Performance And Mechanism Of A Staged Butyrate-Butanol Electro-Fermentation Process With Rice Straw As Substrate

China is a country with huge straw reserves,and the greenhouse gases generated by open burning and pile rotting of straw are as high as about 56 million tons of CO₂equivalent per year,even caused regional air pollution,land degradation,rural environmental management difficulties and other outstanding problems.Vigorously promoting the high-quality conversion technology of straw is an important step for China to achieve the"double carbon"goal on schedule.Modern biorefinery technology is a typical process with characteristics of"carbon neutral",development and promotion of butanol biorefinery technology with lignocellulosic and other non-food biomass as substrates,is a crucial step to crack the ecological and environmental pressure,actively deal with climate change,promote economic development under the new normal background with huge strategic benefits.Aim at addressing the common bottlenecks of lignocellulosic butanol biorefinery technology,such as low biocatalytic conversion efficiency and large amount of cellulose enzyme supply,this paper systematically carries out the construction and targeted regulation research of the"electrochemical-catalytic-enhanced staged butyrate-butanol fermentation process with rice straw as substrate"based on the concept of carboxylate platform and principle of electro-fermentation,providing theoretical guidance and technical reference for the development of efficient lignocellulosic butanol biorefinery technology which does not require large amount of cellulose enzyme.Based on the cultivation of mixed culture DCB which was characterized by butyrate fermentation of rice straw,the cathodic electro-fermentation（CEF）and anodic electro-fermentation（AEF）systems were constructed and comparatively evaluated by butyrate fermentation efficiency.It was found that,under the condition of-0.80 V（vs Ag/Ag Cl）cathodic potential,CEF system significantly increased the abundance of butyrate fermenting bacteria and butyrate conversion efficiency of rice straw.The butyrate production,yield and selectivity in CEF system increased by17.37%,28.13%and 17.93%,respectively,compared with the open-circuit system（OC）without applied potential.Besides,the AEF system and electron mediator neutral red（NR）was not able to ehance the butyrate fermentation efficiency of DCB.After the optimization of CEF system,it showed that suitable cathodic potential（-1.00 V）significantly promoted the growth and metabolism of functional butyrate fermentation flora（Clostridium IV and Clostridium Xl Va）,and fed-batch fermentation mode was more favorable for butyrate accumulation.With-1.00 V cathodic potential,p H 7.0 and 30 g/L initial rice straw dosage,butyrate concentration in the CEF system was as high as 20.35 g/L,however,the butyrate selectivity was only 47.46%.Optimization of DCB by efficient butyrate fermenting bacterium C.tyrobutyricum,multiple gene levels in the mixed microbiome was up-regulated,and the tolerance of microbiome to high concentrations of VFAs was also improved,resulting in butyrate production and selectivity reached 28.02 g/L and 53.58%,respectively,and butyrate yield and production rate increased from 0.31 g/g and 0.97g/（L-d）to 0.41 g/g and 1.33 g/（L-d）,respectively.The butanol CEF system was constructed based on the cultivation of C.beijerinckii NCIMB 8052.With-0.75 V cathodic potential,0.50 mmol/L electron mediator NR,20 g/L glucose,obtained butanol production and yield was 5.49 g/L and0.30 g/g（0.41 mol-C/mol-C）,which was 569.51%and 172.73%higher than that of OC system without NR,respectively.Suitable cathodic potential（-0.60 V）could effectively stimulate the synthesis of intracellular NADH,increase the NADH/NAD⁺level,and rapidly initiate the solvent-producing phase reaction,with less VFAs accumulation,resulted in enhanced butanol fermentation efficiency.The optimization of CEF system showed that,-0.60 V cathodic potential and 0.50 mmol/L methyl viologen（MV）effectively inhibited the hydrogenase activity of C.beijerinckii,reduced H₂ synthesis,and allowed more allocation of electron and carbon source to the butanol synthesis pathway,increasd the butanol production,yield and selectivity to 6.71 g/L,0.30 g/g and 74.75%.With main components of rice straw hydrolysate（glucose,xylose and cellobiose,mass percentages of about 6.23:2.97:1.00）as substrates,C.beijerinckii in the CEF system could efficiently absorb and utilize glucose cellobiose xylose for butanol conversion.When provided with rice straw hydrolysate（30 g/L glucose as a benchmark）as substrate,CEF system could achieved butanol production,yield and selectivity up to 8.53 g/L,0.33 g/g-sugar and 78.55%,respectively.To achieve the integration of butyrate CEF system and butanol CEF system based on substate transformation,the effect of butyrate fermentation broth components（butyrate,acetate and propionate）on butanol fermenation efficiencies of C.beijerinckii NCIMB 8052 was investigated.In comparison,the efficacy of exogenous butyrate was significantly better than acetate and propionate in enhancing butanol fermenation efficiency in the CEF system,and the optimal dose was 4 g/L.At the condition of-0.60 V cathodic potential,0.50 mmol/L MV,the butanol production,yield and selectivity of the CEF system were 13.66 g/L,0.64 mol-C/mol-C and 90.93%,respectively,when fed with 30 g/L glucose and 4 g/L butyrate as co-substrates;With diluted rice straw hydrolysate（30 g/L glucose as a benchmark）and butyrate fermentation broth（4 g/L butyrate as a benchmark）as co-substates,butanol production,yield and selectivity reached 13.89 g/L,0.60 mol-C/mol-C and 89.97%,respectively;With one-time supply of butyrate fermentation broth（to restore butyrate concentration to 4 g/L in the CEF system）during the fermentation process,although no singnificant changes in butanol yield and selectivity were observed,the butanol production could be significantly increased to 16.28 g/L.Moreover,butanol conversion yield and cellulose demand was 121.30 g-butanol/kg-rice straw and 93.87FPU/g-butanol in staged butyrate-butanol electro-fermentation process,which was12.36%higher and 4.31%lower than that of staged butyrate-butanol fermentation process without electrochemical catalysis.A staged butyrate-butanol electro-fermentation process was constructed based on conversion of substrate,the butanol fermentation efficiency was improved with less dosage of cellulose enzyme,and the cost of lignocellulosic butanol biorefinery was also significantly reduced.