ABSTRACT
Recently, much advancement has been made in dark-fermentative hydrogen production. Various types of immobilized systems were developed to achieve high volumetric rates of hydrogen production. Other advances emerged through application of metabolic engineering so that microbes can be manipulated to maximize the yield predicted from the metabolic path. 2.2.1 Hydrogen-Producing MicroorganismsVery few microorganisms are known for their ability of converting biomass to hydrogen. Hydrogen-producing microbes belong to different domains such as facultative anaerobes, obligate anaerobes, methylotrophs, and photosynthetic bacteria (Nandi and Sengupta, 2008). However, fermentation processes contributes toward hydrogen which is present in the biosphere. On the basis of the hydrogen-producing mechanism, microbes are broadly categorized as either dark-fermentative or photofermentative microorganisms. Dark-fermentative hydrogenproduction is more advantageous due to its relatively low cost, low energy demands, moderate operative conditions, and minimal pollution generation (Angenent et al., 2004). Other advantages of dark fermentation over photofermentation are rapid bacterial growth rates, relatively high hydrogen production capacities, no dependency on light sources, no oxygen limitation problems, and low capital costs (Das, 2001). Therefore, dark-fermentative hydrogen production has attracted attention of researches. Among a large number of microbial species, strict anaerobes and facultative anaerobic chemoheterotrophs are efficient hydrogenproducers. The reduction of protons to hydrogen in microorganisms has evolved to dissipate excess electrons within the cells and to allow additional energy steps in metabolism. 2.2.1.1 ClostridiumThe genus Clostridium belongs to the low G+C gram-positive group of bacteria. They are rod-shaped, fermentative, spore-forming, obligate anaerobes. Their size varies from 0.3-2.0 ¥ 1.5-20 µm. They have a lower doubling time and can persevere in unfavorable conditions, where the other anaerobic bacteria fail to survive. This makes the organisms potent for industrial applications. Clostridium sp. came into picture the first time during the First World War
when Clostridium was used in fermentation for solvent and alcohol production (Weizmann and Rosenfeld, 1937). Hydrogen is a common by-product in such fermentation processes. Obligate anaerobic clostridia are potential hydrogen producers and well known for higher hydrogenyield (Kamalaskar et al., 2010; Valdez-Vazquez and Poggi-Varaldo, 2009). C. butyricum, C. welchii, C. pasteurianum, and C. beijerinckii are newly isolated Clostridium sp. that were used individually as well as in synthetic-mixed consortia for hydrogen production. The C. beijerinckii AM21B was isolated from the termite gut and showed the highest hydrogenyield of 1.8 to 2.0 mol per mol of glucose (Taguchi et al., 1996). This strain is capable of utilizing a wide range of other carbohydrates, such as xylose, arabinose, galactose, cellobiose, sucrose, and fructose. Another Clostridium sp. (strain no. 2), also isolated from termites, produces hydrogen more efficiently from xylose and arabinose (13.7 and 14.6 mmol/g or 2.l and 2.2 mol/mol) compared to glucose (11.1 mmol/g or 2.0 mol/mo1) (Taguchi et al., 2011). These results suggest that both Clostridium sp. can be used for hydrogen production using cellulose and hemicellulose as substrates present in plant biomass. Hydrolysis of biomass for the production of a fermentable substrate can be done along with fermentation, where saccharification and fermentation occur simultaneously or in a separate saccharification process preceded by fermentation. Thus, the ability of clostridia to produce hydrogen looks very promising. 2.2.1.2 EnterobacterThe Enterobacter genus belongs to the class Gamma Proteobacteria. They are generally gram-negative, rod-shaped, motile (peritrichous flagellated) or nonmotile, facultative anaerobes. Their size varies from 0.3-1.0 ¥ 1-6 µm. They have high growth rates, are capable of utilization of a wide range of carbon sources, and are resistant to lower traces of dissolved oxygen, and hydrogen production is not inhibited by high hydrogen pressures (Tanisho et al., 1987). However, the yield of hydrogen was lesser in Enterobacter sp. compared to Clostridium sp. when glucose was used as a substrate. Under batch fermentation, a hydrogen yield of l.0 mol per mol of glucose and a production rate of 21 mmol L-1 h-1 was observed (Tanisho, 1998). In a continuous hydrogen production process using a continuous stirred tank reactor
(CSTR), hydrogen production was monitored for 42 days using the same strain and considering molasses as the substrate. The highest hydrogen production rate and yield of 17 mmol L-1 h-1 and 1.5 mol/mol was observed, respectively. In contrast to batch fermentation, the metabolic end product showed prominence of lactate, whereas butyrate and acetic acid were produced in lower amounts (Tanisho and Ishiwata, 1994). It was also observed that flushing the culture medium with argon enhanced the hydrogen yield to 1.6 mol per mol of glucose. To enhance hydrogen production rates, mutants of E. aerogenes and E. cloacae were developed. In these mutants, the production of metabolites such as alcohols and organic acids was blocked. Production of these metabolites competes for reductants (NADH) which are also required for hydrogen production. A double-mutant strain of E. aerogenes was developed, which showed lower production of ethanol and butanediol. This lead to twofold improvement of hydrogen yield compared to the wild type (Rachman et al., 1998). The E. cloacae IIT-BT 08 (presently known as Klebsiella pneumonia IIT-BT 08) strain was isolated from a leaf extract. These bacteria produce hydrogen using a wide ranges of substrates. In batch fermentation, the maximum hydrogen yield of 2.2 mol per mol of glucose was observed (Kumar and Das, 2000). The maximum hydrogen production rate measured was 35 mmol L-1 h-1 using sucrose as a substrate. A double-mutant strain of E. cloacae IIT-BT 08 was also developed for improvement of hydrogen production (Kumar et al., 2001). In batch fermentation, it showed 1.5 times increased hydrogen yield on glucose, that is, 3.4 mol of hydrogen per mol of glucose. In continuous hydrogen production, the E. aerogenes double mutant (Rachman et al., 1998) was reported to give the maximum hydrogen production rate of 58 mmol L-1 h-1 at a dilution rate of 0.67 h-1, which was nearly two times higher compared to the wild type. The whole-cell immobilized system was further investigated in a packed-bed reactor. The column packed with spongy material having immobilized whole cells (E. aerogenes) produced hydrogen on a starch hydrolysate. A maximum hydrogen yield of 1.5 mol per mol of glucose was observed at a dilution rate of 0.1 h-1 (Palazzi et al., 2000).
