| Reed(Phragmites australis(Cav.)Trin.ex Steud.)is widely distributed worldwide and has large biomass.However,due to its high lignocellulose content,the utilization of biomass resources is greatly restricted.The fertilization process based on aerobic composting of reed straw and livestock manure has become an important way to utilize reed resources.The aerobic composting of reed and manure is mainly affected by the type of nitrogen-containing substrate(such as the type of livestock manure),the carbonnitrogen ratio of the substrate(such as the ratio of straw and manure)and additional inputs(such as biochar).How to shape the composition and function of microbial communities during aerobic compost,especially how to regulate the elimination process of antibiotic resistance genes(ARGs)carried in feces,remains unclear,has also become an important scientific question that needs to be answered to reveal the microbial regulation mechanism of aerobic composting and to improve the efficiency and quality of reed fertilization.In order to understand the changes of microbial community composition and the mechanism of ARGs removal during reed straw aerobic composting,outdoor large-scale reed composting experiments were established.Using a combination of active zymography detection technology,amplicon highthroughput sequencing technology and quantitative PCR technology,the effects of different nitrogen-rich substrate additions on lignocellulose-degrading enzymes,bacterial and fungal community composition during reed straw aerobic compost were evaluated.The interaction network of bacterial community and fungal community was constructed.and the key taxa affecting the stability of aerobic composting microbial network were revealed.At the same time,the study tracked the dynamic characteristics of ARGs and mobile genetic elements(MGEs)during reed straw aerobic composting with different substrates C/N,and the effects of environmental factors,microbial communities and MGEs on ARGS were analyzed.Based on the percpective that biochar addition has a great effect on the dynamics of bacterial communities,ARGs and MGEs during aerobic composting,the effect of biochar addition on bacterial community and the profiles of ARGs were investigated,and the rebound of ARGs at late thermophile stage and maturation stage was analyzed during aerobic composting of reed straw.We also evaluated the contribution of different factors to the change of ARGs,and revealed the mechanism of ARGs removal under different treatment conditions.The main conclusions are as follow:1.The activities of lignocellulose-degrading enzymes carried in the substrate were inhibited by high temperature,and the activity of high-temperature tolerant lignocellulose-degrading enzymes secreted by thermophilic microorganisms increased with the composting process.Compared with reed straw cow manure compost(RC)and reed straw mushroom residue compost(RM),the efficient expression of thermophilic lignocellulose-degrading enzymes in reed straw pig manure compost(RP)and reed straw chicken manure compost(RF)were later.The activities of lignocellulose-degrading enzymes in RC and RM were higher than that of RF and RP in early high temperature stage,while the composition of lignocellulose-degrading enzymes were stable at high temperature stage.2.Different nitrogen-rich substrate,substrate C/N and composting process can significantly change the bacterial and fungal community composting during aerobic composting of reed straw,while biochar cannot significantly change the bacterial community composition.During aerobic composting of reed straw,Bacteroidetes and Proteobacteria were the dominant bacterial phyla carried by the initial substrates.Firmicutes used available carbon sources to multiply rapidly and massively,and the resulting high temperature inhibited the initial dominant bacterial phyla.With the increase in the relative content of lignocellulose caused by the consumption of available carbon sources,the Actinobacteria with strong lignocellulose degradation ability increased.In the subsequent stage of maturation,the reduction of heat stress intensity promoted the relative content of Bacteroidetes and Proteobacteria to increase again.Ascomycota is always the dominant fungal phylum during aerobic composting.In terms of microbial genera,Thermopolyspora,Planifilum,Bacillus,Thermobifida and Thermobispora were the main bacterial genera,while Thermomyces,Mycothermus and Talaromyces were the main fungal genera,which have high lignocellulose degradation ability.3.During reed straw aerobic composting,bacteria and fungi mainly formed a complex inter-kingdom interaction network through positive interactions,which was greatly affected by the properties of substrate and rare species.Competition between kingdoms(bacteria-fungi)was stronger than competition within kingdoms(bacteriabacteria,fungi-fungi).Aerobic compost substrate contained more available carbon sources,which helped to maintain the stability of the microbial inter-kingdom interaction network.The complexity of microbial interkingdom network of RC and RM was smaller than that in RF and RP.In addition,key microbial taxa that maintain microbial transboundary interactions networks were rare species.4.Different composting substrate C/N and composting process could significantly change the profiles of ARGs,while biochar addition had no significant effect on ARGs during aerobic composting of reed straw.During aerobic composting of reed straw with cow manure,the removal of ARGs during the high temperature stage acted by inhibiting the antibiotic resistance gene host bacteria Advenella,Tepidimicrobium,Proteiniphilum,Acinetobacter,Pseudomonas,Flavobacteria and Arcbacter.During aerobic composting of reed straw with fowl manure and pig manure,the potential hosts of ARGs Caldicoprobacter,Tepidimicrobium,Pseudogracilibacillus and Ignatzschineria were inhibited by high temperature,resulting in the reduction of total relative abundenace of ARGs.The enrichment of potential hosts of ARGs,such as Actinomadura,Galbibacter,Flavobacterium,Vulgatibacter,Planifilum and Thermobispora during maturation stage resulted in the rebound of total relative abundance of ARGs.5.Bacterial community is a key driver affecting the profiles of ARGs during aerobic composting of reed straw,while fungal community,MGEs and environmental factors had relatively little influence.During aerobic compost of reed straw with cow manure,the change of bacterial community was the predominant reason affecting the profiles of ARGs,and the fungal community indirectly changed the composition of ARGs by affecting the bacterial community.During aerobic compost of reed straw with pig manure and fowl manure,the key factors affecting the profiles of ARGs was MGEs besides the bacterial community.The re-growth of initial microorganisms and the retention of thermophilic microorganisms were the potential reasons for the rebound of the total relative abundance of ARGs.In summary,during aerobic composting of reeds straw and livestock manure,the substrate nutrient status such as nitrogen-containing substrate type,substrate C/N ratio,and carbon composition were the dominant factors affecting the composition of microbial communities,lignocellulose degradation function and the profiles of ARGs,while the effect of exogenous biochar addition was small.Microbial communities and the profiles of ARGs varied greatly during aerobic composting.There is a strong interkingdom interaction network between bacteria and fungi,in which rare species play an important role.The dominant influence factor of ARGs is bacterial community dynamics,which are indirectly affected by fungal communities and environmental.This research reveals the quality of fertilization,characteristics of safe substrate composition and mechanisms of microbial regulation during aerobic composting of reed straw,helps to enrich the microbial ecology theory of plant straw composting,and provides a theoretical reference for the resource utilization of plants with high lignocellulose content. |
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