Biological production of hydrogen is an energy efficient process, and highly economic when produced from waste biomass. Biological process includes microbial conversion of biomass into fuels like ethanol, 1,3-propanediol, hydrogen, methane, biodiesel etc. mainly by two distinct process namely dark fermentation and photo fermentation. Dark fermentation is regarded as the most efficient process due to less hardware requirement, diverse microbial entities and faster hydrogen production rates.
Dark fermentation can be carried out by pure or mixed cultures of facultative and strict anaerobes mostly belonging to Clostridiaceae or Enterobacteriaceae family. Strict anaerobes, which includes Clostridium sps. such as Clostridium acetobutylicum, Clostridium pasteurianum, Clostridium paraputrificum, Clostridium. thermocellum, are more efficient hydrogen producers compared to facultative organisms, which includes Bacillus sps., E. coli, Enterobacter sps.etc. There exists some diversity in the metabolic pathway of hydrogen production in both types of anaerobes. Glycerol metabolism has two phases, viz. acidogenesis (production of organic acids which primarily occurs in exponential growth phase) and solventogenesis (conversion of acid to solvents which occurs in the stationary phase). Glycerol bioconversion commences with conversion of glycerol to dihydroxyacetone (DHA) by enzyme glycerol dehydrogenase followed by further conversion to DHA phosphate, and finally to pyruvate. Pyruvate is then converted to acetyl-coA and then depending on the organism either formate is formed by the PFL (pyruvate formate lyase) pathway or reduced ferredoxin and CO2, through the PFOR (pyruvate ferredoxin oxidoreductase) pathway (Fig. 1.4). Formate is converted to hydrogen and CO2 by formate hydrogen lyase, which mostly contains a [NiFe]
hydrogenase. Only the PFOR organisms can utilize the NADH formed during glycolysis
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to produce hydrogen via NADH dependent [Fe-Fe] hydrogenase. Another class of [Fe- Fe] hydrogenase that produces hydrogen by re-oxidizing ferredoxin, thus called Fd dependent [Fe-Fe] hydrogenase. Reduction of ferredoxin by enzyme hydrogenase results in release of H2.
Like other biofuels originating from fermentation route, unit cost of production of biohydrogen is strongly influenced by the cost of the substrate. Dark fermentation is remarkable for its ability to utilize a wide variety of feedstock. Monomeric hexose/pentose sugars like glucose and xylose are potential substrate for bio-hydrogen production which can be obtained by pretreatment and enzymatic hydrolysis (or saccharification) of lignocellulosic biomass. However, the entire chain of pretreatment, saccharification and fermentation makes it a lengthy and a cost intensive process of biofuel production. Holding these points in mind, this study aims at using crude glycerol, a byproduct of biodiesel industry as a direct substrate for hydrogen production which is devoid of above pre-treatment methods. Conversion of waste glycerol to clean energy is itself an economically viable concept. But due to less diversity in microbial cultures capable of utilizing glycerol, this concept is less studied. Clostridium pasteurianum is a well-known gram-positive bacterium that can utilize glycerol as a substrate and convert to hydrogen and other metabolites (Lo et al., 2013).
Several earlier authors have studied biohydrogen production from fermentation of either pure or crude glycerol. Some authors have also employed mixtures of glycerol (in either pure or crude form) with other substrates such as cheese whey, buffalo slurry, formate and acetate (Table 1.5). As compared to pure glycerol, studies using crude glycerol as substrate are limited. A review of the previous literature on biohydrogen production using pure and crude glycerol is given in Table 1.5.
INTRODUCTION AND LITERATURE
A comprehensive review of literature on microbial hydrogen production by bioconversion of crude glycerol has also been published by Sarma et al. (2012). More recently, Hallenbeck and Liu (2016) have reviewed biohydrogen production by photosynthetic bacteria during photo−fermentative growth. Urbaniec and Bakker (2015) have reviewed hydrogen production by dark fermentation using biomass residues (agricultural and agro-industrial solid waste) and other starchy residues. As seen from Table 1.5, most of the previous studies have employed either mixed cultures (in form of activated sludge) or pure cultures of bacteria of Enterobacteriaceae family for production of biohydrogen. In addition to the microbial culture, an important factor influencing biohydrogen production is the mode of fermentation. As noted in literature review presented in Table 1.5, several previous authors have used fed-batch and continuous mode of fermentation. The yield of biohydrogen has also been defined in different ways by the previous authors.
Figure 1.4. Hydrogen production pathways viz. PFOR (pyruvate:ferredoxin oxidoreductase) pathway and PFL (pyruvate:formate lyase) pathway (adopted from
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1.2.1.1 Clostridium pasteurianum
Clostridium pasteurianumis a rod-shaped, spore forming Gram-positive obligate anaerobic bacteria which was first described in 1895 by Sergei Nikolayevich Winogradsky (Winogradsky, 1895). It belongs to the phylum Firmicutes, class Clostridia, order Clostridiales, family Clostridiaceae and genus Clostridium. C.
pasteurianum was initially known for its ability to fix atmospheric nitrogen in a non- symbiotic process (Zelitch, 1951). However, in recent years it has been exploited for the production of acids, solvents and gas from glycerol and other carbohydrates, which was first mentioned by Nakas et al. (1983). The pathway for glycerol utilization in C.
pasteurianum was proposed by Dabrock et al. (1992) (Fig. 1.5)
Figure 1.5. Metabolic pathway of Clostridium pasteurianum for hydrogen production using glycerol as substrate.
INTRODUCTION AND LITERATURE
1.2.1.2 Dark fermentation in C. pasteurianum
Hydrogen production by the biological processes has been considered as an economical approach and environmentally clean method. Dark fermentation is one of the main biological processes in which microorganisms utilize carbohydrate sources to produce biohydrogen in anaerobic fermentation conditions (Dębowskia et al., 2014). The yield of hydrogen produced depends on metabolic pathway used by microorganisms so that the maximum 4 mol of H2 can be produced theoretically from one mol of hexose (glucose) as carbon source when acetic acid is the main fermentative byproduct as shown in Eq. (1):