International Journal of Environmental Research and Public Health Article Implementation of a Sulfide–Air Fuel Cell Coupled to a Sulfate-Reducing Biocathode for Elemental Sulfur Recovery Enric Blázquez 1, *, David Gabriel 1 , Juan Antonio Baeza 1 , Albert Guisasola 1 , Pablo Ledezma 2 and Stefano Freguia 2,† 1 GENOCOV Research Group, Department of Chemical, Biological and Environmental Engineering, School of Engineering, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; david.gabriel@uab.cat (D.G.); JuanAntonio.Baeza@uab.cat (J.A.B.); albert.guisasola@uab.cat (A.G.) 2 Advanced Water Management Centre, The University of Queensland, Brisbane 4072, Australia; p.ledezma@awmc.uq.edu.au (P.L.); stefano.freguia@unimelb.edu.au (S.F.) * Correspondence: blazquez.enric@gmail.com † Current address: Department of Chemical Engineering, University of Melbourne, Victoria 3010, Australia. Abstract: Bio-electrochemical systems (BES) are a flexible biotechnological platform that can be employed to treat several types of wastewaters and recover valuable products concomitantly. Sulfate- rich wastewaters usually lack an electron donor; for this reason, implementing BES to treat the sulfate and the possibility of recovering the elemental sulfur (S0 ) offers a solution to this kind of wastewater. This study proposes a novel BES configuration that combines bio-electrochemical sulfate reduction in a biocathode with a sulfide–air fuel cell (FC) to recover S0 . The proposed system achieved high   elemental sulfur production rates (up to 386 mg S0 -S L−1 d−1 ) with 65% of the sulfate removed recovered as S0 and a 12% lower energy consumption per kg of S0 produced (16.50 ± 0.19 kWh kg−1 Citation: Blázquez, E.; Gabriel, D.; S0 -S) than a conventional electrochemical S0 recovery system. Baeza, J.A.; Guisasola, A.; Ledezma, P.; Freguia, S. Implementation of a Keywords: bio-electrochemical systems (BES); fuel cells (FC); sulfate removal; resource recovery; Sulfide–Air Fuel Cell Coupled to a Sulfate-Reducing Biocathode for oxygen-reducing cathode Elemental Sulfur Recovery. Int. J. Environ. Res. Public Health 2021, 18, 5571. https://doi.org/10.3390/ ijerph18115571 1. Introduction Bio-electrochemical systems (BES) are a novel technology based on electrochemical Academic Editor: Huan-Tsung Chang processes where the oxidation and/or reduction reactions are catalyzed by microorgan- isms [1]. BES allow for the treatment of various domestic and industrial wastewaters with Received: 18 March 2021 concomitant recovery of reusable products such as heavy metals [2,3], nutrients [4,5] and Accepted: 19 May 2021 organic compounds [6,7]. BES can also facilitate the treatment of sulfate-rich effluents [8] Published: 23 May 2021 with the possibility to recover elemental sulfur (S0 ), a product with a stable worldwide demand of about 70 million tons per year just in the fertilizer market [9]. Publisher’s Note: MDPI stays neutral Sulfate-rich wastewaters are produced by several large-scale industrial activities such with regard to jurisdictional claims in as pulp and paper production, food processing, dye and detergent manufacturing, among published maps and institutional affil- others [10], and if released untreated onto the environment may lead to several environ- iations. mental problems–e.g., due to the biological formation of sulfide when microorganisms use sulfate as terminal electron acceptor [11]–such as odor nuisance, corrosion and toxicity [12]. Sulfate is the highest oxidation form of sulfur and therefore requires a reduction reaction to be removed and recovered as S0 . However, there are several wastewaters with high Copyright: © 2021 by the authors. concentrations of sulfate that lack the electron donors required for this reduction [13,14]. Licensee MDPI, Basel, Switzerland. The lack of sufficient electron donor causes a need for external electron donor supply, This article is an open access article therefore increasing significantly the operational/treatment costs and complexity [15] distributed under the terms and e.g., over-dosing of electron donor and/or dissolved oxygen can result in the undesirable conditions of the Creative Commons formation of sulfide, an inefficiency that happens even in the most carefully operated Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ industrial-scale desulfurization systems [16]. BES offer an alternative to this issue by fa- 4.0/). cilitating the autotrophic biological sulfate reduction in a biocathode with electricity as Int. J. Environ. Res. Public Health 2021, 18, 5571. https://doi.org/10.3390/ijerph18115571 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, x 2 of 12 Int. J. Environ. Res. Public Health 2021, 18, 5571 2 of 11 this issue by facilitating the autotrophic biological sulfate reduction in a biocathode with electricity as the sole electron donor [17–21]. BES-driven sulfate removal has been previ- the soleproven ously electronanddonor coupled[17–21]. BES-driven to anodic sulfidesulfate removal oxidation has [22–27] tobeen obtain previously proven S0 as the predom- and coupled to anodic sulfide oxidationcomplete [22–27] toheterotrophic 0 obtain S as the predominant inant oxidation product. In addition, sulfate reductionoxidation with par- product. In addition, complete heterotrophic sulfate reduction with tial sulfide anodic oxidation to sulfur in a single reactor [28,29] at expenses partial sulfide anodic of external oxidation to sulfur in a single reactor [28,29] at expenses of external organic matter supply, organic matter supply, and the treatment of real industrial effluents such as acid mine and the treatment of real industrial effluents such as acid mine drainage [30] and flue gas drainage [30] and flue gas desulfurization effluents [31] have been also demonstrated. desulfurization effluents [31] have been also demonstrated. Recently, Blázquez et al. [32] integrated an electrolysis cell (EC) into a BES in order Recently, Blázquez et al. [32] integrated an0 electrolysis cell (EC) into a BES in order to reduce the energy losses and improve the S production, in a chamber containing both to reduce the energy losses and improve the S0 production, in a chamber containing both the biocathode for sulfate reduction and the anode for sulfide oxidation. Good results on the biocathode for sulfate reduction and the anode for sulfide oxidation. Good 0results on elemental sulfur production (up to 93% of sulfate reduced converted 0into S ) were ob- elemental sulfur production (up to 93% of sulfate reduced converted into S ) were observed served when the current of the EC was controlled at 7.5 A m−2, but at the expense of low when the current of the EC was controlled at 7.5 A m−2 , but at the expense of low electron electron recoveries and the requirement of power supply. However, as Dutta et al. [33] recoveries and the requirement of power supply. However, as Dutta et al. [33] previously previously demonstrated, sulfide can be spontaneously oxidized in the anode of a fuel cell demonstrated, sulfide can be spontaneously oxidized in the anode of a fuel cell producing producing elemental sulfur as predominant final oxidation product. elemental sulfur as predominant final oxidation product. Theaim The aimofofthis thisstudy studywaswastoto integrate integrate a bio-electrochemical a bio-electrochemical cellcell for for autotrophic autotrophic sul- sulfate fate reduction with a sulfide–air fuel cell to remove sulfate and recover reduction with a sulfide–air fuel cell to remove sulfate and recover elemental sulfur in elemental sulfur in integrated an an integrated single single electrochemical electrochemical reactor reactor configuration(referred configuration (referredhereafter hereafterasasaaBES- BES- FC reactor), therefore reducing capital and operational costs–particularly FC reactor), therefore reducing capital and operational costs–particularly specific energy specific energy requirementsper requirements perkg kgofofsulfate sulfateremoved removedand andSS produced. 0 0produced. 2.2.Experimental Experimental 2.1. 2.1.Reactor ReactorDescription Description The TheBES-FC BES-FCreactor reactor(Figure (Figure1)1)was wasaamodification modificationofofthe thereactor reactorused usedininour ourprevious previous work × ×5 5××22cm 3 work[32] [32]with withthree threeparallel parallelacrylic acrylicframes frames(internal (internaldimensions dimensionsofof 2020 cm3each each frame) frame)bolted bolted together. The TheBESBESconsisted consistedofofanan abiotic abiotic anode anode made made of platinum of platinum wirewire (pu- (purity 99.95%, 0.50 mm diameter × 50 mm long; Advent Research Materials, rity 99.95%, 0.50 mm diameter × 50 mm long; Advent Research Materials, Eynsham, UK) Eynsham, UK) located located in the in the first first frame frame (fromleft (from lefttotoright). right).AA cation-exchange cation-exchange membrane membrane (CMI-7000; (CMI-7000; Membranes Membranes International, USA) was used to separate the biocathodefrom International, USA) was used to separate the biocathode fromthetheanode. anode. Inlet Outlet pH probe Recirculation A I P R L A C S S A E T P I T A C H R O A M D C T E E O S E RE RE M R H Recirculation BES BES FC FC Anode Cathode Anode Cathode Figure1.1.Schematic Figure SchematicofofBES-FC BES-FCsetup. setup.The Theseparator separatorconsisted consisted of of aa plastic plastic mesh (5 × mesh (5 mm22of × 55 mm ofgrid grid and and11mm mmofofthickness). thickness).ItItwas wasreplaced byby replaced rubber after rubber 145145 after days of operation. days CEM: of operation. CEM:cation-exchange cation-ex- change membrane, membrane, RE: reference RE: reference electrode.electrode. Graphite Graphitegranules granuleswith withaadiameter diameterof of66mm mmor orhigher higher(El (ElCarb Carb100; 100;Graphite GraphiteSales, Sales, Nova, Nova, OH, USA) were used as cathode material, which was placed in thesecond OH, USA) were used as cathode material, which was placed in the secondframe. frame. The third frame contained the fuel cell (FC) that consisted of an anode made of reticulated vitreous carbon (RVC) of 19 × 4 × 1 cm3 (45 ppi pore size, Duocel RVC foam; ERG, Int. J. Environ. Res. Public Health 2021, 18, 5571 3 of 11 USA–chosen because it can facilitate spontaneous HS− oxidation without significant S0 deposition/fouling [34]), a plastic mesh separator to avoid short-circuiting between the two electrodes of the FC and a passive gas diffusion air-cathode. The latter consisted of a carbon cloth coated with carbon powder and platinum suspension on the inner side (0.125 mg cm−2 , platinum nominally 40% on high surface area advanced carbon support HiSPEC 4100TM powder; Alfa Aesar, Heysham, UK), whereas the outer side was coated with a polytetrafluoroethylene (PTFE 60% w/w dispersion in H2 O; Sigma-Aldrich, St. Luis, MO, USA) solution (4 layers), which permitted oxygen diffusion into the cell while preventing electrolyte permeation [35,36]. One saturated calomel reference electrode (SCE; RE-2BP KCl sat., equiv. +0.244 V vs. Standard Hydrogen Electrode - SHE - at 25 ◦ C; BASi, USA) was embedded in the biocathode frame to measure/control the biocathode potential of the BES. Current density for BES and FC in A m−2 was defined as the measured current normalized versus the membrane surface area (100 cm2 ). The pH of the biocathode was controlled by addition of 1 M HCl by a pH controller (Liquisys M CPM253; Endress+Hauser, Reinach, Switzerland). 2.2. Biotic and Abiotic Reactions in the BES-FC Process The BES-FC setup undertakes five simultaneous (bio)electrochemical reactions as described in Equations (1)–(5) below [30,32]. Following Figure 1 from left to right, these reactions occur on the surface of the four electrodes as follows: BES anode (abiotic): 4H2 O → 2O2 + 8H+ + 8e− (1) BES-cathode (abiotic): 8H+ + 8e− → 4H2 (2) (biotic): 4H2 + SO4 2− + H+ → HS− + 4H2 O (3) − 0 + − FC-anode (abiotic): HS → S (s) + H + 2e (4) FC cathode (abiotic): O2(g) + 4 H+ + 4e− → 2H2 O (5) 2.3. Operational Conditions The system was inoculated with biomass from a previous reactor containing predomi- nantly Desulfovibrio sp. [19] and was enriched as described in our previous study, using a cultivation mineral medium without organic carbon for the biocathode at fixed potential (100 mV steps from −0.7 to −0.9 V vs. SHE in 15d intervals as a method of biofilm enrich- ment based on sulfate removal rates; see rationale and details in [32]). All electrolytes were recirculated within their respective chambers at 125 mL min−1 for adequate mixing and minimization of mass-transfer limitations. After the inoculation, the BES-FC was operated in a continuation of five experimental phases with their key operating parameters shown in Table 1. Table 1. Operational conditions in each experimental period. Days of Sulfate Influent BES-Cathode Potential HRT Period pH FC Operation Operation [mg S L−1 ] [V vs. SHE] [d] Start-up 0–52 500 −0.7 to −0.9 7.0 3.1–1.1 Off I 52–87 2000 −0.9 7.0 1.1 Off II 87–94 2000 −0.9 7.0 1.1 On III 1 94–145 2000 −0.95 7.5 1.1 On IV 2 145–161 2000 −0.98 ± 0.01 7.5 1.1 On 1 Air-cathode of the FC replaced.2 A non-porous rubber separator was added as physical separation between biocathode and anode, although both chambers remained fluidically connected through the recirculation line (Figure 1). The current density of the BES was controlled at 10 A m−2 . The FC was switched on in experimental period II (i.e., the electrical circuit was closed between RVC anode and air-cathode from this point onwards). The external resistance Int. J. Environ. Res. Public Health 2021, 18, 5571 4 of 11 used in the FC was 95 Ω, which was the optimum value for maximum power transfer according to the polarization results (Figure 2A). Polarization of the FC was performed with a VMP-3 potentiostat/galvanostat (Bio-Logic, Seyssinet-Pariset, France) from the open circuit voltage to 0 V at 1 mV s−1 scan rate. From period II, the current production of the FC was recorded every 60 s using an Agilent 34970A data acquisition unit. Period III started at day 94 with the pH controlled at 7.5 and the cathodic potential of the BES set at −0.95 V vs. SHE to increase the HS proportion up to 70% (according to the pKa of 7 for H2 S) but maintaining a similar current density. In addition, pH of 7.5 lays in the optimum range (from 6 to 8) of sulfate reducing bacteria (SRB) growth [37]. The air-cathode was replaced for a new one in period III to improve its operation and was characterized by Int. J. Environ. Res. Public Health 2021, 18, x 5 of 12 cyclic voltammetry (CV) from −0.2 to +0.5 V vs. SHE at a scan rate of 1 mV s−1 . In the last experimental period (IV), a flat rubber sheet was placed between the biocathode of the BES and the anode of the FC to mitigate the effects of electron-shuttling compounds and the current density output of the BES was controlled at 10 A m−2 . 0.6 0.035 A 0.5 0.030 Power density (W m-2) 0.025 Cell Voltage (V) 0.4 0.020 0.3 0.015 0.2 0.010 0.1 0.005 0.0 0.000 0.0 0.1 0.2 0.3 0.4 Current density (A m-2) 0 B Current density (A m-2) -5 -10 -15 -20 -25 New air-cathode Air-cathode at the end of period II -30 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 Air-cathode potential (V vs. SHE) Figure 2. Electrochemical techniques Figure 2. techniques for for the thecharacterization characterizationofofthe theFC; FC;(A): (A):polarization polarizationcurve curveofof the FC the FC and and (B): cyclic voltammetry of the the air-cathode air-cathode at at the theend endofofperiod periodIIIIand andof ofaanew newpiece pieceofof air-cathode. Scan air-cathode. Scan rate rate of of both both analyses: mV ss−−11. . analyses: 11 mV 2.4. 2.4. Analytical Analytical Methods Methods All All sampleswere samples werefiltered filteredatat0.22 µm 0.22 µm(Millipore, Burlington, (Millipore, MA, Burlington, USA) MA, andand USA) diluted us- diluted ing a sulfide using anti-oxidant a sulfide buffer anti-oxidant (SAOB) buffer solution (SAOB) in order solution to minimize in order sulfide to minimize oxidation sulfide [38]. oxidation Sulfate, sulfite, [38]. Sulfate, total total sulfite, dissolved sulfide dissolved (TDS)(TDS) sulfide and thiosulfate werewere and thiosulfate measured using measured an ion using an chromatograph (IC) with ultra-violet (UV) and conductivity detector (Dionex ion chromatograph (IC) with ultra-violet (UV) and conductivity detector (Dionex ICS- ICS-2000; 2000; Sunnyvale, CA, USA). Polysulfide was assumed to be the difference between the sulfate after H2O2 oxidation and the sum of all sulfur species measured before H2O2 oxi- dation [39]. Elemental sulfur was calculated as the difference between all measured sulfur species of the inlet and all measured sulfur species of the outlet as explained elsewhere Int. J. Environ. Res. Public Health 2021, 18, 5571 5 of 11 Sunnyvale, CA, USA). Polysulfide was assumed to be the difference between the sulfate after H2 O2 oxidation and the sum of all sulfur species measured before H2 O2 oxidation [39]. Elemental sulfur was calculated as the difference between all measured sulfur species of the inlet and all measured sulfur species of the outlet as explained elsewhere [30]. 2.5. Calculations The observed sulfate removal rate (SRR, Equation (6)), sulfide production rate (SPR, Equation (7)), theoretical elemental sulfur production rate (TESPR, Equation (8)) and the elemental sulfur proportion (Equation (9)) were calculated as follows: CSO2− −S,in − CSO2− −S,out 4 4 SRR = (6) HRT CTDS−S,out SPR = (7) HRT CSO2− −S,in − CS species−S,out 4 TESPR = (8) HRT TESPR Elemental sul f ur proportion = ·100 (9) SRR where CSO2− −S,in and CSO2− −S,out are the concentrations of sulfate at the inlet and outlet of 4 4 the system (See Figure 1), CTDS−S,out is the concentration of total dissolved sulfide in the effluent (outlet), CS species−S,out is the sum of all the concentrations of S-species measured in the effluent and HRT is the hydraulic retention time of the medium in the middle chamber (volume: 200 mL). The electron recovery for reducing sulfate was calculated as Equation (10): n ·SRR·V · F Electron recovery = 100 S (10) I · MS where nS is the number of mol of electrons needed to reduce one mole of sulfate to sulfide (= 8 mol e− ), SRR is the sulfate removal rate, V is the reactive volume (sum of BES-cathode and FC-anode chamber volumes), F is Faraday’s constant (96,485 C mol of electrons−1 ), I is the current and MS is the molar weight of sulfur (32.065 g mol−1 ). 3. Results and Discussion 3.1. Effect of Additional Oxygen Input on the Autotrophic Biocathode The BES-FC reactor was operated for 161 days under continuous mode and different operational conditions (see Figure 3). Water oxidation took place in the anode of the BES to supply the electrons for the sulfate reduction at the respective cathode. The BES-FC reactor introduced an extra input of oxygen in the reactor during the whole operation (both when the FC was switched on and off). This is because the modified carbon cloth used as air-cathode allowed for the passive diffusion of oxygen into the reactor from the atmosphere, irrespective of whether the FC electrical circuit was open or closed. The effect of this oxygen permeation through the carbon cloth was evaluated before switching the FC on. During the start-up phase (days 0 to 52, see Figure 1), low electron recoveries for reducing sulfate were observed indicating that there was an additional electron sink in the BES-cathode other than sulfate reduction. This electron flow could be used for: (i) excess H2 production, or (ii) reduction of oxygen reaching the biocathode via permeation through the cation-exchange membrane or from the air-cathode side of the FC. The possible excess of oxygen reaching the biocathode chamber could be also used to fully re-oxidize sulfide to sulfate by sulfide oxidizing bacteria such as Thiobacillus sp. [40]. This last fact could explain the low sulfate removal rate (SRR) and sulfide production rate (SPR) observed. operational conditions (see Figure 3). Water oxidation took place in the anode of th to supply the electrons for the sulfate reduction at the respective cathode. The B reactor introduced an extra input of oxygen in the reactor during the whole ope (both when the FC was switched on and off). This is because the modified carbon Int. J. Environ. Res. Public Health 2021, 18, 5571 6 of 11 used as air-cathode allowed for the passive diffusion of oxygen into the reactor fro atmosphere, irrespective of whether the FC electrical circuit was open or closed. SO42- inlet SO42- outlet TDS outlet S0t outlet FC off FC on 2500 Start-up I II III IV Sulfur species concentration 2000 (mg S L ) -1 1500 1000 500 0 0 20 40 60 80 100 120 140 160 180 Time (d) Figure 3. Evolution Figure 3.ofEvolution sulfur species concentration of sulfur along the BES-FC species concentration operation along the BES-FC phases, operationsulfate in the phases, inletinand sulfate theoutlet, tot dissolved sulfide (TDS) inlet and in the outlet, outlet total and theoretical dissolved elemental sulfide (TDS) sulfurand in the outlet produced (T-Selemental theoretical 0) in the outlet. N.B. the lack of stati sulfur produced tically significant discrepancy (T-S0 ) in the outlet. between N.B. the TDS and lack of S0 at the outlet statistically indicate significant that there between discrepancy is no formation TDS and of Sintermediate 0 at the sulfu products, asoutlet previously shown by Pozo et al. [30]. indicate that there is no formation of intermediate sulfur products, as previously shown by Pozo et al. [30]. Period I started on day 52 achieving a SRR of 646 ± 159 mg SO4 2− -S L−1 d−1 (Figure 4A). In this period, the SPR was 348 ± 48 mg TDS-S L−1 d−1 which meant a putative proportion of elemental sulfur conversion of 46.1 ± 13.9% (Figure 4B). At cathode potential of −0.9 V vs. SHE, the sulfate reduction was higher than sulfide oxidation which can be explained by a low passive oxygen diffusion, so the entire sulfide produced was partially oxidized to elemental sulfur. In addition, the electron recovery for reducing sulfate observed was 83.5 ± 19.6% (Figure 4C). The large standard deviation was potentially caused by different oxygen permeation flows as a result of the carbon cloth becoming covered in salts and/or biofilm [41]. However, the SRR was an order of magnitude higher than the previous investigation of Pozo et al. [42], who achieved 63 mg SO4 2− -S L−1 d−1 in continuous mode, pH 7.3 and graphite granules as biocathode at the same cathode potential (−0.9 V vs. SHE), and nearly double than Blázquez et al. [21], who achieved 358 mg SO4 2− -S L−1 d−1 in batch mode without pH control and graphite-fiber brush as biocathode material. During the whole operation of our BES-FC, the use of a gas diffusion electrode allowed for an extra route of oxygen diffusion into the reactor, which weakened the SRR, but at the same time it improved the theoretical elemental sulfur production rate (TESPR) compared to the results with the BES-EC [32]. As oxygen can be reduced in the BES-cathode to water, the electron recovery is expected to be lower if there is an excess of oxygen. However, the electron recovery in period V was higher than 80%, indicating that the mass transfer of oxygen into the system was minimal, possibly due microorganisms and/or elemental sulfur becoming attached to the carbon cloth cathode [41]. In this period, the SPR was 348 ± 48 mg TDS-S L−1 d−1 which meant a putative proportion of elemental sulfur conversion of 46.1 ± 13.9% (Figure 4B). At cathode potential of −0.9 V vs. SHE, the sulfate reduction was higher than sulfide oxidation which can be explained by a low passive oxygen diffusion, so the entire sulfide produced was partially oxidized to elemental sulfur. In addition, the electron recovery for reducing sulfate observed was Int. J. Environ. Res. Public Health 2021, 18, 5571 7 of 11 83.5 ± 19.6% (Figure 4C). 1000 120 SRR, SPR and TESPR (g S L-1 d-1) A Elemental sulfur proportion (%) FC off FC on FC off FC on B SRR 100 800 SPR TESPR 80 600 60 400 40 200 20 0 0 I II III IV I II III IV Period Period 140 18.0 FC off FC on C 16.0 FC off FC on D 120 Current density (A m-2) 14.0 BES Electron recovery (%) FC 100 12.0 10.0 80 8.0 60 6.0 40 4.0 2.0 20 0.2 0 0.0 I II III IV I II III IV Period Period Figure 4. Figure 4. Average plots plots ofof the thedifferent differentoperational operationalphases phasesofofthe BES-FC the BES-FCafter thethe after start-up on on start-up (A):(A): sulfate reduction sulfate rate reduction (SRR), rate sulfide (SRR), production sulfide productionraterate (SPR) andand (SPR) theoretical elemental theoretical sulfur elemental production sulfur rate rate production (TESPR), (B): (B): (TESPR), elemental sulfur elemental pro- sulfur portion in in proportion relation to to relation sulfate sulfatereduced, reduced,(C): (C):electron electronrecovery recoveryasassulfate sulfatereduced reduced in in the the biocathode and (D): biocathode and (D): current current density density of BES and FC. of BES and FC. 3.2. Microbial Electrochemical Cell and Sulfide–Air Fuel Cell Integration The FC was switched on at day 87 (period II) to evaluate its influence on sulfate treatment, elemental sulfur production and electron recovery. The current density obtained in the FC was 0.09 ± 0.03 A m−2 (Figure 4D), which was negligible compared with the current density of the BES (8.2 ± 0.1 A m−2 ) and, thus, the FC was not able to effectively oxidize the sulfide produced by the BES biocathode. The SRR nevertheless increased up to the highest rate observed of 767 ± 26 mg SO4 2− -S L−1 d−1 and the TESPR up to 386 ± 12 mg S0 -S L−1 d−1 . The observed low improvement on the TESPR with the FC switched on was a consequence of the low FC current density. This in turn could be due to: (i) a low rate of reaction of TDS with the FC-anode or (ii) an inactivation of the FC electrodes because of elemental sulfur precipitation on the air-cathode surface (partially blocking oxygen diffusion) or by fouling of the catalyst (platinum) by sulfide. To check if the low improvement was due to the low reaction of TDS with the FC-anode, the pH increased up to 7.5 in period III (day 94) to increase the HS− speciation fraction from 50% to 70% (the first proton acidity constant for H2 S is pKa = 7.0) [43]. Increasing the pH in the cathode has a negative effect on hydrogen evolution (since hydrogen production is less favorable at higher pH values at a fixed potential, according to the Nernst equation) and this resulted in a current decrease. To compensate for this, the biocathode potential was adjusted to −0.95 V vs. SHE. In addition, a CV of the air-cathode was performed to determine if Int. J. Environ. Res. Public Health 2021, 18, 5571 8 of 11 Int. J. Environ. Res. Public Health 2021, 18, x 9 of 12 there was an observable reduction of catalytic activity due to precipitation and/or fouling (Figure 2B). The CV showed that after 94 days of operation of the reactor with the FC decreasedoff, switched again thetoair-cathode 0.08 ± 0.05 A wasm−2indeed over time. Figure affected, as4demonstrated shows that from by mid-period III to the much lower the end, the current of the FC tended to decrease probably due to the same inactivation current densities attained (Figure 5). For this reason, the air-cathode was replaced at the effects start ofpreviously period III. observed (see Figure 2B). 0.5 I II III IV 0.4 Current density (A m-2) 0.3 0.2 0.1 0.0 0 80 100 120 140 160 180 Time (d) Current density Figure 5. Current density of of the theFC FCduring duringall allthe theexperimental experimentalphases. phases.N.B. N.B.The Thestart-up start-upand andperiod pe- Iriod are Icondensed are condensed because because the FCthewas FC in was in open open circuit circuit duringduring this (no this time timecurrent). (no current). In Theperiod III, the current FC integration did notdensity improveofthe thesystem FC was almost double performance than inmainly as expected periodbe-II cause±of0.06 (0.17 A m−2 ), butofthe a deterioration theSRR decreased air-cathode. The 478 ± grown tobiofilm 126 mgoverSO4the2− -S L−1 d−1 and the air-cathode surface SPR and the reduced TESPR decreased presence significantly. of dissolved oxygen Asinthe thecurrent density bulk liquid andofinthe BES was addition almost better the colum- same (7.7 ± 1.2 A m −2 ), the decrease on the SRR, SPR and TESPR compared with periods I bic efficiencies and current production have been observed as in other studies [40,45]. For and II was very this reason, likely caused the biofilm attachedbyto the thenew air-cathode air-cathode that not should allowed declinea much the FChigher oxygen performance. diffusion The poor which led to complete performance of the FCsulfide might re-oxidation be caused bytoexcess sulfateofagain. oxygen reacting with sul- fide, The by thecurrent of the sulfur elemental FC wasattached still really to low and this poor the air-cathode orperformance by inactivation wasofhypothesized the air-cath- to odebeplatinum caused bycatalyst the effect of electron-shuttling as observed compounds in other metal-based which are catalysts [22].redox mediators that can beA repeatedly membrane next oxidized to theand reduced could air-cathode by thebe electrodes a solutionandforthe thismicroorganisms problem, which [44]. was To minimize this possible effect, a rubber sheet physically separating also studied by Zhao et al. [27]. Other studies tried to remove sulfate heterotrophically the BES-cathode from the oxidize and then FC-anode thewas installed sulfide in the in period anode of aIV (day 145), butreactor, single-chamber keeping both chambers achieving propor- fluidically connected and mixed by separated recirculation. In tions of elemental sulfur between 60 and 75% of the initial sulfate [28,29] butaddition, the current density at lower SRR of the BES was kept constant using galvanostatic mode, controlling and at expenses of an external electron donor supply. Further studies should test the same the current density at system m−2without 10 A but to avoid thefluctuations FC, while stillin the BES performance allowing in period oxygen intrusion throughIV. No operational a gas diffusion changes resulted in a high layer. Additionally, the −current SRR improvement configuration and the should current be density trialed withof the an FC decreased ion-exchange again membraneto 0.08to±replace 0.05 A them 2meshover separator time. Figure 4 shows between that from FC-anode mid-period and cathode, as III atomeans the end,to the current of the FC tended to decrease prevent direct oxidation of sulfide by oxygen. probably due to the same inactivation effects previously observedto(see In comparison ourFigure 2B).study [32], the FC improved significantly the propor- previous The FC integration did not improve the system performance as expected mainly tion of elemental sulfur but with lower SRR and electron recoveries. However, in terms of because of a deterioration of the air-cathode. The biofilm grown over the air-cathode surface energy consumption for sulfur recovery, the BES-FC shows an improvement compared reduced the presence of dissolved oxygen in the bulk liquid and in addition better columbic with the BES-EC that reported an energy consumption per kg of sulfate removed of 9.18 efficiencies and−1current production have been observed as in other studies [40,45]. For this ± 0.80 kWh kg SO42−-S considering the maximum SRR. Converting this result into kg of reason, the biofilm attached to the air-cathode should not decline the FC performance. The sulfur recovered and adding the energy consumption of the EC, this means an energy poor performance of the FC might be caused by excess of oxygen reacting with sulfide, consumption of 18.74 ± 1.81 kWh kg−1 S0-S. With our BES-FC, the energy consumption for by the elemental sulfur attached to the air-cathode or by−1 inactivation of the air-cathode sulfate removed was slightly higher (10.61 ± 0.12 kWh kg SO42−-S) because, as mentioned platinum catalyst as observed in other metal-based catalysts [22]. before, the extra oxygen permeation caused an inherent inefficiency (re-oxidation of some A membrane next to the air-cathode could be a solution for this problem, which was of the sulfide to sulfate). However, as the FC was not consuming energy, the total energy also studied by Zhao et al. [27]. Other studies tried to remove sulfate heterotrophically and consumption for sulfur production was of 16.50 ± 0.19 kWh kg−1 S0-S, a 12% improvement then oxidize the sulfide in the anode of a single-chamber reactor, achieving proportions of in terms ofsulfur elemental energy consumption between 60 andfor75% elemental sulfur of the initial production sulfate [28,29] than but at the use of lower SRRa BES-EC and at and at lower capital investment needs (although the savings can only be determined in larger-scale trials), since the FC does not require a potentiostatic control/power source. Int. J. Environ. Res. Public Health 2021, 18, 5571 9 of 11 expenses of an external electron donor supply. Further studies should test the same system but without the FC, while still allowing oxygen intrusion through a gas diffusion layer. Additionally, the current configuration should be trialed with an ion-exchange membrane to replace the mesh separator between FC-anode and cathode, as a means to prevent direct oxidation of sulfide by oxygen. In comparison to our previous study [32], the FC improved significantly the proportion of elemental sulfur but with lower SRR and electron recoveries. However, in terms of energy consumption for sulfur recovery, the BES-FC shows an improvement compared with the BES-EC that reported an energy consumption per kg of sulfate removed of 9.18 ± 0.80 kWh kg−1 SO4 2− -S considering the maximum SRR. Converting this result into kg of sulfur recovered and adding the energy consumption of the EC, this means an energy consumption of 18.74 ± 1.81 kWh kg−1 S0 -S. With our BES-FC, the energy consumption for sulfate removed was slightly higher (10.61 ± 0.12 kWh kg−1 SO4 2− -S) because, as mentioned before, the extra oxygen permeation caused an inherent inefficiency (re-oxidation of some of the sulfide to sulfate). However, as the FC was not consuming energy, the total energy consumption for sulfur production was of 16.50 ± 0.19 kWh kg−1 S0 -S, a 12% improvement in terms of energy consumption for elemental sulfur production than the use of a BES-EC and at lower capital investment needs (although the savings can only be determined in larger-scale trials), since the FC does not require a potentiostatic control/power source. 4. Conclusions This work demonstrates a BES-FC that can treat synthetic wastewater with high sulfate content obtaining sulfate removal rates up to 767 mg SO4 2− -S L−1 d−1 at −0.9 V vs. SHE. In addition, the BES-FC allowed for a 12% reduction in the energy consumption per kg of elemental sulfur recovered compared with a BES with an electrochemical cell instead of the FC with an air-cathode. The FC improved the proportion of elemental sulfur produced thanks to the oxygen diffusion through the carbon cloth compared with an electrolysis cell, but at expenses of lower sulfate removals. Author Contributions: Conceptualization, E.B., A.G., S.F. and P.L.; methodology, E.B., D.G., J.A.B., A.G., S.F. and P.L.; software, E.B., J.A.B.; formal analysis, E.B., S.F. and P.L.; investigation, E.B., D.G., J.A.B., A.G., S.F. and P.L.; resources, E.B., S.F. and P.L.; data curation, E.B. and P.L.; writing—original draft preparation, E.B. and P.L.; writing—review and editing, E.B., D.G., J.A.B., A.G., S.F. and P.L.; visualization, E.B., A.G.; supervision, D.G., J.A.B., A.G., S.F. and P.L.; project administration, A.G. and S.F.; funding acquisition, A.G. and D.G. All authors have read and agreed to the published version of the manuscript. Funding: Some authors are members of the GENOCOV group (Grup de Recerca Consolidat de la Generalitat de Catalunya, 2017 SGR 1175). E. Blázquez acknowledges a PhD scholarship from AGAUR (2017 FI_B 0121) and to Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya. The authors thank the Australian Research Council for funding support through Discovery Project DP120104415. P. Ledezma furthermore acknowledges an ECR Development Fellowship from The University of Queensland. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article. Conflicts of Interest: The authors declare no conflict of interest. References 1. Jadhav, D.A.; Ghosh Ray, S.; Ghangrekar, M.M. Third generation in bio-electrochemical system research—A systematic review on mechanisms for recovery of valuable by-products from wastewater. Renew. Sustain. Energy Rev. 2017, 76, 1022–1031. [CrossRef] 2. Nancharaiah, Y.V.; Mohan, S.V.; Lens, P.N.L. Metals removal and recovery in bioelectrochemical systems: A review. Bioresour. Technol. 2015, 195, 102–114. [CrossRef] Int. J. Environ. Res. Public Health 2021, 18, 5571 10 of 11 3. Dominguez-benetton, X.; Chandrakant, J.; Pozo, G.; Modin, O.; Ter, A.; Fransaer, J.; Rabaey, K. Progress in Materials Science Metal recovery by microbial electro-metallurgy. Prog. Mater. Sci. 2018, 94, 435–461. [CrossRef] 4. Kelly, P.T.; He, Z. Nutrients removal and recovery in bioelectrochemical systems: A review. Bioresour. Technol. 2014, 153, 351–360. [CrossRef] 5. Nancharaiah, Y.V.; Mohan, S.V.; Lens, P.N.L. Recent advances in nutrient removal and recovery in biological and bioelectrochemi- cal systems. Bioresour. Technol. 2016, 215, 173–185. [CrossRef] 6. Batlle-Vilanova, P.; Puig, S.; Gonzalez-Olmos, R.; Balaguer, M.D.; Colprim, J. Continuous acetate production through microbial electrosynthesis from CO2 with microbial mixed culture. J. Chem. Technol. Biotechnol. 2016, 91, 921–927. [CrossRef] 7. Vassilev, I.; Hernandez, P.A.; Batlle-vilanova, P.; Freguia, S.; Kro, J.O.; Keller, J.; Ledezma, P.; Virdis, B. Microbial Electrosynthesis of Isobutyric, Butyric, Caproic Acids, and Corresponding Alcohols from Carbon Dioxide. ACS Sustain. Chem. Eng. 2018, 6, 8485–8493. [CrossRef] 8. Blázquez, E.; Guisasola, A.; Gabriel, D.; Baeza, J.A. Application of Bioelectrochemical Systems for the Treatment of Wastewaters With Sulfur Species. Microb. Electrochem. Technol. 2019, 641–663. [CrossRef] 9. Cope, G. The calm before the storm. Hydrocarb. Eng. 2012, 10, 82–87. 10. Muyzer, G.; Stams, A.J.M. The ecology and biotechnology of sulphate-reducing bacteria. Nat. Rev. Microbiol. 2008, 6, 441–454. [CrossRef] 11. Lens, P.; Pol, L. Environmental Technologies to Treat Sulfur Pollution—Principles and Applications. Water Intell. Online 2015, 4. [CrossRef] 12. Pol, L.W.H.; Lens, P.N.L.; Stams, A.J.M.; Lettinga, G. Anaerobic treatment of sulphate-rich wastewaters. Biodegradation 1998, 9, 213–224. [CrossRef] 13. Kaksonen, A.H.; Puhakka, J.A. Sulfate reduction based bioprocesses for the treatment of acid mine drainage and the recovery of metals. Eng. Life Sci. 2007, 7, 541–564. [CrossRef] 14. Srivastava, R.K.; Jozewicz, W. Flue Gas Desulfurization: The State of the Art. J. Air Waste Manage. Assoc. 2001, 51, 1676–1688. [CrossRef] 15. Liamleam, W.; Annachhatre, A.P. Electron donors for biological sulfate reduction. Biotechnol. Adv. 2007, 25, 452–463. [CrossRef] 16. Mol, A.; van der Weijden, R.; Klok, J.; Buisman, C. Properties of Sulfur Particles Formed in Biodesulfurization of Biogas. Minerals 2020, 10, 433. [CrossRef] 17. Coma, M.; Puig, S.; Pous, N.; Balaguer, M.D.; Colprim, J. Biocatalysed sulphate removal in a BES cathode. Bioresour. Technol. 2013, 130, 218–223. [CrossRef] [PubMed] 18. Luo, H.; Teng, W.; Liu, G.; Zhang, R.; Lu, Y. Sulfate reduction and microbial community of autotrophic biocathode in response to acidity. Process Biochem. 2017, 54, 120–127. [CrossRef] 19. Pozo, G.; Lu, Y.; Pongy, S.; Keller, J.; Ledezma, P.; Freguia, S. Selective cathodic microbial biofilm retention allows a high current-to-sulfide efficiency in sulfate-reducing microbial electrolysis cells. Bioelectrochemistry 2017, 118, 62–69. [CrossRef] [PubMed] 20. Pozo, G.; Jourdin, L.; Lu, Y.; Keller, J.; Ledezma, P.; Freguia, S. Cathodic biofilm activates electrode surface and achieves efficient autotrophic sulfate reduction. Electrochim. Acta 2016, 213, 66–74. [CrossRef] 21. Blázquez, E.; Gabriel, D.; Baeza, J.A.; Guisasola, A. Evaluation of key parameters on simultaneous sulfate reduction and sulfide oxidation in an autotrophic biocathode. Water Res. 2017, 123, 301–310. [CrossRef] [PubMed] 22. Rabaey, K.; Van de Sompel, K.; Maignien, L.; Boon, N.; Aelterman, P.; Clauwaert, P.; De Schamphelaire, L.; Pham, H.T.; Vermeulen, J.; Verhaege, M.; et al. Microbial Fuel Cells for Sulfide. Environ. Sci. Technol. 2006, 40, 5218–5224. [CrossRef] 23. Gong, Y.; Ebrahim, A.; Feist, A.M.; Embree, M.; Zhang, T.; Lovley, D.; Zengler, K. Sulfide-driven microbial electrosynthesis. Environ. Sci. Technol. 2013, 47, 568–573. [CrossRef] [PubMed] 24. Dutta, P.K.; Rabaey, K.; Yuan, Z.; Rozendal, R.A.; Keller, J. Electrochemical sulfide removal and recovery from paper mill anaerobic treatment effluent. Water Res. 2010, 44, 2563–2571. [CrossRef] 25. Sun, M.; Tong, Z.-H.; Sheng, G.-P.; Chen, Y.-Z.; Zhang, F.; Mu, Z.-X.; Wang, H.-L.; Zeng, R.J.; Liu, X.-W.; Yu, H.-Q.; et al. Microbial communities involved in electricity generation from sulfide oxidation in a microbial fuel cell. Biosens. Bioelectron. 2010, 26, 470–476. [CrossRef] [PubMed] 26. Sun, M.; Mu, Z.X.; Chen, Y.P.; Sheng, G.P.; Liu, X.W.; Chen, Y.Z.; Zhao, Y.; Wang, H.L.; Yu, H.Q.; Wei, L.; et al. Microbe-assisted sulfide oxidation in the anode of a microbial fuel cell. Environ. Sci. Technol. 2009, 43, 3372–3377. [CrossRef] 27. Zhao, F.; Rahunen, N.; Varcoe, J.R.; Chandra, A.; Roberts, A.J.; Avignone-Rossa, C.; Thumser, A.E.; Slade, R.C.T. Activated Carbon Cloth as Anode for Sulfate Removal in a Microbial Fuel Cell. Environ. Sci. Technol. 2008, 42, 4971–4976. [CrossRef] 28. Lee, D.J.; Liu, X.; Weng, H.L. Sulfate and organic carbon removal by microbial fuel cell with sulfate-reducing bacteria and sulfide-oxidising bacteria anodic biofilm. Bioresour. Technol. 2014, 156, 14–19. [CrossRef] 29. Chatterjee, P.; Ghangrekar, M.M.; Rao, S.; Kumar, S. Biotic conversion of sulphate to sulphide and abiotic conversion of sulphide to sulphur in a microbial fuel cell using cobalt oxide octahedrons as cathode catalyst. Bioprocess Biosyst. Eng. 2017, 40, 759–768. [CrossRef] 30. Pozo, G.; Pongy, S.; Keller, J.; Ledezma, P.; Freguia, S. A novel bioelectrochemical system for chemical-free permanent treatment of acid mine drainage. Water Res. 2017, 126, 411–420. [CrossRef] Int. J. Environ. Res. Public Health 2021, 18, 5571 11 of 11 31. Blázquez, E.; Baeza, J.A.; Gabriel, D.; Guisasola, A. Treatment of real flue gas desulfurization wastewater in an autotrophic biocathode in view of elemental sulfur recovery: Microbial communities involved. Sci. Total Environ. 2019, 657, 945–952. [CrossRef] 32. Blázquez, E.; Gabriel, D.; Baeza, J.A.; Guisasola, A.; Freguia, S.; Ledezma, P. Recovery of elemental sulfur with a novel integrated bioelectrochemical system with an electrochemical cell. Sci. Total Environ. 2019, 677, 175–183. [CrossRef] 33. Dutta, P.K.; Rabaey, K.; Yuan, Z.; Keller, J. Spontaneous electrochemical removal of aqueous sulfide. Water Res. 2008, 42, 4965–4975. [CrossRef] 34. Mejia Likosova, E.; Keller, J.; Poussade, Y.; Freguia, S. A novel electrochemical process for the recovery and recycling of ferric chloride from precipitation sludge. Water Res. 2014, 51, 96–103. [CrossRef] [PubMed] 35. Cheng, S.; Liu, H.; Logan, B.E. Increased performance of single-chamber microbial fuel cells using an improved cathode structure. Electrochem. Commun. 2006, 8, 489–494. [CrossRef] 36. Cheng, S.; Liu, H.; Logan, B.E. Power densities using different cathode catalysts (Pt and CoTMPP) and polymer binders (Nafion and PTFE) in single chamber microbial fuel cells. Environ. Sci. Technol. 2006, 40, 364–369. [CrossRef] 37. Hao, O.J.; Chen, J.M.; Huang, L.; Buglass, R.L. Sulfate-reducing bacteria. Crit. Rev. Environ. Sci. Technol. 1996, 26, 155–187. [CrossRef] 38. Keller-Lehmann, B.; Corrie, S.; Ravn, R.; Yuan, Z.; Keller, J. Preservation and Simultaneous Analysis of Relevant Soluble Sulfur Species in Sewage Samples. In Proceedings of the Second International IWA Conference on Sewer Operation and Maintenance, Vienna, Austria, 26–28 October 2006; Volume 26, p. 28. 39. Dutta, P.K.; Rozendal, R.A.; Yuan, Z.; Rabaey, K.; Keller, J. Electrochemical regeneration of sulfur loaded electrodes. Electrochem. Commun. 2009, 11, 1437–1440. [CrossRef] 40. Krayzelova, L.; Bartacek, J.; Díaz, I.; Jeison, D.; Volcke, E.I.P.; Jenicek, P. Microaeration for hydrogen sulfide removal during anaerobic treatment: A review. Rev. Environ. Sci. Biotechnol. 2015, 14, 703–725. [CrossRef] 41. Montpart, N.; Rago, L.; Baeza, A.; Guisasola, A. Oxygen barrier and catalytic effect of the cathodic biofilm in single chamber microbial fuel cells. J. Chem. Technol. Biotechnol. 2018. [CrossRef] 42. Pozo, G.; Jourdin, L.; Lu, Y.; Ledezma, P.; Keller, J.; Freguia, S. Methanobacterium enables high rate electricity-driven autotrophic sulfate reduction. RSC Adv. 2015, 5, 89368–89374. [CrossRef] 43. Moosa, S.; Harrison, S.T.L. Product inhibition by sulphide species on biological sulphate reduction for the treatment of acid mine drainage. Hydrometallurgy 2006, 83, 214–222. [CrossRef] 44. Watanabe, K.; Manefield, M.; Lee, M.; Kouzuma, A. Electron shuttles in biotechnology. Curr. Opin. Biotechnol. 2009, 20, 633–641. [CrossRef] [PubMed] 45. Ou, S.; Zhao, Y.; Aaron, D.S.; Regan, J.M.; Mench, M.M. Modeling and validation of single-chamber microbial fuel cell cathode biofilm growth and response to oxidant gas composition. J. Power Sources 2016, 328, 385–396. [CrossRef]