Rapid and strong biocidal effect of ferrate on sulfidogenic and methanogenic sewer biofilms

Journal Pre-proof Rapid and strong biocidal effect of ferrate on sulfidogenic and methanogenic sewer biofilms Xiaofang Yan, Jing Sun, Ahezhuoli Kenjiahan, Xiaohu Dai, Bing-Jie Ni, Zhiguo Yuan PII:

S0043-1354(19)30982-0

DOI:

https://doi.org/10.1016/j.watres.2019.115208

Reference:

WR 115208

To appear in:

Water Research

Received Date: 29 March 2019 Revised Date:

22 September 2019

Accepted Date: 15 October 2019

Please cite this article as: Yan, X., Sun, J., Kenjiahan, A., Dai, X., Ni, B.-J., Yuan, Z., Rapid and strong biocidal effect of ferrate on sulfidogenic and methanogenic sewer biofilms, Water Research (2019), doi: https://doi.org/10.1016/j.watres.2019.115208. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical Abstract

1

Rapid and Strong Biocidal Effect of Ferrate on Sulfidogenic and

2

Methanogenic Sewer Biofilms

3 4

Xiaofang Yan1, Jing Sun1,2,*, Ahezhuoli Kenjiahan1, Xiaohu Dai1,2,, Bing-Jie Ni1,2,*,

5

Zhiguo Yuan3

6 7

1. State Key Laboratory of Pollution Control and Resources Reuse, College of

8

Environmental Science and Engineering, Tongji University, Shanghai 200092, P.R.

9

China

10

2. Shanghai Institute of Pollution Control and Ecological Security, Shanghai200092,

11

P.R. China

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3. Advanced Water Management Centre (AWMC), The University of Queensland, St.

13

Lucia, QLD 4072, Australia

14 15

*Corresponding Authors:

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Jing Sun, Phone: +86 21 65986849; E-mail: [email protected]

17

Bing-Jie Ni, Phone: +86 21 65986849; E-mail: [email protected]

18

1

19

Abstract

20

For the control of sulfide and methane in sewers, it is favorable to reduce their

21

production rather than to remove them after generation. In this study, we revealed

22

rapid and strong biocidal effect of ferrate (Fe(VI)) on sulfidogenic and methanogenic

23

sewer biofilms, leading to control of sulfide and methane production in sewer. The

24

inactivation of the microorganisms in sewer biofilms by Fe(VI) could be

25

accomplished within 15 min for a single dosing event and the biocidal effect could be

26

enhanced by applying pulse dosing strategy. The microbiological analysis showed that

27

the key functional genes involved in sulfide and methane production, i.e. dsrA and

28

mcrA, in the viable cells after Fe(VI) dosing were decreased substantially by 84.2%

29

and 86.6%, respectively. Significant drops were also observed in the relative

30

abundances of viable sulfide reducing bacteria (SRB) and methanogenic archaea

31

(MA). The direct dosing of Fe(VI) into a sewer reactor led to instant and complete

32

suppression of sulfidogenic and methanogenic activities, and the recovery of the

33

activities resembled the regrowth of residual SRB and MA. The results of this study

34

suggested the feasibility for developing an efficient and cost-effective sulfide and

35

methane control strategy using Fe(VI).

36 37

Keywords: Sewer biofilm; Ferrate; Biocidal effect; Sulfate-reducing bacteria;

38

Methanogenic archaea;

39

2

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1.Introduction

41

Sulfide-induced sewer corrosion and odor have long been considered as major

42

problems in sewer management. The rehabilitation or replacement of corroded sewer

43

pipes always brings heavy financial burdens on water industries and local

44

governments (Pikaar et al. 2014). Meanwhile the malodor caused by sulfide emission

45

can arouse plenty of complaints from the neighborhoods (Jiang et al. 2017, Sun et al.

46

2015b). In addition, high concentration of hydrogen sulfide in sewers may pose health

47

risks to related workers due to its toxicity. A common practice to control sulfide in

48

sewers to date is the dose of chemicals (Jiang et al. 2015a). For example, oxidants

49

such as oxygen/air, nitrate and hydrogen peroxide are usually added into sewers to

50

oxidize sulfide. Metal salts, mainly including ferrous chloride and ferric chloride, are

51

also frequently applied to precipitate sulfide in sewers. Elevation of pH by dosing

52

alkali is another commonly used method to reduce sulfide emission from sewer.

53

However, most of the chemical dosing strategies for sulfide control in sewers involve

54

constant chemical addition to remove sulfide already formed, and therefore resulting

55

in high operational cost due to large chemical consumptions (Ganigue et al. 2011).

56 57

Methane emission from sewers has also been recognized as a potential environmental

58

issue since the last decade. Methane is a potent greenhouse gas with around 21 times

59

global warming potential of carbon dioxide (IPCC 2006). Its emission from sewers

60

could contribute considerably to the overall greenhouse gas emissions from

61

wastewater systems (Guisasola et al. 2008, Liu et al. 2015). Even worse, the release of

62

methane from sewers can pose serious safety risks because of its relatively low

63

explosion limit (lower explosive level is approximately 5%) (Spencer et al. 2006).

64

Moreover, the loss of soluble COD due to methanogenic activity may cause adverse

3

65

effect on biological nutrient removal at the downstream wastewater treatment plants

66

(WWTPs) (Guisasola et al. 2008, Sun et al. 2015a). Therefore, the control of methane

67

emission should also be included in the sewer management.

68 69

To control the sulfide and methane in sewer systems effectively and economically, it

70

is favorable to reduce sulfide and methane production rather than to remove them

71

after their production. In fact, the production of sulfide and methane in sewer is

72

mainly caused by the metabolism of microorganisms in biofilms attached on the

73

sewer pipe walls. Specifically, under the anaerobic conditions, sulfate-reducing

74

bacteria (SRB) in sewer biofilms use various kinds of organic matters and hydrogen

75

as electron donors and sulfate as electron acceptor to produce sulfide (Li et al. 2018).

76

Besides, methane can be generated through the anaerobic respiration of methanogenic

77

archaea (MA), using hydrogen and acetate as common substrates (Sun et al. 2014).

78

Therefore, suppressing the metabolic activities of microorganisms in sewer biofilms,

79

especially these of SRB and MA, is of vital importance for establishing cost-effective

80

sulfide and methane control strategies in sewers.

81 82

To this end, antimicrobial agents, including metabolic inhibitors and broad-spectrum

83

biocides have been under investigation. Chueng and Beech (1996) studied the effect

84

of three different biocides, i.e. formaldehyde, glutaraldehyde and isothiozolone on

85

sessile SRB. They found that activities of SRB biofilm were decreased by 74%-100%

86

after exposure to these three compounds at 400 mg/L for 24 hours. Similarly, Gardner

87

and Stewart (2002) reported that a dose of glutaraldehyde at 500 mg/L for 7 h could

88

completely suppress the sulfide production by a mixed-culture biofilm. Molybdate, a

89

metabolic inhibitor for SRB, has also been used to control sulfide production in swine

4

90

manure, municipal solid wastes and anaerobic digesters (Isa and Anderson 2005,

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Zahedi et al. 2014). Nevertheless, the application of these chemicals in sewer systems

92

could have adverse impact on the downstream WWTPs and receiving water due to

93

their generic toxicity and low biodegradability. Recent studies revealed that free

94

nitrous acid (FNA) was able to decrease the number of viable microorganisms in

95

sewer biofilms substantially, thus suppressing the sulfide and methane production

96

(Jiang et al. 2011). However, to ensure a satisfactory biocidal effect of FNA, a long

97

exposure time (6-24 hours) was often required. It is therefore desirable to develop a

98

new chemical dosing strategy to inactivate sewer biofilm in a more rapid and safe

99

way.

100 101

Ferrate (Fe(VI)), a high-valent tetraoxy iron, has recently come to the forefront as a

102

multifunctional water-treatment chemical (Chen et al. 2018a, Chen et al. 2018b).

103

Fe(VI) could be used as oxidant and disinfectant in water and wastewater treatment,

104

leaving nontoxic Fe(III) oxides/hydroxides after its application, which initiates the

105

process of coagulation (Sharma et al. 2015). In particular, Fe(VI) can effectively kill

106

various bacteria and viruses, such as Escherichia coli, Sphaerotilus, Bacillus,

107

Salmonella and bacteriophage MS2 (Cho et al. 2006, Jiang et al. 2007, Sharma 2007) .

108

It has been observed that Fe(VI) could achieve 99.9% kill rates of total coliforms at

109

the dosage of 0.5-12.5 ppm in water sources collected worldwide (Sharma 2007). In

110

addition, a pioneering study on biofilm showed that a ferrate(VI) concentration of 10-5

111

M can effectively prevent biofilm growth on a condenser with only 5-min contact

112

time every 12 hours (Fagan and Waite 1983). The strong disinfection effect of Fe(VI)

113

suggested the possibility of its use to control the metabolic activities of

114

microorganisms in sewer biofilms within short contact time. Also, as the use of Fe(VI)

5

115

does not produce any mutagenic/carcinogenic by-products (Sharma et al. 2015), its

116

application in sewer system could avoid adverse impact on water environment.

117

Therefore, an in-depth understanding of the biocidal effect of Fe(VI) on the

118

microorganisms situated in sewer biofilms would have great practical significance for

119

sulfide and methane control in sewer management.

120 121

The aim of this study is to investigate the effect of Fe(VI) on sulfidogenic and

122

methanogenic sewer biofilms. The effects of Fe(VI) concentrations, exposure time,

123

dosing modes and pH on the viability of microorganisms in the biofilm were

124

evaluated. The differences in abundances of functional genes of viable cells related to

125

sulfide and methane production and microbial community in the biofilm with and

126

without Fe(VI) treatment were also explored by means of propidum monoazide

127

treatment combined with real-time polymerase chain reactions (PMA-qPCR) and

128

high-throughput sequencing, respectively. In addition, direct dosing of Fe(VI) into the

129

sewer reactor was conducted for assessing its effects on sulfide and methane

130

production activities. The results of this study are expected to lay a fundamental basis

131

for developing efficient and cost-effective sulfide and methane control strategies by

132

applying Fe(VI).

133 134

2. Materials and methods

135

2.1 Sewer reactor setup and operation

136

lab-scale sewer reactors, namely R1, R2 and R3, made of PerspexTM, were set up in

137

parallel to develop sulfidogenic and methanogenic sewer biofilms under anaerobic

138

conditions (Figure S1, Supplementary Information). The reactor has a diameter of 80

139

mm and a height of 200 mm, resulting in an effective volume of 1 L. A small reservoir 6

140

is connected to the top of the reactor to ensure air tightness. Three strings of plastic

141

carriers of approximately 1 cm in diameter were mounted in the reactors to provide

142

additional surface for biofilm growth and to allow the sampling of intact biofilms. The

143

total biofilm area in each reactor, including the inner wall, inner surface of the lid and

144

the carriers, was approximately 0.1 m2, resulting in the area to 100 m2/m3.

145 146

The reactors were fed with real wastewater collected every three days from a wet well

147

in Shanghai, China. The sewage typically contained sulfide at concentration of 1-2

148

mg-S/L, sulfate at concentration between 50-60 mg-S/L, and volatile fatty acid (VFA)

149

at 50-100 mg-COD/L. After the collection, the wastewater was stored immediately in

150

a freezer at 4 oC to minimize the change of water composition. It was then heated up

151

to 20 oC and fed to the reactors through a peristaltic pump every 6 h. During each

152

pumping event, 1 L of wastewater was transferred into the reactor over two minutes.

153

The wastewater in the reactor was mixed with a magnetic stirrer (200 rpm) to produce

154

a moderate shear force and avoid solids settling at the bottom.

155 156

Batch tests were conducted every two weeks to measure the sulfide and methane

157

production activities of each reactor. At the start of the tests, fresh wastewater was

158

pumping into reactors for 6 min to ensure a thorough replacement of liquid in the

159

reactors. Wastewater was sampled at 0, 20, 40, 60 min after the pumping events to

160

analyze sulfide, sulfate and dissolved methane concentrations using method described

161

in Section 2.7. Sulfide and methane production rates (SPR and MPR) were calculated

162

using linear regression of sulfide and methane concentrations. The following

163

experiments were commenced when all three reactors reached the pseudo-steady state,

164

as suggested by the relatively stable SPR and MPR.

7

165 166

2.2 Viability tests on the biocidal effect of Fe(VI)

167

Three sets of viability tests (i.e. Experiment I-III) were carried out to investigate the

168

biocidal effects of Fe(VI) on the sulfidogenic and methogenic sewer biofilms. The

169

Fe(VI) concentration, exposure time, initial pH and dosing mode applied in each test

170

were listed in Table 1. In Experiment I, the effect of Fe(VI) concentration and

171

exposure time on cell viability in biofilms were evaluated. The Fe(VI) concentrations

172

applied on the biofilms varied from 0 to 200 mg-Fe/L and the exposure time ranged

173

from 15 to 60 min, while the initial pH of the wastewater remained unadjusted at ~7.5.

174

The Experiment II explored the effect of pH on the biocidal effect of Fe(VI). In the

175

test, the Fe(VI) concentration was kept at 120 mg-Fe/L, while the initial pH of the

176

wastewater varied from 5-9. The exposure time was kept at 60 min. Experiment III

177

was designed to assess that if pulse dosing mode could enhance the biocial effect of

178

Fe(VI) on the sewer biofilm. In this study, a high-concentration dosing event was spilt

179

into three low-concentration dosing events, with the concentration and exposure

180

duration for each event listed in Table 1.

181 182

The tests were carried in the 50 mL tubes filled with filtered (0.22 µm) wastewater.

183

The pH of wastewater was adjusted by HCl (1 M) when necessary according to the

184

experimental design (Table 1). A plastic carrier with attached biofilm was transferred

185

from the biofilm reactor R1 into each tube. Different volume of stock solution of

186

K2FeO4 (1 g-Fe/L) was added to each tube to achieve the designed Fe(VI)

187

concentrations according to Table 1. Then the tubes were capped to avoid contacting

188

of air and gently mixed by an orbital shaker at 60 rpm. After a certain duration of

189

incubation used in each set of tests as described in Table 1, the biofilm on the carriers 8

190

were sampled for LIVE/DEAD staining, using the method illustrated in Section 2.4.

191

Also, PMA-qPCR and Illumina Miseq sequencing were performed to evaluate the

192

effect of Fe (VI) on the functional genes for sulfide and methane production as well as

193

microbial community in the sewer biofilm, with the method to be further described.

194 195

2.3 Direct Fe(VI) dosing to sewer biofilm reactors

196

Fe(VI) was directly added to the lab-scale sewer reactor R2 and the loss of sulfide and

197

methane production activities was monitored after Fe(VI) dosing during subsequent

198

recovery period. The Fe(VI) dosing strategy in the reactor (Fe concentration and

199

exposure time, dosing mode) was deterimined based on the above vialibility tests

200

results. Specifically, K2FeO4 was immidiately dosed into the reactor after a pumping

201

event to reach a Fe(VI) concentration of 60 mg-Fe/L. The dosing event was repeated

202

for another two times in every 15 min, resulted in a total Fe(VI) dosage of 180

203

mg-Fe/L. The batch tests for determining SPR and MPR in the reactor as described in

204

Section 2.1 were conducted immediately after the Fe(VI) treatment and in the next 60

205

days with intervals of two days to two weeks. The SPR and MPR of R3 (without

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Fe(VI) dosing) were also monitored during the same period, serving as a control. In

207

addition, the volatile suspended solids (VSS) in the influent and effluent of the

208

reactors were also monitored to assess the effect of Fe(VI) dosing on integrity of the

209

biofilm structure.

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2.4 LIVE/DEAD staining

212

The viability of bacterial cells in biofilms was determined using the LIVE/DEAD®

213

Baclight™ bacterial viability kits (Molecular Probes, L-7012). The viability kit

214

involves two nucleic acid stains, namely green-fluorescent SYTO-9 and 9

215

red-fluorescent Propidium Iodide (PI). The SYTO-9 could label all cells while PI

216

could only penetrate cells with damaged membrane and resulted in a reduction in the

217

the SYTO-9 stain fluorescence. As a result, the viable cells are stained green, whereas

218

the dead cells are stained red.

219 220

Before the staining process, the plastic carriers were transferred to filtered (0.22 µm)

221

wastewater and the biofilms were detached through vigorous vortex mixing

222

(VORTEX-5, Kylin-bell®). The staining of cells was conducted based on protocols

223

provided by the manufacturer. Basically, 3 µl mixture of SYTO-9 and PI (volume

224

ratio 1:1) was added into 200 µl of biofilm suspension and mixed thoroughly. The

225

sample was incubated at room temperature in the dark for 15 min. Then 5 µl of the

226

stained biofilm suspension was transferred to a microscope slide and photographed

227

using a fluorescence microscope (Eclipse 80i, Nikon Corp.,Japan) with a halogen

228

lamp (30W, 6V) (Boulos et al. 1999, Hu et al. 2017). Twenty images of randomly

229

chosen areas of the stained biofilm samples were taken for quatification. The ratio of

230

live and dead microorganisms was deterimined by the relative abundance of green and

231

red pixels via the image analysis software, DAIME (version 1.3).

232 233

2.5 PMA-qPCR

234

PMA-qPCR analysis was conducted to evaluate the effect of Fe(VI) on the abundance

235

of functional genes involved in sulfide and methane production in the viable

236

microorganisms in sewer biofilms. PMA can selectively enter dead cells and inhibit

237

the DNA amplification during subsequent qPCR. As a result, only abundacne of genes

238

in living cells will be quantified. The biofilm treated by pulse dosing of Fe(VI) at 60

239

mg-Fe/L for three times as described in Section 2.2 and the biofilms without Fe(VI) 10

240

treatment were examined for this purpose. The abundances of key genes in SRB and

241

MA , i.e. dissimilatory sulfite reductase (dsrA) and methyl-coenzyme M reductase

242

(mcrA) genes were tested. In addition, the 16S rRNA genes of total bacteria and total

243

archaea in living cells were also quantified and compared. The primers used for each

244

targeting gene were listed in Table S1, SI.

245 246

The PMA-qPCR analysis was carried out in the following steps. Firstly, the biofilms

247

were detached from the carries in PBS using the method described in Section 2.4.

248

Subsequently, the samples were centrifuged and resuspended in PBS to allow the VSS

249

concentrations of biofilm suspensions in the recommened range for PMA

250

cross-linking (Tian et al. 2017). The PMA treatment of the samples was then carried

251

out based on protocal described by Taskin et al. (2011) and the details of the protocal

252

were illustrated in SI. Afterwards, DNA extractions were conducted using

253

TIANNAMP Soil DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s

254

instruction. The qPCR was then performed using the StepOnePlus™ real-time PCR

255

dectection system, with the reaction condition further described in SI. Each PCR

256

reaction was run in triplicate for quality assurance and statistical analysis purposes.

257 258

2.6 High-throughput sequencing analysis

259

The high-throughput sequencing was conducted on the same biofilm sample used for

260

PMA-qPCR analysis to explore the effect of Fe(VI) dosing on the viable microbial

261

community struture of sewer biofilm. The samples were firstly undergone with PMA

262

treatment to ensure only viable cells were analyzed. Then, DNA was extracted and a

263

pair of universal primes 515FmodF (5'-GTGYCAGCMGCCGCGGTAA-3') and

264

806RmodR (5'-GGACTACNVGGGTWTCTAAT-3') designed to target the variable 11

265

regions V4-V5 of the microbial 16S rRNA gene was applied for PCR amplification

266

(Walters et al. 2016). The sequencing was performed at Majorbio Bio-pharm

267

Technology Co., Ltd, Shanghai, China on Illumina HiSeq2500 platform. Date analysis

268

was implemented on the online i-sanger sever (http://www.i-sanger.com) of Majorbio

269

Bio-pharmTechnology Co., Ltd. Detailed procedures of the analysis were described in

270

SI. The raw reads were deposited into the NCBI Sequence Read Archive (SRA)

271

database (Accession Number: SRP186539).

272 273

2.7 Chemcial analysis

274

The sulfate concentration was measured by an ion chromatograph with a conductivity

275

dector (DIONEX ICS 1000) and the sulfide concentration was determined by the

276

methylene blue method (Sharma et al. 1997). The wastewater samples were firstly

277

filtered (0.22 µm membrane) before sulfate and sulfide measurement. Dissolved

278

methane concentration in wastewater was analyzed using gas chromatograpy

279

equipped with flame ionization detector (GC-FID) (Shimadzu GC 2010 plus) based

280

on the method previously reported by Sturm et al. (2015). The detection limit of the

281

method is 0.4 µg/L and the recovery ratio is 93%. VFA concentration was measured

282

using gas chromatography (Shimadzu GC 2010 plus) and the VSS of the wastewater

283

were measured according to standard methods (APHA 1998).

284 285

3. Results and discussion

286

3.1 Rapid biocidal effect of ferrate on the sewer biofilm

287

The effects of Fe(VI) concentration and exposure time on the microorganisms’

288

viability in sewer biofilms were illustrated in Figure 1. The results showed that the

12

289

viable cells in biofilms dropped significantly from ~82% to ~35% with the increase of

290

Fe(VI) concentration from 0 to 120 mg-Fe/L at all tested Fe(VI) exposure time.

291

Whereas, a further increase of Fe(VI) dosage to 200 mg-Fe/L did not decrease the

292

viability of microorganisms in biofilms anymore. More importantly, the results

293

showed no significant difference (P>0.05) in the percentage of viable cells in the

294

biofilms with the exposure time decreased from 60 min to 15 min at all tested Fe(VI)

295

dosing rates (Figure 1A). This indicated that Fe(VI) could achieve substantial

296

inactivation rate of microorganisms in sewer biofilms with relatively high efficiency,

297

i.e. within 15 min. The dependency of the percentage of viable microorganisms in the

298

biofilm on the ferrate concentration under different exposure times could be

299

satisfactorily described using the exponential decay model y =

300

shown in Figure 1(B). The estimated parameters with the standard errors were listed

301

in Table S2, SI. The high correlation between experimentally measured results and

302

model predicts (R2>0.95) in all three cases further confirmed that there was no

303

significant difference in microorganism viabilities under different Fe(VI) exposure

304

times.

+

as

305 306

The biocidal effect of Fe(VI) is believed to mainly rely on its oxidation capacity. The

307

redox potential of Fe(VI) (E0=2.20 V) is the highest among the chemicals used for

308

water and wastewater treatment (Jiang 2014). The strong oxidation capacity of Fe(VI)

309

enables it to pose damaging effect on cell wall, protoplasm, genome and other vital

310

microorganism organs, thus leading to the instantaneous death of the microorganisms

311

(Talaiekhozani et al. 2017). In addition, reactive oxygen species of O2 - and H2O2

312

could also be produced during the decomposition of Fe(VI), which could also

313

contribute to the rapid biocidal effect of Fe(VI) (Hu et al. 2012). The high inactivation

13

314

efficiency of Fe(VI) on microorganisms revealed in this study was in accordance with

315

those observed by other researchers. For example, Jiang et al. (2007) found that a

316

Fe(VI) dosage of 8 mg/L can achieve 90% -100% inactivation efficiency on E. coli in

317

model water samples, with exposure times between 5-30 min. Also, it has been

318

reported that exposure time required to obtain 2-log10 inactivation of bacteriophages

319

f2 and Qβ was only ~5 min (Hu et al. 2012). It is worthwhile to note that the

320

minimum exposure time used in this study was 15 min. It is also likely that the

321

exposure time less than 15 min could also achieve the same biocidal effect on the

322

sewer biofilms, which required further investigation.

323 324

In this study, the minimal Fe(VI) dosing amount (through a single dosing event) to

325

achieving lowest viability of microorganisms in sewer biofilms was 120 mg-Fe/L,

326

which was much high than that required for drinking water and wastewater

327

disinfection (~10-50 mg-Fe/L) (Jiang et al. 2006, Talaiekhozani et al. 2017). One

328

possible reason is that the abundance of microorganisms in the sewer biofilm was

329

much higher than that in drinking water or treated wastewater. It may also attribute to

330

the penetration limitation caused by the biofilm matrix (Sun et al. 2014). The

331

microorganisms in drinking water or freshwater were mostly in suspension, so that

332

Fe(VI) can easily get access to them. However, the microorganisms in sewer biofilms

333

were aggregated within a slimy extracellular matrix, which could hamper the

334

chemical penetration. Therefore, a higher dosing rate is required for Fe(VI) to reach

335

the microorganisms located in the inner layer of the biofilms. Moreover, the reactions

336

between Fe(VI) and organic matters in the wastewater are also likely to weaken the

337

biocidal effect of Fe(VI) on sewer biofilm (Deng et al. 2018, Fan et al. 2018). Notably,

338

a further increase of Fe (VI) dosing rate to 200 mg/L did not significantly reduce the

14

339

microbial viability in sewer biofilms. This is possibly due to that Fe(VI) could be fast

340

self-decomposed under high concentration, resulted in a similar effective Fe(VI)

341

amount acting on the biofilm (Lee et al. 2014). Therefore, in order to enhance the

342

biocidal effect of Fe(VI), other approaches rather than simply increasing the Fe(VI)

343

dosing rate should be explored (See details in Section 3.2).

344 345

3.2 Enhancing the biocidal effect of ferrate through pulse dosing strategy

346

The pulse dosing strategy (i.e. split a single high rate dosing event into three low rate

347

dosing events, Table 1) was applied to explore if it could enhance the biocidal effect

348

of Fe(VI) on the sewer biofilms. The viability of the microorganisms in sewer

349

biofilms after pulse dosing was compared with that obtained after a single high rate

350

dosing and the results were summarized in Figure 2. The results clearly show that the

351

percentage of viable microorganisms in sewer biofilms can be significantly reduced

352

by applying the pulse dosing strategy with different Fe(VI) dosing rates from low to

353

high. In particular, the viability of the microorganisms decreased from 61.5% with a

354

single dosing of 60 mg-Fe/L to 36.8% with three dosing of 20 mg-Fe/L. Also, the

355

percentage of viable microorganisms reduced from 41.4% to 33.1% when a single

356

dosage of 120-mg Fe/L was divided into 40 mg-Fe/L for three times dosing. When

357

applying a dosage of 60 mg-Fe/L for three times (180 mg-Fe/L in total) on the biofilm,

358

the overall viability of the microorganisms could further reduced to 17.6%, which is

359

only about half of that achieved with a single dosage of 200 mg-Fe/L.

360 361

The results above suggested that the biocidal effect of Fe(VI) on the sewer biofilm

362

could be reinforced by applying pulse dosing strategy. During the Fe(VI) treatment,

363

its self-decomposition is recognized as an unfavorable factor to its treatment 15

364

effectiveness. It has been reported that the self-decomposition rate of Fe(VI) followed

365

the second-order kinetics with respect to Fe(VI) concentration (Lee et al. 2014, Sarma

366

et al. 2012). This suggested that Fe(VI) will be decomposed more rapidly with a

367

higher initial concentration. Therefore, if the pulse dosing of Fe(VI) with a low

368

concentration was applied, the fast self-decomposition of Fe(VI) under high

369

concentration conditions could be avoid. Based on the second-order kinetics, it could

370

be estimated that the maximum lasting duration of Fe(VI) in the system could be

371

increased about 3 time if a high-rate dosing event being divided into three low-rate

372

dosing event. With a potential longer lasting duration, the actual Fe(VI) loadings

373

taking effect on the biofilm may be increased. As a result, the biocidal effect of Fe(VI)

374

could be enhanced. In addition, as Fe(VI) has been found to change the extracellular

375

polymeric substances (EPS) components in sewage sludge (Zhang et al. 2016), it may

376

also be able to react with the EPS of sewer biofilm and resulted in a loose structure

377

after the first or second dosing event. Therefore, the penetration depth of Fe(VI) into

378

the biofilm could increase during the following treatment and its biocidal effect was

379

consequently improved. The highest inactivation rate achieved by Fe(VI) dosing

380

revealed in this study (viability of 17.6%) was slightly lower than that achieved by

381

FNA dosing (viability <15%) as reported by Jiang et al (2011). However, it should be

382

noted that the optimzation of the pulse dosing strategy was not included in this study.

383

It is quite possible that the viability of the microorganisms in biofilms could further

384

decreased after optimizing the Fe(VI) dosing rate and frequency, which is worthy to

385

be investigated in future.

16

386 387

3.3 The effect of pH on the biocidal effect of Fe(VI)

388

The viability of the microorganisms in biofilms treated with Fe(VI) (120 mg-Fe/L for

389

60 min) at different initial pH was evaluated, with the results presented in Figure 3.

390

Overall, the pH of the wastewater did not significantly affect the viability of the

391

microorganisms at the tested range. The drop of pH from 9 to 6 slightly decreased the

392

percentage of the viable microorganisms in sewer biofilms, while a more notable

393

decrease from 36.3% to 28.0% was observed when the pH is changing from 6 to 5.

394 395

It has been reported that the effectiveness of Fe(VI) treatment under acidic conditions

396

is much better than that under basic conditions (Shin et al. 2018). However, in this

397

study, we did not observe of significant enhancement in biocidal effect of Fe(VI) on

398

sewer biofilm when the pH changing from 9 to 5. This is probably due to the increase

399

of pH of wastewater to basic range after Fe(VI) dosing (Figure S2). Actually, the

400

effectiveness of Fe(VI) treatment was also related to the proportion of different Fe(VI)

401

species in the water. Fe(VI) can be disassociated into different species (FeO42-,

402

HFeO4- and H2FeO4) according to the pH, and the protonated forms of Fe(VI)

403

(HFeO4- and H2FeO4) were found to be more reactive than FeO42- (Cho et al. 2006).

404

The fraction of different Fe(VI) species in the experiments in this section was

405

calculated based on the pH profiles and its disassociation constants (pKa1=3.50 and

406

pKa2=7.23). The results show that the average proportion of HFeO4- was changed in a

407

narrow range between 0.4% to 5 % (Figure 3), resulted in the unobvious enhancement

408

in the biocidal effect of Fe(VI). In fact, in most cases, the inactivation of

409

microorganisms by protonated Fe(VI) was achieved under buffered condition

410

(phosphate buffer) (Cho et al. 2006, Hu et al. 2012). However, to maintain the pH of

17

411

wastewater with phosphate buffer would induce great amount of phosphate into the

412

sewer system. It may consequently increase the burden of phosphorus removal on the

413

downstream WWTPs. As phosphorus is an essential nutrient for organisms, without

414

proper treatment, its discharged could cause eutrophication in receiving water (Jiang

415

and Yuan 2015). Since Fe(VI) could already pose considerable biocidal effect on the

416

sewer biofilm under natural pH through pulse dosing stratagy, to enhance its biocidal

417

effect by adjusting the pH of wastewater is unnecessary.

418

419

3.4 Fe(VI) reduces the abundances of functional genes responsible for sulfate

420

reduction and methanogensis

421

The abundances of bacterial 16S rRNA, archaeal 16S rRNA, dsrA and mcrA genes in

422

the viable cells in sewer biofilms with and without Fe(VI) treatment (60mg Fe/L,

423

three times dosing) were compared in Figure 4. The bacterial 16S rRNA and archaeal

424

16S rRNA genes decreased by 74.4% and 84.2%, respectively, indicating that archaea

425

are more vulnerable to Fe(VI) treatment than bacteria. Overall, the decreases of total

426

viable bacteria and archaea in the sewer biofilms revealed by PMA-qPCR analysis

427

were in good agreement with that suggested by Live/Dead Staining (Figure 2), which

428

further confirmed the strong biocidal effect of Fe(VI) on the sewer biofilm.

429 430

The abundances of dsrA and mcrA genes were also decreased significantly after Fe(VI)

431

treatment. As shown in Figure 4, the abundance of dsrA in the viable microorganism

432

with Fe(VI) treatment was only 15.8% of that without Fe(VI) treatment and the

433

abundance of mcrA remained only 13.4% after Fe(VI) exposure. The dsrA gene

434

encodes the dissimilatory sulfate reductase, which catalyses the dissimilatory

435

reduction of sulfate to sulfite during sulfidogenic processes (Ben-Dov et al. 2007).

18

436

Similarly, mcrA is a key gene responsible for methanogenesis. It encodes the terminal

437

enzyme complex in the methane generation pathway, i.e. methyl coenzyme-M

438

reductase, which catalyses the reduction of a methyl group bound to coenzyme-M,

439

with the concomitant release of methane (Luton et al. 2002). The decreases of dsrA

440

and mcrA abundances in sewer biofilms indicated its sulfidogenic and methanogenic

441

activities were strongly inhibited by Fe(VI) dosing.

442 443

3.5 Relative abundances of viable SRB and MA decreasing after Fe(VI) exposure

444

The microbial communities with viable cells in the sewer biofilms with and without

445

Fe(VI) treatment (60mg-Fe/L, three times dosing) were evaluated through Illumina

446

Miseq sequencing and compared in Figure S3, SI, with their alpha diversity indices

447

listed in Table S3, SI. The relative abundances of viable SRB and MA in sewer

448

biofilms were explored in detail and compared in Figure 5. As shown in Figure 5A,

449

the SRB in sewer biofilms were mainly affiliated within seven genera

450

(Desulforhabdus, Desulfobacter Desulfococcus Desulfobacterium Desulfobulbus

451

Desulfomonile Desulfomicrobium), with Desulforhabdus (1.7%) being predominant in

452

the untreated biofilm. After Fe(VI) treatment, the abundances of five SRB genera

453

(Desulforhabdus, Desulfobacter Desulfococcus Desulfobacterium Desulfomicrobium)

454

were decreased, while the relative abundances of Desulfobulbus and Desulfomonile

455

increased slightly. However, the relative abundances of total SRB were still decreased

456

by 63% as presented in Figure 6B, which indicated that SRB in the biofilms were

457

more sensitive to Fe(VI) treatment than other microorganisms. This is probably due to

458

that SRB are located in the out layer of the sewer biofilm (Sun et al. 2014), where the

459

Fe(VI) concentration was higher than that in the inner layer.

460

19

461

Similarly, the relative abundance of MA was significantly decreased by 90% after

462

Fe(VI) treatment (Figure 5B). Specifically, the proportions of three MA genera found

463

in

464

Methano-methylovorans) were all dropped, with the Methanobacterium became

465

almost negligible after Fe(VI) dosing (Figure 5A). MA usually showed a lower

466

tolerance than bacteria to many chemicals such as oxygen, sulfide and long-chain

467

fatty acids, due to their special compositions of cell membrane (Dong et al. 2019).

468

This is also true in the case of Fe(VI) as suggested by the results of this study.

the

tested

biofilm

(i.e.

Methanobacterium,

Methanosaeta

and

469 470

The decreases of relative abundances of SRB and MA in the sewer biofilm were

471

consistent with that revealed by PMA-qPCR, as numbers of dsrA and mcrA genes

472

decreased more substantially than total bacterial 16S rRNA gene. These results

473

indicated that Fe(VI) posed strong biocidal effect on SRB and MA in sewer biofilms.

474

The complete suppression of sulfate reduction and methane production in sewer

475

biofilms could be achieved even when not all microorganisms were inactivated.

476 477

3.6 Suppression of sulfidogenic and methanogenic activities after Fe(VI) dosage to

478

sewer reactors

479

The sulfide and methane production rates in the Fe(VI) dosed sewer reactor R2

480

relative to the control reactor R3 were shown in Figure 6A. The sulfide and methane

481

production were completely suppressed after Fe(VI) dosage commerced on Day 0.

482

Afterwards, the SPR and MPR were slowly recovered without significant lag phase.

483

The recovery phase could be well described by the Gompertz growth model (Huang

484

2003), suggested that the recovery process was similar to microbial regrowth.

485

According to the model prediction, the time required for 50% recovery (RT50) of SPR 20

486

was 10.1 days, while the RT50 of MPR would took much longer 44.5 days. The RT50

487

of SPR and MPR after Fe(VI) dosage were comparable to that after FNA dosage as

488

reported by Jiang et al.(2011), despite that the viability of the biofilm after Fe(VI)

489

treatment observed in this study was slightly higher. One possible reason is that Fe(VI)

490

dosing may destory the EPS produced by the microorganisms situated in the sewer

491

biofilm and consequently result in a loose biofilm structure (Wu et al. 2015, Zhang et

492

al. 2016), Fe(VI) dosing could cause significant biofilm detachment in the reactor, as

493

suggested by a substaintial increase in the VSS concentration of the effluent after

494

Fe(VI) dosage (Figer 6B). Therefore, the viable microganisms attached on the sewer

495

reactor became less and the recovery period was prolonged.

496 497

3.7 Practical implication

498

This study, for the first time, demonstrated the rapid and strong biocidal effect of

499

Fe(VI) on the sulfidogenic and methanogenic sewer biofilms. The inactivation of

500

microorganims in the sewer biofilm could be accomplished within 15 mins for every

501

single dosing. By innovatively applying the pulse dosing strategy, the biocidal effect

502

could be significantly enhanced and satisfactory inactivation efficiency could be

503

realized. The detailed investigation into SRB and MA revealed decreases in both

504

functional genes copies and relative abundances after Fe(VI) exposure. This indicated

505

that SRB and MA might be more vulnerable to Fe(VI) exposure than other

506

microorganisms in sewer biofilms. All these results indicated that Fe(VI) has a

507

promising perspective in controlling sulfide and methane in sewer systems, and thus a

508

new method for sulfide and methane control using Fe(VI) can be proposed.

509 510

The traditional chemical strategies for sulfide control in sewer mainly rely on the

21

511

oxidation, precipitation and reducing gas-liquid transfer of sulfide already generated

512

and constant dosing is required (Ganigue et al. 2011). The biocidal effect of Fe(VI) on

513

the sewer biofilm can result in a long recovery period for sulfide and methane

514

production, which makes interminttent dosing of Fe(VI) possible. Consequently, the

515

chemical dosing amount could be largely reduced. Besides, as the wastewater in

516

sewers are usually flowing, continous dosing is needed to ensure the exposure time

517

long enough for the inactivation process. The short exposure time required for Fe(VI)

518

treatment suggested that chemcial usage for each dosing event is low, which further

519

guarantees the low chemcial consumption and also improves the ease of operation.

520 521

There are many other potential advantages regarding the Fe(VI) dosing. The reduction

522

of Fe(VI) would produce nontoxic Fe(III) hydroxides, which could precipitate sulfide

523

in sewers at the downstream location (Zhang et al. 2009). Adding iron salts in sewers

524

could also be beneficial to phosphorus removal at the downstream WWTP, where iron

525

sulfide precipitates are oxidised in aeration tanks, regenerating iron phosphate

526

precipitates (Gutierrez et al. 2010). A more recent study also demonstrated iron salts

527

dosed in sewers could decrease sulfide generation in sludge digestions and promote

528

the dewatering performance of anaerobic digested sludge. (Rebosura et al. 2018)

529 530

During the practical application of Fe(VI), its relatively high price used to be a

531

concern. However, based on the intermittent dosing stragegy and short exposure time,

532

the total cost for the chemical is expected to be low. We estimated the chemical cost

533

of Fe(VI) dosing to achieve 80% sulfide control is $ 0.02/m3 or $1.6/kg-S, based on

534

the results in Section 3.5 (total Fe(VI) dosage of 180 mg-Fe/L with an exposure time

535

of 45 min and a dosing interval of 4.5 days for achieving sulfide control efficiency of

22

536

80%). It is much lower than the chemcial costs of many other chemical dosing

537

methods, such as using ferric/ferrous and nitrate (Table S4, SI). Also, studies have

538

suggested that Fe(VI) could be generate in situ through wet chemical method by the

539

reactions of Fe(III) oxides or their salts with hypochlorite (OCl−) in highly alkaline

540

solution (Waite 2012). Alternatively, it could be produced through electrochemical

541

synthesis with the use of iron electrodes (Jiang et al. 2015b, Nikolić-Bujanović et al.

542

2016). By applying these methods, the costs for the chemcial consumption might be

543

further reduced.

544 545

The effects of Fe(VI) dosing on the wastewater characteristics, such as the COD

546

decrease and pH elevation, were not included in this study, which should be fully

547

evaluated in the future. However, it should be noted that Fe(VI) would not be added to

548

the entire sewer network at the same time. By adding Fe(VI) to different sections of a

549

network at different times, the impacts on wastewater characteristics at the

550

downstream WWTPs could be alleviated and the pH would be neutralized. Therefore,

551

further optimization of the Fe(VI) dosing strategy at the network scale should be

552

carried out, to achieve effective sulfide and methane control in sewer with the

553

minimal chemical comsumption rate and avoiding potential adverse impact on

554

downstream WWTPs. Overall, the intermittent dosing of Fe(VI) with pulse dosing

555

strategy can be a cost-effective strategy for sewer corrosion, odour and greenhouse

556

gas control, which bears great application potential in the sewer management.

557 558

4. Conclusions

559

This study evaluated the biocidal effect of Fe(VI) on the sulfidegenic and

560

methanogenic sewer biofilm and the following conclusions can be drawn; 23

561

Fe(VI) has a rapid biocidal effect on the microorganisms in the sewer biofilm,

562

with the inactivation process achieved within 15 min through a single dosing.

563

The biocidal effect of Fe(VI) on the sewer biofilm could be enhanced through

564

pulse dosing strategy and is not affected by the pH of the wastewater.

565

Fe(VI) significantly decreased the functional genes responsible for sulfidogenesis

566

and methanogenesis and also reduced the relative abundances of SRB and MA in

567

sewer biofilms.

568

The rapid and strong biocidal effect of Fe(VI) on the sewer biofilm suggested that

569

it could be intermittently added for controlling sulfide and methane production in

570

sewers, which is a cost-effective strategy for sewer corrosion, odour and

571

greenhouse gas control.

572 573

Acknowledgement

574

This work was partially supported by the Recruitment Program of Global Experts,

575

China; the National Natural Science Foundation of China (51578391, 51608374,

576

51978492 and 51538008); the Program for Young Excellent Talents in Tongji

577

University, the Fundamental Research Funds for the Central Universities (No.

578

2016KJ012) and the State Key Laboratory of Pollution Control and Resource Reuse

579

Foundation, China (No. PCRRK18007).

580

581

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582 583 584 585 586 587 588

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740 741 742

28

Table and Figure Legends

743 744

Table 1. The experimental conditions used in viability tests.

745 746

Figure 1. (A) Percentage of viable microorganisms in biofilms after Fe(VI) treatment

747

with different concentrations and exposure times. (B) The dependency of viable

748

percentages in biofilms on Fe(VI) concentration. Symbols represent for the

749

experimental measurements and the line represents for the modelling result with a

750

3-parameter exponential decay model.

751 752

Figure 2. Microbial viability after being exposed to Fe(VI) with different dosing

753

modes and concentrations, the total reaction time was 60 min.

754 755

Figure 3. Effect of pH on the biocidal effect of Fe(VI) on the sewer biofilm.

756 757

Figure 4. Quantification of bacterial 16S rRNA, archaeal 16S rRNA, dsrA and mcrA

758

genes in the sewer biofilms with and without Fe(VI) treatment (60 mg-Fe/L, three

759

times dosing).

760 761

Figure 5. Relative abundances of different SRB and MA genera (A) and total SRB

762

and MA (B) in the sewer biofilms with and without Fe(VI) treatment (60 mg-Fe/L,

763

three times dosing).

764 765

Figure 6. (A) Sulfide and methane production rates of the reactor treated by Fe(VI),

766

relative to the corresponding control reactor rates. (B) Variation of volatile suspended

767

solids in the effluent of the reactor treated with Fe(VI).

29

Table 1 The experimental conditions used in viability tests Experiment No.

I

II

III

Exposure time (min)

pH

Dosing mode

[Fe(VI)](mg-Fe /L)

15

Original pH

Single doing

30

Original pH

Single doing

60

Original pH

Single doing

0, 30, 60, 90, 120, 150, 200 0, 30, 60, 90, 120, 150, 200 0, 30, 60, 90, 120, 150, 200

60

5, 6, 7, 8, 9

Single dosing

120

60

Original pH

Single dosing,

60, 120, 200

20 for each dosing

Original pH

Three times dosing

20, 40, 60

Figure 1. (A) Percentage of viable microorganisms in biofilms after Fe(VI) treatment with different concentrations and exposure times. (B) The dependency of viable percentages in biofilms on Fe(VI) concentration. Symbols represent for the experimental measurements and the line represents for the modelling result with a 3-parameter exponential decay model.

Figure 2. Microbial viability after being exposed to Fe(VI) with different dosing modes and concentrations, and the total reaction time was 60 min.

Figure 3 Effect of pH on the biocidal effect of Fe(VI) on the sewer biofilm.

Figure 4. Quantification of bacterial 16S rRNA, archaeal 16S rRNA, dsrA and mcrA genes in sewer biofilms with and without Fe(VI) treatment (60 mg-Fe/L, three times dosing).

Figure 5. Relative abundances of different SRB and MA genera (A) and total SRB and MA (B) in the sewer biofilm with and without Fe(VI) treatment (60 mg-Fe/L, three times dosing).

Figure 6. (A) Sulfide and methane production rates of the reactor treated by Fe(VI), relative to the corresponding control reactor rates, (B) Variation of volatile suspended solids in the effluent of the reactor treated with Fe(VI).

Highlights

• • • • •

Fe(VI) could pose a rapid biocidal effect on the sewer biofilm. The biocidal effect of Fe(VI) could be enhanced by pulse dosing. The relative abundances of viable SRB and MA dropped with Fe(VI) dosing. Fe(VI) led to complete suppression of sulfidogenic and methanogenic activities. Fe(VI) dosing strategy is cost-effective for sulfide and methane control in sewers.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: