Impact of Heavy Metals on Transcriptional and ... - ACS Publications

Subscriber access provided by GAZI UNIV

Article

Impact of heavy metals on transcriptional and physiological activity of nitrifying bacteria Vikram Kapoor, Xuan Li, Michael Elk, Kartik Chandran, Christopher Allen Impellitteri, and Jorge W. Santo Domingo Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 26 Oct 2015 Downloaded from http://pubs.acs.org on October 27, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Environmental Science & Technology

1

Impact of heavy metals on transcriptional and

2

physiological activity of nitrifying bacteria

3

Vikram Kapoor1, Xuan Li1, Michael Elk2, Kartik Chandran3, Christopher A. Impellitteri1, and

4

Jorge W. Santo Domingo1*

5

1

6

45268, USA

7

2

Pegasus Technical Services, Inc., Cincinnati, OH 45268, USA

8

3

Department of Earth and Environmental Engineering, Columbia University, New York, NY

9

10027, USA

U.S. Environmental Protection Agency, Office of Research and Development, Cincinnati, OH

10 11

Keywords. Nitrification, wastewater, heavy metals, specific oxygen uptake rate, reverse

12

transcriptase – quantitative polymerase chain reaction, high-throughput sequencing

13 14 15 16 17 18

1 ACS Paragon Plus Environment


19

ABSTRACT

20

Heavy metals can inhibit nitrification, a key process for nitrogen removal in wastewater

21

treatment. The transcriptional responses of amoA, hao, nirK and norB were measured in

22

conjunction with specific oxygen uptake rate (sOUR) for nitrifying enrichment cultures exposed

23

to different metals (Ni(II), Zn(II), Cd(II) and Pb(II)). There was significant decrease in sOUR

24

with increasing concentrations for Ni(II) (0.03-3 mg/L), Zn(II) (0.1-10 mg/L) and Cd(II) (0.03-1

25

mg/L) (p 99%

178

ammonia removal. During steady state nitrification, the average effluent NH4+-N concentration 8 ACS Paragon Plus Environment

Page 8 of 30

Page 9 of 30


179

was 1.77 ± 0.41 mg/L (n=49) and average tCOD was 1923 ± 130 mg/L (n=49). The background

180

levels of AOB 16S rRNA gene copies and amoA gene transcripts in the bioreactor were

181

measured for multiple dates to observe AOB temporal dynamics. The relative abundance of

182

AOB varied between 105 to 106 16S rRNA gene copies/mL (Figure 1). The relative mRNA

183

concentration for amoA was consistent with ammonia oxidation in the bioreactor (Figure 2).

184

Similar profiles for AOB 16S rRNA gene copies and amoA gene transcripts have been observed

185

earlier for nitrifying cultures cultivated in a lab-scale bioreactor having similar configuration as

186

the current study.9

187

The bacterial community composition of the nitrifying bioreactor was also examined to

188

confirm the presence and determine diversity of nitrifying bacteria in the enrichment cultures.

189

More than 200,000 16S rRNA gene sequences were generated from nitrifying biomass DNA

190

extracts obtained on days 5, 10, 15, 25 and 30 of bioreactor operation after attainment of steady-

191

state. These libraries obtained on multiple sampling dates displayed high degrees of similarity,

192

based on a comparison of taxonomic groups identified and their abundance proportions

193

(correlation coefficients, r > 0.9) indicating that the microbial composition of the bioreactor was

194

similar over the experimental period. Analyses of these sequences indicated that most AOB in

195

the bioreactor belonged to the family Nitrosomonadaceae under the class Betaproteobacteria (>

196

60% of total sequences) (Figure 3). Remaining sequences included members related to the

197

groups Bacteroidetes, alphaproteobacteria and gammaproteobacteria. While an additional 10

198

different classes were also identified, they represented less than 1% each. Previous analyses of

199

next generation sequencing data of nitrifying enrichments established from wastewater inoculum

200

also observed that Nitrosomonas-like populations constituted the major group in such

201

enrichments.9,28



202

sOUR - based inhibition. The inhibition of nitrification activity based on the sOUR method was

203

plotted against the total metal dosage for each concentration (Figure 4). The specific ammonium

204

oxidation rate decreased as the applied metal dose to nitrifying biomass increased for both Ni(II)

205

and Cd(II) exposure. For Zn(II) exposure, nitrification inhibition increased with metal

206

concentration up to 3 mg/L Zn(II), after which activity did not increase upon exposure to 10

207

mg/L Zn(II). There was no change observed when the nitrifying biomass was exposed to Pb(II)

208

up to 100 mg/L, however there was 84 % decrease in ammonia oxidation upon exposure to 1000

209

mg/L Pb(II) (Figure 4). An empirical non-competitive inhibition model was used to estimate the

210

half-inhibition coefficient, Ki by minimizing the sum of squared errors between the measured

211

and predicted inhibition using Solver (MS Excel, 2013). The predicted inhibition based on the

212

metal concentration was calculated as follows:

213 214

% inhibition = 100/(1 + Ki /I))

(2)

Based on the empirical estimates of Ki the degree of inhibitory effect of metals toward

215

ammonium oxidation was Cd > Ni > Zn > Pb (Ki values for Cd, Ni and Zn were estimated as

216

0.76 mg/L, 1.9 mg/L and 17.29 mg/L, respectively, based on non-competitive inhibition model).

217

Interestingly, with the exception of Pb, the inhibitory character of the metals tested in our study

218

corresponded well with their sulfide complexation potential series, which follows Pb > Cd >

219

Ni > Zn (stability constants for metal sulfide complexes, log K, 25˚C: -27.5, -27.0, -26.6, -24.7

220

for Pb, Cd, Ni and Zn, respectively).29 In another study18, the molar inhibitory effect toward

221

ammonium oxidation followed: Cu = Zn > Cd > Ni; however their results did not correspond

222

well with the metal-sulfide complexation potential series.

223

The decrease in respiration rates as a response to increasing metal concentrations has

224

previously been observed for nitrifying bacteria batch cultures.17,18 In our study, we observed 10 ACS Paragon Plus Environment

Page 10 of 30

Page 11 of 30


225

that ammonia-dependent oxygen uptake rates were significantly inhibited after a 12-h exposure

226

to Ni(II), Zn(II), or Cd(II). In contrast, cells exposed to Pb(II) did not show significant changes

227

in sOUR for Pb(II) concentrations up to 100 mg/L. Nitrification was only severely inhibited

228

when Pb(II) levels reached 1000 mg/L. This is consistent with previous reports on lack of

229

inhibition of nitrification at Pb concentration as high as 40 mg/L.30 It has been suggested that

230

nitrification inhibition in batch assays with exposure time of up to 12 h continuously increases

231

for Ni, Zn, and Cd, probably due to slow internalization kinetics,11,18 whereas Pb has been shown

232

to be less inhibitory than Ni, Zn and Cd.12

233

Several studies have used respirometry to assess the inhibitory concentrations of heavy

234

metals impacting biological wastewater treatment. Although, ammonia or nitrite may be used as

235

nitrogen substrate to measure oxygen uptake rates by AOB or NOB respectively, we used

236

ammonia-dependent sOUR assays since ammonia oxidation, being the first step of nitrification,

237

is often the rate limiting step and more prone to environmental perturbations.3,31 These methods

238

are considered by many as useful tools for evaluating the effects of organic and inorganic

239

compounds on nitrification. However, a wide range of inhibitory levels have been reported using

240

these methods. For example, using municipal wastewater Juliastuti et al.32 observed complete

241

nitrification inhibition at a Zn(II) concentration of 1.2 mg/L. In another study, more than 3 mg/L

242

of Zn(II) was required to attain greater than 90% inhibition.11 Previously, Cenci and

243

Morozzi33 reported 50% inhibition for Zn2+ concentration of 16 mg/l, which is most close to the

244

value of Ki (i.e. 17.29 mg/L) observed in this study. The differences in exposure time is a likely

245

explanation, although there may be other experimental differences (e.g., biomass used,

246

nutritional growth conditions) that could have contributed to the range of concentrations reported

247

as inhibitory. Thus, alternative methods are required to truly assess nitrification inhibition in



248

WWTPs. A 50% decrease in nitrifying activity has been observed with Cd concentration of 8.3

249

mg/L15 and 13 mg/L14 when using nitrifying enrichment culture and activated sludge,

250

respectively. On the other hand, the concentration of Cu at which 50% inhibition occurred for

251

nitrifying enrichments (173 mg/L)15 was at least an order of magnitude higher than that for

252

mixed liquor (18 mg/L).14 The inhibitory values reported for Cu vary up to more than three-fold

253

using activated sludge,14,34 suggesting that inhibition rates may differ even when the same type

254

of biomass is used in inhibition studies.

255

Transcriptional responses to heavy metals. The amoA transcript levels decreased after

256

exposure to Ni(II), with greatest reduction after the cells were exposed to 3 mg/L Ni(II) (Figure

257

5). The transcript levels of hao decreased with increasing Ni(II) dosages; however, no significant

258

change was observed with 3 mg/L Ni(II) (p > 0.05). The levels of nirK and norB decreased after

259

exposure to 1 mg/L and 3 mg/L Ni(II), respectively. Increased transcription of amoA, hao, and

260

nirK ranging from 0.5 to 1.5-fold, occurred after exposure to 0.3, 1 and 3 mg/L Zn(II). Although

261

the changes in the transcript levels of amoA and hao with Zn(II) exposure were not significant (p

262

> 0.05), amoA and hao transcripts decreased by 0.5-fold when exposed to 10 mg/L Zn(II).

263

Stimulation of norB expression was observed at a Ni(II) dose of 0.3 mg/L for batch nitrifying

264

cultures. There were significant changes in the levels of all four genes when exposed to Cd(II) (p

265

< 0.05). The transcription of amoA was considerably increased at 0.3 mg/L Cd(II), while hao

266

was increased at 1 mg/L Cd(II). Although, amoA transcripts were increased upon exposure to all

267

concentrations of Cd(II), the linear correlation of amoA transcripts with Cd(II) concentrations

268

was not statistically significant (p > 0.05). The transcript levels of amoA, hao, nirK and norB

269

increased by more than half-fold in cells exposed to 100 mg/L Pb(II); but amoA decreased by

270

twofold and hao decreased by one-fold when exposed to 1000 mg/L Pb(II). 12 ACS Paragon Plus Environment

Page 12 of 30

Page 13 of 30

271


Based on 16S rRNA gene sequencing, most AOB detected in this study were closely

272

related to Nitrosomonas group. When Nitrosomonas–like functional genes were measured, on

273

average, we observed that amoA transcript levels decreased when exposed to Ni(II), but such

274

decrease was not as high as compared to the relative decrease in activity as suggested by sOUR

275

data. A more marked discrepancy between molecular data and respirometry was observed for

276

cells exposed to Cd(II) in which amoA transcript levels increased while there was a reduction in

277

sOUR. For Zn(II) and Pb(II), amoA expression was inhibited at 10 and 1000 mg/L respectively

278

(Figure 5). The results suggest that the intracellular RNA content of these functional genes may

279

not strictly correlate with the activity of enzymes under heavy metal exposure. Similar results

280

have been shown in previous studies.7,16 The changes in RNA content of a cell reflect sensitive

281

cellular responses and has been used to estimate the relative growth rates and is an indicator of

282

physiological state of the organism.35,36 However, the lack of correlation between 16S rRNA

283

abundance and physiological activity in AOB under stress conditions has been observed

284

previously,7,37 therefore, monitoring changes in the transcript levels of functional genes such as

285

amoA and hao has been suggested by a few.7,10 Other sentinel genes that are identified to be

286

sensitive to heavy metal inhibition include genes encoding for mercury resistance proteins

287

(merTPCADE),5,38 and genes involved in DNA replication and recombination (such as tnpAR).5

288

The results presented here assist to understand the interplay between physiological and

289

transcriptional responses of genes involved in nitrification for mixed populations under

290

environmental perturbations.

291

The current study focused on acute toxicity (< 24 h exposure) of heavy metals and did not

292

account for chronic toxicity. The chronic effects of heavy metal inhibition to nitrifiers may be

293

underappreciated, considering the ever-increasing industrialization and large-scale mining over



294

recent years and corresponding elevated levels of metals in wastewaters.21,22 Metals may be

295

frequently or even continuously present in activated sludge systems, causing long-term impact

296

on microorganisms. In some cases, bacterial communities may develop resistance to heavy

297

metal inhibition and recover their activities upon long-term exposure to inhibitors.39,40 For

298

instance, Yeung et al.41 evaluated inhibition and adaptation to nickel using nitrifying biomass

299

established from nitrifying activated sludge of a full-scale wastewater treatment plant. They were

300

able to select for nickel-resistant nitrifying cultures obtained by long-term batch incubations of

301

decaying activated sludge with high levels of added inhibitor. After incubating activated sludge

302

with different concentrations of Ni, they observed that nitrifying communities were not impacted

303

with 1 mg/L of Ni, and showed considerably low levels of nitrification inhibition at 5 and

304

10 mg/L Ni. Incubation with 50 mg/L Ni resulted in significant inhibition as reflected by

305

decreased amoA transcript abundance. By contrast, we observed significant down-regulation of

306

amoA transcript levels with 3 mg/L of added Ni(II). The results indicate that, although Ni is

307

inhibitory at concentrations above 1 mg/L, there is considerable variation in reported threshold

308

values due to differences in experimental configurations and biomass source, and adaptation to

309

metals. We used nitrifying enrichment cultures while the aforementioned study used nitrifying

310

activated sludge from the aeration basin of a full-scale wastewater treatment plant as the source

311

of nitrifying biomass for batch incubations. When the tested biomass or sludge are from different

312

sources, or are harvested at varying growth conditions, the nitrifying process could be different

313

since the physiological state of the cells may impact the uptake of inhibitory compounds

314

including metals.11,42 In another study, the inhibitory effect of zinc, copper, nickel and lead on

315

pure cultures was compared with activated sludge.43 It was observed that pure cultures were

316

more sensitive to heavy metal inhibition than nitrifying sludge samples, suggesting that pure


Page 14 of 30

Page 15 of 30


317

culture studies may overestimate true inhibition of a given contaminant. Furthermore, the

318

differences between the chemical and biological properties of nitrifying samples used (pure

319

culture versus activated sludge) represents yet another source of variation in the observed effects

320

of heavy metal inhibition.

321

The transcript levels of amoA, hao, nirK and norB were slightly up-regulated (less than

322

one-fold) at Zn(II) concentration of 3 mg/L, while a decrease in the transcript levels of all the

323

functional genes was observed for a Zn(II) concentration of 10 mg/L. The overall changes in the

324

transcript levels of functional genes were not significant when exposed to Zn(II) (p > 0.05). In a

325

series of studies using nitrifying bacteria pure cultures, the amoA and hao expression were

326

monitored in response to ZnCl2 additions.8,20 The transcriptional responses of Nitrosococcus

327

mobilis, a halophilic nitrifier, to 0.06 and 0.6 mg/L Zn revealed that amoA and hao expression

328

levels were maintained or slightly up-regulated during ZnCl2 additions.20 For N. europaea, a

329

chemolithoautotrophic nitrifier, significant up-regulation of amoA occurred after addition of 30

330

and 90 µM ZnCl2 (1.96 and 5.88 mg/L Zn(II) respectively), while the expression of hao was not

331

significantly inhibited.8 Additionally, both studies suggested that Zn is capable to compete with

332

Cu and replace it in the metal active site in AMO enzyme, thereby inhibiting nitrification.

333

Similar results for amoA were obtained in our study with the exception of hao which was not

334

significantly up-regulated in the previous study.

335

In our study, the impact of Pb exposure, as inferred by sOUR and amoA gene expression,

336

was not statistically different for Pb(II) doses ≤ 100 mg/L Pb (p > 0.05). At 1000 mg/L Pb(II),

337

nitrifying enrichments were significantly inhibited (p < 0.05) based on both sOUR and amoA

338

transcript levels. The addition of Cd(II) resulted in decreased sOUR, however, there was an

339

increase in the transcript levels of amoA, hao, nirK and norB, which is compatible with studies 15 ACS Paragon Plus Environment


340

using N. europaea pure cultures.16 The relative expression of amoA increased in continuously

341

cultured N. europaea cells with each pulse addition of Cd and reached a maximum of 7.6-fold

342

up-regulation after exposure to 60 µM Cd (~6.7 mg/L Cd).16 The overexpression of nirK in N.

343

europaea cells exposed to Cd has been previously reported.5 The up-regulation of amoA

344

observed in the previous study16 and in our study upon Cd exposure suggests that the nitrifying

345

populations may be able to recover from Cd inhibition through the de novo synthesis of AMO

346

enzymes. The preferential synthesis of AMO and HAO mRNAs during energy-limiting

347

conditions has been observed in a previous study, where levels of new AMO and HAO mRNA

348

and de novo AMO enzyme activity correlated with increasing ammonium concentrations.44

349

The influence of heavy metals (e.g. Zn, Ni, Pb, Cd) on nitrifying bacteria at the

350

physiological and transcriptional level is of interest because these metals and their complexes

351

can potentially inhibit nitrification activity by disrupting proteins once they are transported

352

across bacterial cell membranes and interact with proteins functional groups.19,45 The

353

complexation of metals with sulfhydryl functional group has been suggested earlier, and the

354

correspondence between inhibition coefficients and metal sulfide stability constants observed in

355

this study further demonstrates the inhibitory character of the metals. Moreover, active

356

multivalent metal cations may replace essential metals from their metabolic sites and inhibit

357

function of various enzymes. For instance, heavy metals such as Zn have been shown to replace

358

active metal binding cores in enzymes;8,20 Cu2+ and Fe3+ are two redox active multivalent cations

359

present in the active site of AMO. Another possible inhibition mechanism is that the binding of

360

metal cation at a non-active site in the enzyme may alter the structure and function of enzyme.

361

There are limited studies describing the uptake of Cd and Pb by nitrifying bacteria at the

362

molecular level. Nonetheless, they are known to cause nitrification inhibition in pure cultures of


Page 16 of 30

Page 17 of 30


363

AOB and also in activated sludge obtained from WWTPs. In general, it should be considered

364

that heavy metal inhibition to the activated sludge may be influenced by changes in pH,

365

temperature, DO, presence of suspended particles and/or other metals, SRT, and HRT.18,46,47 The

366

aforementioned factors may impact the metal bioavailability and thus strongly alter the observed

367

metal toxicity. Future studies examining the relationship between metal partitioning and

368

microbial toxicity are needed to elucidate the exact mechanism of heavy metal-based nitrification

369

inhibition.

370

Here we applied RNA-based RT-qPCR assays to measure transcript level responses of

371

several functional genes in nitrifying enrichment exposed to different concentrations of heavy

372

metals. The application of RNA-based function specific assays to measure microbial nitrification

373

activity were also employed in previous studies.9,10 While overall the results of these studies

374

show that RNA-based approach may not be a viable indicator of inhibition of nitrification by

375

heavy metals, our results further support that measuring transcript levels may not be directly

376

correlated to responses at the enzyme activity level. Nonetheless, differences in the relative

377

expression of some of these functional genes has been shown to correlate with sOUR rates

378

during imposition of metal inhibition.7 In wastewater treatment systems prone to acute exposure,

379

changes in gene transcription levels may be more dramatic and could supplement sOUR based

380

assays as early-warning indicators to prevent excessive nitrification inhibition. Periodic shocks

381

of heavy metals (or other nitrification inhibitors) at loads much higher than baseline levels are

382

quite common, especially in primarily domestic wastewater treatment plants. As most plants

383

maybe be simultaneously exposed to a mixture of metals, understanding antagonistic or

384

synergistic scenarios will be even more complex and will require a better understanding of

385

mechanistic behavior at the molecular/genetic level.



386

The applicability of sOUR as a sensitive indicator of inhibition has been demonstrated

387

earlier6,7 and in this study. However, it does not allow for discrimination between activities of

388

multiple AOB in mixed populations. Additionally, the metabolic response in a given system to

389

the presence of inhibitory compounds may be largely dependent on characteristics of the

390

bacterial community. Therefore, it is useful to understand how the metabolic pathways (such as

391

transcription of functional genes) of the nitrifying populations can be influenced upon exposure

392

to heavy metals. Another advantage of using expression of key functional genes as a measure of

393

nitrification activity is that this approach could in some cases ‘predict’ impending process upsets

394

or recovery as shown in AOB cultures10 and anaerobic ammonia oxidation reactors previously.48

395

Along this, a critical research need is the ability to identify and quantify inhibition in different

396

steps that involve autotrophic ammonia or nitrite oxidation. Such distinctions are needed when

397

faced with selective inhibitors of nitrification steps. For instance, while certain heavy metals

398

could inhibit ammonia to hydroxylamine oxidation (by displacing essential cations in the active

399

site of amoA), they may not have an impact on hydroxylamine oxidation or nitrite reduction.

400

Such inferences can only be made by targeting functional genes rather than structural genes

401

(such as the 16S rRNA gene). Accordingly, specific biomarkers to quantify the expression of key

402

functional genes involved in nitrification are needed and additional studies are required to

403

implement these molecular approaches in a WWTP scenario.

404

Heavy metal inhibition of nitrifying bacteria has been widely measured in terms of

405

specific ammonia oxidation rates,11,17,18 transcription of several specific genes,8,16,20 as well as by

406

studying whole-genome transcriptional changes.5 However, most of these studies used pure

407

cultures of N. europaea or nitrifying enrichment cultures. By not capturing the diversity of

408

wastewater nitrification systems, the results obtained from pure culture studies may not always


Page 18 of 30

Page 19 of 30


409

reflect the different metal tolerance levels of the various nitrifying populations that co-exist in

410

these systems.49,50 While this may in part explain the range in the reported inhibition values, we

411

must consider that microbial-metal interactions are expected to occur with many other species

412

(i.e., non-nitrifiers) present in any given WWTP. How the latter interactions impact nitrifiers is

413

poorly understood. Consequently, future studies will benefit from examining nitrification

414

inhibition in wastewater using a more holistic (i.e., total community) system approach employing

415

a combination of emerging technologies (metagenomics and metatranscriptomics).

416

417

ASSOCIATED CONTENT

418

Supporting Information. Table of the qPCR primers and probes used in this study and

419

additional details regarding RNA and DNA extraction, qPCR assays and next-generation

420

sequencing processing. This material is available free of charge via the Internet at

421

http://pubs.acs.org.

422

AUTHOR INFORMATION

423

Corresponding Author

424

*E-mail: [email protected]

425

Notes

426

The authors declare no competing financial interest.

427

ACKNOWLEDGEMENTS

428

We are grateful for comments provided by Michael Elovitz. The views expressed in this article

429

are those of the authors and do not necessarily represent the views or policies of the U.S. 19 ACS Paragon Plus Environment


430

Environmental Protection Agency. This research was supported in part by an appointment to the

431

Postdoctoral Research Program at the U.S. Environmental Protection Agency (EPA), Office of

432

Research and Development, Cincinnati, OH, administered by the Oak Ridge Institute for Science

433

and Education through an Interagency agreement between the U.S. Department of Energy and

434

the U.S. Environmental Protection Agency.

435

REFERENCES

436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464

1. Karvelas, M.; Katsoyiannis, A.; Samara, C. Occurrence and fate of heavy metals in the wastewater treatment process. Chemosphere 2003, 53 (10), 1201-1210. 2. Sörme, L.; Lagerkvist, R. Sources of heavy metals in urban wastewater in Stockholm. Sci. Total Environ. 2002, 298 (1), 131-145. 3. Tchobanoglous, G., Burton, F. L., Stensel, H. D., Metcalf & Eddy. Wastewater engineering: treatment and reuse; McGraw-Hill Education, 2003. 4. Hooper, A. B.; Vannelli, T.; Bergmann, D. J.; Arciero, D. M. Enzymology of the oxidation of ammonia to nitrite by bacteria. Anton. van Leeuwen. 1997, 71 (1-2), 59-67. 5. Park, S.; Ely, R. L. Candidate stress genes of Nitrosomonas europaea for monitoring inhibition of nitrification by heavy metals. Appl. Environ. Microbiol. 2008, 74 (17), 5475-5482. 6. Radniecki, T. S.; Dolan, M. E.; Semprini, L. Physiological and transcriptional responses of Nitrosomonas europaea to toluene and benzene inhibition. Environ. Sci. Technol. 2008, 42 (11), 4093-4098. 7. Chandran, K.; Love, N. G. Physiological state, growth mode, and oxidative stress play a role in Cd (II)-mediated inhibition of Nitrosomonas europaea 19718. Appl. Environ. Microbiol. 2008, 74 (8), 2447-2453. 8. Radniecki, T. S.; Semprini, L.; Dolan, M. E. Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions. Biotechnol. Bioeng. 2009, 102 (2), 546-553. 9. Ahn, J. H.; Kwan, T.; Chandran, K. Comparison of partial and full nitrification processes applied for treating high-strength nitrogen wastewaters: microbial ecology through nitrous oxide production. Environ. Sci. Technol. 2011, 45 (7), 2734-2740. 10. Yu, R.; Chandran, K. Strategies of Nitrosomonas europaea 19718 to counter low dissolved oxygen and high nitrite concentrations. BMC Microbiol. 2010, 10 (1), 70. 11. Hu, Z.; Chandran, K.; Grasso, D.; Smets, B. F. Comparison of nitrification inhibition by metals in batch and continuous flow reactors. Water Res. 2004, 38 (18), 3949-3959. 12. Madoni, P.; Davoli, D.; Guglielmi, L. Response of SOUR and AUR to heavy metal contamination in activated sludge. Water Res. 1999, 33 (10), 2459-2464.


Page 20 of 30

Page 21 of 30

465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503


13. Kong, Z.; Vanrolleghem, P.; Willems, P.; Verstraete, W. Simultaneous determination of inhibition kinetics of carbon oxidation and nitrification with a respirometer. Water Res. 1996, 30 (4), 825-836. 14. Elnabarawy, M. T.; Robideau, R. R.; Beach, S. A. Comparison of three rapid toxicity test procedures: Microtox,® polytox,® and activated sludge respiration inhibition. Toxic. Assess. 1988, 3 (4), 361-370. 15. Gernaey, K.; Verschuere, L.; Luyten, L.; Verstraete, W. Fast and sensitive acute toxicity detection with an enrichment nitrifying culture. Water Environ. Res. 1997, 69 (6), 11631169. 16. Radniecki, T. S.; Semprini, L.; Dolan, M. E. Expression of merA, trxA, amoA, and hao in continuously cultured Nitrosomonas europaea cells exposed to cadmium sulfate additions. Biotechnol. Bioeng. 2009, 104 (5), 1004-1011. 17. Hu, Z.; Chandran, K.; Grasso, D.; Smets, B. F. Effect of nickel and cadmium speciation on nitrification inhibition. Environ. Sci. Technol. 2002, 36 (14), 3074-3078. 18. Hu, Z.; Chandran, K.; Grasso, D.; Smets, B. F. Impact of metal sorption and internalization on nitrification inhibition. Environ. Sci. Technol. 2003, 37 (4), 728-734. 19. Nies, D. H. Microbial heavy-metal resistance. Appl. Microbiol. Biotechnol. 1999, 51 (6), 730-750. 20. Radniecki, T. S.; Ely, R. L. Zinc chloride inhibition of Nitrosococcus mobilis. Biotechnol. Bioeng. 2008, 99 (5), 1085-1095. 21. Chipasa, K. B. Accumulation and fate of selected heavy metals in a biological wastewater treatment system. Waste Manage. 2003, 23 (2), 135-143. 22. Üstün, G. E. Occurrence and removal of metals in urban wastewater treatment plants. J. Hazard. Mater. 2009, 172 (2), 833-838. 23. Chandran, K.; Smets, B. F. Applicability of two-step models in estimating nitrification kinetics from batch respirograms under different relative dynamics of ammonia and nitrite oxidation. Biotechnol. Bioeng. 2000, 70 (1), 54-64. 24. Kapoor, V.; Pitkänen, T.; Ryu, H.; Elk, M.; Wendell, D.; Santo Domingo, J. W. Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA-and DNA-Based Quantitative PCR Assays. Appl. Environ. Microbiol. 2015, 81 (1), 91-99. 25. Caporaso, J. G.; Lauber, C. L.; Walters, W. A.; Berg-Lyons, D.; Lozupone, C. A.; Turnbaugh, P. J.; Fierer, N.; Knight, R. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (Supplement 1), 4516-4522. 26. Schloss, P. D.; Westcott, S. L.; Ryabin, T.; Hall, J. R.; Hartmann, M.; Hollister, E. B.; Lesniewski, R. A.; Oakley, B. B.; Parks, D. H.; Robinson, C. J. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 2009, 75 (23), 7537-7541.



504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542

27. Gomez-Alvarez, V.; Revetta, R. P.; Santo Domingo, J. W. Metagenomic analyses of drinking water receiving different disinfection treatments. Appl. Environ. Microbiol. 2012, 78 (17), 6095-6102. 28. Ahn, J. H.; Yu, R.; Chandran, K. Distinctive microbial ecology and biokinetics of autotrophic ammonia and nitrite oxidation in a partial nitrification bioreactor. Biotechnol. Bioeng. 2008, 100 (6), 1078-1087. 29. Martell, A. E.; Smith, R. M. Critical stability constants, Vol. 1; Plenum Press: New York, 1974. 30. You, S. J.; Tsai, Y. P.; Huang, R. Y. Effect of heavy metals on nitrification performance in different activated sludge processes. J. Hazard. Mater. 2009, 165 (1), 987-994. 31. Li, X.; Kapoor, V.; Impelliteri, C.; Chandran, K.; Domingo, J. W. S. Measuring nitrification inhibition by metals in wastewater treatment systems: current state of science and fundamental research needs. Crit. Rev. Environ. Sci. Technol. 2015 (Accepted, in press), DOI:10.1080/10643389.2015.1085234. 32. Juliastuti, S. R.; Baeyens, J.; Creemers, C.; Bixio, D.; Lodewyckx, E. The inhibitory effects of heavy metals and organic compounds on the net maximum specific growth rate of the autotrophic biomass in activated sludge. J. Hazard. Mater. 2003, 100 (1), 271-283. 33. Cenci, G.; Morozzi, G. The validity of the TTC-test for dehydrogenase activity of activated sludges in the presence of chemical inhibitors. Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene. Erste Abteilung Originale. Reihe B: Hygiene, Betriebshygiene, praventive Medizin 1979, 169 (3-4), 320-330. 34. Kong, Z.; Vanrolleghem, P.; Willems, P.; Verstraete, W. Simultaneous determination of inhibition kinetics of carbon oxidation and nitrification with a respirometer. Water Res. 1996, 30 (4), 825-836. 35. Poulsen, L. K.; Ballard, G.; Stahl, D. A. Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms. Appl. Environ. Microbiol. 1993, 59 (5), 1354-1360. 36. Campbell, B. J.; Yu, L.; Heidelberg, J. F.; Kirchman, D. L. Activity of abundant and rare bacteria in a coastal ocean. Proc. Nat. Acad. Sci. 2011, 108 (31), 12776-12781. 37. Bollmann, A.; Schmidt, I.; Saunders, A. M.; Nicolaisen, M. H. Influence of starvation on potential ammonia-oxidizing activity and amoA mRNA levels of Nitrosospira briensis. Appl. Environ. Microbiol. 2005, 71 (3), 1276-1282. 38. Park, S.; Ely, R. L. Genome-wide transcriptional responses of Nitrosomonas europaea to zinc. Arch. Microbiol. 2008, 189 (6), 541-548. 39. Rusk, J. A.; Hamon, R. E.; Stevens, D. P.; McLaughlin, M. J. Adaptation of soil biological nitrification to heavy metals. Environ. Sci. Technol. 2004, 38 (11), 3092-3097. 40. Das, P.; Williams, C. J.; Fulthorpe, R. R.; Hoque, M. E.; Metcalfe, C. D.; Xenopoulos, M. A. Changes in bacterial community structure after exposure to silver nanoparticles in natural waters. Environ. Sci. Technol. 2012, 46 (16), 9120-9128.


Page 22 of 30

Page 23 of 30

543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570


41. Yeung, C. H.; Francis, C. A.; Criddle, C. S. Adaptation of nitrifying microbial biomass to nickel in batch incubations. Appl. Microbiol. Biotechnol. 2013, 97 (2), 847-857. 42. Yu, R.; Lai, B.; Vogt, S.; Chandran, K. Elemental profiling of single bacterial cells as a function of copper exposure and growth phase. PloS one. 2011, 6 (6), e21255. 43. Grunditz, C.; Gumaelius, L.; Dalhammar, G. Comparison of inhibition assays using nitrogen removing bacteria: application to industrial wastewater. Water Res. 1998, 32 (10), 2995-3000. 44. Sayavedra‐Soto, L. A.; Hommes, N. G.; Russell, S. A.; Arp, D. J. Induction of ammonia monooxygenase and hydroxylamine oxidoreductase mRNAs by ammonium in Nitrosomonas europaea. Mol. Microbial. 1996, 20 (3), 541-548. 45. Gadd, G. M.; Griffiths, A. J. Microorganisms and heavy metal toxicity. Microbial Ecol. 1977, 4 (4), 303-317. 46. Battistoni, P.; Fava, G.; Ruello, M. L. Heavy metal shock load in activated sludge uptake and toxic effects. Water Res. 1993, 27 (5), 821-827. 47. Cheng, M. H.; Patterson, J. W.; Minear, R. A. Heavy metals uptake by activated sludge. Journal (Water Pollution Control Federation) 1975, 362-376. 48. Park, H.; Rosenthal, A.; Ramalingam, K.; Fillos, J.; Chandran, K. Linking community profiles, gene expression and N-removal in anammox bioreactors treating municipal anaerobic digestion reject water. Environ. Sci. Technol. 2010, 44 (16), 6110-6116. 49. Juretschko, S.; Timmermann, G.; Schmid, M.; Schleifer, K. H.; Pommerening-Röser, A.; Koops, H. P.; Wagner, M. Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations. Appl. Environ. Microbiol. 1998, 64 (8), 3042-3051. 50. Laanbroek, H. J.; Gerards, S. Competition for limiting amounts of oxygen between Nitrosomonas europaea and Nitrobacter winogradskyi grown in mixed continuous cultures. Arch. Microbiol. 1993, 159 (5), 453-459. 51. Hermansson, A.; Lindgren, P. -E. Quantification of ammonia-oxidizing bacteria in arable soil by real-time PCR. Appl. Environ. Microbiol. 2001, 67 (2), 972-976.

571 572 573 574 575 576 577 578 579 580 581 582 23 ACS Paragon Plus Environment


583

Figure Legends

584

Figure 1. Bioreactor performance as measured by qPCR derived relative abundance of AOB 16S

585

rRNA gene copies and reactor tCOD concentrations.

586

Figure 2. Relative gene transcript concentration of amoA during steady-state operation of the

587

reactor as characterized by > 99% ammonia removal.

588

Figure 3. Bacterial community composition of the nitrifying bioreactor based on 16S rRNA gene

589

sequencing of nitrifying biomass DNA extracts obtained on multiple dates (n=5).

590

Figure 4. Comparison of ammonia oxidation inhibition measured by sOUR as a function of total

591

metal concentration after a 12-h exposure of (a) Ni(II) (0.03 – 3 mg/L), (b) Zn(II) (0.1 – 10

592

mg/L), (c) Cd(II) (0.03 – 1 mg/L) and (d) Pb(II) (1 – 1000 mg/L). Metal effect was measured in two

593

independent experiments for each concentration (experimental duplicates).

594

Figure 5. Relative fold change (in log2 scale) in the transcript levels of amoA, hao, nirK and

595

norB measured by RT-qPCR as a function of metal concentration after 12-h exposure of (a)

596

Ni(II) (0.03 – 3 mg/L), (b) Zn(II) (0.1 – 10 mg/L), (c) Cd(II) (0.03 – 1 mg/L) and (d) Pb(II) (1 –

597

1000 mg/L). Each bar graph represent average of experimental duplicates.


Page 24 of 30

Page 25 of 30


3000 AOB 16S rRNA gene

tCOD 2500

4.00E+06

2000 3.00E+06 1500 2.00E+06 1000 1.00E+06

500

0.00E+00

0 0

5

10

15

20

Time (d)

Figure 1


25

30

tCOD (mg/L)

AOB 16S rRNA gene (copies/ml)

5.00E+06

Page 26 of 30

1

100 amoA/AOB 16S

% ammonia removal

0.8

99.8

0.6

99.6

0.4

99.4

0.2

99.2

0

99 0

5

10

15

20

Time (d)

Figure 2


25

30

Ammonia removal (%)

amoA expression (copies/copies)


Page 27 of 30


Nitrosomonas Alphaproteobacteria Others

Bacteroidetes Gammaproteobacteria

100%

Percent sequences

80%

60%

40%

20%

0% 5

10

15

Time (d)

Figure 3


25

30


Total Ni (mg/L)

a

0.3

Total Zn (mg/L) 3

90 80 70 60 50 40 30 20 10 0 -10

0.1

Inhibition (%)

Inhibition (%)

0.03

b

10

Total Pb (mg/L) 3

1

Inhibition (%)

Inhibition (%)

c

90 80 70 60 50 40 30 20 10 0 -10

0.3

1

90 80 70 60 50 40 30 20 10 0 -10

Total Cd (mg/L) 0.03

Page 28 of 30

d

10

90 80 70 60 50 40 30 20 10 0 -10

Figure 4


100

1000

Page 29 of 30


Relative fold change

amoA

hao

norB

2 1 0 -1 -2 0.03

0.1

0.3

1

3

3

10

Ni (mg/L)

a Relative fold change

nirK

3 2 1 0 -1 -2 0.1

0.3

1 Zn (mg/L)


b

6 4 2 0 -2 0.03

0.1

0.3

1

100

1000

Cd (mg/L)


c 3

2 1 0 -1 -2 -3 1

10 Pb (mg/L)

d

Figure 5



TOC Graphic


Page 30 of 30

Impact of Heavy Metals on Transcriptional and ... - ACS Publications

Recommend Documents