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.

Magnetite particles triggering a faster and more robust syntrophic pathway of methanogenic propionate degradation.

Interspecies electron transfer mechanisms between Bacteria and Archaea play a pivotal role during methanogenic degradation of organic matter in natural and engineered anaerobic ecosystems. Growing evidence suggests that in syntrophic communities electron transfer does not rely exclusively on the exchange of diffusible molecules and energy carriers such as hydrogen or formate, rather microorganisms have the capability to exchange metabolic electrons in a more direct manner. Here, we show that supplementation of micrometer-size magnetite (Fe3O4) particles to a methanogenic sludge enhanced (up to 33%) the methane production rate from propionate, a key intermediate in the anaerobic digestion of organic matter and a model substrate to study energy-limited syntrophic communities. The stimulatory effect most probably resulted from the establishment of a direct interspecies electron transfer (DIET), based on magnetite particles serving as electron conduits between propionate-oxidizing acetogens and carbon dioxide-reducing methanogens. Theoretical calculations revealed that DIET allows electrons to be transferred among syntrophic partners at rates which are substantially higher than those attainable via interspecies H2 transfer. Besides the remarkable potential for improving anaerobic digestion, which is a proven biological strategy for renewable energy production, the herein described conduction-based DIET could also have a role in natural methane emissions from magnetite-rich soils and sediments.

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.

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.

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.

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.

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.

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.

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.

Cable Bacteria and the Bioelectrochemical Snorkel: The Natural and Engineered Facets Playing a Role in Hydrocarbons Degradation in Marine Sediments.

The composition and metabolic traits of the microbial communities acting in an innovative bioelectrochemical system were here investigated. The system, known as Oil Spill Snorkel, was recently developed to stimulate the oxidative biodegradation of petroleum hydrocarbons in anoxic marine sediments. Next Generation Sequencing was used to describe the microbiome of the bulk sediment and of the biofilm growing attached to the surface of the electrode. The analysis revealed that sulfur cycling primarily drives the microbial metabolic activities occurring in the bioelectrochemical system. In the anoxic zone of the contaminated marine sediment, petroleum hydrocarbon degradation occurred under sulfate-reducing conditions and was lead by different families of Desulfobacterales (46% of total OTUs). Remarkably, the occurrence of filamentous Desulfubulbaceae, known to be capable to vehicle electrons deriving from sulfide oxidation to oxygen serving as a spatially distant electron acceptor, was demonstrated. Differently from the sediment, which was mostly colonized by Deltaproteobacteria, the biofilm at the anode hosted, at high extent, members of Alphaproteobacteria (59%) mostly affiliated to Rhodospirillaceae family (33%) and including several known sulfur- and sulfide-oxidizing genera. Overall, we showed the occurrence in the system of a variety of electroactive microorganisms able to sustain the contaminant biodegradation alone or by means of an external conductive support through the establishment of a bioelectrochemical connection between two spatially separated redox zones and the preservation of an efficient sulfur cycling.

Electrolysis-driven bioremediation of crude oil-contaminated marine sediments.

Bioremediation is an effective technology to tackle crude oil spill disasters, which takes advantage of the capacity of naturally occurring microorganisms to degrade petroleum hydrocarbons under a range of environmental conditions. The enzymatic process of breaking down oil is usually more rapid in the presence of oxygen. However, in contaminated sediments, oxygen levels are typically too low to sustain the rapid and complete biodegradation of buried hydrocarbons. Here, we explored the possibility to electrochemically manipulate the redox potential of a crude oil-contaminated marine sediment in order to establish, in situ, conditions that are conducive to contaminants biodegradation by autochthonous microbial communities. The proposed approach is based on the exploitation of low-voltage (2V) seawater electrolysis to drive oxygen generation (while minimizing chlorine evolution) on Dimensionally Stable Anodes (DSA) placed within the contaminated sediment. Results, based on a laboratory scale setup with chronically polluted sediments spiked with crude oil, showed an increased redox potential and a decreased pH in the vicinity of the anode of 'electrified' treatments, consistent with the occurrence of oxygen generation. Accordingly, hydrocarbons biodegradation was substantially accelerated (up to 3-times) compared to 'non-electrified' controls, while sulfate reduction was severely inhibited. Intermittent application of electrolysis proved to be an effective strategy to minimize the energy requirements of the process, without adversely affecting degradation performance. Taken as a whole, this study suggests that electrolysis-driven bioremediation could be a sustainable technology for the management of contaminated sediments.

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.

The "Oil-Spill Snorkel": an innovative bioelectrochemical approach to accelerate hydrocarbons biodegradation in marine sediments.

This study presents the proof-of-concept of the "Oil-Spill Snorkel": a novel bioelectrochemical approach to stimulate the oxidative biodegradation of petroleum hydrocarbons in sediments. The "Oil-Spill Snorkel" consists of a single conductive material (the snorkel) positioned suitably to create an electrochemical connection between the anoxic zone (the contaminated sediment) and the oxic zone (the overlying O2-containing water). The segment of the electrode buried within the sediment plays a role of anode, accepting electrons deriving from the oxidation of contaminants. Electrons flow through the snorkel up to the part exposed to the aerobic environment (the cathode), where they reduce oxygen to form water. Here we report the results of lab-scale microcosms setup with marine sediments and spiked with crude oil. Microcosms containing one or three graphite snorkels and controls (snorkel-free and autoclaved) were monitored for over 400 days. Collectively, the results of this study confirmed that the snorkels accelerate oxidative reactions taking place within the sediment, as documented by a significant 1.7-fold increase (p = 0.023, two-tailed t-test) in the cumulative oxygen uptake and 1.4-fold increase (p = 0.040) in the cumulative CO2 evolution in the microcosms containing three snorkels compared to snorkel-free controls. Accordingly, the initial rate of total petroleum hydrocarbons (TPH) degradation was also substantially enhanced. Indeed, while after 200 days of incubation a negligible degradation of TPH was noticed in snorkel-free controls, a significant reduction of 12 ± 1% (p = 0.004) and 21 ± 1% (p = 0.001) was observed in microcosms containing one and three snorkels, respectively. Although, the "Oil-Spill Snorkel" potentially represents a groundbreaking alternative to more expensive remediation options, further research efforts are needed to clarify factors and conditions affecting the snorkel-driven biodegradation processes and to identify suitable configurations for field applications.

Isolation and quantification of cadmium removal mechanisms in batch reactors inoculated by sulphate reducing bacteria: biosorption versus bioprecipitation.

Biosorbing properties of sulphate reducing bacteria were tested to distinguish the amount of cadmium removed by bioprecipitation from that bound onto biomass surface (biosorption). Experimental results of cadmium abatement in batch growth tests (bioprecipitation tests) were then compared with metabolism-independent binding properties of SRB cell wall surface (biosorption tests performed with dead biomass). Experimental results showed that SRB inoculum removed 59 + or - 5% of sulphates in 21 days even in presence of cadmium (0-36 mmol L(-1)), while non-monotonous kinetic effects were observed for increasing Cd concentrations. Comparison between bioprecipitation and biosorption tests denoted a significant contribution of biosorption (77%) in total Cd removal (0.40 + or - 0.01 mmol g(-1)). Characterisation of bacterial acid-base surface properties by potentiometric titrations and mechanistic modelling denoted that carboxylic, phosphate and amino groups of cell wall are the main responsible of metal removal by biosorption mechanism.