Characteristics And Mechanisms Of Microbial Dark-fermentative Hydrogen Production From Stover Hydrolysate

Due to the advantages of large quantities, lower cost, renewable and easy to obtain, stover-like lignocellulosic materials are considered as potential suitable substrates for dark-fermentation hydrogen (H2) production. Biohydrogen production by stover-like materials can largely decrease H2production cost, making biohydrogen production more economically available. In addition, serious environmental pollution can be avoided by utilizing stover for H2production instead of commonly adopted combustion disposal. However, the higher crystallinity and heterogeneity of stover biomass lead to the lower utilization efficiency of microbes. Therefore, biohydrogen production from stover hydrolysate, which is produced by various pretreatment methods towards stover materials, becomes important route for practical applications. At present, bottleneck problems, such as lower substrate utilization efficiency and H2yield, exist in biohydrogen production from stover hydrolysate by using mixed cultures, preventing the development of practical applications. Furthermore, it is not clear for the molecular mechanisms of substrate degradation and H2production during the process.This study investigated the effects of inoculum pretreatment and fermentation temperature on H2-production performances from stover hydrolysate by different kinds of mixed cultures. Thermoanaerobacterium thermosaccharolyticum W16, which was previously isolated and presented better utilization and H2production from stover hydrolysate, was introduced into mixed cultures for bioaugmentation. Amplified fragment length polymorphism (AFLP) and analysis of lignocellulose degradation genes were carried out to illustrate the molecular mechanisms of T. thermosaccharofyticum-kind microbes. For efficient H2-producing strain W16,[FeFe]-hydrogenase gene complexes were cloned, characterized, and analyzed for the transcriptional level to address the molecular mechanisms of H2production.Among five pretreatment methods of heat, acid, alkali, ultrasonic, and ultraviolet, heat pretreatment presented the better efforts on the inoculums of activated sludge, river sediments and granular sludge, and activated sludge pretreated by heat reached the optimum H2yield of5.03mmol-H2/g-sugar utilized. Among different fermentation temperatures (30℃,37℃,55℃and70℃), H2production reached the optimum at55℃for the three inoculums, in which granular sludge achieved the highest H2yield (7.74mmol-H2/g-sugar utilized). In addition, Fb production at70℃was significantly lower than that at55℃. Inoculum pretreatment and thermophilic condition significantly changed the microbial community structure, from the universal existances of facultive anaerobes such as Enterobacter spp., Escherichia spp. and Klebsiella spp. to the enrichments of some efficient H2-producing microbes such as Clostridium spp., Bacillus spp. and Thermoanaerobacterium spp.The bioaugmentation by the strain W16can significantly increase the H2production from stover hydrolysate by using rotten corn stover and anaerobic sludge, and increase the utilization efficiency to some extent. Bioaugmented rotten corn stover reached the highest H2yield of9.90mmol-H2/g-sugar utilized. AFLP analysis showed that the genomic DNA of T. thermosaccharolyticum-kind microbes was highly reserved, and the2-polyprenylphenol6-hydroxylase gene detected by AFLP might act a key role in the degradation of lignocellulose materials. In addition, the single base located in the-10region (RNA polymerase binding site) of Cellulose1,4-beta-cellobiosidase gene might be relevant to the utilization of cellulose by T. thermosaccharolyticum-kind microbes.For the strain W16, the complete DNA sequences of [FeFe]-hydrogenase hyd and hfs gene complexes were cloned and analyzed, which contained hydⅡ (1308bp), hydC (480bp), hydD (363bp), hydB (1788bp), hydA (1752bp) and hfsA (243bp), hfsB (1716bp), hfsC (1512bp), respectively, in which hydⅡ, hydB, hydA, hfsB, and hfsC were important [FeFe]-hydrogenase genes. The study also discussed the physical and chemical characteristics, secondary and the third level structures of proteins, structural functional zones, and conservative zones of [FeFe]-hydrogenase hyd and hfs gene complexes. While investigating the effects of different gas-phase conditions (initial N2, H2, or CO2pouring) on transcriptional expression of important [FeFe]-hydrogenase genes, the transcription of hydA was significantly inhibited under high H2or CO2partial pressure, hydB was only susceptible for high H2partial pressure, and hydll preformed lower transcriptional activities for all the situations. For hfs complex, hfsB showed higher transcriptional level under high CO2partial pressure and lower transcriptional level under high H2partial pressure, and hfsC displayed the lowest transcriptional level under high CO2partial pressure.