Optimization Of Metabolic Pathways For Biohydrogen Synthesis From Fermentation Of Lignocellulosic Hydrolysate By Klebsiella Sp.

The rapid consumption of fossil fuels and environmental pollution make it urgent to develop new energy sources and reduce environmental pressure.The high efficiency and pollution-free characteristics of hydrogen energy make it the exploitation focus in new energy developing field.Amongst,the microbial fermentative hydrogen production using lignocellulose as substrate is the most noticeable aspect in the field of biological hydrogen production.The conversion of lignocellulose to fermentable sugars and its further application to the regulation of microbial fermentative hydrogen production is often related to microbial strains and their metabolic pathways utilizing reducing sugars for hydrogen production.Researches in this paper were developed based on a high effective hydrogen producing bacterium Klebsiella sp.which was obtained previously in our laboratory.The key enzyme genes,[Ni-Fe]hydrogenase large subunit hycG gene and formate-hydrogen lyase large subunit fhl gene in the biohydrogen synthesis pathway of this strain were cloned and overexpressed,the gene recombinant strain with optimized metabolic pathway and high efficiency for hydrogen production from rice straw hydrolysate would be obtained,and the effects of the two gene overexpression on the metabolic flow distribution of biohydrogen synthesis related pathways would also be explained.The research contents and achievements are as follows:(1)The effect of nickel ion on hydrogen production of Klebsiella sp.was firstly studied,the results showed that the cumulative hydrogen production could be enhanced via adding a certain concentration of Ni2+to the hydrogen production system from fermentation of lignocellulosic hydrolysate by Klebsiella sp.,confirming that Ni2+could promote the biohydrogen synthesis by changing the[Ni-Fe]hydrogenase activity.Therefore,we cloned the[Ni-Fe]hydrogenase large subunit hycG gene,and successfully constructed the prokaryotic expression vector pET-28a-hycG,thus the homologous overexpression of this gene in Klebsiella sp.was further realized.As this recombinant strain RT-H used for hydrogen production from fermentation of rice straw hydrolysate,the cumulative hydrogen production reached 4034.1 mL/L,and increased by 14.5%as compared to the wild strain WT.The dynamically fitted results of the cumulative hydrogen production also showed that there was a higher hydrogen production potential P and a higher maximum hydrogen production rate Rm during the fermentation process of hydrogen generated by RT-H,which indicated that the expression of hycG gene favored the enhancement of strain's hydrogen production.The monitoring results of hydrogenase activity also verified the expression of hycG gene was beneficial for improving the hydrogenase activity thus promote the generation of hydrogen gas.Moreover,the expression of hycG gene also caused the shift of metabolite profile and metabolic flux distribution during different fermentation stages,especially caused the pyruvate metabolic rate and formate consumption rate in RT-H significantly higher than WT,illustrating this gene expression might increase the metabolic flux distribution in the biohydrogen synthesis branch.(2)The formate-hydrogen lyase large subunit fhl gene of Klebsiella sp.was cloned based on the analysis of its metabolic pathway for hydrogen production.The prokaryotic expression vector pET-28a-fhl was successfully constructed,and the recombinant strain RT-F with fhl gene overexpression was obtained.This strain gained the strong hydrogen producing ability to generate hydrogen gas from fermentation of rice straw hydrolysate,its cumulative hydrogen production increased by 24.8%as compared with WT,which achieved to 4396.4 mL/L.The results of dynamically fitted cumulative hydrogen production also confirmed that the strain had higher hydrogen production potential P and maximum hydrogen production rate Rm than WT,which could reached 4289.6 mL·L-1,89.47 mL·L-1·h-1 respectively.The formate-hydrogen lyase activity of RT-F reached the peak at fermenting 24 h and was significantly higher than that of WT,indicating that fhl gene expression led to the improvement of the formate-hydrogen lyase activity in the early fermentation stage,which was conducive to the synthesis of biohydrogen in the early fermentation stage.In addition,the consumption rates of intracellular and extracellular formate of RT-F were significantly higher than that of WT,indicating that the fhl gene expression may lead to the rapid splitting of formic acid to produce hydrogen gas,which is beneficial to the early synthesis of biohydrogen.(3)A recombinant strain RT-FH was constructed with co-expression of the[Ni-Fe]hydrogenase large subunit hycG gene and the formate-hydrogen lyase large subunit fhl gene.This strain was used for hydrogen production from fermentation of rice straw hydrolysate,the results revealed that the cumulative hydrogen production obtained by RT-FH increased by 19.7%comparing to WT,which reached 4227.6 mL/L.Moreover,the recombinant strain RT-FH obtained high hydrogen producing rate in the early fermentation stage,thus 96?of the total hydrogen production has been accumulated as fermenting to 72 h.Besides,the co-expression of fhl and hycG gene also changed the metabolite profile and metabolic flux distribution in different fermentation stages.As compared with WT,RT-FH obtained a lower ethanol metabolic rate,and the increased intracellular formate production and extracellular formate consumption.