Metabolic Regulation Of Klebsiella Sp. HQ-3and Optimization Of Fermentation Conditions For Hydrogen Production

Fermentative hydrogen production has advantages of rapid hydrogen production rate,simple operation, mild reaction condition and bioconversion feasibility from variousorganic wastes in comparison with other hydrogen production technologies. Therefore, ithas been receiving increased attention in recent years. However, low hydrogen-productionyield of wild strains blocks industrialization of biohydrogen. Till now, genetic engineeringstrategies such as knocking-out the key enzyme of the competing metabolic pathway orthe negative regulator, over-expressing the positive regulator genes were used as powerfultools for improving hydrogen production in Escherichia coli and Enterobacter aerogenes.In addition, the optimization of fermentation parameters is the most direct way to improvethe microbial hydrogen production in order to achieve the best state of hydrogenproduction.Klebsiella sp., a facultative anaerobic bacterium, grows fast and has high potential forhydrogen-production. Many works have been carried out to enhance the hydrogenproduction mainly by optimizing the fermentative conditions in current, whereas, itscapability of hydrogen is far from industrial needs. To our knowledge, there were fewreports about the regulation of metabolic network of Klebsiella sp. on hydrogenproduction till now. Therefore, it is significant to elucidate and control the hydrogenevolving pathway for improving the hydrogen yield in Klebsiella sp..In this study, in order to improve the hydrogen yield of Klebsiella sp. HQ-3, a keyenzyme of succinate pathway, which competes with hydrogen production in anaerobicfermentation, was inactived by gene deletion. Moreover, NAD synthetase from E.aerogenes was over-expressed in Klebsiella sp. HQ-3. Based on the genetic engineering,hydrogen fermentation conditions of recombinant strain HQ-3-P (Δppc) was optimized byresponse surface method (RSM). The hydrogen yield of HQ-3-P was increased to1.125mol H2/mol glucose finally. The main work and the results were as follows:1. The knockout of ppc gene in Klebsiella sp. HQ-3. The gene ppc encodingphosphoenolpyruvate carboxylase (PEPC), which is a key enzyme in metabolic pathwayof succinate production, was amplified from Klebsiella sp. HQ-3by PCR. Then the gene ppc was knocked out from Klebsiella sp. HQ-3by Red recombination system. And theresulting recombinant strain was named HQ-3-P. The result of hydrogen fermentationexperiments indicated that the efficiency of hydrogen production of engineering bacteriaHQ-3-P was0.762mol H2/mol glucose, increased by24.21%than the wild strain.2. Over-expression of NAD synthetase from E. aerogenes in Klebsiella sp. HQ-3andHQ-3-P. Expression vector pET28-pkan-nadE, including the NAD synthetase gene (nadE),was transformed into HQ-3and HQ-3-P by electroporation. After the resistance screeningand validation of molecular biology, heterologous expression strain HQ-3-N (nadE) andHQ-3-P/N (Δppc/nadE) were obtained. The hydrogen production experiments showed thatthe growth rates of NAD synthetase expressing strains were increased comparing withoriginal strains. And the hydrogen yield of HQ-3-N was increased from0.581to0.620mol H2/mol glucose. While the hydrogen yield of HQ-3-P/N was increased from0.762to0.786mol H2/mol glucose.3. Optimization the key parameters in the fermentation conditions. The analysis ofsingle-factor experiments showed that inoculum, initial pH and temperature weresignificant factors of hydrogen production by HQ-3-P. Experimental results indicated thatoptimum culture inoculum, initial pH and temperature respectively were20%,7.0and33℃without considering the premise of the interaction. The experimental conditions weredesigned by Central Composite Design (CCD) based on the results from single-factorexperiment. The response values were analyzed by statistical software Design-Expert (DE)8.0.5and the out-put of DE suggested that optimal inoculum, initial pH and temperaturewere21.03%,7.14and31.81℃, respectively. The predicted value of the yield ofhydrogen-production was1.111mol H2/mol glucose in the optimal condition. Theconfirmatory experiments were carried out and the maximum production of hydrogen was1.125mol H2/mol glucose under the optimized condition. This result indicated that themodel has good precision and reliability.