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Dichloromethane (DCM, methylene chloride) is a toxic, high-volume industrial pollutant of long-standing. Anaerobic biodegradation is crucial for its removal from contaminated environments, yet prevailing mechanisms remain unresolved, especially concerning dehalogenation. In this study, we obtained an assembled genome of a novel DCM-degrading strain, Dehalobacterium formicoaceticum strain EZ94, from a stable DCM-degrading consortium, and we analyzed its proteome during degradation of DCM. A gene cluster recently predicted to play a major role in anaerobic DCM catabolism (the mec cassette) was found. Methyltransferases and other proteins encoded by the mec cassette were among the most abundant proteins produced, suggesting their involvement in DCM catabolism. Reductive dehalogenases were not detected. Genes and corresponding proteins for a complete Wood-Ljungdahl pathway, which could enable further metabolism of DCM carbon, were also found. Unlike for the anaerobic DCM degrader “Ca. F. warabiya,” no genes for metabolism of the quaternary amines choline and glycine betaine were identified. This work provides independent and supporting evidence that mec-associated methyltransferases are key to anaerobic DCM metabolism.

Dehalobacter (Firmicutes) encompass obligate organohalide-respiring bacteria used for bioremediation of groundwater contaminated with halogenated organics. Various aspects of their biochemistry remain unknown, including the identities and interactions of respiratory proteins. Here, we sequenced the genome of Dehalobacter sp. strain 8M and analysed its protein expression. Strain 8M encodes 22 reductive dehalogenase homologous (RdhA) proteins. RdhA D8M_v2_40029 (TmrA) was among the two most abundant proteins during growth with trichloromethane and 1,1,2-trichloroethane. To examine interactions of respiratory proteins, we used blue native gel electrophoresis together with dehalogenation activity tests and mass spectrometry. The highest activities were found in gel slices with the highest abundance of TmrA. Protein distributions across gel lanes provided biochemical evidence that the large and small subunits of the membrane-bound [NiFe] uptake hydrogenase (HupL and HupS) interacted strongly and that HupL/S interacted weakly with RdhA. Moreover, the interaction of RdhB and membrane-bound b-type cytochrome HupC was detected. RdhC proteins, often encoded in rdh operons but without described function, migrated in a protein complex not associated with HupL/S or RdhA. This study provides the first biochemical evidence of respiratory protein interactions in Dehalobacter, discusses implications for the respiratory architecture and advances the molecular comprehension of this unique respiratory chain.

We used C-Cl dual isotope analysis and microcosm studies for elucidating the origin and fate of the common groundwater pollutant dichloromethane (DCM) in two different multi-contaminant field sites in Catalonia, Spain; where DCM contamination could be the result of direct solvent releases and/or chloroform (CF) transformation. Known commercial solvents isotopic compositions as well as characteristic C-Cl dual isotope slopes from our anaerobic enrichment culture containing Dehalobacterium sp., capable of fermenting DCM, and other bacteria from the literature were used for field data interpretation. © The Authors, published by EDP Sciences, 2019.

The contribution of the sorption processes in the elimination of pharmaceuticals (PhACs) during the fungal treatment of wastewater has been evaluated in this work. The sorption of four PhACs (carbamazepine, diclofenac, iopromide and venlafaxine) by 6 different fungi was first evaluated in batch experiments. Concentrations of PhACs in both liquid and solid (biomass) matrices from the fungal treatment were measured. Contribution of the sorption to the total removal of pollutants ranged between 3% and 13% in relation to the initial amount. The sorption of 47 PhACs in fungi was also evaluated in a fungal treatment performed in 26 days in a continuous bioreactor treating wastewater from a veterinary hospital. PhACs levels measured in the fungal biomass were similar to those detected in conventional wastewater treatment (WWTP) sludge. This may suggest the necessity of manage fungal biomass as waste in the same manner that the WWTP sludge is managed. © 2017 Elsevier B.V.

1,1,2-Trichloroethane (1,1,2-TCA) is a non-flammable organic solvent and common environmental contaminant in groundwater. Organohalide-respiring bacteria are key microorganisms to remediate 1,1,2-TCA because they can gain metabolic energy during its dechlorination under anaerobic conditions. However, all current isolates produce hazardous end products such as vinyl chloride, monochloroethane or 1,2-dichloroethane that accumulate in the medium. Here, we constructed a syntrophic co-culture of Dehalogenimonas and Dehalococcoides mccartyi strains to achieve complete detoxification of 1,1,2-TCA to ethene. In this co-culture, Dehalogenimonas transformed 1,1,2-TCA via dihaloelimination to vinyl chloride, whereas Dehalococcoides reduced vinyl chloride via hydrogenolysis to ethene. Molasses, pyruvate, and lactate supported full dechlorination of 1,1,2-TCA in serum bottle co-cultures. Scale up of the cultivation to a 5-L bioreactor operating for 76 d in fed-batch mode was successful with pyruvate as substrate. This synthetic combination of bacteria with known complementary metabolic capabilities demonstrates the potential environmental relevance of microbial cooperation to detoxify 1,1,2-TCA.

1,1,2-Trichloroethane (1,1,2-TCA) is a non-flammable organic solvent and common environmental contaminant in groundwater. Organohalide-respiring bacteria are key microorganisms to remediate 1,1,2-TCA because they can gain metabolic energy during its dechlorination under anaerobic conditions. However, all current isolates produce hazardous end products such as vinyl chloride, monochloroethane or 1,2-dichloroethane that accumulate in the medium. Here, we constructed a syntrophic co-culture of Dehalogenimonas and Dehalococcoides mccartyi strains to achieve complete detoxification of 1,1,2-TCA to ethene. In this co-culture, Dehalogenimonas transformed 1,1,2-TCA via dihaloelimination to vinyl chloride, whereas Dehalococcoides reduced vinyl chloride via hydrogenolysis to ethene. Molasses, pyruvate, and lactate supported full dechlorination of 1,1,2-TCA in serum bottle co-cultures. Scale up of the cultivation to a 5-L bioreactor operating for 76 d in fed-batch mode was successful with pyruvate as substrate. This synthetic combination of bacteria with known complementary metabolic capabilities demonstrates the potential environmental relevance of microbial cooperation to detoxify 1,1,2-TCA. © 2017 Elsevier B.V.

The use of the ligninolytic fungi Trametes versicolor for the degradation of micropollutants has been widely studied. However, few studies have addressed the treatment of real wastewater containing pharmaceutically active compounds (PhAC) under non-sterile conditions. The main drawback of performing such treatments is the difficulty for the inoculated fungus to successfully compete with the other microorganisms growing in the bioreactor. In the present study, several fungal treatments were performed under non-sterile conditions in continuous operational mode with two types of real wastewater effluent, namely, a reverse osmosis concentrate (ROC) from a wastewater treatment plant and a veterinary hospital wastewater (VHW). In all cases, the setup consisted of two parallel reactors: one inoculated with T. versicolor and one non-inoculated, which was used as the control. The main objective of this work was to correlate the operational conditions and traditional monitoring parameters, such as laccase activity, with PhAC removal and the composition of the microbial communities developed inside the bioreactors. For that purpose a variety of biochemical and molecular biology analyses were performed: phospholipid fatty acids analysis (PLFA), quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE) followed by sequencing. The results show that many indigenous fungi (and not only bacteria, which were the focus of the majority of previously published research) can successfully compete with the inoculated fungi (i.e., Trichoderma asperellum overtook T. versicolor in the ROC treatment). We also showed that the wastewater origin and the operational conditions had a stronger impact on the diversity of microbial communities developed in the bioreactors than the inoculation or not with T. versicolor. © 2016 Elsevier B.V.

An on-farm biopurification system (BPS) biomixture, with proven capacity to degrade carbofuran (CBF) was used as inoculum for a selective-enrichment process to obtain a bacterial consortium capable of using CBF as a sole source of carbon. Bacterial strains that comprise the consortium were identified as members of the genera Cupriavidus, Achromobacter and Pseudomonas. The consortium was tested in batch bioreactors, with initial CBF concentrations ranging from 50 to 200 mg L−1, in which complete degradation was attained in 16–17 h. Continuous operation of the bioreactor was optimized to achieve conditions of complete removal of up to 100 mg L−1 of CBF at a HRT of 23 h by the microbial consortium. Cross-degradation assays showed that the consortium was also capable of degrading high concentrations of other carbamates such as aldicarb, methiocarb and methomyl in batch bioreactors. The use of this consortium for the treatment of CBF and other carbamates at reactor scale represents a potential approach for the removal of these pesticides from agricultural wastewater, as an alternative to BPS. © 2016 Elsevier Ltd

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well. © 2015, Springer-Verlag Berlin Heidelberg.

Ketoprofen is a nonsteroidal anti-inflammatory drug that has been detected in the environment in the range of ng L-1-µg L-1 due to its low degradability in some wastewater treatment plants. In this study, the use of the white-rot fungus Trametes versicolor to effectively degrade ketoprofen in a defined liquid medium was assessed. The fungus eliminated ketoprofen to nondetectable levels in 24 h when it was added at 10 mg L-1 whereas at low concentration of 40 µg L-1 it was almost completely removed (95%) after 5 h. Low extracellular laccase activity was detected in the T. versicolor cultures but the addition of the laccase-mediator system did not lead to ketoprofen oxidation. The cytochrome P-450 inhibitor 1-aminobenzotriazole reduced ketoprofen oxidation. These data suggest that the first oxidation step is cytochrome P450 mediated. During time-course degradation experiments, three intermediates were structurally elucidated and quantified by HPLC-DAD-MS and NMR: 2-[3-(4-hydroxybenzoyl)phenyl]-propanoic acid, 2-[(3-hydroxy(phenyl)methyl)phenyl]-propanoic acid, and 2-(3-benzoyl-4-hydroxyphenyl)-propanoic acid. The latter was reported for the first time in biological systems. After 7 d of incubation, only small amounts of 2-[(3-hydroxy(phenyl)methyl)phenyl]-propanoic acid (0.08 mg) remained in the liquid medium in comparison with the initial ketoprofen dose (1.0 mg), suggesting possible mineralization of ketoprofen.

The white-rot fungus Trametes vesicolor degraded naproxen (10 mg L-1) in a liquid medium to non-detectable levels after 6 h. When naproxen was added in the range of concentrations typically found in the environment (55 µg L-1), it was almost completely degraded (95%) after 5 h. In vitro degradation experiments with purified laccase and purified laccase plus mediator 1-hydroxybenzotriazol showed slight and almost complete naproxen degradation, respectively. A noticeable inhibition on naproxen degradation was also observed when the cytochrome P450 inhibitor 1-aminobenzotriazole was added to the fungal cultures. These data suggest that both enzymatic systems could play a role in naproxen degradation. 2-(6-hydroxynaphthalen-2-yl)propanoic acid and 1-(6-methoxynaphthalen-2-yl)ethanone were structurally elucidated by HPLC-DAD-MS and NMR as degradation intermediates of naproxen. After 6 h of incubation, both parent compound and intermediates disappeared from the medium. The non-toxicity of the treated medium was confirmed by Microtox test.

Biological advanced oxidation of the pharmaceuticals clofibric acid (CA), carbamazepine (CBZP), atenolol (ATL) and propranolol (PPL) is reported for the first time. Extracellular oxidizing species were produced through a quinone redox cycling mechanism catalyzed by an intracellular quinone reductase and any of the ligninolytic enzymes of Trametes versicolor after addition of the lignin-derived quinone 2,6-dimethoxy-1,4-benzoquinone (DBQ) and Fe3+-oxalate in the medium. Time-course experiments with approximately 10 mg L-1 of initial pharmaceutical concentration resulted in percent degradations above 80% after 6 h of incubation. Oxidation of pharmaceuticals was only observed under DBQ redox cycling conditions. A similar degradation pattern was observed when CBZP was added at the environmentally relevant concentration of 50 µg L-1. Depletion of DBQ due to the attack of oxidizing agents was assumed to be the main limiting factor of pharmaceutical degradation. The main degradation products, that resulted to be pharmaceutical hydroxylated derivatives, were structurally elucidated. The detected 4- and 7-hydroxycarbamazepine intermediates of CBZP degradation were not reported to date. Total disappearance of intermediates was observed in all the experiments at the end of the incubation period.

Degradation of diclofenac sodium, a nonsteroidal anti-inflammatory drug widely found in the aquatic environment, was assessed using the white-rot fungus Trametes versicolor. Almost complete diclofenac removal (=94%) occurred the first hour with T. versicolor pellets when the drug was added at relatively high (10 mg L-1) and environmentally relevant low (45 µg L-1) concentrations in a defined liquid medium. In vivo and in vitro experiments using the cytochrome P450 inhibitor 1-aminobenzotriazole and purified laccase, respectively, suggested at least two different mechanisms employed by T. versicolor to initiate diclofenac degradation. Two hydroxylated metabolites, 4'-hydroxydiclofenac and 5-hydroxydiclofenac, were structurally elucidated by nuclear magnetic resonance as degradation intermediates in fungal cultures spiked with diclofenac. Both parent compound and intermediates disappeared after 24 h leading to a decrease in ecotoxicity calculated by the Microtox test. Laccase-catalyzed transformation of diclofenac led to the formation of 4-(2,6-dichlorophenylamino)-1,3-benzenedimethanol, which was not detected in in vivo experiments probably due to the low laccase activity levels observed through the first hours of incubation.

The biodegradation of Grey Lanaset G, which consists of a mixture of metal complexed dye, was studied. Experiments were carried out in a bioreactor with retained pellets of the fungus Trametes versicolor that was operated under conditions of laccase production. Although decolorization was highly efficient (90%), no direct relationship to extracellular enzyme was apparent. Moreover, the extracellular enzyme was found to be unable to degrade the dye in vitro. The process involves several steps. Thus, the initial adsorption of the dye and its transfer into cells is followed by breaking of the metal complex bond in the cells release of the components. The metal (Cr and Co) contents of the biomass and treated solutions, and their closer relationship to intracellular enzyme and degradation of the dye, confirm the initial hypothesis.