Isolation Of Hydrogen Producing Bacterium And Investigation On Mechanism Of Hydrogen Production From Lignocellulose By Thermophilic Bacteria

The shortage of fossil fuels and environmental concerns caused by burning of fossil fuels have attracted widespread attention in many countries,and the development of alternative clean energy sources has become a matter of urgency.Biomass is the most abundant material in the world and the valorization of waste biomass has been studied by lots of scholars,which can not only solve the environmental pollution problems but also add value to waste biomass.Hydrogen is one of the cleanest sources of energy with a high energy density?143 GJ/t?.It is a kind of promising energy carrier.The biohydrogen production from biomass is an environment friendly technology,which could avoid the shortcomings of traditional technology.The high cost of biohydrogen production impedes the industirial application of biohydrogen production technology.Selection of distinctive hydrogen-producing strain and exploitation of novel fermentation process are hot research topics,which can improve the efficiency and competitiveness of biohydrogen production.A new strain named MJ1 was isolated from cultures of paper sludge,which was identified as Thermoanaerobacterium thermosaccharolyticum by 16S rDNA gene analysis.T.thermosaccharolyticum MJ1 can utilize glucose,xylose and cellobiose for hydrogen production,but can not utilize sucrose.The analysis of metabolites indicated the hydrogen was produced by the biosynthetic pathway of acetic acid and butyric acid.It was worth mentioning that MJ1 could efficiently utilize pretreated sugarcane bagasse hydrolysate for hydrogen production without detoxication,showing an excellent tolerance to inhibitors.The hydrogen production reached 39.64,105.42,111.75 and 110.44 mM at 20,40,60 and 80%of pretreated sugarcane bagasse hydrolysate,respectively.Supplementation of CaCO₃ enhanced the hydrogen production by 21.32%than control.Furthermore,a wide pH range was suitable for hydrogen production by MJ1,but not a simple buffer system.The dilute acid pretreated sugarcane bagasse is the typical representative of biomass.Here,efficient biohydrogen production?277.4 mM?6.2 L-H₂/L??from non-detoxified sugarcane bagasse?NDSCB?was achieved through a novel two-stage anaerobic fermentation.Interestingly,the hydrogen production of the second stage fermentation?167.8 mM?was significantly higher than that of the first stage fermentation?109.6 mM?.Metabolite analysis showed that the metabolic flux had been redirected and Butyrate/Acetate ratio was increased in the second stage fermentation.Simulation of the second-stage fermentation condition showed that acetate and butyrate had positive impact on hydrogen production,depending on varying substrate,and acetate?3 g/L?and butyrate?2 g/L?could significantly enhance the hydrogen production from NDSCB by 50.7%and 27.8%,respectively.The linear correlation between acetate concentration and biomass with hydrogen production was observed.There were various inhibitors in pretreated sugarcane bagasse hydrolysate.In order to characterize the the effect of inhibitors on hydrogen production by MJ1,eight kinds of inhibitors were selected to test with DSM1313 used as a control.The results showed that the MJ1 had a better tolerance to inhibitors than DSM1313 and the growth of MJ1 was significantly affected by ferulic acid and p-coumaric acid.The growth of MJ1 was completely restrained by 1.5 g/L ferulic acid and 0.5 g/L p-coumaric acid.0.25 g/L p-coumaric acid led to a lag phase of 10.7 h,while the other inhibitors showed no significant effect on lag phase.The hydrogen productions decreased with increasing inhibitor concentrations,showing the similar trend with growth.While vanillin and syringaldehyde could significantly inhibit the hydrogen production,showing little effect on growth.Metabolite analysis showed that the total metabolite production was not significantly affect by vanillin and syringaldehyde,but the metabolic flux had been redirected.The lactate production increased with decreasing productions of butyrate and acetate,which was against the hydrogen production.Given the above,some inhibitors decreased the hydrogen production by inhibiting growth,while others decreased the hydrogen production by redirecting the metabolic flux.There was a synergistic effect among different inhibitor mixtures and metabolite analysis showed that the lactate production increased with increasing mixture concentrations,indicating that the inhibitory effect in mixture was mainly resulted from vanillin and syringaldehyde.?-glucosidases?10 U/g-substrate?could significantly enhanced hydrogen production by Clostridium thermocellum,leading to an increase of hydrogen production by 37.1%.Additionally,?-glucosidase and Triton X-100 had a synergistic effect on biological saccharification,resulted in an accumulation of 5.1 g/L reducing sugars with an increase by28.1%.However,fusion?-glucosidase showed no improvement on hydrogen production and biological saccharification.The results indicated the fusion of?-glucosidase to cellulosome could not promote the degradation of lignocellulose.The fermentation inhibitors resulted from diluted acid pretreatment process could completely suppress the growth of DSM1313.Three popular detoxification methods were explored to evaluate their effects on biohydrogen production.The results showed laccase and NaBH₄ could slightly relieve the toxicity,leading to a hydrogen production of below 7 mM.As for detoxification by washing,the biohydrogen production reached 44 mM,nearly 10times higher than the non-detoxified sample.Additionally,co-culture strategy was further adopted to enhance the biohydrogen production.The maximum biohydrogen production?118.7 mM?was obtained by optimizing the fermentation conditions,which was 2.7 times higher than monoculture and 25 times higher than non-detoxified sample.Although laccase and NaBH₄ were suitable for the ethanol fermentation by yeast,they were not suitable for the hydrogen fermentation by bacteria.The results indicated that washing had a widespread application in detoxification.The biomass of DSM1313 could be promoted by supplement of CaCO₃,which was beneficial to degradation of cellulose.The genes involved in protein synthesis,amino acid synthesis,acetyl-Co A synthesis,glycolysis,transcription and other pathways could be up-regulated by CaCO₃,while the genes involved in flagellum assembly,chemotaxis and other pathways could be down-regulated.The results of transcriptome indicated the CaCO₃ could enhance the metabolic pathways and maintain the physiological activity of cells.