Electrical stimulation improves microbial salinity-resistance and ...

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Mar 27, 2015 - accordance with an enrichment of halophilic organisms in the BES. ... recommended for salinity wastewater treatment (5, 6), high salinity may ...
AEM Accepted Manuscript Posted Online 27 March 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.04066-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Electrical stimulation improves microbial salinity-resistance and

1

organofluorine removal in bioelectrochemical systems

2 3 4

Huajun Feng1,2,3, Xueqin Zhang1, Kun Guo3, Eleni Vaiopoulou3, Dongsheng Shen1,2,

5

Yuyang Long1,2, Jun Yin 1,2, Meizhen Wang1,2*

6 7 8

1

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Hangzhou 310012, China; 2Zhejiang Provincial Key Laboratory of Solid Waste

10

Treatment and Recycling, Hangzhou 310012, China; 3Laboratory of Microbial

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Ecology and Technology, Ghent University, Coupure Links 653, B-9000, Ghent,

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Belgium

School of Environmental Science and Engineering, Zhejiang Gongshang University,



Corresponding author. Mailing address: School of Environmental Science And Engineering,

Zhejiang Gongshang University, Hangzhou 310012, China. Tel.: +86 571 87397126; fax: +86 571 87397126. E-mail address: [email protected] 1

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ABSTRACT: Fed batch bioelectrochemical systems (BESs) based on electrical

14

stimulation were used to treat p-fluoronitrobenzene (p-FNB) wastewater at high

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salinities. At a NaCl concentration of 40 g/L, p-FNB was removed 100% in 96 h in

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the BES, whereas in the biotic control (BC) (absence of current), p-FNB removal was

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only 10 %. By increasing NaCl concentrations from 0g/L to 40 g/L, defluorination

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efficiency decreased around 40% in the BES and in the BC it was completely ceased.

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p-FNB was mineralized by 30% in the BES and hardly in the BC. Microorganisms

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were able to store 3.8 and 0.7 times more concentration of K+ and Na+ intracellularly

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in the BES rather than in the BC. Following the same trend, the ratio of protein to

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soluble polysaccharide increased from 3.1 to 7.8 as the NaCl increased from 0 to 40

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g/L. Both trends rise speculations that on an electrical stimulation driving microbial

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preference of K+ and protein accumulation to tolerate salinity. These findings are in

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accordance with an enrichment of halophilic organisms in the BES. Halobacterium

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dominated in the BES by 56.8% at the NaCl concentration of 40 g/L, while its

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abundance was found as low as 17.5% in the BC. These findings propose a new

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method of electrical stimulation to improve microbial salinity-resistance.

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INTRODUCTION

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Organofluorine compounds, especially fluorinated aromatic compounds, are

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widely used in the production of adhesives, pesticides, dyes, pharmaceuticals,

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refrigerants, and surfactants (1). They were found to inhibit enzymes, modify

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cell-to-cell communication, and disrupt membrane transport as well as energy

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generation processes (2). Due to their high toxicity and recalcitrance, conventional

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biological methods fail to efficiently remove organofluorine from wastewaters (1, 3).

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On the other hand, bioelectrochemical treatment has been proved to be an effective

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method

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p-fluoronitrobenzene-contaminated organofluorine wastewater (4).

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Salinity

to

poses

minimize

another

refractory

serious

challenge

properties

to

the

of

treatment

typical

of

such

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organofluorine-containing wastewater. Typically, strong acid or alkali addition to

41

adjust the pH during production processes results in high salt concentrations in

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organofluorine wastewaters. As an example, salinity concentrations from an

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organofluorine industry effluent in China typically fluctuate between 2 and 3 %, to a

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maximum of 5 %. Conventional physicochemical treatment processes for salinity

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wastewater are energy-intensive and costly. Although biological processes have been

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recommended for salinity wastewater treatment (5, 6), high salinity may cause cell

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plasmolysis and even the death of microorganisms due to osmotic pressure increase (7,

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8). To address this issue, wastewaters are always diluted, which results in a

49

meaningless fresh water consumption and increases operational cost (5). An

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enhancement of microbial salinity-resistance would enable implementation of

3

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biological methods for salinity wastewater treatment, which could be succeeded by

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acclimatization and enrichment of haloduric or halophilic strains (5). However,

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gradual increase of salinity to enrich halophilic microorganisms showed that it is not

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such an easy task (5, 7).

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Application of an electrical stimulation to stimulate microbial respiratory

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processes as well as microbial consortia evolution has been applied for

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waste-treatment and remediation (9, 10). An electrical stimulation can directly or

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indirectly play a role depending on whether there is hydrogen/oxygen evolution or

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soluble mediator involvement. Direct effect refers to energy gain for organisms

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attached via a biofilm or not on the electrode surface from that electrode. On the

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contrary, indirect effect involves electron transfer from a working electrode to

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microorganisms either through a soluble mediator or a gas (usually hydrogen or

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oxygen produced by electrolysis of water) (9). Poising electrodes at specific potentials

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has been shown to activate key metabolic enzymes for degradation of recalcitrant

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compounds. Zhang et al. (11) found that application of a fixed potential induced

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microbial

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2,4-dichlorophenoxyacetic acid was biologically degraded. In agreement, electrical

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stimulation has been also shown to spur microbial growth (12) and enhance the cell

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density (13). Cell density of bacterium Enterobacter dissolvens was found to be

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significantly improved and microbial activity was increased two-fold (14). More

71

studies show that electrical stimulation may result in specific community evolution

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that are able to adapt to unique environments (15) or in the development of specific

oxidoreductase

production

4

for

electron

transfer

when

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microbial functions (16). These findings open new horizons for the application of

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electrical stimulation to facilitate degradation of recalcitrant compounds in harsh

75

environments.

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On this frame, objectives of this study were to investigate 1) the effects of direct

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electrical stimulation in the absence of both hydrogen evolution and soluble mediator

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on microbial salinity-resistance; 2) whether the adverse effects of high salinity could

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be ameliorated; 3) to characterize the microbial community that is able to

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organofluorine removal in BESs under high salinity.

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MATERIALS AND METHODS

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Microbial inoculum and growth medium

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Activated sludge obtained from a chemical industrial wastewater treatment plant in

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Linhai (Zhejiang, China) was used as the inoculum of the biocathode. Total

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Suspended Solids (TSS) was measured approximately 3000 mg·L-1 and the ratio of

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Volatile Suspended Solids to Total Suspended Solids (VSS/TSS) was 55%. The basic

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nutrient medium used in the anode and cathode chambers contained 3.4 g L−1 K2HPO4,

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4.4 g L−1 KH2PO4, 0.1 g L−1 NH4Cl, 2 g L−1 NaHCO3, 0.24 g L−1 MgSO4·7H2O, and

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trace elements, as previously described (17). A series amount of NaCl was added to

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the medium to adjust the salinity namely 0, 15, 30, and 40 g/L, giving final salinities

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of about 1%, 2.5%, 4% and 5%, respectively. p-FNB was added to the cathode

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chamber with an initial concentration of 0.4 mmol L−1.

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Bioelectrochemical experiment

94

Studies were carried out at 30°C in potentiostat-poised, dual-chambered

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bioelectrochemical systems (BESs) as previously described (18) (Supporting

96

Information, Fig. S1). Nafion 117 (DuPont) proton exchange membrane was used to

97

separate the anode and cathode chambers. Graphite felt (Beijing Sanye Carbon Co.,

98

Ltd., Beijing, China) was used as both anode and cathode electrode. No additional

99

mediators were introduced to the anodic and cathodic electrolyte. The working

100

volume of the cathode chamber was 100 mL and the headspace was 41 mL. The BES

101

core was a biocathode and cathode compartment was unsealed so that ambient air

102

could enter the headspace through the sampling ports on the top of reactors. The

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bioelectrochemical reactors were operated in batch mode and with a hydraulic

104

retention time of 4 days. An external power source (1.8 V) was applied to the abiotic

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reactors and BESs, with the cathode potential kept at about -0.78V vs (Ag/AgCl).

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This potential was chosen to avoid H2 evolution under each salinity condition.

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Two types of control reactors were operated under identical conditions, namely

108

abiotic control (AC) (without bacteria) and biotic control (BC) (no applied voltage).

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The performance of BES, AC, and BC were compared at different salinities for

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kinetic experiments and confirmatory experiments (Supporting Information, Table S1).

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The p-FNB removal efficiency for each cycle was determined by measuring the

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p-FNB concentration of the effluent from the reactor. Steady state conditions were

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reached when the difference of p-FNB removal efficiency among 3 consecutive

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batches was less than 5%. Then, sampling took place to determine p-FNB removal,

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defluorination and mineralization.

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Chemicals and Analytical Methods

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p-FNB (99% purity) was purchased from Aladdin Chemical Ltd. (Shanghai,

118

China). Methanol used was of HPLC grade, while all other chemicals were of

119

analytical grade. Samples were taken from reactors using a 10 mL syringe and were

120

filtered through a 0.22 µm filter. The p-FNB and fluoride ion concentrations were

121

determined using a HPLC (Waters Corp., Milford, MA, USA) and a IC plus ion

122

chromatograph (Metrohm AG, Herisau, Switzerland), with methods described in

123

previous publication (4). The total organic carbon (TOC) content was measured using

124

a TOC analyzer (Shimadzu, Kyoto, Japan).

125

The

primary

characteristic

metabolites

were

identified

using

an

126

Agilent 6890N GC/5975B MSD (Agilent, Corp. USA) according to the previously

127

published method (19). VSS and TSS were determined according to standard methods

128

(20). Total protein and soluble polysaccharide were determined in sludge extracts.

129

Cellular lysates of sludge were obtained from a washed sample by adding fresh

130

normal saline and sonicating it to 20 MHz ultrasound (using a Sonics Ultra Cell

131

instrument; Sonics and Materials, Inc., Newtown, CT, USA) at a power of 97.5 W for

132

5 s each time, 50 times, with a 10 s gap between each time. Then cellular lysates were

133

used for total protein and soluble polysaccharide determination, according to the

134

methods described respectively by Lowry et al. (21) and the phenol–sulfuric method

135

described by Dubois et al. (22).

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Sludge-intracellular ion concentration of sodium and potassium was determined

137

by ion release extracellularly by boiling to cause cell rupture. The steps of this process

138

included the following sequence; A 10 mL aliquot of the well-mixed sludge was

7

139

collected and washed with magnesium chloride solution, the concentration of which

140

was determined by the principle of keeping the osmotic pressure of magnesium

141

chloride solution similar to that of medium where sludge was fed. The washed sludge

142

was collected in 25 mL ultrapure water and intracellular ions were extracted by

143

boiling in a water bath for 20 min. Then the K+ and Na+ concentrations were

144

measured by an atomic absorption spectrophotometry (ZEEnit700, Analytik Jena,

145

Germany).

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Microbial Community Analysis

147

At the end of kinetic performance experiments (96 d), sludge samples were

148

collected from BES and BC reactors for community structure analysis. The 16S rRNA

149

gene sequences of dominant bacterial population at high salinity (40 g/L NaCl)

150

determined in this study were deposited in the GenBank database under accession

151

numbers KP091460-KP091467 and KP893254-KP893255. Details of the genomic

152

DNA extraction, PCR amplification and statistical analyses are given in the

153

supplementary data.

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RESULTS AND DISCUSSION

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Effects of Salinity on p-FNB Treatment

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p-FNB Removal. In general, the p-FNB removal efficiency in different systems

157

followed the order BES > AC > BC, in spite of variations in salinity (Fig. 1). BESs

158

are previously reported to remove recalcitrant pollutants more efficiently than

159

conventional electrochemical treatments because of microbial catalysis (10, 23). In

160

agreement, these results suggest that biological activity is well maintained in the BES,

8

161

even at high salinity.

162

When the NaCl concentration increased from 0 to 15 g/L, the constant of p-FNB

163

removal rate (kp-FNB) in the BES increased from 0.106 h-1 to 0.125 h-1, and declined to

164

0.103 h-1 when the concentration sequentially increased to 40 g/L (Table 1). However,

165

kp-FNB in the AC consistently increased from 0.041 h-1 to 0.074 h-1 as salinity increased.

166

This can be attributed to the fact that increase of salinity improves solution

167

conductivity, which further faciltates electron generation and transportation in the

168

circuit. This concept applies also to the BES, as our previous study revealed that

169

p-FNB was readily metabolized in the BES due to interaction between

170

microorganisms and the electrode (4). On one hand, p-FNB removal rate in the BES

171

grew with the salinity increase. On the other hand, inhibition to microbial activity was

172

aggravated at the same time, resulting in a decreased p-FNB removal (Fig. 1). This

173

implies that there must be a critical salinity concentration above which the microbial

174

degradation of p-FNB is inhibited.

175

p-FNB removal efficiency in the BC declined significantly from 85 % to 10 % at

176

96 h as the NaCl concentration increased from 0 g/L to 40 g/L (Fig. 1a and 1d). These

177

findings indicated severe inhibition of the microbial metabolism caused by high

178

salinity damage. A decrease of 88 % in p-FNB removal efficiency was observed in the

179

BC as the NaCl concentration increased from 0 g/L to 40 g/L, while the decrease was

180

only 3.1% in the BES, indicating a relief from hyperhaline inhibition in the BES.

181

p-FNB Defluorination. The effects of salinity on defluorination performance were

182

also shown in Fig. 1. BES exhibited the highest defluorination efficiency among all

9

183

tested systems, and the fluoride ion accumulation in 4 days decreased from

184

0.249±0.005 mmol/L to 0.149±0.020 mmol/L as the NaCl concentration increased

185

from 0 g/L to 40 g/L.

186

Electrochemical defluorination in the AC and BC was found to be weak at all

187

salinities whereas fluoride ion concentration was always increasing in the BES (Fig.

188

1). As the NaCl concentration increased from 0g/L to 40 g/L, defluorination efficiency

189

decreased by 40.2% (from 62.2% to 37.3%) in the BES, while it was totally inhibited

190

(from 27.1% to 0) in the BC. These findings indicate that microorganisms in the BES

191

formed a better capacity to resist salinity and degrade target pollutants than those in

192

the BC.

193

To theoretically evaluate the chemical form of fluorine speciation for p-FNB

194

degradation, a mass balance of fluorine was performed by quantifying fluoride ion

195

from p-FNB, p-FA and free fluoride ion from other intermediates (Supporting

196

Information, Fig. S2). In the AC, the fluoride ion accounted for a very small

197

proportion (less than 10%) of the total fluorine under all salinity conditions. A

198

previous report has proved that the direct electrochemical reduction of nitroaromatics

199

tends to generate intermediates such as nitrosobenzene, azobenzene, and

200

azoxybenzene, which are more toxic or resistant to biodegradation (24).

201

Nitroaromatics have the same functional group of nitro as p-FNB, thus, the large

202

proportion of other unspecified intermediates for p-FNB degradation may indicate

203

that there were considerable side-reactions in the AC.

204

The fluoride ion was the dominant fluorine species in the BES. At 96 h, the

10

205

maximum percentage of 62% fluorine ion was produced at 0 g/L NaCl, and then it

206

gradually decreased to around 37% when the NaCl concentration increased to 40 g/L

207

(Supporting Information, Fig. S2). Additionally, the unspecified fluorine species

208

percentage went up as salinity increased. It reached a peak value of 11.5% at 0 g/L

209

NaCl, whereas at the highest salinity of 40 g/L NaCl, it reached a maximum value of

210

about 43%. These results suggest that biological defluorination in the BES is inhibited

211

by high salinity, and that undesired side-reactions exacerbated this, which does not

212

promote p-FNB degradation.

213

p-FNB Mineralization. TOC removal efficiency was measured to determine the

214

extent of p-FNB mineralization. As shown in Fig. 2, when the NaCl concentration

215

increased from 0 g/L to 40 g/L, TOC removal efficiency both in the BES and BC

216

gradually decreased from 69% to 30% and from 22% to 3.3%, respectively. On the

217

contrary, salinity gradient had a positive effect on TOC removal efficiency in the AC,

218

as increased from 7.9% to 13.0%.

219

The TOC removal performance in the tested systems was found to be different

220

but was consistent with the defluorination rate regardless of the system type or salinity.

221

Considering the strengthening effect of fluorine substituent and nitro group on the

222

recalcitrant properties of p-FNB, a high nitro reduction and rapid defluorination rate

223

can contribute to an enhanced mineralization performance in the BES. As described

224

above, defluorination in the BC was tremendously inhibited at high salinity, which

225

was also observed in the case of p-FNB mineralization. At 40 g/L NaCl, the TOC

226

removal efficiency in the BES was about 83% higher than the sum of both efficiencies

11

227

in the BC and AC, demonstrating that BES is a good alternative technology for

228

hyperhaline wastewater treatment.

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Electrical Stimulation on Biological Salinity-Resistance

230

Biomass characterization. High salinity inhibited microbial metabolism resulting in

231

cell plasmolysis and even in lethal effects on microorganisms, which directly

232

impacted biomass concentrations. Fig. 3 shows that TSS in the BES always remained

233

constant at the inoculation level of about 3000 mg/L, whereas TSS gradually declined

234

in the BC as salinity increased, reaching a loss of about 35% at 40 g/L NaCl.

235

Simultaneously, VSS/TSS ratio decreased to 37% and 27% in the BES and BC, with a

236

corresponding maximum loss of 32% and 51%, respectively (Fig. 3). These findings

237

support our assumption that despite high salinity inhibition, organisms in the BES are

238

less vulnerable to salinity variations.

239

Effect of intracellular Na+ and K+ role on salinity resistance. High osmotic

240

pressure stems from the density gradient between the intracellular and extracellular

241

environment. The strategy of “salt-in” is considered an important mechanism to resist

242

high-salinity that in principple is based on the ability of many organisms to uptake

243

inorganic ions from the extracellular environment and accumulate salts in high

244

concentrations within their cells to balance osmotic pressure (25). As sodium and

245

potassium were the dominant cations present in the medium of the extracellular

246

environment, their intracellular concentrations were investigated at different salinities

247

to verify whether they were accumulated or not.

248

Although

the

extracellular

ion

density

12

should

influence

intracellular

249

osmoregulation in theory (25), under an electrical field, initial concentrations of Na+

250

and K+ in the extracellular medium are not constant, because these two cations may

251

move from the anode to the cathode chamber. In this case, microorganisms in the

252

cathode chamber may experience different salinity effects from the ones expected. To

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eliminate this concern, their concentration was determined in the bulk and it was

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found that Na+ and K+ in the anode and the cathode chamber were maintained at

255

almost their initial level due to low current applied (Supporting Information, Fig. S3).

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As shown in Table 2, intracellular K+ and Na+ concentrations in the BES and

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BC both tended to increase as the NaCl concentration increased from 0 g/L to 40 g/L.

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Intracellular K+ concentration in the BES increased almost 20 times (from 0.042 to

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0.817 mmol/g SS), which is much higher than that of intracellular Na+ concentration

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(5.5 times). At the maximum NaCl concentration of 40 g/L, the intracellular K+

261

concentration in the BES is 3.8 times higher than that in the BC, while the

262

intracellular Na+ concentration was only 0.7 times higher. These results indicate that

263

K+ was preferentially accumulated by microorganisms in the BES to regulate osmotic

264

pressure. K+ accumulation contributes to the maintenance of cellular activities at high

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salinity. Microorganisms can directly adjust the cytoplasm concentration by K+

266

accumulation to regulate osmotic pressure; besides extracellular K+ may promote the

267

accumulation of some other soluble organic osmoticum, thus regulating osmotic

268

pressure indirectly (26, 27). Accordingly, maintaining a higher intracellular K+ here is

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likely a useful strategy for inducing salinity-resistance in the BES.

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Effect of protein and soluble polysaccharide on salinity resistance. Most haloduric

13

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microorganisms prefer to accumulate some soluble intracellular organics as a counter

272

measure to balance the osmotic pressure; this is another strategy so-called

273

“compatible-solute” (28). Microbial protein and polysaccharides are the typically

274

preferred intracellular organics, whose variation can help elucidate the mechanisms of

275

salinity tolerance. Results showed that the concentrations of total protein and soluble

276

polysaccharide in the BC remained relatively stable within the salinity variation

277

(Table 3). However, when the NaCl concentration increased from 0 to 40 g/L, the

278

protein and soluble polysaccharide intracellular concentrations in the BES increased

279

by 3 and 1.3 times, respectively. This suggests that microorganisms can be electricity

280

driven to accumulate intracellularly organics so that they can regulate osmotic

281

pressure. Moreover, the ratio of protein to soluble polysaccharide increased from 3.1

282

to 7.8 as the salinity increased, implying that microorganisms in the BES relied

283

strongly on protein accumulation to adapt to high salinity.

284

Microbial community structure

285

Further confirmatory experiments indicated that a specific microbial community with

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the ability to mineralize p-FA and resist to high salinity is likely to be selected by a

287

long-term electrical stimulation (Supporting Information, Fig. S4). Thus, microbial

288

community structure in terms of bacteria and archaea dynamics were investigated

289

here.

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Bacteria dynamics

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Compared with the inoculum, Bacteroidetes, Chloroflexi, Firmicutes and

292

Spirochaetae were enriched and their abundance was improved as the salinity

14

293

increased in the BES (Supporting Information, Fig. S5a). Bacteroidetes, Chloroflexi,

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Firmicutes and Spirochaetae have been reported to be common and abundant during

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the treatment of high-salinity wastewater (6, 29, 30, 31), suggesting that these bacteria

296

may play specific roles in salinity-resistance in the BES for p-FNB treatment. The

297

dominant phyla in the BES were also observed to be abundant in the BC, but at

298

different proportions, demonstrating that haloduric bacteria surviving in the BC

299

showed considerable homology to those enriched in the BES.

300

Classes

of

Alphaproteobacteria,

Betaproteobacteria,

Anaerolineae,

301

Deltaproteobacteria, VandinHA17, Spirochaetes, Clostridia and Bacteroidia were

302

enriched compared with the inoculum and became relatively dominant in the BES

303

(Supporting Information, Fig. S5b). These classes were also found to be relatively

304

abundant in the BC, and bacterial consortia at the class level showed considerable

305

homology. Moreover, the initial microbial community shift with and without electrical

306

stimulation was mostly based on the proportional imparity of each population.

307

16S rRNA gene sequences of microbial consortia at 40 g/L NaCl in the BES

308

were clustered into OTUs, and the dominant as well as unique bacterial OTUs are

309

listed in Table 4. The most dominant bacterial OTU population was close to

310

uncultured Spirochaeta sp. (6.9%), which was once isolated from an oil field, growing

311

optimally with a NaCl concentration of 5% and being adapted to a variety of

312

substrates (32). This optimal NaCl concentration was similar to the salinity conditions

313

in our experiments. Finding of the same species under similar condition shows that

314

Spirochaeta sp. may play an important role in pollutant mineralization at high salinity.

15

315

Additionally, an anaerobic MO-CFX2 bacterium (6.7%), known to specifically

316

degrade halogenated aromatic compounds (33), was also uniquely detected in the

317

BES.

318

Archaea dynamics

319

Evolution in the archaea consortia can be demonstrated by variations in the

320

archaea community structure. As shown in Fig. 4a, archaea classes in all samples

321

consisted initially of Halobacteria and Methanomicrobia. As the NaCl concentration

322

increased from 0 g/L to 40 g/L, the abundance of Halobacteria in the BES increased

323

from 24.2% to 66.4%, while Methanomicrobia abundance dropped from 59.3% to

324

0.2%. However, the relative proportion of the two populations showed opposite

325

tendencies in the BC, with Halobacteria and Methanomicrobia abundances of 17.5%

326

and 65.6%, respectively, being observed at 40 g/L NaCl.

327

Halobacterium is the dominant archaeum genus in the BES, and its abundance

328

changed with variations in salinity (Fig. 4b). With salinity increased, Halobacterium

329

abundance ascended from 17.4% to 56.8% in the BES, whereas it descended from

330

65.5% to 17.5% in the BC. Halobacteria is a typical halophilic archaeum with

331

specifically adaptive capacity for hyperhaline environments (34). Phylogenetic

332

Halobacterium is a type of Halobacteria that has been extensively investigated for its

333

ability to live in saline environments and its roles in saline wastewater treatment

334

processes (31, 35). A significant community shift and the proportion decreasing of

335

Halobacterium at 40 g/L NaCl in the BC indicated that BS was severely impacted by

336

salt damage, which complies with VSS/TSS decrease in the BC. On the contrary, the

16

337

same population was further enriched at high salinity in the BES, which was most

338

probably an adaptation outcome. The same Halobacterium population exhibited

339

different adaptive behavior to salinity in the BES and BC, indicating that different

340

abilities to tolerate salinity evolved in the two systems. Given the system differences,

341

this discrepancy is likely caused by the effect of electrical stimulation. Halobacterium

342

has been shown to store K+ and simultaneously excrete Na+ to regulate high osmotic

343

pressure (36). This strategy involves active transport of K+ and is dependent on energy

344

consumption. Electrical stimulation might provide microbial energy through a specific

345

strategy of electron transport (9, 10). Thus, K+ uptake was probably stimulated in the

346

BES, and salinity-resistance was thus promoted.

347

Implications of practice

348

BES improved microbial salinity-resistance and enhanced organofluorine removal.

349

The improved performance of the BES was attributed to direct electrical stimulation.

350

Based on the microbial metabolism and community evolution results, two possible

351

mechanisms of electrical stimulation mechanisms were proposed: (1) electrical

352

stimulation provides some specific organisms with energy in the form of electrons to

353

spur microbial metabolism in terms of K+ uptake as well as protein and soluble

354

polysaccharide accumulation; (2) microbial communities able to tolerate high salinity

355

and degrade organofluorides was adapted by electrical stimulation to the hyperhaline

356

environment. These are observations derived from the data recorded experimentally

357

and they are reported as indications of electricity driving enhanced microbial

358

organofluoride degradation in halophilic conditions. Further experiments are still

17

359

needed to provide direct evidences on a cell level to support these two proposed

360

mechanisms.

361

In agreement to our work, previous studies (11, 12, 13, 14, 37) have linked

362

enhanced microbial degradation in BES. Long-term electrical application resulted in

363

selection of salt-adapted and specific p-FNB mineralizing microorganisms in the BES.

364

Indeed, the robustness of BESs, especially under harsh conditions, has been

365

confirmed to be due to the selection of microorganisms with specific functions (38, 39,

366

40). The understanding of such mechanisms will further promote the development and

367

application of electrical stimulation crossing the limitations of current systems.

368

ACKNOWLEDGEMENT

369

This research was supported by the National Natural Science Foundation of China

370

(51478431), a Science and Technology Planning Project from the Science and

371

Technology Department in Zhejiang Province (2013C33004 and 2014C33028), a

372

Postgraduate Technology Innovation Project from Zhejiang Gongshang University

373

(1260XJ1513144), and a project from the Zhejiang Province education department

374

(2014R408087).

18

375

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24

490

TABLE CAPTIONS

491

TABLE 1. p-FNB removal kinetics in the bioelectrochemical system (BES) and

492

abiotic control (AC) system under different salinity conditions (a, b, c, d respectively

493

correspond to salinity condition of 0 g/L, 15 g/L, 30 g/L and 40 g/L)

494

TABLE 2. Intracellular K+ and Na+ concentration in the bioelectrochemical system

495

(BES) and biotic control (BC) system under different salinity conditions on day 96

496

TABLE 3. Total protein and soluble polysaccharide concentrations in the

497

bioelectrochemical system (BES) and biotic control (BC) system under different

498

salinity conditions on day 96

499

TABLE 4. Dominant bacterial and archaea populations in the bioelectrochemical

500

system at 40 g/L NaCl concentration

25

501

FIGURE CAPTIONS

502

FIG. 1. Removal and defluorination of p-FNB under different salinity conditions in

503

the fed batch bioelectrochemical system, abiotic control (AC) system and biotic

504

control (BC) system (a, b, c, d correspond to the salinity of 0 g/L, 15 g/L, 30 g/L, 40

505

g/L NaCl concentration). Test conditions: ambient temperature (30 ± 2°C); initial

506

p-FNB concentration of 0.4 mmol/L.

507

FIG. 2. TOC removal efficiency under different salinity conditions in the fed batch

508

bioelectrochemical system (BES), abiotic control (AC) system and biotic control (BC)

509

system. Initial p-FNB concentration of 0.4 mmol; hydraulic retention time: 96 h.

510

FIG. 3. Characterization of sludge concentration and biomass at different salinity

511

conditions in the bioelectrochemical system (BES), abiotic control (AC) system and

512

biotic control (BC) system. Operation period: 96 d.

513

FIG. 4. Archaeal community structure of class (a) and genus (b) distribution in the

514

initial inoculums (Seed), bioelectrochemical system (BES) and biotic control (BC)

515

system at different salinity. “0”, “15” and “40” stand for salinity condition at 0 g/L, 15

516

g/L and 40 g/L NaCl concentration, respectively.

26

TABLE 1. p-FNB removal kinetics in the bioelectrochemical system (BES) and abiotic control (AC) system under different salinity conditions (a, b, c, d respectively correspond to salinity condition of 0 g/L, 15 g/L, 30 g/L and 40 g/L) Kinetic equation

Rate constant K (h−1)

Half life (h)

Correlation coefficient R2

BESa

lnC=-0.106t-0.9163

0.106

6.53

0.9991

BESb

lnC=-0.125t-0.9163

0.125

5.55

0.9996

c

lnC=-0.112t-0.9163

0.112

6.19

0.9992

d

lnC=-0.103t-0.9163

0.103

6.74

0.9954

ACa

lnC=-0.041t-0.9163

0.041

16.82

0.9696

ACb

lnC=-0.057t-0.9163

0.057

12.18

0.9834

ACc

lnC=-0.065t-0.9163

0.065

10.70

0.9805

0.074

9.38

0.9850

BES BES

ACd

lnC=-0.074t-0.9163

The p-FNB removal kinetics was characterized by fitting p-FNB concentrations measured in batch experiments as a function of time.

TABLE 2. Intracellular K+ and Na+ concentration in the bioelectrochemical system (BES) and biotic control (BC) system under different salinity conditions on day 96 NaCl

Intracellular K+ concentration

Intracellular Na+ concentration

concentration

(mmol /g SS)

(mmol /g SS)

(g/L)

BES

BC

BES

BC

0

0.042

0.053

0.383

0.373

15

0.290

0.096

0.996

0.807

30

0.578

0.137

1.523

0.978

40

0.817

0.171

2.088

1.226

TABLE 3. Total protein and soluble polysaccharide concentrations in the bioelectrochemical system (BES) and biotic control (BC) system under different salinity conditions on day 96

NaCl

protein

soluble polysaccharide

Protein/soluble

concentration

(mg prot/g VSS)

(mg /g VSS)

polysaccharide

(g/L)

BES

BC

BES

BC

BES

BC

0

88.9±2.5

83.4±1.8

28.8±2.4

21.1±6.3

3.1

4.0

15

133.1±2.5

106.5±4.3

34.0±0.7

31.1±2.1

3.9

3.4

30

252.3±9.8

85.8±5.4

36.2±0.1

28.2±3.4

7.0

3.0

40

287.6±12.9

84.5±13.8

36.9±0.4

27.6±0.9

7.8

3.1

TABLE 4. Dominant bacterial and archaea populations in the bioelectrochemical system at 40 g/L NaCl concentration Accession OUT ID

Relative

Accession

Similarity

abundance (%)

no.

(%)

Closest relative no.

B1

KP091460

Uncultured Spirochaeta sp.

6.9

EU809870

98.2

B2

KP091461

Anaerobic bacterium MO-CFX2 gene

6.7

AB598278

98.3

B3

KP091462

Uncultured Clostridium sp.

2.6

HQ183781

99.3

B4

KP091463

Uncultured Anaerolineaceae bacterium

2.2

HE974801

96.9

B5

KP091464

Uncultured bacterium clone

1.5

KC796715

99.6

B6

KP091465

Uncultured Firmicutes bacterium

1.3

JQ012313

96.7

B7

KP091466

Pseudomonas sp.

1.2

AB836756

99.6

B8

KP091467

Uncultured Chloroflexi bacterium clone

1.1

JQ919721

96.7

B9

KP893254

Uncultured archaeon clone

56.6

JQ795002

96.3

B9

KP893255

Methanobacterium sp.

1.4

KF697731

100.0