A Membrane Reactor for Simultaneous Production of Anaerobic

4 A Membrane Reactor for Simultaneous Production of Anaerobic Single-Cell Protein and Methane R. K. Finn and E. Ercoli

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: July 11, 1986 | doi: 10.1021/bk-1986-0314.ch004

School of Chemical Engineering, Cornell University, Ithaca, NY 14853

Single-cell protein can be produced from agricultural residue anaerobically in yields of about 20% (wt. cells per wt. substrate) by using a mixed culture of rumen bacteria. Even higher cell yields should be possible. To achieve high cell densities, it is proposed that acidic end products be removed by cyclic microfiltration into a methanogenic fermentor. Preliminary experiments suggest that such a tandem fermentation should be feasible on a continuous basis. More data are needed for an economic evaluation. Very l i t t l e attention has been given to the p o s s i b i l i t i e s for anaerobic production of s i n g l e - c e l l protein (SCP) from cheap carbohydrate residues (1,2). The reason for dismissing any anaerobic process is that c e l l y i e l d s , according to c l a s s i c a l Embden-Meyerhof catabolism, are only 10 to 15% of the substrate fermented. In cont r a s t , aerobic c e l l yields of 50 to 60% are easily obtainable. However, there are highly e f f i c i e n t anaerobic c e l l s . These include the acetogens, propionic bacteria, and above a l l the various rumen bacteria. The l a t t e r can attain c e l l yields on carbohydrate of 30 to 35 dry weight (3^4). Such y i e l d values are already corrected for any polysaccharide formation, and in fact the protein content of rumen bacteria is about 60% (2). We therefore have been considering t h e i r use as a protein feecT supplement for monogastric animals l i k e chickens or pigs. The ruminant animal and rumen microorganisms exist in a r e c i procally beneficial relationship, in which cellulose and other plant carbohydrates are fermented by the rumen microbes to form c h i e f l y C0 and v o l a t i l e fatty acids (VFA). The microorganisms are adapted to l i v e between pH 5.5 and 7.0, in the absence of oxygen, 1

2

These high yields result from a combination of factors including low maintenance energy, higher than normal c e l l yields per mol of ATP, and f i n a l l y excess ATP production, which can involve "anaerobic respiration" with cytochrome b in a fumarate c y c l e . x

0097-6156/ 86/ 0314-0043506.00/ 0 © 1986 American Chemical Society

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

44

SEPARATION, RECOVERY, AND PURIFICATION IN BIOTECHNOLOGY

at temperatures of 39° to 40°C, and in the presence of moderate concentrations of fermentation products. These v o l a t i l e fatty acids, c h i e f l y acetic and propionic, are absorbed through the rumen wall to provide energy for the animal. Removal of the acids is essential because at concentration above about 0.3% they do i n h i b i t c e l l growth. Within the rumen though, microorganisms grow very e f f i c i e n t l y , and thereby provide s i n g l e - c e l l protein for t h e i r host animals. Our challenge as engineers is to duplicate in v i t r o such performance. Some of the basic ideas of the anaerobic SCP process we are developing at Cornell are summarized below. Anaerobic SCP Process


1) 2) 3) 4) 5)

Rapid growth of mixed rumen bacteria (e.g. y = 0.16 hr"" on starch) to high c e l l densities at c e l l yields of 30-35% Membrane removal of inhibitory acid products The acids feed slower growing methanogenic bacteria in a separate fermentor The CH^ generated thus can be used to dry the SCP product Rapid interchange between the two fermentors is possible with alternating pulsed m i c r o f i 1 t r a t i o n . 1

The key to economic c e l l production is rapid growth to cell d e n s i t i e s l i k e those in the rumen, namely 1 0 or 1 0 cells/ml. Acidic end-products are used to feed a methane generator, so that most of the carbon is recovered in a useful form. An unusual feature of this process is that rapid u l t r a f i l t r a t i o n rather than slow d i a l y sis can be used to feed the methane fermentor. Insoluble substrates such as starch, hemicel1ulose or cellulose are retained within the rumen fermentor by appropriate membranes. The rapid i n terchange of soluble acids between the two fermentors allows only a low steady-state concentration to develop in the rumen fermentor because conversion to methane proceeds simultaneously in the second fermentor. Additional features of the process are l i s t e d below. 10

1) 2) 3) 4)

5)

11

Broad range of insoluble carbohydrates fermented...crude mixed cultures of defined consortia of rumen bacteria. S t e r i l e operation may be unnecessary because rumen conditions select for a very specialized mixed population. Safety and nutritional value of the SCP product for use as an animal feed has already been proven by studies on ruminant animals. A lower productivity than in an aerobic process is to be expected because of a somewhat lower c e l l y i e l d . Overall costs may s t i l l be competitive because of simpler equipment and energy savings. Fed-batch or continuous operation seems f e a s i b l e .


4.

FINN AND ERCOLI

45

Simultaneous Production of SCP and Methane


We h a v e d o n e o n l y p r e l i m i n a r y e x p e r i m e n t a l w o r k u s i n g m a i n l y g l u cose and sugar beet pulp as s u b s t r a t e s . We h a v e n o t y e t c o m b i n e d t h e m e t h a n o g e n i c s t e p ; i n s t e a d we h a v e u s e d a b u f f e r e d s a l t s m e d i u m i n t h e s e c o n d chamber t o remove i n h i b i t o r y e n d p r o d u c t s . Procedures. T h e b a s a l medium c o n t a i n e d m i n e r a l s a l t s , y e a s t e x t r a c t (0.1 g/Jl), t r y p t i c a s e ( 0 . 1 g/λ), c y s t e i n e h y d r o c h l o r i d e (0.4 g/£) a s a r e d u c i n g a g e n t , a n d r e a z u r i n a s a r e d o x i n d i c a t o r . T h e b a s e u s e d t o m a i n t a i n a c o n s t a n t pH w a s s o d i u m c a r b o n a t e . Some m e d i a i n c l u d e d h e m i n ( 2 t o 6 mg/V) b e c a u s e m o s t s t r a i n s o f B a c t e r o i d e s r u m i n i c o l a , a m a j o r t y p e o f rumen b a c t e r i a , a r e s t i m u l a t e d b y t h e a d d i t i o n o f s m a l l amounts o f h e m i n t o t h e medium ( 5 ) . F o r example, t h e molar growth y i e l d o f B a c t e r o i d e s f r a g i l e s subsp f r a g i l i s i n c r e a s e d f r o m 17.9 t o 4 7 . 0 ( g d r y w e i g h t c e l l p e r m o l o f g l u c o s e ) w h e n 2 mg/I o f h e m i n w e r e a d d e d ( 6 ) . M e d i a w e r e i n o c u l a t e d w i t h f r e s h r u m e n f l u i d t a k e n f r o m a cow f e d w i t h g r a i n a n d hay. The samples were used w i t h i n two h o u r s a f t e r r e m o v a l . P r o t e i n was d e t e r m i n e d b y t h e m e t h o d o f L o w r y ( 7 ) a f t e r h y d r o l y s i s w i t h 0.2N NaOH (100°C, 15 m i n ) . T o t a l n i t r o g e n was m e a s u r e d by t h e m i c r o - K j e l d a h l m e t h o d w i t h s u l f u r i c a c i d / h y d r o g e n p e r o x i d e r e a g e n t j t h e a m m o n i a was d e t e c t e d w i t h N e s s l e r s r e a g e n t . Glucose was m e a s u r e d b y s t a n d a r d c o l o r i m e t r i c a s s a y u s i n g d i n i t r o s a l i c y l i c acid. S t a r c h was h y d r o l y z e d w i t h c o n c e n t r a t e d HC1 a n d t h e n d e t e r mined as sugar. f

Results. T o e s t a b l i s h o p t i m u m g r o w t h c o n d i t i o n s , we u s e d i n t h e e a r l y experiments a low c o n c e n t r a t i o n o f the carbon source. Re moval o f t h e i n h i b i t o r y a c i d s i s then unnecessary. T a b l e I shows r e s u l t s f o r a m i x e d p o p u l a t i o n o f rumen b a c t e r ia. T h e f e r m e n t a t i o n s w e r e c o m p l e t e ( e s s e n t i a l l y no r e s i d u a l g l u cose) a f t e r 6 t o 7 hours. Table I . Substrate Cone.

G r o w t h o f Rumen B a c t e r i a o n G l u c o s e Hemin

(mg/*)

Growth r a t e (h ) l

(g/«

(g/λ) 5 5 15

Nitrogen* Fixed

6

-

0.61 0.66 0.60

0.071 0.090 0.126

Est'd c e l l * * yield (g/g s u b s t r . ) 0.15 0.19 0.08

(0.18)

*Net u t i l i z a t i o n o f s o l u b l e n i t r o g e n f r o m t h e medium, i . e . c o n v e r ted i n t o biomass. **The c e l l y i e l d w a s e s t i m a t e d f r o m t h e n i t r o g e n f i x e d , a s s u m i n g 50% p r o t e i n c o n t e n t f o r t h e c e l l s , 15% n u c l e i c a c i d s . The v a l u e s h o w n i n p a r e n t h e s i s was m e a s u r e d d i r e c t l y b y d r y w e i g h t . The r e s u l t s i n T a b l e I show t h a t c e l l y i e l d s a r e l o w e r t h a n e x p e c t e d b u t t h a t added hemin s t i m u l a t e s g r o w t h . The l o w y i e l d o f c e l l s a t h i g h e r g l u c o s e c o n c e n t r a t i o n may b e c a u s e d b y a c c u m u l a t e d a c i d s suppressing growth while a l l o w i n g fermentation t o proceed.


46


Using starch as substrate yields were also low. An explanation may be that Streptococcus bovis, a homofermentative l a c t i c acid organism common in the rumen grows rapidly on starch with low y i e l d of biomass (&). Table n shows some of the results for fermentation of sugar beet pulp (SBP). Table

II.

Substrate Cone.

Growth of Rumen Bacteria on Sugar Beet Pulp, (5% inoculum) Hemi η (mg/£)


(9/A)

5 5 5 5 10 20

_

2 4 6 6

-

Nitrogen fixed (g/A) 0.067 0.075 0.074 0.086 0.123 0.203

E s f d cell yield (g/g subst. supplied) 0.14 0.16 0.16 0.18 0.12 0.10

The y i e l d values in Table II are based on substrate supplied; the quantity unfermented could not be readily measured. The incorpora tion of nitrogen from the medium is similar to that observed for glucose. Growth rates on sugar beet pulp were slower than on glucose, as indicated by comparison of the solid lines in Figure 1, where nitrogen in the solids fraction provides a measure of biomass. The rate of acid production (broken lines in Figure 1) was also slower for the sugar beet pulp; from the slopes of such lines one can estimate the rate at which i t will be necessary to remove acids from a fermentation with higher substrate concentrations. Early experiments were done using simple d i a l y s i s in a 2-chambered membrane apparatus constructed from 3-inch glass pipe (Figure 2). Starch was added p e r i o d i c a l l y to the rumen bacteria so as to simulate a fed-batch operation. A d i a l y s i s membrane was held between flanges separating the two chambers. The rate of acid removal was too slow, however, in this simple d i a l y s i s apparatus. Consequently, a microfi1tration chamber was arranged as shown in the diagram of Figure 3. Plugging or fouling of the m i c r o f i l t r a tion membrane can be avoided by changing the pressure periodically on either side of the membrane. Flow then o s c i l l a t e s across the membrane. Such cycling can be accomplished without changing the pressure in either of the fermentors by using two small centrifugal pumps and the valve arrangement shown at the top of the diagram. Both pumps operated continuously. The solenoid valves were a l t e r nately opened and closed on 30-second repeat cycles. Back-pressure valves were set to provide c i r c u l a t i o n through the membrane cham bers while maintaining also a positive pressure for m i c r o f i l t r a tion. Each of the fermentation chambers had a working volume of 1.5 l i t e r s and there was an additional holdup of 0.5 l i t e r s on each side of the f i l t r a t i o n chamber. A single membrane of polytetraf1uoroethylene was used. It had a pore size of 0.22 micrometers and an area of 155 cm . Preliminary experiments have indicated



FINN AND ERCOLI

Simultaneous Production of SCP and Methane

Time, h F i g u r e 1. T i m e c o u r s e o f t h e r u m e n f e r m e n t a t i o n . Solid lines show n i t r o g e n i n t h e s o l i d s f r a c t i o n f o r 5 g / l i t e r g l u c o s e ( s o l i d c i r c l e s ) and f o r 5 g / l i t e r sugar beet pulp (open circles). The h i g h i n i t i a l value f o r sugar beet pulp r e p r e sents i t s p r o t e i n content as r e c e i v e d . B r o k e n l i n e s show t h e c o r r e s p o n d i n g a m o u n t s o f 2 5 % a q u e o u s Na2CÛ3 u s e d f o r m a i n t a i n i n g c o n s t a n t pH d u r i n g f e r m e n t a t i o n o f the glucose o r sugar beet pulp.

American Chemical Society Library 1155 16th St., N.W. Washington, D.C. 20036 Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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STARCH

RUMEN

METHANOGENIC

BACTERIA

BACTERIA

F i g u r e 2. D i a g r a m o f a p p a r a t u s u s i n g t w o 3 - i n c h g l a s s e l b o w s w i t h a d i a l y s i s membrane a t t h e f l a n g e s e p a r a t i n g t h e t w o chambers. No f o r c e d f l o w t h r o u g h t h e membrane.

1

BACK-PRESSURE VALVES

2

S GLENOID VALVES

METHANOGENIC

RUMEN

BACTERIA

BACTERIA

1

A Membrane Reactor for Simultaneous Production of Anaerobic

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