Effects Of Biological Pretreatment Of Stover On Microbial And Methane Production In Anaerobic Digestion

China's stovers biogas project is facing some problems while it is developing rapidly,one of which is to improve stovers pretreatment technology.The characteristics of biological pretreatment are more suitable for the needs of ecological sustainable development.Microorganisms with the ability to degrade lignocellulose are continuously excavated for pretreatment to increase biogas production,and the mechanism of action of microorganisms in the pretreatment process is deeply studied.Although the functional mechanism of microorganisms in anaerobic digestion has been elucidated,the effects of biological pretreatment on microorganisms in anaerobic digestion remain to be further explored,which is exactly what this paper elaborated.A batch test was set up to optimize the pretreatment time to obtain a better effect of biological pretreatment.Five different pretreatment times were set for 2 d,4 d,6 d,8 d and 10 d respectively.The results show that the pretreatment time is significantly correlated with the degradation rate of lignocellulose and the accumulation of volatile fatty acids.The accumulation of acetic acid was the highest in pretreatment for 6 days,which was 971.5 mg/L.There was also a significant difference in daily biogas production and total biogas production by the test groups.Among them,the biogas production was the highest in the pretreatment for 6 days,and the biogas and methane production increased by 51.04 % and 43.09 %,respectively.The pretreatment 6d was selected as the treatment,and the control was still untreated.The sampling time is set at the peak and stable period of the most significant difference in daily biogas production.The DNA and RNA of the sample were extracted,and the diversity and species abundance of the microbial community structure and the active microbial community were obtained by high-throughput sequencing and based on the 16 S r RNA gene annotation.The highest proportion of abundance in the pretreatment period was bacteria,which was Ruminofilibacter(7.12 %).The three microorganisms with the highest abundance in the two periods that in untreated were the same as in the pretreatment.The active microbial community based on RNA showed that the microorganisms with the highest abundance in the peak periods was methanosaeta(10.82 %).Methanosarcina(5.21 %)was the most abundant methanogenic archaea in the untreated.The results of microbial function prediction showed that the relative abundance of the acetoclastic methanogenesispathway in pretreatment was significantly higher than that of untreated.Based on the KEGG database,the differences in microbial functional gene expression and metabolic pathways between the two samples were analyzed.Compared with the untreated samples,the number of Unigenes up-regulated in the pretreatment was 4323,and the number of down-regulation was only 1548.The relative abundances of methane metabolism and phosphorus oxide metabolism pathways in energy metabolism were 13.55 % and 4.16 %,respectively,which were significantly higher than 9.04 % and 3.01 % of untreated samples.In the methane metabolic pathway,the pretreated samples contained three unique KOs associated with the synthesis or transformation of Coenzyme F420,Acetyl-Co A,Malyl-Co A,and Glyoxylate,respectively.The KO unique to the untreated sample was associated with the conversion of formaldehyde and formate,but the other KOs of the pathway were not annotated.By annotating the relative expression of KO,it was found that there were 11 KOs with significantly up-regulated expression of pre-treated samples relative to untreated samples.These enzymes are involved in various pathways of methane metabolism,which play an important role in the initial stages of different methanogenic metabolic pathways such as acetic acid activation and transformation,CO2 conversion and methylation,among which mcr A is the key gene for the final step of methane conversion.Analysis of the above results shows that the continuous and stable operation of anaerobic digestion after pretreatment feed is derived from a relatively complex reaction system.Changes in substrate composition have a significant impact on the active microbial community,with Methanosaeta,which prefers this change,a dominant microorganism,stronger substrate capture capacity and methane conversion capacity,and a unique metabolic pathway that significantly enhances methane production.The microbial changes in the anaerobic digestion of real functional performers during biogas production were analyzed,and the relationship between this change and the significant increase in methane production.This facilitates the reverse optimization of the conditions of the biological pretreatment to achieve higher biogas production and methanogenic efficiency.