Microbial Communities And Functional Adaptation Mechanisms In High Temperature Compost And Low Temperature Plateau Investigated Based On Multi-omics Method

Extreme microorganisms and the extreme enzymes they produce have shown great potential in industrial applications.Exploring the physiology,biochemistry and omics characteristics of extremophiles in extreme environments is of great significance for exploring the scientific problems of "species evolution" and "limits of life",and also provides new research ideas for the development of extremophiles and extremophiles with special uses.The natural high-temperature environment represented by aerobic composting and the low-temperature environment represented by the soil of the Sanjiangyuan of the Qinghai-Tibet Plateau are important resource pools for screening thermophilic/cold microorganisms and thermophilic/cold enzymes.Therefore,based on the integrated multi-omics analysis technology,the changes of microbial community and function under the two opposite extremes of compost high-temperature environment and low-temperature environment on the Qinghai-Tibet Plateau were studied,and the pH of compost was changed by adding excipients in high-temperature composting environment,and structural proteomics was introduced to analyze the molecular structure adaptation mechanism of enzyme molecules involved in material transformation to environmental factor changes.The following results were achieved:1.By using the method of integrated multi-omics,the effects of high-nitrogen mushroom residue on the microbial community and function of natural corn straw cow manure compost were explored,and Planifilum fulgidum was determined to be the main functional microorganism in the thermophilic phase.The results showed that there was no significant change in the microbial community in the compost at the gate level after adding high-nitrogen mushroom residue to the natural corn straw cow manure compost raw material.Fungi were dominated by Ascomycetes,and bacteria were dominated by Firmicutes,Proteobacteria,Actinobacteria and Bacteroidetes.The hightemperature phase of composting promoted the succession of bacterial community structure from the predominance of gram-negative bacteria(Bacteroidetes,Proteobacteria)to the predominance of gram-positive bacteria(Firmicutes,Actinobacteria).The addition of mushroom residue with high nitrogen content accelerated the composting process,and the substrate was effectively degraded within 14 days.Macroproteomics results showed that the dominant microorganism P.fulgidum secreted a large number of S8,M17 and M32 family proteases,which can efficiently degrade macromolecular protein substrates after the addition of mushroom residue.The dominant thermophilic bacteria Thermobifida fusca and Melanocarpus albomyces,which were not affected by mushroom residue addition,synergistically degraded hemicellulose,cellulose,and proteins with P.fulgidum.2.The substrate degradation preference of the dominant functional microorganism P.fulgidum,was explored by integrated omics,and it was proved that the main activity of the bacteria was to degrade protein substrates.The study showed that the optimum optimum growth temperature of P.fulgidum is 60℃,indicating that it is a thermophilic bacterium.Genomic analysis found that the bacterium had a total of 88 protease genes and 19 carbohydrate active enzyme genes.When induced in culture containing a single nitrogen source,carbon source and natural substrate,it was found to grow rapidly on a single nitrogen source dominated by small protein molecules(yeast extract,soluble corn gluten powder,casein amino acids,etc.)and natural substrates(corn stover,wheat straw,bean husk,okara,etc.).Further mass spectrometry identified extracellular functional enzyme systems mainly including 33 proteases and 7 lignocellulose side chain degrading enzymes of xylan or glucosidases.Extracellular proteases mainly relied on two S8 family serine endopeptidases,one M28 metalloexopeptidase and S13 and S41A family carboxypeptidases to synergistically degrade proteins.Therefore,in natural compost habitats,P.fulgidum mainly secretes proteases to degrade small molecular weight proteins.3.The addition of sulfur powder lowered the pH value during the compost fermentation process,and the dominant bacteria for protein degradation were replaced by Planifilum by Novibacillus.The effects of different sulfur-containing compounds(S,(NH4)2SO4,chicken feathers)on microbial community succession and functional enzymes in natural maize straw manure compost were explored by a multi-omics technique integrating metagenomics,metaproteomics and structural proteomics.The results showed that the addition of sulfur powder induced sulfur oxidizing microorganisms with SOX system to oxidize S0 to S2O32-and further oxidized to SO42-,which reduced the pH of the sulfur powder group to 7.5 on day 7,about 1 lower than that of the control group.However,there was no significant change in pH of ammonium sulfate and chicken feather treatments.The decrease in pH in the sulfur powder addition group caused Planifilum(which mainly secretes S8,M17 and M32 family proteases)to lose its competitive advantage,while Novibacillus(which mainly secretes M14 and M19 family metalloproteinases)became the dominant bacterium.Structural proteomics showed that Novibacillus-secreted proteases had more negatively charged acidic amino acid residues on the surface(the total surface charge averages-39.46)than Planifilum,the high acidic amino acid content on the protein surface contributed to the stability of the protease at low pH.4.The effect of thiourea residue,a highly saline chemical waste,on microbial community composition in compost was explored,indicating that the addition of a certain amount of thiourea sludge to corn stover cow dung compost could achieve the cofermentation of compost.Thiourea residue is a solid waste in the process of thiourea processing and production,which is strongly alkaline and has a pH of 13 in the water extract.Thiourea residue was added to corn stover and cow manure compost at a mass ratio of 10%,the results showed that the addition of thiourea residue led to the thermophilic phase after 7 days,which was delayed by 4-5 days compared to the control,and the initial higher pH(13)and EC(6 ms/cm)gradually tended to the control group after entering the thermophilic phase,and the functional proteins were secreted,indicating that the compost with high-saline-alkali thiourea residue could achieve the physicochemical properties of ordinary compost through the biological process of composting.Through microbial community analysis,it was found that the addition of thiourea residue introduced more anaerobic salt-alkali-tolerant Firmicute bacteria such as Alkalibacterium and Sporosarcina,which were gradually replaced by thermophilic bacteria with high-temperature aerobic fermentation,and the dominant community Planifilum,Novibacillus,and Thermobifida tended to be the same as that of the control group.Structural proteomic analysis of extracellular functional enzymes annotated by the genome of initial saline-resistant bacteria Sporosarcina revealed a high total surface charge(-2.6),basic amino acid residues and hydrophobic amino acid surface area which may be the structural basis for enhancing the stability of enzymes in the early stage of fermentation in a high salinity.5.The microbial communities and carbon and nitrogen degradation functions in the low-temperature environment of the Sanjiangyuan area of the Qinghai-Tibetan Plateau were analyzed by metagenomics,and the degradation mechanism and limiting factors in low-temperature habitats were preliminarily elucidated.Sanjiangyuan Nature Reserve is located in the core area of the Qinghai-Tibet Plateau,with alpine swamps,meadows and grasslands as the main ecosystem types,and the diversity sequencing of bacteria and fungi in the Sanjiangyuan area of the Qinghai-Tibet Plateau,including native grasslands and their degraded and artificially restored ecosystems.The results showed that in the extreme environment of low temperature,low nitrogen and low oxygen on the plateau,Acidobacteria,Proteobacteria,Actinobacteria and Chloroflexi were the main microbial communities in the soil,and the influence of moisture content on the structure of soil microbial communities was greater than that of degradation and artificial restoration.Through metagenomic functional annotation analysis,it was found that plateau microorganisms lacked the functional enzyme system for comprehensive and efficient degradation of organic carbon and nitrogen and other biomolecules,and mainly relying on GH enzymes encoded by Solirubrobacter and Phycicoccus(GH3,GH74,GH109)of Actinobacteria,oxidative degrading enzymes(AA1,AA3,AA7)and esterase(CE4,CE7)degraded hemicellulose branch chains and their acetylation modifications,and the degradation of cellulose backbones mainly depended on the energy-saving mode of phosphorylase,so the short-term degradation of vegetation did not affect the basic community composition of soil microorganisms.The low plateau ecological biomass is closely related to the lack of nitrogen,and the abundance of Bacillus of Firmicutes with a more complete protease system(S8,S11,S26,M24)is lower in the Tibetan Plateau,which limits the mineralization of organic nitrogen to ammonium.In summary,this paper explored the composition of microbial communities and functional enzyme systems in the two extreme habitats of high-temperature compost and low-temperature plateau and the molecular mechanism of coping with environmental changes.The genome and proteome analysis located the dominant microorganisms and functional enzymes in the complex natural habitat.Structural omics was introduced to quantitatively analyze the surface properties of the enzyme molecular structure by changing the nutrients and pH key environmental factors of the compost raw materials,and clarified that the change of environmental factors selected the enzyme molecule with adaptive structure,and then made the microorganisms with secretase molecules become the dominant bacteria.This paper provided a research basis for the mining of extremophiles and their enzymes in extreme habitats for bioindustrial technology.