Environmental Science and Ecotechnology 12 (2022) 100199 Contents lists available at ScienceDirect Environmental Science and Ecotechnology journal homepage: www.journals.elsevier.com/environmental-science-and- ecotechnology/ Bioelectrochemically-assisted degradation of chloroform by a co- culture of Dehalobacter and Dehalobacterium ndez-Verdejo a, Pilar Corte David Ferna nquez a, *, s b, Albert Guisasola c, Paqui Bla Ernest Marco-Urrea a a Biorem UAB, Department of Chemical, Biological and Environmental Engineering, School of Engineering, Universitat Auto noma de Barcelona, 08193, Bellaterra, Barcelona, Spain b Departament de Gen etica i de Microbiologia, Facultat de BioCi noma de Barcelona, 08193, Bellaterra, Barcelona, Spain encies, Universitat Auto c GENOCOV, Department of Chemical, Biological and Environmental Engineering, School of Engineering, Universitat Auto noma de Barcelona, 08193, Bellaterra, Barcelona, Spain a r t i c l e i n f o a b s t r a c t Article history: Using bioelectrochemical systems (BESs) to provide electrochemically generated hydrogen is a promising Received 20 April 2022 technology to provide electron donors for reductive dechlorination by organohalide-respiring bacteria. In Received in revised form this study, we inoculated two syntrophic dechlorinating cultures containing Dehalobacter and Dehalo- 17 June 2022 bacterium to sequentially transform chloroform (CF) to acetate in a BES using a graphite fiber brush as the Accepted 20 June 2022 electrode. In this co-culture, Dehalobacter transformed CF to stoichiometric amounts of dichloromethane (DCM) via organohalide respiration, whereas the Dehalobacterium-containing culture converted DCM to Keywords: acetate via fermentation. BES were initially inoculated with Dehalobacter, and sequential cathodic po- Dehalobacter Dehalobacterium tentials of 0.6, 0.7, and 0.8 V were poised after consuming three CF doses (500 mM) per each po- Chloroform tential during a time-span of 83 days. At the end of this period, the accumulated DCM was degraded in Dichloromethane the following seven days after the inoculation of Dehalobacterium. At this point, four consecutive Bioelectrochemistry amendments of CF at increasing concentrations of 200, 400, 600, and 800 mM were sequentially trans- Bioremediation formed by the combined degradation activity of Dehalobacter and Dehalobacterium. The Dehalobacter 16S rRNA gene copies increased four orders of magnitude during the whole period. The coulombic effi- ciencies associated with the degradation of CF reached values > 60% at a cathodic potential of 0.8 V when the degradation rate of CF achieved the highest values. This study shows the advantages of combining syntrophic bacteria to fully detoxify chlorinated compounds in BESs and further expands the use of this technology for treating water bodies impacted with pollutants. © 2022 The Authors. Published by Elsevier B.V. on behalf of Chinese Society for Environmental Sciences, Harbin Institute of Technology, Chinese Research Academy of Environmental Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Due to the typically anoxic conditions of contaminated groundwater, applying anaerobic bioremediation treatments cata- Chloroform (CF) is a chlorinated compound usually employed as lyzed by organohalide-respiring bacteria (OHRB) is a potential so- a solvent and in the chemical synthesis of fluorocarbons [1]. Due to lution to remediate sites impacted with chlorinated pollutants improper handling and disposal practices, CF can be discharged [3,4]. To date, bacteria belonging to the genera Dehalobacter and into the subsurface environment, which constitutes a health risk to Desulfitobacterium are the only OHRB described using CF as a ter- humans. According to the 2019 ATSDR Substance Priority List, CF is minal electron acceptor, dechlorinating CF to predominantly ranked 11th (out of 275) based on a combination of frequency, dichloromethane (DCM) [5,6]. Dehalobacter is a strict hydro- toxicity and potential for human exposure [2]. genotroph; therefore, an external and continuous supply of * Corresponding author. nquez). E-mail address: paqui.blanquez@uab.cat (P. Bla https://doi.org/10.1016/j.ese.2022.100199 2666-4984/© 2022 The Authors. Published by Elsevier B.V. on behalf of Chinese Society for Environmental Sciences, Harbin Institute of Technology, Chinese Research Academy of Environmental Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ndez-Verdejo, P. Cortes, A. Guisasola et al. D. Ferna Environmental Science and Ecotechnology 12 (2022) 100199 hydrogen in contaminated groundwaters is required to promote The system was sealed with butyl rubber septa and aluminum the dechlorination of CF by this genus [7]. The direct injection of crimp caps, allowing liquid samples to be added and withdrawn fermentable organic substrates, such as lactate, ethanol and from the anodic and cathodic vessels. molasses, is the common procedure to deliver hydrogen for enhanced anaerobic bioremediation. Still, some disadvantages of 2.2. Cultivation of the Dehalobacter and Dehalobacterium- this procedure include the rapid degradation of the soluble sub- containing cultures in serum bottles strates and the subsequent need for repetitive reinjections that could lower the pH in groundwater [8]. The enriched cultures containing Dehalobacter used in this study In the last years, intensive research with bioelectrochemical were derived from groundwater contaminated with CF [30]. The systems (BESs) has been performed as a novel strategy to supply inoculum for the Dehalobacterium-containing consortia was ob- hydrogen as the electron donor to promote anaerobic reductive tained from slurry samples of a membrane bioreactor from a dechlorination of chlorinated pollutants in groundwaters [9e14]. wastewater plant and subsequently enriched in the laboratory [25]. BESs consist of two-chamber devices where an oxidation reaction The Dehalobacter sp. contained in the culture, denominated Deha- occurs in the anode, and a reduction reaction occurs in the cathode, lobacter sp. strain 8 M, transformed CF to DCM via organohalide with one or both reactions catalyzed by bacteria. BESs aiming to respiration [30], whereas the Dehalobacterium-containing culture produce hydrogen in cathodes are not thermodynamically spon- transformed DCM to acetate in a three-step process [26]. Both taneous; thus, an additional voltage is required to drive the process. cultures were maintained in 100 mL glass bottles containing 65 mL In previous studies, a correlation has been observed between more of reduced bicarbonate-buffered medium (pH ¼ 7) as described negative cathodic potentials and hydrogen production, followed by elsewhere [31]. In brief, the growth medium of Dehalobacterium a noticeable increase in the degradation rates of dechlorinating contained trace elements, tungsten (22.8 mM), selenium (24.2 mM), bacteria [9,11]. However, this increase has been at the cost of an vitamins, yeast extract (200 mg L1), Na2S$9H2O, and L-cysteine excess of hydrogen production and a concomitant decrease in the (0.2 mM each) as reducing agents and sodium bicarbonate (12 mM) coulombic efficiency of the process. To date, the OHRB studied in as a buffering agent. This same medium was used for the Dehalo- BESs belonged to the genera Dehalococcoides, Geobacter, and bacter microcosms but included sodium acetate (5 mM) as an Dehalogenimonas and degraded contaminants as 1,2- additional carbon source. Butyl rubber septa and aluminum crimp dichloroethane, tetrachloroethene, trichloroethene, cis-dichlor- caps were used to seal the bottles. Dehalobacter cultures were oethene, or 2,3,4,5-tetrachlorobiphenyl [9,13,15e18]. gassed with N2/CO2 (4:1 at 0.2 bar of overpressure) and H2 (0.4 bar To detoxify CF-impacted sites, the next step after the dechlori- of overpressure), while Dehalobacterium cultures were gassed with nation of CF by Dehalobacter is the remediation of DCM, which is N2 (0.4 bar of overpressure). still a toxic compound of environmental concern [19,20]. To date, only two DCM fermentative bacteria, all belonging to the Pepto- coccaceae family, have been identified to ferment DCM: “Candida- 2.3. Operation of BESs tus Formimonas warabiya” (formerly referred to as strain DCMF) and Dehalobacterium formicoaceticum [21e24]. The fermentation of Cathodic and anodic vessels were filled with 130 mL of the DCM by D. formicoaceticum produces acetate and formate, which anaerobic medium of Dehalobacter described above. Cathodic are environmentally friendly products [25e27]. Additionally, compartments were initially spiked with 500 mM CF and inoculated “Candidatus Dichloromethanomonas elyuquensis” was recently with 3 mL of the Dehalobacter-containing culture (concentration of found to completely mineralize DCM to H2 and CO2 [28,29]. Dehalobacter ~9.38  104 ± 2.38  104 16S rRNA gene copies per The study aims to assess the feasibility of coupling the dechlo- mL). Biotic open circuit BESs without electrodes were included as rination potential of two mixed cultures, with one containing controls to assess biodegradation of CF without electrochemically Dehalobacter and the other Dehalobacterium, in the cathodic generated H2. In addition, abiotic BESs with the cathodes poised at chamber of a BES to transform CF into non-toxic final products for certain potentials were included to assess that CF and DCM were the first time. The sequential dechlorination proceeds through the not transformed electrochemically. The cathodic potentials were organohalide respiration of CF to DCM via Dehalobacter, followed by initially adjusted to 0.6 V vs. Standard Hydrogen Electrode (SHE) the fermentation of DCM to acetate and formate via Dehalobacte- against an Ag/AgCl reference electrode (RE-1B, BAS Inc., þ197 mV rium. The application of sequentially decreasing cathodic potentials vs. SHE) by applying a potentiostatic control using a power source was tested to balance the electrochemical production of hydrogen (Quad Potentiostat, Whistonbrook Software). All the potential to the growth of the Dehalobacter population to maximize at the values mentioned are V vs. Standard Hydrogen Electrode (SHE) same time the degradation rates and the coulombic efficiency of unless otherwise stated. the process. This work expands the number of bacteria and con- Each cell configuration was operated with three parallel repli- taminants degraded utilizing BES-based technologies while cates. CF was re-spiked with 500 mM whenever depleted. After combining for the first time the degradation of CF in a two-step consuming three CF amendments at 0.6 V, the cathodic potential dechlorination process in a single BES. was sequentially reduced to 0.7 and 0.8 V after degrading three CF amendments at each potential. Afterward, the systems were 2. Materials and methods inoculated with 6 mL of the Dehalobacterium-containing culture to proceed with the consumption of the DCM produced. At this point, 2.1. Bioelectrochemical cells and operation several CF amendments of increasing concentration were periodi- cally added to degrade both CF and DCM sequentially. During the The bioelectrochemical system used in this study was described whole BES operation, 1-mL liquid samples were periodically taken previously [18]. In brief, it consisted of two glass vessels with to monitor pH, and it was adjusted to pH 7 by adding HCl from an 165 mL of total volume separated by a cation-exchange membrane anaerobic stock solution (1 M). The liquid medium removed for (CMI-7000, Membranes International INC, USA) with an aperture analytical measurements (see section 2.5) was replaced with the diameter of 4 cm. A titanium sheet and a graphite brush were used addition of a fresh medium. The hydraulic retention time was as the anodic and cathodic electrodes, respectively. Mixing of the longer than the length of the experiments, so the BESs were liquid medium of each vessel was performed by a magnetic stirrer. considered to operate in fed-batch mode. 2 ndez-Verdejo, P. Cortes, A. Guisasola et al. D. Ferna Environmental Science and Ecotechnology 12 (2022) 100199 2.4. Coulombic efficiency and energy consumption Xgen Y¼ (4) The coulombic efficiency (CE) after the consumption of each CF CFdeg amendment was calculated using equation (1): where Xgen is the increase of Dehalobacter 16S rRNA gene copies 2$V$F$½CFdeg and CFdeg is the mmol of CF degraded during a certain time period. CE ¼ ð (1) The maximum specific CF utilization rate by Dehalobacter (q, IðtÞdt mmol CF degraded per 16S rRNA gene copy generated per day) can be calculated as follows: where 2 is the number of electrons required to dechlorinate a m molecule of CF to DCM, V is the liquid volume at the cathodic vessel q¼ (5) (L), F is Faraday's constant (96,485 A s mol1), [CF]deg is the con- Y centration of CF degraded (M) and !I(t)dt is the integration of the monitored intensity throughout the experimental time (A s). Also, the energetic input (EI) required for each mol of CF 3. Results and discussion degraded at each experiment was calculated as follows: ð 3.1. Effect of the cathodic potential on CF dechlorination by VA $ IðtÞdt Dehalobacter EI ¼ (2) V$½CFdeg BESs inoculated with Dehalobacter successfully dechlorinate several doses of CF operating at sequential cathodic potentials where VA is the applied voltage in the system (V). of 0.6, 0.7, and 0.8 V (Fig. 1). CF was not bioelectrochemically transformed in BESs operating at the non-hydrogen producing 2.5. Analytical methods potential of 0.3 V, discarding direct electron transfer from the graphite brush electrode surface to Dehalobacter (Fig. S2). In this set The concentrations of CF and DCM were determined by trans- of experiments, neither the dechlorination of CF nor the continuous ferring 1 mL of the liquid medium from the cathode to a 10 mL vial production of DCM was observed in three different set-ups: a sealed with Teflon-coated stoppers that contained 5.5 mL of conventional BES system, abiotic controls and biotic open circuit deionized water. The vials were placed in a headspace sampler controls during 20 days of operation (Fig. S2). The CF concentration (Agilent 7964) and heated for 15 min to 85  C. Automatically, 1 mL steadily decreased for all the experimental replicates at a similar headspace gas sample from the vials was injected into an Agilent rate. The measured residual concentrations of DCM (less than 6890 N gas chromatograph provided with an Agilent DB-624 col- 60 mM) did not amount to the total CF lost and were probably umn (30 m  0.32 mm with 0.25 mm film thickness) and a flame caused by traces of hydrogen. Therefore, CF decrease could only be ionization detector following a method previously described [25]. explained by its diffusion through the cationic exchange membrane The calibration of both compounds was based on aqueous external and not any kind of electrochemical or biological degradation. standards with the same liquid and gas volumes as to the experi- mental BES. Details for the calculation of CF and DCM concentra- tions are provided in the Supplementary Information (SMM1). Hydrogen concentration was measured on 0.1-mL headspace samples from the cathode using an Agilent 7820A GC equipped with a thermal conductivity detector as previously described [26]. 2.6. Real-time PCR (qPCR) Liquid samples (3 mL) were obtained from the cathode vessel at different time points, centrifuged at 9000 g for 20 min and extracted as previously described [18]. An experimental procedure quantifying the 16S rRNA gene copies of Dehalobacter by qPCR previously used in another study was employed [30]. The employed calibration curve was depicted in Fig. S1. 2.7. Cell growth kinetic calculations The maximum specific growth rates (m, d1) were calculated by selecting the exponential growth phase period according to the 16S rRNA gene concentrations of Dehalobacter obtained by qPCR and using the following equation: 1 dX m¼ $ (3) Fig. 1. a, Degradation profile of CF in experimental BES inoculated with Dehalobacter X dt and poised with a cathodic potential, abiotic controls, and biotic open circuits. The time lapse between days 70 and 130 was amplified in subpanel b for best readability of where X is the 16S rRNA gene copies concentration in the BESs (16S measurements. Changes in the concentration of DCM were only depicted in the sub- rRNA gene copies per mL). panel b with blue circles. Numbers indicate the number of CF amendments in the The biomass growth yield (Y, 16S rRNA gene copies generated experimental BES. Values plotted are the average of triplicate BES, and error bars per mmol CF degraded) was calculated as follows: indicate standard deviation. 3 ndez-Verdejo, P. Cortes, A. Guisasola et al. D. Ferna Environmental Science and Ecotechnology 12 (2022) 100199 The dechlorination of CF was almost immediately detected After the degradation of nine CF doses, the experimental BESs when the cathode was poised at 0.6 V, with the concomitant were inoculated with an enriched culture containing Dehalobacte- production of DCM (Fig. 1). The BESs were sequentially operated rium on day 83 of operation. At this point, the DCM accumulated in at 0.6, 0.7, and 0.8 V after the consumption of three doses of CF the BES was 1,128 ± 616 mM. DCM was completely degraded in the per cathodic potential (Fig. 1). The decrease of the poised cathodic following seven days at a degradation rate of 481 ± 141 mM d1 potential showed a significant increase in both the CF degradation (Fig. 1). Once DCM was degraded, CF was spiked at increasing rate and the DCM production rate. There were statistical differences concentrations of approximately 200, 400, 600 and 800 mM to (p-value < 0.02) for all three potentials, as shown with a Brown- couple both Dehalobacter and Dehalobacterium dechlorination ca- Forsythe and Welch ANOVA test for multiple comparisons. As pabilities (Fig. 1). At lower concentrations of CF (200 and 400 mM), observed in Fig. 2a, CF degradation and DCM production rates the degradation of CF and DCM proceeded rapidly (Fig. 1, arrow increased one order of magnitude when moving from 0.6 V numbers 10 and 11). However, when CF was added at higher con- to 0.8 V, reaching values of 10.3 ± 7.3 and 10.1 ± 8.5 mM d1 centrations (600 and 800 mM), DCM accumulated (Fig. 1, arrow (at 0.6 V cathodic potential) and 131.6 ± 64.9 and numbers 12 and 13), and degradation only started after CF was 125.2 ± 58.9 mM d1 (at 0.8V cathodic potential), respectively almost exhausted. This agrees with a recent study showing that CF (Fig. 2a). at ~800 mM inhibited the DCM degradation activity of Dehalo- DCM production rates were almost identical to CF degradation bacterium, and the activity could be recovered when the culture rates indicating that molar balance can be considered closed was transferred to a CF-free medium [26]. The production of acetate (Fig. 2a). The low basal CF degradation rate obtained in the abiotic derived from DCM fermentation was not quantified because it was controls was 2.82 ± 0.47, 2.92 ± 1.19, and 2.51 ± 1.90 mM d1 for masked by the high concentrations of acetate in the medium and cathodic potentials of 0.6, 0.7 and 0.8 V, respectively, and used as a carbon source by Dehalobacter (Fig. S3). confirmed that CF degradation was biologically-mediated in BESs. On the other hand, the first dose of CF was rapidly depleted in the biotic open circuit controls (Fig. 1), but the second amendment of 3.2. Dehalobacter growth and degradation kinetics in BESs CF was not completely degraded after ~70 days, indicating that the first amendment was probably consumed linked to the residual Periodic samples from the cathodic vessel were withdrawn, and hydrogen dissolved in the inoculum. Dechlorination of CF was not the DNA was extracted to quantify the increase of 16S rDNA gene inhibited by the DCM accumulated in the cathode, and this is in copies during the BES operation period (Fig. S4). Dehalobacter 16S accordance with previous studies showing an inhibitory threshold rRNA gene copies increased over four orders of magnitude from an of 2,500 mM DCM for Dehalobacter [1]. initial concentration of 4.33  103 ± 1.00  103 to 1.96  107 ± 1.01  107 16S rRNA genes per mL after 126 days of operation (Fig. S4). Assuming five 16S rRNA gene copies per genome [32], a cell density of 3.92  106 ± 2.02  106 Dehalobacter cells per mL was obtained. The growth yield was calculated for each BES set-up by deter- mining the changes in Dehalobacter 16S rDNA gene copies produced during the consumption of each CF dose (Fig. 3). The average growth yield was 7.76  106 ± 3.60  106 Dehalobacter 16S rRNA gene per mmol of CF degraded (or Dehalobacter 16S rRNA gene per mmol Cl). The average yield values reported in this study are in the same order as the magnitude of those previously reported for other Dehalobacter sp. respiring CF (3.6  106 ± 2.6  106 and 2.5  107 ± 0.9  107 16S rRNA gene copies per mmol Cl) [1,33]. The obtained growth yield is one order of magnitude lower than the one obtained for the CF-respiring Desulfitobacterium sp. strain PR (1.16  107 ± 0.16  107 cells per mol of chlorine released) [6]. The growth yield values obtained for Dehalo- bacter respiring CF are between one or two orders of magnitude lower compared with other chlorinated compounds as 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, or 1,2,4-trichlorobenzene [33e36]. The exponential growth phase from each experimental BES was Fig. 2. Degradation rate of CF and production rate of DCM (a) and CF degradation rate and DCM production rate normalized by the 16S rRNA gene copies (b) for each dose of CF consumed in experimental BESs. The numbers refer to the dose number depicted in Fig. 1. The grey background represents the average CF degradation rate (a) and average CF normalized degradation rate (b) for each applied potential. Asterisks indicate CF Fig. 3. Correlation between the moles of CF consumed and changes in the 16S rRNA doses where the formation of DCM accumulation was not detected due to a fast gene copy numbers for the experimental BES triplicates. degradation by Dehalobacterium. 4 ndez-Verdejo, P. Cortes, A. Guisasola et al. D. Ferna Environmental Science and Ecotechnology 12 (2022) 100199 selected from the qPCR results and linearized. From their slope, the degradation potential of the culture was not used, which would maximum specific growth rates and their corresponding doubling explain the decrease of the specific dechlorination activity of CF at times were calculated, reaching a value of 0.218 ± 0.097 d1 and lower cathodic potentials (Fig. 2b). Overall, operating BESs with a 3.81 ± 2.13 d, respectively. The doubling times observed in this sequential increase in the cathodic potential is a feasible strategy to study are approximately 60% lower than those obtained in previous control the maximum degradation rate values attained and reduce enriched cultures of Dehalobacter growing with CF (9.9 ± 1.2 d) [33], energetic costs during bioremediation processes assisted with suggesting that Dehalobacter sp. 8 M present in BES grows at a electrochemically generated hydrogen. Several parameters other faster rate. A maximum specific CF utilization of 2.83  108 ± than the cathodic potential should be studied in view of upscaling 0.12  108 mmol of CF degraded per Dehalobacter 16S gene copy BES reactors. The reactor configuration should aim at reducing the generated per day was obtained by combining the maximum overpotentials; therefore, anodic and cathodic areas should be growth rate and the growth yield. maximized and placed as close as possible. A possible alternative to Fig. 2b displays the normalized degradation rates, referring to reduce the overall energy requirements is to replace water oxidation the concentration of Dehalobacter 16S rDNA gene copies. The as an anodic reaction with another more favorable reaction, such as highest normalized degradation rates were obtained for the highest the (bio)electrochemical oxidation of another pollutant. The cathodic potentials, as also observed in a previous study with BES coupled oxidation of toluene and the reduction of trichloroethene inoculated with a Dehalogenimonas-containing culture respiring have recently been reported [37]. Moreover, this high-scale config- 1,2-dichloropropane [18]. uration should be able to operate under continuous mode. From an economic point of view, the cost of the electrode materials to drive 3.3. Coulombic efficiency and energetic input of the process hydrogen reduction should also be decreased. The current intensities decreased when the cathodic potential 4. Conclusions was lowered, obtaining average current density values of 0.050 ± 0.060, 0.110 ± 0.029, and 0.325 ± 0.043 mA m2 for This study demonstrates the full dechlorination of CF by potentials 0.6, 0.7, and 0.8 V, respectively (Fig. S5). These values combining Dehalobacter and Dehalobacterium in a BES for the first were used to assess the coulombic efficiency of the CF degradation time. The application of a steadily decreasing cathodic potential process, which was 42.33 ± 28.22% when working at a cathodic instead of a fixed one allowed to adjust the supply of hydrogen and potential of 0.6 V and increased to values between 60% and 70% maximize the coulombic efficiency of the process and the degra- when the potential decreased to 0.7 and 0.8 V (Fig. 4). The values dation rates obtained even at the most negative cathodic potential of coulombic efficiency and required energetic input to degrade one poised (0.8 V). We operated the BESs in a fed-batch mode for 126 mol of CF were similar between the two most negative potentials days, obtaining Dehalobacter concentrations up to 107 16S rRNA poised in this study. As expected, the energetic input required for gene copies per mL after increasing in four orders of magnitude the degrading a certain amount of contaminant was inversely correlated initial Dehalobacter concentration. This study provides a basis to to the coulombic efficiency. In this study, a higher amount of energy use BESs as on-site bioreactors to deliver enriched OHRB and to degrade a mol of contaminant was required when working at hydrogen in contaminated groundwater requiring biostimulation potential 0.6 V and decreased the required value at potentials 0.7 and bioaugmentation. The organohalide respiration of CF and the and 0.8 V. This observation does not correlate with the results of fermentation of DCM proceeded almost simultaneously at con- previous studies working with OHRB in BESs which showed that the centrations of CF up to 400 mM, which would avoid the accumu- decrease of cathodic potential produced faster degradation rates at lation of DCM in the environment. At higher concentrations of CF, the cost of severely decreasing the coulombic efficiency of the pro- Dehalobacterium is inhibited but recovers the DCM fermenting ac- cess [9]. This could be explained by the sequential decrease of the tivity when CF reaches lower concentrations. In all, these results cathodic potential applied in this study that is accompanied by a show the potential of the constructed co-culture and BESs to continuous growth of Dehalobacter that would potentially consume completely dechlorinate CF, which cannot be fully dechlorinated by the hydrogen supplied as it is produced. This observation is sup- a single anaerobic bacterium to date. ported by gas measurements from the headspace of the BESs, which showed no hydrogen accumulation (Fig. S6). Conflict of interest The absolute degradation rates (Fig. 2a) presented similar values during the three CF doses at each poised cathodic potential value The authors declare no conflict of interest. despite the 16S rRNA gene concentrations increasing over time (Fig. S4). Thus, the observed maximum degradation rate was limited Declaration of interests by the hydrogen bioavailability and not cell density. The maximum The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the Spanish Ministry of Science, Innovation and Universities projects CTM2017-91879-EXP and PID2019-103989RB-100. D. Fern andez-Verdejo acknowledges a predoctoral grant from UAB (PIF 2017e2018). Appendix A. Supplementary data Fig. 4. Coulombic efficiencies and energetic inputs per mol of CF degraded obtained Supplementary data to this article can be found online at for each cathodic potential. https://doi.org/10.1016/j.ese.2022.100199. 5 ndez-Verdejo, P. Cortes, A. Guisasola et al. D. 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