The Key Biological Transformation Process From Aerobic To Anaerobic Sludge And The Function Of Simplified Methanogenesis System

The alternation of aerobic and anaerobic processes is a common process in nature and water treatment.Organic compounds are mineralized to carbon dioxide（CO₂）by aerobic bacteria,protozoa,and fungi under aerobic conditions,whereas in anoxic or anaerobic environments（e.g.,fermentation systems）,when CO₂ or other carbon-based compounds produced by fermentation act as the main terminal electron acceptor,a complex food network catalyzed jointly by different microbial metabolism is required to fully mineralize the organic compounds.For domestic wastewater with low organic load,it is usually treated by the aerobic activated sludge process to achieve clean wastewater.For wastewater with high organic load（pig manure wastewater/kitchen waste）,it is usually treated through anaerobic digestion,during which substances such as CO₂,acetic acid and methyl small molecules are used as the main terminal electron acceptors.Its advantage is that it can realize resource utilization and produce clean energy（methane）and the main actors are hydrolytic,acidifying bacteria and methanogens.The study of aerobic and anaerobic evolution is of great significance to elucidate the biogeochemical carbon morphologic transformation.However,the microbial communities and biological processes have undergone fundamental changes in aerobic and anaerobic processes,but the specific key links that have undergone changes are currently lacking in-depth studies.In addition,there are a variety of bacteria in the anaerobic methanogenic system,but the culturability of the anaerobic pure culture is less than 0.1%,and most of the anaerobic microorganisms belong to unknown functional bacteria and non-independent trophic bacteria.Metagenomics can provide an efficient way to analyze the function of unknown microbial communities,but most metagenomics-based studies have used actual raw materials such as faeces,wastewater,and organic waste,creating rich niches for extremely diverse microbial communities and complex metabolic activities.It is difficult to determine the relevance of specific microbes and their functions.Therefore,it is necessary to simplify the flora and function of methanogenic system to reveal the functional role of key species.In this study,aerobic sludge was cultured under anaerobic conditions and pig manure waste water was used as the natural substrate.Using physical and chemical properties determination,16S rRNA amplicon sequencing,and macro-transcriptome techniques,we analyzed the gas properties,microbial flora,and functional transitions during the critical transition from aerobic to anaerobic sludge and explained the key biological transition processes of the aerobic sludge to anaerobic system.After that,anaerobic cultivated sludge was used as seed sludge,and common substrates and intermediate metabolites in fermentation were used as single carbon sources to construct a simplified methanogenesis system.Combined with metagenomic sequencing,the simplified methanogenesis system of different single carbon sources were studied from the overall fermentation performance to the structure and function of microbial community.Finally,based on the simplified methanogenesis systems,the high-quality metagenomes assemble genomes（MAGs）were further assembled using the new method constructed in our laboratory,and the relationships between individual microorganisms and their functions in the methanogenesis system were revealed.The main conclusions and results obtained are as follows:（1）Through the study of the key biological transformation process from aerobic sludge to anaerobic system,it was found that it took about 20 days to form a completely anaerobic state from aerobic.On the sixth day,aerobic bacteria such as Arenimonas and Mycobacterium dramatically declined,resulting in a dramatic reduction in microbial diversity.At the same time,microorganisms in the system secreted large amounts of secondary metabolites,decomposed substrates,and produced large amounts of acetic acid and carbon dioxide,thus recruiting and enriching Methanosarcina with diverse substrate metabolism as new cooperators in anaerobic conditions.The stable methanogenesis of aerobic to anaerobic acclimation went through three key stages:first,the single methanogenic bacteria combat（d6-d24）,then the intersymbiosis of methanogenic bacteria and fatty acid oxidizing bacteria（d30）,and finally the efficiency of intersymbiosis was improved（d63）.The efficiency of methane production was low in individual combat stage,and the fatty acid oxidizing bacteria and Methanosarcina degrade a large number of fatty acids in the intercropping stage,and the methane concentration tended to be stable（d30）.On the 63rd day,the refractory substrate was degraded in large quantities,the gas production tended to stabilize,and the intersymbiosis efficiency improved.The methanogenesis process significantly enhanced the acetoclastic methanogenesis pathway（d24）,hydrogentrophic methanogenesis pathway（d30）and methylotrophic methanogenesis pathway（d63）in turn.The facultative bacteria rapidly metabolized organic matter at the initial stage and produced a large amount of acetic acid,resulting in a significantly enhanced methanogenesis pathway of acetoclastic at d24.At the same time as methane production,there was also a build-up of carbon dioxide,which subsequently resulted in a significantly enhanced methanogenesis pathway of hydrogentrophic at d30.With the progress of fermentation,the accumulation of difficult degradation substrates such as benzene resulted in significant enhancement of methylotrophic methanogenesis pathway（d63）,which may be related to the degradation of benzene substances into methyl substances.（2）By constructing a natural carbon source to a single carbon source simplified methanogenesis system,we revealed the differences in fermentation performance and function of different carbon sources in anaerobic digestion.The stability of fermentation system fed by the single carbon source was much lower than that of mixture.With the exception of proteins and amino acids that did not require artificial p H regulation,the rest of the fermentation without artificial regulation were difficult to maintain the long-term progress.Gas production rates were best in the mixed carbon source and protein systems,and methane concentrations were highest in the oil system.Butyric acid was the dominant mode of acidification in starch and glucose system,acetic acid was the main acidizing dominant mode of acidification in the protein,glutamic acid and oil system,and ethanol could convert into acetic acid and butyric acid in the fermentation system.The methanogenesis potential of different carbon sources varied greatly,but the carbon source had little effect on the methanogenesis pathway.The methanogenesis pathway was mainly based on the hybrid methanogenesis pathway of hydrogentrophic and acetoclastic.The methanogenesis potential was lipid>protein>starch;amino acid>glucose;butyric acid>propionate+valerate;acetic acid≈ethanol.Bacteria（Clostridium and g＿＿unclassified＿o＿＿Bacteroidales）and archaea（Methanosarcina and Methanothrixi）are widely distributed in different single substrate systems.The dominant bacteria in each system were significantly reduced and multiple functional bacteria were successfully enriched with substrates,i mproving the likelihood of obtaining genomic information of potentially unknown bacteria.（3）Based on the simplified methanogenesis system,a total of 425 metagenomic assembly genomes（MAGs）were reconstructed,of which 265 MAGs were unknown species.Among these unknown species,105 potentially high-quality unknown species that are difficult to isolate and culture have obtained more than 90%of their full genome information through metagenomic assembly,and 5have obtained their full genome complete information.A total of 16 MAGs with the acetoclastic methanogenic pathway,13 with the hydrogentrophic methanogenic pathway,31 with the methanogenic pathway of methanogenic methanol,and 4 with methyl methanogenic pathway were i dentified.In addition,the key steps of methanogenesis（carbohydrate hydrolysis,protein hydrolysis,li pohydrolysis,volatile fatty acid（VFA）oxidation,methanogenesis）were thoroughly analyzed,and th e complete anabolic network functional flora from the degradation of glycolipoprotein to m ethanogenesis was analyzed.In some MAGs,a new function was found.Currently,only M ethanothrix and Methanosarcina are acetoclastic methanogens.However,this study found that ba cteria C9＿bin（p＿＿Armatimonadota;c＿＿UBA5829）had a complete functional module of ac etoclastic pathway.Only one functional module（1 bm）was missed from archaea Methanobacterium,M ethanospirillum,Methanofollis and Methanoculleus,which provided the potential possibility that a va riety of bacteria can also produce methane with acetic acid.This study greatly expands our un derstanding of the individual functions of methanogenic microorganisms.In this study,16S rRNA amplicon sequencing and macrotranscriptome techniques were used to reveal the key biological transition processes from aerobic sludge to anaerobic acclimation system.A simplified methanogenesis system was constructed by switching the fermentation substrate from natural carbon sources to single carbon sources,and the differences in overall performance,microbial structure and function of different single carbon source fermentation systems were analyzed.Finally,a new method constructed in our laboratory was used to obtain high-quality assembled genomes that reveal the relevance of individual microorganisms and their functions in the single carbon source systems.The experimental results extended the understanding of the biogeochemical carbon cycle and provided effective fundamental data for the study of interactions between individual microorganisms and their environment using synthetic strains.