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Continuous CH4 Production from H2 and CO2 by Methanobacterium thermoautotrophicum in a fixed-bed reactor
[J. Ferment. Technol., Vol. 66, No. 2, 235--238. 1988]
Note
Continuous CH4 Production from H2 and CO2 by
Methanobacterium thermoautotrophicum in a Fixed-Bed Reactor HAE SUNG JEE, NAOMICHI NISHIO*, and SHIRO NAGAI
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Sa~jo-cho, Higashi-Hiroshima 724, Japan Methanation of H2 and COs by Methanobacterium thermoautotrophicumAH was carried cut in a fixed-bed reactor (31 m_m~b× 180 mm H) packed with granular diatomaceous earth clay (2-3 mm~b) as a support material. The maximum methane production rate obtained was 5.2 l/l bed volume]h, while a conversion rate reaching up to some 80% of the theoretical value was achieved by controlling the feed rate of the substrate gas throughout the culture period. After cultivation methanogen cells were fixed almost homogeneously on the whole of the support material, giving 30 mg dry cell[era s packed support.
CH4, a c a l o r i e - r i c h gas, c a n be p r o d u c e d by b i o m e t h a n a t i o n f r o m H~ a n d CO~ as a n a l t e r n a t i v e e n e r g y source to fossil fuels, l-s) 4H2+CO~=CH4+2HsO
(1)
I n p r a c t i c a l b i o m e t h a n a t i o n , an efficient supply o f H~ a n d C O s to the c u lt u r e m e d i u m is the most significant factor in the process, due to e x t r e m e l y l o w solubility to water.4-e) T h e r e f o r e , as r e p o r t e d in a previous paper,2) we designed a b i o r e a c t o r using a c y l i n d r i c a l c e r a m i c s u p p o rt instead o f the c o n v e n t i o n a l s u b m e r g e d culture, in o r d e r to facilitate mass transfer of the substrate gases. S u c h a reactor gave high methane productivity, b u t the conversion rate was still low (ca.
60%). I n the present study, a fixed-bed r e a c t o r p a c k e d w i t h a g r a n u l a r s u p p o r t was constructed, w h i c h it was t h o u g h t m i g h t facilitate the mass transfer o f the gaseous substrate to a m e t h a n o g e n , M . thermoautotrophicum AH. * Corresponding author
Ease in c h a n g i n g the r e a c t o r size a n d a r e d u c t i o n in r e a c t o r c o n s t r u c t i o n cost ar e two a d v a n t a g e s o f using g r a n u l a r supports. A feedback control system w i t h a supply of gaseous substrate w a s also a t t e m p t e d in o r d e r to get a h i g h conversion rate, ca. 8 0 % , o f the gaseous substrate to m e t h a n e . Materials and Methods Bacterial strain Methanobacteriumthermoautotrophicum AH (DSM 1053) from the Deutsche Sammlung yon Mikrooganismen (G6ttingen, FRG) was used. The euiture medium was the same as described previously. 1) All culture manipulations were carried out under oxygen-free conditions, as described previously, x) Preculture in a 125-ml serum vial (20ml working volume, H8[CO2=80]20, v/v in the gas phase) was carried out anaerobically for 20 h at 65°C. The mixed gas (H~[CO2=80]20, v/v) adjusted by two mass flow controllers was supplied at 1.5kg]cm~ before cultivation. Cultivation in a fixed-bed reactor As supports of the fixed-bed reactor, granular cellulose acetate (1.7-2.8mm~), granular diatomaceous earth clay (2-3, 5-6 mm~) and cylindrical diatomaceous earth
236
JzE, NISHIO, and NAOAX
[J. Ferment. Technol.,
are the flow rates of the respective gases, in l/h. The conversion rate, YCHo can also be calculated by the gas composition in the exit gas line. 5PcH, YCH,= 5 P c L 4 7 , c o , _ p ,
2
(6)
Where, PCH,, Pco2 and PH2 are the partial pressures of the respective gases of the exit gas. To achieve a YCH4 of more than 0.8, Freed was controlled on the basis of the Fexit value as a feedback control signal. This can be achieved by the substitution of YcH,~_0.8 into Eq. 5, from which the following relationship between Freed and Fexit can be derived. Ffeed<:2.78 Fextt
Fig. 1. Scheme of continuous culture apparatus for methanation of I-Is and CO2 with M. thermoautotrophicum fixed on granular support. 1, medium reservoir; 2, pump; 3, t-I2; 4, COs; 5, mass flow controller; 6, flow meter; 7, methanation reactor; 8, granular support; 9, water bath.
clay (3.5 m m ~ × 3 4 ram) were used. The supports of cellulose acetate and diatomaceous earth clay were obtained from Daicel Chem. Ind. Co., Tokyo and Nagao Soda Co., Ltd., Okayarna, respectively. The volume of packed support was 52.8 ems (7.54 cm 2 × 70ram) in a glass column ( 3 1 m m ~ × l l 0 m m ) . After packing, the reactor was flushed with the mixed gas at a feed rate of 20 ml/min overnight to maintain anaerobic conditions. Fixation of methanogen cells was carried out by passage of the preculture (44 ml), and through this procedure ca. 0.5 mg cell of the methanogen was fixed on the support. Cultivation was started by supplying the inorganic medium (16 ml/ h) via a peristaltic pump and the mixed gas (2.8 l/h) through a flow meter. The medium and the mixed gas were supplied through a hand-made nozzle at the top of the reactor (see Fig. 1). In the methanation process, based on Eq. 1, the conversion rate of H~ and COs to CH4, YCH4, can be calculated as follows. F feea= (F H*+Fc02) feed Fexit = ( F ~ + F c o 2 ) e x i t + F c H , F f eed -- F ext t = 5F c n , - - F c H~= 4 F cH6
(2) (3) (4)
Hence, YCH4 can be expressed by, 5FcH4 5 / Ffeed--Fexit YCH,= P~eeT = 4 - ~ Freed )
(5)
where, Fexit and Freed are the gas flow rates at the exit and inlet of the reactor (l/h). Faz, Fco2 and FcH,
(7)
Based on Eq. 7, if F~eed is controlled at less than 2.78 times of gexit, the methanation from Freed ( H 2 / C O s : 4 / 1 ) can be attained at a rate more than 0.8 of YCH~. Analysis To estimate the methanogen cell mass adhering onto the supports after cultivation, the support in the column reactor was carefully withdrawn and subdivided horizontally into 7 sections, 1 cm s of the support from the each section was crushed in 1 1V[ NaOH solution, and the eluted whole-cell protein was determined by a dye-binding method. 7,s) The hydrogen, carbon dioxide and methane contents of the exhaust gas were analysed by gas chromatography with a thermal conductivity detector under the conditions described previously. 1) The volume of exhaust gas from the reactor was measured with a gas flow meter and corrected to standard temperature conditions.
Results and Discussion Selection of support for fixed-bed reactor T o s e l e c t a s u i t a b l e s u p p o r t for the
fixed-bed
reactor
of M. thermoautomaterials were tested for t h e i r i n f l u e n c e o n t h e r n e t h a n a t i o n o f H~ and CO2. A f t e r f i x i n g t h e m e t h a n o g e n cells, cultivation was started by supplying the m i x e d g a s (2.6 l / h ) a n d t h e m e d i u m (16 m l / h). The highest rate of methanogenesis was observed with granular diatomaceous earth c l a y (size, 2 - 3 m m ~ ) (see F i g . 2 a ) . After 20 h o f c u l t i v a t i o n , m e t h a n a t i o n o f H s a n d COs reached a steady state at which the methane content of the exhaust gas was 15.5% (see Fig. 2 b ) , a n d t h e m e t h a n e p r o d u c t i o n r a t e w a s 280 m l / h a t t h e s t e a d y state. T h i s v a l u e c o r r e s p o n d s to 5.3 l C H 4 / I trophicum A H , g r a n u l a r
Vol. 66, 1988]
!3°°I
Biomethanation of Hs and CO2
a}
237
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Fig. 3. Methanation of t~s and COs by M. thermoautotrophicum ceils fixed on granular diatomaceous earth clay (2-3 mm~b). Culture conditions: 0 2 0 4 0 60 8 0 I00 120 mixed gas feed rate, 3.2 l/h; medium feed rate, C u l f u r e time (h) 18 ml/h; inoculura, 1.0 rag cells; volume of packed support, 105.6 cms (7.54 cm 2 × 140 mm Fig. 2. Methanation of Hs and COs by M. thermoH). Other culture conditions: See Fig. 2. Comautotrophicum cells fixed on various supports. position of exit gas: See Fig. 2. Culture conditions: volume of packed support, 52.8 ems (7.54 em* × 70 mm) ; inoculum, 0.5 mg cells; mixed gas (Hs/COs=80/20, v/v) feed rate, from a m a x i m u m v a l u e o f 540 m l / h to 2.6l/h; raedium feed rate, 16 ral/h; culture temperature, 65°C. a) l , granular diatomaceous 470 m l / h a t the end o f the culture. I n this earth clay (2-3 mm~b) ; Fq, granular diatomaceous run, the conversion r a t e of the s u b s t r a t e gas earth clay (5-6 mm~); /% cylindrical diatomace- to m e t h a n e d e c r e a s e d from 8 7 % at 25 h to ous earth clay (3.5 mm~ × 3 4 ram) ; A, cellulose 72O//o at the end, p r o b a b l y d u e to c h a n n e l i n g acetate (1.7-2.8mra~). b) Support: granular of the s u b s t r a t e flow b y the excess cell mass, diatomaceous earth clay (2-3 mm~). Comw h i c h caused a decrease in the c o n t a c t a r e a position of exit gas: ~ , I-Is; ~ , CO2; O, CH4. b e t w e e n the substrate gas a n d the m e t h a n o g e n cells. Next, a f e e d b a c k control system based on bed volume/h. Methanation with ceils fixed on granuF,,~t was set u p to get a h i g h conversion A f e e d b a c k control lar diatomaceous earth clay T o in- rate, YcH,, e.g., 8 0 % . crease the conversion r a t e of m e t h a n a t i o n o f r u n could be c o n d u c t e d b y c o n t r o l l i n g the I-I2 a n d CO2 b y i n c r e a s i n g r e a c t o r v o l u m e , feed r a t e of the m i x e d gas, Ffeed~__2.78 Fex~t (see Fig. 4). By this f e e d b a c k control, a b o u t a l o n g e r c o l u m n r e a c t o r (H, 180 m m ; 4, 31 m m ; c e r a m i c v o l u m e , 1 0 5 . 6 c m 8) was 8 0 % of YcH, (by Eq. 5) was o b t a i n e d t h r o u g h used. I n the m e t h a n a t i o n , the feed rates of o u t the culture, a v a l u e w h i c h c o i n c i d e d well the m i x e d gas a n d the m e d i u m to the r e a c t o r w i t h t h a t c a l c u l a t e d from the d i r e c t analysis were 3.2 l/h a n d 1 8 m l / h , respectively. I n of the exit gas (Eq. 6). After 260-h cultim e t h a n a t i o n w i t h the l o n g e r fixed-bed re- vation, the d i s t r i b u t i o n o f m e t h a n o g e n cells actor, the m e t h a n e c o n t e n t o f the exit gas inside the s u p p o r t was m e a s u r e d ; ca. 30 m g increased u p to 5 8 % at 25 h of c u l t i v a t i o n , cells/cm 8 p a c k e d s u p p o r t was found, w i t h then d e c r e a s e d g r a d u a l l y w i t h c u l t u r e t i m e , a h o m o g e n e o u s d i s t r i b u t i o n t h r o u g h the a n d showed a low v a l u e o f 3 4 % at the e n d o f r e a c t o r (see Fig. 5). I n a d d i t i o n , m e t h a n o the c o n t i n u o u s c u l t u r e (160 h, see Fig. 3). gen cell l e a k a g e in the effluent m e d i u m was T h e m e t h a n e p r o d u c t i o n r a t e also decreased v e r y low, ca. 40 mg/l c o m p a r e d w i t h the ',.9
0
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0 20 40 60 80 I00120 140 Deplh of pocked support (turn)
Fig. 5. Cell mass distribution of M. thermoautotrophicum in the fixed bed reactor. After 260 h continuous culture (see Fig. 4), the methanogen adhering on granular diatomaceous earth clay was horizontally subdivided into 7 sections (20 mm thick), and 1 cma of each section was crushed in 25 ml of 1 M NaOH solution to determine the cell mass concentration.
1.6 ,
0
.
40 80 120 160 200240 280 Culture time (h)
Fig. 4. Methanation of H2 and CO2 by M. thermoautotrophicum cells fixed on granular diatomaceous earth clay (2-3 mm4). Culture conditions: See Figs. 2 and 3; volume of packed support, see Fig. 3. Arrow indicates start of control of gas feed rate. Conversion rate: U], calculated from Eq. 5; O, calculated from Eq. 6. Composition of exit gas: See Fig. 2. A, gas flow rate at inlet; A, gas flow rate at exit. figure inside the reactor of 30 mg/cmS, a n d the p H of the c u l t u r e m e d i u m showed a n almost c o n s t a n t v a l u e of 6.6 t h r o u g h o u t the c o n t i n u o u s culture (data n o t shown). I n m e t h a n a t i o n using the reactor, the m e t h a n e p r o d u c t i o n rate decreased g r a d u a l l y with c u l t u r e t i m e (see Fig. 4), p r o b a b l y due to c h a n n e l i n g of the substrate flow b y the excess m e t h a n o g e n cells. F u r t h e r studies o n imp r o v i n g the stability of the system, as well as on a n a l t e r n a t i v e process, are n o w i n progress. I n conclusion, it is believed that a fixed-bed reactor using a support such as g r a n u l a r d i a t o m a c e o u s e a r t h clay could be a p p l i c a b l e to the c o n t i n u o u s p r o d u c t i o n of m e t h a n e from H2 a n d CO2 b y M . thermoautotrophicum
AH, a h i g h m e t h a n e conversion rate of more t h a n 8 0 % b e i n g o b t a i n e d w h e n Ffe0d was controlled b y F,xit as a feedback control signal. F o r this gas substrate fermentation, the conversion rate c a n easily be estimated by r e a d i n g the two gas flow rates at the inlet a n d exit w i t h o u t the need for chemical analysis of the exit gas b y gas c h r o m a t o g r a p h y . Acknowledgments The authors are grateful to Nagao Soda Co., Ltd., for providing diatomaceous earth clay, and Daicel Chem. Ind. Co. for providing cellulose acetate support. References 1) Yano, T., Jee, H.S., Nishio, N., Nagai, S.: J. Ferment. Technol., 64, 383 (1986). 2) Jee, H. S., Yano, T., Nishio, N., Nagai, S.: J. Ferment. Technol., 65, 413 (1987). 3) Wise, D.L., Cooney, C.L., Augenstein, D.C.: Biotechnol. Bioeng., 20, 1153 (1978). 4) Lange, N.A.: Handbook of Chemistry, Handbook Publishers, Inc., Ohio (1952). 5) Hamstra, R.S., Muftis, M.R., Tramper, J.: Biotechnol. Bioeng., 29, 884 (1987). 6) Ariga, O., Okumura, T., Taya, M., Kobayashi, T.: J. Chem. Eng. Japan, 17, 577 (1984). 7) Hippe, H., Caspari, D., Fiebig, K., Gottsehalk, G.: Proc. Natl. Acad. Sci. USA, 76, 494 (1979). 8) Bradford, M. Nf.: Anal. Biochem., 72, 248 (1976). (Received September 7, 1987)
Note
Continuous CH4 Production from H2 and CO2 by
Methanobacterium thermoautotrophicum in a Fixed-Bed Reactor HAE SUNG JEE, NAOMICHI NISHIO*, and SHIRO NAGAI
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Sa~jo-cho, Higashi-Hiroshima 724, Japan Methanation of H2 and COs by Methanobacterium thermoautotrophicumAH was carried cut in a fixed-bed reactor (31 m_m~b× 180 mm H) packed with granular diatomaceous earth clay (2-3 mm~b) as a support material. The maximum methane production rate obtained was 5.2 l/l bed volume]h, while a conversion rate reaching up to some 80% of the theoretical value was achieved by controlling the feed rate of the substrate gas throughout the culture period. After cultivation methanogen cells were fixed almost homogeneously on the whole of the support material, giving 30 mg dry cell[era s packed support.
CH4, a c a l o r i e - r i c h gas, c a n be p r o d u c e d by b i o m e t h a n a t i o n f r o m H~ a n d CO~ as a n a l t e r n a t i v e e n e r g y source to fossil fuels, l-s) 4H2+CO~=CH4+2HsO
(1)
I n p r a c t i c a l b i o m e t h a n a t i o n , an efficient supply o f H~ a n d C O s to the c u lt u r e m e d i u m is the most significant factor in the process, due to e x t r e m e l y l o w solubility to water.4-e) T h e r e f o r e , as r e p o r t e d in a previous paper,2) we designed a b i o r e a c t o r using a c y l i n d r i c a l c e r a m i c s u p p o rt instead o f the c o n v e n t i o n a l s u b m e r g e d culture, in o r d e r to facilitate mass transfer of the substrate gases. S u c h a reactor gave high methane productivity, b u t the conversion rate was still low (ca.
60%). I n the present study, a fixed-bed r e a c t o r p a c k e d w i t h a g r a n u l a r s u p p o r t was constructed, w h i c h it was t h o u g h t m i g h t facilitate the mass transfer o f the gaseous substrate to a m e t h a n o g e n , M . thermoautotrophicum AH. * Corresponding author
Ease in c h a n g i n g the r e a c t o r size a n d a r e d u c t i o n in r e a c t o r c o n s t r u c t i o n cost ar e two a d v a n t a g e s o f using g r a n u l a r supports. A feedback control system w i t h a supply of gaseous substrate w a s also a t t e m p t e d in o r d e r to get a h i g h conversion rate, ca. 8 0 % , o f the gaseous substrate to m e t h a n e . Materials and Methods Bacterial strain Methanobacteriumthermoautotrophicum AH (DSM 1053) from the Deutsche Sammlung yon Mikrooganismen (G6ttingen, FRG) was used. The euiture medium was the same as described previously. 1) All culture manipulations were carried out under oxygen-free conditions, as described previously, x) Preculture in a 125-ml serum vial (20ml working volume, H8[CO2=80]20, v/v in the gas phase) was carried out anaerobically for 20 h at 65°C. The mixed gas (H~[CO2=80]20, v/v) adjusted by two mass flow controllers was supplied at 1.5kg]cm~ before cultivation. Cultivation in a fixed-bed reactor As supports of the fixed-bed reactor, granular cellulose acetate (1.7-2.8mm~), granular diatomaceous earth clay (2-3, 5-6 mm~) and cylindrical diatomaceous earth
236
JzE, NISHIO, and NAOAX
[J. Ferment. Technol.,
are the flow rates of the respective gases, in l/h. The conversion rate, YCHo can also be calculated by the gas composition in the exit gas line. 5PcH, YCH,= 5 P c L 4 7 , c o , _ p ,
2
(6)
Where, PCH,, Pco2 and PH2 are the partial pressures of the respective gases of the exit gas. To achieve a YCH4 of more than 0.8, Freed was controlled on the basis of the Fexit value as a feedback control signal. This can be achieved by the substitution of YcH,~_0.8 into Eq. 5, from which the following relationship between Freed and Fexit can be derived. Ffeed<:2.78 Fextt
Fig. 1. Scheme of continuous culture apparatus for methanation of I-Is and CO2 with M. thermoautotrophicum fixed on granular support. 1, medium reservoir; 2, pump; 3, t-I2; 4, COs; 5, mass flow controller; 6, flow meter; 7, methanation reactor; 8, granular support; 9, water bath.
clay (3.5 m m ~ × 3 4 ram) were used. The supports of cellulose acetate and diatomaceous earth clay were obtained from Daicel Chem. Ind. Co., Tokyo and Nagao Soda Co., Ltd., Okayarna, respectively. The volume of packed support was 52.8 ems (7.54 cm 2 × 70ram) in a glass column ( 3 1 m m ~ × l l 0 m m ) . After packing, the reactor was flushed with the mixed gas at a feed rate of 20 ml/min overnight to maintain anaerobic conditions. Fixation of methanogen cells was carried out by passage of the preculture (44 ml), and through this procedure ca. 0.5 mg cell of the methanogen was fixed on the support. Cultivation was started by supplying the inorganic medium (16 ml/ h) via a peristaltic pump and the mixed gas (2.8 l/h) through a flow meter. The medium and the mixed gas were supplied through a hand-made nozzle at the top of the reactor (see Fig. 1). In the methanation process, based on Eq. 1, the conversion rate of H~ and COs to CH4, YCH4, can be calculated as follows. F feea= (F H*+Fc02) feed Fexit = ( F ~ + F c o 2 ) e x i t + F c H , F f eed -- F ext t = 5F c n , - - F c H~= 4 F cH6
(2) (3) (4)
Hence, YCH4 can be expressed by, 5FcH4 5 / Ffeed--Fexit YCH,= P~eeT = 4 - ~ Freed )
(5)
where, Fexit and Freed are the gas flow rates at the exit and inlet of the reactor (l/h). Faz, Fco2 and FcH,
(7)
Based on Eq. 7, if F~eed is controlled at less than 2.78 times of gexit, the methanation from Freed ( H 2 / C O s : 4 / 1 ) can be attained at a rate more than 0.8 of YCH~. Analysis To estimate the methanogen cell mass adhering onto the supports after cultivation, the support in the column reactor was carefully withdrawn and subdivided horizontally into 7 sections, 1 cm s of the support from the each section was crushed in 1 1V[ NaOH solution, and the eluted whole-cell protein was determined by a dye-binding method. 7,s) The hydrogen, carbon dioxide and methane contents of the exhaust gas were analysed by gas chromatography with a thermal conductivity detector under the conditions described previously. 1) The volume of exhaust gas from the reactor was measured with a gas flow meter and corrected to standard temperature conditions.
Results and Discussion Selection of support for fixed-bed reactor T o s e l e c t a s u i t a b l e s u p p o r t for the
fixed-bed
reactor
of M. thermoautomaterials were tested for t h e i r i n f l u e n c e o n t h e r n e t h a n a t i o n o f H~ and CO2. A f t e r f i x i n g t h e m e t h a n o g e n cells, cultivation was started by supplying the m i x e d g a s (2.6 l / h ) a n d t h e m e d i u m (16 m l / h). The highest rate of methanogenesis was observed with granular diatomaceous earth c l a y (size, 2 - 3 m m ~ ) (see F i g . 2 a ) . After 20 h o f c u l t i v a t i o n , m e t h a n a t i o n o f H s a n d COs reached a steady state at which the methane content of the exhaust gas was 15.5% (see Fig. 2 b ) , a n d t h e m e t h a n e p r o d u c t i o n r a t e w a s 280 m l / h a t t h e s t e a d y state. T h i s v a l u e c o r r e s p o n d s to 5.3 l C H 4 / I trophicum A H , g r a n u l a r
Vol. 66, 1988]
!3°°I
Biomethanation of Hs and CO2
a}
237
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0 20 40 60 80 I00 120 140 160 Culture t i m e (h)
Fig. 3. Methanation of t~s and COs by M. thermoautotrophicum ceils fixed on granular diatomaceous earth clay (2-3 mm~b). Culture conditions: 0 2 0 4 0 60 8 0 I00 120 mixed gas feed rate, 3.2 l/h; medium feed rate, C u l f u r e time (h) 18 ml/h; inoculura, 1.0 rag cells; volume of packed support, 105.6 cms (7.54 cm 2 × 140 mm Fig. 2. Methanation of Hs and COs by M. thermoH). Other culture conditions: See Fig. 2. Comautotrophicum cells fixed on various supports. position of exit gas: See Fig. 2. Culture conditions: volume of packed support, 52.8 ems (7.54 em* × 70 mm) ; inoculum, 0.5 mg cells; mixed gas (Hs/COs=80/20, v/v) feed rate, from a m a x i m u m v a l u e o f 540 m l / h to 2.6l/h; raedium feed rate, 16 ral/h; culture temperature, 65°C. a) l , granular diatomaceous 470 m l / h a t the end o f the culture. I n this earth clay (2-3 mm~b) ; Fq, granular diatomaceous run, the conversion r a t e of the s u b s t r a t e gas earth clay (5-6 mm~); /% cylindrical diatomace- to m e t h a n e d e c r e a s e d from 8 7 % at 25 h to ous earth clay (3.5 mm~ × 3 4 ram) ; A, cellulose 72O//o at the end, p r o b a b l y d u e to c h a n n e l i n g acetate (1.7-2.8mra~). b) Support: granular of the s u b s t r a t e flow b y the excess cell mass, diatomaceous earth clay (2-3 mm~). Comw h i c h caused a decrease in the c o n t a c t a r e a position of exit gas: ~ , I-Is; ~ , CO2; O, CH4. b e t w e e n the substrate gas a n d the m e t h a n o g e n cells. Next, a f e e d b a c k control system based on bed volume/h. Methanation with ceils fixed on granuF,,~t was set u p to get a h i g h conversion A f e e d b a c k control lar diatomaceous earth clay T o in- rate, YcH,, e.g., 8 0 % . crease the conversion r a t e of m e t h a n a t i o n o f r u n could be c o n d u c t e d b y c o n t r o l l i n g the I-I2 a n d CO2 b y i n c r e a s i n g r e a c t o r v o l u m e , feed r a t e of the m i x e d gas, Ffeed~__2.78 Fex~t (see Fig. 4). By this f e e d b a c k control, a b o u t a l o n g e r c o l u m n r e a c t o r (H, 180 m m ; 4, 31 m m ; c e r a m i c v o l u m e , 1 0 5 . 6 c m 8) was 8 0 % of YcH, (by Eq. 5) was o b t a i n e d t h r o u g h used. I n the m e t h a n a t i o n , the feed rates of o u t the culture, a v a l u e w h i c h c o i n c i d e d well the m i x e d gas a n d the m e d i u m to the r e a c t o r w i t h t h a t c a l c u l a t e d from the d i r e c t analysis were 3.2 l/h a n d 1 8 m l / h , respectively. I n of the exit gas (Eq. 6). After 260-h cultim e t h a n a t i o n w i t h the l o n g e r fixed-bed re- vation, the d i s t r i b u t i o n o f m e t h a n o g e n cells actor, the m e t h a n e c o n t e n t o f the exit gas inside the s u p p o r t was m e a s u r e d ; ca. 30 m g increased u p to 5 8 % at 25 h of c u l t i v a t i o n , cells/cm 8 p a c k e d s u p p o r t was found, w i t h then d e c r e a s e d g r a d u a l l y w i t h c u l t u r e t i m e , a h o m o g e n e o u s d i s t r i b u t i o n t h r o u g h the a n d showed a low v a l u e o f 3 4 % at the e n d o f r e a c t o r (see Fig. 5). I n a d d i t i o n , m e t h a n o the c o n t i n u o u s c u l t u r e (160 h, see Fig. 3). gen cell l e a k a g e in the effluent m e d i u m was T h e m e t h a n e p r o d u c t i o n r a t e also decreased v e r y low, ca. 40 mg/l c o m p a r e d w i t h the ',.9
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0 20 40 60 80 I00120 140 Deplh of pocked support (turn)
Fig. 5. Cell mass distribution of M. thermoautotrophicum in the fixed bed reactor. After 260 h continuous culture (see Fig. 4), the methanogen adhering on granular diatomaceous earth clay was horizontally subdivided into 7 sections (20 mm thick), and 1 cma of each section was crushed in 25 ml of 1 M NaOH solution to determine the cell mass concentration.
1.6 ,
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.
40 80 120 160 200240 280 Culture time (h)
Fig. 4. Methanation of H2 and CO2 by M. thermoautotrophicum cells fixed on granular diatomaceous earth clay (2-3 mm4). Culture conditions: See Figs. 2 and 3; volume of packed support, see Fig. 3. Arrow indicates start of control of gas feed rate. Conversion rate: U], calculated from Eq. 5; O, calculated from Eq. 6. Composition of exit gas: See Fig. 2. A, gas flow rate at inlet; A, gas flow rate at exit. figure inside the reactor of 30 mg/cmS, a n d the p H of the c u l t u r e m e d i u m showed a n almost c o n s t a n t v a l u e of 6.6 t h r o u g h o u t the c o n t i n u o u s culture (data n o t shown). I n m e t h a n a t i o n using the reactor, the m e t h a n e p r o d u c t i o n rate decreased g r a d u a l l y with c u l t u r e t i m e (see Fig. 4), p r o b a b l y due to c h a n n e l i n g of the substrate flow b y the excess m e t h a n o g e n cells. F u r t h e r studies o n imp r o v i n g the stability of the system, as well as on a n a l t e r n a t i v e process, are n o w i n progress. I n conclusion, it is believed that a fixed-bed reactor using a support such as g r a n u l a r d i a t o m a c e o u s e a r t h clay could be a p p l i c a b l e to the c o n t i n u o u s p r o d u c t i o n of m e t h a n e from H2 a n d CO2 b y M . thermoautotrophicum
AH, a h i g h m e t h a n e conversion rate of more t h a n 8 0 % b e i n g o b t a i n e d w h e n Ffe0d was controlled b y F,xit as a feedback control signal. F o r this gas substrate fermentation, the conversion rate c a n easily be estimated by r e a d i n g the two gas flow rates at the inlet a n d exit w i t h o u t the need for chemical analysis of the exit gas b y gas c h r o m a t o g r a p h y . Acknowledgments The authors are grateful to Nagao Soda Co., Ltd., for providing diatomaceous earth clay, and Daicel Chem. Ind. Co. for providing cellulose acetate support. References 1) Yano, T., Jee, H.S., Nishio, N., Nagai, S.: J. Ferment. Technol., 64, 383 (1986). 2) Jee, H. S., Yano, T., Nishio, N., Nagai, S.: J. Ferment. Technol., 65, 413 (1987). 3) Wise, D.L., Cooney, C.L., Augenstein, D.C.: Biotechnol. Bioeng., 20, 1153 (1978). 4) Lange, N.A.: Handbook of Chemistry, Handbook Publishers, Inc., Ohio (1952). 5) Hamstra, R.S., Muftis, M.R., Tramper, J.: Biotechnol. Bioeng., 29, 884 (1987). 6) Ariga, O., Okumura, T., Taya, M., Kobayashi, T.: J. Chem. Eng. Japan, 17, 577 (1984). 7) Hippe, H., Caspari, D., Fiebig, K., Gottsehalk, G.: Proc. Natl. Acad. Sci. USA, 76, 494 (1979). 8) Bradford, M. Nf.: Anal. Biochem., 72, 248 (1976). (Received September 7, 1987)