The Breeding Of Thermophilic Anaerobic Cellulolytic And Hydrogen-producing Strains And Study On The Enhanced Mechanisms Of Anaerobic Fermentative Performance By Biochar Amendment

The resource and energy utilization of organic solid waste are of great significance to alleviate the energy and environmental crisis and the reasonable disposal of waste.The biological conversion of organic waste through anaerobic fermentation technology for hydrogen production has the advantages of simple operation process and environmental friendliness,which has attracted wide attention.However,the development of its industrial scale is still limited by the problems of low yield of target product and low conversion rate of substrate.In this study,sugarcane bagasse(SCB)was used as a model lignocellulosic substrate,with the goal of improving its anaerobic bioconversion efficiency,The breeding of thermophilic anaerobic cellulolytic and hydrogen-producing strains and study on the enhanced mechanisms of anaerobic fermentative performance by biochar amendment were carried out.Firstly,SCB pretreatment process based on potassium peroxymonosulfate(PMS)combined with deacetylation was established.Pretreated SCB(PSCB)contained holocellulose as high as 83.5% and the inhibitors in SCB for enzymatic hydrolysis and fermentation were both effectively removed.The evaluation results of simultaneous saccharification and cofermentation(SSCF)showed that the highest productions of ethanol and succinic acid reached120.8 g/L and 34.8 g/L,respectively,at a substrate loading as high as 330 g/L,indicating that a SCB substrate with excellent degradability was obtained by the constructed pretreatment process.Consolidated bioprocessing(CBP)based on cellulolytic and hydrogen-producing bacteria could simplify the production process of biological hydrogen to the greatest extent.Thermophilic bacteria have relatively more vigorous growth and metabolism activities and more efficient capacities for cellulose degradation and hydrogen production.Taking paper sludge as the source of target functional bacteria,stable thermophilic cellulolytic consortium MC1 with hydrogen-producing capacity was obtained through continuous passage and enrichment,which contained high abundance of Ruminiclostridium with cellulolytic function and Thermoanaerobacterium with high-efficiency hydrogen production capacity.Clostridium thermocellum MJC1 with cellulolytic and saccharification ability was isolated with cellulose as carbon source.The hydrogen production was 45.1 and 47.7 m M with 5 g/L of cellulose and cellobiose as substrate,respectively.Thermoanaerobacterium thermosaccharolyticum MJ2 that can efficiently use fermentable sugars to produce hydrogen was isolated from MC1 using glucose as carbon source.The hydrogen production by MJ2 using 10 g/L glucose,xylose,and cellobiose was 146.8,156.7,and 183.7 m M,respectively.The hydrogen production of MJ2 decreased significantly when the glucose was increased to 15 g/L.The isolated functional strains laid a good foundation for constructing efficient CPB process for SCB degradation and hydrogen production.By adding biochar,the problem of poor fermentative performance of MJ2 under high glucose concentration was solved.Biochar amendment significantly reduced the lag period of fermentation,and enhanced the maximum rate and the potential of hydrogen production by 2.9times by 2.8 times,respectively.Physiological and biochemical analysis results showed that the synthesis of cell biomass and biofilm were effectively improved by biochar amendment while the intracellular metabolic activity was not.Transcriptome analysis results showed that the expression levels of genes related to carbon metabolism were not promoted by biochar amendment.However,biochar amendment significantly up-regulated flagellar genes involved in biofilm formation and cell immobilization,nitrogenase/Ech hydrogenase genes involved in hydrogen production and simultaneously down-regulated Hnd hydrogenase genes related to hydrogen-consuming,and the immobilized cells showed more significant differential expression than free cells in the same biochar amended system.In addition,by biochar amendment,the potential and the maximum rate of hydrogen production by MJC1 were effectively increased by 101.8% and 113.1%,respectively.The enhanced hydrogen production by MJC1 were attributed to the increased cell biomass,the reduced oxidation-reduction potential(ORP)of the fermentation system,the improved cellulase activity and the adjusted carbon flow balance with biochar amendment.Exploring the coculture system of MJC1 and MJ2 for hydrogen production,it was found that the maximum hydrogen production was obtained at an initial inoculation ratio of 2:1 and the highest level of hydrogen production reached 177.5 m M at a PSCB loading of 10 g/L.The effect of biochar on hydrogen fermentation by the co-cultured system was further explored and the results showed that the production of acetic acid and butyric acid were significantly increased with an increase of final hydrogen production by 33.1%.The enhanced mechanisms of biochar amendment were further studied in a dark fermentation system,in which the PSCB was degraded by a mesophilic microbial community for hydrogen production.The analysis of physicochemical properties showed that different types of biochar present significant differences in element composition,specific surface area and electrical conductivity.The results showed that biochar could significantly increase the level of intracellular and extracellular reducing power,improve cell biomass and the activity of key enzymes,stimulate the synthesis of extracellular polymeric substance(EPS),improve the electron transfer efficiency of fermentation system and the abundance of functional bacteria and significantly enhance the maximum rate and potential of hydrogen production by 5.8 times and 3.0 times,respectively.The formed spatial ecological niche may contribute to the microbial synergistic interaction during the degradation of PSCB and hydrogen production.The methane fermentation by anaerobic digestion from hydrogen-producing effluent was explored and it was found that biochar amendment significantly improved the degradation rate of substrate and the final methane production of up to 41.2%.The addition of biochar significantly reduced the secretion of extracellular electroactive shuttles and promoted the synthesis of humic acid.Relevantly stable microbial community was established and maintained on biochar by tight adsorption and functional microbes were selectively enriched or colonized in biochar-amended systems at the initial stage of fermentation.In addition,the abundance of functional archaea/bacteria were improved and the selection preference of microbes for specific spatial locations was observed in biochar-amended systems.The SCB pretreatment method constructed in this study provides a reference for the pretreatment process of lignocellulosics.The isolated T.thermosaccharolyticum MJ2 provides a high-quality candidate for the research of biological hydrogen production.The hydrogen fermentation strategy based on co-cultivation and biochar amendment provides a technical solution for efficient hydrogen production from lignocellulosics.Combining multi-angle physiological/biochemical,electrochemical and transcriptome analysis provides strong theoretical basis and new insights in revealing the potential mechanisms of enhanced performance of anaerobic fermentation by biochar amendment.