| Butanol is the most promising biofuel,and with the gradual depletion of fossil fuels and the increasing amount of agricultural waste,the production of butanol from lignocellulosic biomass has received widespread attention from scholars both domestically and abroad.However,butanol-producing bacteria cannot directly use lignocellulosic biomass as a substrate.Therefore,it needs a large number of hydrolytic enzymes or acid-base reagents to hydrolyze lignocellulosic biomass in the traditional butanol production technology.This greatly increases the cost of butanol production and restricts the development and promotion of butanol production technology.The purpose of this research was to solve the problem of raw material pretreatment and develop more efficient butanol production technology.This study was based on the natural hydrolysis ability of rumen microbial systems to lignocellulosic biomass and proposes a stepwise fermentation route for butyric acid and butanol production.This route firstly used rumen microbes to ferment lignocellulosic biomass for producing a mixed carboxylic acid mainly composed of butyric acid,and then the mixed carboxylic acid was used as a substrate for ABE fermentation to produce butanol.Rumen microbes could directly use lignocellulosic biomass for acid fermentation.Thus,the pretreatment of lignocellulose was not required.This not only saved the use of hydrolytic enzyme preparations,but also greatly reduced the cost of butanol fermentation.This study provided a reference for the development of more economical and efficient butanol production technology.The main results of this study are as follows:（1）An anaerobic digestion system was established to produce butyric acid with rumen microorganism as inoculant and rice straw as substrate,and the production conditions of the system were optimized.The highest concentration and yield of butyric acid were achieved when the p H of the system was 5.5 and the loading was 6 gvs·（L·d）-1,and reached 74 mmol/L and15.79%,respectively,which were 13 and 5.35 times higher than those before optimization.Based on the analysis of carbon metabolism under optimal conditions,it was found that about36.9%of the carbon flow was directed towards the pathway of butyric acid production.It indicated that the system had a strong ability to produce butyric acid.After condition optimization,the total abundance of Rikenellaceae＿RC9＿gut＿group,Lachnospiraceae,and Prevotellaceae significantly increased from 3.78%to 25.39%.This suggested that a new microbial community relationship was formed after optimization,which was beneficial for enhancing the anaerobic acid production ability.（2）The conversion of acetic acid to butyric acid was achieved by adding exogenous electron donor to increase the process of carbon chain extension reaction in rumen anaerobic digestion system.The results showed that the highest conversion rate of acetic acid to butyric acid was achieved when 70 mmol/L ethanol and 60 mmol/L lactate（M3）were used as mixed electron donors,reaching 80.64%.The concentration and yield of butyric acid were the highest,reaching 178.14 mmol/L and 37.52%,respectively,which were 1.40 and 1.38 times higher than those in the control group.Butyric acid accounted for over 70%of the liquid products and became the main product.The activities of Butyryl Co A/Co A,butyrate kinase,and phosphobutyryltransferase were maintained at the highest level in M3 group.The abundance of common CE functional bacterial genera,such as Lachnospiraceae,Oscillospira,Eubacterium and Clostridium＿sensu＿stricto＿12,reached the maximum.In addition,compared with the control group,the relative abundance of key functional genes,such as ACCA2,HADH,ECHS1,and fad H,in group M3 increased by 95.8%,139.83%,163.85%,and 51.83%,respectively,which facilitated the expansion of the butyric acid metabolism pathway and strengthened butyric acid production.（3）A butanol fermentation system was established using rumen fermentation fluid as the substrate.The best butanol production was achieved when the concentration of butyric acid was80 mmol/L,the inoculation amount of the strain was 9%,and the dilution ratio was 50%.The butanol yield and B/A reached 103.68 mmol/L and 3.28,respectively,which were 1.8 times and1.66 times higher than the control group（traditional ABE fermentation using glucose and starch as substrates）,and the yield reached 35.64%.Through the analysis of the transcription levels of key enzyme genes before and after fermentation,the transcription levels of key enzyme encoding genes buk,adc related to butyric acid absorption and conversion,and ctfa and adh E encoding genes related to butanol generation were significantly upregulated,increased by 2.05to 6.45 times,which was conducived to promoting the absorption and conversion of butyric acid and the production of butanol. |
