| Microorganisms drive important biogeochemical cycles and are closely related to the conversion and removal of various pollutants in the natural environment.Microorganisms in nature exist in the form of community,so exploring the microbial community structure and its corresponding functional groups in polluted environment is very important.It can not only enable us to know which microorganisms and functional genes are involved in the pollutant’s transformation process,but also understand the final trend of pollutants,providing theoretical basis for pollution control.The continuous development of omics technology and highthroughput sequencing technology have provided more possibilities for in-depth study of microbial community structure,function,and interaction at the community level.However,there are still many problems to be solved in studying different microbial groups.In this study,fungi and bacterial were selected as research objects.To solve the problems of non-standard primer evaluation process and inconsistent primer selection in fungal research,a comprehensive primer evaluation system was constructed,and the amplification characteristics of primers in different ITS regions were investigated.For the lack of functional gene database and unknow functional gene diversity in bacterial microbial communities,a new database of aromatic ringhydroxylating dioxygenase functional genes was established,the diversity of functional genes was analyzed and the existing primers were systematically evaluated.On this basis,the succession of diversity,functions,and interactions of the fungal and bacterial microbial communities under the stress of aromatic hydrocarbons,as well as the stability mechanism of the community structure and function were investigated.The interdomain interaction between fungi and bacteria and their role in the synergistic degradation of pollutants were further discussed.Main research contents are as follows:Three widely used or newly designed ITS region primers for fungi were systematically investigated by in silico simulation,two sets of mock communities and two sets of environmental samples.Results from in silico simulation showed that species composition of three primers was different.Through the even and staggered mock communities,it was found that the proportion of chimeras and mismatch sequences,and the species composition obtained among three primers was significantly different.Further,results from environmental samples showed that primers had a greater impact on community composition and diversity than experimental treatments.Among the three pairs of primers,5.8S-Fun/ITS4-Fun could capture fungal diversity more accurately.For environmental samples,only 5.8S-Fun/ITS4-Fun showed significant difference between control and treatment,indicating high sensitivity and specificity.This study revealed the need for various evaluation methods,and showed how primers determine the diversity and composition of observed fungal communities,which have important guiding significance for the evaluation and selection of primers in future studies.Activated sludge samples from wastewater treatment plant were collected and three groups of sequencing batch reactors were built.Benzene,naphthalene and both of benzene and naphthalene were added to examine the diversity,function,and interaction succession pattern of fungal communities under the continuous stress of aromatics,and to explore the changes of biodegradation function in community levels and inherent mechanism to maintain the stability of the community function.Results showed that the succession of diversity,functional characteristics and network properties were consistent under different aromatics stress.With the addition of aromatics,diversity decreased significantly,and the differences among communities increased with time.Community assembly process gradually changed from stochastic process to deterministic process.Relative abundances of some functional genera such as Exophiala,Candida,and Fusarium increased.Laccase,peroxidase,monooxygenase,and dioxygenase may play an important role in the biodegradation process and overall multifunctionality at the community level.Under the stress of aromatic hydrocarbons,the cooperative behavior within community members,and the more organized network structure together ensured the structural and functional stability of the fungal community in activated sludge.The protein sequences of aromatic ring-hydroxylating dioxygenase α subunit were collected from bacterial microbes and the seed sequences were obtained after quality control.A new database of aromatic ring-hydroxylating dioxygenase functional genes was established,the diversity of functional genes was assessed and the coverage of existing primers was evaluated.Results showed that 103 seed sequences were obtained,including 72 bacterial genera,34 genes and 17 substrates.The wide distribution of species and substrate types of seed sequences indicated a high diversity of dioxygenase α subunit.The number of highly level sequences in the constructed database was 6368,which was 61.8 times the number of original seed sequences.The database contained 407 different bacterial genera and 38 different substrates.There was a complex many-to-many relationship between the substrate type and the species present in the database.The same genus can degrade multiple aromatic compounds,and the same aromatic compounds can be degraded by multiple bacteria.The primers were evaluated according to the constructed database,and results showed that the coverage of the reported primers was low and the degeneracy was generally high.Future application of highthroughput sequencing technology to explore the degradation potential of aromatic compounds in various environments remains a great challenge.The diversity,function,and interaction of bacterial communities under the continuous stress of aromatic hydrocarbons were investigated,and the changes of the bacteria-fungi interdomain network were further explored to reveal the different roles of fungi and bacteria in the synergistic degradation of pollutants.Results showed that the diversity,function,and network property of bacterial community had a relatively consistent succession process under the stress of different aromatics,and the pattern was consistent with that of fungal community.Relative abundances of various degrading bacteria increased.Functional prediction analysis indicated that the abundance of aromatic degradation genes increased.Network analysis showed that although both fungi and bacteria maintained stable community structure and function,they had different internal mechanisms.For fungi,the increase of positive interactions and the cooperation between species played an important role in community stability.For bacteria,their tolerance to aromatic hydrocarbons was low,so the environmental filtering effect led to niche differentiation and further maintained the stability of the community.Results of the bacteria-fungi interdomain network showed that fungi played an increasingly role over time.Proteobacteria and Ascomycota were the most important taxa in bacteria and fungi,respectively,in maintaining structural and functional stability. |
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