Microecological Effects Of Typical Inhibitors During Anaerobic Transformation

Biomass waste refers to the solid waste derived from animals and plants,which can be degraded by microorganisms,and it is a major component of domestic waste,municipal sludge and agricultural solid waste.According to biodegradability,biowaste can be divided into two types:easily biodegradable biowaste and refractory biowaste.Anaerobic digestion is a typical valorization of easily degradable biowaste,but this process is prone to accumulation of organic acids and ammoniacal nitrogen,namely acetate inhibition and ammonium inhibition.The valorization of refractory/non-biodegradable biowaste and synthetic waste（i.e.plastic and rubber）can be achieved by integrating thermochemical and anaerobic microbial conversion,but microbes can be inhibited by CO during this process.Therefore,clarifying the metabolic response and regulation of anaerobic microbes to the above inhibitors is essential for the technical optimization of valorization.Available researches on anaerobic digestion inhibition focus on the variation of physicochemical parameters and the succession of the overall community structure under inhibitory conditions.For example,a large number of studies have found that Methanosarcina is a dominant methanogenic genus under inhibitory conditions.However,few studies have demonstrated that how anaerobic microbes regulate their metabolism to adapt to inhibitory conditions,especially methanogens,which are the most vulnerable.To explore the metabolic regulation of dominant methanogens under inhibitory conditions,the dominant multi-trophic Methanosarcina barkeri was used in this paper.Acetate inhibition was built where acetate concentrations were 10,25 and 50m M,and ammonium inhibition was also built where total ammoniacal nitrogen concentrations were 1,4 and 7 g-N/L.Pure cultivation,microscope,conventional transcriptomics,strand-specific transcriptomics,isobaric tag for relative and absolute quantitation（i TRAQ）proteomics were employed to explore the regulation trends of cell proliferation,stress response,quorum sensing,element transmembrane transport,methanogenesis and nitrogen fixation of the M.barkeri under inhibitory conditions.The main conclusions are as follows:1)Under both acetate and ammonium inhibition,with the strength of inhibition increased,the multicellular aggregates of M.barkeri gradually shrank and disaggregated into free single cells,the cell morphology also changed from plump sphericity to irregular.Meanwhile,quorum sensing system was limited by insufficient energy conservation.These changes counteracted the advantages of multicellular aggregates in protecting inside cells and shortening the distance of signaling molecules.However,50 m M acetate and 7 g-N/L ammonium conditions would accelerate the adaptation of M.barkeri to inhibition.These findings suggest that M.barkeri can be pre-stimulated with high inhibitory concentrations and the immobilization of M.barkeri can be performed using polyvinyl alcohol,thereby rapidly enhancing the resistance of M.barkeri.The above processes can be used as effective pretreatments for the bioaugmentation using M.barkeri.2)In the initial stages of acetate inhibition and ammonium inhibition,with the increase of inhibition intensity,M.barkeri enhanced the synthesis of reduced nitrogen sources,glutamine and glutamate,thereby maintaining the balance of intracellular p H and osmotic pressure,and ensuring the normal progress of basic physiological activities such as amino acid synthesis.Meanwhile,the transport of Fe²⁺was also enhanced.Additionally,M.barkeri also significantly enhanced the uptake of K⁺in the initial stage of ammonium inhibition to maintain osmotic balance.These findings suggest that the biological regulation strategy by adding reduced nitrogen sources or elements can be used to ensure the normal physiological activities,apart from the traditional regulation strategies by stripping,diluting materials and adding carbon-based materials.The studies about anaerobic digestion inhibition focus on complicated mixed microbial cultures,but the available researches on CO conversion and inhibition mostly concern pure cultivation or the mixed microbial cultures with simpler structures.Due to the lack of the researches on unknown carboxydotrophy and their interaction in a complicated mixed microbial culture,the inhibitory conditions with initial CO partial pressures of 35 and 95 k Pa were built.Artificial stable isotope tracing technology,stable isotope-labeled metabolite analysis,16S r RNA gene sequencing and metagenomics were employed to compare the product characteristics of the mixed carboxydotrophic cultures under different CO partial pressures,and to reveal the community structure and the ecological niche in the metabolic flux of CO bioconversion.The main conclusions are as follows:1)Without the addition of methanogenic inhibitors,more CO can be converted into short-and medium-chain carboxylate by maintaining the initial CO partial pressure as 95 k Pa.The functional potential analysis also confirmed the above findings.Compared with 35 k Pa CO,the mixed cultures under 95 k Pa CO had higher potential in butyrate metabolism,propionate metabolism and fatty acid synthesis,which were beneficial to the synthesis of the carboxylate with longer carbon chains.In this study,the variation of physicochemical indicators was analyzed from the dimension of functional potential analysis,providing the basis for the selection of CO partial pressure and analyzing the evolution of microbial cultures and metabolic functions to adapt to waste-derived syngas composition.2)The appearance of novel Rhodoplanes sp.broadens the phylogenetic diversity of mesophilic carboxydotrophic hydrogenogens,highlighting the previously overlooked anaerobic CO-oxidizing capacity of Proteobacteria.The novel Desulfitobacterium＿A sp.indicates that mesophilic carboxydotrophs also have hydrogenesis and acid synthesis potential simultaneously.From the perspective of genome,the appearance of this Desulfitobacterium＿A sp.proved that a single species can independently complete carboxydotrophic acid synthesis and subsequent carbon chain elongation.The emergence of several novel Propionicimonas spp.reveals the important role of Propionicimonas in carboxydotrophic propionate biosynthesis.The novel Methanobacterium＿A sp.and Methanobacterium＿C sp.not only have potential for carboxydotrophic methanogenesis,but may also participate in acetate synthesis.Genome reconstruction analysis reveals that most of the microbes participating in the metabolism of CO or CO-derived products have multiple metabolic potentials.These findings label the important taxonomy that lacks attention for subsequent CO bioconversion researches,and also provide a basis for species selection and combination for defined mixed cultures,ensuring functional redundancy and improving conversion efficiency.Furthermore,these findings provide an optimized direction for the development of one-carbon gas sequestration technology to improve its substrate utilization and conversion efficiency.