o f b r o t h c o n t a i n e d i n a 200 m l b e a k e r . M e t h a n o l i s a t o x i c s u b s t r a t e f o r b a c t e r i a ; e v e n f o r metha n o l u t i l i z i n g o r g a n i s m s a c o n c e n t r a t i o n b e l o w l.O/ may i n h i b i t t h e g r o w t h o f many s t r a i n s (12,13). T h e r e f o r e t h e e f f e c t o f meth a n o l c o n c e n t r a t i o n o n t h e g r o w t h o f M. mucosa was s t u d i e d i n shake f l a s k s . To do t h i s , 250 m l p o r t i o n s o f l o w p h o s p h a t e m e d i um ( b a s a l medium b u t w i t h o n l y h a l f t h e amount o f p h o s p h a t e ) i n 1 - l i t e r i n d e n t e d f l a s k s were i n o c u l a t e d w i t h s e e d f r o m a chemos t a t o p e r a t i n g a t a d i l u t i o n r a t e o f 0.25 h r " a n d a t a s t e a d y s t a t e e f f l u e n t m e t h a n o l c o n c e n t r a t i o n o f 1.0 v/v$>. M e t h a n o l c o n c e n t r a t i o n s i n t h e r a n g e 0.1k t o 2.0/o ( v / v ) were i n v e s t i g a t e d . S p e c i f i c g r o w t h r a t e s a t 30°C a n d 350 RPM r o t a t i o n o f t h e s h a k e r i n c u b a t o r were d e t e r m i n e d i n t h e t i m e p e r i o d when t h e maximum change i n t h e m e t h a n o l c o n c e n t r a t i o n was l e s s t h a n 10$> o f t h e i n i t i a l value i n the f l a s k . The s p e c i f i c g r o w t h r a t e a s a f u n c t i o n o f m e t h a n o l c o n c e n t r a t i o n was t h e n p l o t t e d .


0

1

R e s u l t s and

Discussion

K i n e t i c s o f Growth. The e x p o n e n t i a l g r o w t h d a t a f r o m shake f l a s k s i n d i c a t e t h a t m e t h a n o l c o n c e n t r a t i o n s above 1$ v / v a r e i n h i b i t o r y ( F i g u r e 1). F u r t h e r m o r e , a L i n e w e a v e r - B u r k p l o t shows t h a t a t c o n c e n t r a t i o n s l e s s t h a n 1$ t h e d a t a f i t a Monod m o d e l f o r s u b s t r a t e - l i m i t e d g r o w t h . The e x t r a p o l a t e d m a x i m a l s p e c i f i c g r o w t h r a t e , \x ., f r o m F i g u r e 2 i s 0.725 h r " , ( e q u i v a l e n t t o a g e n e r a t i o n t i m e o f 0.956 h r ) . T h i s i s a b o u t 3 t i m e s h i g h e r t h a n t h e a v e r a g e v a l u e f o r most o f t h e m e t h a n o l u t i l i z i n g b a c t e r i a r e p o r t e d i n t h e l i t e r a t u r e (11), a n d i s a b o u t t w i c e t h a t o f P s e u domonad C ( ώ ) , t h e f a s t e s t g r o w i n g m e t h a n o l b a c t e r i a r e p o r t e d . S u c h a f a s t g r o w t h r a t e makes M. mucosa a t t r a c t i v e a s a n o t h e r bacterium f o r s i n g l e - c e l l p r o t e i n production. The h i g h s p e c i f i c g r o w t h r a t e o b s e r v e d i n s h a k e f l a s k s was l a t e r c o n f i r m e d b y a c e l l w a s h o u t e x p e r i m e n t i n a c h e m o s t a t , where t h e m a x i m a l s p e c i f i c g r o w t h r a t e was m e a s u r e d a s 0.719 h r " . The o t h e r k i n e t i c c o n s t a n t , Ks i n t h e Monod m o d e l , was f o u n d t o b e 0.20 M m e t h a n o l . T h i s v a l u e i s two o r d e r s o f m a g n i t u d e l a r g e r t h a n t h e v a l u e o f 0.00375M (120 m g / l ) r e p o r t e d f o r H a n s e n u l a p o l y m o r p h a - a t h e r m o p h i l i c m e t h a n o l - u t i i i z i n g y e a s t whose g r o w t h k i n e t i c s a l s o f i t t h e Monod m o d e l (15). Recent s t u d i e s on the growth o f Candida b o i d i n i i , another m e t h a n o l - u t i l i z i n g yeast, show a K v a l u e a s h i g h a s 0.02M ( l 6 ) . No o t h e r l i t e r a t u r e v a l u e s o f K s f o r m e t h a n o l - a s s i m i l a t i n g b a c t e r i a a r e a v a i l a b l e f o r compar ison. The v a l u e o f K o b t a i n e d i s a l s o much h i g h e r t h a n v a l u e s o b t a i n e d f o r m i c r o b i a l g r o w t h o n o t h e r c a r b o n s o u r c e s , w h i c h gen e r a l l y r a n g e f r o m 1 t o 50 m g / l (17). S i n c e M. mucosa i s sub c u l t u r e d i n yjo m e t h a n o l - s a l t s medium w h i c h i s i n h i b i t o r y f o r most o t h e r m e t h a n o l - a s s i m i l a t i n g b a c t e r i a , t h e b a c t e r i u m must h a v e d e v e l o p e d a t r a n s p o r t mechanism t h a t r e g u l a t e s a slow p e r m e a t i o n of substrate i n t o the c e l l i n order t o reduce the i n h i b i t o r y 1

m

1

s

s



60

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Figure 1. Specific growth rate at different initial sub strate concentrations

Figure 2. Lineweaver-Burk plot for the specific growth rate data

-L

( % Μ · ! Η α η · Ι )r l


5.

ΤΑΜ

A N D FINN

Polysaccharide

Formation

by a Methylomonas

61

e f f e c t o f t h e methanol. A l s o t h e p o l y s a c c h a r i d e s l i m e i s an a d d i t i o n a l b a r r i e r f o r t h e d i f f u s i o n o f m e t h a n o l i n t o t h e c e l l . The h i g h K i m p l i e s t h a t a low o v e r a l l a f f i n i t y f o r methanol should be e x p e c t e d . The good agreement o f t h e e x t r a p o l a t e d μ w i t h t h e w a s h o u t datum adds c o n f i d e n c e t o t h e a c c u r a c y o f t h e k i n e t i c c o n stants. The i m p l i c a t i o n o f s u c h a h i g h v a l u e f o r K g i s t h a t a s t a b l e r e a c t o r c a n be o p e r a t e d a t a d o u b l i n g t i m e as s h o r t as 1.8 h r f o r M. mucosa i n a c a r b o n - l i m i t e d c h e m o s t a t . F i g u r e 3 shows t h a t d a t a f o r t h e s u b s t r a t e - i n h i b i t o r y r e g i o n f i t t h e model, s


ηι

μ

where

μ

=

1 + S/K.

= 6.05 h r "

(

1

)

1

Κ. = 1 8 Λ m M o l a r ι The f a c t t h a t d a t a f i t t h e t w o - p a r a m e t e r m o d e l s does n o t t e l l u s t h e e x a c t mechanism o f i n h i b i t i o n o r growth s t i m u l a t i o n a t t h e molecular l e v e l . However, n o f u r t h e r e x p e r i m e n t s were done t o e l u c i d a t e t h e mechanism o r s i t e o f i n h i b i t i o n b e c a u s e t h e p r i m e o b j e c t i v e o f d e t e r m i n i n g t h e safe o p e r a t i o n range f o r a carbonl i m i t e d c h e m o s t a t was o b t a i n e d i n t h i s s e t o f e x p e r i m e n t s . Respiration Kinetics. From t h e d e p l e t i o n r a t e o f d i s s o l v e d o x y g e n a n d a n a v e r a g e c e l l mass o f 0.152 mg i n t h e Y e l l o w S p r i n g s D i s s o l v e d Oxygen m o n i t o r i n g chambers, t h e s p e c i f i c r e s p i r a t i o n r a t e s were c a l c u l a t e d f o r d i f f e r e n t i n i t i a l m e t h a n o l c o n c e n t r a tions. I n t h e absence o f s u b s t r a t e i n h i b i t i o n , M i c h a e l i s - M e n t e n k i n e t i c s f i t t h e r e s p i r a t i o n r a t e d a t a as i n d i c a t e d b y t h e s t r a i g h t l i n e i nt h e Lineweaver-Burk p l o t (Figure 4). The c e l l s demonstrate a h i g h a f f i n i t y f o r m e t h a n o l as s u g g e s t e d b y t h e l o w v a l u e o f Κ , 8.1 pmolar methanol. The maximum r e s p i r a t i o n r a t e ( e x t r a p o l a t e d ) i s 33 mMole 0 / ( g c e l l , h r ) , w h i c h i s s l i g h t l y h i g h e r t h a n t h e a v e r a g e v a l u e o f 2 6 . 6 mMole 0 / ( g , h r ) o b t a i n e d from an oxygen balance i n t h e chemostat o p e r a t i n g w i t h a s t e a d y s t a t e e f f l u e n t m e t h a n o l c o n c e n t r a t i o n b e t w e e n 0.6$ a n d 1.5$ (ν/ )· The l o w e r c h e m o s t a t v a l u e o f V m i g h t be due t o s u b s t r a t e i n h i b i tion. Compared w i t h l i t e r a t u r e v a l u e s ( T a b l e I ) , M. mucosa h a s a K i n t h e same o r d e r o f m a g n i t u d e a s H y p h o m i c r o b i u m . The m a x i m a l s p e c i f i c r e s p i r a t i o n r a t e i s about t w i c e t h e h i g h e s t r a t e l i s t e d i n t h e Table. A h i g h e r r e s p i r a t i o n r a t e i s e x p e c t e d f o r M. mucosa because o f i t s v e r y h i g h s p e c i f i c growth r a t e . Another piece o f evidence t h a t agrees w i t h t h e e x t r a p o l a t e d s p e c i f i c r e s p i r a t i o n r a t e comes f r o m s e p a r a t e b a t c h e x p e r i m e n t s . A t t h e p o i n t when t h e d i s s o l v e d o x y g e n r e a c h e s z e r o , t h e o x y g e n demand c a l c u l a t e d f r o m t h e s p e c i f i c r e s p i r a t i o n r a t e s h o u l d j u s t equal t h e o x y g e n 2

2

ν

m

r


MICROBIAL

POLYSACCHARIDES


EXTRACELLULAR


5.

ΤΑΜ

A N D FINN

Polysaccharide

Formation

63

by a Methylomonas

supplied. The o x y g e n s u p p l y , b a s e d o n g a s c h r o m a t o g r a p h i c a n a l y s i s o f t h e i n f l u e n t a n d e f f l u e n t g a s , was k6k0 mg 0 / h r , a n d t h e p r e d i c t e d o x y g e n demand b a s e d o n t h e above r e s p i r o m e t e r d a t a ( 33 mMole 0 p e r g c e l l p e r h r ) was 4780 mg Cg/hr. The endogeneous r e s p i r a t i o n r a t e i n m e t h a n o l - f r e e medium i s 1.21 ± 0.05 mMole 0 / ( g c e l l , h r ) w h i c h a g r e e s w i t h t h e a v e r a g e endogeneous r a t e o f 1.3 ± 0.3 mMole 0 / ( g c e l l , h r ) o b t a i n e d i n t h e Y e l l o w S p r i n g s D i s s o l v e d - O x y g e n M o n i t o r Chamber, u s i n g c e l l s grown i n d i f f e r e n t shake f l a s k s . 2

2

2


2

Table I Michaelis-Menten K i n e t i c Constants f o r the R e s p i r a t i o n o f B a c t e r i a Growing i n Methanol Reference

Κ (μΜ)

Organism

Harrison

(l8)

Harrison

(l8)

Γ

nrg, h r

Pseudomonas e x t o r q u e n s

20Λ

10.5

methane u t i l i z i n g

50.0

7

4.15

Pseudomonad Wilkinson

(19)

Hyphomi c r o b i u m

Wilkinson

(19)

mixed c u l t u r e

T h i s work Kim

& R y u (20)

8.53 29400

M e t h y l o m o n a s mucosa

8.1

0.0215 0.024 33.0 18.0

Methylomonas sp.

C a r b o n - l i m i t e d Chemostat. The s t r a i g h t l i n e i n t h e L i n e weaver-Burk p l o t f o r t h e s p e c i f i c growth r a t e i n t h e chemostat, w i t h m e t h a n o l as t h e l i m i t i n g s u b s t r a t e , s u g g e s t s t h a t Monod-type growth k i n e t i c s f i t t h e data ( F i g u r e 5).

s = 1.43 h r

where Κ

.1

=0.583 Molar

However, t h e s e c o n s t a n t s do n o t a g r e e w i t h t h e s h a k e f l a s k d a t a , where ^ = 0.725 h r " , a n d K = 0.20 M o l a r . Such a d i s c r e p a n c y i s t o o l a r g e t o be e x p l a i n e d b y t h e s h o r t - c i r c u i t o f t h e f l o w o r o t h e r e x p e r i m e n t a l e r r o r s . The o n l y l o g i c a l e x p l a n a t i o n i s t h a t b y c o n t i n u o u s l y c u l t i v a t i n g M. mucosa i n t h e c h e m o s t a t f o r o v e r a week, some v a r i a n t was s e l e c t e d t h a t h a d a l o w e r a f f i n i t y f o r methanol and a f a s t e r growing r a t e . When t h e s p e c i f i c m e t h a n o l u t i l i z a t i o n r a t e (Q^) i s p l o t t e d a g a i n s t d i l u t i o n r a t e (D), a s t r a i g h t l i n e i s o b t a i n e d ( F i g u r e 6) 1

s



Figure 6.

Specific substrate utilization rate correlation

0.1

0.2

Dilution

0J

Rat* ,

1

Hr*


5.

Polysaccharide

Τ Α Μ A N D FINN

Formation

by a

65

Methylomonas

This r e s u l t confirms t h e v a l i d i t y o f the e m p i r i c a l equation b y P i r t (17) a n d N a g a i e t a l . (21 ) :

(3) be

F o r s t e a d y - s t a t e i n a chemostat w i t h no r e c y c l e , e q u a t i o n comes Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 6, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch005

used

x/s where

m = maintenance c o e f f i c i e n t f o r methanol = 0.26 g methanol/(g c e l l , h r ) Υ ^ = 0.3*1-5 g c e l l / g m e t h a n o l χ

3

The e x t r a p o l a t e d m i s t h e same o r d e r o f m a g n i t u d e as t h a t r e p o r t e d f o r A. v i n e l a n d i i (22 ), b u t a n o r d e r o f m a g n i t u d e h i g h e r t h a n t h a t f o r other microorganisms (Table II). U n l i k e most o f t h e m i c r o o r g a i s m s l i s t e d , ( w i t h t h e e x c e p t i o n o f A. v i n e l a n d i i w h i c h f o r m s p o l y - b e t a - h y d r o x y b u t y r i c a c i d ) M. mucosa p r o d u c e s e x t r a c e l l u l a r polysaccharides i n a d d i t i o n t o c e l l t i s s u e s and carbon d i o x i d e . The f o r m a t i o n o f e x t r a s t o r a g e p r o d u c t o r p o l y m e r r e q u i r e s more carbon uptake, and t h e r e f o r e a h i g h e r value o f t h e c e l l mainte n a n c e c o e f f i c i e n t s h o u l d b e e x p e c t e d f o r A. v i n e l a n d i i a n d M. mucosa a s i n d i c a t e d i n T a b l e 2. The e x p e r i m e n t a l y i e l d c o e f f i c i e n t Y / = 0.3^5 g c e l l / g CH3OH i s q u i t e r e a s o n a b l e , b e c a u s e M a t e l e s e t a l . r e p o r t e d Y / = 0.31 f o r t h e i r polymer-producing Pseudomonad C i n shake f l a s k s (24) a n d Y / = 0 . 5 4 i n a c h e m o s t a t t h a t f a v o r e d c e l l p r o d u c t i o n (iJT). The c e l l y i e l d s o f o t h e r m e t h a n o l - u t i l i z i n g b a c t e r i a , w i t h no polymer p r o d u c t i o n , range f r o m 0 . 2 t o 0 . 4 (11). H a g g s t r o m (2£) e s t i m a t e d t h a t f o r h i s m e t h a n o l - u t i l i z i n g b a c t e r i a , t h e e f f i c i e n c y o f transforming the carbon from methanol i n t o t h e c a r b o n i n c e l l s w o u l d be 4 l $ ( C M c - m a s s / m e t h a n o l ) · we assume t h e c o m p o s i t i o n o f M. mucosa i s C 5 H 8 O 3 N a n d c a l c u l a t e the e f f i c i e n c y o f carbon t r a n s f o r m a t i o n t o biomass from the exper i m e n t a l y i e l d Y / = Ο.345, t h e e f f i c i e n c y i s 42.7$, about t h e same a s t h e number a s o b t a i n e d b y H a g g s t r o m . The e m p i r i c a l f o r mula C 5 H 8 O 3 N i s used i n s t e a d o f the formula o f C H80 N based on Hamer a n d J o h n s o n s d a t a b e c a u s e t h e l a t t e r p r e d i c t s 13-7$ i n t h e c e l l , w h e r e a s t h e n i t r o g e n c o n t e n t o f M. mucosa i s 11.0 + 0 . 5 $ a v a l u e i n c l o s e r agreement w i t h t h e f o r m u l a C 5 H 8 O 3 N (Table 3). The a v e r a g e y i e l d c o e f f i c i e n t s Y n / = 0.175 a n d Y"c0 /s = 0.483 a r e c o n s t a n t w i t h i n t h e r a n g e o r d i l u t i o n r a t e s s t u d i e d (Figure 7). I f t h e hypothesis i s c o r r e c t , t h a t t h e carbon i n t h e methanol o n l y t u r n s i n t o c e l l s , polymer and carbon d i o x i d e then a c a r b o n b a l a n c e b a s e d o n t h e sum o f t h e t h r e e y i e l d c o e f f i c i e n t s x

S

x

S

x

S

c

x

I

S

4

2

1

S

2


f


methane

methanol

Methane m i x b a c t e r i a (23)

Methylomonas mucosa

oxygen

glycerol

glucose

(22)

(22)

Saccharomyces c e r e v i s i a e (22)

Azotobacter vinelandii

Aerobacter aerogenes

Organism

Limiting Factor

methanol

methane

glucose

glucose

glycerol

Substrate

0.26

0.12

0.02

0.15

0.08

m g sub — cell, hr

Growth Y i e l d and Maintenance C o e f f i c i e n t s and O t h e r M i c r o o r g a n i s m s

Table I I

0.345

0.7

O.5O

0.26

0.56

x/s g cell g sub.

f o r M^ mucosa

2

0.039

0.06

0.02

0.18

0.10

g c e l l , hr

g o

ο


0.425

Ο.38

1.10

0.4l

2

cell

g o 0.94

g

M

o

>

η

>

CO

o

S W >

53

>

r r

ο M

>


Haggstrom

Harrison et a l .

2.4

7.1

6.15

11.4

11.0

io.9

io.8

45.Ο

47.5

46.2

methanol

average :

methanol

36.9

32.12

ash

48.0

4.0

methanol

30.1

7.0

11.0

5

8

3

(28)

(27)

(23)

(26)

assuming a f o r m u l a of C H 0 N

(25)

Hamer e t a l .

Sheehan e t a l .

47-9

Ρ

methane

1.62

29.44

7.14

11.7

50.1

Vary & Johnson

Reference

methane

-

36.72

7.1

Other Elements

9.48

0

Η

Ν

46.7

C

methane

Substrate

E l e m e n t a l A n a l y s e s o f Methane a n d M e t h a n o l U t i l i z i n g Bacteria

Table I I I


68

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

s h o u l d add up t o u n i t y . I n o r d e r t o do t h e c a r b o n b a l a n c e , t h e f o l l o w i n g two a s s u m p t i o n s were made: l ) t h e r e i s U6.2# c a r b o n i n t h e c e l l s as i n d i c a t e d b y t h e e m p i r i c a l f o r m u l a C5H8O3ÏÏ and 2 ) t h e r e i s 4θ$> c a r b o n i n t h e p o l y m e r . Since the polymer i s a h e t e r o p o l y s a c c h a r i d e , t h e g e n e r a l f o r m u l a f o r c a r b o h y d r a t e CH 0 i s a good a p p r o x i m a t i o n . The o v e r a l l c a r b o n b a l a n c e comes o u t t o b e (Figure 7): 2

ν fys Ô 3 7 5


ο

Λ

ν 0^62 . V s Ô375

ο

+

v Y

0.2725 C 0 / s 0.375

n =

2

s °* Q

R

9 6 5

S t r i p p i n g o f t h e v o l a t i l e m e t h a n o l o r a t r a c e amount o f b y p r o d uct f o r m a t i o n d u r i n g f e r m e n t a t i o n , s u c h as t h e y e l l o w p i g m e n t , w i l l a c c o u n t f o r t h e 3· 5$ d i s c r e p a n c y i n t h e c a r b o n b a l a n c e . Thus t h e d a t a a r e i n t e r n a l l y c o n s i s t e n t . To a c c o u n t f o r a l l t h e y i e l d d a t a , t h e f o l l o w i n g s t o i c h i o m e t r i c e q u a t i o n can be w r i t t e n ( l l ) 22

CH3OH+I5.5 0 + 2 N 0 ~ 2 H 2

^ 2 C H 0 N + 4 CH 0+33 H 0

+

3

5

8

2

3

2

T h i s e q u a t i o n p r e d i c t s Y / = 0.171, Y / = Ο . 3 6 9 , Y c o / s = 0 . 5 0 . These numbers a g r e e w i t h t h e Y / = 0.175, Y / = 0.3^5, Y C 0 / s = 0.483 o b t a i n e d f r o m t h e e x p e r i m e n t . The e x p e r i m e n t a l p o l y m e r y i e l d o f 17· 5$ i - "too l°w £ ° a n y p r a c t i c a l polymer p r o d u c t i o n process. However, p r e v i o u s s h a k e f l a s k experiments performed w i t h n i t r o g e n l i m i t a t i o n suggested t h a t t h e p o l y m e r y i e l d c o u l d be i m p r o v e d a t t h e e x p e n s e o f c e l l yield (ll). The f e a s i b i l i t y o f a c o n t i n u o u s p o l y m e r p r o d u c t i o n scheme w i t h n i t r o g e n a s t h e l i m i t i n g s u b s t r a t e w i l l be i n v e s t i gated i n the f o l l o w i n g section. p

S

x

p

S

S

2

x

S

2

s

r

N i t r o g e n - l m i t e d C h e m o s t a t . To a c h i e v e n i t r o g e n - l i m i t e d g r o w t h , 1 g/L N a N 0 was u s e d i n t h e f e e d and t h e f l o w r a t e o f medium was a d j u s t e d s o t h a t t h e c e l l d e n s i t y was 1.63 ± 0 . 0 3 g c e l l / L f o r a l l t h e d i l u t i o n r a t e s ( 0 . l 4 t o Ο . 3 2 h r " i ) . The g r o w t h o f t h e c e l l s was n o t o x y g e n l i m i t e d s i n c e t h e d i s s o l v e d oxygen,D. 0., was a l w a y s more t h a n t h a t e q u i v a l e n t t o 30$ a i r s a t u ration. The s p e c i f i c m e t h a n o l u t i l i z a t i o n r a t e , Qj^, r e m a i n e d c o n s t a n t i n s t e a d o f i n c r e a s i n g l i n e a r l y w i t h d i l u t i o n r a t e as was t h e c a s e f o r c a r b o n - l i m i t e d g r o w t h ( F i g u r e 6 ) . The a v e r a g e i s 0.97 + 0.015 g m e t h a n o l / ( g c e l l , h r ) . S i n c e t h e c e l l c o n c e n t r a t i o n a n d t h e Çfa were e s s e n t i a l l y c o n s t a n t f o r a l l t h e d i l u t i o n r a t e s i n v e s t i g a t e d , an i n c r e a s e i n r e s i d e n c e time i m p l i e d t h a t more m e t h a n o l w o u l d be c o n v e r t e d i n t o p o l y m e r . Thus t h e s p e c i f i c p o l y m e r p r o d u c t i o n r a t e , Qp, s h o u l d i n c r e a s e l i n e a r l y w i t h r e s i d e n c e t i m e , and t h i s i s shown i n F i g u r e 8 where p o l y m e r f o r m a t i o n i s e x p r e s s e d b o t h as g l u c o s e e q u i v a l e n t , Qg, and a l s o as d r y w e i g h t , Q ,. The d a t a f o r Q a r e s c a t t e r e d b e c a u s e o f e r r o r s i n d r i e d weight determinations. However, t h e s l o p e s o f Qg and Q s h o u l d b e t h e same, and a t z e r o r e s i d e n c e t i m e , n o 3

p

p

p



5.

Τ Α Μ AND

Polysaccharide Formation

FINN

.3 Dilution

Figure 7.

.4 rat» ,

Hr."

RISIDENCI

Figure 8.

by a Methylomonas

TIM!

69

Yield coefficients for the car bon-limited chemostat

(hr)

9

Polymer production in nitrogen-limited chemostat


70

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

p o l y m e r s h o u l d be p r o d u c e d . A s t r a i g h t l i n e w i t h s l o p e p a r a l l e l t o Qg a n d w i t h z e r o i n t e r c e p t i s drawn t h r o u g h t h e d a t a f o r t h e s p e c i f i c polymer p r o d u c t i o n r a t e . F o ra given c e l l population (X g / l ) , t h e t o t a l amount o f p o l y m e r f o r m e d u n d e r n i t r o g e n - l i m i t ed c o n d i t i o n s i s g i v e n b y : t

Op d0 = 0.035X ( t | - t f ) ( g p o l y m e r / 1 )

X Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 6, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch005

2

Jtx The y i e l d s o f c a r b o n d i o x i d e b a s e d o n m e t h a n o l consumed d i d n o t v a r y w i t h d i l u t i o n r a t e , D. The a v e r a g e v a l u e o f Y c o / s °.47 a g r e e s w e l l w i t h t h e a v e r a g e v a l u e o f 0.48 f o r t h e c a r t o n - l i m i t i n g c a s e . However Yp/s a n d Υχ/s v a r i e d i n v e r s e l y w i t h e a c h o t h e r as d i l u t i o n r a t e was c h a n g e d ( F i g u r e 9)· A t D=0, t h e e x t r a p o l a t e d Υχ/s i s z e r o a n d Yp/s i s Ο . 5 6 , w h i c h s u g g e s t s t h a t t h e maximum y i e l d f o r t h e p o l y m e r i s a b o u t 56$ o f t h e m e t h a n o l consumed. This a p p a r e n t h i g h p r o j e c t e d y i e l d m i g h t n o t be a t t a i n a b l e i n p r a c t i c e because o f d i s s o l v e d oxygen l i m i t a t i o n d u r i n g t h e polymer forma t i o n phase. E v e n i f t h e s y s t e m were o p e r a t e d a t h a l f t h e maximum y i e l d , s a y a t Y / = 0 . 2 8 , t h e p e r f o r m a n c e w o u l d s t i l l be b e t t e r t h a n f o r t h e c a r o o n - l i m i t e d c a s e where Y / s 0-175The y i e l d data s t r o n g l y suggest use o f a n i t r o g e n - l i m i t i n g process f o r p o l y mer p r o d u c t i o n . A c h e c k f o r c o n s i s t e n c y o f t h e d a t a was made b y t a k i n g a c a r b o n b a l a n c e w i t h t h e same a s s u m p t i o n s a s b e f o r e , i . e . 4 6 . 2 $ c a r b o n i n c e l l s a n d 40$ c a r b o n i n p o l y m e r . =

2

p

S

=

p

Y

p / s

(i.o 8) 5

+

x

x / s

(i.2 2) 5

+

Y

C

0

2

/

S

(O.T26)

=c

The a v e r a g e v a l u e f o r C t u r n e d o u t t o be 0.990 i n s t e a d o f 0. 965 as i n t h e c a r b o n - l i m i t i n g c a s e . I n o t h e r w o r d s , t h e r e was o n l y 1$ e r r o r i n t h e c a r b o n b a l a n c e . When Yp/s i s z e r o , t h e e x t r a p o l a t e d maximum c e l l y i e l d Y / s i s 0-555Assuming t h a t t h e carbon d i o x i d e y i e l d remains c o n s t a n t a t 0.47 i n t h e a b s e n c e o f p o l y m e r f o r m a t i o n , a c a r b o n b a l a n c e g i v e s a v a l u e o f C a s 1.02, i . e . 2$ e r r o r i n t h e c a r b o n b a l a n c e when o n l y c e l l s a n d C 0 a r e formed. The c o i n c i d e n c e o f t h e m a x i mum v a l u e s f o r t h e y i e l d c o e f f i c i e n t s Υχ/s = 0.56 a n d Yp/s = 0.555 s u g g e s t s t h a t t h e e n e r g y d e r i v e d f r o m c a t a b o l i c p r o c e s s e s i s u s e d w i t h a p p r o x i m a t e l y t h e same maximum e f f i c i e n c y f o r t h e b i o s y n t h e s i s o f e i t h e r c e l l s o r polymer. I n f a c t , these y i e l d d a t a agree c l o s e l y w i t h t h e p r e d i c t i o n s based on " t h e o r e t i c a l " m o l a r g r o w t h y i e l d s f r o m A T P (29). From gas c h r o m a t o g r a p h i c a n a l y s i s , t h e e f f l u e n t a i r h a d a n a v e r a g e c o m p o s i t i o n o f Ο . 6 7 ± 0.02$ c a r b o n d i o x i d e a n d 19-4 ± 0.15$ oxygen. By an oxygen and carbon d i o x i d e b a l a n c e , t h e r e s p i r a t o r y q u o t i e n t (R.Q.) was f o u n d t o be 0 . 4 l 8 m o l e C 0 / m o l e 0 . The a v e r a g e s p e c i f i c o x y g e n c o n s u m p t i o n r a t e was 2 6 . 6 m m o l e x

2

2


2

5.

ΤΑΜ

Polysaccharide

A N D FINN

Formation

by a Methylomonas

71

°2/(g c e l l , h r ) , w h i c h comes c l o s e t o t h e e x t r a p o l a t e d maximum v a l u e o f 33 m m o l e 0 / ( g c e l l , h r ) f r o m t h e p r e v i o u s r e s p i r a t i o n study. An i n t e r e s t i n g f l o c c u l a t i o n phenomenon was o b s e r v e d a t t h e h i g h d i l u t i o n r a t e s (F>1.2 l / h r ) . C e l l s tended t o f l o c c u l a t e and s e t t l e much f a s t e r u p o n s t a n d i n g i n a t e s t t u b e a t room t e m p e r a ture. However, a f t e r a s h i f t t o l o w d i l u t i o n r a t e where more p o l y m e r was p r o d u c e d , t h e f l o c c u l a t i n g phenomenon d i s a p p e a r e d . There are two p o s s i b l e e x p l a n a t i o n s : e i t h e r a mutant i s formed o r t h e f l o c c u l a t i o n i s due t o a c o n c e n t r a t i o n e f f e c t o f t h e p o l y m e r . Only a t a p a r t i c u l a r c o n c e n t r a t i o n o f the a n i o n i c polymer t h a t t h e i n t e r a c t i o n b e t w e e n t h e f i x e d amount o f c e l l a n d t h e c o l l o i d a l p h o s p h a t e c a t i o n c o m p l e x i n t h e b a s a l medium w o u l d b r i n g t h e system t o the i s o e l e c t r i c p o i n t and r e s u l t i n agglomeration and precipitation. A g a r p l a t e s i n o c u l a t e d w i t h t h e p r e c i p i t a t i n g c e l l s gave t h e same t y p e o f c o l o n y as t h e n o r m a l c e l l s . A l s o the r a p i d r e v e r s i b i l i t y o f t h e c o a g u l a t i n g phenomenon a c h i e v e d b y c h a n g i n g t h e d i l u t i o n r a t e ( i . e . t h e amount o f p o l y m e r f o r m e d ) s u g g e s t s t h a t the second reason provides a b e t t e r e x p l a n a t i o n .


2

Hon-growth A s s o c i a t e d C o e f f i c i e n t . I t i s apparent from the n i t r o g e n - l i m i t e d growth data t h a t polymer p r o d u c t i o n i s nongrowth a s s o c i a t e d . I n order t o t e s t the e x t r a p o l a t e d polymer y i e l d d a t a (Yp/s °·56) f o r a non-growth s i t u a t i o n ( Y / = θ ) and to f i n d c o e f f i c i e n t f o r non-growth a s s o c i a t e d polymer p r o d u c t i o n i n t h e L u e d e k i n g (30 ) e q u a t i o n , d P / d t = a d X / d t + bX, a shake f l a s k e x p e r i m e n t was. done u s i n g w a s h e d c e l l s . D i f f e r e n t amounts o f w a s h e d c e l l s s u s p e n d e d i n p h o s p h a t e b u f f e r were u s e d t o i n o c u l a t e n i t r o g e n - f r e e b r o t h i n i n d e n t e d f l a s k s c o n t a i n i n g 1.29$ m e t h a n o l . The p o l y m e r p r o d u c t i o n r a t e s were l i n e a r f o r t h e f i r s t s i x t o e i g h t h o u r s b u t d e c r e a s e d when t h e t i m e o f i n c u b a t i o n i n c r e a s e d beyond f o u r generation times. The i n i t i a l p o l y m e r p r o d u c t i o n r a t e was p l o t t e d a g a i n s t t h e d r i e d c e l l w e i g h t . A s t r a i g h t l i n e was o b t a i n e d as shown i n F i g u r e 10. The n o n - g r o w t h a s s o c i a t e d c o e f f i c i e n t , b, o b t a i n e d from t h e s l o p e i s Ο.39 g polymer ( g c e l l , h r ) . The a v e r a g e p o l y m e r y i e l d f o r t h e f i v e f l a s k s was 0.59 ± 0.15 w h i c h a g r e e s w e l l w i t h t h e e x t r a p o l a t e d v a l u e o f 0.56 f r o m F i g u r e 9. The r e l a t i v e l y l a r g e e r r o r i n t h e Yp/s c a l c u l a t i o n i s due t o t h e s m a l l q u a n t i t i e s o f m e t h a n o l consumed i n t h e f i r s t s i x h o u r s ; a d i f f e r e n c e o f 0.01$ m e t h a n o l c o n t e n t w o u l d g i v e 1 0 $ e r r o r i n =

x

S

V*· N i t r o g e n - l i m i t i n g Batch. Based on the p r e v i o u s o b s e r v a t i o n s , a p o l y m e r p r o d u c t i o n scheme w i t h p e r i o d i c n i t r o g e n s t a r v a t i o n was investigated. A b a s a l medium c o n t a i n i n g 1.5 g/L N a N 0 was u s e d and t w o p u l s e s o f a d d i t i o n a l c a r b o n a n d n i t r o g e n (65 m l m e t h a n o l and 2.15 g ammonium s u l f a t e ) were a d d e d a t 9 l / h o u r s a n d a t 23 hours a f t e r the i n i t i a t i o n o f the batch run. For these e x p e r i ments t h e Magnaferm f e r m e n t o r h a d a n a e r a t i o n r a t e o f 5 l i t e r s o f 3

2


72

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Cproducts


Cmethanol

Figure

9.

Yield

coefficients in nitrogen-limited chemostat

.25,

Figure

10. Polymer formation washed cell suspension

rate in

Dried Cell

(g/i)


5.

ΤΑΜ

AND

Polysaccharide

FINN

Formation

by

a Methylomonas

73

a i r p e r m i n u t e , and a s t i r r e r s p e e d o f 800 REM. For t h e e x p o n e n t i a l growth phase, the s p e c i f i c c e l l growth r a t e was 0.278 h r " (ta. = 2.5 h r ) w h i c h s h i f t e d t o 0.102 h r " u p o n the a d d i t i o n o f t h e f i r s t p u l s e o f carbon and n i t r o g e n . The l o w c e l l p r o d u c t i o n r a t e was l a r g e l y due t o d i s s o l v e d o x y g e n l i m i t a t i o n , as shown i n F i g u r e 11. The c o n c e n t r a t i o n o f d i s s o l v e d o x y gen r e m a i n e d z e r o a f t e r t h e c o n s u m p t i o n o f lfo m e t h a n o l . The f i r s t - o r d e r r a t e c o n s t a n t f o r g l u c o s e p r o d u c t i o n i s 0.24 hr" . A l a g o f a b o u t two h o u r s was o b s e r v e d b e f o r e p r o d u c t i o n o f polymer resumed a f t e r t h e p u l s e a d d i t i o n o f c a r b o n and n i t r o g e n . S i n c e ammonium s u l f a t e was u s e d as a n i t r o g e n s o u r c e , no n i t r i t e should accumulate t o i n h i b i t polymer p r o d u c t i o n . Perhaps i t t a k e s t i m e f o r M. mucosa t o a d j u s t t o t h e c o n c e n t r a t i o n s h o c k p r o d u c e d b y a s t e p i n c r e a s e o f m e t h a n o l t o an i n h i b i t i n g l e v e l . The i m p o r t a n t t h i n g t o note here i s t h a t the r a t e o f polymer p r o d u c t i o n does n o t d e c r e a s e a p p r e c i a b l y i n a n o n - g r o w t h s i t u a t i o n b e t w e e n t h e l 4 t h a n d 23rd h o u r s . A f t e r t h e d i s s o l v e d o x y g e n c o n t e n t r e a c h e d z e r o , t h e mass t r a n s f e r c o e f f i c i e n t Kjja, as c a l c u l a t e d f r o m o x y g e n b a l a n c e , was about c o n s t a n t . The a v e r a g e K a was 165 h r " i . With the aeration a n d s t i r r i n g p a r a m e t e r s k e p t c o n s t a n t , t h e e f f e c t o f t h e foam b r e a k e r on t h e o x y g e n mass t r a n s f e r c o e f f i c i e n t c o u l d be s e e n b y a sudden d e c r e a s e i n K^a f r o m 165 t o 120 h r " when t h e s u r f a c e o f t h e b r o t h f a i l e d t o r e a c h t h e foam b r e a k e r ( a t t h e 34th h o u r ) . The t e r m i n a t i o n o f p o l y m e r p r o d u c t i o n c o i n c i d e d w i t h t h e d r o p i n K^a v a l u e . T h i s o b s e r v a t i o n s u g g e s t s t h a t mass t r a n s f e r o f d i s s o l v e d o x y g e n may l i m i t p o l y m e r p r o d u c t i o n o r e l s e t h e r e i s a c c u mulation of t o x i c by-products i n the batch. A n o t h e r i n t e r e s t i n g p o i n t was t h e c o n t i n u e d m e t h a n o l consump t i o n a t a l i n e a r r a t e o f Ο.332 g m e t h a n o l / ( 1 , h r ) a f t e r b o t h g r o w t h and polymer s y n t h e s i s had stopped. T h i s decrease i n methanol c o u l d p e r h a p s be a c c o u n t e d f o r b y t h e c e l l m a i n t e n a n c e r e q u i r e m e n t and t h e s t r i p p i n g l o s s . The f i n a l y i e l d d a t a a t t h e e n d o f t h e 56 h o u r s w e r e : Y / = 0.118, Y / = 0.4θ8, a n d 1.897fo s o l i d p r o d u c e d f o r 4.55$> m e t h a n o l consumed. The maximum y i e l d c o e f f i c i e n t s f o r p o l y m e r p r o d u c t i o n i n t h e b a t c h s h o u l d be c a l c u l a t e d a t t h e 38th h o u r when p o l y m e r p r o d u c t i o n was t e r m i n a t e d . Thus, d i s r e g a r d i n g t h e methanol l o s s i n c e l l maintenance and s t r i p p i n g d u r i n g t h e l a s t 18 h o u r s o f i n c u b a t i o n , t h e y i e l d c o e f f i c i e n t s s h o u l d be Υχ/s =.0.122 a n d Y / = 0.452 w h i l e t h e t o t a l s o l i d y i e l d s h o u l d be 0.574. The p o l y m e r y i e l d a p p r o a c h e d t h e e x t r a p o l a t e d maximum o f Ο.56 ( F i g u r e 8 ) . 1

1


1

L

1

x

S

p

p

S

S

S e m i - c o n t i n u o u s F e r m e n t a t i o n . An a t t e m p t t o c u l t u r e t h e b a c t e r i a c o n t i n u o u s l y a t l o w d i l u t i o n r a t e was n o t v e r y s u c c e s s f u l . The y i e l d o f p o l y m e r d e c r e a s e d a f t e r one week o f c o n t i n u o u s f e r mentation. C o n t a m i n a t i o n a t l o w s t e a d y - s t a t e m e t h a n o l l e v e l and/ o r p o s s i b l y c u l t u r e d e g e n e r a t i o n became t h e m a j o r o b s t a c l e s t o s u c c e s s f u l o p e r a t i o n at low d i l u t i o n rate. Operation o f a carbonl i m i t e d chemostat a t a h i g h e r d i l u t i o n r a t e had p r e v i o u s l y r e -


MICROBIAL

POLYSACCHARIDES


EXTRACELLULAR



RPM

dt

dS

jo

hr

CH3OH

dP g g l u c o s e d t 1, h r

dX g c e l l d t 1, h r

Time, h r

.37 t o

500

.065

.033

.16

0 t o 32

Semicontinuous

First

0.4 t o

.175

500

500

350

.175

hO

.018

.014

30 t o

.038

.057

.Oik

3 t o 47

.054

hi

Semi - c o n t i n u o u s

Second C y c l e Batch

.175

550

.045

.031

.044

42 t o 64

Fermentor

.049

.109

0

33 t o

Shake flask

Cycle

Rate Constants f o r the Semi-continuous Operation i n the l4 L

Table IV



76

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

s u i t e d i n s e l e c t i n g a f a s t g r o w i n g v a r i a n t t h a t was a p o o r p o l y m e r former. I n o r d e r t o a v o i d p u t t i n g s e l e c t i v e p r e s s u r e on M. mucosa and t o i m p r o v e t h e p r o c e s s e c o n o m i c s , a s e m i - c o n t i n u o u s o p e r a t i o n scheme was t h e r e f o r e c o n s i d e r e d . The s e t - u p c o n s i s t e d o f t w o t a n k s i n s e r i e s ; t h e f i r s t f e r m e n t o r was t h e Magnaferm w h i c h s e r v e d as a c o n t i n u o u s c e l l p r o p a g a t o r o p e r a t i n g a t r e l a t i v e l y h i g h d i l u t i o n r a t e (0.42 l / h r ) . I n t h i s t a n k t h e s t e a d y s t a t e m e t h a n o l c o n c e n t r a t i o n was k e p t above lfo s o a s t o p r e v e n t g r o w t h o f c o n t a m i n a n t s . B a s a l medium w i t h 3$ m e t h a n o l a n d 2.5 g / l N a N 0 was f e d t o t h e Magnaferm c o n t i n u o u s l y . The e f f l u e n t f r o m t h e f i r s t t a n k was d i r e c t e d i n t o t h e l 4 - l f e r m e n t o r where n i t r o g e n - l i m i t e d g r o w t h began. The n i t r o g e n l i m i t a t i o n not o n l y f a v o r e d polymer formation but a l s o h e l p e d t o prevent the growth o f contaminants. A f t e r a f i x e d volume h a d a c c u m u l a t e d i n t h e s e c o n d t a n k , e f f l u e n t f r o m t h e f i r s t t a n k was w a s t e d ( o r d i v e r t e d i n t o another fermentor i n a c t u a l p l a n t o p e r a t i o n ) w h i l e t h e s e c o n d t a n k was a l l o w e d t o r u n a s a b a t c h p r o c e s s . A pulse o f 1.7 g N a N 0 a n d 84 m l m e t h a n o l was a d d e d t o t h e l 4 - l i t e r f e r m e n t o r when c o n t i n u o u s f e e d s t o p p e d ( F i g u r e 12). A t t h e 33**d h o u r o f o p e r a t i o n , t h e s e c o n d t a n k was e m p t i e d a n d 250 m l o f t h e b r o t h was p u t i n a n i n d e n t e d f l a s k a n d i n c u b a t e d a t 350 REM i n a c o n s t a n t t e m p e r a t u r e (30°C) s h a k e r . Then t h e s e c o n d c y c l e o f t h e c o n t i n u o u s f e e d t o t h e s e c o n d t a n k b e g a n a n d no a d d i t i o n a l n i t r a t e was a d d e d i n t h i s c y c l e . The r e s u l t s o f t h e t w o c y c l e s o f s e m i - c o n t i n u o u s o p e r a t i o n f o r t h e s e c o n d t a n k a r e shown i n F i g u r e s 12 a n d 13 a n d t h e r a t e c o n s t a n t s a r e summarized i n TableIV. I n t h e f i r s t c y c l e , 2.776 g / l N a N 0 was consumed i n 33 h o u r s a n d 4.22 g c e l l / l was p r o d u c e d . I f a l l t h e a v a i l a b l e n i t r o g e n h a d ended up i n t h e c e l l s , t h e p e r c e n t a g e o f n i t r o g e n i n t h e c e l l s w o u l d be 10.82$> w h i c h a g r e e s w i t h t h e v a l u e o f 10.8ofo p r e d i c t e d b y t h e e m p i r i c a l f o r m u l a C H s 0 N . The m e a s u r e d c e l l a n d p o l y m e r p r o d u c t i o n r a t e s were a l l l i n e a r (Table 4). I f t h e b r o t h were a l l o w e d t o i n c u b a t e l o n g e r t h a n 30 h o u r s i n t h e a e r a t e d t a n k , t h e r a t e o f p o l y m e r p r o d u c t i o n s h o u l d s l o w down t o a b o u t o n e - t h i r d o f t h e i n i t i a l v a l u e as s u g g e s t e d b y d a t a i n t h e s e c o n d c y c l e ( F i g u r e 12). However, t h e r a t e o f p o l y m e r p r o d u c t i o n a c t u a l l y i n c r e a s e d f r o m Ο.Ο65 t o 0.109 g g l u c o s e / l , h r i n t h e shake f l a s k ( a t 350 RFM). T h i s i n d i c a t e d t h a t f o r t h e v i s c o u s b r o t h , a shake f l a s k h a d b e t t e r a e r a t i o n , a n d t h a t a K L a v a l u e o f 98 h r " i n t h e a e r a t e d t a n k was n o t h i g h enough t o meet t h e o x y g e n demand. A n o t h e r p i e c e o f e v i d e n c e f o r o x y g e n l i m i t a t i o n was f o u n d i n t h e s e c o n d c y c l e o f t h e o p e r a t i o n i n the second tank. A 10fo i n c r e a s e i n t h e a g i t a t i o n r a t e o f t h e i m p e l l e r , f r o m 500 t o 550 RPM, r a i s e d t h e p o l y m e r f o r m a t i o n r a t e f r o m 0.018 t o 0.031 g g l u c o s e / ( L , h r ) , a n d t h e c e l l p r o d u c t i o n r a t e f r o m 0.014 t o 0.044 g c e l l / ( L , h r ) . However, t h e K a showed l i t t l e o b s e r v a b l e change a n d r e m a i n e d c o n s t a n t a t 120 h r - i . The r e s p i r a t o r y q u o t i e n t (R.Q.) i s a v e r y s e n s i t i v e p a r a m e t e r t h a t t e l l s t h e age d i s t r i b u t i o n o f t h e p o p u l a t i o n b e c a u s e t h e d e mand f o r o x y g e n a n d t h e e v o l u t i o n o f c a r b o n d i o x i d e a r e n o t c o n 3

3

3

5

1

L


3

Polysaccharide

ΤΑΜ A N D FINN


5.

Formation

by a Methylomonas

HOUR Figure 12.

I

Ο

.

ι

10

First cycle semi-continuous fermentation

J—Λ

20

,

ι

30

,

i _ U

40

1

50

.

•

60

•

I

70

HOUR

Figure 13.

Second cycle semi-continuous fermentation


77


78

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

s t a n t d u r i n g t h e d i f f e r e n t phases o f the b a c t e r i a l growth c y c l e . For example, i n t h e f i r s t c y c l e o f t h e s e m i - c o n t i n u o u s o p e r a t i o n , a p r e s s u r e l e a k was d e v e l o p e d i n t h e f i r s t t a n k r e s u l t i n g i n no o v e r f l o w i n t o t h e second t a n k and b r o t h a c c u m u l a t i o n i n t h e c e l l p r o p a g a t o r . A b o u t a n h o u r l a t e r (10-1/2 h o u r i n F i g u r e 1 2 ) t h e p r e s s u r e was r e a d j u s t e d a n d a b o u t 0 . 6 l i t e r o f c e l l s f r o m t h e e x p o n e n t i a l g r o w t h p h a s e was f o r c e d i n t o t h e s e c o n d t a n k . The R. Q. i m m e d i a t e l y jumped f r o m 0.2 t o 0-31 as i n d i c a t e d b y t h e d a s h e d l i n e i n F i g u r e 12. The maximum i n t h e R. Q. c u r v e a l s o i n d i c a t e d t h a t a r e l a t i v e l y l a r g e p o r t i o n o f e x p o n e n t i a l l y growing c e l l s was i n t h e p o p u l a t i o n d u r i n g t h e c o n t i n u o u s f e e d i n g s t a g e i n the second c y c l e ( F i g u r e 12). T h e o r e t i c a l l y , t h e maximum r e s p i r a t o r y q u o t i e n t (R. Q. = 0 . 6 6 ) o c c u r s when c a r b o n i n m e t h a n o l s e r v e s o n l y as a n e n e r g y s o u r c e a n d i s c o m p l e t e l y o x i d i s e d t o c a r b o n d i o x i d e and w a t e r . 2

CH3OH

+

3 0

> 4H 0 + 2 C 0

2

2

3^7 K c a l / m o l e CH 0H

2

3

However, when p a r t o f t h e c a r b o n i s d i v e r t e d t o c e l l a n d p o l y m e r s y n t h e s i s , l e s s c a r b o n d i o x i d e s h o u l d be f o r m e d a n d t h e R. Q. s h o u l d be l e s s t h a n 0.66. S i n c e p o l y m e r i z a t i o n r e q u i r e s l e s s energy than c e l l s y n t h e s i s , the r e s p i r a t o r y quotient s h o u l d de c r e a s e m o n o t o n i c a l l y as more p o l y m e r a n d f e w e r c e l l s a r e formed. The e x p e r i m e n t a l R. Q. d a t a f a l l b e t w e e n 0 . 4 t o 0.1 a n d t h e d e c r e a s e i n t h e r e s p i r a t o r y q u o t i e n t w i t h t h e i n c r e a s e i n t h e amount o f p o l y m e r f o r m e d does i n f a c t a g r e e w i t h t h e p r e d i c t e d g e n e r a l trend. The f i n a l y i e l d d a t a f o r t h e s e m i - c o n t i n u o u s e x p e r i m e n t a r e s u m m a r i z e d i n T a b l e V. Table V F i n a l Y i e l d Data f o r the Semi-continuous

Yield Constant Y

t o t a l solid/s

Y

,

T

a

n

k

Experiment

2

F i r s t Cycle

Second C y c l e

·3

·3*

2

(-W

.121». ( . ι 8 )

.128 (.160)

.196 (.229)

.212 (.264)

5

Χ/ S

Y^

g

Numbers i n t h e b r a c k e t s show t h e y i e l d c o n s t a n t s , c o r r e c t e d f o r l o s s due t o m e t h a n o l s t r i p p i n g a t a r a t e o f 0.12 g M e t h a n o l / ( 1 , h r ) f o r 60 h o u r s . The y i e l d d a t a a r e l o w e r t h a n t h e b e s t o b s e r v e d v a l u e s o f Y / = 0.122, Y p / = ΟΛ52 a n d Y t o t a l s o l i d / s = Ο . 5 6 i n t h e n i t r o g e n - l i m i t e d b a t c h ( F i g u r e 11). The p o o r e r y i e l d i s due t o t h e x

S

S



5.

ΤΑΜ

A N D FINN

Polysaccharide

Formation

by a Methylomonas

79

i n f e r i o r o x y g e n t r a n s f e r c a p a c i t y o f t h e s e c o n d t a n k and, more i m p o r t a n t l y , t o t h e n i t r o g e n dosage scheme. I n t h e n i t r o g e n - l i m i t e d b a t c h c u l t u r e , a d d i t i o n a l n i t r o g e n s o u r c e was a d d e d i n a way t h a t avoided p r o l o n g e d p e r i o d s o f n i t r o g e n exhaustion, and conse q u e n t l y t h e r e was a r e l a t i v e l y l a r g e p o p u l a t i o n o f y o u n g c e l l s i n the broth. The f a c t t h a t t h e R. Q. was b e t w e e n 0.4 a n d 0.33 i n t h e f i r s t 50 h o u r s o f t h e b a t c h o p e r a t i o n a s compared t o t h e a v e r a g e R. Q. o f l e s s t h a n 0.2 i n t h e s e m i - c o n t i n u o u s o p e r a t i o n , s u p p o r t s t h e above argument. I n c o n c l u s i o n , a s e m i - c o n t i n u o u s o p e r a t i o n seems f e a s i b l e b e cause i t i s r e p r o d u c i b l e and because i t m i n i m i z e s problems o f contamination o r c u l t u r e degeneration. The p r e s e n t o p e r a t i o n a l p r o c e d u r e i s n o t t h e o p t i m a l one. Improvements i n a e r a t i o n b y i n s t a l l i n g a foam b r e a k e r a n d o p e r a t i n g a t a h i g h e r s t i r r e r s p e e d w i l l h e l p t o b r i n g t h e K ^ a v a l u e above 150 h r " * . A l s o t h e mode o f n i t r o g e n dosage c a n be r e v i s e d s o a s t o m a i n t a i n a l a r g e r p o r t i o n o f y o u n g c e l l s i n t h e s e c o n d f e r m e n t o r (R. Q. b e t w e e n 0.3 t o 0 . 4 ) . 1

Literature Cited 1. Bikales, Ν. M. (ed.) in "Water Soluble Polymers", pp 227-42, Plenum Publishing Corp., New York, N.Y., 1973. 2. McNeely, W. H. in "Microbial Technology", H. Peppler (ed.), 381-402, Reinhold Publishing Corp., New York, N.Y., 1967. 3. MacWilliams, D.C., Rogers, J. Η., and West, T. J. in "Ency clopedia of Polymer Science and Technology", Vol. II, pp 105-126, Wiley-Interscience, New York, N.Y., 1973. 4. Moraine, R. A. and Rogovin, P., Biotechnol. Bioeng. (1971), 13, 381-91. 5. Tannahill, Alex L. and Finn, R. Κ., U.S. Patent 3,878,045, April 15, 1975. 6. Finn, R. Κ., Tannahill, Alex L . , and Laptewicz, J. E. Jr., U.S. Patent 3,923,782, Dec. 2, 1975. 7. Herbert, D., Phipp, P. J., and Strange, R. E. in "Methods in Microbiology", Norris, J. R. and Ribbons, D. W. (eds.), Vol. 5B, pp 249-51, Academic Press, London, 1971. 8. Dubios, M. et al., Anal. Chem. (1956), 28, 350-56. 9. Johnson, M. J., Borkowski, J., and Engblom, C., Biotechnol. Bioeng. (1964), 6, 457-68. 10. ibid. 9, 635-39. 11. Tam, K. T., Ph.D. Thesis, Cornell University, Ithaca, Ν. Υ., 1975. 12. Van Dijken, J. P. and Harder, W., J. Gen. Microbiol. (1974), 84, 409-11. 13. Whittenburg, R., Phillips, K. C., and Wilkinson, J. F., J. Gen. Microbiol. (1970), 6 1, 205-18. 14. Battat, E . , Goldberg, I . , and Mateles, R. I., Appl. Microbiol. (1974), 28, 906-11. 15. Levine, P. W. and Cooney, C. L . , Appl. Microbiol. (1973), 26, 982-90.



80

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

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Polysaccharide Formation by a

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