BioH2 Production in Protein to Community Level Perspective

Excessive usage of traditional energy reserves leading to increased environmental pollution and global warming have strongly urged for alternative sustainable energy source. Due to non-polluting nature and high energy yields, H₂ gas is considered as an ideal candidate for alternative fuel. Microbe-assisted H₂ production from bioconversion of organic wastes is a cost-effective and sustainable approach, addressing energy production through organic waste disposal. Organic wastes such as lignocellulosic biomass and industrial glycerol, a by-product of biodiesel manufacturing process, have been recently investigated for their bioconversion potential. However, bioconversion of such organic wastes is a challenge due to the presence of impurities, toxic degradation products and complex nature. In comparison to pure bacterial strains, natural microflora could be an ideal inoculum choice offering better substrate utilization efficiency and bioconversion rates. Another challenge to ensure efficient fermentation is to optimize physico-chemical factors such as pH, temperature, substrate concentration, H₂ partial pressure and media nutrients and supplements due to individual and interactive effects on microbial growth, metabolism and hydrogenase enzyme.

Hydrogenases are metalloenzymes that reversibly catalyzes proton reduction to H₂, and are divided into three classes based on the metal cofactor at the active site, [Fe-Fe], [Ni-Fe] and [Fe] hydrogenase. Among the hydrogenase classes, [Fe-Fe] hydrogenases exhibit highest catalytic activity involving mostly in H₂ production. Apart from their pivotal role in fermentative H₂ production, [Fe-Fe] hydrogenases promise an alternative catalyst choice in fuel cells. However, in spite of their preference towards hydrogen production, [Fe-Fe] hydrogenases are extremely prone to catalytic inactivation upon oxygen exposure. This is the major challenge, at the protein level, that hinders a cost-effective approach for biotechnological applications and suggests the requirement of targeted tools to investigate the inactivation process at the molecular level.

The purpose of the present study was to investigate bioH₂ production in protein to community level perspective. More specifically the aims were (1) to establish anaerobic biopanning procedure to enrich molecular binders’ specific against Clostridial [Fe-Fe] hydrogenase protein, (2) to develop and standardize a novel enrichment system and implement the technique to enrich functional inoculum capable of degrading complex substrates, (3) to enrich crude glycerol fermenting microbial community and finally, (4) to optimize the physico-chemical factors influencing fermentative H₂ production for efficient bioprocess.

In the present study, phage display and anaerobic biopanning aided the enrichment of molecular binders specific towards hydrogenase catalytic states. The binders also showed good binding specificity towards hydrogenases from Clostridium acetobutylicum and Clostridium butyricum crude lysates. The study aided in identifying unique anti-hydrogenase antibodies specific towards C. acetobutylicum and C. butyricum [Fe-Fe] hydrogenases and catalytically inactive [Fe-Fe] hydrogenase. Based on the results from this study, it could be reasoned that the molecular binders can offer a simple platform to monitor the structural changes during [Fe-Fe] hydrogenase inactivation process.

The study progresses in investigating bioH₂ production in perspective of microbial community. The novel microbial enrichment system was developed and the proof-of-principle experiments conducted using an artificial mixed microbial community and varied selection criteria allowed the enrichment of the best H₂ producer from the microbial consortium. The system was implemented in enriching cellobiose degrading H₂ producer from an environmental sample. The bacterial strain isolated by spread plate technique on agar plates containing CMC was affiliated with Citrobacter sp. and named as Citrobacter sp. CMC-1. Citrobacter sp. CMC-1 utilized glucose, cellobiose and CMC and followed mixed-acid fermentation profile producing H₂ and CO₂ as gaseous metabolites and acetate, formate, lactate and ethanol as liquid metabolites. Optimized values of cultivation conditions such as pH and cultivation temperature were observed to be 6.0 and 34 ËšC, respectively. Except for arabinose, the isolate efficiently fermented monomeric hemi-cellulose sugars to H₂. An increase in cultivation period improved the biomass and H₂ yield followed with observations of reduced formate accumulation in the medium, indicating that Citrobacter sp. CMC-1 produced H₂ from formate breakdown via the FHL complex.

Microbial community, pre-dominated with Clostridial sp., enriched from activated sludge fermented crude glycerol mainly to H₂, CO₂, acetate, butyrate and ethanol. Optimal bioprocess conditions for the enriched inoculum were experimentally observed to be pH 6.5, 40 ËšC and 1 g/L crude glycerol. At high crude glycerol concentrations, substrate utilization and H₂ production were limited due to the presence of impurities in the crude glycerol fraction. Incomplete substrate utilization was observed even at increased cultivation periods suggesting the utilization of non-glycerol organic matter in the crude glycerol. The bioconversion of crude glycerol to H₂ was further improved by statistical optimization of the cultivation media components. Initial screening with Plackett – Burman design identified NH₄Cl, K₂HPO₄ and KH₂PO₄ with individual and interactive effects on H₂ yield. Among the three identified media components, NH₄Cl and KH₂PO₄ imparted the maximal significance and were optimized in scrutiny. A series of statistical models and central composite face centered design together with response surface methodology identified the optimal media composition for improved hydrogen yield from crude glycerol fermentation. The optimization experiments were successful in improving the hydrogen yield by 29%.

Depletion of petroleum-based energy resources, global warming, increased environmental pollution and strict emission regulations have strongly urged for an alternative energy source (Balat 2008). Currently, as shown in Figure 1.1, energy derived from fossil fuels covers majority of total global fuel consumption (IEA Key world energy statistics, 2012). Hydrogen (H₂) is considered as an alternative fuel due to high specific energy content (Yılmaz et al. 2012), less energy intensive (Das, Veziroǧlu 2001) and carbon neutral combustion products (Balat, Kırtay 2010). Though H₂ fulfills all the essential requirements of an ideal fuel, currently it is produced by chemical methods from coal, oil, natural gas (Cheong, Hansen 2006, Valdez-Vazquez, Poggi-Varaldo 2009), naptha (Valdez-Vazquez, Poggi-Varaldo 2009) or physical methods like electrolysis, photolysis (Das, Veziroǧlu 2001), electrolysis of water (Oh, Van Ginkel & Logan 2003) and thermolysis (Das, Veziroǧlu 2001). Current major commercial modes of H₂ production are steam-methane reforming (48%), oil reforming (18%), gasification of coal (30%) and water electrolysis (4%) (Mazloomi, Gomes 2012).

Figure 1.1. Fuel share percentage from global energy consumption in 2010 (Modified from IEA Key world energy statistics, 2012)

Since current H₂ production methods are expensive and non-sustainable, biological hydrogen production offers a sustainable alternative. Thus intensive research has been concentrated in the field of biological hydrogen production. Investigations on biohydrogen production have been reported for both pure strains and mixed microbial community. Kleerebezem and Loosdrecht (2007) reported the importance of ‘Mixed culture Biotechnology’ in the field of biotechnology and bioprocess technology (Kleerebezem, van Loosdrecht 2007). Diversity of microbial consortia in natural samples helps in the complete degradation of organic substrates or biowastes and

directing towards the production of H₂ and other interested compounds. Biological production of H₂ can be performed by various processes like dark fermentation, photo fermentation, direct and indirect biophotolysis and systems utilizing the efficiency of both fermentative and photosynthetic organisms.

Direct and indirect biophotolysis

Green algae and cyanobacteria are able to produce H₂ from water, either by direct or indirect biophotolysis (Figure 1.2). In direct biophotolysis, the light energy absorbed by photosystem II (PSII) and photosystem I (PSI) assists in electron transport from water to ferredoxin. The reduced ferredoxin is re-oxidized by transferring the electrons to hydrogenases and/or nitrogenases. (For reviews, (Eroglu, Melis 2011, Bičáková, Straka 2012) Though direct biophotolysis relies on inexpensive substrate and studies on this technology have reported high energy conversion efficiencies (Melis 2009), one major limitation is the presence of oxygen produced from PSII mediated water oxidation which irreversibly inactivates both hydrogenase and nitrogenase. Melis et al (2007) have studied the effect of sulfur deprivation on reducing oxygen production by PSII and improving H₂ production in Chlamydomonas reinharditii (Melis 2007). The authors report a decreased PSII activity under sulfur limiting conditions thereby lowering the possibility of oxygen mediated hydrogenase inactivation. This drawback led to immense attention towards indirect biophotolysis, wherein the nitrogenase is present in heterocysts.

In indirect biophotolysis, CO₂ is fixed to synthesize sugars via Clavin cycle and stored sugars are fermented to H₂ by nitrogenase (McKinlay, Harwood 2010). In indirect biophotolysis, the nitrogenases are present in heterocysts, spatially and temporally separated from oxygenic photosynthesis. Apart from spatial separation of nitrogenases and [Fe-Fe] hydrogenases in nitrogen fixing organisms, cyanobacteria can also produce H₂ without nitrogen fixing (Bičáková, Straka 2012). Non-nitrogen fixing cyanobacteria such as Synechocystis, Synechococcus, Gloebacter can produce H₂ through oxygen tolerant bidirectional [NiFe] hydrogenases (Baebprasert, Lindblad & Incharoensakdi 2010); (Das, Veziroglu 2008). (For review, (Kim, Kim 2011)

Figure 1.2. A schematic representation of direct and indirect biophotolysis. Figure from (McKinlay, Harwood 2010)

Photo fermentation

Photo fermentation is a process in which photo-fermentation bacteria obtain electrons required for H₂ production are obtained from organic (acetate, butyrate and lactate) or inorganic (H₂S or Fe²⁺) sources with utilization of solar energy (McKinlay, Harwood 2010, Kim, Kim 2011). One of the best studied species in photo fermentation is purple non-sulfur bacteria (PNSB) due to proficient utilization of solar energy, high substrate conversion efficiencies and lacks PSII therefore eliminating O₂ mediated inhibition (Golomysova, Gomelsky & Ivanov 2010), wide substrate utilization range and requirement of less free energy when compared to biophotolysis (Eroglu, Melis 2011). PNSB produces H₂ via Mo-nitrogenase under nitrogen limiting conditions. Oxidation of organic or inorganic substrates produces CO₂, H⁺ and electrons. The electrons are transferred to PSI via NAD reduction and ubiquinone, in turn undergoes electron transfer to nitrogenase enzyme by reduced ferredoxin. Figure 1.3 depicts a schematic representation of photo fermentation. Photo fermentation is an important process that contributes towards bioconversion of agricultural and industrial wastes. (For review, (Hay et al. 2013)

Photo fermentation process is greatly hindered due to low specific activity of nitrogenase enzyme (Bičáková, Straka 2012, Hay et al. 2013). Several attempts have been reported in improving the catalytic activity by engineering nitrogenase enzyme at gene or protein level. Uptake hydrogenase present in PNSB lowers the H₂ production by utilizing the H₂ towards ATP synthesis (McKinlay, Harwood 2010). Strategies have focused in deactivating the enzyme by random mutagenesis or by gene knock-out. Photo fermenting H₂ production can be improved by eliminating the uptake hydrogenase. Photo fermentation also requires careful optimization of light intensity, cultivation temperature and growth supplements to maximize H₂ production.

Figure 1.3. Schematic representation of photo fermentation. Figure from (Kim, Kim 2011)

Biological water-gas shift reaction

Certain gram-negative bacteria, such as Rhodospirillum rubrum, Rubrivax gelatinosus and Rhodobacter sphaeroides, and non-photosynthetic gram positive Carboxydothermus hydrogenoformans can produce H₂ and CO₂ by carbon monoxide (CO) oxidation (Saxena, Adhikari & Goyal 2009, Ni et al. 2006). PNSB such as Rubrivax gelatinosus and Rhodospirillum rubrum produces H₂ in darkness through CO-mediated water-gas shift reaction. Under anaerobic conditions, CO induces the synthesis of CO dehydrogenase, iron-sulfur protein and CO-tolerant hydrogenase. CO dehydrogenase oxidizes CO to CO₂ and the reducing equivalents produced are transferred to hydrogenase via Fe-S cofactor. Kerby et al (1995) have reported the requirement of nickel in the growth medium and optimal CO partial pressure for efficient hydrogen production by Rhodospirillum rubrum (Kerby, R.L, Ludden, P.W, Roberts, G.P 1995). However, due to slow bacterial growth when grown with CO as sole carbon source, low gas-liquid mass transfer rates, slow kinetics and highly sensitive to O₂, biological water-gas shift method is not yet considered to be economical (Kim, Kim 2011, Haryanto et al. 2005).

Dark fermentation

Dark fermentation, conversion of organic substrates to bioH₂ in absence of light, is achieved by obligate or facultative anaerobes under anaerobic conditions and is associated with the action of hydrogenases. Dark fermentation is considered as a practical route towards biological hydrogen production due to high H₂ production rate (Levin, Pitt & Love 2004b)(Wu et al. 2006), simple bioreactor set-up and ability for bioconversion of organic wastes (Chong et al. 2009a), thus addressing both energy production and waste disposal issues.

The pivotal step in dark fermentation process is the production of acetyl-CoA from pyruvate degradation (Ntaikou I, Antonopoulou G, Lyberatos G 2010). Pyruvate breakdown and subsequent H₂ production is catalyzed either by pyruvate-formate lysase (PFL) and formate-hydrogen lyase (FHL) or pyruvate-ferredoxin oxidoreductase (PFOR), ferredoxin reduction by NADH-ferredoxin oxidoreductase (NFOR) and bioH₂ production through hydrogenases (Kim, Kim 2011)(Kim, Kim 2011, Hay et al. 2013).

PFL and FHL enzyme systems are observed in facultative anaerobes in Enterobacteriaceae family. In mixed acid fermentation, the pyruvate formed from glycolysis is broken down to H₂ and CO₂ as shown in the reaction below (Figure 1.4). FHL complex comprises of cytoplasmic formate dehydrogenase-H and membrane bound [Ni-Fe] hydrogenase (hydrogenase-3) (Axley, Grahame & Stadtman 1990, Sawers 1994). Maximal theoretical yield of 2 mole H₂ per mole of glucose consumed can be achieved from formate degradation. (Hay et al. 2013, Ntaikou I, Antonopoulou G, Lyberatos G 2010, Manish, Banerjee 2008)

In obligate anaerobes, such as Clostridia species, acetyl-CoA production from pyruvate is catalyzed by PFOR, enzyme involving ferredoxin reduction, to acetyl-CoA. Electron transfer towards hydrogenase is also catalyzed by ferredoxin reduced via NFOR (Figure 1.4). (Kim, Kim 2011, Hay et al. 2013, Levin, Pitt & Love 2004b)

The reduced ferredoxin is re-oxidized by donating electrons to hydrogenase enzyme, thus producing hydrogen.

When compared with formate degradation, breakdown of acetyl-CoA produces increased H₂ production. Acetyl-CoA degradation follows two routes either towards acetate or butyrate production. Theoretically, acetate production route yields 4 moles H₂ per consumed glucose and 2 moles of H₂ are produced with butyrate as the main end-metabolite. The additional H₂ formed with acetate production is catalyzed by the reduction of ferredoxin and proton transfer to hydrogenase through electrons donated from oxidation of NADH to NAD⁺. The electron transfer is thermodynamically favorable during low H₂ partial pressure conditions. In the case that butyrate is the final product; the electrons are required for conversion of acetoacetyl-CoA to butyrate. (Ntaikou I, Antonopoulou G, Lyberatos G 2010)

Efficiency of bioH₂ production is affected by several operational parameters such as pH, temperature, H₂ partial pressure, substrate type and concentration, inoculum type,

inorganic nutrients, growth supplements, hydraulic retention time and bioreactor configuration (for in-detail review see Section 2).

Figure 1.4. Biological H₂ production routes through dark fermentation. In Enterobacteriaceae, pyruvate is converted to acetyl-CoA and formate, which is degraded to hydrogen by FHL together with [Ni-Fe] hydrogenase or by formate-dependant hydrogenase. Pyruvate breakdown via PFOR pathway produces acetyl-CoA and reduced ferredoxin. Electron transfer between reduced ferredoxin and [FeFe] hydrogenase results in evolution of molecular hydrogen. During low H₂ partial pressure, NADH mediated ferredoxin reduction assists in production of additional H₂ via NFOR or acts as a co-substrate with ferredoxin (red) to produce H₂ from NADH-dependent [Fe-Fe] hydrogenase. Figure from (Hallenbeck, Abo-Hashesh & Ghosh 2012)

Hybrid fermentation

Hybrid fermentation is a promising technique for efficient H₂ production as the method solves the negative effect of metabolic end products from dark fermentation and problem of electron donors in photo fermentation. Moreover, in theory, 12 moles of H₂ can be produced from 1 mole of glucose through hybrid fermentation. (Bičáková, Straka 2012)(Tekucheva, Tsygankov 2012)

The initial step in this combined approach, involves bioconversion of sugars and organic wastes to H₂ and organic end products. These liquid end products are then converted to H₂ by photosynthetic bacteria. (Figure 1.5) Yokoi et al (2002) have reported a H₂ yield of 7.2 mol / mol hexose with Clostridium butyricum, Enterobacter aerogenes HO-39 (dark fermentation) and Rhodobacter spp. M-19 (photo fermentation) and sweet potato starch residue as the carbon source (Yokoi et al. 2002). Sucrose fermentation by Clostridium pasteurianum CH₄ and Rhodobacter palustris WP3-5 resulted in a H₂ yield of 10.1 mol/mol hexose with 72.0% COD removal (Chen et al. 2008). Another approach in hybrid fermentation is the usage phototrophic accumulation of polysaccharides in microalgal biomass as the substrate for the two-stage fermentation. Kawaguchi et al (2002) investigated the effect of starch in Dunaliella tertiolecta biomass as the carbon source for hybrid fermentation involving Lactobacillus amylovorus and Rhodobium marinum A-501 (Kawaguchi et al. 2001). The authors reported a H₂ yield of 8.3 mol/mol hexose. Though feasible, factors such as concentration of nitrogenous components, presence of end-metabolites and availability of the organic substrates are important factors that required careful optimization (Tekucheva, Tsygankov 2012).