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Enhanced hydrogen production in altered mixed acid fermentation of glucose by Enterobacter aerogenes
JOURNAL
BIOENOINEERIING
OPFERMENTATIONAND
Vol. 83, No. 4, 358-363. 1991
Enhanced Hydrogen Production in Altered Mixed Acid Fermentation of Glucose by Enterobacter aerogenes MAHYUDIN
ABDUL RACHMAN, YOSHINORI FURUTANI, YUTAKA NAKASHIMADA, TOSHIHIDE KAKIZONO, AND NAOMICHI NISHIO”
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 1-4-I Kagamiyama, Higashi Hiroshima 739, Japan Received 26 September 1996/Accepted 22 January 1997 Hydrogen (Hz) production by mutants of Enterobacter aerogenesHU-101, a strain isolated and characterized in our laboratory, was found to be enhanced compared with that of HU-101 due to blockage of production of other metabolites. The mutants were isolated by the ally1 alcohol (AA) and/or proton suicide method. Among the AA resistant mutants isolated after NTG treatment of E. aerogenes HU-101, strain A-l produced 78 mmol of Hz/Z medium together with no 2,3-butanedlol and reduced ethanol formation, compared to 52.5 mmol Hz/l medium by HU-101 when a complex medium containing 20 g/Z glucose, 5 g/Z yeast extract and 5 g/Z tryptone was used for the cultivation. However, the amounts of lactate and acetate produced by A-l were higher than those of HU-101. Subsequently, mutants incapable of producing these acids were isolated by a proton suicide method. A mutant (HZ-3) derived from HU-101 showed reduced acid production and increased Hz production, to 1.5-fold higher than that of HU-101. Furthermore, the production of acidic and alcoholic metabolites by the mutant AY-2, obtained by treatment of A-l with the proton suicide method was found to be lower, and its production of H2 at 101.5 mmol/Z medium, to be 2.0-fold higher, than those of HU-101. [Key words: hydrogen production, mutation, Enterobacteraerogenes] Hydrogen (Hz) expected in recent years to be used as a clean energy is generally synthesized photosynthetically and fermentatively by a rather large group of microorganisms under anaerobic conditions (1). For industrial use, enhancement of Hz production by microorganisms is being achieved through extensive research. Enterobacter aerogenes strain HU-101, isolated from sludge for methane fermentation by the authors in this study, has a high growth rate and several advantageous properties similar to those of other members of the Enterobacteriaceae, such as the ability to utilize a wide range of carbon sources, facultative anaerobicity and lack of au inhibitory effect on Hz generation of high H2 pressure (2). However, the yield of Hz from glucose of this bacterium is less than 1 mol/mol glucose, which is lower than that Rhodobacter sp. (3) and for other bacteria including Clostridium sp. (4). For industrial-scale H2 production by E. aerogenes, enhancement of its H2 production ability is needed. Although it is said that bacteria which exhibit mixed acid fermentation produce Hz through formate decomposition, Tanisho et al. reported that Hz is also produced via the NADH pathway in E. aerogenes (5). The NADH is generated during the fermentative conversion of glucose to pyruvate, called glycolysis. On the other hand, E. aerogenes produces 2,3-butanediol (BD), ethanol and organic acids (lactate, acetate and formate) besides Hz (6, 7). Of these metabolites, ethanol, BD and lactate are formed by coupling of oxidation of NADH. Therefore, it would be possible to enhance Hz production by blocking the pathways from pyruvate to ethanol, BD and lactate. Mutants carrying defects in the structural genes for butanediol dehydrogenase (BDDH) and alcohol dehydrogenase (ADH) can be selected by the ally1 alcohol (AA) method (8). In this method, since AA is oxidized
by ADH and/or BDDH to a toxic aldehyde (acrolein), mutants deficient in these enzymes survive. A number of Saccharomyces cerevisiae X2180-1A and X2180-IB (9), Escherichia coli (N-12), and Clostridium acetobutylicum mutants (13) have been isolated by this method. Furthermore, non- or low acid producers can be isolated by a proton suicide method (14, 15) based on lethal effects of bromine and bromite produced from a mixture of NaBr and NaBr03 during production of acids such as lactate and acetate. This method is simple and rapid, and approximately 50% of the survivors show recognizable defects in sugar metabolism. In this study, therefore, whether Hz production by E. aerogenes HU-101 could be enhanced by blocking of formation of alcoholic and acidic metabolites by use of the AA and/or proton suicide method was determined. Furthermore, the glucose metabolic characteristics of the mutants obtained were analyzed stoichiometrically for determination of the metabolic orientation of the mutants. MATERIALS
AND METHODS
The microMicroorganism and culture conditions organism used was E. aerogenes HU 101 isolated in this study (see Results and Discussion). Methanogenic sludge for methane fermentation of glucose developed in our laboratory (16) was used for isolation of this microorganism. By repeated batch culture over several weeks in the presence of 2-bromoethane sulfonic acid (ErBS) as an inhibitor of growth of methanogenic bacteria in glucose minimal medium, methanogenic bacteria were removed from the sludge. Chemostat culture of the sludge, with pH and temperature controlled at 7.0 and 37”C, respectively, was then performed with changing of the dilution rate for isolation of microorganisms that exhibit high growth and Hz production rates, and then a single colony was picked by the roll tube method (17).
* Corresponding author. 358
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For the characterization of an isolated microorganism, IMViC reactions such as indole formation, methyl red test, Voges-Proskauer reaction and citrate utilization were used. The morphology of the colonies, Gram staining and oxygen requirement were also investigated (18, 19). Complex medium was used for all experiments (20) except those for the stoichiometric analyses, in which medium without yeast extract and tryptone (glucose minimal medium) was used. The complex medium contained (per liter): 20g glucose, 5.Og yeast extract, 5.Og tryptone, 7.0 g K2HP04, 5.5 g KH2POI, 0.5 g L-cysteine-HC1.H20, l.Og (NH&SO.,, 0.25 g MgS04.7Hz0, 0.021 g CaC&. 2Hz0, 0.029 g Co(N0&.6Hz0, 0.039 g Fe(NH.&S04. 6H20, 2.0mg nicotinic acid, 0.172 mg Na2Se03, 0.02mg NiC& and 10ml of trace element solution containing 0.5 g MnC12.4Hz0, 0.1 g H3B03, 0.01 g AlK(SO&-HzO, 0.001 g CuC&-2Hz0 and 0.5 g NazEDTA per liter. For plating, 2% agar was added into medium. A modified Hungate technique in combination with the serum bottle technique (21) was used to culture the bacterium. The medium without glucose, cysteine and phosphate buffer was boiled for 20min, cooled in ice water with continuous bubbling of Nz gas, dispensed into serum bottles sealed with black butyl rubber stoppers and then sterilized (18min, 121°C). Concentrated aqueous solutions of glucose, cystein and phosphate buffer autoclaved separately were then injected into the medium using a hypodermic syringe. After inoculation of 2.5 ml of seed culture into serum bottles (approximately 125 ml in volume containing 50 ml of culture medium) and adjustment of the pH to 6.8, the bottles were incubated at 37°C without shaking. Mutagenesis and mutant isolation Aliquots (1 ml) of the cultures of E. uerogenes (about lo8 cells/ml) in the exponential growth phase were transferred to plastic Eppendorf tubes to be centrifuged and cells were washed twice with 0.1 M citrate buffer (pH, 5.5). After mutagenesis with 600 pg/ml NTG in 0.1 M phosphate buffer (pH, 7.0) for 90min and centrifugation, the cells were washed twice with 0.1 M phosphate buffer (pH, 7.0), incubated in the complex medium at 37°C for 24 h and spread to the agar medium containing 0.5 mM AA which was added to the agar medium at 45°C just before pouring into petri dishes for obtaining AA-resistant mutants (8). Bromo cresol purple (O.O4g/c) was also added to the agar medium and only purple colonies were picked for isolation of low acid producers (13) among the AAresistant mutants. Mutants in the proton suicide method were isolated on agar medium at pH 4.8 containing equimolar concentrations (32.5 mM) of NaBr and NaBr03 as a selection agent (see Results and Discussion) and 2,3,5-triphenyl tetrazolium chloride as an indicator (14, 15). Anaerobic growth was performed in simple anaerobic culture using an Anaero Pack (Mitsubishi Gas Chemical Co. Inc., Tokyo). The cell growth was measured in terms of Analysis optical density (OD) at 680 nm; 1 OD unit was estimated as being equivalent to 0.74 g/l of dry cell mass. Ethanol and BD were determined by gas chromatography (GC 14 A, Shimadzu, Kyoto) with a flame ionization detector. Gas production was determined by a method of displacement of saturated aqueous NaCl in a graduated cylinder. The contents of CO2 and Hz were determined by gas chromatography (GC 8A, Shimadzu) with a thermal conductivity detector (22). Lactate, acetate, formate, pyru-
BY E. AEROGENES
359
and acetoin were determined by HPLC as previously described (23). D-Glucose was determined by the glucose-oxidase method (Gluci-net, Tournei, Belgium) (20).
vate
RESULTS AND DISCUSSION
After elimiScreening and identification of HU-101 nation of methanogenic bacteria by repeated batch cultures in the presence of BrES in the glucose minimal medium, chemostat cultures were carried out with increasing dilution rate for isolation of microorganisms which grow at a high rate under the conditions used (data not shown). By this method, strain HU-101 was isolated at a dilution rate of 6.84 d-r. The microbiological characteristics of HU-101 are summarized in Table 1. The cells of HU-101 are gram negative, rod-shaped, facultatively anaerobic and glucose fermenting. The M-R test result was negative, and the V-P test result was positive. These findings demonstrate that this strain belongs to group II of the family Enterobacteriaceae (18). Since the results for all of the tests for membership in the genus Enterobacter were positive except for the lack of utilization of adonitol, HU-101 was concluded to be a member of the genus Enterobacter, species aerogenes. Figure 1 shows the time courses of the batch culture of HU-101 in the complex medium at pH6.8 and 37°C. The cell growth was rapid for the initial 6 h of culture and then slowed, although glucose remained in the medium (Fig. 1A). Since acetate and lactate were present in the medium and the culture pH decreased to 4.9 at 24 h TABLE 1.
Diagnostic characteristics and carbon source utilization test results for HU-101
a. IMViC reactions, microscopic test results and production hydrogen _ Gram staining _ Spore formation Facultatively anaerobic + _ Indole formation _ Methyl red test Acetoin formation + Citrate utilization + Hz formation + b. Fermentation of carbon sources Pentose Arabinose + Xylose Methyl pentose + Rhamnose Galactose t Fructose Hexose + Glucose Sorbose + CeLlobiose Mannose Disaccharide + Lactose Sucrose t Maltose Trehalose + Melibiose Polysaccharide + Soluble starch Glycoside t Inositol Esculin Sugar alcohol + Sorbitol Mannitol _ Adonitol Organic acid + Fumarate Succinate
of
+ +
+ + + +
+ + _
+ , Growth and acid formation; - , no growth on respective carbon source (20 g/l, pH 7.0) after 3 d.
360
RACHMAN ET AL.
0
4
8
J. FERMENT.BIOENG.,
12
16
20
24
Time (h)
Time (h)
FIG. 1. Time courses of growth of and glucose consumption by (A) and production of fermentative metabolites by (B) E. aerogenes HU-101. Symbols: 0, dry cell mass; ??, glucose; A, Hz; 0, ethanol; 7, 2,3-butanediol; ? ?, lactate; V , acetate. Cultivation conditions: complex medium (glucose=20g/l) at initial pH of 6.8, 37°C without shaking in serum bottles (approximately 125 ml in volume 50ml of medium).
FIG. 3. Time courses of growth of and glucose consumption by (A) and production of fermentative metabolites by (B) ally1 alcoholresistant mutant A-l. Symbols and cultivation conditions: see Fig. 1 legend. A-l was isolated as an AA-resistant mutant on a plate containing 0.5 mM AA in a complex medium using HU-101 as a parent strain.
(data not shown), the growth rate was assumed to have decrease because of the decrease in pH due to the accumulation of acid metabolites. The production of Hz reached approximately 55 mmol Hz/l medium at 24 h culture. As to the formation of other metabolites, HU-101 synthesized ethanol, BD, acetate and lactate at 43, 23, 12 and 51 mmol/l medium, respectively, in 24 h of incubation (Fig. 1B). Subsequently, the isolation of mutants showing altered metabolites production characteristics and characterization of their HZ production abilities were performed.
When the AA method was used for the isolation of mutants showing blocked formation of alcohols, two strains (A-l and AA-l) resistant to AA at 0.5 mM were obtained. As shown in Fig. 2, HU-101 did not grow on the same plate containing AA. In subsequent experiments, A-l was characterized with respect to metabolite formation during anaerobic cultivation in the glucose complex medium (glucose= 111 mM). There was no significant difference in both growth and glucose consumption between the HU-101 and A-l (Fig. 1A and Fig. 3A). The Hz production of A-l, however, was found to be 78 mmol/l medium at 24 h culture, which is 1.5-fold higher than that of HU-101. A-l formed less than
Isolation of ally1 alcohol (AA)-resistant mutants
A
FIG. 2. Colony formation of E. uerogenes HU-101 and ally1 alcohol-resistant with (B) ally1 alcohol at 0.5 mM.
HU-101 mutants on plates without (A) ally1 alcohol (AA) and
ENHANCED HYDROGEN PRODUCTION
VOL. 83, 1997
TABLE 2. Lethal concentrations of NaBr and NaBrOr at various pHs for modified proton suicide method for E. uerogenes Growth at initial DH
Reagent (mM) NaBr NaBrOS
6.9
6.0
5.5
5.0
t+t ttt +t 4 + Y c
i+t ‘it +lt +t tc + _
ttt tt+ it tt + + _
+I+ + tL + + _ _
0 25 27.5 30 32.5 35 40 50 55
0 25 27.5 30 32.5 35 40 50 55
4.8 4t +t + + _
_
ttt, Normal lawn; 4, translucent colonies; - , no growth.
lawn; +, translucent
isolated
30mmol/[ medium of ethanol and no BD but more acetate (25 mmol/l medium) and lactate (75 mmol/l medium) than HU-101 as shown in Fig. 3B, indicating that pyruvate was metabolized to lactate and acetate instead of alcohols with improved Hz production like mutants deficient in butanol dehydrogenase and ethanol dehydrogenase as reported previously (12, 13). Isolation of non- or low acid producing mutants by proton suicide method Next, the proton suicide
method was used for obtaining non- or low acid producing mutants. For determination of the optimum conditions for the proton suicide method, the combined effect of bromide plus bromate on the growth of HU-101 was determined at various pHs as shown in Table 2. The
361
lethal concentration decreased when the initial pH was lowered. Since low concentrations of bromate and bromide were better than high concentrations for avoiding osmotic inhibition of cell growth, a solid medium containing a selection agent at 32.5 mM with pH adjusted to 4.8 was used for obtaining non- or low acid producers in subsequent mutation experiments. Figure 4 shows the time courses of batch culture of the mutant HZ-3 obtained in the proton suicide method using HU101 as a parent strain. Although the specific growth rate of HZ-3 was slightly lower than that of HU-101, the Hz production of HZ-3 was 86 mmol Hz/l medium. The ethanol and BD productions were higher, at 56.4 and 49.8 mmol/l medium, respectively, than those of HU101, while the acetate and lactate productions were lower, at 0 and 26.1 mmol/Z medium, respectively. These metabolite profiles are opposite to those for the AAresistant mutant A-l. Double mutation by AA and proton suicide methods
The Hz production by the mutants isolated by the AA (A-l and AA-l) or proton suicide method (HZ-3) was higher than that of the HU-101. However, the production of metabolites such as acetate and lactate in the AA method and ethanol and BD in the proton suicide method were also higher. This led to a decrease in the concentration of NADH which is indispensable for Hz production in the cells. Therefore, double mutation by the AA and proton suicide methods was performed to block production of both alcoholic and acidic metabolites. In the proton suicide method using the AA-resistant strain A-l as a parent, strain AY-2 was obtained. In Fig. 5, time
120
120
T 2100 8
BY E. AEROGENES
2 2100
50
!
3 EeO
s
(B)
-
20- -
LO-
E Q40
E.
-
640
%2u P
0 0
4
8
12
16
20
24
Time(h) FIG. 4. Time courses of growth of and glucose consumption by (A) and production of fermentative metabolites by (B) low acid producing mutant HZ-3. Symbols and cultivation conditions: see Fig. 1 legend. HZ-3 was isolated by the proton suicide method on a plate containing 32.5 mM bromide and 32.5 mM bromate in a complex medium using HU-101 as a parent strain.
0
. 4
i
12
16
20
i4
ImW)
FIG. 5. Time courses of growth of and glucose consumption by (A) and production of fermentative metabolites by (B) low acid and low alcohol producing mutant AY-2. Symbols and cultivation conditions: see Fig. 1 legend. Isolation method for AY-2 was the same as that described in the legend of Fig. 4 except for the use of an AAresistant mutant (A-l) as a parent strain.
362
RACHMAN ET AL. TABLE 3.
J. FERMENT. BIOENG..
Stoichiometric analyses of end products of fermentation by E. uerogenes HU-101 and three mutantsa grown on glucose minimal medium Yield (mol/mol glucose)
Strain HU-101 A-l HZ-3 AY-2
Cell GH7NOz
Ethanol
0.13 0.14 0.13 0.15
0.49 0.32 0.54 0.34
C2I-W
BDb C4H1002
0.37 0.0 0.46 0.04
Acetoin C4&02
0.01 0.19 0.0 0.16
Formate H2C02
0.16 0.11 0.14 0.18
Recovery (%)
Lactate
Acetate
C3H603
C2H402
0.29 0.55 0.16 0.31
0.17 0.48 0.13 0.14
Pyruvate
C-,
H2 2
Carbon
Electron
94.3 99.0 96.5 87.7
97.1 98.3 104.1 83.8
C3H403
0.02 0.08 0.09 0.14
1.08 0.88 1.07 1.22
0.56 0.84 0.83 1.17
Cultivation conditions: glucose minimal medium (glucose=20g/l) at initial pH of 6.8, at 37°C for 48 h without shaking in serum bottles (approximately 125 ml with 50 ml of medium). a Screening method for strains A-l. HZ-3 and AY-2: see leeends for Figs. 3-5. b BD, 2,3-iutanediol.
courses of batch culture of AY-2 are shown. The specific growth rate after a 2-h lag phase was found to be higher than that of A-l. No BD and little acetate were formed during fermentation, and the amount of lactate formed was reduced to 50% compared to that for A-l. The Hz production reached 101.5 mmol Hz/l medium, which was the maximum value in this study. Stoichiometric conversion of glucose Stoichiometric conversion of glucose to metabolites in HU-101 and isolated mutants in the glucose minimal medium instead of the complex medium was analyzed in detail and the results are summarized in Table 3. Since glucose is the sole carbon source in the glucose minimal medium, recoveries of carbon and electron can be calculated from yields of metabolites and cells to glucose consumed. The cellular composition of CSH7N02 was used for calculation (24). The yield of Hz of the AA-resistant mutant A-l was 0.84 mol/mol glucose, compared to 0.56 mol/mol glucose for HU-101. The yields of lactate and acetate of Al were also higher than those of HU-101 although yields of alcoholic metabolites and CO2 were lower, with accumulation of acetoin in A-l instead of lack of formation of BD. The activities of enzymes for conversion of pyruvate to acetoin would still remained in A-l although an enzyme (butanediol dehydrogenase) for conversion of acetoin to BD would be completely inactivated. On the other hand, the mutant HZ-3 obtained by the proton suicide method produced Hz at almost the same yield, 0.83 mol/mol glucose, as but less acetate and lactate, and more ethanol, BD and pyruvate than A-l. The H2 yield of AY-2, obtained by double mutation with the AA and proton suicide methods, reached 1.17 mol/mol glucose, which is 2.1-fold higher than that of HU-101. The level of pyruvate accumulation in the AY-2 cultures was higher than that in HU-101 cultures. There was not much difference in formate production among the wildtype and mutant strains. The carbon and electron recoveries in AY-2 decreased to 87.7% and 83.8X, respectively, compared to those in HU-101, indicating that intermediate(s) not detected in this study might have accumulated in the cells or culture medium of AY-2. The yields of alcoholic and acidic metabolites could be reduced by the AA method and the proton suicide method, respectively, compared to those in the case of HU-101. However, the yields of alcoholic metabolites in the case of AY2 were higher than those in the case of A-l, and the yields of acidic metabolites were higher in the case of AY-2 than in that of HZ-3. A limitation of hydrogenase activity in AY-2 might cause production of other metabolites via NADH not oxidized by hydrogenase. Accordingly, enhancement of the hydrogenase activity of AY-2
would be needed for further tion.
enhancement
of H2 produc-
REFERENCES 1. Alvin, I. K.: Hydrogenase: properties and application. Enzyme Microb. Technol., 1. 165-172 (1979). 2 Tank&o, S., Snzoki; Y., and w&o, N.: Fermentative hydrogen evolution by Enferobacter aerogenes strain E.82005. Int. J. Hydrogen Energy, 12, 623-627 (1987). 3. Mlyake, Y., Mao, X.-Y., and Knwamura, S.: Photoproduction of hydrogen from glucose by a co-culture of a photosynthetic bacterium and Clostridium butyricum. J. Ferment. Technol., 62, 531-535 (1984). 4. Tagnchi, F., Mizukami, N., Hasegawa, K., and Saito-Taki, T.: Microbial conversion of arabinose and xylose to hydrogen by a newly isolated Clostridium sp. no. 2. Can. J. Microbial., 40, 228-233 (1994). 5. Tanisho, S., Kamiya, N., and Wakao, N.: Hydrogen evolution of Enterobucter aerogenes depending on culture pH: mechanism of hydrogen evolution from NADH by means of membrane-bound hydrogenase. Biochim. Biophys. Acta, 973, l-6 (1989). 6. Magee, R. J. and Kosaric, N.: The microbial production of 2,3-butanediol. Adv. Appl. Microbial., 32, 89-159 (1987). 7. Johansen, L., Bryn, K., and Stormer, F. C.: Physiological and biochemical role of butanediol pathway in Enterobucter uerogenes. J. Bacterial., 123, 1124-l 130 (1975). 8. Diirre, P., Kuhn, A., and Gottschalk, G.: Treatment with ally1 alcohol selects specially for mutants of Clostridium acetobutylicum defective in butanol synthesis. FEMS Microbial. Lett., 36, 77-81 (1986). 9. Wills, C. and Phelps, J.: A technique for the isolation of yeast alcohol dehydrogenase mutants with altered substrate specificity. Arch. Biochem. Bioohvs., 167. 627-637 (1975). 10. bark, D. and Cronan; j. E., J;.: Acetaldehydk coenzyme A dehydrogenase of Escherichia coli. J. Bacterial., 144, 179-184 (1980). 11. MatJan, F., Klswar, Y. A., and Clark, D.: Mutants of E. coli deficient in the fermentative lactate dehydrogenase. J. Bacteriol., 171, 342-348 (1989). 12. Lorowits, W. and Clark, D.: Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase. J. Bacterial., 152, 935-938 (1982). 13. Rogers, P. and Palosaari, N.: Clostridium acetobutylicum mutants that produce butylaldehyde and altered quantities of solvents. Appl. Environ. Microbial., 53, 2761-2766 (1987). 14. Winkelman, J. W. and Clark, D.: Proton suicide: general method for direct selection of sugar transport- and ferrnentation-defective mutants. J. Bacterial., 160, 687-690 (1984). 15. Pablo, H. C. and Me’ndez, B. S.: Direct selection of Clostridiurn acetobutylicum fermentation mutants by a proton suicide method. ADDI. Environ. Microbial., 56. 578-580 (1990). 16. Chang, Y.ij., Nisklo, N., Maruta, -H.,‘and Nagi, S.:’Characteristics of granular methanogenic sludge grown on glucose in a UASB reactor. J. Ferment. Bioeng., 75, 430-434 (1993).
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17. Hungate, R. E.: A roll tube method for cultivation of strict anaerobes, p. 117-132. In Norris, J. R. and Ribbons, D. W. (ed.), Methods in microbiology, 3 B. Academic Press Inc., London and New York (1969). 18. Murray, R. G. E., Breiner, J. D., Bryant, M. P., and John, G. H.: Facultatively anaerobic gram-negative rods, p. 175-289. In Tansiil, B. and Nolley, C. S. (ed.), Bergey’s manual of determinative bacteriology, 9th ed. Williams and Wilkins Co., Baltimore and London (1989). 19. Schlegel, H. G. and Schmidt, K.: Formic acid fermentation and Enterobacteriaceae, p. 285-305. In Kogut, M. (ed.), General microbiology, 6th ed. Cambridge University Press Inc., London, New York and Melbourne (1986). 20. Koesnandar, Ago, S., Nishio, N., and Nagai S.: Production of extra cellular 5aminolevulinic acid by Clostridium thermoaceti-
21. 22.
23.
24.
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cum grown in minimal medium. Biotechnol. Lett., 11, 567-572 (1989). Miller, T. L. and Wolin, M. J.: A serum bottle modification of the Hungate technique for cultivating obligate anaerobes. Appl. Microbial., 27, 985-987 (1974). Nishio, N., Eguchi, S. Y., Kawashima, H., and Nagai, S.: Mutual conversion between Hz plus COz and formate by a formate-utilizing methanogen. J. Ferment. Technol., 61, 557561 (1983). Fukuzaki, S., Chang, Y-J., Nishio, N., and Nagai, S.: Characteristics of granular methanogenic sludge grown on lactate in a UASB reactor. J. Ferment. Bioeng., 72, 465-472 (1991). Moser, A.: The principles of bioprocess technology, p. 25-40. In Manor, P. (ed.), Bioprocess technology kinetics and reactors. Springer-Verlag, New York (1988).
BIOENOINEERIING
OPFERMENTATIONAND
Vol. 83, No. 4, 358-363. 1991
Enhanced Hydrogen Production in Altered Mixed Acid Fermentation of Glucose by Enterobacter aerogenes MAHYUDIN
ABDUL RACHMAN, YOSHINORI FURUTANI, YUTAKA NAKASHIMADA, TOSHIHIDE KAKIZONO, AND NAOMICHI NISHIO”
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 1-4-I Kagamiyama, Higashi Hiroshima 739, Japan Received 26 September 1996/Accepted 22 January 1997 Hydrogen (Hz) production by mutants of Enterobacter aerogenesHU-101, a strain isolated and characterized in our laboratory, was found to be enhanced compared with that of HU-101 due to blockage of production of other metabolites. The mutants were isolated by the ally1 alcohol (AA) and/or proton suicide method. Among the AA resistant mutants isolated after NTG treatment of E. aerogenes HU-101, strain A-l produced 78 mmol of Hz/Z medium together with no 2,3-butanedlol and reduced ethanol formation, compared to 52.5 mmol Hz/l medium by HU-101 when a complex medium containing 20 g/Z glucose, 5 g/Z yeast extract and 5 g/Z tryptone was used for the cultivation. However, the amounts of lactate and acetate produced by A-l were higher than those of HU-101. Subsequently, mutants incapable of producing these acids were isolated by a proton suicide method. A mutant (HZ-3) derived from HU-101 showed reduced acid production and increased Hz production, to 1.5-fold higher than that of HU-101. Furthermore, the production of acidic and alcoholic metabolites by the mutant AY-2, obtained by treatment of A-l with the proton suicide method was found to be lower, and its production of H2 at 101.5 mmol/Z medium, to be 2.0-fold higher, than those of HU-101. [Key words: hydrogen production, mutation, Enterobacteraerogenes] Hydrogen (Hz) expected in recent years to be used as a clean energy is generally synthesized photosynthetically and fermentatively by a rather large group of microorganisms under anaerobic conditions (1). For industrial use, enhancement of Hz production by microorganisms is being achieved through extensive research. Enterobacter aerogenes strain HU-101, isolated from sludge for methane fermentation by the authors in this study, has a high growth rate and several advantageous properties similar to those of other members of the Enterobacteriaceae, such as the ability to utilize a wide range of carbon sources, facultative anaerobicity and lack of au inhibitory effect on Hz generation of high H2 pressure (2). However, the yield of Hz from glucose of this bacterium is less than 1 mol/mol glucose, which is lower than that Rhodobacter sp. (3) and for other bacteria including Clostridium sp. (4). For industrial-scale H2 production by E. aerogenes, enhancement of its H2 production ability is needed. Although it is said that bacteria which exhibit mixed acid fermentation produce Hz through formate decomposition, Tanisho et al. reported that Hz is also produced via the NADH pathway in E. aerogenes (5). The NADH is generated during the fermentative conversion of glucose to pyruvate, called glycolysis. On the other hand, E. aerogenes produces 2,3-butanediol (BD), ethanol and organic acids (lactate, acetate and formate) besides Hz (6, 7). Of these metabolites, ethanol, BD and lactate are formed by coupling of oxidation of NADH. Therefore, it would be possible to enhance Hz production by blocking the pathways from pyruvate to ethanol, BD and lactate. Mutants carrying defects in the structural genes for butanediol dehydrogenase (BDDH) and alcohol dehydrogenase (ADH) can be selected by the ally1 alcohol (AA) method (8). In this method, since AA is oxidized
by ADH and/or BDDH to a toxic aldehyde (acrolein), mutants deficient in these enzymes survive. A number of Saccharomyces cerevisiae X2180-1A and X2180-IB (9), Escherichia coli (N-12), and Clostridium acetobutylicum mutants (13) have been isolated by this method. Furthermore, non- or low acid producers can be isolated by a proton suicide method (14, 15) based on lethal effects of bromine and bromite produced from a mixture of NaBr and NaBr03 during production of acids such as lactate and acetate. This method is simple and rapid, and approximately 50% of the survivors show recognizable defects in sugar metabolism. In this study, therefore, whether Hz production by E. aerogenes HU-101 could be enhanced by blocking of formation of alcoholic and acidic metabolites by use of the AA and/or proton suicide method was determined. Furthermore, the glucose metabolic characteristics of the mutants obtained were analyzed stoichiometrically for determination of the metabolic orientation of the mutants. MATERIALS
AND METHODS
The microMicroorganism and culture conditions organism used was E. aerogenes HU 101 isolated in this study (see Results and Discussion). Methanogenic sludge for methane fermentation of glucose developed in our laboratory (16) was used for isolation of this microorganism. By repeated batch culture over several weeks in the presence of 2-bromoethane sulfonic acid (ErBS) as an inhibitor of growth of methanogenic bacteria in glucose minimal medium, methanogenic bacteria were removed from the sludge. Chemostat culture of the sludge, with pH and temperature controlled at 7.0 and 37”C, respectively, was then performed with changing of the dilution rate for isolation of microorganisms that exhibit high growth and Hz production rates, and then a single colony was picked by the roll tube method (17).
* Corresponding author. 358
VOL. 83. 1997
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For the characterization of an isolated microorganism, IMViC reactions such as indole formation, methyl red test, Voges-Proskauer reaction and citrate utilization were used. The morphology of the colonies, Gram staining and oxygen requirement were also investigated (18, 19). Complex medium was used for all experiments (20) except those for the stoichiometric analyses, in which medium without yeast extract and tryptone (glucose minimal medium) was used. The complex medium contained (per liter): 20g glucose, 5.Og yeast extract, 5.Og tryptone, 7.0 g K2HP04, 5.5 g KH2POI, 0.5 g L-cysteine-HC1.H20, l.Og (NH&SO.,, 0.25 g MgS04.7Hz0, 0.021 g CaC&. 2Hz0, 0.029 g Co(N0&.6Hz0, 0.039 g Fe(NH.&S04. 6H20, 2.0mg nicotinic acid, 0.172 mg Na2Se03, 0.02mg NiC& and 10ml of trace element solution containing 0.5 g MnC12.4Hz0, 0.1 g H3B03, 0.01 g AlK(SO&-HzO, 0.001 g CuC&-2Hz0 and 0.5 g NazEDTA per liter. For plating, 2% agar was added into medium. A modified Hungate technique in combination with the serum bottle technique (21) was used to culture the bacterium. The medium without glucose, cysteine and phosphate buffer was boiled for 20min, cooled in ice water with continuous bubbling of Nz gas, dispensed into serum bottles sealed with black butyl rubber stoppers and then sterilized (18min, 121°C). Concentrated aqueous solutions of glucose, cystein and phosphate buffer autoclaved separately were then injected into the medium using a hypodermic syringe. After inoculation of 2.5 ml of seed culture into serum bottles (approximately 125 ml in volume containing 50 ml of culture medium) and adjustment of the pH to 6.8, the bottles were incubated at 37°C without shaking. Mutagenesis and mutant isolation Aliquots (1 ml) of the cultures of E. uerogenes (about lo8 cells/ml) in the exponential growth phase were transferred to plastic Eppendorf tubes to be centrifuged and cells were washed twice with 0.1 M citrate buffer (pH, 5.5). After mutagenesis with 600 pg/ml NTG in 0.1 M phosphate buffer (pH, 7.0) for 90min and centrifugation, the cells were washed twice with 0.1 M phosphate buffer (pH, 7.0), incubated in the complex medium at 37°C for 24 h and spread to the agar medium containing 0.5 mM AA which was added to the agar medium at 45°C just before pouring into petri dishes for obtaining AA-resistant mutants (8). Bromo cresol purple (O.O4g/c) was also added to the agar medium and only purple colonies were picked for isolation of low acid producers (13) among the AAresistant mutants. Mutants in the proton suicide method were isolated on agar medium at pH 4.8 containing equimolar concentrations (32.5 mM) of NaBr and NaBr03 as a selection agent (see Results and Discussion) and 2,3,5-triphenyl tetrazolium chloride as an indicator (14, 15). Anaerobic growth was performed in simple anaerobic culture using an Anaero Pack (Mitsubishi Gas Chemical Co. Inc., Tokyo). The cell growth was measured in terms of Analysis optical density (OD) at 680 nm; 1 OD unit was estimated as being equivalent to 0.74 g/l of dry cell mass. Ethanol and BD were determined by gas chromatography (GC 14 A, Shimadzu, Kyoto) with a flame ionization detector. Gas production was determined by a method of displacement of saturated aqueous NaCl in a graduated cylinder. The contents of CO2 and Hz were determined by gas chromatography (GC 8A, Shimadzu) with a thermal conductivity detector (22). Lactate, acetate, formate, pyru-
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359
and acetoin were determined by HPLC as previously described (23). D-Glucose was determined by the glucose-oxidase method (Gluci-net, Tournei, Belgium) (20).
vate
RESULTS AND DISCUSSION
After elimiScreening and identification of HU-101 nation of methanogenic bacteria by repeated batch cultures in the presence of BrES in the glucose minimal medium, chemostat cultures were carried out with increasing dilution rate for isolation of microorganisms which grow at a high rate under the conditions used (data not shown). By this method, strain HU-101 was isolated at a dilution rate of 6.84 d-r. The microbiological characteristics of HU-101 are summarized in Table 1. The cells of HU-101 are gram negative, rod-shaped, facultatively anaerobic and glucose fermenting. The M-R test result was negative, and the V-P test result was positive. These findings demonstrate that this strain belongs to group II of the family Enterobacteriaceae (18). Since the results for all of the tests for membership in the genus Enterobacter were positive except for the lack of utilization of adonitol, HU-101 was concluded to be a member of the genus Enterobacter, species aerogenes. Figure 1 shows the time courses of the batch culture of HU-101 in the complex medium at pH6.8 and 37°C. The cell growth was rapid for the initial 6 h of culture and then slowed, although glucose remained in the medium (Fig. 1A). Since acetate and lactate were present in the medium and the culture pH decreased to 4.9 at 24 h TABLE 1.
Diagnostic characteristics and carbon source utilization test results for HU-101
a. IMViC reactions, microscopic test results and production hydrogen _ Gram staining _ Spore formation Facultatively anaerobic + _ Indole formation _ Methyl red test Acetoin formation + Citrate utilization + Hz formation + b. Fermentation of carbon sources Pentose Arabinose + Xylose Methyl pentose + Rhamnose Galactose t Fructose Hexose + Glucose Sorbose + CeLlobiose Mannose Disaccharide + Lactose Sucrose t Maltose Trehalose + Melibiose Polysaccharide + Soluble starch Glycoside t Inositol Esculin Sugar alcohol + Sorbitol Mannitol _ Adonitol Organic acid + Fumarate Succinate
of
+ +
+ + + +
+ + _
+ , Growth and acid formation; - , no growth on respective carbon source (20 g/l, pH 7.0) after 3 d.
360
RACHMAN ET AL.
0
4
8
J. FERMENT.BIOENG.,
12
16
20
24
Time (h)
Time (h)
FIG. 1. Time courses of growth of and glucose consumption by (A) and production of fermentative metabolites by (B) E. aerogenes HU-101. Symbols: 0, dry cell mass; ??, glucose; A, Hz; 0, ethanol; 7, 2,3-butanediol; ? ?, lactate; V , acetate. Cultivation conditions: complex medium (glucose=20g/l) at initial pH of 6.8, 37°C without shaking in serum bottles (approximately 125 ml in volume 50ml of medium).
FIG. 3. Time courses of growth of and glucose consumption by (A) and production of fermentative metabolites by (B) ally1 alcoholresistant mutant A-l. Symbols and cultivation conditions: see Fig. 1 legend. A-l was isolated as an AA-resistant mutant on a plate containing 0.5 mM AA in a complex medium using HU-101 as a parent strain.
(data not shown), the growth rate was assumed to have decrease because of the decrease in pH due to the accumulation of acid metabolites. The production of Hz reached approximately 55 mmol Hz/l medium at 24 h culture. As to the formation of other metabolites, HU-101 synthesized ethanol, BD, acetate and lactate at 43, 23, 12 and 51 mmol/l medium, respectively, in 24 h of incubation (Fig. 1B). Subsequently, the isolation of mutants showing altered metabolites production characteristics and characterization of their HZ production abilities were performed.
When the AA method was used for the isolation of mutants showing blocked formation of alcohols, two strains (A-l and AA-l) resistant to AA at 0.5 mM were obtained. As shown in Fig. 2, HU-101 did not grow on the same plate containing AA. In subsequent experiments, A-l was characterized with respect to metabolite formation during anaerobic cultivation in the glucose complex medium (glucose= 111 mM). There was no significant difference in both growth and glucose consumption between the HU-101 and A-l (Fig. 1A and Fig. 3A). The Hz production of A-l, however, was found to be 78 mmol/l medium at 24 h culture, which is 1.5-fold higher than that of HU-101. A-l formed less than
Isolation of ally1 alcohol (AA)-resistant mutants
A
FIG. 2. Colony formation of E. uerogenes HU-101 and ally1 alcohol-resistant with (B) ally1 alcohol at 0.5 mM.
HU-101 mutants on plates without (A) ally1 alcohol (AA) and
ENHANCED HYDROGEN PRODUCTION
VOL. 83, 1997
TABLE 2. Lethal concentrations of NaBr and NaBrOr at various pHs for modified proton suicide method for E. uerogenes Growth at initial DH
Reagent (mM) NaBr NaBrOS
6.9
6.0
5.5
5.0
t+t ttt +t 4 + Y c
i+t ‘it +lt +t tc + _
ttt tt+ it tt + + _
+I+ + tL + + _ _
0 25 27.5 30 32.5 35 40 50 55
0 25 27.5 30 32.5 35 40 50 55
4.8 4t +t + + _
_
ttt, Normal lawn; 4, translucent colonies; - , no growth.
lawn; +, translucent
isolated
30mmol/[ medium of ethanol and no BD but more acetate (25 mmol/l medium) and lactate (75 mmol/l medium) than HU-101 as shown in Fig. 3B, indicating that pyruvate was metabolized to lactate and acetate instead of alcohols with improved Hz production like mutants deficient in butanol dehydrogenase and ethanol dehydrogenase as reported previously (12, 13). Isolation of non- or low acid producing mutants by proton suicide method Next, the proton suicide
method was used for obtaining non- or low acid producing mutants. For determination of the optimum conditions for the proton suicide method, the combined effect of bromide plus bromate on the growth of HU-101 was determined at various pHs as shown in Table 2. The
361
lethal concentration decreased when the initial pH was lowered. Since low concentrations of bromate and bromide were better than high concentrations for avoiding osmotic inhibition of cell growth, a solid medium containing a selection agent at 32.5 mM with pH adjusted to 4.8 was used for obtaining non- or low acid producers in subsequent mutation experiments. Figure 4 shows the time courses of batch culture of the mutant HZ-3 obtained in the proton suicide method using HU101 as a parent strain. Although the specific growth rate of HZ-3 was slightly lower than that of HU-101, the Hz production of HZ-3 was 86 mmol Hz/l medium. The ethanol and BD productions were higher, at 56.4 and 49.8 mmol/l medium, respectively, than those of HU101, while the acetate and lactate productions were lower, at 0 and 26.1 mmol/Z medium, respectively. These metabolite profiles are opposite to those for the AAresistant mutant A-l. Double mutation by AA and proton suicide methods
The Hz production by the mutants isolated by the AA (A-l and AA-l) or proton suicide method (HZ-3) was higher than that of the HU-101. However, the production of metabolites such as acetate and lactate in the AA method and ethanol and BD in the proton suicide method were also higher. This led to a decrease in the concentration of NADH which is indispensable for Hz production in the cells. Therefore, double mutation by the AA and proton suicide methods was performed to block production of both alcoholic and acidic metabolites. In the proton suicide method using the AA-resistant strain A-l as a parent, strain AY-2 was obtained. In Fig. 5, time
120
120
T 2100 8
BY E. AEROGENES
2 2100
50
!
3 EeO
s
(B)
-
20- -
LO-
E Q40
E.
-
640
%2u P
0 0
4
8
12
16
20
24
Time(h) FIG. 4. Time courses of growth of and glucose consumption by (A) and production of fermentative metabolites by (B) low acid producing mutant HZ-3. Symbols and cultivation conditions: see Fig. 1 legend. HZ-3 was isolated by the proton suicide method on a plate containing 32.5 mM bromide and 32.5 mM bromate in a complex medium using HU-101 as a parent strain.
0
. 4
i
12
16
20
i4
ImW)
FIG. 5. Time courses of growth of and glucose consumption by (A) and production of fermentative metabolites by (B) low acid and low alcohol producing mutant AY-2. Symbols and cultivation conditions: see Fig. 1 legend. Isolation method for AY-2 was the same as that described in the legend of Fig. 4 except for the use of an AAresistant mutant (A-l) as a parent strain.
362
RACHMAN ET AL. TABLE 3.
J. FERMENT. BIOENG..
Stoichiometric analyses of end products of fermentation by E. uerogenes HU-101 and three mutantsa grown on glucose minimal medium Yield (mol/mol glucose)
Strain HU-101 A-l HZ-3 AY-2
Cell GH7NOz
Ethanol
0.13 0.14 0.13 0.15
0.49 0.32 0.54 0.34
C2I-W
BDb C4H1002
0.37 0.0 0.46 0.04
Acetoin C4&02
0.01 0.19 0.0 0.16
Formate H2C02
0.16 0.11 0.14 0.18
Recovery (%)
Lactate
Acetate
C3H603
C2H402
0.29 0.55 0.16 0.31
0.17 0.48 0.13 0.14
Pyruvate
C-,
H2 2
Carbon
Electron
94.3 99.0 96.5 87.7
97.1 98.3 104.1 83.8
C3H403
0.02 0.08 0.09 0.14
1.08 0.88 1.07 1.22
0.56 0.84 0.83 1.17
Cultivation conditions: glucose minimal medium (glucose=20g/l) at initial pH of 6.8, at 37°C for 48 h without shaking in serum bottles (approximately 125 ml with 50 ml of medium). a Screening method for strains A-l. HZ-3 and AY-2: see leeends for Figs. 3-5. b BD, 2,3-iutanediol.
courses of batch culture of AY-2 are shown. The specific growth rate after a 2-h lag phase was found to be higher than that of A-l. No BD and little acetate were formed during fermentation, and the amount of lactate formed was reduced to 50% compared to that for A-l. The Hz production reached 101.5 mmol Hz/l medium, which was the maximum value in this study. Stoichiometric conversion of glucose Stoichiometric conversion of glucose to metabolites in HU-101 and isolated mutants in the glucose minimal medium instead of the complex medium was analyzed in detail and the results are summarized in Table 3. Since glucose is the sole carbon source in the glucose minimal medium, recoveries of carbon and electron can be calculated from yields of metabolites and cells to glucose consumed. The cellular composition of CSH7N02 was used for calculation (24). The yield of Hz of the AA-resistant mutant A-l was 0.84 mol/mol glucose, compared to 0.56 mol/mol glucose for HU-101. The yields of lactate and acetate of Al were also higher than those of HU-101 although yields of alcoholic metabolites and CO2 were lower, with accumulation of acetoin in A-l instead of lack of formation of BD. The activities of enzymes for conversion of pyruvate to acetoin would still remained in A-l although an enzyme (butanediol dehydrogenase) for conversion of acetoin to BD would be completely inactivated. On the other hand, the mutant HZ-3 obtained by the proton suicide method produced Hz at almost the same yield, 0.83 mol/mol glucose, as but less acetate and lactate, and more ethanol, BD and pyruvate than A-l. The H2 yield of AY-2, obtained by double mutation with the AA and proton suicide methods, reached 1.17 mol/mol glucose, which is 2.1-fold higher than that of HU-101. The level of pyruvate accumulation in the AY-2 cultures was higher than that in HU-101 cultures. There was not much difference in formate production among the wildtype and mutant strains. The carbon and electron recoveries in AY-2 decreased to 87.7% and 83.8X, respectively, compared to those in HU-101, indicating that intermediate(s) not detected in this study might have accumulated in the cells or culture medium of AY-2. The yields of alcoholic and acidic metabolites could be reduced by the AA method and the proton suicide method, respectively, compared to those in the case of HU-101. However, the yields of alcoholic metabolites in the case of AY2 were higher than those in the case of A-l, and the yields of acidic metabolites were higher in the case of AY-2 than in that of HZ-3. A limitation of hydrogenase activity in AY-2 might cause production of other metabolites via NADH not oxidized by hydrogenase. Accordingly, enhancement of the hydrogenase activity of AY-2
would be needed for further tion.
enhancement
of H2 produc-
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