1. Clean fuel generation from agro-waste by a novel isolate IODB-O3
Arundhati Debroy, Anshu Mathur, Deepak Kumar Tuli and Anil Kumar Patel*
DBT-IOC Centre for Advanced Bio-Energy Research, Indian Oil Corporation Ltd; R&D Centre, Sector-13, Faridabad , India
Objective: Aim of the study was to isolate, characterize and identify a potent H2 producing bacterial isolate. It was focused to utilize wheat straw pre-hydrolysate (WSPH) by isolate IODB-O3 to reduce economic constrain on the overall cellulosic ethanol process and also establish a sustainable biohydrogen production process.
Introduction: World’s dependence on fossil fuels for energy is dominant. Hence development of alternate energy sources is inevitable. Bio-H2 is foreseen as one of the major alternative for it. H2 has been demonstrated as a sustainable and clean biofuel carrier for future as it produces only H2O when combusted. It has high calorific value by mass 122kJ/g which is significantly higher than gasoline. Bio-H2 finds application as energy source in aviation, space, automobile, refining, welding and metal fabrication industries.
Bio-H2 production from pre-treated biomass has not been fully explored as a complete process. Pre-treatment via steam-explosion generates ample amount of WSPH which remains unutilized for bioethanol production due to non-availability of efficient C5-fermenting microorganisms.
Culture condition: Bio-H2 production media (BPM) contains (mg/L): K2HPO4- 450; KH2PO4- 450; (NH4)2SO4- 900; NaCl- 900; MgSO4.7H2O- 90; CaCl2- 90; Hemin ; Yeast-extract- 3000; Na2CO ; L-cystein.HCl- 500 (reducing agent); Resazurin (redox indicator); Vitamins: biotin- 4.0×10-2 and p-Amino benzoic acid- 1.0×10-2 and trace elements: MnSO4.H2O- 3.0×10-3; FeSO4.6H2O- 8.0×10-3 and CoCl2.6H2O-3.0×10-3.
A 50ml BPM was taken in 100ml serum bottle, N2 was used for media reduction.
A 10g/L WS Enzyme hydrolysate (WHEH) or WSPH concentrations were used.
Strain characteristics & Identification: IODB-O3 isolated from sludge was gram+ve, rod-shaped, obligate anaerobic bacilli growing optimally at 37oC and pH 8.5. A 16S rDNA gene analysis revealed its 98% similarity with Clostridium sp. Nucleotide sequence of IODB-O3 isolate has been submitted to the NCBI gene bank under accession number KM
Pre-treatment and Hydrolysis: Wheat straw (WS) pre-treatment was conducted in 10 L steam-explosion reactor. 500 g of WS was impregnated with 1% (w/w) H2SO4 at 30oC for 30 min then cooked with100 bar at 200ºC for 10 min. WS solid fraction was hydrolysed by in-house cellulase produced under submerged fermentation by Penicillium janthinellum EMS-UV-8. Twenty FPUs per gram biomass of cellulase was added at 10% solid loading. Liquid WSPH was used after pH adjustment.
Process optimization: Effect of below factors on H2 production were studied using glucose as C-source in batch fermentation.
temperature: 25 to 45 oC (Fig 1)
Initial pH: with pH 0.5 interval (Fig 2)
incubation time: 3-36h (Fig 3a).
Figure 1
Figure 2
Figure 3a
Figure 3b
Multiple feed ability test: H2 production from various C-sources including WSPH (composed of mainly xylose) and WSEH (composed of mainly glucose) by isolate IODB-O3 were studied (Fig 4) .
Results:
Maximum H2 was produced at 37oC and pH 8.5 in 24h.
Maximum H2 yield was obtained 2.54±0.2 mol-H2/mol-reducing sugar from
WSPH and 2.61±0.1 mol-H2/mol-reducing sugar from WSEH.
The cumulative H2 production (ml/L), 3680±105 and 3270±100, H2 production
rate (ml/L/h), 153±5 and 136±5, and specific H2 production (ml/g/h), 511±5 and
681±10 with WSPH and WSEH were obtained respectively.
Conclusion: In this study, bio-H2 production process was developed using low-cost agro-waste. Preferred utilization of xylolytic low value liquid fraction (WSPH) of pre-treated biomass by isolate IODB-O3 is of special economic significance. This isolate has potential to produce H2 from both C5 and C6 sugars obtained from biomass pre-treatment. Thus, it can add value to cellulosic ethanol process by co-producing hydrogen in appreciable amounts.
Acknowledgement: All authors acknowledge Department of Biotechnology, Govt. of India for providing financial support. AKP thanks DBT for DBT-Energy Bioscience Overseas Fellowship. Refining technology division at IOCL R&D centre is duly acknowledged for helping in gas samples analysis.
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Figure 4