Anaerobic treatment of groundwater co-contaminated by toluene and copper in a single chamber bioelectrochemical system.

Addressing the simultaneous removal of multiple coexisting groundwater contaminants poses a significant challenge, primarily because of their different physicochemical properties. Indeed, different chemical compounds may necessitate establishing distinct, and sometimes conflicting, (bio)degradation and/or removal pathways. In this work, we investigated the concomitant anaerobic treatment of toluene and copper in a single-chamber bioelectrochemical cell with a potential difference of 1 V applied between the anode and the cathode. As a result, the electric current generated by the bioelectrocatalytic oxidation of toluene at the anode caused the abiotic reduction and precipitation of copper at the cathode, until the complete removal of both contaminants was achieved. Open circuit potential (OCP) experiments confirmed that the removal of copper and toluene was primarily associated with polarization. Analogously, abiotic experiments, at an applied potential of 1 V, confirmed that neither toluene was oxidized nor copper was reduced in the absence of microbial activity. At the end of each experiment, both electrodes were characterized by means of a comprehensive suite of chemical and microbiological analyses, evidencing a highly selected microbial community competent in the biodegradation of toluene in the anodic biofilm, and a uniform electrodeposition of spherical Cu2O nanoparticles over the cathode surface.

Enrichment of Aerobic and Anaerobic Hydrocarbon-Degrading Bacteria from Multicontaminated Marine Sediment in Mar Piccolo Site (Taranto, Italy).

Marine sediments act as a sink for the accumulation of various organic contaminants such as polychlorobiphenyls (PCBs). These contaminants affect the composition and activity of microbial communities, particularly favoring those capable of thriving from their biodegradation and biotransformation under favorable conditions. Hence, contaminated environments represent a valuable biological resource for the exploration and cultivation of microorganisms with bioremediation potential. In this study, we successfully cultivated microbial consortia with the capacity for PCB removal under both aerobic and anaerobic conditions. The source of these consortia was a multicontaminated marine sediment collected from the Mar Piccolo (Taranto, Italy), one of Europe's most heavily polluted sites. High-throughput sequencing was employed to investigate the dynamics of the bacterial community of the marine sediment sample, revealing distinct and divergent selection patterns depending on the imposed reductive or oxidative conditions. The aerobic incubation resulted in the rapid selection of bacteria specialized in oxidative pathways for hydrocarbon transformation, leading to the isolation of Marinobacter salinus and Rhodococcus cerastii species, also known for their involvement in aerobic polycyclic aromatic hydrocarbons (PAHs) transformation. On the other hand, anaerobic incubation facilitated the selection of dechlorinating species, including Dehalococcoides mccartyi, involved in PCB reduction. This study significantly contributes to our understanding of the diversity, dynamics, and adaptation of the bacterial community in the hydrocarbon-contaminated marine sediment from one sampling point of the Mar Piccolo basin, particularly in response to stressful conditions. Furthermore, the establishment of consortia with biodegradation and biotransformation capabilities represents a substantial advancement in addressing the challenge of restoring polluted sites, including marine sediments, thus contributing to expanding the toolkit for effective bioremediation strategies.

Toluene-driven anaerobic biodegradation of chloroform in a continuous-flow bioelectrochemical reactor.

Subsurface co-contamination by multiple pollutants can be challenging for the design of bioremediation strategies since it may require promoting different and often antagonistic degradation pathways. Here, we investigated the simultaneous degradation of toluene and chloroform (CF) in a continuous-flow anaerobic bioelectrochemical reactor. As a result, 47 µmol L-1 d-1 of toluene and 60 µmol L-1 d-1 of CF were concurrently removed, when the anode was polarized at +0.4 V vs. Standard Hydrogen Electrode (SHE). Analysis of the microbial community structure and key functional genes allowed to identify the involved degradation pathways. 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 inhibition effects. Overall, this study proved the efficacy of the developed bioelectrochemical system in simultaneously treating multiple groundwater contaminants, paving the way for the application in real-world scenarios.

Enhancing the Anaerobic Biodegradation of Petroleum Hydrocarbons in Soils with Electrically Conductive Materials.

Anaerobic bioremediation is a relevant process in the management of sites contaminated by petroleum hydrocarbons. Recently, interspecies electron transfer processes mediated by conductive minerals or particles have been proposed as mechanisms through which microbial species within a community share reducing equivalents to drive the syntrophic degradation of organic substrates, including hydrocarbons. Here, a microcosm study was set up to investigate the effect of different electrically conductive materials (ECMs) in enhancing the anaerobic biodegradation of hydrocarbons in historically contaminated soil. The results of a comprehensive suite of chemical and microbiological analyses evidenced that supplementing the soil with (5% w/w) magnetite nanoparticles or biochar particles is an effective strategy to accelerate the removal of selected hydrocarbons. In particular, in microcosms supplemented with ECMs, the removal of total petroleum hydrocarbons was enhanced by up to 50% relative to unamended controls. However, chemical analyses suggested that only a partial bioconversion of contaminants occurred and that longer treatment times would have probably been required to drive the biodegradation process to completion. On the other hand, biomolecular analyses confirmed the presence of several microorganisms and functional genes likely involved in hydrocarbon degradation. Furthermore, the selective enrichment of known electroactive bacteria (i.e., Geobacter and Geothrix) in microcosms amended with ECMs, clearly pointed to a possible role of DIET (Diet Interspecies Electron Transfer) processes in the observed removal of contaminants.

Coupling of bioelectrochemical toluene oxidation and trichloroethene reductive dechlorination for single-stage treatment of groundwater containing multiple contaminants.

Bioremediation of groundwater contaminated by a mixture of aromatic hydrocarbons and chlorinated solvents is typically challenged because these contaminants are degraded via distinctive oxidative and reductive pathways, thus requiring different amendments and redox conditions. Here, we provided the proof-of-concept of a single-stage treatment of synthetic groundwater containing toluene and trichloroethene (TCE) in a tubular bioelectrochemical reactor, known as a "bioelectric well". Toluene was degraded by a microbial bioanode (up to 150 µmol L-1 d-1) with a polarized graphite anode (+0.2 V vs. SHE) serving as the terminal electron acceptor. The electric current deriving from microbially-driven toluene oxidation resulted in (abiotic) hydrogen production (at a stainless-steel cathode), which sustained the reductive dechlorination of TCE to less-chlorinated intermediates (i.e., cis-DCE, VC, and ETH), at a maximum rate of 500 µeq L-1 d-1, in the bulk of the reactor. A phylogenetic and functional gene-based analysis of the "bioelectric well" confirmed the establishment of a microbiome harboring the metabolic potential for anaerobic toluene oxidation and TCE reductive dechlorination. However, Toluene degradation and current generation were found to be rate-limited by external mass transport phenomena, thus indicating the existing potential for further process optimization.

Insights into the syntrophic microbial electrochemical oxidation of toluene: a combined chemical, electrochemical, taxonomical, functional gene-based, and metaproteomic approach.

Biodegradation of aromatic hydrocarbons in anoxic contaminated environments is typically limited by the lack of bioavailable electron acceptors. Microbial electrochemical technologies (METs) are able to provide a virtually inexhaustible electron acceptor in the form of a solid electrode. Recently, we provided first experimental evidence for the syntrophic degradation of toluene in a continuous-flow bioelectrochemical reactor known as the "bioelectric well". Herein, we further analyzed the structure and function of the electroactive toluene-degrading microbiome using a suite of chemical, electrochemical, phylogenetic, proteomic, and functional gene-based analyses. The bioelectric well removed 83 ± 7 % of the toluene from the influent with a coulombic efficiency of 84 %. Cyclic voltammetry allowed to identify the formal potentials of four putative electron transfer sites, which ranged from -0.2 V to +0.1 V vs. SHE, consistent with outer membrane c-type cytochromes and pili of electroactive Geobacter species. The biofilm colonizing the surface of the anode was indeed highly enriched in Geobacter species. On the other hand, the planktonic communities thriving in the bulk of the reactor harbored aromatic hydrocarbons degraders and fermentative propionate-producing microorganisms, as revealed by phylogenetic and proteomic analyses. Most likely, propionate, acetate or other VFAs produced in the bulk liquid from the degradation of toluene were utilized as substrates by the electroactive biofilm. Interestingly, key-functional genes related to the degradation of toluene were found both in the biofilm and in the planktonic communities. Taken as a whole, the herein reported results highlight the importance of applying a comprehensive suite of techniques to unravel the complex cooperative metabolisms occurring in METs.

Passive electrobioremediation approaches for enhancing hydrocarbons biodegradation in contaminated soils.

Electrobioremediation technologies hold considerable potential for the treatment of soils contaminated by petroleum hydrocarbons (PH), since they allow stimulating biodegradation processes with no need for subsurface chemicals injection and with little to no energy consumption. Here, a microbial electrochemical snorkel (MES) was applied for the treatment of a soil contaminated by hydrocarbons. The MES consists of direct coupling of a microbial anode with a cathode, being a single conductive, non-polarized material positioned suitably to create an electrochemical connection between the anoxic zone (the contaminated soil) and the oxic zone (the overlying oxygenated water). Soil was also supplemented with electrically conductive particles of biochar as a strategy to construct a conductive network with microbes in the soil matrix, thus extending the radius of influence of the snorkel. The results of a comprehensive suite of chemical, microbiological and ecotoxicological analyses evidenced that biochar addition, rather than the presence of a snorkel, was the determining factor in accelerating PH removal from contaminated soils, possibly accelerating syntrophic and/or cooperative metabolisms involved in the degradation of PH. The enhancement of biodegradation was mirrored by an increased abundance of anaerobic and aerobic microorganisms known to be involved in the degradation of PH and related functional genes. Plant ecotoxicity assays confirmed a reduction of soils toxicity in treatments receiving electrically conductive biochar.

Snorkels enhance alkanes respiration at ambient and increased hydrostatic pressure (10 MPa) by either supporting the TCA cycle or limiting alternative routes for acetyl-CoA metabolism.

The impact of piezosensitive microorganisms is generally underestimated in the ecology of underwater environments exposed to increasing hydrostatic pressure (HP), including the biodegradation of crude oil components. Yet, no isolated pressure-loving (piezophile) microorganism grows optimally on hydrocarbons, and no isolated piezophile at all has a HP optimum <10 MPa (e.g. 1000 m below sea water level). Piezosensitive heterotrophs are thus largely accountable for oil clean up < 10 MPa, however, they are affected by such a mild HP increase in ways which are not completely clear. In a first study, the application of a bioelectrochemical system (called "oil-spill snorkel") enhanced the alkane oxidation capacity in sediments collected at surface water but tested up to 10 MPa. Here, the fingerprint left on transcript abundance was studied to explore which metabolic routes are 1) supported by snorkels application and 2) negatively impacted by HP increase. Transcript abundance was comparable for beta-oxidation across all treatments (also at a taxonomical level), while the metabolism of acetyl-CoA was highly impacted: at either 0.1 or 10 MPa, snorkels supported acetyl-CoA oxidation within the TCA cycle, while in negative controls using non-conductive rods several alternative routes for acetyl-CoA were stimulated (including those leading to internal carbon reserves e.g. 2,3 butanediol and dihydroxyacetone). In general, increased HP had opposite effects as compared to snorkels, thus indicating that snorkels could enhance hydrocarbons oxidation by alleviating in part the stressing effects imposed by increased HP on the anaerobic, respiratory electron transport chain. 16S rRNA gene analysis of sediments and biofilms on snorkels suggest a crosstalk between oil-degrading, sulfate-reducing microorganisms and sulfur oxidizers. In fact, no sulfur was deposited on snorkels, however, iron, aluminum and phosphorous were found to preferentially deposit on snorkels at 10 MPa. This data indicates that a passive BES such as the oil-spill snorkel can mitigate the stress imposed by increased HP on piezosensitive microorganisms (up to 10 MPa) without being subjected to passivation. An improved setup applying these principles can further support this deep-sea bioremediation strategy.

An underappreciated DIET for anaerobic petroleum hydrocarbon-degrading microbial communities.

Direct interspecies electron transfer (DIET) via electrically conductive minerals can play a role in the anaerobic oxidation of petroleum hydrocarbons in contaminated sites and can be exploited for the development of new, more effective bioremediation approaches.

Parallel artificial and biological electric circuits power petroleum decontamination: The case of snorkel and cable bacteria.

Degradation of petroleum hydrocarbons (HC) in sediments is often limited by the availability of electron acceptors. By allowing long-distance electron transport (LDET) between anoxic sediments and oxic overlying water, bioelectrochemical snorkels may stimulate the regeneration of sulphate in the anoxic sediment thereby accelerating petroleum HC degradation. Cable bacteria can also mediate LDET between anoxic and oxic sediment layers and thus theoretically stimulate petroleum HC degradation. Here, we quantitatively assessed the impact of cable bacteria and snorkels on the degradation of alkanes in marine sediment from Aarhus Bay (Denmark). After seven weeks, cable bacteria and snorkels accelerated alkanes degradation by +24 and +25%, respectively, compared to control sediment with no cable bacteria nor snorkel. The combination of snorkels and cable bacteria further enhanced alkanes degradation (+46%). Higher degradation rates were sustained by LDET-induced sulphide removal rather than, as initially hypothesized, sulphate regeneration. Cable bacteria are thus overlooked players in the self-healing capacity of crude-oil contaminated sediments, and may inspire novel remediation treatments upon hydrocarbon spillage.

Microbial electrochemistry for bioremediation.

Lack of suitable electron donors or acceptors is in many cases the key reason for pollutants to persist in the environment. Externally supplementation of electron donors or acceptors is often difficult to control and/or involves chemical additions with limited lifespan, residue formation or other adverse side effects. Microbial electrochemistry has evolved very fast in the past years - this field relates to the study of electrochemical interactions between microorganisms and solid-state electron donors or acceptors. Current can be supplied in such so-called bioelectrochemical systems (BESs) at low voltage to provide or extract electrons in a very precise manner. A plethora of metabolisms can be linked to electrical current now, from metals reductions to denitrification and dechlorination. In this perspective, we provide an overview of the emerging applications of BES and derived technologies towards the bioremediation field and outline how this approach can be game changing.

Microbiomes in Soils Exposed to Naturally High Concentrations of CO2 (Bossoleto Mofette Tuscany, Italy).

Direct and indirect effects of extremely high geogenic CO2 levels, commonly occurring in volcanic and hydrothermal environments, on biogeochemical processes in soil are poorly understood. This study investigated a sinkhole in Italy where long-term emissions of thermometamorphic-derived CO2 are associated with accumulation of carbon in the topsoil and removal of inorganic carbon in low pH environments at the bottom of the sinkhole. The comparison between interstitial soil gasses and those collected in an adjacent bubbling pool and the analysis of the carbon isotopic composition of CO2 and CH4 clearly indicated the occurrence of CH4 oxidation and negligible methanogenesis in soils at the bottom of the sinkhole. Extremely high CO2 concentrations resulted in higher microbial abundance (up to 4 × 109 cell g-1 DW) and a lower microbial diversity by favoring bacteria already reported to be involved in acetogenesis in mofette soils (i.e., Firmicutes, Chloroflexi, and Acidobacteria). Laboratory incubations to test the acetogenic and methanogenic potential clearly showed that all the mofette soil supplied with hydrogen gas displayed a remarkable CO2 fixation potential, primarily due to the activity of acetogenic microorganisms. By contrast, negligible production of acetate occurred in control tests incubated with the same soils, under identical conditions, without the addition of hydrogen. In this study, we report how changes in diversity and functions of the soil microbial community - induced by high CO2 concentration - create peculiar biogeochemical profile. CO2 emission affects carbon cycling through: (i) inhibition of the decomposition of the organic carbon and (ii) promotion of CO2-fixation via the acetyl-CoA pathway. Sites naturally exposed to extremely high CO2 levels could potentially represent an untapped source of microorganisms with unique capabilities to catalytically convert CO2 into valuable organic chemicals and fuels.

Bioelectrochemical treatment of groundwater containing BTEX in a continuous-flow system: Substrate interactions, microbial community analysis, and impact of sulfate as a co-contaminant.

Microbial electrochemical technologies (MET) are increasingly being considered for in situ remediation of contaminated groundwater. However, their application potential for the simultaneous treatment of complex mixtures of organic and inorganic contaminants, has been only marginally explored. Here we have analyzed the performance of the 'bioelectric well', a previously developed bioelectrochemical reactor configuration, in the treatment of benzene, toluene, ethyl-benzene and xylenes (BTEX) mixtures. Although to different extents, all BTEX were found to be degraded in the bioelectrochemical system, operated using a continuous-flow of groundwater at a hydraulic retention time of 8.8 h, with the graphite anode potentiostatically controlled at +0.200 V vs. the standard hydrogen electrode. In the case of toluene and ethyl-benzene, biodegradation was further confirmed by the GC-MS identification of fumarate-addition metabolites, previously shown to be involved in the activation of these contaminants under anaerobic conditions. Degradation rates were higher for toluene (31.3 ±â€¯1.5 mg/L d) and lower for benzene (6.1 ±â€¯0.3 mg/L d), ethyl-benzene (3.3 ±â€¯0.1 mg/L d), and xylenes (4.5 ±â€¯0.2 mg/L d). BTEX degradation was linked to electric current generation, with coulombic efficiencies falling in the range 53-69%, although methanogenesis also contributed to contaminant degradation. Remarkably, the system also allowed removal of sulfate simultaneously with toluene. Sulfate removal was likely driven by the hydrogen abiotically generated at the cathode. Taken as a whole, these findings highlight the remarkable potential of this innovative reactor configuration for application in a variety of contamination scenarios.

Anode potential selection for sulfide removal in contaminated marine sediments.

Sulfate reducing microorganisms are typically involved in hydrocarbon biodegradation in the sea sediment, with their metabolism resulting in the by-production of toxic sulfide. In this context, it is of utmost importance identifying the optimal value for anodic potential which ensures efficient toxic sulfide removal. Along this line, in this study the (bio)electrochemical removal of sulfide was tested at anodic potentials of -205 mV, +195 mV and +300 mV (vs Ag/AgCl), also in the presence of a pure culture of the sulfur-oxidizing bacterium Desulfobulbus propionicus. Current production, sulfide concentration and sulfate concentration were monitored over time. At the end of the experiment sulfur deposition on the electrodes and the microbial communities were characterized by SEM-EDS and by next generation sequencing of the 16S rRNA gene respectively. Results confirmed that current production was linked to sulfide removal and D. propionicus promoted back oxidation of deposited sulfur to sulfate. The highest electron recovery was observed at +195 mV vs Ag/AgCl, and the lowest sulfur deposition was obtained at -205 mV vs Ag/AgCl anode polarization.

The bioelectric well: a novel approach for in situ treatment of hydrocarbon-contaminated groundwater.

Groundwater contamination by petroleum hydrocarbons (PHs) is a widespread problem which poses serious environmental and health concerns. Recently, microbial electrochemical technologies (MET) have attracted considerable attention for remediation applications, having the potential to overcome some of the limiting factors of conventional in situ bioremediation systems. So far, field-scale application of MET has been largely hindered by the limited availability of scalable system configurations. Here, we describe the 'bioelectric well' a bioelectrochemical reactor configuration, which can be installed directly within groundwater wells and can be applied for in situ treatment of organic contaminants, such as PHs. A laboratory-scale prototype of the bioelectric well has been set up and operated in continuous-flow regime with phenol as the model contaminant. The best performance was obtained when the system was inoculated with refinery sludge and the anode potentiostatically controlled at +0.2 V versus SHE. Under this condition, the influent phenol (25 mg l-1 ) was nearly completely (99.5 ± 0.4%) removed, with an average degradation rate of 59 ± 3 mg l-1 d and a coulombic efficiency of 104 ± 4%. Microbial community analysis revealed a remarkable enrichment of Geobacter species on the surface of the graphite anode, clearly pointing to a direct involvement of this electro-active bacterium in the current-generating and phenol-oxidizing process.

Enhancing methane production from food waste fermentate using biochar: the added value of electrochemical testing in pre-selecting the most effective type of biochar.

BACKGROUND: Recent studies have suggested that addition of electrically conductive biochar particles is an effective strategy to improve the methanogenic conversion of waste organic substrates, by promoting syntrophic associations between acetogenic and methanogenic organisms based on interspecies electron transfer processes. However, the underlying fundamentals of the process are still largely speculative and, therefore, a priori identification, screening, and even design of suitable biochar materials for a given biotechnological process are not yet possible. RESULTS: Here, three charcoal-like products (i.e., biochars) obtained from the pyrolysis of different lignocellulosic materials, (i.e., wheat bran pellets, coppiced woodlands, and orchard pruning) were tested for their capacity to enhance methane production from a food waste fermentate. In all biochar-supplemented (25 g/L) batch experiments, the complete methanogenic conversion of fermentate volatile fatty acids proceeded at a rate that was up to 5 times higher than that observed in the unamended (or sand-supplemented) controls. Fluorescent in situ hybridization analysis coupled with confocal laser scanning microscopy revealed an intimate association between archaea and bacteria around the biochar particles and provided a clear indication that biochar also shaped the composition of the microbial consortium. Based on the application of a suite of physico-chemical and electrochemical characterization techniques, we demonstrated that the positive effect of biochar is directly related to the electron-donating capacity (EDC) of the material, but is independent of its bulk electrical conductivity and specific surface area. The latter properties were all previously hypothesized to play a major role in the biochar-mediated interspecies electron transfer process in methanogenic consortia. CONCLUSIONS: Collectively, these results of this study suggest that for biochar addition in anaerobic digester operation, the screening and identification of the most suitable biochar material should be based on EDC determination, via simple electrochemical tests.

Bridging spatially segregated redox zones with a microbial electrochemical snorkel triggers biogeochemical cycles in oil-contaminated River Tyne (UK) sediments.

Marine sediments represent an important sink for a number of anthropogenic organic contaminants, including petroleum hydrocarbons following an accidental oil spill. Degradation of these compounds largely depends on the activity of sedimentary microbial communities linked to biogeochemical cycles, in which abundant elements such as iron and sulfur are shuttled between their oxidized and reduced forms. Here we show that introduction of a small electrically conductive graphite rod ("the electrochemical snorkel") into an oil-contaminated River Tyne (UK) sediment, so as to create an electrochemical connection between the anoxic contaminated sediment and the oxygenated overlying water, has a large impact on the rate of metabolic reactions taking place in the bulk sediment. The electrochemical snorkel accelerated sulfate reduction processes driven by organic contaminant oxidation and suppressed competitive methane-producing reactions. The application of a comprehensive suite of chemical, spectroscopic, biomolecular and thermodynamic analyses suggested that the snorkel served as a scavenger of toxic sulfide via a redox interaction with the iron cycle. Taken as a whole, the results of this work highlight a new strategy for controlling biological processes, such as bioremediation, through the manipulation of the electron flows in contaminated sediments.