Respiratory protein interactions in Dehalobacter sp. strain 8M revealed through genomic and native proteomic analyses.

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.

Proteogenomics of the novel Dehalobacterium formicoaceticum strain EZ94 highlights a key role of methyltransferases during anaerobic dichloromethane degradation.

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.

Assessment of aerobic biodegradation of lower-chlorinated benzenes in contaminated groundwater using field-derived microcosms and compound-specific carbon isotope fractionation.

Biodegradation of lower chlorinated benzenes (tri-, di- and monochlorobenzene) was assessed at a coastal aquifer contaminated with multiple chlorinated aromatic hydrocarbons. Field-derived microcosms, established with groundwater from the source zone and amended with a mixture of lower chlorinated benzenes, evidenced biodegradation of monochlorobenzene (MCB) and 1,4-dichlorobenzene (1,4-DCB) in aerobic microcosms, whereas the addition of lactate in anaerobic microcosms did not enhance anaerobic reductive dechlorination. Aerobic microcosms established with groundwater from the plume consumed several doses of MCB and concomitantly degraded the three isomers of dichlorobenzene with no observable inhibitory effect. In the light of these results, we assessed the applicability of compound stable isotope analysis to monitor a potential aerobic remediation treatment of MCB and 1,4-DCB in this site. The carbon isotopic fractionation factors (ε) obtained from field-derived microcosms were -0.7‰ ± 0.1 ‰ and -1.0‰ ± 0.2 ‰ for MCB and 1,4-DCB, respectively. For 1,4-DCB, the carbon isotope fractionation during aerobic biodegradation was reported for the first time. The weak carbon isotope fractionation values for the aerobic pathway would only allow tracing of in situ degradation in aquifer parts with high extent of biodegradation. However, based on the carbon isotope effects measured in this and previous studies, relatively high carbon isotope shifts (i.e., ∆δ13C > 4.0 ‰) of MCB or 1,4-DCB in contaminated groundwater would suggest that their biodegradation is controlled by anaerobic reductive dechlorination.

Water resource recovery coupling microalgae wastewater treatment and sludge co-digestion for bio-wastes valorisation at industrial pilot-scale.

This case study is part of a circular bioeconomy project for a winery company aiming to integrate a microalgae-based system within the existing facilities of the winery WWTP, promoting nutrient recovery and transformation into valuable products and bioenergy. Microalgae were used for wastewater treatment, removing N-NH4+ (97%) and P-PO4-3 (93%). A pilot anaerobic reactor was used for batch anaerobic mono-digestion of secondary sludge (WAS) and for co-digestion of WAS and algal biomass. The methane yield using WAS from two different wine production seasons was 155.4 and 132.9 NL CH4 kg VS-1. Co-digestion led to the highest methane yield (225.8 NL CH4 kg VS-1). The application of the bio-wastes for fertilization was assessed through plant growth bioassays: mono- and co-digestion digestates and dry algal biomass enhanced plant biomass accumulation (growth indexes of 163%, 155% and 121% relative to those of the control - commercial amendment, respectively), demonstrating a lack of phytotoxicity.

Evaluation of an outdoor pilot-scale tubular photobioreactor for removal of selected pesticides from water.

This work assesses the capacity of a microalgae-based system to remove three highly to medium polar pesticides typically found in freshwater: acetamiprid, bentazone, and propanil. Degradation of the pesticides was firstly studied individually at batch lab-scale reactors and abiotic and heated-killed controls were employed to clarify their removal pathways. At lab-scale, propanil and acetamiprid were completely removed after 7 days whereas bentazone was not removed. Four and two transformation products (TPs) were generated in the biodegradation process for acetamiprid and propanil, respectively. Then, the simultaneous removal of the pesticides was assessed in an outdoor pilot photobioreactor, operated with a hydraulic residence time of 8 days. During the steady-state, high removal efficiencies were observed for propanil (99%) and acetamiprid (71%). The results from batch experiments suggest that removal is mainly caused by algal-mediated biodegradation. Acetamiprid TPs raised throughout the operational time in the photobioreactor, while no propanil TP was detected at the pilot-scale. This suggests complete mineralization of propanil or residual formation of its TPs at concentrations below the analytical method detection limit. Aiming at biomass valorization, diverse microalgae harvesting methods were investigated for biomass concentration, and the effect of residual pesticides on the biogas yield was determined by biochemical methane potential tests. Anaerobic digestion was not inhibited by the pesticides as verified by the digestion performance. The results highlight the potential of microalgae-based systems to couple nutrient removal, biomass production, micropollutant biodegradation, and biofuel production.

Trichloromethane dechlorination by a novel Dehalobacter sp. strain 8M reveals a third contrasting C and Cl isotope fractionation pattern within this genus.

Trichloromethane (TCM) is a pollutant frequently detected in contaminated aquifers, and only four bacterial strains are known to respire it. Here, we obtained a novel Dehalobacter strain capable of transforming TCM to dichloromethane, which was denominated Dehalobacter sp. strain 8M. Besides TCM, strain 8M also completely transformed 1,1,2-trichloroethane to vinyl chloride and 1,2-dichloroethane. Quantitative PCR analysis for the 16S rRNA genes confirmed growth of Dehalobacter with TCM and 1,1,2-trichloroethane as electron acceptors. Carbon and chlorine isotope fractionation during TCM transformation was studied in cultured cells and in enzymatic assays with cell suspensions and crude protein extracts. TCM transformation in the three studied systems resulted in small but significant carbon (εC = -2.7 ± 0.1‰ for respiring cells, -3.1 ± 0.1‰ for cell suspensions, and - 4.1 ± 0.5‰ for crude protein extracts) and chlorine (εCl = -0.9 ± 0.1‰, -1.1 ± 0.1‰, and - 1.2 ± 0.2‰, respectively) isotope fractionation. A characteristic and consistent dual CCl isotope fractionation pattern was observed for the three systems (combined ΛC/Cl = 2.8 ± 0.3). This ΛC/Cl differed significantly from previously reported values for anaerobic dechlorination of TCM by the corrinoid cofactor vitamin B12 and other Dehalobacter strains. These findings widen our knowledge on the existence of different enzyme binding mechanisms underlying TCM-dechlorination within the genus Dehalobacter and demonstrates that dual isotope analysis could be a feasible tool to differentiate TCM degraders at field studies.

Integration of enzymatic pretreatment and sludge co-digestion in biogas production from microalgae.

Integration of microalgae-based systems with conventional wastewater treatment plants provides an effective alternative to waste stream management. In this work, alkaline and enzymatic pretreatments of a microalgal culture mainly constituted by Chlorella sp. and Scenedesmus sp. and cultivated in wastewater from an industrial winery wastewater treatment plant were assessed. Microalgal enzymatic pretreatments were expected to overcome algal recalcitrancy before anaerobic digestion. pH-induced flocculation at pH 10 and 11 did not enhance microalgal harvesting and solubilisation, achieving a performance similar to that of natural sedimentation. Enzymatic hydrolysis of algal biomass was carried out using three commercial enzymatic cocktails (A, B and C) at two enzymatic doses (1% and 2% (v/v)) over 3 h of exposure time at 37 °C. Since pretreatments at a 1% dose for 0.5 h and 2% dose for 2 h achieved higher solubilisation, they were selected to evaluate the influence of the pretreatment on microalgal anaerobic digestibility. Biochemical methane potential tests showed that the pretreatments increased the methane production of the raw algal biomass 3.6- to 5.3-fold. The methane yield was 9-27% higher at the lower enzyme dose. Hence, microalgae pretreated with enzymes B and C at a 1% dose were co-digested with waste activated sludge (WAS). Even when the enzyme increased the methane yield of the inoculum and the WAS, the methane yield of the raw microalgae and WAS mixture was not significantly different from that obtained when algae were enzymatically pretreated. Nonetheless, co-digestion may achieve the goals of a waste recycled bio-circular economy.

Biodegradation of hydrophobic pesticides by microalgae: Transformation products and impact on algae biochemical methane potential.

Intensive and extensive use of pesticides has contributed to their wide distribution in soil, air, and water. Due to their detrimental effects on non-target organisms, different technologies have been considered for their removal. In this work, three hydrophobic pesticide active compounds, namely, chlorpyrifos, cypermethrin, and oxadiazon, were selected to study the potential for their removal from aqueous media by a microalgae consortium. An abiotic and a killed control (thermally inactivated dead microalgae biomass) were employed to clarify their removal pathways, and pesticide content was quantified in liquid and biomass phases for 7 days. At the final time, total degradation (biodegradation plus photodegradation) contributed to the removal of 55% of oxadiazon, 35% of chlorpyrifos, and 14% of cypermethrin. Furthermore, more than 60% of chlorpyrifos and cypermethrin were removed by sorption onto microalgae biomass. Overall, the three pesticides showed high removal from the liquid phase. O,O-diethyl thiophosphate was identified in the liquid phase as a transformation product of chlorpyrifos formed by microalgae degradation. Phycoremediation was coupled with anaerobic degradation of the microalgae biomass containing the retained pesticides by sorption through biochemical methane potential tests. Anaerobic digestion was not inhibited by the pesticides as verified by methane production yields. The removal efficiency of the pesticides in the digestate was as follows: chlorpyrifos > cypermethrin > oxadiazon. These results highlight the potential of low-cost algal-based systems for the treatment of wastewater or effluents from agrochemical industries. The integration of wastewater treatment with biogas production through anaerobic digestion is a biorefinery approach that facilitates the economic feasibility of the process.

Interspecies interaction and effect of co-contaminants in an anaerobic dichloromethane-degrading culture.

An anaerobic stable mixed culture dominated by bacteria belonging to the genera Dehalobacterium, Acetobacterium, Desulfovibrio, and Wolinella was used as a model to study the microbial interactions during DCM degradation. Physiological studies indicated that DCM was degraded in this mixed culture at least in a three-step process: i) fermentation of DCM to acetate and formate, ii) formate oxidation to CO2 and H2, and iii) H2/CO2 reductive acetogenesis. The 16S rRNA gene sequencing of cultures enriched with formate or H2 showed that Desulfovibrio was the dominant population followed by Acetobacterium, but sequences representing Dehalobacterium were only present in cultures amended with DCM. Nuclear magnetic resonance analyses confirmed that acetate produced from 13C-labelled DCM was marked at the methyl ([2-13C]acetate), carboxyl ([1-13C]acetate), and both ([1,2-13C]acetate) positions, which is in accordance to acetate formed by both direct DCM fermentation and H2/CO2 acetogenesis. The inhibitory effect of ten different co-contaminants frequently detected in groundwaters on DCM degradation was also investigated. Complete inhibition of DCM degradation was observed when chloroform, perfluorooctanesulfonic acid, and diuron were added at 838, 400, and 107 µM, respectively. However, the inhibited cultures recovered the DCM degradation capability when transferred to fresh medium without co-contaminants. Findings derived from this work are of significant relevance to provide a better understanding of the synergistic interactions among bacteria to accomplish DCM degradation as well as to predict the effect of co-contaminants during anaerobic DCM bioremediation in groundwater.

Integrative isotopic and molecular approach for the diagnosis and implementation of an efficient in-situ enhanced biological reductive dechlorination of chlorinated ethenes.

Based on the previously observed intrinsic bioremediation potential of a site originally contaminated with perchloroethene (PCE), field-derived lactate-amended microcosms were performed to test different lactate isomers and concentrations, and find clearer isotopic and molecular parameters proving the feasibility of an in-situ enhanced reductive dechlorination (ERD) from PCE-to-ethene (ETH). According to these laboratory results, which confirmed the presence of Dehalococcoides sp. and the vcrA gene, an in-situ ERD pilot test consisting of a single injection of lactate in a monitoring well was performed and monitored for 190 days. The parameters used to follow the performance of the ERD comprised the analysis of i) hydrochemistry, including redox potential (Eh), and the concentrations of redox sensitive species, chlorinated ethenes (CEs), lactate, and acetate; ii) stable isotope composition of carbon of CEs, and sulphur and oxygen of sulphate; and iii) 16S rRNA gene sequencing from groundwater samples. Thus, it was proved that the injection of lactate promoted sulphate-reducing conditions, with the subsequent decrease in Eh, which allowed for the full reductive dechlorination of PCE to ETH in the injection well. The biodegradation of CEs was also confirmed by the enrichment in 13C and carbon isotopic mass balances. The metagenomic results evidenced the shift in the composition of the microbial population towards the predominance of fermentative bacteria. Given the success of the in-situ pilot test, a full-scale ERD with lactate was then implemented at the site. After one year of treatment, PCE and trichloroethene were mostly depleted, whereas vinyl chloride (VC) and ETH were the predominant metabolites. Most importantly, the shift of the carbon isotopic mass balances towards more positive values confirmed the complete reductive dechlorination, including the VC-to-ETH reaction step. The combination of techniques used here provides complementary lines of evidence for the diagnosis of the intrinsic biodegradation potential of a polluted site, but also to monitor the progress, identify potential difficulties, and evaluate the success of ERD at the field scale.

Promoting circular economy in the surroundings of an organic fraction of municipal solid waste anaerobic digestion treatment plant: Biogas production impact and economic factors.

The Anaerobic Digestion and Composting Plant of the Vallès Oriental Waste Treatment Centre processes source-selected organic fraction of municipal solid wastes generated in its surrounding area. To promote Circular Economy between Municipal Solid Waste and industrial waste management systems, the Treatment Centre is looking for complementary wastes to be valorised through co-digestion with its main substrate. The study includes waste characterization and a complete treatment cost analysis, that jointly with the biogas potential and the mass balance of the Plant allows to calculate the price of each waste to be treated in the Plant. Up to 13 industrial wastes have been characterised for its biogas potential and its treatment cost calculated. Treatment prices ranged between 83 and 51 € t-1.

Multi-method assessment of the intrinsic biodegradation potential of an aquifer contaminated with chlorinated ethenes at an industrial area in Barcelona (Spain).

The bioremediation potential of an aquifer contaminated with tetrachloroethene (PCE) was assessed by combining hydrogeochemical data of the site, microcosm studies, metabolites concentrations, compound specific-stable carbon isotope analysis and the identification of selected reductive dechlorination biomarker genes. The characterization of the site through 10 monitoring wells evidenced that leaked PCE was transformed to TCE and cis-DCE via hydrogenolysis. Carbon isotopic mass balance of chlorinated ethenes pointed to two distinct sources of contamination and discarded relevant alternate degradation pathways in the aquifer. Application of specific-genus primers targeting Dehalococcoides mccartyi species and the vinyl chloride-to-ethene reductive dehalogenase vcrA indicated the presence of autochthonous bacteria capable of the complete dechlorination of PCE. The observed cis-DCE stall was consistent with the aquifer geochemistry (positive redox potentials; presence of dissolved oxygen, nitrate, and sulphate; absence of ferrous iron), which was thermodynamically favourable to dechlorinate highly chlorinated ethenes but required lower redox potentials to evolve beyond cis-DCE to the innocuous end product ethene. Accordingly, the addition of lactate or a mixture of ethanol plus methanol as electron donor sources in parallel field-derived anoxic microcosms accelerated dechlorination of PCE and passed cis-DCE up to ethene, unlike the controls (without amendments, representative of field natural attenuation). Lactate fermentation produced acetate at near-stoichiometric amounts. The array of techniques used in this study provided complementary lines of evidence to suggest that enhanced anaerobic bioremediation using lactate as electron donor source is a feasible strategy to successfully decontaminate this site.

An automated on-line turbulent flow liquid-chromatography technology coupled to a high resolution mass spectrometer LTQ-Orbitrap for suspect screening of antibiotic transformation products during microalgae wastewater treatment.

The evaluation of wastewater treatment capabilities in terms of removal of water pollutants is crucial when assessing water mitigation issues. Not only the monitoring of target pollutants becomes a critical point, but also the transformation products (TPs) generated. Since these TPs are very often unknown compounds, their study in both wastewater and natural environment is currently recognized as a tedious task and challenging research field. In this study, a novel automated suspect screening methodology was developed for a comprehensive assessment of the TPs generated from nine antibiotics during microalgae water treatment. Three macrolides (azithromycin, erythromycin, clarithromycin), three fluoroquinolones (ofloxacin, ciprofloxacin, norfloxacin) and three additional antibiotics (trimethoprim, pipemidic acid, sulfapyridine) were selected as target pollutants. The analysis of samples was carried out by direct injection in an on-line turbulent flow liquid chromatography-high resolution mass spectrometry (TFC-LC-LTQ-Orbitrap-MS/MS) system, followed by automatic data processing for compound identification. The screening methodology allowed the identification of 40 tentative TPs from a list of software predicted intermediates created automatically. Once known and unknown TPs were identified, degradation pathways were suggested considering the different mechanisms involved on their formation (biotic and abiotic). Results reveal microalgae ability for macrolide biotransformation, but not for other antibiotics such as for fluoroquinolones. Finally, the intermediates detected were included into an in-house library and applied to the identification of tentative TPs in real toilet wastewater treated in a microalgae based photobioreactor (PBR). The overall approach allowed a comprehensive overview of the performance of microalgae water treatment in a fast and reliable manner: it represents a useful tool for the rapid screening of wide range of compounds, reducing time invested in data analysis and providing reliable structural identification.

Effect of cultivation conditions on ß-estradiol removal in laboratory and pilot-plant photobioreactors by an algal-bacterial consortium treating urban wastewater.

The use of microalgal consortia for urban wastewater treatment is an increasing trend, as it allows simultaneous nutrient removal and biomass production. Emerging contaminants proposed for the list of priority substances such as the hormone 17ß-estradiol are commonly found in urban wastewater, and their removal using algal monocultures has been accomplished. Due to the inherent potential of algae-based systems, this study aimed to assess the capability of native photobioreactor biomass to remove 17ß-estradiol under indoor and outdoor conditions. At the same time, the microbial community changes in regular and bioaugmented operations with Scenedesmus were assessed. The results show that almost complete removal (>93.75%) of the hormone 17ß-estradiol can be attained in the system under favourable seasonal conditions, although these conditions greatly influence biomass concentrations and microbial diversity. Even under the harsh conditions of low temperatures and solar irradiation, the established consortium removed more than 50% of the pollutant in 24 h. While species from genus Chlorella were stable during the entire operation, the microbial diversity analysis revealed that assorted and evenly distributed populations stimulate the removal rates. Bioaugmentation assays proved that the input of additional biomass results in higher overall removal and decreases the yield per mg of biomass.

Fungal treatment for the removal of endocrine disrupting compounds from reverse osmosis concentrate: Identification and monitoring of transformation products of benzotriazoles.

The removal of 27 endocrine-disrupting compounds and related compounds (suspect effect) from a reverse osmosis concentrate using an alternative decontamination method based on a fungal treatment involving Trametes versicolor was assessed. In addition to chemical analysis, the toxicity of the treated water during the treatment was monitored using a bioluminescence inhibition test and estrogenic and anti-estrogenic tests. The compounds 1H-benzotriazole (BTZ) and two tolyltriazoles (TTZs), 4-methyl-1H-benzotriazole (4-MBTZ) and 5-methyl-1H-benzotriazole (5-MBTZ), were present in the reverse osmosis concentrate at the highest concentrations (7.4 and 12.8 µg L-1, respectively) and were partially removed by the fungal treatment under sterile conditions (58% for BTZ and 92% for TTZs) and non-sterile conditions, although to lesser extents (32% for BTZ and 50% for TTZs). Individual biotransformation studies of BTZ and the TTZs by T. versicolor in a synthetic medium and further analysis via on-line turbulent flow chromatography coupled to an HRMS-Orbitrap allowed the tentative identification of the transformation products (TPs). Six TPs were postulated for BTZ, two TPs were postulated for 4-MBTZ, and four TPs were postulated for 5-MBTZ. Most of these TPs are suggested to have been generated by conjugation with some sugars and via the methylation of the triazole group. Only TP 148 A, postulated to be derived from the biotransformation of BTZ, was observed in the effluent of the bioreactor treating the reverse osmosis concentrate.

Ciprofloxacin removal during secondary domestic wastewater treatment in high rate algal ponds.

This study investigated the removal of antibiotic ciprofloxacin during the treatment of real wastewater using high rate algal ponds (HRAP). When spiked at 2 mg/L into primary domestic wastewater, ciprofloxacin (CPX) was efficiently removed from laboratory scale photobioreactors continuously operated under various durations of artificial illumination and hydraulic residence times. Subsequent batch tests conducted with reactor microcosms showed CPX removal was mainly caused by photodegradation during daytime, and sorption to biomass during night time. These findings were confirmed during an experiment conducted in a 1000 L pilot HRAP operated outdoors, as well as during outdoor batch assays conducted using pilot HRAP microcosms. While these results highlight a potentially interesting treatment capacity in comparison to conventional biological treatment, further research must confirm these findings at relevant pollutant concentration (ng-µg/L) and determine the fate and potential toxicity of degradation products.

Performance of a microalgal photobioreactor treating toilet wastewater: Pharmaceutically active compound removal and biomass harvesting.

In this study, a 1200L outdoor pilot scale microalgal photobioreactor (PBR) was used for toilet wastewater (WW) treatment and evaluate its ability to remove pharmaceutically active compounds (PhACs). The PBR was operated at two different hydraulic retention times (HRTs), which were 8 and 12days, during Period I (September-October) and Period II (October-December), respectively. Algal biomass concentrations varied by operating period because of seasonal changes. Nutrients (ammonia, nitrogen and total phosphorous) and chemical oxygen demand (COD) were monitored and efficiently removed in both periods (>80%), attaining the legislation limits. At the theoretical hydraulic steady state in both periods, pharmaceutical removal reached high levels (>48%). Two harvesting techniques were applied to the PBR microalgae effluent. Gravity sedimentation was efficient for biomass removal (>99% in 7min) in Period I when large particles, flocs and aggregates were present. In contrast, a longer sedimentation time was required when biomass was mainly composed of single cells (88% clarification in a 24h in Period II). The second harvesting technique investigated was the co-pelletization of algal biomass with the ligninolytic fungus Trametes versicolor, attaining >98% clarification for Period II biomass once pellets were formed. The novel technology of co-pelletization enabled the complete harvesting of single algae cells from the liquid medium in a sustainable way, which benefits the subsequent use of both biomass and the clarified effluent.

Detoxification of 1,1,2-trichloroethane to ethene in a bioreactor co-culture of Dehalogenimonas and Dehalococcoides mccartyi strains.

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 76d 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.

Pretreatment of vinasse from the sugar refinery industry under non-sterile conditions by Trametes versicolor in a fluidized bed bioreactor and its effect when coupled to an UASB reactor.

BACKGROUND: During hydrous ethanol production from the sugar refinery industry in Mexico, vinasse is generated. Phenolic compounds and melanoidins contribute to its color and make degradation of the vinasse a difficult task. Although anaerobic digestion (AD) is feasible for vinasse treatment, the presence of recalcitrant compounds can be toxic or inhibitory for anaerobic microorganism. Therefore, this study presents new data on the coupled of the FBR (Fluidized Bed Bioreactor) to the UASB (Upflow Anaerobic Sludge Blanket) reactor under non-sterile conditions by T. versicolor. Nevertheless, for an industrial application, it is necessary to evaluate the performance in this kind of proposal system. RESULTS: Therefore, this study used a FBR for the removal of phenolic compounds (67%) and COD (38%) at non-sterile conditions. Continuous operation of the FBR was successfully for 26 days according to the literature. When the FBR was coupled to the UASB reactor, we obtained a better quality of effluent, furthermore methane content and yield were 74% and 0.18 m3 CH4/ kg CODremoval respectively. CONCLUSIONS: This study demonstrated the possibility of using for an industrial application the coupled of the FBR to the UASB reactor under non-sterile conditions. Continuous operation of the FBR was carried out successfully for 26 days, which is the highest value found in the literature.

Molecular and carbon isotopic characterization of an anaerobic stable enrichment culture containing Dehalobacterium sp. during dichloromethane fermentation.

Biodegradation of dichloromethane (DCM) under reducing conditions is of major concern due to its widespread detection in contaminated groundwaters. Here, we report an anaerobic enrichment culture derived from a membrane bioreactor operating in an industrial wastewater treatment plant, capable of fermenting DCM and the brominated analogue dibromomethane (DBM). Comparative analysis of bacterial 16S rDNA-DGGE profiles from fresh liquid medium inoculated with single colonies picked from serial dilution-to-extinction agar vials showed that cultures degrading DCM contained a predominant band belonging to Dehalobacterium, however this band was absent in cultures unable to degrade DCM. Analysis of the microbial composition of the enrichment by bacterial 16S rRNA gene amplicon paired-end sequencing confirmed the presence of Dehalobacterium together with three additional phylotypes belonging to Acetobacterium, Desulfovibrio, and Wolinella, representing all four operational taxonomic units >99.9% of the retrieved sequences. The carbon isotopic fractionation (ε) determined for DCM degradation in this culture was -27±2‰. This value differs from the ε previously reported for the DCM-fermentative bacteria Dehalobacter (-15.5±1.5‰) but they are both significantly different from those reported for facultative methylotrophic organisms (ranging from -45 to -61‰). This significant difference in the ε allows differentiating between hydrolytic transformation of DCM via glutathione-dependent dehalogenases and fermentation pathway. CAPSULE: The carbon isotopic fractionation of dichloromethane by an enriched Dehalobacterium-containing culture has significant potential to monitor biodegradation of DCM in groundwaters.