Excessive usage of traditional energy reserves leading to increased environmental pollution and global warming have strongly urged for alternative sustainable energy sources. Due to non-polluting nature and high energy yields, hydrogen (H₂) gas is considered as an ideal candidate for alternative fuel. Biohydrogen (bioH₂) production from organic wastes is a 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 adaptability, substrate utilization efficiency and bioconversion rates. Another challenge to ensure efficient fermentation is to optimize various physico-chemical factors such as pH, temperature, substrate selection and concentration, medium compounds, and H₂ removal and collection 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 H₂ 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 to (1) establish an anaerobic biopanning procedure to enrich antibody binders specific against clostridial [Fe-Fe] hydrogenase protein, (2) develop and standardize a novel enrichment system, (3) implement the enrichment technique to enrich functional inoculum capable of degrading complex substrates, (4) enrich crude glycerol fermenting microbial community and finally, (5) optimize the physico-chemical factors influencing fermentative H₂ production for efficient bioprocess.
In the present study, biopanning with synthetic ‘mixed’ single chain variable fragment (scFv) libraries against active and inactive clostridial [Fe-Fe] hydrogenases aided the enrichment of anti-hydrogenase antibodies. Out of ninety four (from inactive hydrogenase) and ninety two (from active hydrogenase) random clones screened, nine potential antibody clones with recognition specificity towards Clostridium acetobutylicum [Fe-Fe] hydrogenase were selected. The enriched binders also recognized [Fe-Fe] hydrogenase from C. butyricum. Based on the results from this study, it could be reasoned that the binders with generic specificity against closely related clostridial [Fe-Fe] hydrogenases can be used as novel molecular tools for quantitative monitoring [Fe-Fe] hydrogenases at the protein level. Another of-note observation was the specificity of the antibody binders towards active and inactive hydrogenases. Preliminary experiments indicated 7Ac binder (enriched against active hydrogenase) specificity towards the catalytically active [Fe-Fe] hydrogenase rather to the inactive state and 48In (enriched against inactive hydrogenase) recognized both catalytic states. These findings indicate the possibility to apply the isolated antibody clones for functional detection of clostridial [Fe-Fe] hydrogenases.
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 artificial mixed microbial community and varied selection criteria allowed the enrichment of the best H₂ producer. 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 carbon dioxide (CO₂) as gaseous metabolites and acetate, formate, lactate and ethanol as liquid metabolites. At optimized values of cultivation conditions (pH 6.0 and 34 ˚C) the H₂ yield was 1.82 mol-H₂/mol-glucose. The isolate efficiently fermented monomeric hemi-cellulose sugars to H₂ (mol-H₂/mol-substrate): Galactose, 1.18; Mannose, 1.23; Xylose, 1.22; Arabinose, 0.94 and Rhamnose, 1.01). Except for arabinose, an increase in cultivation period improved the biomass and H₂ yield (mol-H₂/mol-substrate): Galactose, 1.68; Mannose, 1.93 and Xylose, 1.63) 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 Clostridium spp. 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˚C and 1g/L crude glycerol. The H₂ yield from raw glycerol at optimal cultivation conditions was 1.1 mol-H₂/mol-glycerol consumed . At elevated crude glycerol concentrations, substrate utilization and H₂ production were limited due to the presence of impurities in the crude glycerol fraction. The bioconversion of crude glycerol to H₂ was further improved by statistical optimization of the growth medium composition.
Initial screening with Plackett – Burman design identified NH₄Cl, K₂HPO 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 identified the optimal media composition for improved H₂ production from crude glycerol fermentations and were successful in improving the H₂ yield by 29% (1.42 mol-H₂/mol-glycerol consumed ) in comparison to previously reported value (1.1 mol-H₂/mol-glycerol consumed ).