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
9
Hangzhou 310012, China; 2Zhejiang Provincial Key Laboratory of Solid Waste
10
Treatment and Recycling, Hangzhou 310012, China; 3Laboratory of Microbial
11
Ecology and Technology, Ghent University, Coupure Links 653, B-9000, Ghent,
12
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
13
ABSTRACT: Fed batch bioelectrochemical systems (BESs) based on electrical
14
stimulation were used to treat p-fluoronitrobenzene (p-FNB) wastewater at high
15
salinities. At a NaCl concentration of 40 g/L, p-FNB was removed 100% in 96 h in
16
the BES, whereas in the biotic control (BC) (absence of current), p-FNB removal was
17
only 10 %. By increasing NaCl concentrations from 0g/L to 40 g/L, defluorination
18
efficiency decreased around 40% in the BES and in the BC it was completely ceased.
19
p-FNB was mineralized by 30% in the BES and hardly in the BC. Microorganisms
20
were able to store 3.8 and 0.7 times more concentration of K+ and Na+ intracellularly
21
in the BES rather than in the BC. Following the same trend, the ratio of protein to
22
soluble polysaccharide increased from 3.1 to 7.8 as the NaCl increased from 0 to 40
23
g/L. Both trends rise speculations that on an electrical stimulation driving microbial
24
preference of K+ and protein accumulation to tolerate salinity. These findings are in
25
accordance with an enrichment of halophilic organisms in the BES. Halobacterium
26
dominated in the BES by 56.8% at the NaCl concentration of 40 g/L, while its
27
abundance was found as low as 17.5% in the BC. These findings propose a new
28
method of electrical stimulation to improve microbial salinity-resistance.
2
29
INTRODUCTION
30
Organofluorine compounds, especially fluorinated aromatic compounds, are
31
widely used in the production of adhesives, pesticides, dyes, pharmaceuticals,
32
refrigerants, and surfactants (1). They were found to inhibit enzymes, modify
33
cell-to-cell communication, and disrupt membrane transport as well as energy
34
generation processes (2). Due to their high toxicity and recalcitrance, conventional
35
biological methods fail to efficiently remove organofluorine from wastewaters (1, 3).
36
On the other hand, bioelectrochemical treatment has been proved to be an effective
37
method
38
p-fluoronitrobenzene-contaminated organofluorine wastewater (4).
39
Salinity
to
poses
minimize
another
refractory
serious
challenge
properties
to
the
of
treatment
typical
of
such
40
organofluorine-containing wastewater. Typically, strong acid or alkali addition to
41
adjust the pH during production processes results in high salt concentrations in
42
organofluorine wastewaters. As an example, salinity concentrations from an
43
organofluorine industry effluent in China typically fluctuate between 2 and 3 %, to a
44
maximum of 5 %. Conventional physicochemical treatment processes for salinity
45
wastewater are energy-intensive and costly. Although biological processes have been
46
recommended for salinity wastewater treatment (5, 6), high salinity may cause cell
47
plasmolysis and even the death of microorganisms due to osmotic pressure increase (7,
48
8). To address this issue, wastewaters are always diluted, which results in a
49
meaningless fresh water consumption and increases operational cost (5). An
50
enhancement of microbial salinity-resistance would enable implementation of
3
51
biological methods for salinity wastewater treatment, which could be succeeded by
52
acclimatization and enrichment of haloduric or halophilic strains (5). However,
53
gradual increase of salinity to enrich halophilic microorganisms showed that it is not
54
such an easy task (5, 7).
55
Application of an electrical stimulation to stimulate microbial respiratory
56
processes as well as microbial consortia evolution has been applied for
57
waste-treatment and remediation (9, 10). An electrical stimulation can directly or
58
indirectly play a role depending on whether there is hydrogen/oxygen evolution or
59
soluble mediator involvement. Direct effect refers to energy gain for organisms
60
attached via a biofilm or not on the electrode surface from that electrode. On the
61
contrary, indirect effect involves electron transfer from a working electrode to
62
microorganisms either through a soluble mediator or a gas (usually hydrogen or
63
oxygen produced by electrolysis of water) (9). Poising electrodes at specific potentials
64
has been shown to activate key metabolic enzymes for degradation of recalcitrant
65
compounds. Zhang et al. (11) found that application of a fixed potential induced
66
microbial
67
2,4-dichlorophenoxyacetic acid was biologically degraded. In agreement, electrical
68
stimulation has been also shown to spur microbial growth (12) and enhance the cell
69
density (13). Cell density of bacterium Enterobacter dissolvens was found to be
70
significantly improved and microbial activity was increased two-fold (14). More
71
studies show that electrical stimulation may result in specific community evolution
72
that are able to adapt to unique environments (15) or in the development of specific
oxidoreductase
production
4
for
electron
transfer
when
73
microbial functions (16). These findings open new horizons for the application of
74
electrical stimulation to facilitate degradation of recalcitrant compounds in harsh
75
environments.
76
On this frame, objectives of this study were to investigate 1) the effects of direct
77
electrical stimulation in the absence of both hydrogen evolution and soluble mediator
78
on microbial salinity-resistance; 2) whether the adverse effects of high salinity could
79
be ameliorated; 3) to characterize the microbial community that is able to
80
organofluorine removal in BESs under high salinity.
81
MATERIALS AND METHODS
82
Microbial inoculum and growth medium
83
Activated sludge obtained from a chemical industrial wastewater treatment plant in
84
Linhai (Zhejiang, China) was used as the inoculum of the biocathode. Total
85
Suspended Solids (TSS) was measured approximately 3000 mg·L-1 and the ratio of
86
Volatile Suspended Solids to Total Suspended Solids (VSS/TSS) was 55%. The basic
87
nutrient medium used in the anode and cathode chambers contained 3.4 g L−1 K2HPO4,
88
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
89
trace elements, as previously described (17). A series amount of NaCl was added to
90
the medium to adjust the salinity namely 0, 15, 30, and 40 g/L, giving final salinities
91
of about 1%, 2.5%, 4% and 5%, respectively. p-FNB was added to the cathode
92
chamber with an initial concentration of 0.4 mmol L−1.
93
Bioelectrochemical experiment
94
Studies were carried out at 30°C in potentiostat-poised, dual-chambered
5
95
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
103
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
105
reactors and BESs, with the cathode potential kept at about -0.78V vs (Ag/AgCl).
106
This potential was chosen to avoid H2 evolution under each salinity condition.
107
Two types of control reactors were operated under identical conditions, namely
108
abiotic control (AC) (without bacteria) and biotic control (BC) (no applied voltage).
109
The performance of BES, AC, and BC were compared at different salinities for
110
kinetic experiments and confirmatory experiments (Supporting Information, Table S1).
111
The p-FNB removal efficiency for each cycle was determined by measuring the
112
p-FNB concentration of the effluent from the reactor. Steady state conditions were
113
reached when the difference of p-FNB removal efficiency among 3 consecutive
114
batches was less than 5%. Then, sampling took place to determine p-FNB removal,
115
defluorination and mineralization.
116
Chemicals and Analytical Methods
6
117
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).
136
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).
146
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.
154
RESULTS AND DISCUSSION
155
Effects of Salinity on p-FNB Treatment
156
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.
229
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
253
eliminate this concern, their concentration was determined in the bulk and it was
254
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).
256
As shown in Table 2, intracellular K+ and Na+ concentrations in the BES and
257
BC both tended to increase as the NaCl concentration increased from 0 g/L to 40 g/L.
258
Intracellular K+ concentration in the BES increased almost 20 times (from 0.042 to
259
0.817 mmol/g SS), which is much higher than that of intracellular Na+ concentration
260
(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
265
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
269
likely a useful strategy for inducing salinity-resistance in the BES.
270
Effect of protein and soluble polysaccharide on salinity resistance. Most haloduric
13
271
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
286
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.
290
Bacteria dynamics
291
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,
294
Firmicutes and Spirochaetae have been reported to be common and abundant during
295
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
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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
