Partnership of Arthrobacter and Pimelobacter in Aerobic Degradation

Environ. Sci. Technol. , Article ASAP. DOI: 10.1021/acs.est.7b05913. Publication Date (Web): January 29, 2018. Copyright © 2018 American Chemical Soc...
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Partnership of Arthrobacter and Pimelobacter in aerobic degradation of sulfadiazine revealed by metagenomics analysis and isolation Yu Deng, Yulin Wang, Yanping Mao, and Tong Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05913 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018

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Partnership of Arthrobacter and Pimelobacter in

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aerobic degradation of sulfadiazine revealed by

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metagenomics analysis and isolation

5 Yu Deng1, Yulin Wang1, Yanping Mao1,2 and Tong Zhang1,3

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1

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University of Hong Kong, Pokfulam Road, Hong Kong, China

Environmental Biotechnology Laboratory, Department of Civil Engineering, The

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2

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Shenzhen, China

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3

13

University of Science and Technology, Shenzhen, China

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Corresponding author, +852-28578551; fax:+852-25595337; [email protected] and/or [email protected]

College of Chemistry and Environmental Engineering, Shenzhen University,

International Center for Antibiotics Resistance in the Environments, Southern

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ABSTRACT

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In this study, metagenomic analyses were combined with cultivation-based techniques

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as a nested approach to identify functionally significant bacteria for sulfadiazine

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biodegradation within enrichment communities. The metagenomic investigations

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indicated that our previously isolated sulfadiazine degrader, Arthrobacter sp. D2, and

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another Pimelobacter bacterium concomitantly occurred as most abundant members

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in the communities of an enrichment culture that performed complete sulfadiazine

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mineralization for over two years. Responses of the enriched populations to sole

25

carbon source alternation further suggested the ability of this Pimelobacter member to

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grow on 2-aminopyrimidine, the most prominent intermediate metabolite of

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sulfadiazine. Taking advantage of this propensity, additional cultivation procedures

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have enabled the successful isolation of Pimelobacter sp. LG209, whose genomic

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sequences exactly matched that of the dominant Pimelobacter bacterium in the

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sulfadiazine enrichment culture. Integration of metagenomic investigations with the

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physiological characteristics of the isolates conclusively demonstrated that the

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sulfadiazine mineralization in a long-running enrichment culture was prominently

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mediated by primary sulfadiazine-degrading specialist strain Arthrobacter sp. D2 in

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association with the 2-aminopyrimidine-degrading partner strain Pimelobacter sp.

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LG209. Here, we provided the first mechanistic insight into microbial interactions in

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steady sulfadiazine mineralization processes, which will help develop appropriate

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bioremediation strategies for sulfadiazine-contaminated hotspot sites.

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INTRODUCTION

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Sulfonamides were one of the most commonly purchased antibiotics on the market,

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mainly for the intensification of food animal production in agriculture1. Survey data

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reported that sulfonamides accounted for about 11 to 23 percent of the annual sales of

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veterinary antibiotics in 25 European countries in 20112, 3. Sulfonamides excreted

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from animals or human were generally insufficiently treated by wastewater treatment

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plants4-7, and sulfonamide residues were ubiquitously found in various environmental

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compartments (e.g. surface water8-10, soil11-13 and groundwater14-17) in ng/L to µg/L

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concentrations. The selection pressures due to the presence of sulfonamide antibiotics

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in the open environment may promote the dissemination of sulfonamide resistant

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genes in clinically relevant pathogens18, and potentially threaten the therapy

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effectiveness of sulfonamides. The implementation of sulfonamide degraders in

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sulfonamide-contaminated hotspot sites (e.g. discharge points for pharmaceutical

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wastewater, livestock farm wastewater and hospital wastewater) through biological

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wastewater treatment is a pragmatic remediation method. This method can combat

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sulfonamide resistance by directly reducing sulfonamide concentrations in the

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pollution sources, and thus mitigate sulfonamide exposure level to the pathogenic

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microorganisms. Since these sulfonamide degraders are also sulfonamide-resistant

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microorganisms, to avoid their distribution from the biological treatment reactors

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(such as activated sludge reactors) to the open environment, treatment should be

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carried out within enclosed engineered systems instead of natural environment which

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is more difficult to control. In addition, post-control strategies like membrane

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separation of biologically treated effluent coupled with incineration of the biomass

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sludge should be applied.

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Preliminary attempts to identify the key degraders within sulfonamide-degrading

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consortia have seen variable selections on disparate species19-23. This could mainly

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due to perturbations imposed by different carbon source amendments (e.g. sodium

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acetate or yeast extract) during sulfonamide exposures. Enrichment culturing

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strategies featuring sulfonamide as the sole carbon source should facilitate more

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explicit interpretations of the community responses that were actually driven by

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sulfonamide catabolic activities. Particularly, a few studies24, 25 examined the overall

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microbial populations that were capable of subsisting on sulfonamides, only using

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16S rRNA-based molecular methods. The function of individual microorganisms and

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ecology of the community, however, are difficult to be fully understood and

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characterized just based on phylogenetic classifications.

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Cultivation-based techniques have already taken a prominent leap forward through

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efforts in isolation of the sulfonamide-degrading specialist bacteria. A diverse array of

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bacterial isolates in the phyla Proteobacteria26-30 and Actinobacteria31-33 have been

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demonstrated the capabilities to grow on different classes of sulfonamides. Despite

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the sulfonamide catabolic efficiency measured in particular single isolate were

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promising

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sulfonamide-degrading bacteria in near-field conditions failed to increase the

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sulfonamide biodegradation rates34, 35, most likely owing to the poor survival or low

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activity of the inoculated specialists in the natural environment. Improved

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understanding on how the key degraders establish and maintain their memberships

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with other participating bacteria during the biodegradation processes can aid further in

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sufficiently exploiting the catabolic activities of the sulfonamide-degrading isolates.

under

microcosm-scale

laboratory

experiments,

addition

of

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Driven by the hypothesis that structurally similar sulfonamides being sole carbon

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source would lead to the enrichment of similar key degraders possessing prevalent

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sulfonamide degradation strategy (such as ipso-hydroxylation coupling subsequent

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fragmentation of the mother molecule36), we established the enrichment cultures of

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two frequently encountered sulfonamides (sulfadiazine (SDZ) and sulfamethoxazole

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(SMX)). Metagenomic approaches can bypass the cultivation needs, and have been

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demonstrated highly informative in profiling microbial structures of natural37, 38 and

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engineered39,

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communities, two metagenomic analysis strategies, i.e. the reconstructed 16S rRNA

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gene analyses and binned genome analyses, were used to catalog species-level

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diversities of these two long-running sulfonamide enrichment cultures. In addition,

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our previous study characterized the SDZ enrichment community at day 285 with 16S

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rRNA PCR cloning approach, and isolated two Arthrobacter strains, with strain D2

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exhibiting distinct sulfadiazine degradation abilities41. Though the SDZ degradation

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processes by isolate D2 have been extensively documented in our previous study, its

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role in the ecological network of microbial community of SDZ enrichment culture has

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yet to be elucidated. Here, the SDZ enrichment culture was specifically used as a

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model community to illustrate how metagenomic analyses can be incorporated with

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pure culture physiological characterizations to reveal the roles of the isolated degrader,

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and to understand its interactions with other community members in the SDZ

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biodegradation process.

40

microbiomes. With an aim to compare these two microbial

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MATERIAL AND METHODS

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Culture enrichment and metagenomic sequencing and assembly

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Two sulfonamide-degrading cultures were inoculated with the same activated sludge

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sampled from a local sewage treatment plant (Shek Wu Hui Sewage Treatment Work,

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Hong Kong). These two cultures were maintained in 500 mL serum bottles, in a

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semi-batch mode, with mineral salts medium (MSM)41 containing SDZ or SMX as

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sole carbon source for total 812 days (Figure S1). To select 2-aminopyrimidine (2-AP,

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the most prominent intermediate during SDZ mineralization by SDZ enrichment

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culture) degraders, we therefore established the 2-AP enrichment seeded with

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subculture of SDZ enrichment (at day 767). 20 mL the SDZ enrichment cultures were

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transferred into 180 mL fresh MSM supplied with 100 ppm SDZ and 100 ppm 2-AP

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(SDZ+2-AP enrichment). After three draw-and-fill cycles (a total of 23 days), the

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carbon source was changed to sole 2-AP with 200 ppm concentration, and was

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maintained for another three cycles. At the sampling time (SDZ enrichment at day 812,

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SMX enrichment at day 812, SDZ+2-AP enrichment at day 23, and 2-AP enrichment

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at day 45), approximately 100 mL of enrichment liquid were taken, and were

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subsequently subjected to DNA extraction using FastDNA SPIN Kit for Soil (MP

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Biomedicals, Solon, OH). Four genomic DNA samples (SDZ enrichment, SMX

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enrichment, SDZ+2-AP enrichment, and 2-AP enrichment) were sent to BGI

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(Shenzhen, China) for paired-end library construction (insert size of 350 bp) and

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sequencing via Illumina HiSeq-2500 platform. The yield 150 bp paired-end reads

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were filtered and assembled independently with CLC Genomics Workbench software

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(CLC bio, Denmark). Only the contigs with length over than 500 bp were included for

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downstream analyses.

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Community profiling and detection of sulfonamide resistance genes

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Initially, MetaPhlAn2 (v.2.6.0) was applied to assess the overall taxonomic

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compositions of the enrichment communities by mapping total raw reads to around 1

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million taxonomy-specific marker genes42. Since a majority of the clean reads were

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assembled into contigs, we also used the protein-coding sequences located on the

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contigs to further characterize the microbial community compositions. All assembled

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contigs were predicted into open reading frames (ORFs) by Prodigal43, and were

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subjected to homologous screening against the proteins in the NCBI-RefSeq database

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using RAPSearch244 (E-value ﹤10-5). Taxonomy of each ORF was assigned by

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MEGAN (v 5.10.3)45 using the default parameters of the lowest common ancestor

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algorithm. Reconstruction of the near full-length 16S rRNA gene sequences from

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metagenomic datasets were performed by EMIRGE algorithm46 with 80 iterations.

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DECIPHER47 was used to detect the chimera among EMIRGE reconstructed

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sequences. Phylogenetic classifications of the resulting sequences were determined by

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two approaches: taxonomic assignments of SILVA (SINA online)48 with 95%

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minimum identity and 10 neighbors per query, and online nucleotide BLAST against

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NCBI nr database for query sequences that were unclassified by SILVA database. The

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relative abundance of annotated query sequences was derived from EMIRGE

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algorithm on the basis of prior probabilities of the read coverage. For

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EMIRGE-reconstructed 16S rRNA genes, similarity percentage analyses (SIMPER)

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was used to identify taxa contributing to dissimilarities among two enrichment groups

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through Bray–Curtis distances. The detection of sulfonamide resistance genes in the

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raw datasets of enrichment cultures was performed via an online detection pipeline for

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antibiotic resistance genes (available at http://smile.hku.hk/SARGs)49.

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Population genome binning, annotation and analysis

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All four metagenomes were subjected to co-assembly for binning of population

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genomes. Following the reported mmgenome workflow50, population genomes were

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reconstructed based on the differences in GC content, tetranucleotide frequencies and

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sample coverage. Genome quality assessment was calculated with CheckM (v1.0.5)51.

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Binned genome sequences were submitted to IMG/ER52 for annotation and data

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sharing (see Table S1 for accession numbers). The 16S rRNA genes presented in the

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genome were identified through RNAmmer53. Genome phylogeny was evaluated by a

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genome tree which was constructed by a concatenated set of 16 benchmarked

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ribosomal proteins54 and visualized in iTOL55. The 2563 reference genomes included

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in the genome tree were downloaded from public datasets including NCBI, IMG/ER

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and ggKbase.

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Pure culture isolation and batch experiments

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Dilutions of small volume of the 2-AP enrichment cultures were spread on mineral

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salts agar plates supplemented with 200 ppm 2-AP as sole carbon source. After one

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week, single colonies grown on the plate corresponding to the most diluted fraction

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were picked, followed by restreaking of selected single colonies on corresponding

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plates, and the purification procedures were repeated for five times. 16S rRNA genes

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in the purified colonies were amplified by polymerase chain reaction (PCR) using the

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universal primes 27F and 1492R, and were sent to BGI (Shenzhen, China) for Sanger

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sequencing. The extraction and sequencing of the genomic sample of the identified

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strain LG209 were performed as the same as the metagenomic samples. Assembled

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draft genome sequences of strain LG209 were deposited in GenBank under the

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accession numbers NHTS00000000.

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The activity of the new strain LG209 on SDZ and 2-AP were tested in 100-ml serum

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bottles in duplicates under three different nutrient conditions: (1) MSM supplemented

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with 200 ppm SDZ/2-AP; (2) MSM supplemented with 200 ppm SDZ or 2-AP

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together with 2000 ppm glucose; (3) LB medium amended with 50 ppm SDZ or 2-AP.

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Liquids were taken at specified intervals. The dissipation of mother compound and

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generation of metabolites were measured by ultra-performance liquid chromatograph

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coupling double mass detector (UPLC-MS/MS) using the detection methods we

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reported before41.

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RESULTS AND DISCUSSION

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Enrichment of SDZ-degrading consortia

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The SDZ-degrading culture examined here was derived from the same activated

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sludge sampled from a local municipal wastewater treatment plant (Shek Wu Hui

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Sewage Treatment Work, Hong Kong). Consecutive transfers of portions (10-20%) of

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these cultures were performed over 2 years in MSM supplemented with SDZ as the

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sole carbon source in incrementally increasing concentrations (50, 100 and 200 mg/L).

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SDZ degradation activity can be recovered repeatedly from the dilutions of these

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enrichment cultures, as indicated by the periodically stable reduction of the total

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organic carbon (TOC) in the medium (Figure S1), suggesting sufficient enrichment of

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SDZ-degrading consortia. In order to address the roles of uncharacterized or

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uncultivated microorganisms presented in the long-running enrichment cultures,

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biomass from SDZ enrichment at day 812 were sampled for metagenomic analyses.

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At the sampling point, SDZ biodegradation occurred at respective degradation rate of

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0.12 mgL-1h-1 (half-life time was 5.84 h, Figure S2). Meanwhile the corresponding

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TOC reduction rate was 92% for SDZ enrichment culture, respectively, achieving

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almost complete mineralization.

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To understand the microbial interactions underpinning sulfonamide biodegradation

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processes, we further detected important intermediates during SDZ degradation by

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SDZ enrichment culture using UPLC-MS/MS as we previously reported41. Consistent

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production of 2-aminopyrimidine (2-AP) was observed as the most prominent

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intermediate before complete TOC reduction in SDZ enrichment culture. It is notable

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that a majority of the sulfonamide-degrading isolates have been shown their limited

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abilities to the degradation of the heterocyclic moiety of sulfonamides27, 28, 31, 32, only

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a part of the organic carbon derived from sulfonamides can be mineralized by these

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isolates to CO2 or biomass. Thus, the nearly complete SDZ mineralization observed in

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the enrichment cultures were presumably mediated either by primary SDZ degraders

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in association with 2-AP-degrading partner populations, or, contrastingly, by single

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microorganism that occupy the ecological niches required for complete SDZ

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mineralization. To track the abundance changes of microbial populations in response

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to sole carbon source alternation, metagenomic samples from this enrichment were

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collected at two time points (Day 23, SDZ+2-AP enrichment; Day 45, 2-AP

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enrichment) when substantial TOC reductions were demonstrated (Figure S1).

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Microbial community overview

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Four metagenomic datasets (28.7 Gb total raw reads) were individually assembled and

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annotated for taxonomic profiling. On average, 90.2% of the raw reads from each

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metagenome could be mapped onto the contigs from the assembly, indicating the

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efficient usage of the raw reads. The microbial communities of four enrichments were

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primarily evaluated by read-based taxonomic assignments via MetaPhlAn2 and

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phylogenic classifications of total functional proteins via MEGAN. Both approaches

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identified bacteria as the dominating microorganisms across all samples, with little

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eukaryotic (< 5.2%) and archaeal (< 0.1%) detected (Table S2). Representative

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bacterial populations were further determined by EMIRGE-reconstructed 16S rRNA

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gene sequences. In addition, we also checked the sulfonamide resistance gene

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abundance in the raw datasets of SDZ enrichment cultures. The sulfonamide

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resistance gene abundance were found as 1.1 copy per cell in SDZ enrichment

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cultures, largely increased during the enrichment process compared with the level in

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the seed sludge (0.05 copy per cell).

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Not surprisingly, the SDZ enrichment community showed high similarity to another

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sulfonamide enrichment community (SMX enrichment), at genus level, shared taxa by

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these two communities comprised over 90% of the total communities (Figure 1a).

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Specifically, the relative abundance of EMIRGE-reconstructed 16S rRNA genes

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revealed that Actinobacteria was by far the most abundant phylum (80.9%) in the

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community of SDZ enrichment (Table S3), among which the genera Arthrobacter

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(59.9%) and Pimelobacter (18.2%) were notably dominant (Figure 1b). The other

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genera (including Dokdonella, Hydrogenophaga, Achromobacter and Leadbetterella,

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Figure 1c) constituted a small fraction of the shared taxa between SDZ enrichments,

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accounting for 15.1% in the SDZ enrichment community.

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On the other hand, occurring as microbial responses to sole carbon source alternation,

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evidently differentiated community structures were observed in SDZ and 2-AP

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enrichment cultures, though the inoculum for the 2-AP enrichment was derived from

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the SDZ enrichment. The Pimelobacter similarly dominated in the 2-AP enrichment

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community (23.3%) and the relative abundance of Arthrobacter in the 2-AP

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enrichment quickly declined from 32.7% (day 23) to 0 (day 45) over the period of 22

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days. To identify the contribution of certain taxa to the differences between these two

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communities, a similarity percentage (SIMPER) analysis56 was performed (Table S4

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and S5). Intriguingly, the observed compositional variations between SDZ and 2-AP

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enrichments were primarily driven by the changes of Arthrobacter populations in the

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communities (> 44% of variation at genus level). The absence of Arthrobacter

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populations in the 2-AP enrichment may result from its inability to use 2-AP as

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carbon source.

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Arthrobacter and Pimelobacter populations in the SDZ-degrading consortia

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16S rRNA genes may not allow enough resolution at genome level for the analyses of

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functional roles of individual members in the consortia. To investigate the roles of

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individual members in SDZ biodegradation, genome-centric exploration of the

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microbial communities were further performed for metagenomes of SDZ enrichment,

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SDZ+2-AP enrichment, and 2-AP enrichment. The three metagenomic datasets were

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thus subjected to combined assembly and differential coverage-based binning,

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resulting in the recovery of 23 bacterial population genomes (Table S1 and Figure S3)

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that were nearly complete (72%~98% completeness and 0%~8% contaminations).

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According to the percentage of reads mapping39, these 23 draft genomes accounted

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for the majority of the microbial communities (74.7%, 61.9%, and 56.2% for SDZ,

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SDZ-2-AP, and 2-AP datasets, respectively). A genome tree, inferred from 16

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previously benchmarked ribosomal proteins54, showed that the recovered 23

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population genomes represented organisms of the phyla Proteobacteria (nine

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genomes),

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Armatimonadetes (one) and Deinococcus-Thermus (one) (Figure 2 and Table S6).

Bacteroidetes

(six),

Actinobacteria

(four),

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Acidobacteria

(two),

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Similar to EMIRGE 16S rRNA gene analyses, Actinobacteria genomes constituted

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the most abundant fraction in SDZ-degrading consortia. Specifically, Pimelobacter

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bin_SDZ1 and Arthrobacter bin_SDZ2 were estimated to be the two highest

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abundance genomes based on average coverage of the binned contigs57. These two

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organisms totaled to respective 52% relative abundance in the SDZ enrichment

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communities (Figure 3a), highly enriched from the seed sludge, in which the relative

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abundance for Pimelobacter bin_SDZ1 and Arthrobacter bin_SDZ2 were only 0%

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and 0.02%. The organism Pimelobacter bin_SDZ1 had the type strain Pimelobacter

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simplex ATCC 694658 as the closest relative. Notably, the genome tree places

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Arthrobacter bin_SDZ2 genome in the same genus-level lineage with our previously

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isolated SDZ-degrading Arthrobacter sp. D2. Arthrobacter bin SDZ2 had 100%

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average amino acid (AAI) and nucleotide acid identities (ANI)59, to the strain

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Arthrobacter sp. D2, indicating they were the same strain. Also included in

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SDZ-degrading communities were low abundance ( 5% relative abundance in both

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samples), rare shared (present at < 5% relative abundance in both samples)

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or unshared.

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nob ac

teria

A Ar cti th no ro ba ba c ct ter er ia sp bin D4 SD Z

2

Acti

Ar ch ae a

p_ Fi rm ic ut es

p_ Te ne ric ut

p_ Prote Ca. Mup roteo obact Prote ba o e s_Ach bacteria ria bin S cteria b romob D acterin SDZ22 Z5 xylo

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ns g_Bord etella

0 D2 1 p YN rs K te r A c e ba ct ro ba th ro ) Ar rth yla ph A 0 irae ) (10 yla la C1 ph .N rosp .Phy 4 a ( C it Ca yla ia p_ p_N p_ Ph acter . 0 ) Ca dab DZ2 hyla p_ in S SDZ16 .Da la (2 p ia b Ca p_ a.Phy acter ria bin b p_C cido obacte A cid A bacter aceae ri ri a.Ko obacte C _ g Acid f_ bia micro si lu tes p_ E eneden Hydrog p_Ca.

p_Ca.Ph yla (2 ph yla) p_Ca.R aymon p_Ca dbacte . Zix ria ibacte ria p_C a p_C .Phyla (3 p hloro h y bi la) f_S apr p_ osp irac Bacte roid eae Ba ete s g_ B cte Baacte roideg_ N Chitin r ia ct o te o s f_ Cy f_C eroididete s bin tella phag a Ba top yto ete s b SD s b in S Z21 f_ cte hag pha in DZ Cy ro g a SD 18 to ide les ales Z1 ph te 2 ag s b in ac SD g _ ea Z1 g_ So e 1 Sp lit hi ale ng a ob ac te riu m

7 Z1 SD

ria te ac ob te Po

o_ En te

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te r ia le

s

n bi s r te te de ac oi b er do c t Pe Ba g_

es onadal osom o_Nitr s llales riale llione _Neisse a G _ ales o o phil thylo e s M le o_ la acil Z6 hiob SD 4 cidit bin SDZ ia o_A r te ia bin e c a r ea b ia ac teo acte tia ter Pro teob uran onad ac ria ob cte Prouria a hom e t ba ro nt te o p a a ria ales r e a t F te f_X s_ mm apro ac ich les Ga ob iotr ella m c_ te m h a o r T on G ap o_ egi c_ m L am o_ G c_

756 757 758 759

Figure 2 Phylogenetic placements of 23 population genomes in relative to 2563 genomes using 16 concatenated ribosomal proteins.

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a

Pimelobacter bin_SDZ1

b

Frateuria bin_SDZ4 Achromobacter bin_SDZ5 Frateuria bin_SDZ6 Nitrobacter bin_SDZ7 Fimbriimonas bin_SDZ8

Truepera bin_SDZ10 Cytophagaceae bin_SDZ11 Chitinophagaceae bin_SDZ12 Rhodobacter bin_SDZ13 Nitrobacter bin_SDZ14 Bradyrhizobiaceae bin_SDZ15 Acidobacteriaceae bin_SDZ16 Pedobacter bin_SDZ17 Chitinophagaceae bin_SDZ18 Micrococcaceae bin_SDZ19 Acidobacteriaceae bin_SDZ20 SDZ enrichment bin 2-AP enrichment bin

Chitinophagaceae bin_SDZ21 Achromobacter bin_SDZ22 Micrococcaceae bin_SDZ23

0

762

10 20 Relative abundance (%)

−20 −10 0 10 Difference in relative abundance (%)

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Figure 3 (a) Relative abundance of recovered population genomes in SDZ and 2-AP enrichment communities. Relative abundance was estimated based on

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average coverage of the binned contigs. (b) Difference in genome relative

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abundance between SDZ and 2-AP enrichment communities. Three most

768 769

significant contributors to the abundance difference were identified by SIMPER analysis, and were highlighted with in red (overrepresented in

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SDZ enrichment) or green (overrepresented in 2-AP enrichment).

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Figure 4 (a) Degradation and mineralization of SDZ by strain D2 alone (OD595 for D2 was 0.31±0.02) and the co-culture of strain D2 and LG209 (OD595 for

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D2 and LG209 were 0.32±0.01 and 0.29±0.01, respectively). (b) Schematic

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diagram illustrating community membership based on carbon flow in the

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SDZ enrichment. Symbol size is proportional to the genome relative

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abundance in the community. Color represents phylum-level taxonomy.

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Strain D2 was largely responsible for the initial breakdown of SDZ,

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producing 2-AP as the predominant metabolites and a small amount of

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aniline, and strain LG209 further mineralized 2-AP. 54±1.3% TOC from

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Environmental Science & Technology

783

SDZ was removed by these two strains, while other less abundant members

784

participate in the SDZ mineralization by consuming catabolic by-products

785

presumably derived from primary degraders.

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