Isolation Of A Hydrogen-producing Bacterium Klebsiella Sp. WL1316 And Metabolic Regulation Of The Hydrogen Production From Fermentation Of Cotton Stalk Hydrolysate By Isolated Strain

Mankind enters the 21st century,as the energy crisis intensifying,and the petroleum based traditional fossil energy exhausting,the energy production based on lignocellulosic biomass has attracted worldwide attention.Hydrogen,the cleanest energy on earth,has presently become a renewable energy resource developed by all countries in the world.In resent years,the biological hydrogen production using lignocellulose as substrate has become the important hydrogen production mode due to the renewability of the feedstock.In the dark fermentation of lignocellulosic hydrogen production,the fermentative microorganisms play the leading role,however,the hydrogen yield produced by single pure wild strain is relatively low in nature,so many works focused on isolation of efficient hydrogen production strains for a long time.Meanwhile,the components of the lignocellulose or its hydrolysate in the straw biomass are always complex,the xylose can not be utilized by most hydrogen-producing bacteria,so it's particularly important to isolate hydrogen-producing strains with the ability to hydrolyze the lignocellulose or metabolize the hydrolysate.Cotton stalk is the most widely distributed lignocellulosic feedstock in Xinjiang,China with huge yield of 6 to 8 million tons per year.Transforming the lignocellulose of cotton stalk into high-value products has been of interest in recent years,and exploitation of high-value products has also presented diversification,some researches have been reported to coverse cotton stalk hydrolysate into high-value chemicals,such as bioethanol,xylitol and single cell lipid.However,there is few report concerning the biohydrogen production from fermentation of cotton stalk hydrolysate by hydrogen-producing bacteria.In order to obtain efficient hydrogen-producing bacteria for hydrogen production from fermentation of cotton stalk hydrolysate,this thesis isolated a hydrogen-producing bacterium from the intestine of wild carp(Cyprinus carpio L.)of the Tarim River Basin,Xinjiang,China,which was used for hydrogen production from fermentation of cotton stalk hydrolysate.The fermentation conditions were screened,and the fermentation process was scaled-up and regulated,as a result,coutilization of glucose and xylose in cotton stalk hydrolysate and high yield of biohydrogen |were obtained.Moreover,the strain's metabolic network coupling glucose and xylose utilization,and fermentative hydrogen production was rebuilt based on the genome information of Klebsiella sp.WL1316,combining the analysis of key enzymes and flux analysis of key note metabolites,the metabolic basis for fermentative hydrogen production of this hydrogen-producing bacterium was illuminated.The main results were demonstrated as follows:1.A hydrogen-producing bacterium WL1316 was isolated from the intestine of wild carp(Cyprinus carpio L.)of the Tarim River Basin,Xinjiang,China,which was identified as Klebsiella sp.based on examination of morphological,physiological and biochemical characteristics and 16S rDNA gene sequencing.It was discovered that this strain possessed the ability to produce hydrogen gas with glucose and xylose coutilized.Using cotton stalk hydrolysate as sugar substrate with fermentation time of 24 h,the isolate obtained hydrogen production,hydrogen production rate and hydrogen yield of 554.0 ± 22.6 mL/L,23.1 ± 0.9 mL/(L·h),0.33 ± 0.01 mL/(L·mol sugarconsumed),respectively,indicating the strain possessed the potential to utilize cotton stalk hydrolysate for efficient biohydrogen production.The optimum fermentation conditions for hydrogen production were further determined as follows:initial sugar concentration of 40 g/L,fermentation temperature of 37 ? and initial pH value of 8.0,and the dynamical parameters Rm and P reached the maximum using modified Gompertz equation fitting the cumulative hydrogen production under such fermentation conditions,representing Klebsiella sp.WL1316 obtained high fermentative hydrogen production rate and hydrogen productin potential.2.The amplification fermentation was conducted in a 5 L fermenter using optimized parameters mentioned aboved.Higher productivities with maximum daily hydrogen production of 937.0 ± 41.0 mL/(L·d),cumulative hydrogen production of 2908.5 ± 47.4 mL/L,viable cells of(20.2 ± 0.6)×108 CFU/mL,and hydrogen yield of 1.44 ± 0.08 mol/mol sugarconsumed were obtained.Higher glucose and xylose utilization were also obtained,showing the ability of the strain to converse glucose and xylose of cotton stalk hydrolysate into biohydrogen.Alternative pH regulation could effectively alleviated the dramatic decrease of pH value induced by acidic metabolites generation,and significantly improve the daily hydrogen production and cumulative hydrogen production,and enhance the glucose and xylose utilization and the bacterial growth.In contrast,the alternative temperature regulation couldn't significantly enhance the daily hydrogen production and cumulative hydrogen production,while could promote the glucose and xylose utilization and the bacterial growth to some extent.3.The genome sequencing of Klebsiella sp.WL1316 was developed.Based on annotation of Klebsiella sp.genomic data,the strain's KEGG metabolic pathway was primarily analyzed,and the main metabolic pathway for glucose utilization and fermentative pathway for hydrogen production using pyruvate as intermediate metabolite were obtained.Furthermore,the COG classified functional proteins were analyzed,and the xylose can be isomerizated to be xylulose,furthermore phosphorylate as xylulose-5-phosphate,and then entered into pentose phosphate pathway for metabolism.In addition,this strain also obtained various hydrogenases and formate hydrogen lyases,which had superior quantity distribution over some closely related bacterial species.On this basis,the metabolic network of the strain involving glucose,xylose utilization and fermentative hydrogen production was reconstructed.Based on the reconstructed metabolic network,the activities of enzymes in key metabolic branch concerning biohydrogen synthesis of Klebsiella sp.WL1316 were examined and analyzed,and flux analysis for key node metabolites was also developed.The results showed that the glucose and xylose effectively metabolized,which promoted the biomass synthesis and fermentative process.The pyruvate rapidly conversed as per decarboxylation,and provided carbon skeleton for other metabolic branches.The formate lysed for biohydrogen synthesis in fermentation time of 48 h.When fermenting 24?48 h,the metabolic flux originating from pyruvate node partly flowed to Krebs cycle.The lactate synthesis branch turned to be the competitive branch in the fermentation time of 48?72 h.The metabolic branch of acetate and ethanol synthesis exhibited main competition for biohydrogen synthesis during the late fermentation period.The constructed quasi-steady-state based metabolic network also proved the metabolic pathways mentioned above.In summary,it could be considered that the competitive branch of biohydrogen synthesis should be effectively regulated in the middle and later fermentation periods(48?96 h)to efficiently improve the biohydrogen production.