Study On The Green Synthesis Of Metal Nanoparticles And Their Transcriptional Regulation Mechanism For Enhancement Of Fermentative Hydrogen Production By Klebsiella Sp.

Energy shortage and environmental deterioration have become the major obstacles to restrict economic development and productivity progress.Hydrogen is a kind of green energy with high energy storage,and obtained clean and renewable merit,which can be used as a substitute of traditional fossil fuel.Amongst,the dark fermentative hydrogen production has attracted wide attention due to its advantages of wide raw materials,low cost and simple operation.Fe and Ni,as the important activating elements combining to the active center of hydrogenase,were prepared into different types of nanoparticles in recent years and added to the dark fermentation hydrogen production system.Such metal nanoparticles,in addition to be used as hydrogenase active center binding elements,both their surface effect and quantum size effect can promote the synthesis of biohydrogen.Therefore,regulation of the metal nanoparticles addition to the dark fermentative hydrogen production process has become an attractive research topic in the field of biological hydrogen production.In this study,the water extract of hyacinth was used as raw material to react with Fe and Ni ions to prepare the corresponding metal oxide nanoparticles,and supplement to the hydrogen production system from fermentation of lignocellulosic hydrolysate by Klebsiella sp.,to investigate their regulatory effect on hydrogen production process.The regulatory effect of Fe and Ni oxide nanoparticles on the dark fermentative hydrogen production by Klebsiella sp.was studied through monitoring the biohydrogen yield,bacterial growth and hydrolysate substrate consumption during the fermentation process,and the mechanism of these nanoparticles regulating dark fermentative hydrogen production would be elucidated via fluorescence quantitative PCR analysis.The research contents are as follows:(1)Metal oxide nanoparticles were prepared via the reaction using water hyacinth extract as raw material and a certain concentration of iron and nickel ions.According to XRD,TEM and SEM analysis,the nickel oxide nanoparticles prepared by two procedures exhibited the same morphology,but the aggregation state and particle size distribution are different,the average particle size of N6NPs is 9.3 nm,and the average particle size of N8NPs is 9.5 nm;The iron oxide nanoparticle was characterized as magnetite through XRD analysis,and the iron oxide nanoparticle was identified as spherical,magnetic,and aggregated Fe3O4 by TEM and SEM detection,and the average diameter was calculated as 13,0 nm.(2)The effects of different concentrations of Fe2+on the fermentative hydrogen production characteristics was firstly studied,the results showed that the addition of Fe2+ with concentration of 10-20 mg/L can promote the synthesis of biohydrogen by Klebsiella sp.for hydrogen production from fermentation of cotton stalk hydrolysate,the highest cumulative hydrogen production was obtained under the addition Fe2+with concentration of 20 mg/L,which was reached higher than 2200 mL/L,and increased by 20%as compared to the no addition treatment.The effects of Fe3O4NPs with different particle sizes on the fermentative hydrogen production characteristics were further studied,the results revealed that as the particle size was 10 nm,the cumulative hydrogen production was the highest,which could reach higher than 5 300 mL/L,and increased by 61.8%as compared to the no addition treatment.Except for promoting the synthesis of biohydrogen,this treatment could also favor the reducing sugar utilization and the bacterial growth.In addition,the regulatory effects of different concentrations of green synthesized Fe and Ni oxides NPs on the fermentative hydrogen production were investigated,the results showed that the NiONPs prepared by the two procedures exhibited different effects on the hydrogen production at different adding concentrations.When the concentration of N6NPs is 5-30 mg/L,it is beneficial for fermentative hydrogen production.Especially the highest cumulative hydrogen production was obtained at the concentration of 20 mg/L,which reached 4742 mL/L and increased by about 44.2%comparing to the control treatment.In the treatment with addition of N8NPs,only the treatment with addition of 20 mg/L N8NPs showed the regulatory effects of significantly improving the hydrogen production,which obtained the highest cumulative hydrogen production(4100 mL/L),while too high or too low concentrations would inhibit the fermentative hydrogen production.As the green-synthesized Fe3O4NPs supplemented in the range of low concentration(10-20 mg/L),the cumulative hydrogen production was higher than that of the control treatment after 120 h fermentation,especially,the highest cumulative hydrogen production higher than 4 000 mL/L was obtained at the Fe3O4NPs adding concentration of 20 mg/L,which was 20%higher than that of the control treatment.(3)The hydrogenase activity of Klebsiella sp.was detected under different nanoparticles addition treatments,the results showed that the peak value of hydrogenase activity in each treatment could be obtained at fermenting 24 h,especially,the N6NPs treatment gained the most significant effect,which obtain the hydrogenase activity 2 times higher than the control treatment.The real-time fluorescence quantitative PCR technology was applied to study the variation of the hydrogenase gene expression level under additions of different green synthesized metal oxide nanoparticles.The results showed that as the above three kinds of green synthesized metal oxide nanoparticles added to the fermentation medium at the optimal concentration,the hydrogenase gene expression level significantly enhanced within fermenting 24 h by Klebsiella sp.,especially,the treatment adding with 20 mg/L N8NPs exhibited the most significant effect,which obtained the increased times of 7.4 of hydrogenase gene expression level.