Screening Of Efficient Hydrogen-producing Strains And Optimization Of Anaerobic Fermentation Conditions

Owing to its high efficiency, cleanness and renewability, hydrogen is widely used in many fields, such as chemical synthesis, aerospace, metal metallurgy, electronics, etc. At present, production of hydrogen has attracted more and more attention in the world, and is becoming one of the hot spots in biological science and engineering. Hydrogen production from anaerobic fermentation technology, being relatively simple, renewable raw materials, and power saving, has been extensively explored. So far, a number of technologies have been put into pilot trial, but the low efficiency of hydrogen production and high cost still obstructs their industrialization.The main approaches to further improve the production efficiency are to sieve more efficient hydrogen-producing bacteria, to optimize fermen tation conditions, and to innovate techniques, such as employing metabolic regulation. In this study, we have screened two efficient hydrogen-producing strains, optimized hydrog en-producing fermentation conditions, and further identified the metabolic pathways. The main work is listed below:(1) The riverbed sludge samples were collected from sewage channel in Huazhong University of Science and Technology. By primary plate screening and secondary flask assay, two strains with highly efficient hydrogen production were obtained. They were identified using 16Sr DNA sequence and phylogency analysis, and termed as Klebsiella sp. and Serratia sp., and named the strain of Klebsiella sp. as Klebsiella sp. HQ-3 and the other one Serratia sp. B-3.(2) Through monofactorial anaerobic fermentation experiments, it was found that the best carbon and nitrogen sources for hydrogen production were glucose, peptone; the opti mum pH was 8.0, and the optimal glucose, peptone, KH₂PO₄ were 24.0 g/L, 27.0 g/L and 8.0 g/L, respectively. In order to optimize the liquid hydrogen-producing culture medium, Plackett-Burrman design was applied to screen the principal factors from Na₂HPO₄·12H₂O, KH₂PO₄, MgSO₄·7H₂O, glucose, peptone, yeast extract, pH in hydrogen production medium. PH, glucose, peptone, KH₂PO₄ were determined as the principal factors. Then, the path of steepest ascent experiments design was adopted to determine the experimental center factor value of the principal factors. The anaerobic fermentation liquid medium was further optimized with Response Surface Methodology by Modeling tools MATLAB. The optimal fermentation conditions and predicted maximal hydrogen-producing value were determined. In order to verify the pedicted value, the experimental was measured under the optimal fermentation medium, the obtained value of hydrogen production was 826.3 mL/L, coinciding perfectly with the predictive value of 806.0 mL/L. Subsequently, we employed monofactorial experiments to determine the optimum fermentation temperature, inoculum size, and shaking speed. Thus, the optimum culture conditions were: yeast extract 8.0g/L, Na₂HPO₄ 15.0 g/L, MgSO₄ 3.0 g/L, initial pH 8.3, KH₂PO₄ 7.7 g/L, peptone 18.1 g/L, glucose 21.4 g/L, temperature 32℃, inoculum loading 3%, shaking speed 60 rpm. In the optimum culture conditions, the hydrogen production volume measured was up to 865.0 mL/L, 39% more than the initial volume of 621.0 mL/L.(3) In order to determine the type of anaerobic fermentation, under optimum fermentation condition, we measured the liquid phase of the fermentation products ethanol, formic acid, acetic acid, propionic acid. The results showed that ethanol, formic acid, acetic acid occupied ca 93% of the total liquid product, suggesting the fermentation type belong to a mixed acid pathway.