Effect Of Magnetic Peach Gum Immobilization On Hydrogen Production Process Of Dark Fermentation By Klebsiella Sp. And The Regulatory Mechanism

Hydrogen production by microbial dark fermentation has become an important way of hydrogen energy production in the 21st century due to its diversity of raw materials,reproducibility and cheapness.The addition of metal nanoparticles and immobilization of microbial cells have been reported as important regulatory methods to promote dark fermentative hydrogen production in recent years.However,there are few reports on the coupling regulation of these two methods.Peach gum is a natural raw material with high polysaccharide content and strong adsorption performance,which shows the basic characteristics of embedding carrier and is expected to be used as a new immobilization carrier.Moreover,the rich biological active substances in peach gum also make it a natural raw material for green synthesis of metal nanoparticles.On the basis,this study synthesized magnetic Fe₃O₄ nanoparticles using natural peach gum as raw material,and coupled with peach gum to immobilize the Klebsiella sp.cells,in order to investigate the regulation of biohydrogen synthesis and utilization of lignocellulosic hydrolysate.Further,the mechanism of the novel hydrogen-producing regulation mode was preliminarily revealed by monitoring the strain’s hydrogenase activity,comparative analysis of the number of living cells and real-time quantitative PCR detection of key enzyme gene expression.The specific research content is as follows:1.First of all,using natural peach gum as raw material,adding a certain concentration of Fe²⁺,Fe³⁺for reaction,the magnetic Fe₃O₄nanoparticles were prepared,and the results showed that XRD characterization analysis,the diffraction peak in the sample was sharp,and there was no other diffraction peak,which clearly showed that the prepared product was highly crystalline,and JCPDS The standard magnetite XRD pattern of No.19-0629 is good.SEM and TEM show Fe₃O₄ nanoparticles in a spherical shape with slight agglomeration,possibly due to magnetic attraction between nanoparticles.2.Secondly,the effect of magnetic Fe₃O₄ nanoparticles on the hydrogen production characteristics of Klebsiella sp.fermentation was studied at different concentrations,and the results showed that the addition of 10-50 mg/L magnetic Fe₃O₄ nanoparticles could promote the increase of hydrogen production of strains,but the hydrogen production was different from the addition concentration,especially at 20 mg/L,which had a strong promoting effect on Klebsiella sp.fermentation hydrogen production,with a maximum hydrogen production of 4455.21±31.25 m L/L.Compared with the control treatment（no nanoparticle addition）,it was about 29.61%higher;and the OD₆₀₀ added value(△OD₆₀₀)and biohydrogen synthesis efficiency Y（H₂/S）were the best at nanoparticle addition concentrations of20 mg/L,which were increased by 9.87%and 30.12%,respectively.Thus,PGP-Fe₃O₄-NP was established to improve the growth rate,reducing sugar conversion rate and hydrogen production efficiency of bacteria in the hydrogen synthesis pathway,showing that Klebsiella sp.had a high fermentation hydrogen production potential at a concentration of 20 mg/L nanoparticles.3.Based on the prepared magnetic Fe₃O₄ nanoparticles,a certain proportion of peach gum and sodium alginate were added to the Klebsiella sp.for cell gel bead fixation.SEM characterization results revealed that,its immobilized gel beads have many independent or combined honeycomb pores,and the bacterial body is evenly embedded in its honeycomb pores.The effects of magnetic Fe₃O₄ nanoparticles and Klebsiella sp.immobilization on the hydrogen production characteristics of fermentation were studied at different concentrations,and the results showed that the viability of bacteria was prolonged under immobilization treatment,and the number of living cells decreased rapidly after 48 h,which was 24 h longer than that of immobilization.And the hydrogen production capacity has been significantly improved,the addition of 5-25 mg/L nanoparticles coupled fermentation can promote the production of biological hydrogen,the addition of concentration of 10 mg/L is the best,the cumulative hydrogen production of up to 5903±85 m L/L,compared with the control group increased by about 71.73%,reducing sugar consumption rate of more than 96.06%.In contrast,Klebsiella sp.further promoted the utilization of reducing sugars under the coupling immobilization treatment of peach gum nanoparticles.4.The change of Klebsiella sp.number of live bacteria over time during the production of hydrogen by dark fermentation organisms was determined.The results showed that the number of viable bacteria in a single gel bead（PCS）reached the highest at 24 h under immobilization treatment,which was（2.21±0.02）×10¹⁰ cfu/pcs,which was much higher than that of the control group,indicating that immobilization had a delaying effect on the decline of bacteria cells.Through the detection of hydrogenase and hydrogen formate lyase enzyme activity,it was determined that the addition of 10 mg/L nanoparticles for immobilization had a high impact on enzyme activity,and the hydrogenase and formate lyase enzyme activities were 13.32±0.23μg/L/h and 93.32±1.63μg/L/h,which were 35.50%and 19.15%higher than the immobilization treatment without nanoparticles.Therefore,by using real-time quantitative PCR（RT-PCR）detection technology,the detection and analysis of 4 hydrogenase genes,3 hydrogen formate lyase genes of Klebsiella sp.showed that the addition of 20 mg/L Fe₃O₄-NPs for peach gum immobilization increased the expression of hydrogenase genes by a maximum of 34.69±1.97 times compared with the control,and led to changes in gene expression of hydrogenase and key enzymes,promotes hydrogen production at different stages.It can be seen that Fe₃O₄ nanoparticle coupled peach gum immobilization has a certain role in promoting the expression of Klebsiella sp.hydrogenase gene,and then regulates the synthesis of biological hydrogen.