Immobilized Microbial Cells - American Chemical Society

2 Pore Dimensions for Accumulating Biomass R. A. MESSING, R. A. OPPERMANN, and F. B. KOLOT Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 20, 2016 | http://pubs.acs.org Publication Date: August 16, 1979 | doi: 10.1021/bk-1979-0106.ch002

Corning Glass Works, Sullivan Park, Corning, NY 14830

Such processes as the production of cells (single cell proteins), fermentation for secondary metabolites, and waste conversion require high concentrations of cells. Generally speaking, to produce a large number of cells such as is required f o r single cell proteins, one must pass through the lag phase and operate within the log phase. The greater the number of cells per unit volume, the more progeny will be produced per volume provided that the cells are neither nutrient l i m i t e d nor gas l i m i t e d . The requirement for high concentration of cells or accumulations of biomass i n the production of secondary metabolites i s even more apparent than for cell production. Secondary metabolites are generally produced i n the stationary phase; the greater the concent r a t i o n s of cells, the greater the production of secondary metabolites per unit volume and per unit time. The accumulation and r e t e n t i o n of biomass lends itself r e a d i l y to the employment of continuous single pass reactors such as plug-flow or fluidized-bed reactors. When high q u a n t i t i e s of biomass are retained, greater t o t a l q u a n t i t i e s of nutrients may be delivered and greater q u a n t i t i e s of waste products may be removed per unit time. Any mechanism that can be offered to r e t a i n the c e l l s i n high concentrations, d e l i v e r the nutrients r a p i d l y , and remove waste products should o f f e r a h i g h l y e f f i c i e n t reactor. We have found a r e l a t i o n s h i p between the accumul a t i o n of stable and v i a b l e biomass and the pore morphology of a dimensionally stable inorganic c a r r i e r . That r e l a t i o n s h i p i s dependent upon the mode of reproduction of the s p e c i f i c microbe. 0-8412-0508-6/79/47-106-013$05.00/0 © 1979 American Chemical Society Venkatsubramanian; Immobilized Microbial Cells ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

14

IMMOBILIZED MICROBIAL CELLS

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 20, 2016 | http://pubs.acs.org Publication Date: August 16, 1979 | doi: 10.1021/bk-1979-0106.ch002

M a t e r i a l s and Methods. The c o n t r o l l e d - p o r e c e r a m i c s and the f r i t t e d g l a s s e s employed i n t h e s e s t u d i e s were manufactured by C o r n i n g G l a s s Works. The b o r o s i l i c a t e g l a s s i s a p r o d u c t o f C o r n i n g G l a s s Works. A DuPont Biometer, Model 760, was employed t o determine the v i a b l e microbe count f o r a l l organisms e x c e p t Streptomyces olivochromogenes and Pénicillium chrysogenum. We employed a p r o t e i n d e t e r m i n a t i o n u t i l i z i n g the F o l i n r e a g e n t f o r d e t e r m i n i n g the l o a d i n g s o f the l a t t e r two m i c r o b e s . f

Determination of M i c r o b i a l Loadings(Biomass). We were not a b l e t o employ the c o n v e n t i o n a l p l a t e c o u n t i n g t e c h n i q u e s t o determine l o a d i n g s due t o the f a c t t h a t m i c r o b i a l l o a d i n g s (biomass a c c u m u l a t i o n s ) i n v o l v e d measuring t h e number o f microbes bonded w i t h i n the p o r e s o f v a r i o u s porous s u p p o r t s . I n s t e a d , t h e microbe counts or the r e l a t i v e q u a n t i t y o f b i o mass were determined by employing the Biometer which d e t e r m i n e s the r e l a t i v e number o f v i a b l e microbes o r v i a b l e biomass based upon the amount o f ATP p r e s e n t i n a g i v e n sample. The a c t u a l p r o c e d u r e employed was as f o l l o w s : t o a p p r o x i m a t e l y 10-20 mg o f i m m o b i l i z e d microbe composite, 0.5 ml o f 90% d i m e t h y l s u l f o x i d e i n water was added and the s u s p e n s i o n was mixed v i g o r o u s l y f o r 10 seconds. The s u s p e n s i o n was a l l o w e d t o e x t r a c t f o r 20 minutes and then 4 ml o f 0.01M morpholinopropane s u l f o n i c a c i d b u f f e r , pH 7.4, was d e l i v e r e d and t h e s u s p e n s i o n was mixed t h o r o u g h l y and s t o r e d i n i c e u n t i l p r e p a r a t i o n s were complete f o r the ATP d e t e r m i n a t i o n s . P r i o r t o t h i s d e t e r m i n a t i o n , t h e l u c i f e r i n - l u c i f e r a s e m i x t u r e was p r e p a r e d a c c o r d i n g t o the Biometer p r o c e d u r e ! . A f t e r 0.1 ml o f enzyme-substrate m i x t u r e was d e l i v e r e d t o t h e r e a c t i o n c u v e t t e , 10 m i c r o l i t e r s o f t h e above DMSO e x t r a c t was added t o t h e c u v e t t e c o n t a i n i n g the l u c i f e r i n - l u c i f e r a s e m i x t u r e . The l i g h t e m i s s i o n measurement was t h e n made and t h e c o r r e l a t e d v a l u e t o v i a b l e c e l l q u a n t i t y was r e c o r d e d . S i n c e i t i s i m p o s s i b l e t o r e l a t e i n d i v i d u a l c e l l s of microbes that generate m y c e l i a t o ATP p r e s e n t , we e l e c t e d t o r e p o r t t h e mass o f ATP i n femtograms ( 1 0 " gms) as r e p r e s e n t a t i v e o f 1 5

Venkatsubramanian; Immobilized Microbial Cells ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


2. MESSING ET AL.

15

Biomass Accumulation

the v i a b l e biomass w i t h r e s p e c t t o l o a d i n g / g r a m f o r each i n d i v i d u a l c a r r i e r . The d e t e r m i n a t i o n o f biomass p r o t e i n f o r both S. ο1ivochromogenes and P. chrysogenum was performed on 0.5 gms o f c a r r i e r which c o n t a i n e d t h e i m m o b i l i z e d biomass. A f t e r t h e l o a d i n g s and/or subsequent growth, the 0.5 gm o f c a r r i e r sample was washed 3 t i m e s w i t h 10 ml o f phosphate b u f f e r , pH 7.0. The c a r r i e r was then e x t r a c t e d w i t h 3 ml o f phosphate b u f f e r by g r i n d i n g t h e r e w i t h . Subsequently, 3 ml o f 1 Ν sodium h y d r o x i d e were added, t h e temperature o f t h e m i x t u r e was r a i s e d t o 60° and m a i n t a i n e d a t t h a t temperature f o r 1 hour i n o r d e r t o h y d r o l y z e t h e m y c e l i a . T h i s procedure r e s u l t s i n q u a n t a t a t i v e r e l e a s e o f the p r o t e i n from t h e m y c e l i a . To f u r t h e r e x t r a c t p r o t e i n , 3 ml o f e t h y l a l c o h o l were added t o t h e m i x t u r e i n t h e case o f S.. o l i v o c h r o mogenes w h i l e 2 ml o f t h e a l c o h o l were added i n t h e case o f P.. chrysogenum and t h i s was a l l o w e d t o r e a c t at room temperature f o r o n e - h a l f hour. After centrif u g a t i o n , t h e s u p e r n a t a n t f l u i d was decanted and t h e p r o t e i n c o n t e n t o f t h e f l u i d was determined a c c o r d i n g t o t h e p r o c e d u r e o f H i l l e t a L ? and H a u s c h k a l . Table I C a r r i e r Parameters Average Carrier Pore Number Diameter (u) 1 2 3 4 5 6 7 8 9 10 11

1.1 3.0 3.1 3.5 4.5 10 13 19 40 195 non-porous

Pore Diameter( μ) Distribution 0.8-1.8 1.5-6 1.5-4 1.5-4.5 3-6 2-19 8-20 17-35 18-100 170-220

Carrier Composition F r i t t e d glass Cordierite Fritted glass Fritted glass F r i t t e d glass Cordierite F r i t t e d glass Z i r c o n i a Ceramic Fritted glass F r i t t e d glass Borosilicate glass


16



Bioaccumulation of E s c h e r i c h i a

coli.

Samples o f Ε . c o l i ( h a v i n g major dimensions o f 1-6μ) were i m m o b i l i z e d t o t h e v a r i o u s c a r r i e r s , u s i n g s t e r i l e t e c h n i q u e on b o t h a v i r g i n c a r r i e r s u r f a c e and a s u r f a c e t h a t had been c o n v e r t e d t o a t e r m i n a l amine by s i l a n i z a t i o n . The bonding by a d s o r p t i o n t o t h e v i r g i n s u r f a c e was a c c o m p l i s h e d by r e a c t i n g 3 gm p o r t i o n s o f 18-25 mesh p a r t i c l e s o f t h e i n d i c a t e d c a r r i e r s f o r 3 hours a t 22°C w i t h 20 ml o f s u s p e n s i o n o f Ε . coli cells. The bonding t o t h e t e r m i n a l amine s u r f a c e was i n i t i a t e d f i r s t by r e a c t i n g 2 gms o f 18-25 mesh p a r t i c l e s w i t h 20 ml o f 10% γ-aminopropyltriethoxys i l a n e i n water f o r 2 h o u r s a t 100°C. The t r e a t e d c a r r i e r was s u b s e q u e n t l y d r i e d and r e a c t e d w i t h 20 ml o f s u s p e n s i o n o f E. c o l i c e l l s o v e r n i g h t a t 22°C. B a c t e r i a l l o a d i n g s (number/gram o f support) were t h e n r e c o r d e d v i a ATP measurement a p p r o x i m a t e l y 18 hours a f t e r the p r e p a r a t i o n o f t h e i m m o b i l i z e d m i c r o b e s . The r e s u l t s a r e summarized i n F i g u r e s l a and l b . B i o a c c u m u l a t i o n o f S e r r a t i a marcescens by P o l y i s o c y a nate

Coupling.

S. marcescens, h a v i n g major dimensions o f 0.6-2μ was c o u p l e d t o g l a s s s u r f a c e s w i t h p o l y i s o c y a n a t e (PAPI 901, Upjohn Company, Kalamazoo, MI) . The c a r r i e r d e r i v a t i v e was p r e p a r e d by s h a k i n g a t 100 RPM 0.5 gms o f c a r r i e r i n 10 ml o f 0.5% p o l y i s o c y a n a t e i n acetone f o r 45 minutes a t room t e m p e r a t u r e . The c o u p l i n g s o l u t i o n was decanted and r e p l a c e d w i t h 10 ml o f a c e l l s u s p e n s i o n c o n t a i n i n g 3 χ 10^ c e l l s / m l . The c e l l s were r e a c t e d w i t h t h e d e r i v a t i z e d c a r r i e r f o r 3 h o u r s f o l l o w i n g which t h e excess c e l l s were poured o f f and t h e c a r r i e r was washed 3 t i m e s w i t h 0.1 M phosphate b u f f e r a t pH 7.2. The r e s u l t s o f t h i s s t u d y w i t h v a r i o u s c a r r i e r s a r e p l o t t e d i n F i g u r e 2. The

I m m o b i l i z a t i o n o f B a c i l l u s s u b t i l i s by A d s o r p t i o n ,

The c u l t u r e c e l l s were s i z e d p r i o r t o i m m o b i l i zation. The major d i m e n s i o n o f t h e c e l l s were found t o be between 3 and 4μ w i t h some l o n g double c e l l s o f a p p r o x i m a t e l y 7μ i n l e n g t h .



2.

MESSING ET AL.


17

P r i o r t o use, t h e c a r r i e r s were d r y a u t o c l a v e d . B a c i l l u s s u b t i l i s c e l l s were grown i n 1 l i t e r b r a i n h e a r t i n f u s i o n b r o t h f o r 36 hours, c e n t r i f u g e d , a n d t h e c e l l s were washed 2 times w i t h s t e r i l e phosphate b u f f e r Ten ml o f t h e washed c e l l s u s p e n s i o n was added t o each f l a s k which c o n t a i n e d 0.5 gms o f c a r r i e r . A f t e r 3 h o u r s o f c o n t a c t time, t h e c a r r i e r s were washed 3 times and m a i n t a i n e d a t 8°C o v e r n i g h t . The c e l l mass i m m o b i l i z e d on t h e c a r r i e r s was determined by t h e B i o m e t e r . The r e s u l t s o f t h i s study a r e r e p o r t e d i n F i g u r e 3. Bioaccumulation

o f Yeast.

The y e a s t c e l l s employed i n t h i s study were a l l grown i n shake f l a s k s i n n u t r i e n t b r o t h p l u s 1% dext r o s e f o r a p e r i o d o f 36-40 h o u r s a t room t e m p e r a t u r e . The c e l l s u s p e n s i o n was c e n t r i f u g e d and washed 3 times w i t h sodium-potassium phosphate b u f f e r , pH 7.2. The washed s u s p e n s i o n o f c e l l s was added t o t h e c a r r i e r and a g i t a t e d by s h a k i n g f o r 3 h o u r s o f c o n t a c t t i m e . For a d s o r p t i o n , t h e c a r r i e r s were ground t o an 1825 mesh, s t e r i l i z e d d r y , and p l a c e d i n a 37°C i n c u b a t o r o v e r n i g h t t o produce a d r y c a r r i e r . A q u a n t i t y o f 0.5 gms o f each c a r r i e r was added t o 50 ml f l a s k s . Ten ml of c o n c e n t r a t e d y e a s t c e l l s u s p e n s i o n was added t o t h e c a r r i e r i n the f l a s k . A t t h e end o f 3 h o u r s o f c o n t a c t time, t h e e x c e s s c e l l s were poured o f f , t h e c a r r i e r was washed 3 times with phosphate b u f f e r and s t o r e d i n t h e r e f r i g e r a t o r p r i o r to the loading determinations. The c o u p l i n g p r o c e d u r e i n v o l v e d t h e a d d i t i o n o f 0.5 gms o f each c a r r i e r t o s e p a r a t e 50 ml f l a s k s . The f l a s k s , w i t h c o n t e n t s , were d r y a u t o c l a v e d and p l a c e d i n a 37°C i n c u b a t o r o v e r n i g h t t o m a i n t a i n t h e c a r r i e r dry. Ten ml o f a 0.5% p o l y i s o c y a n a t e i n acetone s o l u t i o n was added t o each c a r r i e r . The f l a s k s w i t h c o n t e n t s were shaken f o r 45 minutes a t room temperature a f t e r which t h e c o u p l i n g s o l u t i o n was d e c a n t e d . Ten ml o f c o n c e n t r a t e d y e a s t c e l l s u s p e n s i o n was then added t o each f l a s k and t h e f l a s k s were shaken f o r 3 h o u r s a f t e r which t h e e x c e s s c e l l s were d e c a n t e d and t h e immobil i z e d p r e p a r a t i o n was washed 3 times w i t h phosphate buffer. The ATP d e t e r m i n a t i o n s o f c e l l l o a d i n g s were p e r formed w i t h t h e Biometer as p r e v i o u s l y d e s c r i b e d .




0%

io h 8

10

0 50 100 150 200 AVG PORE DIAMETER (μ)

b

AVG. PORE DIAMETER {μ)

Figure 1.

(a and b) Bioaccumulation of E. coli




MESSING ET AL.

0%'

J

I

I L

8 16 24 32 190 AVG. PORE DIAMETER (μ)

Figure 2.

Bioaccumulation of S. marcescens



20


The y e a s t , Saccharomyces c e r e v i s i a e , was s i z e d p r i o r t o i m m o b i l i z a t i o n . The average c e l l dimensions were 4 χ 5.5μ (2.5-4 χ 4-7) and about 20% o f t h e c e l l s were 4.5 χ 7μ. The r e s u l t s o f t h e b i o a c c u m u l a t i o n o f t h i s microbe by a d s o r p t i o n a r e p l o t t e d i n F i g u r e 4. The y e a s t , Saccharomyces amurcae, p r i o r t o immo b i l i z a t i o n were found t o have dimensions o f 5.5 χ 7μ (3-7 χ 6-9) i n terms o f s m a l l , s i n g l e c e l l s w h i l e an a d d i t i o n a l p o p u l a t i o n o f l a r g e r , double c e l l s which c o n s t i t u t e d a p p r o x i m a t e l y 75% o f t h e p o p u l a t i o n were found t o be 6-8 χ 13-18μ. These c e l l s were i m m o b i l i z e d by c o u p l i n g w i t h p o l y i s o c y a n a t e and t h e r e s u l t s o f t h i s s t u d y a r e p l o t t e d i n F i g u r e 5. Bioaccumulation of A s p e r g i l l u s niger. The s p o r e s o f t h i s organism were observed t o range from 3-5μ. These s p o r e s were e l u t e d from a mature growth i n a B l a k e b o t t l e w i t h a s t e r i l e phosphate buffer. The s u s p e n s i o n was made t o 100 ml w i t h b u f f e r . Ten ml o f t h e spore s u s p e n s i o n was added t o each 50 ml m i c r o f e r n b a c k f l a s k which had been d r y a u t o c l a v e d w i t h 1 gram o f c a r r i e r and d r i e d o v e r n i g h t p r i o r t o i t s u s e . A f t e r 3 hours o f s h a k i n g a t room temperature, t h e excess s p o r e s were poured o f f , t h e c a r r i e r was washed 3 t i m e s w i t h phosphate b u f f e r and s t o r e d o v e r n i g h t a t 8°C. The q u a n t i t y o f s p o r e s t h a t was adsorbed by t h e v a r i o u s c a r r i e r s was determined by ATP measurement and the r e s u l t s a r e p l o t t e d i n F i g u r e 6. In o r d e r t o determine t h e optimum pore diameter range f o r m y c e l i a l growth, 0.5 grams o f each c a r r i e r w i t h t h e i m m o b i l i z e d spores was p l a c e d i n 75 ml o f Sabouraud d e x t r o s e b r o t h and t h e composite was a l l o w e d t o shake on a shaker a t room temperature. A t t h e end o f 27 hours, a sample o f each c a r r i e r was taken and t h e amount o f ATP was determined as a measure o f m y c e l i a l growth. The r e s u l t s a r e r e c o r d e d i n F i g u r e 6. B i o a c c u m u l a t i o n o f Streptomyces

olivochromogenes.

E q u a l amounts o f s p o r e s i n phosphate b u f f e r were added t o 0.5 grams o f each o f t h e c a r r i e r s . The s p o r e s and c a r r i e r s were a l l o w e d t o r e a c t t o g e t h e r f o r 48 hours. The n o n - r e a c t e d s p o r e s were decanted and t h e c a r r i e r was washed 3 t i m e s w i t h 2 ml a l i q u o t s o f


MESSING ET AL.


21


2.

I0 5 7

^5

^ ^5 (so Î8Ô 210 Figure 6. Bioaccumulation of A. niger AVERAGE PORE DIA. (μ) in controlled pore inorganics



22


s t e r i l e phosphate b u f f e r . These n o n - r e a c t e d s p o r e s p l u s the washings were c o l l e c t e d , the volume determined and t h e n a n a l y z e d f o r p r o t e i n c o n t e n t . The q u a n t i t y of p r o t e i n adsorbed i n the c a r r i e r was c a l c u l a t e d as t h e d i f f e r e n c e between t h e i n i t i a l c o n t e n t o f p r o t e i n i n t h e r e a c t i n g volume c o n t a i n i n g the s p o r e s and t h a t c o n t a i n e d i n the n o n - r e a c t e d s p o r e s p l u s t h e washings. The washed c a r r i e r c o n t a i n i n g the s p o r e s was s e p a r a t e l y t r a n s f e r r e d t o a f l a s k c o n t a i n i n g 50 ml o f Emerson b r o t h medium. Mycelium f o r m a t i o n w i t h i n t h e c a r r i e r p o r e s was e v a l u a t e d a f t e r i n c u b a t i o n w i t h s h a k i n g f o r 24 and 48 hours a t room temperature. At the end o f t h e s t a t e d p e r i o d s o f i n c u b a t i o n , t h e c a r r i e r was s e p a r a t e d from t h e medium v i a c e n t r i f u g a t i o n and washed 3 t i m e s w i t h 10 ml o f phosphate b u f f e r , pH 7.0. The p r o t e i n c o n t e n t w i t h i n t h e c a r r i e r was determined by the p r o c e d u r e d e s c r i b e d p r e v i o u s l y . The r e s u l t s o f both the spore a c c u m u l a t i o n and the m y c e l i a l growth w i t h i n t h e c a r r i e r a r e r e p o r t e d i n F i g u r e 7. B i o a c c u m u l a t i o n o f Pénicillium chrysogenum. The t e c h n i q u e s f o r t h e i m m o b i l i z a t i o n o f the s p o r e s and t h e h a n d l i n g o f t h e c a r r i e r m a t e r i a l s was e s s e n t i a l l y the same as t h o s e d e s c r i b e d f o r t h e olivochromogenes e x p e r i m e n t s . The washing o f t h e c a r r i e r s and t h e d e t e r m i n a t i o n o f p r o t e i n c o n t e n t were conducted i n the same manner as p r e v i o u s l y d e s c r i b e d . Other than t h e m o d i f i c a t i o n o f a l c o h o l q u a n t i t y f o r e x t r a c t i n g the p r o t e i n , t h e o n l y o t h e r change was the medium f o r m y c e l i a l growth. In p l a c e o f the Emerson b r o t h , 50 ml o f an aqueous medium h a v i n g a pH o f 6 . 3 w i t h the f o l l o w i n g c o m p o s i t i o n was u t i l i z e d f o r the growth o f P. chrysogenum; 2% l a c t o s e , 1% g l u c o s e , 0.2%KH2PO4, 0.125% N H 4 N O 3 , 0.05% N a S 0 , 0.025%MgSO , 0.002% MnS04, 0.00025% CuSO4, and 0.002% ZnS04. A s i n g l e i n c u b a t i o n of 48 hours was employed. The r e s u l t s o b t a i n e d w i t h P^ chrysogenum a r e r e c o r d e d i n F i g u r e 8. 2

4

4

R e s u l t s and D i s c u s s i o n s Upon p e r u s a l o f t h e f i g u r e s , i t becomes r a t h e r c l e a r t h a t a t l e a s t one optimum i s noted f o r the bioaccumu-


tr icr

Immobilized Microbial Cells - American Chemical Society

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