Biological production of hydrogen from cellulose by natural anaerobic microflora

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JOURNAL OFFERMENTATION ANDBIOENOIN~ERING Vol. 79, No. 4, 395-397.1995

Biological Production

of Hydrogen from Cellulose by Natural Anaerobic Microflora

YOSHIYUKI UENO,* TATSUSHI KAWAI, SUSUMU SATO, SEIJI OTSUKA, ANDMASAYOSHI MORIMOTO Kajima Technical Research Institute, Chofu, Tokyo 182, Japan

Received23 June 1994/Accepted22 December1994 The capability of natural anaerobic microflora to produce hydrogen was examined with artificial wastewater containing cellulose. The microflora in sludge compost was found to produce a significant amount of hydrogen (2.4 mol/mol-ltexose). Among the fermentation products other than hydrogen and carbon dioxide, the lower fatty acids, mainly acetate and butyrate, constituted more than approximately 90% of the total soluble metabolites. [Key words:

hydrogen production, anaerobic bacteria, microflora, cellulose] argon gas. To inhibit activity of photosynthetic bacteria, the culture was carried out without illumination. The culture medium was sampled and analyzed periodically. The evolved gas was collected in a gas collection bag (Ohmi Odo Air Service Co., Shiga) and the volume of evolved gas was determined by the water displacement method in graduated cylinders prefilled with water which was adjusted to pH 3 or less in order to prevent dissolution of the gas. The gas composition was determined using a gas chromatograph (Shimadzu Co., Kyoto, GC9A) under the following conditions: column, Molecular sieve and Porapack-Q; carrier gas, argon; flow rate, 30 ml/min; column temperature, 40°C; injection temperature, 50°C; detector temperature, 50°C; detector, TCD. Soluble total organic carbons (S-TOC) of filtrates of the sampled medium were analyzed using a total organic carbon analyzer (Shimadzu Co., TOC-500). Lower fatty acids of CZ to Cs were determined using a gas chromatograph (Shimadzu Co., GC-14A) under the following conditions: column, Unisole F-200; carrier gas, helium; flow rate, 30ml/min; column temperature, 140°C; injection temperature, 22O’C; detector temperature, 220°C; detector, FID by the method described by Aramata and Nagasaka (10). The concentration of cellulose powder in the medium was determined by the phenol-H,SO, method after the washout treatment of cell mass as described by Minato et al. (11). The time course of the fermentation pattern from cellulose during the cultivation of the anaerobic microflora is shown in Fig. 1. In the case of the anaerobic digestion sludge, gas evolution began to occur gradually after 24 h of cultivation and 1,375 ml/l-culture of gas was evolved at 120 h of cultivation. A decrease in pH and an increase in S-TOC were observed in parallel with gas evolution. The increase in S-TOC was caused by the formation of organic acids during fermentation of cellulose powder. There were no noticeable variations in after 120 h. The evolved gas consisted of hydrogen, carbon dioxide and methane, the ratio of which varied with time; the composition was 33%, 50% and 17%, respectively, at the final stage (see Table 1). Since the microflora was obtained from a stable methane fermentation process, only methane and carbon dioxide were produced in the 24 h period immediately after the start of the experiment. After 24 h, it is thought that hydrogen was accumulated because acid formation became dominant. Renewed

Hydrogen is a clean and renewable energy resource, not contributing to the greenhouse effect. Biological production of hydrogen using wastewater and other biomass as raw materials has been attracting attention as an environmentally friendly process that does not consume fossil fuels. Hydrogen production by microorganisms can be divided into two main categories: one is by photosynthetic bacteria cultured under anaerobic light conditions, and the other is by other anaerobic bacteria. The latter possess the ability to produce hydrogen without photoenergy. There have been many studies on the conversion of biomass to hydrogen by anaerobic bacteria. These have been carried out using pure cultures of different strains (l-5). The present study is aimed at developing hydrogen production during the treatment process of organic wastewater. Generally, natural microflora is used in various wastewater treatment processes since a sterilization process is not necessary in this case and since it can be adapted to various kinds of components in the wastewater. The main form of anaerobic wastewater treatment up to now was methane fermentation (6, 7). Hydrogen production by anaerobic microflora has been reported by Minoda et at. (8) and Sykes and Kirsch (9). In their experiments, hydrogen production resulted from inhibition of methane formation from hydrogen, and microflora which produces hydrogen with high efficiency has not been obtained. In this study, experiments were carried out in order to select the anaerobic microflora suitable for the production of hydrogen with a medium containing cellulose as a model of wastewater. Two types of microflora were used. One was anaerobic digestion sludge that was obtained in a thermophilic methanogenic fermentation process of kitchen wastewater. The other was sludge compost that was manufactured from aerobic activated sludge by forced aeration. Cellulose powder was used as a substrate for hydrogen production. The culture medium contained the following materials (g/c): KH2P04, 1.5; Na2HP0.,. 12H20, 4.2; NH&l, 0.5; MgC12-6Hz0, 0.18; yeast extract, 2.0; cellulose powder (Funakoshi Co., Funacel SF), 10. Fifteen g of the microflora was inoculated into 3 I of culture medium and cultured at 200 rpm and 60°C under anaerobic conditions where gas and liquid phases were replaced by * Correspondingauthor. 395

396

UENO ET AL.

J. FERMENT.BIOENG.,

(b)

24

48 72 96 Cultivation time 01)

FIG. 1. Time course of fermentation sludge; (b) microflora in sludge compost. n , cellulose.

120

Microflora

48 12 96 Cultivation time (h)

120

pattern from cellulose during the cultivation of the microflora. (a) Microflora in anaerobic digestion Symbols: 0, H2 gas; 0, CO2 gas; A, CH4 gas; x , pH; A, S-TOC products; 0, acetate; l , butyrate;

formation of methane observed after 48 h might be due to methane formation from hydrogen and carbon dioxide as substrates, but after 96 h the formation of methane became negligible. This might be due to the reduced activity of methanogens caused by the decrease in pH. In the case of the sludge compost, after 120 h of cultivation, 3,325 ml/l-culture of gas had evolved. The composition of the gas was 58% hydrogen and 42% carbon dioxide, and no methane was detected. The gas composition ratio did not vary with time. An increase in S-TOC and decrease in pH were observed with gas evolution. Noticeable variations in the reaction were not observed in after 120 h. As an explanation for the absence of methane in the evolved gas, it is speculated that the sludge compost, being made from aerobic activated sludge by rapid composting by forced aeration, contained scarcely any methanogens, and/or, since pH was not controlled in these batch cultures, methanogen activity declined causing the proliferation of hydrogen-producing acidogens and leading to the dominance of acid formation. As observed in the anaerobic digestion sludge, even if methanogens existed, a decrease of methane formation TABLE 1.

24

Fermentation

Gas composition Gas (%) evolved (ml/l-ctdture) Hz CO2 CH.,

was observed along with a decrease in pH. For each cultivation, significant production of acetate and butyrate was observed (see Table 1). The amounts of lower fatty acids and S-TOC produced are shown in terms of carbon content (see Table 1). Among the fermentation products other than hydrogen and carbon dioxide produced by the microflora in sludge compost, the lower fatty acids, mainly 1,724 mg/l of acetate and 1,281 mg/l of butyrate, constituted more than approximately 90% of the total soluble metabolites. The production levels of the other lower fatty acids were equivalent to approximately 50mg/l. The remainders are assumed to be metabolites such as ethanol and lactate which are also known to be by-products in the anaerobic decomposition of hexose (4). Table 1 lists the digestion ratios of the cellulose powder and the amounts of fermentation products produced per hexose consumed in the anaerobic microflora. The cellulose digestion ratio of the microflora in sludge compost was relatively high compared to that of the microflora in anaerobic digestion sludge, and it is suggested that sludge compost contained a large number of bacteria with higher cellulose digestion capabilities. The hydrogen production level of the microflora in

products produced from cellulose by anaerobic microflora

VFA VFA in Cellulose S-TOC? (mg/l) (mg,r) S-TOCb digestion (%) (%) Acetate Butyrate

Fermentation productsC Carbon (mol/mol-hexose) recovery (%) Hz CO2 CH4 Acetate Butyrate

Anaerobic digestion sludge

1375

33

50

17

1154

630

1034

77.8

38.5

0.9

1.3

0.4

0.8

0.3

88

Sludge compost

3325

58

42 N.D.

1724

1281

1530

90.7

58.0

2.4

1.7 N.D.

0.8

0.4

83

Data were obtained after 120 h of cultivation in the medium (cellulose: 10 g/f). Cellulose was calculated as hexose [(C,HIOOO,),]. N.D.: Not detected. B S-TOC: Soluble TOC produced. b Calculated from produced acetate and butyrate in terms of carbon contents. c Production yields per decomposed cellulose. d Calculated from produced S-TOC including acetate and butyrate as main products and evolved gas.

NOTES

VOL. 79, 1995

sludge compost was high, with no production of methane. The production of acetate and butyrate from hexose under anaerobic conditions can be explained by the following chemical equations (12). C6HLZOs+ 2H,0+2CH3COOH + 2C02 + 4H, t (1) +CH3CH2CH2COOH GH ~0, +2C02+2H2 t (2) Hydrogen acts as an acceptor of surplus electrons during acetate/butyrate formation. If the measured values of formed acetate and butyrate for the microflora in sludge compost are substituted into Eqs. 1 and 2, the calculated value of the formed hydrogen (2.4 mol/molhexose) is in good agreement with the measured value. The surplus electrons formed by the microflora in the sludge compost were recovered as the hydrogen gas. On the other hand, surplus electrons are apparently not present in a stable digestion process under anaerobic conditions. In the case of the microflora in anaerobic digestion sludge, the amount of hydrogen gas recovered was lower (0.9moVmoLhexose) than in the case of the microflora in sludge compost, due to the consumption of formed hydrogen as a reducing agent in the formation of methane. Acidogenic fermentation of hexose by microflora usually leads to butyrate fermentation under appropriate conditions. However, other groups of bacteria contributing to the fermentation process may become dominant if culture conditions are changed, resulting in formation of different fermentation products (13-16). The microorganisms that perform the reaction with Eqs. 1 and 2 are dominant in the microflora in sludge compost. The anaerobic spore-forming bacteria form an important part of the acidogenic population performing acetate/butyrate fermentation (14). These anaerobic bacteria were not deactivated during the forced aeration in the rapid composting and existed in the sludge compost. Clostridium butyricum (17) and Ruminococcus albus (18) are known to possess high hydrogen-producing capabilities, corresponding to 2.4 mol/mol-hexose and 2.6 mol/mol-hexose, respectively. Although the culture conditions differ in the composition of media and cultivation systems, it is stressed that the microflora in sludge compost in the present experiment has hydrogen production capabilities nearly equivalent to those of C. butyricum and R. afbus. Those are not thermophilic bacteria grown under the present conditions. Hydrogen production by the microflora in sludge compost might be due to the presence of thermophilic acetate/butyrate-producing acidogens which have not been identified here. There have been no previous reports on natural anaerobic microflora that converts hexose to hydrogen with high efficiency (2.4 mol/mol-hexose). Thus, the microflora in sludge compost may be the most useful microorganisms in the anaerobic production of hydrogen from biomass resources such as cellulose.

Earth (RITE) as a part of the Research & Development Project on Environmentally Friendly Technology for the Production of Hydrogen supported by the New Energy and Industrial Technology Development Organization (NEDO). REFERENCES 1.

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We are grateful to Dr. Yasuo Igarashi, Graduate School of Agriculture and Life Sciences of The University of Tokyo. for his helpful suggestion in this study. We also thank to Miss Mixue Yoshida, Miss Ikuko Sakamoto and Miss Tamai Hatano for their analytical support. This work was performed under the management of the Research Institute of Innovative Technology for the

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