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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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Environmental Science & Technology

1 2

Partnership of Arthrobacter and Pimelobacter in

3

aerobic degradation of sulfadiazine revealed by

4

metagenomics analysis and isolation

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

6 7 8

1

9

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

Environmental Biotechnology Laboratory, Department of Civil Engineering, The

10

2

11

Shenzhen, China

12

3

13

University of Science and Technology, Shenzhen, China

14 15

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

ACS Paragon Plus Environment


16

ABSTRACT

17 18

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

19

as a nested approach to identify functionally significant bacteria for sulfadiazine

20

biodegradation within enrichment communities. The metagenomic investigations

21

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

22

another Pimelobacter bacterium concomitantly occurred as most abundant members

23

in the communities of an enrichment culture that performed complete sulfadiazine

24

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

26

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

27

sulfadiazine. Taking advantage of this propensity, additional cultivation procedures

28

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

29

sequences exactly matched that of the dominant Pimelobacter bacterium in the

30

sulfadiazine enrichment culture. Integration of metagenomic investigations with the

31

physiological characteristics of the isolates conclusively demonstrated that the

32

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

34

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

35

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

36

steady sulfadiazine mineralization processes, which will help develop appropriate

37

bioremediation strategies for sulfadiazine-contaminated hotspot sites.


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INTRODUCTION

39 40

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

41

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

42

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

43

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

44

from animals or human were generally insufficiently treated by wastewater treatment

45

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

46

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

47

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

48

in the open environment may promote the dissemination of sulfonamide resistant

49

genes in clinically relevant pathogens18, and potentially threaten the therapy

50

effectiveness of sulfonamides. The implementation of sulfonamide degraders in

51

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

52

wastewater, livestock farm wastewater and hospital wastewater) through biological

53

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

54

sulfonamide resistance by directly reducing sulfonamide concentrations in the

55

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

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microorganisms. Since these sulfonamide degraders are also sulfonamide-resistant

57

microorganisms, to avoid their distribution from the biological treatment reactors

58

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

59

carried out within enclosed engineered systems instead of natural environment which

60

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.

63



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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

66

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

67

acetate or yeast extract) during sulfonamide exposures. Enrichment culturing

68

strategies featuring sulfonamide as the sole carbon source should facilitate more

69

explicit interpretations of the community responses that were actually driven by

70

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

71

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

72

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

73

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

74

characterized just based on phylogenetic classifications.

75 76

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

79

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

80

the sulfonamide catabolic efficiency measured in particular single isolate were

81

promising

82

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

84

activity of the inoculated specialists in the natural environment. Improved

85

understanding on how the key degraders establish and maintain their memberships

86

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

87

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

under

microcosm-scale

laboratory

experiments,

addition

of

88 89

Driven by the hypothesis that structurally similar sulfonamides being sole carbon


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90

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

92

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

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two frequently encountered sulfonamides (sulfadiazine (SDZ) and sulfamethoxazole

94

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

95

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

98

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

99

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

100

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

104

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

105

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

106

model community to illustrate how metagenomic analyses can be incorporated with

107

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

110 111

MATERIAL AND METHODS

112 113

Culture enrichment and metagenomic sequencing and assembly

114 115

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



116

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

117

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

118

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,

120

the most prominent intermediate during SDZ mineralization by SDZ enrichment

121

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

124

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

125

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

126

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

130

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

131

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

132

(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

134

were filtered and assembled independently with CLC Genomics Workbench software

135

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

136

downstream analyses.

137 138

Community profiling and detection of sulfonamide resistance genes

139 140

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

141

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

143

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

144

contigs to further characterize the microbial community compositions. All assembled

145

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

147

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

148

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

149

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

150

metagenomic datasets were performed by EMIRGE algorithm46 with 80 iterations.

151

DECIPHER47 was used to detect the chimera among EMIRGE reconstructed

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sequences. Phylogenetic classifications of the resulting sequences were determined by

153

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

154

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

155

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

156

relative abundance of annotated query sequences was derived from EMIRGE

157

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

158

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

159

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

160

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

161

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

162

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

163 164

Population genome binning, annotation and analysis

165 166

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

167

genomes. Following the reported mmgenome workflow50, population genomes were



168

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

169

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

170

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

171

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

172

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

174

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

175

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

176

and ggKbase.

177 178

Pure culture isolation and batch experiments

179 180

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

181

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

182

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

183

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

184

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

187

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

188

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

189

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

190

accession numbers NHTS00000000.

191 192

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

193

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

195

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

196

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

197

generation of metabolites were measured by ultra-performance liquid chromatograph

198

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

199

reported before41.

200 201

RESULTS AND DISCUSSION

202 203

Enrichment of SDZ-degrading consortia

204 205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

uncultivated microorganisms presented in the long-running enrichment cultures,

215

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

216

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

217

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

218

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

219

almost complete mineralization.



220 221

To understand the microbial interactions underpinning sulfonamide biodegradation

222

processes, we further detected important intermediates during SDZ degradation by

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

the enrichment cultures were presumably mediated either by primary SDZ degraders

231

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

233

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

234

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

235

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

236

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

237 238

Microbial community overview

239 240

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

241

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

242

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

243

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

244

primarily evaluated by read-based taxonomic assignments via MetaPhlAn2 and

245

phylogenic classifications of total functional proteins via MEGAN. Both approaches


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246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

the seed sludge (0.05 copy per cell).

254 255

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

256

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

257

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

258

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

259

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

260

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

261

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

262

genera (including Dokdonella, Hydrogenophaga, Achromobacter and Leadbetterella,

263

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

264

accounting for 15.1% in the SDZ enrichment community.

265 266

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

267

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

268

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

269

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

271

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



Page 12 of 34

272

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

273

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

274

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

275

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

276

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

277

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

278

carbon source.

279 280

Arthrobacter and Pimelobacter populations in the SDZ-degrading consortia

281 282

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

283

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

284

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

285

microbial communities were further performed for metagenomes of SDZ enrichment,

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

previously benchmarked ribosomal proteins54, showed that the recovered 23

294

population genomes represented organisms of the phyla Proteobacteria (nine

295

genomes),

296

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

Bacteroidetes

(six),

Actinobacteria

(four),

297


Acidobacteria

(two),

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298

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

299

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

300

bin_SDZ1 and Arthrobacter bin_SDZ2 were estimated to be the two highest

301

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

302

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

303

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

304

abundance for Pimelobacter bin_SDZ1 and Arthrobacter bin_SDZ2 were only 0%

305

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

306

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

307

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

308

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

309

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

310

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

311

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

754

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

755

or unshared.


Page 31 of 34


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

es

soxida

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

ro ba c

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.

760 761



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 (%)

763 764 765

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

766

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

767

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

770

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

771


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772 773 774 775

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

776

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

777

diagram illustrating community membership based on carbon flow in the

778

SDZ enrichment. Symbol size is proportional to the genome relative

779

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

780

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

781

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

782

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



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.


Page 34 of 34

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