Electrical Stimulation Improves Microbial Salinity Resistance and Organofluorine Removal in Bioelectrochemical Systems

Abstract

Fed batch bioelectrochemical systems (BESs) based on electrical stimulation were used to treat p-fluoronitrobenzene (p-FNB) wastewater at high salinities. At a NaCl concentration of 40 g/liter, p-FNB was removed 100% in 96 h in the BES, whereas in the biotic control (BC) (absence of current), p-FNB removal was only 10%. By increasing NaCl concentrations from 0 g/liter to 40 g/liter, defluorination efficiency decreased around 40% in the BES, and in the BC it was completely ceased. p-FNB was mineralized by 30% in the BES and hardly in the BC. Microorganisms were able to store 3.8 and 0.7 times more K⁺ and Na⁺ intracellularly in the BES than in the BC. Following the same trend, the ratio of protein to soluble polysaccharide increased from 3.1 to 7.8 as the NaCl increased from 0 to 40 g/liter. Both trends raise speculation that an electrical stimulation drives microbial preference toward K⁺ and protein accumulation to tolerate salinity. These findings are in accordance with an enrichment of halophilic organisms in the BES. Halobacterium dominated in the BES by 56.8% at a NaCl concentration of 40 g/liter, while its abundance was found as low as 17.5% in the BC. These findings propose a new method of electrical stimulation to improve microbial salinity resistance.

INTRODUCTION

Organofluorine compounds, especially fluorinated aromatic compounds, are widely used in the production of adhesives, pesticides, dyes, pharmaceuticals, refrigerants, and surfactants (1). They were found to inhibit enzymes, modify cell-to-cell communication, and disrupt membrane transport as well as energy generation processes (2). Due to their high toxicity and recalcitrance, conventional biological methods fail to efficiently remove organofluorine from wastewaters (1, 3). On the other hand, bioelectrochemical treatment has been proved to be an effective method to minimize refractory properties of typical p-fluoronitrobenzene-contaminated organofluorine wastewater (4).

Salinity poses another serious challenge to the treatment of such organofluorine-containing wastewater. Typically, addition of strong acid or alkali to adjust the pH during production processes results in high salt concentrations in organofluorine wastewaters. As an example, salinity concentrations from an organofluorine industry effluent in China typically fluctuate between 2 and 3%, with a maximum of 5%. Conventional physicochemical treatment processes for salinity wastewater are energy intensive and costly. Although biological processes have been recommended for salinity wastewater treatment (5, 6), high salinity may cause cell plasmolysis and even the death of microorganisms due to osmotic pressure increases (7, 8). To address this issue, wastewaters are always diluted, which results in a meaningless freshwater consumption and increases operational cost (5). An enhancement of microbial salinity resistance would enable implementation of biological methods for salinity wastewater treatment, which could be succeeded by acclimatization and enrichment of haloduric or halophilic strains (5). However, gradual increase of salinity to enrich halophilic microorganisms showed that it is not such an easy task (5, 7).

Application of an electrical stimulation to stimulate microbial respiratory processes as well as microbial consortium evolution has been applied for waste treatment and remediation (9, 10). An electrical stimulation can directly or indirectly play a role depending on whether there is hydrogen or oxygen evolution or soluble-mediator involvement. A direct effect would be an energy gain for organisms attached via a biofilm or not on the electrode surface from that electrode. In contrast, an indirect effect involves electron transfer from a working electrode to microorganisms through either a soluble mediator or a gas (usually hydrogen or oxygen produced by electrolysis of water) (9). Poising electrodes at specific potentials has been shown to activate key metabolic enzymes for degradation of recalcitrant compounds. Zhang et al. (11) found that application of a fixed potential induced microbial oxidoreductase production for electron transfer when 2,4-dichlorophenoxyacetic acid was biologically degraded. In agreement, electrical stimulation has been also shown to spur microbial growth (12) and enhance the cell density (13). The cell density of the bacterium Enterobacter dissolvens was found to be significantly improved and microbial activity was increased 2-fold (14). More studies show that electrical stimulation may result in evolution of specific communities that are able to adapt to unique environments (15) or in the development of specific microbial functions (16). These findings open new horizons for the application of electrical stimulation to facilitate degradation of recalcitrant compounds in harsh environments.

In light of these studies, the objectives of this study were to (i) investigate the effects of direct electrical stimulation in the absence of both hydrogen evolution and soluble mediator on microbial salinity resistance, (ii) investigate whether the adverse effects of high salinity could be ameliorated, and (iii) characterize the microbial community that is able to remove organofluorines in batch bioelectrochemical systems (BESs) under high salinity.

MATERIALS AND METHODS

Microbial inoculum and growth medium.

Activated sludge obtained from a chemical industrial wastewater treatment plant in Linhai, Zhejiang, China, was used as the inoculum of the biocathode. Total suspended solids (TSSs) measured approximately 3,000 mg · liter⁻¹, and the ratio of volatile suspended solids to total suspended solids (VSS/TSS) was 55%. The basic nutrient medium used in the anode and cathode chambers contained 3.4 g liter⁻¹ of K₂HPO₄, 4.4 g liter⁻¹ of KH₂PO₄, 0.1 g liter⁻¹ of NH₄Cl, 2 g liter⁻¹ of NaHCO₃, 0.24 g liter⁻¹ of MgSO₄·7H₂O, and trace elements, as previously described (17). A series of amounts of NaCl were added to the medium to adjust the salinity, namely, 0, 15, 30, and 40 g/liter, giving final salinities of about 1%, 2.5%, 4%, and 5%, respectively. p-Fluoronitrobenzene (p-FNB) was added to the cathode chamber with an initial concentration of 0.4 mmol liter⁻¹.

Bioelectrochemical experiment.

Studies were carried out at 30°C in potentiostat-poised, dual-chambered BESs as previously described (18) (see Fig. S1 in the supplemental material). A Nafion 117 (DuPont) proton exchange membrane was used to separate the anode and cathode chambers. Graphite felt (Beijing Sanye Carbon Co., Ltd., Beijing, China) was used as both anode and cathode electrodes. No additional mediators were introduced to the anodic and cathodic electrolytes. The working volume of the cathode chamber was 100 ml, and the headspace was 41 ml. The BES core was a biocathode, and the cathode compartment was unsealed so that ambient air could enter the headspace through the sampling ports on the top of reactors. The bioelectrochemical reactors were operated in batch mode and with a hydraulic retention time of 4 days. An external power source (1.8 V) was applied to the abiotic reactors and BESs, with the cathode potential kept at about −0.78 V versus Ag/AgCl. This potential was chosen to avoid H₂ evolution under each salinity condition.

Two types of control reactors were operated under identical conditions, namely, abiotic control (AC) (without bacteria) and biotic control (BC) (no applied voltage). The performances of BES, AC, and BC were compared at different salinities for kinetic experiments and confirmatory experiments (see Table S1 in the supplemental material). The p-FNB removal efficiency for each cycle was determined by measuring the p-FNB concentration of the effluent from the reactor. Steady-state conditions were reached when the difference of p-FNB removal efficiency among 3 consecutive batches was less than 5%. Then, sampling took place to determine p-FNB removal, defluorination, and mineralization.

Chemicals and analytical methods.

p-FNB (99% purity) was purchased from Aladdin Chemical Ltd. (Shanghai, China). Methanol used was of high-performance liquid chromatography (HPLC) grade, while all other chemicals were of analytical grade. Samples were taken from reactors using a 10-ml syringe and were filtered through a 0.22-μm filter. The p-FNB and fluoride ion concentrations were determined using an HPLC (Waters Corp., Milford, MA) and an IC plus ion chromatograph (Metrohm AG, Herisau, Switzerland), with methods described in a previous publication (4). The total organic carbon (TOC) content was measured using a TOC analyzer (Shimadzu, Kyoto, Japan).

The primary characteristic metabolites were identified using an Agilent 6890N GC/5975B MSD (Agilent Corp., USA) according to a previously published method (19). VSS and TSS were determined according to standard methods (20). Total protein and soluble polysaccharide were determined in sludge extracts. Cellular lysates of sludge were obtained from a washed sample by adding fresh normal saline and sonicating it to a 20-MHz ultrasound (using a Sonics Ultra Cell instrument; Sonics and Materials, Inc., Newtown, CT) at a power of 97.5 W for 5 s each time for a total of 50 times, with a 10-s gap between each time. Then cellular lysates were used for total protein and soluble polysaccharide determination, according to the methods described by Lowry et al. (21) and the phenol-sulfuric method described by Dubois et al. (22).

Sludge intracellular ion concentration of sodium and potassium was determined by ion release extracellularly by boiling to cause cell rupture. The steps of this process included the following sequence. A 10-ml aliquot of the well-mixed sludge was collected and washed with magnesium chloride solution, the concentration of which was determined by the principle of keeping the osmotic pressure of magnesium chloride solution similar to that of medium where sludge was fed. The washed sludge was collected in 25 ml of ultrapure water, and intracellular ions were extracted by boiling in a water bath for 20 min. Then the K⁺ and Na⁺ concentrations were measured by an atomic absorption spectrophotometry (ZEEnit700; Analytik Jena, Germany).

Microbial community analysis.

At the end of kinetic performance experiments (96 days), sludge samples were collected from BES and BC reactors for community structure analysis. Details of the genomic DNA extraction, PCR amplification, and statistical analyses are given in the supplemental material.

Nucleotide sequence accession numbers.

The 16S rRNA gene sequences of the dominant bacterial population at high salinity (40 g/liter of NaCl) determined in this study were deposited in the GenBank database under accession numbers KP091460 to KP091467, KP893254, and KP893255.

RESULTS AND DISCUSSION

Effects of salinity on p-FNB treatment. (i) p-FNB removal.

In general, the p-FNB removal efficiency in different systems followed the order BES > AC > BC, in spite of variations in salinity (Fig. 1). BESs have been previously reported to remove recalcitrant pollutants more efficiently than conventional electrochemical treatments because of microbial catalysis (10, 23). In agreement, these results suggest that biological activity is well maintained in the BES, even at high salinity.

FIG 1.

Removal and defluorination of p-FNB under different salinity conditions in the fed batch bioelectrochemical system (BES), abiotic control (AC) system, and biotic control (BC) system (panels a, b, c, and d correspond to NaCl concentrations of 0 g/liter, 15 g/liter, 30 g/liter, and 40 g/liter, respectively). The test conditions were ambient temperature (30 ± 2°C) and an initial p-FNB concentration of 0.4 mmol/liter.

When the NaCl concentration was increased from 0 to 15 g/liter, the constant of p-FNB removal rate (k_p-FNB) in the BES increased from 0.106 h⁻¹ to 0.125 h⁻¹, and it declined to 0.103 h⁻¹ when the concentration sequentially increased to 40 g/liter (Table 1). However, k_p-FNB in the AC consistently increased from 0.041 h⁻¹ to 0.074 h⁻¹ as salinity was increased. This can be attributed to the fact that an increase of salinity improves solution conductivity, which further facilitates electron generation and transportation in the circuit. This concept applies also to the BES, as our previous study revealed that p-FNB was readily metabolized in the BES due to interaction between microorganisms and the electrode (4). On one hand, the rate of p-FNB removal in the BES grew with the salinity increase. On the other hand, inhibition of microbial activity was aggravated at the same time, resulting in decreased p-FNB removal (Fig. 1). This implies that there must be a critical salinity concentration above which the microbial degradation of p-FNB is inhibited.

TABLE 1.

p-FNB removal kinetics in the bioelectrochemical system and abiotic control system under different salinity conditions

System and NaCl concn (g/liter)	Kinetic equationa	kp-FNB (h−1)	Half-life (h)	Correlation coefficient
BES
0	lnC = −0.106t − 0.9163	0.106	6.53	0.9991
15	lnC = −0.125t − 0.9163	0.125	5.55	0.9996
30	lnC = −0.112t − 0.9163	0.112	6.19	0.9992
40	lnC = −0.103t − 0.9163	0.103	6.74	0.9954
AC
0	lnC = −0.041t − 0.9163	0.041	16.82	0.9696
15	lnC = −0.057t − 0.9163	0.057	12.18	0.9834
30	lnC = −0.065t − 0.9163	0.065	10.70	0.9805
40	lnC = −0.074t − 0.9163	0.074	9.38	0.9850

^aThe p-FNB removal kinetics was characterized by fitting p-FNB concentrations measured in batch experiments as a function of time (t).

p-FNB removal efficiency in the BC declined significantly, from 85% to 10%, at 96 h as the NaCl concentration was increased from 0 g/liter to 40 g/liter (Fig. 1a and d). These findings indicated severe inhibition of the microbial metabolism caused by damage by high salinity. A decrease of 88% in p-FNB removal efficiency was observed in the BC as the NaCl concentration was increased from 0 g/liter to 40 g/liter, while the decrease was only 3.1% in the BES, indicating a relief from hyperhaline inhibition in the BES.

(ii) p-FNB defluorination.

The effects of salinity on defluorination performance are shown in Fig. 1. BES exhibited the highest defluorination efficiency among all tested systems, and the fluoride ion accumulation in 4 days decreased from 0.249 ± 0.005 mmol/liter to 0.149 ± 0.020 mmol/liter as the NaCl concentration was increased from 0 g/liter to 40 g/liter.

Defluorination in the AC and BC was found to be weak at all salinities, whereas the fluoride ion concentration was always increasing in the BES (Fig. 1). As the NaCl concentration was increased from 0 g/liter to 40 g/liter, defluorination efficiency decreased by 40.2% (from 62.2% to 37.3%) in the BES, while it was totally inhibited (from 27.1% to 0) in the BC. These findings indicate that microorganisms in the BES formed a better capacity to resist salinity and degrade target pollutants than those in the BC.

To theoretically evaluate the chemical form of fluorine speciation for p-FNB degradation, a mass balance of fluorine was performed by quantifying fluoride ion from p-FNB, p-fluoroaniline (p-FA; the dominant intermediate in p-FNB degradation), and free fluoride ion from other intermediates (see Fig. S2 in the supplemental material). In the AC, the fluoride ion accounted for a very small proportion (less than 10%) of the total fluorine under all salinity conditions. A previous report has proved that the direct electrochemical reduction of nitroaromatics tends to generate intermediates such as nitrosobenzene, azobenzene, and azoxybenzene, which are more toxic or resistant to biodegradation (24). Nitroaromatics have the same functional nitro group as p-FNB; thus, the large proportion of other unspecified intermediates for p-FNB degradation may indicate that there were considerable side reactions in the AC.

The fluoride ion was the dominant fluorine species in the BES. At 96 h, the maximum percentage of 62% fluorine ion was produced at 0 g/liter of NaCl, and then it gradually decreased to around 37% when the NaCl concentration was increased to 40 g/liter (see Fig. S2 in the supplemental material). Additionally, the unspecified fluorine species percentage went up as salinity was increased. It reached a peak value of 11.5% at 0 g/liter of NaCl, whereas at the highest salinity, 40 g/liter NaCl, it reached a maximum value of about 43%. These results suggest that biological defluorination in the BES is inhibited by high salinity and that undesired side reactions exacerbated this, which does not promote p-FNB degradation.

(iii) p-FNB mineralization.

TOC removal efficiency was measured to determine the extent of p-FNB mineralization. As shown in Fig. 2, when the NaCl concentration was increased from 0 g/liter to 40 g/liter, TOC removal efficiency in both the BES and BC gradually decreased, from 69% to 30% and from 22% to 3.3%, respectively. In contrast, the salinity gradient had a positive effect on TOC removal efficiency in the AC, as it increased from 7.9% to 13.0%.

FIG 2.

TOC removal efficiency under different salinity conditions in the fed batch bioelectrochemical system (BES), abiotic control (AC) system, and biotic control (BC) system. The initial p-FNB concentration was 0.4 mmol; the hydraulic retention time was 96 h.

The TOC removal performances in the tested systems were found to be different but were consistent with the defluorination rate regardless of the system type or salinity. Considering the strengthening effect of fluorine substituent and nitro group on the recalcitrant properties of p-FNB, a high nitro reduction and rapid defluorination rate can contribute to an enhanced mineralization performance in the BES. As described above, defluorination in the BC was tremendously inhibited at high salinity, which was also observed in the case of p-FNB mineralization. At 40 g/liter of NaCl, the TOC removal efficiency in the BES was about 83% higher than the sum of both efficiencies in the BC and AC, demonstrating that BES is a good alternative technology for hyperhaline wastewater treatment.

Electrical stimulation on biological salinity resistance. (i) Biomass characterization.

High salinity inhibited microbial metabolism, resulting in cell plasmolysis and even in lethal effects on microorganisms, which directly impacted biomass concentrations. Figure 3 shows that TSS in the BES always remained constant at an inoculation level of about 3,000 mg/liter, whereas TSS gradually declined in the BC as salinity was increased, reaching a loss of about 35% at 40 g/liter of NaCl. Simultaneously, the VSS/TSS ratios decreased to 37% and 27% in the BES and BC, with corresponding maximum losses of 32% and 51%, respectively (Fig. 3). These findings support our assumption that despite high-salinity inhibition, organisms in the BES are less vulnerable to salinity variations.

FIG 3.

Characterization of sludge concentration and biomass under different salinity conditions in the bioelectrochemical system (BES), abiotic control (AC) system, and biotic control (BC) system. The operation period was 96 days.

(ii) Effect of intracellular Na⁺ and K⁺ on salinity resistance.

High osmotic pressure stems from the density gradient between the intracellular and extracellular environment. The strategy of “salt-in” is considered an important mechanism to resist high salinity that in principle is based on the ability of many organisms to take up inorganic ions from the extracellular environment and accumulate salts in high concentrations within their cells to balance osmotic pressure (25). As sodium and potassium were the dominant cations present in the medium of the extracellular environment, their intracellular concentrations were investigated at different salinities to verify whether they were accumulated.

Although the extracellular ion density should influence intracellular osmoregulation in theory (25), under an electrical field, initial concentrations of Na⁺ and K⁺ in the extracellular medium are not constant, because these two cations may move from the anode to the cathode chamber. In this case, microorganisms in the cathode chamber may experience different salinity effects from the ones expected. To eliminate this concern, their concentration was determined in the bulk and it was found that Na⁺ and K⁺ in the anode and the cathode chamber were maintained at almost their initial level due to the low current applied (see Fig. S3 in the supplemental material).

As shown in Table 2, intracellular K⁺ and Na⁺ concentrations in the BES and BC both tended to increase as the NaCl concentration was increased from 0 g/liter to 40 g/liter. The intracellular K⁺ concentration in the BES increased almost 20 times (from 0.042 to 0.817 mmol/g of SS), which is much higher than the intracellular Na⁺ concentration (5.5 times). At the maximum NaCl concentration of 40 g/liter, the intracellular K⁺ concentration in the BES was 3.8 times higher than that in the BC, while the intracellular Na⁺ concentration was only 0.7 times higher. These results indicate that K⁺ was preferentially accumulated by microorganisms in the BES to regulate osmotic pressure. K⁺ accumulation contributes to the maintenance of cellular activities at high salinity. Microorganisms can directly adjust the cytoplasm concentration by K⁺ accumulation to regulate osmotic pressure; besides, extracellular K⁺ may promote the accumulation of some other soluble organic osmoticum, thus regulating osmotic pressure indirectly (26, 27). Accordingly, maintaining a higher intracellular K⁺ here is likely a useful strategy for inducing salinity resistance in the BES.

TABLE 2.

Intracellular K⁺ and Na⁺ concentrations in the bioelectrochemical system and biotic control system under different salinity conditions on day 96

NaCl concn (g/liter)	Intracellular K+ concn (mmol/g of SS)		Intracellular Na+ concn (mmol/g of SS)
	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

(iii) Effect of protein and soluble polysaccharide on salinity resistance.

Most haloduric microorganisms prefer to accumulate some soluble intracellular organics as a countermeasure to balance the osmotic pressure; this is another strategy to acquire so-called “compatible solutes” (28). Microbial protein and polysaccharides are the typically preferred intracellular organics, whose variation can help elucidate the mechanisms of salinity tolerance. Results showed that the concentrations of total protein and soluble polysaccharide in the BC remained relatively stable within the salinity variation (Table 3). However, when the NaCl concentration was increased from 0 to 40 g/liter, the protein and soluble polysaccharide intracellular concentrations in the BES increased 3 and 1.3 times, respectively. This suggests that microorganisms can be electricity driven to accumulate organics intracellularly so that they can regulate osmotic pressure. Moreover, the ratio of protein to soluble polysaccharide increased from 3.1 to 7.8 as the salinity was increased, implying that microorganisms in the BES relied strongly on protein accumulation to adapt to high salinity.

TABLE 3.

Total protein and soluble polysaccharide concentrations in the bioelectrochemical system and biotic control system under different salinity conditions on day 96

NaCl concn (g/liter)	Concn of protein (mg/g of VSS)a		Concn of soluble polysaccharide (mg/g of VSS)a		Protein/soluble polysaccharide ratio
	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

^aValues are means ± SDs.

Microbial community structure.

Further confirmatory experiments indicated that a specific microbial community with the ability to mineralize p-FA and resist to high salinity is likely to be selected by a long-term electrical stimulation (see Fig. S4 in the supplemental material). Thus, microbial community structure in terms of bacterial and archaeal dynamics was investigated in this study.

Bacterial dynamics.

Compared with the inoculum, Bacteroidetes, Chloroflexi, Firmicutes, and Spirochaetae were enriched, and their abundance was improved as the salinity was increased in the BES (see Fig. S5a in the supplemental material). Bacteroidetes, Chloroflexi, Firmicutes, and Spirochaetae have been reported to be common and abundant during the treatment of high-salinity wastewater (6, 29, 30, 31), suggesting that these bacteria may play specific roles in salinity resistance in the BES for p-FNB treatment. The dominant phyla in the BES were also observed to be abundant in the BC, but at different proportions, demonstrating that haloduric bacteria surviving in the BC showed considerable homology to those enriched in the BES.

Alphaproteobacteria, Betaproteobacteria, Anaerolineae, Deltaproteobacteria, vadinHA17 (an uncultured eubacterium), Spirochaetes, Clostridia, and Bacteroidia were enriched compared with the inoculum and became relatively dominant in the BES (see Fig. S5b). These classes were also found to be relatively abundant in the BC, and bacterial consortia at the class level showed considerable homology. Moreover, the initial microbial community shift with and without electrical stimulation was mostly based on the proportional imparity of each population.

16S rRNA gene sequences of microbial consortia at 40 g/liter of NaCl in the BES were clustered into operational taxonomic units (OTUs), and the dominant as well as unique bacterial OTUs are listed in Table 4. The most dominant bacterial OTU population was close to an uncultured Spirochaeta sp. (6.9%), which was once isolated from an oil field, growing optimally with a NaCl concentration of 5% and being adapted to a variety of substrates (32). This optimal NaCl concentration was similar to the salinity conditions in our experiments. Finding of the same species under similar condition shows that Spirochaeta may play an important role in pollutant mineralization at high salinity. Additionally, an anaerobic MO-CFX₂ bacterium (6.7%), known to specifically degrade halogenated aromatic compounds (33), was also uniquely detected in the BES.

TABLE 4.

Dominant bacterial and archaeal populations in the bioelectrochemical system at 40 g/liter of NaCl

OTU identifier	GenBank accession no.	Closest relative	Relative abundance (%)	GenBank accession no. of closest relative	Similarity (%)
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 bacterial 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 bacterial clone	1.1	JQ919721	96.7
B9	KP893254	Uncultured archaeal clone	56.6	JQ795002	96.3
B9	KP893255	Methanobacterium sp.	1.4	KF697731	100.0

Archaeal dynamics.

Evolution in the archaeal consortia can be demonstrated by variations in the archaeal community structure. As shown in Fig. 4a, archaeal classes in all samples consisted initially of Halobacteria and Methanomicrobia. As the NaCl concentration was increased from 0 g/liter to 40 g/liter, the abundance of Halobacteria in the BES increased from 24.2% to 66.4%, while Methanomicrobia abundance dropped from 59.3% to 0.2%. However, the relative proportion of the two populations showed opposite tendencies in the BC, with Halobacteria and Methanomicrobia abundances of 17.5% and 65.6%, respectively, being observed at 40 g/liter of NaCl.

FIG 4.

Archaeal community structure of class (a) and genus (b) distributions in the initial inoculums (seed), bioelectrochemical system (BES), and biotic control (BC) system at different salinities. “0,” “15,” and “40” stand for salinities corresponding to 0 g/liter, 15 g/liter, and 40 g/liter of NaCl, respectively.

Halobacterium was the dominant archaeal genus in the BES, and its abundance changed with variations in salinity (Fig. 4b). With increased salinity, Halobacterium abundance ascended from 17.4% to 56.8% in the BES, whereas it descended from 65.5% to 17.5% in the BC. Halobacteria is a typical halophilic archaeon with specifically adaptive capacity for hyperhaline environments (34). Phylogenetic Halobacterium is a type of Halobacteria that has been extensively investigated for its ability to live in saline environments and its roles in saline wastewater treatment processes (31, 35). A significant community shift and the proportion decreasing of Halobacterium at 40 g/liter of NaCl in the BC indicated that the BC was severely impacted by salt damage, which is consistent with a VSS/TSS decrease in the BC. In contrast, the same population was further enriched at high salinity in the BES, which was most probably an adaptation outcome. The same Halobacterium population exhibited different adaptive behaviors in response to salinity in the BES and BC, indicating that different abilities to tolerate salinity evolved in the two systems. Given the system differences, this discrepancy is likely caused by the effect of electrical stimulation. Halobacterium has been shown to store K⁺ and simultaneously excrete Na⁺ to regulate high osmotic pressure (36). This strategy involves active transport of K⁺ and is dependent on energy consumption. Electrical stimulation might provide microbial energy through a specific strategy of electron transport (9, 10). Thus, K⁺ uptake was probably stimulated in the BES, and salinity resistance was thus promoted.

Implications of practice.

BES improved microbial salinity resistance and enhanced organofluorine removal. The improved performance of the BES was attributed to direct electrical stimulation. Based on the microbial metabolism and community evolution results, two possible mechanisms of electrical stimulation mechanisms were proposed: (i) electrical stimulation provides some specific organisms with energy in the form of electrons to spur microbial metabolism in terms of K⁺ uptake as well as protein and soluble polysaccharide accumulation, and (ii) microbial communities able to tolerate high salinity and degrade organofluorides adapted by electrical stimulation to the hyperhaline environment. These are observations derived from the data recorded experimentally, and they are reported as indications of electricity driving enhanced microbial organofluoride degradation under halophilic conditions. Further experiments are still needed to provide direct evidence on a cell level to support these two proposed mechanisms.

In agreement with our work, previous studies (11, 12, 13, 14, 37) have linked enhanced microbial metabolism in the BES. Long-term electrical application resulted in selection of salt-adapted and specific p-FNB-mineralizing microorganisms in the BES. Indeed, the robustness of BESs, especially under harsh conditions, has been confirmed to be due to the selection of microorganisms with specific functions (38, 39, 40). The understanding of such mechanisms will further promote the development and application of electrical stimulation crossing the limitations of current systems.