Chemosphere 338 (2023) 139467 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Toluene-driven anaerobic biodegradation of chloroform in a continuous-flow bioelectrochemical reactor Matteo Tucci a, 1, David Fernández-Verdejo b, 1, Marco Resitano a, Pamela Ciacia a, Albert Guisasola c, Paqui Blánquez b, Ernest Marco-Urrea b, Carolina Cruz Viggi a, Bruna Matturro a, d, Simona Crognale a, d, Federico Aulenta a, d, * a Water Research Institute (IRSA), National Research Council (CNR), Via Salaria km 29.300, 00015, Monterotondo, RM, Italy b Biorem UAB, Department of Chemical, Biological and Environmental Engineering, School of Engineering, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain c GENOCOV, Department of Chemical, Biological and Environmental Engineering, School of Engineering, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain d National Biodiversity Future Center (NBFC), Palermo, Italy H I G H L I G H T S G R A P H I C A L A B S T R A C T • Toluene and chloroform biodegradation in single-stage bioelectrochemical reactor. • Nearly complete removal of the target contaminants via distinct pathways. • Chloroform metabolite dichlorometane was also removed via fermentation. • Acetate negatively impacted the removal of the contaminants. • Taxonomical and functional gene-based analysis revealed a competent microbiome. A R T I C L E I N F O A B S T R A C T Handling Editor:A Adalberto Noyola Subsurface co-contamination by multiple pollutants can be challenging for the design of bioremediation stra­ tegies since it may require promoting different and often antagonistic degradation pathways. Here, we investi­ Keywords: gated the simultaneous degradation of toluene and chloroform (CF) in a continuous-flow anaerobic Microbial electrochemical technologies bioelectrochemical reactor. As a result, 47 μmol L− 1 d− 1 of toluene and 60 μmol L− 1 d− 1 of CF were concurrently Groundwater remediation removed, when the anode was polarized at +0.4 V vs. Standard Hydrogen Electrode (SHE). Analysis of the Bioremediation * Corresponding author. E-mail address: federico.aulenta@irsa.cnr.it (F. Aulenta). 1 These authors contributed equally to the work. https://doi.org/10.1016/j.chemosphere.2023.139467 Received 31 March 2023; Received in revised form 12 June 2023; Accepted 9 July 2023 Available online 10 July 2023 0045-6535/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). M. Tucci et al. Chemosphere 338 (2023) 139467 Toluene microbial community structure and key functional genes allowed to identify the involved degradation pathways. Chloroform Interestingly, when acetate was supplied along with toluene, to simulate the impact of a readily biodegradable substrate on process performance, toluene degradation was adversely affected, likely due to competitive inhi­ bition effects. Overall, this study proved the efficacy of the developed bioelectrochemical system in simulta­ neously treating multiple groundwater contaminants, paving the way for the application in real-world scenarios. 1. Introduction oxidation at the anode and chlorinated compounds reduction at the cathode with MECs is still very limited (Tucci et al., 2022b). Due to their widespread usage, chlorinated compounds and aromatic Recently, we proposed a prototype of MEC named “Bioelectric well” hydrocarbons are among the most frequent groundwater and soil con­ (Palma et al., 2018b): its tubular design allows insertion in groundwater taminants (Rivett et al., 2011). Their occurrence in subsurface envi­ wells and piezometers for in-situ groundwater treatment. Moreover, the ronments is mainly caused by industrial activities, surface runoff, absence of membrane between anode and cathode minimizes the ohmic accidental spills from tanks and pipes, improper handling or disposal losses and facilitates its upscaling. Recent works proved that the practices (Blázquez-Pallí et al., 2019; Collins et al., 2002). Due to their bioelectric well is able to remove at the same time oxidable (toluene, toxicity and persistence in the environment, these contaminants pose TPH) and reducible (sulfate, TCE) contaminants from a synthetic high risks to human health and the ecosystem (Chary and groundwater (Tucci et al., 2021; Viggi et al., 2022). Fernandez-Alba, 2012). In this study we performed the simultaneous degradation of toluene, Bioremediation is considered a very effective approach to treat these as a model aromatic hydrocarbon, and chloroform (CF), a highly toxic kinds of pollutants, having in general lower requirements in terms of and possibly carcinogen chlorinated pollutant (Yamamoto et al., 2002), costs, equipment, labor and energy, and a lower environmental impact in a continuously fed bioelectric well operated for over 190 days. compared with conventional physicochemical techniques, such as air Importantly, CF biodegradation has never been previously studies in a sparging and activated carbon adsorption, which also typically result in continuous-flow bioelectrochemical reactor. Here, toluene was oxidized a phase transfer of contaminants without an effective degradation by the electroactive microbial consortium at the anode, while CF was (Lhotský et al., 2017). However, the presence of multiple types of con­ converted to harmless end products via a two-step process involving first taminants in the same site may complicate operations and increase the a reductive hydrogenolysis step leading to dichloromethane (DCM) costs of bioremediation, since different treatments need to be imple­ followed by the fermentation to acetate and formate. mented in different times and space to meet the distinct metabolic needs of the different microbial degraders (Megharaj et al., 2011; Rabus et al., 2. Materials and methods 2016). As aromatic hydrocarbons are preferentially degraded via oxidative pathways, the microbial community requires availability of 2.1. Chemicals and electrode potentials high potential electron acceptors such as oxygen or nitrate (Allard and Neilson, 1997; Jabbar et al., 2022). On the contrary, the reductive All chemicals used for the experiments were of analytical grade. All dechlorination of chlorinated compounds, carried out by of them were purchased from Merck KGaA (Germany). De-ionized water organohalide-respiring bacteria (OHRB), requires an electron donor (i.e. (Millipore, Germany) was used to prepare the mineral medium and all molecular hydrogen) while the contaminant serves as the respiratory other solutions. All potentials reported in this work are referred to the electron acceptor (Farhadian et al., 2008). standard hydrogen electrode (SHE). Theoretically, it would be possible to exploit the oxidation of aro­ matic hydrocarbons as source of electrons for the reduction of chlori­ 2.2. Reactor setup and operations nated compounds. However, since most OHRB are restricted to using H2 as electron donor, the possibility of carrying out the simultaneous The bioelectric well was set up as previously reported by Tucci et al. removal of both types of pollutants in a single-step reaction is very (2021): the anode consisted of a cylinder made of 8 contiguous graphite limited (Soder-Walz et al., 2022). rods (purity: 99.995%, length: 30 cm, ø: 0.6 cm; Merck KGaA, Germany), Microbial electrochemical systems (MESs) are being proposed as a whereas the cathode was a stainless steel mesh (dimensions: 3 × 30 cm; promising alternative to conventional bioremediation strategies. They type 304, Alpha Aesar, USA) (Fig. 1A). Anode and cathode were con­ are able to provide inexhaustible source and/or sink of electrons to tained in a 250 mL glass cylinder and were kept separated by a poly­ sustain microbial metabolism in form of a solid electrode, thus avoiding ethylene mesh (ø: 1 cm, length 30 cm; Fig. 1B), which allowed free the drawbacks connected to the injection of air, oxygen or other circulation of the electrolyte. The distance between the anodic and the chemicals in the aquifer (He and Su, 2015). Many studies on bioreme­ cathodic surfaces was 2 mm. The two electrodes were connected to an diation with MESs focused on Microbial Fuel Cells, which can sponta­ external circuit with titanium wires (ø: 0.81 mm Alfa Aesar, USA). neously oxidize organic compounds at the anode by using oxygen as the During the experiments, the anode was polarized at +0.4 V vs. SHE by terminal electron acceptor at the cathode (Kronenberg et al., 2017). means of an IVIUMnSTAT potentiostat (IVIUM Technologies, The However, in contaminated subsurface environments, the access to O2 is Netherlands). This potential was chosen according to the findings of strongly limited due to rapid depletion, thus hindering the biodegra­ previous studies with the bioelectric well, where the bioelectrochemical dation of pollutants (Wartell et al., 2021). oxidation peak of toluene on graphite ranged between 0.2 and 0.4 V In Microbial Electrolysis Cells (MECs), a potential difference is (Tucci et al., 2022c). The counter electrode potential was periodically applied to the electrodes in order to catalyze sluggish or thermody­ measured throughout the experiment. A saturated Ag/AgCl electrode namically unfavorable oxidation reactions at the (bio)anode and/or (+0.198 V vs. SHE; AMEL, Italy) was used as reference electrode. reduction reactions at the (bio)cathode (Kadier et al., 2014). Several To ensure the presence of microbes able to degrade toluene, the studies demonstrated the possibility to use MECs to treat aromatic hy­ reactor was inoculated with 0.25 L of real groundwater contaminated drocarbons at the anode (Friman et al., 2013; Marzocchi et al., 2020; with petroleum hydrocarbons collected from a petrochemical site in Palma et al., 2018a, 2019; Tucci et al., 2021, 2022c, 2022a; Zhang et al., Italy. 2010) or chlorinated compounds at the cathode (Aulenta et al., 2011; Lai During the experiment, the reactor was fed in continuous mode with et al., 2017; Leitão et al., 2015; Verdini et al., 2015) in anaerobic con­ mineral medium (pH 7) in which O2 was eliminated through N2 ditions. Nevertheless, the research on coupling aromatic hydrocarbon sparging. The medium composition is reported in the supporting 2 M. Tucci et al. Chemosphere 338 (2023) 139467 information (Table S1). The medium was spiked with the contaminants Table 1 toluene and CF as summarized in Table 1. Acetate was also added to the Operational conditions for all the different runs operated with the bioelectric medium to study the impact of a readily available substrate on the well. reactor performances. The inlet was stored in 5 L collapsible Tedlar® gas Run Days Toluene Chloroform Acetate Polarization (V bags and pumped in the reactor through the bottom port (flow rate: 0.63 (μmol L− 1) (μmol L− 1) (μmol L− 1) vs. SHE) L d− 1, HRT 11 h) by means of a peristaltic pump (120 S, Watson Marlow, 1 0–13 260 / 920 +0.4 Falmouth, UK). The treated effluent was discharged from the upper port 2 14–36 260 120 920 +0.4 by passive overflow. Flow-through sampling cells (volume: 25 mL) were 3 37–46 390 120 920 +0.4 4 47–80 160 120 920 installed at the inlet and at the outlet of the reactor for liquid and gas +0.4 5 81–114 130 120 920 OCP monitoring. The liquid phase of the reactor was constantly recycled with 6 115–127 100 120 / OCP another peristaltic pump (flow rate: 192 mL min− 1; model: 323, Watson 7 128–190 100 120 / +0.4 Marlow, Falmouth, UK) to avoid concentration gradients of substrates, products and/or biomass. The tubings were made of Viton® (Merk KGaA, Germany) to minimize volatilization losses and adsorption of system was reconnected to the potentiostat and polarized. organic contaminants. During the whole study the system was main­ tained at room temperature (i.e., 24 ± 3 ◦ C). 2.3. Cyclic voltammetries The experiment lasted 190 days, during which different conditions were tested (Table 1). The first run was an acclimation phase for the The bioanode and (bio)cathode developments were monitored with toluene degrading consortium in absence of CF. Then, CF was added to Cyclic voltammetries (CVs, scan rate: 1 mV s− 1) using an IVIUMnSTAT the mineral medium at a concentration of 14 mg L− 1 and the reactor was potentiostat (IVIUM Technologies, The Netherlands). The stainless-steel inoculated (10% vol/vol) with two microbial cultures: one enriched cathode and the graphite anode were used as counter electrode during with Dehalobacter spp. and the other with Dehalobacterium for­ the anodic and cathodic CVs, respectively. For all CVs, an Ag/AgCl micoaceticum, capable to perform the reductive hydrogenolysis of CF to electrode (+0.198 V vs. SHE; AMEL, Italy) was used as reference. DCM and the subsequent fermentation of DCM to acetate and formate, respectively (Fernández-Verdejo et al., 2022; Trueba-Santiso et al., 2.4. Gas-chromatographic analyses 2017, 2020). At this point three different concentrations of toluene (in the range of The quantification of O2, H2 and CH4 was performed using a gas- 15–36 mg L− 1) were tested from runs 2 to 4. In runs 5 and 6 the elec­ chromatograph (Agilent 8860, GC system USA) equipped with a ther­ trodes were disconnected from the potentiostat to study the removal of mal conductivity detector (TCD); the quantification of toluene, CF and contaminants at open circuit potential (OCP). Finally, in runs 6 and 7 acetate was performed with a gas-chromatograph (Agilent 8860, GC acetate was omitted from the mineral medium and during run 7 the system USA) equipped with a flame ionization detector (FID). The Fig. 1. The bioelectric well: A) schematic representation of the setup used for the experiments, where WE is the working electrode, CE is the counter electrode and RE is the reference electrode; B) cross-sectional view showing the relative position of electrodes. 3 M. Tucci et al. Chemosphere 338 (2023) 139467 methods for the GC, the calibration ranges and detection limits (LOD) toluene or CF measured in the influent (CIn) and the concentration are reported in the Supporting Information (Table S2). Gas-phase con­ measured in the effluent (Cout), Vr (L) is the empty volume of the reactor centrations were converted into liquid-phase concentrations using and Q (L d− 1) is the flow rate of the influent. tabulated Henry’s Law constants (Sander, 2015). The relative removal q% of toluene and CF were calculated as follows: 2.5. High-throughput rRNA gene sequencing and bioinformatic analysis ΔConc q% = × 100 (2) Cin The liquid effluent (15 mL) and the biofilm grown on the graphite rods were collected at the end of the operation, filtered through poly­ 4. Results and discussion carbonate membranes (pore size 0.2 mm, 25 mm diameter, Nuclepore) and immediately stored at − 20 ◦ C. The DNA extraction was performed 4.1. Reactor performances by using the DNeasy PowerSoil Pro Kit (QIAGEN - Germantown, MD) following manufacturer’s instructions. The genomic DNA was utilized as The bioelectric well was operated for 190 days in different conditions template for the amplification of the V1–V3 region of 16 S rRNA gene of (Table 1). In Fig. 2A the current profile for all the different runs is re­ Bacteria (27F 5′-AGAGTTTGATCCTGGCTCAG-3’; 534R 5′- ported. After a start-up phase with toluene as the only contaminant (run ATTACCGCGGCTGCTGG-3′) following the procedure for library prepa­ 1), the current started to increase and CF was added (run 2). Then, the ration and sequencing described elsewhere (Crognale et al., 2019). The current reached a steady level with an average value of 3.4 mA (runs 3 samples were paired end sequenced (2 × 301bp) on a MiSeq platform and 4). During run 5 and 6 the system was kept in OCP, and thereafter re- (Illumina) using a MiSeq Reagent kit v3, 600 cycles (Illumina, USA) connected in run 7. It can be noticed how, as soon as the electrodes were following the standard guidelines for preparing and loading samples. polarized again, the current increased until it reached values similar to Phix control library was spiked at a concentration of 20%. Bioinfor­ those of the previous polarized runs. The recorded CVs clearly point to a matics analysis were performed using QIIME2 v. 2018.2 (Bolyen et al., gradual formation of an electroactive biofilm on the anode, starting from 2019) following the procedure reported elsewhere (Crognale et al., T0 to the end of run 3 (Fig. 2B). Indeed, it can be noticed the formation 2021). High-throughput sequencing of the V1–V3 region of the bacterial of peaks associated with a redox couple (red line) and a time-dependent 16 S rRNA gene yielded a total of 16,934 sequence reads after quality increase of the oxidative current. The cathodic reductive current also control and bioinformatics processing that resolved into 150 amplicon increased overtime, probably due to the accumulation of hydrogenase- sequence variants (ASVs). containing microorganisms at the electrode, which can facilitate H2 formation at the cathode (Fig. 2C) (Aulenta et al., 2012). Furthermore, 2.6. Quantification of key-functional genes the measured cathodic potential maintained a value of 0.77 ± 0.07 V vs. SHE throughout the experiment, which is suitable for H2 generation. The genomic DNA was also used as template for the quantification of The current generation is strictly linked to the degradation of functional genes involved in the upper pathway of anaerobic toluene toluene, as it can be observed in Fig. 3A. At the beginning of the degradation (bssA, bcrC, bzdN, bamB), CF to DCM reduction (cfrA) and experiment, electroactive bacteria started to degrade toluene at the DCM fermentation (mecE, mecF). 16 S rRNA of Dehalobacter spp. and anode in presence of acetate. However, even at the lowest influent D. formicoaceticum were also quantified. Absolute quantification assays toluene concentration (i.e. 160 μmol L− 1), only a fraction of the toluene were performed via Digital Droplet PCR (ddPCR) with the QX200™ load was removed. Conversely, during run 7, where acetate was not Droplet Digital™ PCR System (Bio-rad, United States) as describe else­ present in the inlet, almost 100% of toluene was degraded. During OCP where (Di Franca et al., 2022). The primers used have been listed in (runs 5 and 6), as expected, negligible toluene degradation was Table S3. observed. The steps for the quantification assays included: i) preparation of the The consumption of acetate was also monitored (Fig. S1): acetate PCR reaction mixture for each targeted gene (22 μl total volume: ddPCR was completely consumed during the polarized runs (1–4), while almost EvaGreen Supermix ® (Bio-Rad, United States), 3 μl of DNA as a tem­ no removal was observed during OCP (run 5). When acetate was omitted plate, and 900 nM of each primer); ii) droplets generation (20 μl of PCR from the inlet (runs 6 and 7), no residual amounts were detected in the mixture and 70 μl of Droplet Generation Oil® (Bio-Rad, United States) reactor. with the QX200 Droplet Generator (Bio-Rad, United States); iii) PCR Regarding CF, the degradation started quickly as soon as it was amplification on 40 μl of the droplets’ mixture with a T100 thermal added to the reactor (Fig. 3B). As a result, the dechlorination product cycler (Bio-Rad, United States) (cycling conditions: 5 min at 95 ◦ C, 39 DCM (deriving from the reductive hydrogenolysis of CF) started to being cycles for 30 s at 95 ◦ C and 1 min at 60 ◦ C (ramping rate set to 2 ◦ C/s), 5 produced. At the same time, DCM fermentation commenced, hence min at 4 ◦ C, 5 min at 90 ◦ C, ending at 4 ◦ C); iv) quantitative data reading slowly decreasing its concentration in the reactor. During OCP, the with QX200 Droplet Reader (Bio-Rad, United States) to determine the dechlorination of CF slowed down but it was still present, being prob­ positive and negative fluorescent droplets and calculate the targeted ably sustained by DCM fermentation and the endogenous decay of gene concentrations. The data were analyzed using the QuantaSoft® biomass. Indeed, it is well documented that the slow release of electron software (Bio-Rad, United States) by calculating the ratio of the positive donors generated by fermentation processes is effective in sustaining droplets over the total droplets in each sample. Quantitative data have reductive dechlorination processes (Amanat et al., 2022; Yang and been reported as gene copies L− 1 of liquid effluent or gene copies cm− 1 McCarty, 2000). When the system was polarized again (run 7), the of graphite rod (95% confidence intervals). previous level of CF removal was restored. It is worth noticing that, towards the end, almost all CF and its metabolite DCM were removed. 3. Calculations Furthermore, no adverse effects on toluene degradation were observed with the addition of CF. The removal rate r (μmol L− 1 d− 1) of toluene and CF were calculated Methanogenesis started at the beginning of run 1, sustained by using the following equation: electrochemically produced H2 (Fig. 3C). However, methane generation decreased over time after CF addition, probably due to the inhibitory r= ΔConc. Q (1) effect caused by this contaminant on methanogens (Yu and Smith, Vr 2000). During OCP runs (5 and 6) no hydrogen was generated, and thus no CH4 was produced. Once almost all CF was removed by the where ΔConc. (μmol L− 1) is the difference between the concentration of 4 M. Tucci et al. Chemosphere 338 (2023) 139467 Fig. 2. A) Current trend of the bioelectric well during the different runs. B) CVs of the (bio)anode at T0, in run 1 and 3. C) CVs of the (bio)cathode at T0, in run 1 and 3. dehalogenating bacteria (run 7), methanogenesis started again. lower when acetate was added to the inlet, both in absolute terms and as During the polarized run, in absence of acetate (run 7), 47 μmol L− 1 percentage of the contaminant load in the inlet. This phenomenon is d of toluene and 60 μmol− 1 d− 1 of CF were removed, corresponding to − 1 probably caused by the fact that both toluene and acetate competed for 96% and 94% of the contaminant load, respectively (Table 2). This the active catalytic sites at the anodic biofilm, with acetate likely being proves the effectiveness of the bioelectrochemical system, since only 0.1 preferentially metabolized compared to toluene (Edwards and μmol L− 1 d− 1 of toluene (i.e. 0,2%) and 47 μmol L− 1 d− 1 of chloroform (i. Grbic-Galic, 1994; Zhang et al., 2010). It is worth noticing that higher e. 77%) were removed during OCP. Nevertheless, the degradation rate of toluene degradation in absence of acetate indirectly resulted in higher toluene obtained in this work is considerably lower than the ones ob­ CF removal. However, the mechanism behind this phenomenon is un­ tained in previous studies with the bioelectric well, which were as high clear and warrants further investigation. It is worth noticing that only in as 336 and 150 μmol L− 1 d− 1 when sulfate and TCE were used as co- the absence of acetate (run 7), it was possible to reach an almost com­ contaminants, respectively (Tucci et al., 2021; Viggi et al., 2022). It is plete degradation of both contaminants. likely that the co-contamination with acetate and CF limited the pres­ ence of toluene-degrading microorganisms within the microbial com­ munity, the first one through competition and the second through 4.3. Characterization of the mixed microbial communities inhibition. As a matter of fact, CF has well documented toxic effects on bacteria, including anaerobic consortia (Pollice et al., 2001). As a result, The amplicon sequencing of the 16 S rRNA gene revealed a highly the microbial community showed a reduced toluene degradation po­ selected microbial community both in the liquid effluent and in the tential and thus a lower removal rate. biofilm taken at the end of the operation of the reactor (Fig. 6). In The maximum CF consumption rate obtained in this experiment (60 particular, the liquid effluent was mostly constituted by members of the μmol L− 1 d− 1) was lower as compared to the one obtained in previous genus Propionicicella (∼ 2% of total reads) capable to produce acetic and experiments conducted with a two-vessel BES (132 μmol L− 1 d− 1) propionic acids. Some Propionicicella strains (i.e. P. superfundia (Bae (Fernández-Verdejo et al., 2022). The reason behind this decrease in CF et al., 2006)), have been isolated from groundwater contaminated with degradation rate could be the lower supplied concentration of CF in the chlorinated ethanes and vinyl chloride and are facultative anaerobe system, leading to overall lower reaction kinetics. A similar phenome­ capable of utilizing fermentative metabolic strategies. Candidatus non was observed with toluene in previous studies conducted with the Endomicrobium, unidentified members of family Lentimicrobiaceae and bioelectric well where a clear dependency of bioreaction rates on sub­ Acetobacterium were also found (≥8%, each), the latter known to pro­ strate concentration, in the mg/L range, was observed (Tucci et al., duce acetate and formate when grown in mixed bacterial culture with 2022c). DCM and H2 (Trueba-Santiso et al., 2017). Furthermore, reads affiliated with families Anaerolineaceae, Bur­ kholderiaceae, Geobacteraceae, and Rikenellaceae counted together up to 4.2. Effects of toluene concentration and acetate addition 6.5% of total reads suggesting the potentialities of the effluent’s microbiome in hydrocarbon degradation (Weelink et al., 2010). Bur­ The influent toluene concentration is a key-factor influencing the kholderiaceae is considered a key family of toluene degradation with kinetics of the anodic bio-oxidation (Fig. 4A). In this study, toluene several members, as for example Ralstonia pickettii, involved in the removal increased with the inlet concentration up to 280 μmol L− 1, complete toluene degradation pathway via two successive where the system became apparently saturated. This is in contrast with ring-hydroxylating reactions (Lünsmann et al., 2016). The dominant previous studies, where a direct correlation between the amount of role of this family can be due to occurrence of toluene monooxygenase toluene and its removal rate was observed in a similar system up to 434 and dioxygenases-encoding genes, which would allow the microorgan­ μmol L− 1 (Tucci et al., 2022c). Once more, the effect of the isms harboring this type of enzymes to be adapted to grow on some co-contamination with CF may be the cause for the limitations in the aromatic compounds (Martínez-Lavanchy et al., 2015). toluene removal efficacy. Conversely, CF removal rate did not seem Differently from the liquid effluent, the microbiome composition of correlated to the concentration of toluene (Fig. 4B). This is likely due to the biofilm grown on graphite rods was mostly represented by uniden­ the fact that, during cycles 2, 3 and 4, electrons were also generated by tified members of Actinobacteria order OPB41 (41.0%). The direct the oxidation of acetate, thus masking a possible correlation with the involvement of members of this order in toluene degradation was not concentration of toluene. discussed so far even though they were reported as a component of In Fig. 5 it is possible to see how the degradation of toluene was microbial community in previous works concerning hydrocarbons 5 M. Tucci et al. Chemosphere 338 (2023) 139467 Fig. 3. A) Trend of toluene concentration in the inlet and in the outlet of the reactor. B) Trend of CF concentration in the inlet and in the outlet of the reactor and DCM formation in the outlet. C) Trend of H2 and CH4 formation in the outlet of the reactor. degradation (Chen et al., 2016; Laso-Pérez et al., 2019). In line with Table 2 previous evidence in similar bioelectrochemical studies (Tucci et al., Reactor performances in terms of toluene and CF removal during polarized mode 2022a, 2022c), the enrichment of the electroactive microbiome in the vs. OCP mode in absence of acetate. biofilm was suggested by the presence of members of family Anaeroli­ Polarized (Run 7) OCP (Run 6) naceae (19.0%) and genus Geobacter (8.6%). Furthermore, 6.5% of total Tol. rem. Rate (μmol L¡1 d¡1) 47 ± 3 0.1 ± 0.2 reads were affiliated with family Lentimicrobiaceae, comprising strictly Tol. rem. % 96 ± 1 0.2 ± 0.2 anaerobic gram-negative bacteria able to form a consortium with other CF rem. Rate (μmol L¡1 d¡1) 60 ± 1 47 ± 2 CF rem. % 94 ± 1 77 ± 1 electroactive microorganisms to convert acetate into electric energy (Xiao et al., 2015; Zhu et al., 2022). The key functional enzymes involved in the initial two steps of the anaerobic toluene and CF degradation pathways (Lueders, 2017; Tang 6 M. Tucci et al. Chemosphere 338 (2023) 139467 Fig. 4. Effect of the toluene concentration on the toluene A) and CF B) removal rates calculated as averages of the cycles 2,3 and 4. Fig. 5. Effect of acetate on the removal of toluene and CF in terms of rate A) and percentage B) of the contaminant load in the influent. are the benzylsuccinate synthase (bssA), encoding for the enzyme that catalyzes the anaerobic toluene activation by fumarate addition (Winderl et al., 2007); the bcrC, bzdN genes encoding for the ATP-dependent class I benzoyl-CoA reductases (Kuntze et al., 2011); the bamB gene encoding for the ATP-independent class II benzoyl CoA reductase (Löffler et al., 2011). Tese genes were highly abundant both in the effluent and in the biofilm of the graphite rods (Fig. 7A). In the liquid effluent, genes involved in the toluene degradation accounted for 1, 73E+09 (bcrC), 4,62E+08 (bzdN), 4,95E+08 (bamB) and 2,75E+08 (bssA) gene copies L− 1 (Fig. 7A). Similarly, in the biofilm of the graphite rods, the same concentration trend was observed, with bcrC gene as the most abundant one (1,1E+07 gene copies cm− 1 graphite rod). Regarding the CF degradation, the presence of the anaerobic bacteria Dehalobacter spp., responsible for the reductive hydrogenolysis of CF to DCM, and of D. formicoaceticum involved in the fermentation of DCM to acetate and formate, were also ascertained both in the liquid effluent and on the graphite surface, although not spotted by the 16 S rRNA gene amplicon sequencing (Grostern et al., 2010; Justicia-Leon et al., 2014). Moreover, process-specific biomarker genes were quantified to track CF to DCM (cfrA gene) transformation and DCM fermentation (mecE and mecF homologous genes belonging to the methylene chloride catabolism Fig. 6. - Bacterial community composition revealed by the 16 S rRNA gene gene cassette, recently identified as prognostic and diagnostic tools amplicon sequencing. Data are expressed as relative abundance (% of total supporting bioremediation of matrices impacted by DCM (Murdoch reads) of genera (≥1% in at least one sample) in the liquid effluent of the et al., 2022)). reactor and biofilm grown on graphite rods. 16 S rRNA of Dehalobacter (dre gene in Fig. 7B) was found with 1,63E+09 16 S rRNA gene copies L− 1 effluent and 4,15E+06 gene copies and Edwards, 2013; Von Netzer et al., 2016) have been quantified both cm− 1 graphite. cfrA gene was also found at high concentration in the in the liquid effluent and on the biofilm established on the graphite rods liquid effluent (1.74E+09 gene copies L− 1) and on the graphite surface (Fig. 7). (1,18E+06 gene copies cm− 1). These results are in line with the The functional genes involved in the anaerobic toluene degradation observed performances of the reactor and previous evidence (Tucci 7 M. Tucci et al. Chemosphere 338 (2023) 139467 Fig. 7. Quantification of functional genes involved in toluene degradation (bssA, bcrC, bzdN, bamB) (7A), in CF to DCM degradation (Dehalobacter spp., cfrA) and in DCM fermentation (D. formicoaceticum, mecE and mecF genes). et al., 2022a, 2022c) suggesting that, though likely catalyzed by Declaration of competing interest different microorganisms, toluene utilization in the liquid medium and on the graphite’s biofilm shared the same initial degradation steps likely The authors declare that they have no known competing financial commencing with fumarate addition and involving benzoyl-CoA as a interests or personal relationships that could have appeared to influence central intermediate. Moreover, D. formicoaceticum was highly abundant the work reported in this paper. in the liquid effluent (3,54E+08 gene copies L− 1) and in the biofilm (4, 15E+06 gene copies L-cm). Similarly, mecE and mecF homologous genes Data availability were found both in the liquid effluent and on the graphite surface with high concentrations (Fig. 7B). Data will be made available on request. 5. Conclusion Aknowledgements For the first time, the removal of toluene was coupled with CF This study was supported by the European Union’s Horizon 2020 degradation in a single-stage bioelectrochemical reactor. The bioelectric project ELECTRA (www.electra.site) under grant agreement No. well proved once again to be effective in treating complex mixtures of 826244. D. Fernández-Verdejo acknowledges a predoctoral grant from contaminants and exploiting both the oxidation and the reduction re­ UAB (PIF 2017–2018) and financial support for the research stay per­ action simultaneously. Furthermore, under optimal conditions, the formed (ESTPIF2022-04). reactor was able to achieve almost complete removal of the target contaminants. 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