Identification of Anaerobic Aniline-Degrading Bacteria at a

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Identification of anaerobic aniline-degrading bacteria at a contaminated industrial site Weimin Sun, Yun Li, Lora McGuinness, Shuai Luo, Weilin Huang, Lee Kerkhof, Elizabeth Erin Mack, Max M. Haggblom, and Donna E Fennell Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02166 • Publication Date (Web): 17 Aug 2015 Downloaded from http://pubs.acs.org on August 23, 2015

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Environmental Science & Technology

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Identification of anaerobic aniline-degrading bacteria at a contaminated industrial site

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Weimin Sun1,2, Yun Li1, Lora R. McGuinness3, Shuai Luo1, Weilin Huang1, Lee J. Kerkhof3, E. Erin

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Mack4, Max M. Häggblom2, and Donna E. Fennell1*

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1

Department of Environmental Sciences, Rutgers University, New Brunswick, NJ, USA

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2

Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, USA

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3

DepartmentofMarine and Coastal Sciences, Rutgers University, New Brunswick, NJ, USA

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4

DuPont, Corporate Remediation Group, Wilmington, DE, USA

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*Corresponding author

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14 College Farm Road

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New Brunswick, NJ 08901

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Office: +01-848-932-5748

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Email: [email protected]

16 17 18 19 20 21 22 23 24 25 26 27 28 29

ACS Paragon Plus Environment


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Abstract: Anaerobic aniline biodegradation was investigated under different

31

electron-accepting conditions using contaminated canal and groundwater aquifer

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sediments from an industrial site. Aniline loss was observed in nitrate- and

33

sulfate-amended

34

methanogenic conditions. Lag times of 37 days (sulfate amended) to more than 100

35

days (methanogenic) were observed prior to activity. Time-series DNA-stable isotope

36

probing (SIP) was used to identify bacteria that incorporated 13C-labeled aniline in the

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microcosms established to promote methanogenic conditions. In microcosms from

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heavily contaminated aquifer sediments, a phylotype with 92.7% sequence similarity

39

to Ignavibacterium album was identified as a dominant aniline degrader as indicated

40

by incorporation of 13C-aniline into its DNA. In microcosms from contaminated canal

41

sediments, a bacterial phylotype within the family Anaerolineaceae, but without a

42

match to any known genus, demonstrated the assimilation of 13C-aniline. Acidovorax

43

spp. were also identified as putative aniline degraders in both of these two treatments,

44

indicating that these species were present and active in both the canal and aquifer

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sediments. There were multiple bacterial phylotypes associated with anaerobic

46

degradation of aniline at this complex industrial site, which suggests that anaerobic

47

transformation of aniline is an important process at the site. Furthermore, the aniline

48

degrading phylotypes identified in the current study are not related to any known

49

aniline-degrading bacteria.

50

expands current knowledge regarding the potential fate of aniline under anaerobic

51

conditions.

microcosms

and

in

microcosms

established

to

promote

The identification of novel putative aniline degraders

52 53

Key

words:

anaerobic

aniline

degradation,

54

Anaerolineaceae, stable isotope probing

Ignavibacterium,

Acidovorax,

2


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Introduction Aniline is a basic industrial chemical for production of dyes, pesticides and

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pharmaceutical compounds, and is a pollutant commonly detected in soils and

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groundwater 1. Aniline is considered as a potential carcinogen2 and is toxic to human

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and aquatic life3, 4. Aniline enters the environment from releases during chemical

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manufacturing5, by the use of crude/synthetic oils and coal-based fuels6, and via

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biodegradation of pesticides in soils7, 8. Accidental spillage remains an important

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source of aniline contamination to the environment. Recently, two massive aniline

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spills occurred in northern China that posed health risks to exposed populations. The

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Tianji coal chemical industry chemical spill of 20129 introduced more than 39 tons of

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aniline into the Zhuozhang River and caused a water crisis for more than a million

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people living in downstream cities. In early 2013, aniline leaked into the water supply

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of a village in Hebei province, China and killed at least 700 chickens10. Aniline

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contamination may remain as an environmental threat because global aniline

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consumption grew by 3% annually between 2006–2010, and by nearly 10% per year

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from 2010 to 201211. Therefore, it is important to understand the fate of aniline in the

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environment.

72 73

The removal of aniline from contaminated aquifers, sediments and wastewater has

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been attempted by both biological and abiological means12-14. Biodegradation of

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aniline is an important remedial process in soil and aquatic environments13 and has

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been observed over several decades14-19. Current understanding of aniline

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biodegradation pathways come primarily from pure isolates or defined consortia

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grown under aerobic conditions20-23. Information about aniline degradation by bacteria

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in anoxic environments is rather limited. Aniline loss under denitrifying conditions

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has been reported in aquatic sediments and digester sludge24, anoxic reactors25, and 3



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inflow columns26. Aniline degradation was also observed under Fe(III)-reducing

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conditions in river sediments27. In contrast, aniline concentrations remained

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unchanged for more than 200 days under methanogenic conditions in sediments and

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digester sludge24. Further, few anaerobic aniline-degrading bacteria have been

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identified. In 1989, Desulfobacterium anilini was isolated from marine sediment with

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aniline as the sole carbon source and sulfate as the electron acceptor28 and its

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degradation pathway was characterized29. Later, strain HY99, an aerobic aniline

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degrader phylogenetically similar to Delftia acidovorans, was reported to degrade

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aniline under both aerobic and nitrate-reducing conditions30. Unfortunately, our

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current understanding of anaerobic aniline biodegradation is based entirely on these

91

limited studies.

92 93

In the current study, microcosms established from aniline-contaminated canal and

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groundwater aquifer sediments were used to examine aniline biodegradation under

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anoxic conditions where nitrate, sulfate, or CO2 was added. In addition, we used

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DNA-SIP with

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taxa that were able to incorporate

98

results indicated that members of Ignavibacterium, Anaerolineaceae, and Acidovorax

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were associated with anaerobic degradation of aniline. These findings suggest that

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anaerobic transformation of aniline can be an important process and could be used to

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enhance bioremediation of aniline in contaminated sites.

13

C-labeled aniline to discern the phylogenetic identity of bacterial 13

C carbons from aniline into their DNA. The

102 103

Materials and Methods

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Contaminated site description

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The study site is a chemical manufacturing facility in continuous operation since the

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mid-1890s located along the Delaware River in southern New Jersey, USA. The site is

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contaminated

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monochlorobenzene, dichlorobenzene, polycyclic aromatic hydrocarbons (PAHs), and

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dyes as described previously31-33. Aniline concentrations at the site ranged from 0.06

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to 660 µM (6 to 61,000 µg/L), according to historical sampling data. Samples were

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taken from four different locations at the site as shown in Figure 1. The first location

112

is a heavily contaminated groundwater (HCGW) aquifer with aniline concentrations

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exceeding 530 µM (50,000 µg/L). Geochemistry of the groundwater near the HCGW

114

location included: neutral pH, alkalinity, 275 to 400 mg/L; dissolved oxygen (DO) < 2

115

mg/L; nitrate not detected; methane, 14,000 µg/L; and sulfate, 125 to 150 mg/L. The

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second location is a lightly contaminated groundwater (LCGW) aquifer.

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Geochemistry of the groundwater near the LCGW location included: pH, 6.1 to 6.8;

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alkalinity 40 to 50 mg/L; DO < 5 mg/L; nitrate < 1 mg/L; methane, 50 to 220 µg/L;

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and sulfate, 20 to 40 mg/L. Two other site locations are from a freshwater canal with

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heavily contaminated canal sediments (HCFW) adjacent to the contaminated aquifer

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and lightly contaminated canal sediments (LCFW) upstream of the contaminated

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aquifer. Monitoring wells screened in the canal sediments indicated: neutral pH;

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alkalinity 275 to 400 mg/L; DO < 1 mg/L; nitrate not detected; methane, 3400 to 6900

124

µg/L; and sulfate, 72 to 210 mg/L. Sediment cores were obtained from each location

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and were cut, capped and labeled in the field prior to shipment to Rutgers University

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on ice. Specifically, aquifer cores of 0.9 m × 0.04 m diameter from 0 to 3 m below

127

ground surface were obtained from LCGW and HCGW; and sediment cores of 1.5 m

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× 0.08 m diameter were obtained from LCFW and HCFW. Groundwater and canal

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water was also obtained from each location at the time of sampling. Core materials

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(saturated zone only for LCGW and HCGW) were composited in a disposable

with

mixtures

of

different

chemicals

including

aniline,

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glovebag under a sterile nitrogen purge and stored in sterile glass jars before

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dispensing into microcosms. All materials were stored at 4 °C until use.

133 134

Set up of sediment microcosms

135

Sediment microcosms were established under anoxic conditions in triplicate with a

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total volume of 100 mL (with 20 g sediment and balance of the volume canal water or

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groundwater) under a headspace of N2/CO2 (70:30 vol/vol). [The microcosm study is

138

described in detail by Li, 201434 and is also briefly described in the Supporting

139

Information.] Twelve treatments were established as shown in Table S1. Briefly,

140

treatments amended with Na2SO4 (20 mM) (denoted S) and KNO3 (30 mM) (denoted

141

N) were established to promote sulfate-reducing and nitrate-reducing conditions,

142

respectively, while treatments amended with CO2 were intended to promote

143

methanogenic conditions (denoted M). Microcosms were amended with aniline

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(Sigma Aldrich, St. Louis, USA) and monitored for 1400 days. Aniline was initially

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spiked to a concentration of 100 µM in stage 1 from day 0 to 340; and to a

146

concentration of 1500 µM during stage 2 from day 340 to 1400. The increase of

147

aniline concentration was implemented to increase the selective pressure on the

148

sediment communities.

149 150

Microcosms for SIP

151

Microcosms from treatments established to promote methanogenic conditions and

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showing substantial loss of aniline, i.e., HCGW-M and LCFW-M, were selected as

153

parent microcosms to set up daughter microcosms for SIP analysis (see Table S2).

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Briefly, 20 mL slurry from parent microcosms was transferred to 60 mL serum bottles

155

and mixed with 20 mL groundwater or canal water in an anaerobic chamber (Coy

6


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Laboratory Products Inc., Grass Lake, USA). Triplicate cultures were amended with

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either 0.3 to 0.4 mM uniformly labeled 13C-aniline (Cambridge Isotope Laboratories,

158

Inc. Andover, MA, USA) or 12C-aniline (Sigma Aldrich, St. Louis, USA).

159 160

Analytical techniques

161

Sediment slurry (1 mL each for aniline and ion analyses) was removed from

162

microcosms using sterile syringes flushed with sterile N2/CO2. The sediment slurry

163

was then extracted with 1 mL acetonitrile and filtered extracts were analyzed using an

164

Agilent 1100 high performance liquid chromatography (HPLC) system (Agilent,

165

Santa Clara, USA). Nitrate and sulfate were analyzed in filtered, supernatant diluted

166

20-fold using an ICS-1000 ion chromatograph (Dionex, Sunnyvale, USA). Headspace

167

samples (250 µL) were analyzed for methane content using a gas chromatography

168

system (Agilent 6890N G1530N network GC system) with flame ionization detection.

169

Additional details for aniline and methane measurements are provided in the

170

Supporting Information.

171 172

Molecular analyses

173

For

174

extraction at two different time points (an early time point, T1 (21 days) and a later

175

time point, T2 (48 days)) during aniline depletion. For unlabeled aniline treatments,

176

DNA was extracted only at one time point during aniline depletion, denoted as T3.

177

The amount of aniline that had been consumed by T1 and T2 varied for each

178

treatment as shown in Table S2.

13

C-labeled aniline treatments, one replicate microcosm was sampled for DNA

179 180

Total genomic DNA was extracted from ~0.25 g sediment slurry using the PowerSoil

7



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DNA extraction kit (MO BIO Laboratories, Inc. Carlsbad, USA) based on the

182

manufacturer’s protocol. Purified DNA (~300 ng) was subjected to cesium chloride

183

(CsCl) isopycnal centrifugation (225,000 g for 48 h) after mixing with 100 ng of

184

archaeal (Halobacterium salinarum) DNA as described previously35-37. After

185

ultracentrifugation, two DNA bands were observed, i.e. a

186

genomic DNA from the resident microbial community and a

187

containing newly synthesized DNA from active microorganisms assimilating

188

aniline. The

189

DNA fractions for each

190

(5’-AGAGTTTGATCMTGGCTCAG, 5’ end-labeled with carboxyfluorescine) and

191

1100R (5’-AGGGTTGCGCTCGTTG) (Sigma Aldrich, St. Louis, USA) for

192

generating bacterial amplicons for TRFLP analysis. Fifteen ng PCR product was

193

digested with MnlI endonuclease (New England Biolab, Beverly, USA). All digests

194

were performed in 20 µL for 6 h at 37°C. Precipitation of digested DNA was

195

performed as described previously38. T-RFLP fingerprinting was carried out on an

196

ABI 310 genetic analyzer (Applied Biosystems, Foster City, USA) using Genescan

197

software.

12

12

C band (top) containing 13

C band (bottom)

C and 13C-DNA bands were collected by pipette35, 37. The 12C and treatment

13

C

13

C

were PCR-amplified using 27F-FAM

198 13

199

To identify the main T-RFs, clone libraries were generated from

C bacterial SSU

200

amplicons derived from DNA extracted at T2. For detailed information regarding

201

cloning and sequencing, please refer to the Supporting Information. The Ribosomal

202

Database Project (RDP) Classifier analysis tool was utilized to assign taxonomic

203

identity. Phylogenetic trees for the partial 16S rRNA gene sequences along with the

204

closest matches in Genbank were obtained by the neighbor-joining method using

205

MEGA 5.0 software. The 16S rRNA gene sequences were deposited with GenBank

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under accession numbers KP682336-KP682352.

207 208

Results and Discussion

209

Long-term monitoring of aniline biodegradation

210

Aniline loss without a lag was observed in microcosms LCGW-N, LCFW-N and

211

HCFW-N under nitrate-amended conditions (Figure S1). No aniline loss was observed

212

in HCGW-N. Loss of nitrate was observed in microcosms concomitant with the loss

213

of aniline (Figure S2). In contrast to nitrate-amended microcosms, sulfate-amended

214

and methanogenic microcosms experienced a longer lag time before aniline loss was

215

observed (Figure 2). In addition, some replicates behaved differently despite being set

216

up at the same time and identical conditions. Depletion of aniline was observed in

217

LCGW-S and LCFW-S. Substantial loss of aniline occurred in one of the triplicate

218

HCGW-S microcosms after 300 days, but no aniline loss was observed in the other

219

two replicates. Substantial loss of aniline was observed in all killed microcosms from

220

HCFW, likely indicating sorption to sediment. Therefore，it was difficult to determine

221

whether aniline loss could be attributed to biodegradation in HCFW-S.

222 223

To examine whether aniline loss was coupled to reduction of the electron acceptors

224

under each condition, predicted values of electron donor loss based on stoichiometric

225

equations (excluding cellular yield) were computed to compare to those measured in

226

aniline-degrading microcosms. The comparison between predicted values and actual

227

values of selected treatments is summarized in Table S4. Most treatments, with the

228

exception of HCFW-S, consumed more nitrate and sulfate than predicted, indicating

229

that natural organic matter in the sediment, or perhaps other contaminants, in addition

230

to aniline, were also consumed. Actual methane production was larger than predicted

9



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values for HCGW-M (Table S4), indicating that carbon sources other than aniline

232

contributed to methane production. Methane detected in microcosms under all redox

233

conditions is shown in Table S5. In general when nitrate or sulfate was present, there

234

was little methane production indicating that methanogens were outcompeted under

235

these conditions.

236 237

Loss of aniline was observed in methanogenic microcosms. All triplicate microcosms

238

from HCGW-M and LCFW-M demonstrated aniline depletion after more than 200

239

days incubation. LCFW-M was the most active treatment with repeated loss of aniline

240

upon re-amendment. All triplicates from HCGW-M demonstrated aniline loss during

241

stage 1. It is noteworthy that similar losses (e.g. similar lag period) of aniline in live

242

and killed microcosms were also observed in HCFW-M, indicating aniline loss may

243

not be wholly attributable to biodegradation in that system. In LCGW-M and

244

HCFW-M aniline depletion was observed in only one replicate but the remaining live

245

microcosms did not show substantial aniline loss.

246 247

Degradation of aniline under conditions established to promote methanogenesis

248

occurred most readily at HCGW, LCFW and HCFW (Table S3). Geochemical data

249

indicated that the highest concentrations of methane were detected at the HCGW

250

(14,000 µg/L), and LCFW and HCFW sites (3,400-6,900 µg/L), indicating that these

251

locations are more reduced. In contrast, the LCGW site had lower methane (50 to 220

252

µg/L) and was likely less reduced. In contrast, aniline degradation under

253

nitrate-amended conditions did not occur at HCGW.

254

loss under sulfate-amended conditions, however for HCGW, only one replicate

255

showed activity.

All locations exhibited aniline

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At stage 2, increased aniline loading (1500 µM) may have had some detrimental

257

effect on aniline biodegradation. As shown in Table S4, a comparison between aniline

258

loss in stage 1 and stage 2 clearly demonstrated that some microcosms showing

259

aniline loss in stage 1 at lower aniline concentrations did not exhibit aniline loss at

260

stage 2 when higher aniline concentrations were imposed. All triplicates from

261

HCGW-M showed aniline loss at stage 1 but only one microcosm showed aniline loss

262

at stage 2. However, microcosms from LCFW demonstrated a consistent degradation

263

pattern. All triplicates in LCFW-N, LCFW-S, and LCFW-M showed aniline loss at

264

both stage 1 and stage 2. The inhibition of aniline biodegradation by increased aniline

265

loading could be challenging for remediation of extensive aniline spills, which may

266

involve high environmental concentrations.

267 268

Aniline biodegradation under nitrate-reducing conditions was reported in previous

269

studies30. Our current observation confirmed that substantial and rapid aniline loss

270

was observed in all nitrate-amended treatments except HCGW-N, suggesting that

271

aniline might be readily degradable under nitrate-reducing conditions at these sites.

272

Substantial aniline loss was also observed in two sulfate-amended treatments

273

(LCGW-S and LCFW-S). It is notable that decrease in sulfate concentration was also

274

observed in these two treatments (Figure 3), indicating the aniline loss may be

275

coupled to sulfate reduction. Substantial aniline loss was observed in two locations for

276

the methanogenic treatments, HCGW-M and LCFW-M. Methanogenic treatments

277

LCGW-M, LCFW-M, and HCFW-M exhibited substantial methane production,

278

indicating that methanogenic conditions were indeed established, however, HCGW-M

279

did not produce substantial methane (Table S4 and S5). Overall, long-term monitoring

280

indicated that aniline was depleted under a variety of redox conditions, even under

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methanogenic conditions, in contrast to prior reports in anaerobic sludge and estuarine

282

sediment24. The most rapid degradation rates observed for aniline under the different

283

redox conditions are shown in Table S6. The observation that aniline biodegradation

284

did not occur at all sites or in all replicates under methanogenic conditions, and that

285

long incubation periods were sometimes required, suggests that the microbes

286

responsible for this activity may have been present at lower abundances in the

287

composited sediments than those that were active under the nitrate-amended

288

conditions.

289 290

Stable isotope probing of two anaerobic aniline-degrading treatments

291

Since information is currently lacking regarding aniline degradation under

292

methanogenic conditions, we selected two active treatments that were intended to

293

promote methanogenic conditions (HCGW-M and LCFW-M) for SIP analysis.

294

Daughter microcosms seeded from HCGW-M and LCFW-M, denoted GW-M and

295

FW-M, respectively, demonstrated aniline loss after re-amendment of aniline (Figure

296

S3). However, only FW-M generated substantial methane during SIP incubation

297

(Figure S3). Consistent with long-term monitoring of the original microcosms, GW-M

298

did not produce substantial amounts of methane. Thus GW-M cannot be explicitly

299

referred to as methanogenic but was an anoxic treatment that was amended with no

300

electron acceptors (other than CO2).

301 302

Identification of anaerobic aniline-degrading bacteria

303

The incorporation of

304

T-RFs enriched in

305

carbon source by bacteria in the enrichment cultures. No T-RFs were observed in

13

C-labeled aniline into DNA as evidenced from a number of

13

C-DNA fractions confirms that aniline was being utilized as a

12


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306

13

307

minimize the effect of cross-feeding in our study, we performed SIP at two time

308

points, T1 and T2. T1 represents an early biodegradation time point, while T2

309

represents a time point when ~50% aniline was degraded. No obvious differences

310

were observed in TRFLP profiles derived from 13C-fractions at the two time points for

311

GW-M and FW-M, indicating that bacteria assimilating labeled carbons were similar

312

at early and middle stages of biodegradation.

C-DNA fractions of corresponding

12

C-aniline amended controls (Figure S4). To

313 13

314

The clone library retrieved from the

C-fraction of GW-M (135 clones) was

315

dominated by the phyla Chlorobi (37%) and Betaproteobacteria (40%). In addition,

316

Actinobacteria (7%), Firmicutes (7%) and Deltaproteobacteria (6%) were also

317

present in the clone library at a relatively small proportion (Table 1). In FW-M, 150

318

bacterial clones were screened from the 13C-DNA fraction and eight OTUs detected in

319

the 13C-fraction were identified in the clone library. As in GW-M, members of phylum

320

Betaproteobacteria (50%) were predominant in FW-M. However, unlike GW-M, the

321

Chloroflexi (29%) were predominant while the Chlorobi were not present in large

322

numbers in the library derived from FW-M.

323 324

Analysis of the 13C TRFLP profiles demonstrated similarities and differences between

325

the two microcosms. In GW-M, two T-RFs, 239 bp and 274 bp, were dominant in

326

13

327

239 bp T-RF was unique to the GW-M microcosms and was solely enriched in

328

13

329

12

330

profiles indicated that several bacteria were associated with assimilation of 13C-aniline

C-DNA TRFLP profiles with relative abundances greater than 20% (Figure S5). The

C-DNA fractions, whereas the 274 bp T-RF showed high abundance in both 13C- and C-DNA fractions. The enrichment of multiple T-RFs in the

13

C-DNA TRFLP

13



331

derived

carbon

under

methanogenic

332

Betaproteobacteria, and Chlorobi.

conditions

including

Page 14 of 37

Chloroflexi,

333 334

The 239 bp T-RF unique to GW-M was identified as a phylotype within the genus

335

Ignavibacterium39. The Ignavibacterium-related phylotypes identified in this study

336

were most closely related to two cultivated strains, i.e., 92.7% sequence similarity to

337

Ignavibacterium album (CP003418) and 87.5% sequence similarity to Melioribacter

338

roseus (CP003557), a low level of similarity. The genus Ignavibacterium branches

339

deeply in the phylum Chlorobi. Ignavibacterium album is a strictly anaerobic,

340

moderately thermophilic, neutrophilic and obligately heterotrophic bacterium39.

341

Genomic analysis revealed that I. album is a chemoheterotroph with a versatile

342

metabolism, suggesting that it could live under oxic and anoxic conditions because of

343

the presence of genes encoding oxidases and reductases40. Another closely related

344

cultivated isolate, Melioribacter roseus, is a facultatively anaerobic and obligately

345

organotrophic bacterium which was isolated from a microbial mat from a deep oil

346

exploration well. The Melioribacter strain grew on polysaccharides by aerobic

347

respiration or by reducing different electron acceptors41. Bacteria closely related to

348

Ignavibacterium were recently detected in a microbial enrichment established from

349

geothermal springs in Armenia42, in an anammox membrane bioreactor43, in microbial

350

fuel cells44, and in an upflow anaerobic sludge blanket reactor45. The ecological and

351

functional role of Ignavibacterium spp. in these natural and built habitats is still

352

unexplored. Our study is the first to indicate that members of Ignavibacterium may

353

have the potential to degrade aniline, suggesting a potential role for Ignavibacterium

354

in bioremediation.

355

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13

356

In FW-M, three T-RFs, 121 bp, 208bp and 274 bp, were enriched in the

C-DNA

357

TRFLP profiles (Figure 4). Bacterial phylotypes represented by the 121 bp T-RF

358

belonged to another poorly-classified bacterial lineage. The RDP classifier placed this

359

phylotype in the family Anaerolineaceae. The closest cultivated isolate is Anaerolinea

360

thermolimosa (85% sequence similarity), which was isolated from methanogenic

361

sludge granules46.

362

GQ406185) was found in submerged Lake Huron sinkholes inundated with hypoxic,

363

sulfate-rich groundwater47. Members of Anaerolinea are present at high frequency in

364

many

365

contaminated aquifers, sediments, and soils48. Anaerolinea were also enriched in

366

methanogenic oil-degrading microcosms amended with North Sea crude oil46,

367

suggesting a link with methanogenic hydrocarbon degradation. Anaerolineaceae

368

sequences were frequently detected in sulfidogenic crude oil-degrading microcosms

369

when sulfate was depleted and n-alkane degradation had occurred49. These previous

370

studies suggest that members of Anaerolineaceae may be correlated with

371

biodegradation of hydrocarbon contaminants under strictly anaerobic conditions. Here,

372

a direct link between Anaerolineaceae and methanogenic aniline degradation was

373

demonstrated using SIP, providing evidence for an additional novel physiological trait

374

to be attributed to Anaerolineaceae.

The closest uncultured clone (97% sequence similarity,

hydrocarbon-impacted

environments

including

petroleum

reservoirs,

375 376

The 208 bp T-RF was represented by a phylotype belonging to the order

377

Burkholderiales. The closet cultivated isolate was Methylibium petroleiphilum PM1

378

(93% sequence similarity)50. Strain PM1 is notable for its capability of using methyl

379

tert-butyl ether (MTBE) as a sole carbon and energy source under aerobic conditions51.

380

The genus Methylibium contains some species that are able to degrade other organic

15



Page 16 of 37

381

compounds52, 53. Sequences corresponding to the 208 bp T-RF were not related to any

382

known aniline-degrading bacteria, suggesting that these phylotypes might be novel

383

anaerobic aniline-degrading bacteria. The 274 bp T-RF was predominant in the

384

13

385

Acidovorax-related bacteria. This phylotype was most closely related to Acidovorax

386

defluvii strain BSB411 (98% sequence similarity)54. Members of Acidovorax were

387

also

388

hydrocarbon56, nitrobenzene and mono-nitrophenol57. Some species of Acidovorax,

389

including Acidovorax delafieldii and Acidovorax facilis, are facultative bacteria58,

390

59

391

FW-M produced methane (indicative of a strictly anaerobic environment), it is

392

hypothesized that the detected Acidovorax phylotypes are facultatively anaerobic

393

aniline degraders or that they may degrade aniline derived metabolites. Bacteria

394

represented by the 208 bp and 274 bp T-RFs were present in both 12C- and 13C-DNA

395

fractions (Figure S5). Both these two phylotypes belonged to the order

396

Burkholderiales. Burkholderiales have been detected in many environments

397

contaminated by PAHs60-65. In addition, members of Burkholderiales were associated

398

with toluene and m-xylene degradation under denitrifying and aerobic conditions66-68.

C-DNA fractions of both the GW-M and FW-M and was identified as

responsible

for

degradation

of

chlorobenzene55,

polycyclic

aromatic

. Since GW-M and FW-M were established under anoxic conditions, and since

399 400

Other phylotypes identified in clone library derived from 13C-fractions

401

Some phylotypes not enriched in

402

13

403

clone library derived from GW-M, but the corresponding T-RF was not enriched in

404

13

405

related (99% sequence similarity) to a putative Fe(III)-reducing benzene-degrading

13

C-DNA TRFLP profiles were detected in the

C-DNA fraction clone libraries. Members of Peptococcaceae were identified in the

C-DNA fractions. The Peptococcaceae clones identified in this study were closely

16


Page 17 of 37


406

bacterium previously identified by DNA-SIP69. No exogenous Fe3+ was provided to

407

the microcosms, and prolonged incubation under reduced conditions would be

408

expected to result in reduction of naturally-occurring Fe3+. However, it appears that

409

Peptococcaceae might be responsible for consuming some of the labeled aniline in

410

this system. Phylotypes related to the order Syntrophobacterales were detected in

411

clone libraries of GW-M and FW-M. Syntrophobacterales consists of syntrophic

412

bacteria capable of degrading organic compounds such as acetate, propionate or

413

butyrate70-72. Further, it has also been proposed that Syntrophobacterales (Smithella)

414

can degrade hydrocarbons73. Detection of Syntrophobacterales phylotypes as active

415

organisms in aniline-degrading microcosms suggest that they are either directly

416

involved in the initial degradation of aniline, or that they degrade intermediates in the

417

aniline degradation pathway. Several studies reported that aromatic compounds such

418

as benzene and toluene were degraded by syntrophic interactions of primary

419

biodegraders with syntrophic bacteria and/or hydrogen consuming species69,

420

Anaerobic aniline degradation in the current study is likely to be a syntrophic process.

74-76

.

421 422

Aniline loss was observed in long-term microcosms from an industrial contaminated

423

site incubated under different redox conditions. A limited number of studies have

424

reported aniline loss under different anoxic redox conditions27,

425

identify the bacteria responsible for biodegradation. Here, we report that aniline loss

426

at two sites within our study area, with different histories of contaminant exposure

427

and environmental conditions (aquifer versus aquatic sediment), is mediated by

428

location-specific microbes within this industrial site. Using DNA-SIP the taxonomic

429

groups that incorporated

430

from known aniline degraders (Figure 5). In particular, Ignavibacterium-affiliated

29, 30

but did not

13

C carbons during aniline loss were shown to be different

17



Page 18 of 37

431

bacteria have not previously been associated with biodegradation of aromatic

432

compounds. The active microbial consortia also included bacteria related to those

433

known to be involved in syntrophic associations. Future work should explore the roles

434

of these phylotypes in field-based studies of natural attenuation of aniline at this and

435

other aniline-contaminated sites.

436 437

Acknowledgements

438

The authors gratefully acknowledge funding from E. I. du Pont de Nemours and

439

Company to D.E.F. and W.H. for the long-term microcosm study. We thank the New

440

Jersey Department of Environmental Protection, Office of Science, for funding to

441

D.E.F and M.M.H. in support of SIP analyses.

442

(formerly DuPont) and all environmental support personnel at the site for enabling

443

this study. We thank Kathy West of AECOM for assistance with site materials and

444

information.

We thank Edward Lutz of Chemours

445 446

Supporting Information Available

447

This information is available free of charge via the Internet at http://pubs.acs.org.

448 449

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Jones, D.; Adams, J.; Larter, S., The quantitative significance of Syntrophaceae and

675

syntrophic partnerships in methanogenic degradation of crude oil alkanes. Environ.

676

Microbiol. 2011,13, (11), 2957-2975.

677

(74) Sun, W.; Sun, X.; Cupples, A. M., Identification of Desulfosporosinus as

678

toluene-assimilating microorganisms

679

Biodeterior. Biodegradation 2014,88, 13-19.

680

(75) Herrmann, S.; Kleinsteuber, S.; Chatzinotas, A.; Kuppardt, S.; Lueders, T.;

681

Richnow,

682

benzene-degrading enrichment culture by DNA stable isotope probing. Environ.

metabolism

H.

under

H.; Vogt,

C.,

sulfate-reducing

from a

conditions

in

a

methanogenic consortium. Int.

Functional characterization

of an anaerobic

27



Page 28 of 37

684

Microbiol. 2010,12, (2), 401-411. A (76) van der Zaan, B. M.; Saia, F. T.; Stams, A. J.; Plugge, C. M.; de Vos, W. M.;

685

Smidt, H.; Langenhoff, A. A.; Gerritse, J., Anaerobic benzene degradation under River

686

denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence

687

for a syntrophic process. Environ. Microbiol. 2012,14, (5), 1171-1181.

683

Site Boundary

Aquifer

Road

688 Bridge

689 690

Canal

691 692

B b

693

c

d

e LCGW

a HCGW

694 695

Canal

696 697

HCFW LCFW

698 699

Direction of Canal Water Flow

700 701 702 703 704 705 706 707 708 28


Page 29 of 37


709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724

Figure 1 Sampling locations at a large chemical manufacturing site in southern New

725

Jersey. Map A demonstrates the relative locations of sampling sites. Map B shows the

726

aniline concentration contour near each location. Symbols in contour map: a: >50,000

727

µg/L; b: 25,000 to 50,000 µg/L; c: 5,000 to 25,000 µg/L; d: 1,000 to 5,000 µg/L; e: 5

728

to 1,000 µg/L. HCGW, highly contaminated ground water aquifer; LCGW, lightly

729

contaminated ground water aquifer; HCFW, highly contaminated freshwater canal

730

sediments; LCFW, lightly contaminated freshwater canal sediment.

731 732 733

29



Page 30 of 37

734 735

A

B

C

D

E

F

G

H

736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758

Figure 2. Aniline concentrations in sulfidogenic and methanogenic microcosms including

30


Page 31 of 37


759

LCGW-S (A), LCGW-M (B), HCGW-S (C), HCGW-M (D), LCFW-S (E), LCFW-M (F),

760

HCFW-S (G), and HCFW-M (H). Arrows indicate re-amendment of aniline. Note

761

different axis scales. For killed control, symbols are averages of triplicate cultures and

762

error bars represent one standard deviation. LCGW-S, lightly contaminated groundwater

763

aquifer sulfidogenic treatment; LCGW-M, lightly contaminated groundwater aquifer

764

methanogenic

765

sulfidogenic

766

methanogenic treatment; LCFW-S, lightly contaminated freshwater canal sulfidogenic

767

treatment; LCFW-M, lightly contaminated freshwater canal methanogenic treatment;

768

HCFW-S, highly contaminated freshwater aquifer sulfidogenic treatment; HCFW-M,

769

highly contaminated freshwater aquifer methanogenic treatment.

treatment; treatment;

HCGW-S,

highly

contaminated

groundwater

aquifer

HCGW-M,

highly

contaminated

groundwater

aquifer

770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 31



Page 32 of 37

787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810

Figure 3. Sulfate concentrations in sulfate-amended treatments (A) LCGW-S; (B)

811

LCFW-S; and (C) HCFW-S. Note that axis scales are different. Only one microcosm

812

from HCGW-S demonstrated aniline degradation. No sulfate loss occurred in

813

HCGW-S.

814 815 816 817 32


Page 33 of 37


GW-M (13C aniline)

T1-12C top band

T1-13C bottom band

GW-M (13C aniline)

239 bp

GW-M (13C aniline)

T2-12C top band

T2-13C bottom band

274 bp

T1-12C top band

T1-13C bottom band

GW-M (13C aniline)

FW-M (13C aniline)

120 bp

FW-M (13C aniline)

208 bp

T2-12C top band

FW-M (13C aniline)

T2-13C bottom band

274 bp

FW-M (13C aniline)

818 819 820 821 822 823

Figure 4. TRFLP profiles from different 13C-aniline amended treatments at different

824

time points. The treatment, time point, and

825

arrows and numbers indicate the fragment length of the enriched T-RFs in

826

fractions.

12

C or

13

C fractions are indicated. The 13

C-DNA

33



Page 34 of 37

Flectobacillus lacus strain R73022 (HM032866.1) Acidovorax defluvii strain BSB411 (NR 026506.1) 75 274 bp T-RF enriched in heavy fractions of GW-M and FW-M 92 100 Uncultured Acidovorax sp. clone 5_0_B1_b (JQ087024.1) Diaphorobacter sp. J5-51 (GU017974.1) Delftia sp. AN3 (AY052781.1) 100 Aniline-degrading bacterium HY99 (AF210313.1)* Rhodoferax sp. Asd M2A1 (FM955857.1) Mitsuaria sp. H24L1C (EU714910.1) 208 bp T-RF enriched in heavy fractions of FW-M 100 Uncultured bacterium clone AN162 in PCB dechlorinating sediments (GQ859926.1) Methylibium petroleiphilum strain PM1 (NR 041768.1) 100 Methylibium fulvum (AB649013.1) Erwinia amylovora strain HSA 6 (GQ222272.1)* Rhizobium borbori strain DN316 (EF125187.1)* Desulfobacterium anilini strain Ani1 (NR 025348.1)* Melioribacterroseus P3M-2 (NR 074796.1) 239 bp T-RF enriched in heavy fractions of GW-M 100 100 Uncultured Chlorobi bacterium clone Aug-CD246 (JQ795235.1) 100 Ignavibacterium album JCM 16511 (NR 074698.1) 100 Ignavibacterium album strain Mat9-16 (NR 112875.1) 100 120 bp T-RF enriched in heavy fractions of FW-M Uncultured Chloroflexi bacterium clone 4.26 (GQ183434.1) Anaerolinea thermolimosa strain IMO-1 (NR 040970.1) 99 Anaerolinea thermophila UNI-1 (NR 074383.1) 59

86 88

100

44

65

79

56

88

100

0.05

34


Page 35 of 37


Figure 5. Phylogenetic tree using partial 16S rRNA gene sequences of the major aniline-degrading phylotypes, with the closest matches within GenBank and the closest type strain, constructed with MEGA 5.0 software using the neighbor-joining method. 656 bp unambiguously aligned positions were used for analysis. *indicates aniline-degrading isolates identified in previous studies. Acronyms: GW-M, anoxic microcosms seeded from highly contaminated groundwater aquifer sediments; FW-M, methanogenic microcosms seeded from lightly contaminated canal sediments.

35



Page 36 of 37

1

Table 1. Phylogenetic groups and number of bacterial 16SrRNA gene clones in clone

2

libraries from 13C-fractions in two treatments and their corresponding Mnl I terminal

3

restriction fragment sizes as determined by in silico enzyme digestion.

4

Phylogenetic group Acidobacteria Gp3 Actinobacteria Rhodococcus Betaproteobacteria Burkholderiales Acidovorax Rhodocyclaceae Methylibium Deltaproteobacteria Syntrophaceae Chlorobi Ignavibacterium Chloroflexi Anaerolineaceae Firmicutes Peptococcaceae Fusibacter Unclassified Bacterium Total number of clones

Aquifer sediments, Canal Sediments, Anaerobic (GW-M) Methanogenic (FW-M) number of clones number of clones

Mnl I digested fragment size (bp)

-

10

170

9

12

179

14 39 2

3 34 10 28

168 277 146 211

8

5

259

51

5

243, 285

3

43

54,120

6 3

-

130 388 N/A

2

135

150

5 6 7 8 9 10 11 12

36


Page 37 of 37


13C-aniline

12C

DNA

+ Active aniline degraders

DNA

Substrate addition

DNA extraction

13C

DNA +carrier DNA Separation of labeled and unlabeled DNA


TRFLP + Clone library analysis

Identification of Anaerobic Aniline-Degrading Bacteria at a

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