Enhancing Hydrogen Production Of Enterbacter Aerogenes Via Metabolic Engineering And The Pretreatment Method Of Its Renewable Biomass Substrates

Hydrogen is regarded as one of the sustainable fuels in the future. Biohydrogen is receiving more and more attentions because it has the advantages of sustainable production, mild reaction conditions and environment-friendly and can be coupled with the pretreatment of pollutants. However, so far, although the studies of biohydrogen have been made increasing progress, the efficiency of biohydrogen production is still very low for industrial application. Therefore, the key problems of elucidating the mechanism for bioproduction of hydrogen and improving the efficiency of biohydrogen production still need to be thoroughly solved.In this study, facultative anaerobe hydrogen-producing bacterium Enterobacter aerogenes AB91102 was investigated due to it that E. aerogenes grows very fast and its metabolic pathway can be easily modified. Furthermore, it has a high potential for large scale hydrogen production because its maximum theoretical hydrogen evolution rate may attain 10 mol/mol glucose, but its wild strain can only produce approcimately 1 mol hydrogen per mol glucose. Thus, the key principal factors, uptake hydrogenase and the key enzymes of competitive pathways of hydrogen production, were focused on, and their genes were knocked out for enhancing the yield of hydrogen in E. aerogenes. Subsequently, the NAD synthetase was homologously overexpressed for further promoting hydrogen production efficiency, and a deeper understanding about the effects on the distribution of carbon resources was attempted via altering intracellular NADH concentration. Then, the pretreatment method of renewable waste feedstock was preliminarily explored for future industrial application. Eventually, the anaerobic batch fermentations for biohydrogen from microalgal hydrolysate were operated and its key parameters were optimized using response surface methodology. The main contents and results of this study were summarized below.1. Effects of knocking out ppc and ldh genes encoding key enzymes of competitive pathways on hydrogen production of E. aerogenes. The ppc gene encoding PEPC (the key enzyme of succinate pathway) and ldh gene encoding LDH (the key enzyme of the lactate pathway) were cloned from E. aerogenes. Then, the PCR products of these genes were recombinated with the chromosome mediated by Red recombinase system, and the results of PCR verification, antibiotic resistance experiments and analysis of metabolites showed that ppc and ldh genes were successfully deleted from the chromosome. The specific hydrogen production rates (SHPRs) of the AB91102-L(?ldh) and AB91102-P(?ppc) were respectively increased 12.4% and 32.2%, and the yield of hydrogen were decreased 4.5% and 6.1% compared with the wild strain. The lactate pathway of AB91102-L was completely inactivated and the yield of succinate of AB91102-P was decreased 94.6%, the double knockout mutant AB91102-LP (?ldh, ?ppc) grew slowly and its yield of hydrogen was very low.2. Effects of knocking out hybO gene encoding small subunit of uptake hydrogenase on hydrogen production of E. aerogenes. The PCR product of hybO gene was homologously recombined with the chromosomes of wild strain, AB91102-L and AB91102-P using the Red system, yielding three mutants AB91102-O (?hybO), AB91102-OL (?hybO/Aldh) and AB91102-OP (?hybO/Appc). The SHPRs were respectively enhanced 24.0% and 68.8% in AB91102-O and AB91102-OP, and the yields of hydrogen were increased 21.3% and 27.6% compared with the wild strain. The significant increase of SHPR in AB91102-OP suggests that the two strategies, reducing hydrogen consumption and inactivating competitive pathways for hydrogen production exhibits a positively synergistic effect on hydrogen production.3. Promoting hydrogen production of E. aerogenes and its mutants by overexpression of NAD synthetase. The nadE gene encoding NAD synthetase was cloned from the wild strain. A plasmid pET28kan modified from plasmid pET-28(+) was constructed for homologous expression of NAD synthetase in E. aerogenes. The PCR product of nadE gene was inserted into the vector pET28kan to create the plasmid pET28kan-nadE, which was transformed into wild strain and the mutants for overexpression of NAD synthetase. The overexpression of NAD synthetase was confirmed by SDS-PAGE and Western blot analysis. Two mutants, AB91102-O/N (?hybOlnadE), AB91102-OP/N (?hybO/Appc/ nadE) and a control strain AB91102-OC were obtained. The resultes of the chemostat experiments showed that the total NAD(H) pool size in AB91102-O/N and AB91102-OP/N respectively increased 91.2% and 109.7% compared with the control strain AB91102-OC, but the NADH/NAD+ ratio decreased to 0.83 and 0.94 from 1.11. The SHPRs and the hydrogen yield of AB91102-O/N increased 66.0% and 165.8%, while they increased 149.5% and 301.1% in AB91102-OP/N, respectively, and the maximum hydrogen yield of AB91102-OP/N reached 5.1 L/L. Through the analyses of the changes of metabolism concentrations, NADH utilization rate (NADH)UH/Glu and specific activities of ADH, LDH and BDDH, the results indicated that the triple modification strategies, reducing hydrogen consumption, increasing NADH utilization rate, and enchancing the total concentration of NAD significantly exhibits positive synergistic effects on hydrogen production.4. Studies on the pretreatment method for hydrolysis of lipid extracted microalgal biomass residues (LMBRs). LMBRs were treated by the proteases in the autolysate of lipase-extracted yeast biomass residues (LYBRs) at 48? for 24 h, and the total nitrogen of the treated LMBRs decreased 45.0%. Then, the treated and untreated LMBRs were hydrolyzed by thermo-alkaline method, and the best effect was obtained at 100?. The maximum yields of reducing sugar, total nitrogen and amino acids were 189.3 mg/g LBMRs,36.56 mg/g LBMRs and 102.8 mg/g LBMRs, respectively. The reducing sugar hydrolyzed from the untreated LBMRs was only 87.1% compared with the treated LBMRs. The results indicated that the pretreatment by the enzymes of LYBRs coupling with thermo-alkaline method was an effective and simple method for the hydrolysis of LMBRs.5. Optimizing the paremeters of anaerobic batch fermentation for hydrogen production from LMBRs. The central composite designs (CCDs) were performed, and a quadratic regression model based on temperature, pH, inoculum and hydrogen yield was obtained from the triplicate experimental data. The analysis of variances (ANOVAs) suggested that the model was significant and reliability. The predicted maximum yield 54.22 H2 mL/g LMBRs of hydrogen production should be obtained when temperature, pH and inoculum were respevtively at 37.55?,5.95 and 12.25%. The confirmatory experiments were conducted, and the mutant evolved 54.61 mL H2/g LMBRs under the optimal conditions. The coincident result verified the practicability of the model.