Enhanced Hydrogen Production Of Klebsiella Sp. HQ-3from Organic Waste By Metabolic Regulation

With the depletion of fossil energy, deterioration of environment pollution, andshortage of natural resources becoming increasingly prominent, hydrogen as anenvironmentally friendly alternative energy is attracting worldwide attention. Darkhydrogen fermentation is a promising method due to its high hydrogen-producing rate,mild reaction conditions, low-cost equipment and bioconversion feasibility from therecycling organic wastes. However, low hydrogen production of wild strains and high costof the substrate are the main bottlenecks of biohydrogen industrialization. Therefore,investigating mechanism and regulating metabolic pathway of hydrogen production arebecoming the hot field for practical biohydrogen production. Meanwhile, more researchesare focusing on the exploration of new substrate to reduce fermentation cost.In this paper, several genes involving in the metabolic pathway of hydrogenproduction were homologously overexpressed in wild-type strain Klebsiella sp.HQ-3andrecombinant strain HQ-3-P (Δppc),whose phosphoenolpyruvate carboxylase gene (ppc)had been deleted in our previous studies. Eight mutants were achieved by homologouslyexpressing three forward regulatory factors of formate hydrogenlyase pathway (fhlA, fnr,fdhF) and a cofactor of NADH pathway (pncB), respectively. Furthermore, hydrogenfermentation parameters of recombinant strain HQ-3-P/fhlA were optimized by centralcomposite design-response surface method (CCD-RSM). Finally, different pretreatmentmethods were used for hydrolysate of lipid-extracted microalgal biomass residues(LMBRs), and was used as a carbon source for dark fermentation of hydrogen production.The main work and results were listed as follows:1. Three forward regulatory factors and a cofactor were homologously overexpressedin Klebsiella sp. HQ-3and HQ-3-P. The fhlA gene encoding formate hydrogenlyasesystem transcriptional activator (FHLA), fnr gene encoding the global transcriptionalfactor (Fnr), fdhF gene encoding the formate dehydrogenase H (FDHH), and pncB geneencoding the nicotinic acid phosphoribosyl-transferase (NAPRase) were amplified fromthe genomic DNA of Klebsiella sp. HQ-3by genomic walking using degenerate primers.The PCR products of genes were respectively introduced into the modified expression plasmid pET28a-Pkan. Then, the four recombinant plasmids were electroporated into wildKlebsiella sp. HQ-3and HQ-3-P, respectively, and eight recombinant strains wereobtained. The yields of hydrogen production of HQ-3-fdhF, HQ-3-pncB, HQ-3-fnr andHQ-3-fhlA were increased by7.28%,11.62%,12.26%,15.45%compared with that ofwild strain, respectively. Meanwhile, the hydrogen yield of HQ-3-P/fdhF, HQ-3-P/pncB,HQ-3-P/fnr and HQ-3-P/fhlA were improved by12.33%,15.58%,19.13%,29.35%compared with that of recombinant strain HQ-3-P, respectively.2. The fermentation conditions of the recombinant HQ-3-P/fhlA were optimized. Thesingle-factor analysis showed that significant factors were initial inoculum, temperatureand initial pH, and the optimal conditions were20%,33°C, and6.5, respectively. Basedon the single-factor analysis, a three-level-three-factor CCD-RSM were performed by thesoftware (Design-Expert V.8.0.5) to further analyze the effect of significant factors onhydrogen production. The experimental results indicated that optimal initial inoculum,temperature and intial pH were21.26%,31.75°C and6.67, respectively. In theconfirmatory experiments, the maximum production of hydrogen was3.214mol H2/molglucose, which coincided with the predicted value3.236mol H2/mol glucose. The resultindicates that the model has good precision and reliability.3. The hydrogen production of the recombinant strain HQ-3-P/fhlA was preliminaryinvestigated by using the organic waste LMBRs. LMBRs was first treated by the methodsof ultrasonic, dilute acid, dilute alkaline and ammonia soaking, respectively. Afterneutralization, the resulting LMBRs was further treated with cellulase and used as thecarbon source to produce hydrogen. The results of "pretreatment-enzymatic" analysisshowed that the strategy of alkali-enzyme pretreatment obtained the highest reducingsugar yield. More importantly, its hydrogen production reached652mL/L when thealkali-enzyme LMBRs was used as the dark-fermentation subsrate. The residues ofLMBRs pretreatment, containing undegradeble proteins and trace minerals, were thencondegradation with lipid-extracted yeast biomass residues (LYBRs).