ABSTRACT
Æ (3.3)C H O CH CH COOH+ 2CO + 2H6 12 6 3 2 2 2Æ (3.4) The catalysis of pyruvate oxidation to acetyl-CoA and formate performed by pyruvate formate lyase (PFL) is the second type of mechanism which occurs in few facultative anaerobic bacteria, such as Escherichia coli (Knappe and Sawers, 1990) (Eq. 3.5, Fig. 3.1): Pyruvate +CoA Acetyl-CoA +FormateÆ (3.5) The formate hydrogen lyase (FHL) then cleaves this formate, resulting in the production of hydrogen and CO2 (Eq. 3.6, Fig. 3.1). This pathway was first reported in 1932 by Stephenson and Stickland. HCOOH CO HÆ +
Figure 3.1 Metabolic pathways involved in dark-fermentative hydrogen production. In case propionic acid, lactic acid, or ethanol is the end product of pyruvate oxidation, no hydrogen is produced. Besides, in the
presence of alternate electron acceptors like nitrate, fumarate, etc., hydrogen production is hindered because it causes a biochemical shift of reactions from fermentation to anaerobic respiration, especially in facultative anaerobes like enteric bacteria. Thus, studies focusing on hydrogen production from facultative anaerobes should ensure that the production medium is devoid of any such electron acceptors. The much-gained attention toward dark fermentation for hydrogen production is due to its potential to utilize a wide variety of substrates. Furthermore, utilization of different kinds of wastewater as a substrate has broadened the scope of the process, hence providing dual benefits of waste bioremediation along with hydrogen production. For example, the use of industrial wastewater as a substrate suffices the basic yardsticks of substrate selection, viz., biodegradability, cost, and availability (Cai et al., 2009). For biohydrogen production from wastewater via dark fermentation, researchers have explored the potential for the following wastewater types: palm oil mill wastewater (Pandu and Joseph, 2012), wheat straw wastes, molasses-based distillery effluents (Das et al., 2008), rice spent wash from wineries (Nath and Das, 2004), wastewater from processed food industries (Cheong and Hansen, 2006), cellulose-and pentose-rich paper mill effluents (Cheng et al., 2011), starchy wastewater from households (Chen et al., 2008b), wastewater generated from cattle-based industries (Chen et al., 2008a), and chemical wastewater (Cakir et al., 2010). These reports have majorly worked upon mixed cultures in acidophilic conditions after selective enrichment. Due to the nonsterile and heterogeneous environment of wastewater, utilization of mixed microflora is crucial and is more relevant for the dark fermentation process. Most of these wastes, however, require pretreatment before being used as a substrate source. For instance, plant biomass is rich in cellulose and must undergo physicochemical pretreatment before use in fermentors. The pretreatment method can range from treating it with an acid or alkali to the oxidative steam explosion method carried out at high temperatures. This pretreatment strategy of lignocellulosic material yields a mixture of pentose and hexose sugars, thus allowing microbial fermentation of these hydrolysis products. Hence while using plant biomass for hydrogen production, pretreatment plays a major role. However, fermentation of this mixture of hexose and pentose sugars is usually
avoided as in the presence of glucose, catabolic repression causes lower conversion of pentose sugar and hence decreases the overall yield of hydrogen (Strobel, 1993; Aberu et al., 2010). Besides, the different fermentation pathways in organisms affect the efficiency of hydrogen production from sugars (Abreu et al., 2012). Dark fermentation yields organic acids that can be further used as substrates by photofermentative bacteria and produce CO2 and hydrogen. Thus, by combination of photofermentation and dark fermentation into a two-stage (hybrid) process, higher hydrogen yields can be obtained. This hybrid process can theoretically generate around 12 mol of hydrogen per mol of glucose (Nath and Das, 2008). 2CH3COOH + 4H2O Æ 8H2 + 4CO2 (3.7) But photofermentation is marred by many operational constrains. The major bottleneck is low photosynthetic light conversion efficiency of photofermentative microorganisms. Moreover, a low rate of hydrogen production and the shading effect of pigments produced by the photofermentative microorganisms also undermine the potential of photofermentation. Another hybrid system that propped up in recent times is two-stage biohydrogen followed by the biomethanation process. This could be achieved either in the same reactor or in a different bioreactor. During dark-fermentative hydrogen production, only 12%–18% of the total energy available from the feedstock could be extracted. To maximize gaseous energy extraction, another hybrid system has been developed in which hydrogen production would be followed by second-stage methanogenesis. Theoretically, 4 mol of hydrogen are obtained from 1 mol of glucose. The theoretical maximum was almost achieved; however, the energy trapped in 2 mol of acetic acid generated could not be recovered. The energy trapped in acetic acid could be recovered by acetoclastic methanogens as the seed to form methane. C6H12O6 + 2H2O Æ 4H2 + 2CH3COOH + 2CO2
(Stage I: Biohydrogen production) (3.8) 2CH3COOH Æ 2CH4 + + 2CO2(Stage II: Biomethane production) (3.9)
The biomethanation process is a well-established process. So, integration of the biohydrogen and biomethane production processes, known as the biohythane process, could prove to be a better option for gaseous energy recovery compared to the photofermentative process. 3.2.1 Enhancement of Hydrogen Production by
Metabolic EngineeringCurrent advances in metabolic engineering with development of techniques like expression analysis, gene technology, and genome sequencing have improved the possibilities of engineering microorganisms for introduction, deletion, or modification of desired metabolic pathways. The results of quantitative and systematic analysis of metabolic pathways are integrated with genomic approaches and molecular biology via metabolic engineering. The alteration of the metabolic pathway could increase the production of a native or nonnative product (Stephanopoulos, 1988; Wiechert, 2002), such as biohydrogen and biomethane. The effect of limiting factors in biohydrogen production pathways can be diminished by metabolic engineering to divert and concentrate the flow of electrons toward hydrogen-evolving pathways. The approach can also be used for increasing substrate consumption and making oxygen-tolerant hydrogenases, which will ultimately improve the efficiency of the process. In dark-fermentative hydrogen production, the metabolic engineering approach can be employed at different stages for overall process improvement. Metabolic engineering can be employed either for introducing new pathways for hydrogen production or for altering existing pathways so that hydrogen production can be improved in terms of hydrogen yields and/or rates. 3.2.1.1 Metabolic engineering approach for improvement of
hydrogen-producing obligate anaerobesIn strict anaerobes, hydrogen is produced by the enzyme [Fe-Fe] hydrogenase that mediates transfer of electrons from ferredoxin (Fd) to a proton (H+ ion). The metabolic pathway and possible target enzymes for metabolic engineering to enhance hydrogen production in obligate anaerobes are shown in Fig. 3.2. In such organisms,
pyruvate produced in glycolysis is converted to acetyl-CoA and CO2 through PFOR. This oxidation of pyruvate requires ferredoxin, which later transfers its electron to a proton facilitated by [Fe-Fe] hydrogenase to evolve hydrogen. The whole process results in a maximum yield of 2 mol of hydrogen per mol of glucose metabolized. However, strict anaerobes are known to produce higher yields of hydrogen, that is, up to 4 mol of hydrogen per mol of glucose. Another 2 mol of hydrogen can be produced by the oxidation of NADH formed during glycolysis. The NADH undergoes oxidation and transfers its electron to ferredoxin (Fd) through NADH ferredoxin oxidoreductase (NFOR). Thus, the overall maximum yield of 4 mol of hydrogen per mol of glucose consumed can be obtained in strict anaerobes via metabolic engineering. However, the reason for lower hydrogen yields is also due to its dependence on the other competing pathways and metabolites, for example, acetate, butyrate, lactate, or ethanol produced during fermentation. The maximum yield of hydrogen is achieved when acetate is the end product. Thermophilic fermentation is an interesting approach to direct the reaction toward hydrogen production because of more favorable thermodynamic conditions (Kadar et al., 2004; van Niel et al., 2002). High temperatures shift the equilibrium point of the hydrogen pathways in the direction of hydrogen production by a factor of up to 4.5, which results in higher hydrogen yields (Kongjan and Angelidaki, 2011; Veit et al., 2008). It has been reported that under thermophilic conditions, a higher hydrogen production rate (1050 ± 63 mmol h-1 at 60°C) is exhibited (Ahn et al., 2005) and a lesser variety of fermentation end products are obtained (Schonheit and Schafer, 1995). Moreover, the H+ ion produced inside the cell during fermentation is expelled outside the cell. This generates a proton motive force that further influences hydrogen production and ATP generation (Hakobyan et al., 2012). Different approaches applied to increase hydrogen production in obligate anaerobes have been summarized in Table 3.1. To increase hydrogen production in strict anaerobes overexpression of [Fe-Fe] hydrogenase has been reported in some studies. The [Fe-Fe] hydrogenases are known to evolve hydrogen and are found in strict anaerobes such as Clostridia, Thermotoga, and Desulfovibrio sp. The hydrogenase gene from different microorganisms has been characterized, and genetic modifications of this gene have been
performed to increase hydrogen production. In strict anaerobes, hydA encoding the [Fe-Fe] hydrogenase can be overexpressed homologously as well as heterologously. Morimoto et al. reported that homologous overexpression of the hydA gene in Clostridium paraputrificum M-21 resulted in 1.7-fold increased hydrogen production compared to the wild strain with no lactic acid production and increased acetic acid production (Morimoto et al., 2005). Homologous overexpression of the [Fe-Fe] hydrogenase gene, hydA in C. tyrobutyricum JM1, resulted in a 1.7-fold and 1.5-fold enhancements in the hydrogenase activity and hydrogen yield, respectively, against the wild-type strain (Jo et al., 2010). Earlier attempts of heterologous overexpression of [Fe-Fe] hydrogenase were in vain, and later it was shown that for successful functional expression of hydrogenase, co-expression of three maturation genes, hydE, hydF, and hydG, is necessary. These maturation genes are required for maturation and insertion of the H-cluster in organisms where these genes are absent (Posewitz et al., 2008). Therefore, the reported work of hydrogenase overexpression where these accessory genes were not co-expressed (Karube et al., 1983; Subudhi et al., 2011). For co-expression of maturation genes along with hydA, cloning vectors having two cloning sites are available (Duet vectors). Co-expressing hydE, hydF, and hydG genes from C. acetobutylicum in E. coli has shown a stable hydrogen production. Co-expression of C. acetobutylicum maturation proteins along with various algal and bacterial [Fe-Fe] hydrogenase results in enzymes with similar specific activity as the ones purified from native sources. Hence, it can be inferred that for the catalytically active [Fe-Fe] hydrogenase to be biosynthesized, it is essential that the Hyd maturation proteins and catalytic domain of the hydrogenase be present (King et al., 2006). However, if somehow the host genome is encoded for hydrogen-producing proteins, accessory genes will not be required and heterologous expression will be possible. During dark-fermentative hydrogen production, one of the major bottlenecks encountered is the accumulation of end metabolites that inhibit hydrogen production. Accessory metabolic pathways operating during fermentative hydrogen production also compete for NADH, diluting the pool of NADH required for the hydrogen production via the NFOR pathway, resulting in decreased hydrogen production. An alternative approach to increase hydrogen production
is to block the existing competing pathways that utilize the pool of NADH. Integration mutagenesis was used to inactivate acetate kinase-encoding ack gene so that the acetate-forming pathway can be blocked in C. tyrobutyricum (Liu et al., 2006). The objective of this study was to increase butyrate and hydrogen production. The wild strain of C. tyrobutyricum produces acetate, butyrate, and hydrogen, while it was reported that the ack-deleted mutant strain produced 50% more hydrogen from glucose and increased butyrate production. Another study showed that in the C. saccharoperbutylacetonicum strain N1-4 down-regulation of the hupCBA gene cluster using antisense RNA strategy improved the hydrogen production by 3.1-fold compared to the wild strain. HupCBA proteins are responsible for uptake of hydrogen in Clostridium sp. (Nakayama et al., 2008). Directed mutagenesis is an important approach in molecular biology, and most of the mutations in Clostridium sp. are still constructed through a homologous recombination. Recently, a ClosTron system has been developed, which allows directed construction of a stable mutant in Clostridium sp. using a bacterial group II intron (Heap et al., 2007). The ltrB gene present in Lactococcus lactis encodes for a mobile group II intron, which lays the foundation for development of the ClosTron system. This mutagenic system was developed to function in Clostridium sp. hosts. With the help of the RNA-mediated “retrohoming” mechanism, the group II mobile intron elements from the ltrb gene of L. lactis inserts into their specific target gene. Base pairing between the excised intron lariat RNA and target site DNA helps in target site recognition (Mohr et al., 2000). Intron target specificity can be reprogrammed by alteration of the DNA sequence encoding for the target part of the intron. Recently, several improvements have been made in the ClosTron system so that multiple mutation-carrying strains can be constructed. Automated ClosTron design bioinformatics tools are available online (https://clostron.com). The group II intron (TargeTron gene knockout system)-mediated mutagenesis was used to delete the lactate dehydrogenase-encoding ldh gene in the C. pefringens strain W11, and it was reported that in the ldh-deleted mutant strain W13, the hydrogen yield increased by 51% with almost zero lactate production (Wang et al., 2011). This work was further extended by inactivating the acetyl-CoA acetyltransferase
gene (atoB) so that butyrate formation could be blocked by use of the TargeTron gene knockout system. The resultant mutant strain W15 showed decreased hydrogen production, revealing the importance of butyrate formation pathway for hydrogen production in C. pefringens (Yu et al., 2013). Group II intron-based mutation has successfully worked in C. difficle, C. acetobutylicum, C. sporogenes, C. botulinum, C. beijerinckii, and C. pefrigenes. This newly developed system has opened up a new avenue for engineering of pathways for improved production of biofuels, including hydrogen production.
