New concepts in anaerobic digestion processes: recent advances and biological aspects.

Waste treatment and the simultaneous production of energy have gained great interest in the world. In the last decades, scientific efforts have focused largely on improving and developing sustainable bioprocess solutions for energy recovery from challenging waste. Anaerobic digestion (AD) has been developed as a low-cost organic waste treatment technology with a simple setup and relatively limited investment and operating costs. Different technologies such as one-stage and two-stage AD have been developed. The viability and performance of these technologies have been extensively reported, showing the supremacy of two-stage AD in terms of overall energy recovery from biomass under different substrates, temperatures, and pH conditions. However, a comprehensive review of the advantages and disadvantages of these technologies is still lacking. Since microbial ecology is critical to developing successful AD, many studies have shown the structure and dynamics of archaeal and bacterial communities in this type of system. However, the role of Eukarya groups remains largely unknown to date. In this review, we provide a comprehensive review of the role, abundance, dynamics, and structure of archaeal, bacterial, and eukaryal communities during the AD process. The information provided could help researchers to select the adequate operational parameters to obtain the best performance and biogas production results.

Degradation of bisphenol A and acute toxicity reduction by different thermo-tolerant ascomycete strains isolated from arid soils.

Four different laccase-producing strains were isolated from arid soils and used for bisphenol A (BPA) degradation. These strains were identified as Chaetomium strumarium G5I, Thielavia arenaria CH9, Thielavia arenaria HJ22 and Thielavia arenaria SM1(III) by internal transcribed spacer 5.8 S rDNA analysis. Residual BPA was evaluated by HPLC analysis during 48 h of incubation. A complete removal of BPA was observed by the whole cell fungal cultures within different times, depending on each strain. C. strumarium G5I was the most efficient degrader, showing 100% of removal within 8 h of incubation. The degradation of BPA was accompanied by the production of laccase and dye decolorizing peroxidase (DyP) under degradation conditions. The presence of aminobenzotriazole (ABT) as an inhibitor of cytochrome P450s monooxygenases (CYP) demonstrated a slight decrease in BPA removal rate, suggesting the effective contribution of CYP in the conversion. The great involvement of laccase in BPA transformation together with cell-associated enzymes, such as CYP, was supported by the identification of hydroxylated metabolites by ultra-high performance liquid chromatography-mass spectroscopy (UHPLC-MS). The metabolic pathway of BPA transformation was proposed based on the detected metabolites. The acute toxicity of BPA and its products was investigated and showed a significant reduction, except for T. arenaria SM1(III) that did not caused reduction of toxicity (IC50 < 8%), possibly due to the presence of toxic metabolites. The results of the present study point out the potential application of the isolated ascomycetes in pollutant removal processes, especially C. strumarium G5I as an efficient degrader of BPA.

Paenibacillus etheri sp. nov., able to grow on media supplemented with methyl tert-butyl ether (MTBE) and isolated from hydrocarbon-contaminated soil.

A bacterial strain, designated SH7T, was isolated from the hydrocarbon-contaminated soil of a pilot plant (Granada, Spain). The strain was selected for its capacity to grow in media supplemented with methyl tert-butyl ether (MTBE) as sole energy and carbon source. Strain SH7T was a Gram-stain-positive, facultatively anaerobic, spore-forming, rod-shaped bacterium. Phylogenetic analysis using 16S rRNA gene sequences showed that strain SH7T belongs to a cluster comprising species of the genus Paenibacillus and was closely related to Paenibacillus borealis KK19T (97 % 16S rRNA gene sequence similarity) and Paenibacillus odorifer TOD45T (98 %). DNA-DNA hybridization tests showed low relatedness of strain SH7T with the type strains of Paenibacillus borealis (16.9 ± 1.5 %) and Paenibacillus odorifer (16.6 ± 2.1 %). The cell wall of strain SH7T contained meso-diaminopimelic acid. The predominant respiratory quinone was MK-7, and anteiso-C15 : 0 (32.9 %) and C16 : 0 (29.0 %) were the predominant cellular fatty acids. Phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and three unknown aminophospholipids were the major phospholipids. The DNA G+C content was 44.3 mol%. Data obtained in this study indicate that strain SH7T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus etheri sp. nov. is proposed. The type strain is SH7T ( = CECT 8558T = DSM 29760T).

Massilia umbonata sp. nov., able to accumulate poly-ß-hydroxybutyrate, isolated from a sewage sludge compost-soil microcosm.

A bacterial strain, designated strain LP01(T), was isolated from a laboratory-scale microcosm packed with a mixture of soil and sewage sludge compost designed to study the evolution of microbial biodiversity over time. The bacterial strain was selected for its potential ability to store polyhydroxyalkanoates (PHAs) as intracellular granules. The cells were aerobic, Gram-stain-negative, non-endospore-forming motile rods. Phylogenetically, the strain was classified within the genus Massilia, as its 16S rRNA gene sequence had similarity of 99.2 % with respect to those of Massilia albidiflava DSM 17472(T) and M. lutea DSM 17473(T). DNA-DNA hybridization showed low relatedness of strain LP01(T) to the type strains of other, phylogenetically related species of the genus Massilia. It contained Q-8 as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) as the major fatty acid(s). It was found to contain small amounts of the fatty acids C18 : 0 and C14 : 0 2-OH, a feature that served to distinguish it from its closest phylogenetic relatives within the genus Massilia. The DNA G+C content was 66.0 mol%. Phylogenetic, phenotypic and chemotaxonomic data obtained in this study suggest that strain LP01(T) represents a novel species of the genus Massilia, for which the name Massilia umbonata sp. nov. is proposed. The type strain is LP01(T) ( = CECT 7753(T) = DSM 26121(T)).

Constitutive expression of the cytochrome P450 EthABCD monooxygenase system enables degradation of synthetic dialkyl ethers in Aquincola tertiaricarbonis L108.

In Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005, and Gordonia sp. strain IFP 2009, the cytochrome P450 monooxygenase EthABCD catalyzes hydroxylation of methoxy and ethoxy residues in the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). The expression of the IS3-type transposase-flanked eth genes is ETBE dependent and controlled by the regulator EthR (C. Malandain et al., FEMS Microbiol. Ecol. 72:289-296, 2010). In contrast, we demonstrated by reverse transcription-quantitative PCR (RT-qPCR) that the betaproteobacterium Aquincola tertiaricarbonis L108, which possesses the ethABCD genes but lacks ethR, constitutively expresses the P450 system at high levels even when growing on nonether substrates, such as glucose. The mutant strain A. tertiaricarbonis L10, which is unable to degrade dialkyl ethers, resulted from a transposition event mediated by a rolling-circle IS91-type element flanking the eth gene cluster in the wild-type strain L108. The constitutive expression of Eth monooxygenase is likely initiated by the housekeeping sigma factor σ(70), as indicated by the presence in strain L108 of characteristic -10 and -35 binding sites upstream of ethA which are lacking in strain IFP 2001. This enables efficient degradation of diethyl ether, diisopropyl ether, MTBE, ETBE, TAME, and tert-amyl ethyl ether (TAEE) without any lag phase in strain L108. However, ethers with larger residues, n-hexyl methyl ether, tetrahydrofuran, and alkyl aryl ethers, were not attacked by the Eth system at significant rates in resting-cell experiments, indicating that the residue in the ether molecule which is not hydroxylated also contributes to the determination of substrate specificity.

Simultaneous Heavy Metal-Polycyclic Aromatic Hydrocarbon Removal by Native Tunisian Fungal Species.

Multi-contamination by organic pollutants and toxic metals is common in anthropogenic and industrial environments. In this study, the five fungal strains Chaetomium jodhpurense (MH667651.1), Chaetomium maderasense (MH665977.1), Paraconiothyrium variabile (MH667653.1), Emmia lacerata, and Phoma betae (MH667655.1), previously isolated in Tunisia, were investigated for the simultaneous removal and detoxification of phenanthrene (PHE) and benzo[a]anthracene (BAA), as well as heavy metals (HMs) (Cu, Zn, Pb and Ag) in Kirk's media. The removal was analysed using HPLC, ultra-high performance liquid chromatography (UHPLC) coupled to a QToF mass spectrometer, transmission electron microscopy, and toxicology was assessed using phytotoxicity (Lepidium sativum seeds) and Microtox® (Allivibrio fisherii) assays. The PHE and BAA degradation rates, in free HMs cultures, reached 78.8% and 70.7%, respectively. However, the addition of HMs considerably affected the BAA degradation rate. The highest degradation rates were associated with the significant production of manganese-peroxidase, lignin peroxidase, and unspecific peroxygenase. The Zn and Cu removal efficacy was considerably higher with live cells than dead cells. Transmission electron microscopy confirmed the involvement of both bioaccumulation and biosorption processes in fungal HM removal. The environmental toxicological assays proved that simultaneous PAH and HM removal was accompanied by detoxification. The metabolites produced during co-treatment were not toxic for plant tissues, and the acute toxicity was reduced. The obtained results indicate that the tested fungi can be applied in the remediation of sites simultaneously contaminated with PAHs and HMs.

Assembled mixed co-cultures for emerging pollutant removal using native microorganisms from sewage sludge.

The global pharmaceutical pollution caused by drug consumption (>100,000 tonnes) and its disposal into the environment is an issue which is currently being addressed by bioremediation techniques, using single or multiple microorganisms. Nevertheless, the low efficiency and the selection of non-compatible species interfere with the success of this methodology. This paper proposes a novel way of obtaining an effective multi-domain co-culture, with the capacity to degrade multi-pharmaceutical compounds simultaneously. To this end, seven microorganisms (fungi and bacteria) previously isolated from sewage sludge were investigated to enhance their degradation performance. All seven strains were factorially mixed and used to assemble different artificial co-cultures. Consequently, 127 artificial co-cultures were established and ranked, based on their fitness performance, by using the BSocial analysis web tool. The individual strains were categorized according to their social behaviour, whose net effect over the remaining strains was defined as 'Positive', 'Negative' or 'Neutral'. To evaluate the emerging-pollutant degradation rate, the best 10 co-cultures, and those which contained the social strains were then challenged with three different Pharmaceutical Active compounds (PhACs): diclofenac, carbamazepine and ketoprofen. The co-cultures with the fungi Penicillium oxalicum XD-3.1 and Penicillium rastrickii were able to degrade PhACs. However, the highest performance (>80% degradation) was obtained by the minimal active microbial consortia consisting of both Penicillium spp., Cladosporium cladosporoides and co-existing bacteria. These consortia transformed the PhACs to derivate molecules through hydroxylation and were released to the media, resulting in a low ecotoxicity effect. High-throughput screening of co-cultures provides a quick, reliable and efficient method to narrow down suitable degradation co-cultures for emerging PhAC contaminants while avoiding toxic metabolic derivatives.

High-Throughput Microbial Community Analyses to Establish a Natural Fungal and Bacterial Consortium from Sewage Sludge Enriched with Three Pharmaceutical Compounds.

Emerging and unregulated contaminants end up in soils via stabilized/composted sewage sludges, paired with possible risks associated with the development of microbial resistance to antimicrobial agents or an imbalance in the microbial communities. An enrichment experiment was performed, fortifying the sewage sludge with carbamazepine, ketoprofen and diclofenac as model compounds, with the aim to obtain strains with the capability to transform these pollutants. Culturable microorganisms were obtained at the end of the experiment. Among fungi, Cladosporium cladosporioides, Alternaria alternata and Penicillium raistrickii showed remarkable degradation rates. Population shifts in bacterial and fungal communities were also studied during the selective pressure using Illumina MiSeq. These analyses showed a predominance of Ascomycota (Dothideomycetes and Aspergillaceae) and Actinobacteria and Proteobacteria, suggesting the possibility of selecting native microorganisms to carry out bioremediation processes using tailored techniques.

Comparative analysis of microbial DNA extraction protocols for groundwater samples.

A comparative analysis of four different DNA extraction protocols was performed to determine the best choice for groundwater microbial diversity studies using temperature gradient gel electrophoresis (TGGE) analysis. The methods used were a chelex-based method, a modified salting out procedure (MSOP), and the commercial kits Epicentre and FastDNA. Both commercial kits exhibited the greatest reproducibility in their methods; however, their band patterns were very different. The protocol that showed the highest diversity was the chelex-based method, and the one that showed the lowest diversity was the FastDNA kit.

Biological treatment of two-phase olive mill wastewater (TPOMW, alpeorujo): polyhydroxyalkanoates (PHAs) production by Azotobacter strains.

Azotobacter chroococcum H23 (CECT 4435), Azotobacter vinelandii UWD, and Azotobacter vinelandii (ATCC 12837), members of the family Pseudomonadaceae, were used to evaluate their capacity to grow and accumulate polyhydroxyalkanoates (PHAs) using two-phase olive mill wastewater (TPOMW, alpeorujo) diluted at different concentrations as the sole carbon source. The PHAs amounts (g/l) increased clearly when the TPOMW samples were previously digested under anaerobic conditions. The MNR analysis demonstrated that the bacterial strains formed only homopolymers containing beta-hydroxybutyrate, either when grown in diluted TPOMW medium or diluted anaerobically digested TPOMW medium. COD values of the diluted anaerobically digested waste were measured before and after the aerobic PHA-storing phase, and a clear reduction (72%) was recorded after 72 h of incubation. The results obtained in this study suggest the perspectives for using these bacterial strains to produce PHAs from TPOMW, and in parallel, contribute efficiently to the bioremediation of this waste. This fact seems essential if bioplastics are to become competitive products.

Biodegradation and toxicity reduction of nonylphenol, 4-tert-octylphenol and 2,4-dichlorophenol by the ascomycetous fungus Thielavia sp HJ22: Identification of fungal metabolites and proposal of a putative pathway.

Research on the biodegradation of emerging pollutants is gained great focus regarding their detrimental effects on the environment and humans. The objective of the present study was to evaluate the ability of the ascomycetes Thielavia sp HJ22 to remove the phenolic xenobiotics nonylphenol (NP), 4-tert-octylphenol (4-tert-OP) and 2,4-dichlorophenol (2,4-DCP). The strain showed efficient degradation of NP and 4-tert-OP with 95% and 100% removal within 8 h of incubation, respectively. A removal rate of 80% was observed with 2,4-DCP within the same time. Under experimental conditions, the degradation of the tested pollutants concomitantly increased with the laccase production and cytochrome P450 monooxygenases inhibition. This study showed the involvement of laccase in pollutants removal together with biosorption mechanisms. Additionally, results demonstrated the participation of cytochrome P450 monooxygenase in the elimination of 2,4-DCP. Liquid chromatography-mass spectrometry analysis revealed several intermediates, mainly hydroxylated and oxidized compounds with less harmful effects compared to the parent compounds. A decrease in the toxicity of the identified metabolites was observed using Aliivibrio fischeri as bioindicator. The metabolic pathways of degradation were proposed based on the identified metabolites. The results point out the potential of Thielavia strains in the degradation and detoxification of phenolic xenobiotics.

Social microbial inocula confer functional stability in a methyl tert-butyl ether extractive membrane biofilm bioreactor.

Methyl tert-butyl ether (MTBE) degradation technologies based on two-phase partitioning systems such as extractive membrane biofilm reactors (EMBFR) permit separation of biological and contaminant compartments, thus allowing optimization of the biological section. In this study, we set-up an EMBFR with three MTBE-degrading and cooperating strains (termed social biofilm: Agrobacterium sp. MS2, Paenibacillus etheri SH7T and Rhodococcus ruber EE6). The removal efficiency of the social-biofilm EMBFR was 80%, and functional stability was observed in the reactor, i.e. more efficient than previous studies (single-strain inoculated EMBFR, <50% removal efficiency and unstable function). Metabolite tert-butyl alcohol was not observed, and the EC50 values were higher than those observed in single-strain EMBFRs. Comparative analysis of the MTBE enzymatic pathway and the social-biofilm was performed, where the mechanism of cooperation observed within the social-biofilm is likely due to enzymatic redundancy. Functional outcomes were equal to previous batch tests, hence 100% scalability was obtained. Overall, higher functional and stability outcomes are obtained with the use of the social-biofilm in an MTBE-EMBFR.

Evaluation of diclofenac biodegradation by the ascomycete fungus Penicillium oxalicum at flask and bench bioreactor scales.

Diclofenac (DFC) is a common anti-inflammatory drug, and has attracted the significant attention due to its massive use around the world and its environmental impact. In this work, we describe for the first time the use of Penicillium oxalicum, an ascomycetes fungus, for the biotransformation of DFC at flask and bench bioreactor scales. We present a complete study of the role of enzymes, metabolic pathway, acute toxicity assays and comparison between free and immobilised biomass. Pellets of P. oxalicum degraded 100 µM of DFC within 24 h, and the activity of CYP450 enzymes was key for the elimination of the drug. The scaling-up to bench bioreactor was optimised by the reduction of nutrients, and characterising the actions of free pellets, polyurethane foam- and plastic K1-immobilised biomass revealed free pellets to be the most efficient DFC removal system (total elimination occurred in 36 h). Hydroxylated metabolites were detected during the process, suggesting that a mixture of biological and physical processes were involved in the elimination of DFC. The use of P. oxalicum reduced the acute toxicity of the medium supplemented with diclofenac and represents a novel and attractive alternative for the elimination of pharmaceutical compounds.

Selection and identification of bacterial strains with methyl-tert-butyl ether, ethyl-tert-butyl ether, and tert-amyl methyl ether degrading capacities.

Nine bacterial strains isolated from two hydrocarbon-contaminated soils were selected because of their capacity for growth in culture media amended with 200 mg/L of one of the following gasoline oxygenates: Methyl-tert-butyl ether (MTBE), ethyl-tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). These strains were identified by amplification of their 16S rRNA gene, using fDl and rD1 primers, and were tested for their capacity to grow and biotransform these oxygenates in both mineral and cometabolic media. The isolates were classified as Bacillus simplex, Bacillus drentensis, Arthrobacter sp., Acinetobacter calcoaceticus, Acinetobacter sp., Gordonia amicalis (two strains), Nocardioides sp., and Rhodococcus ruber. Arthrobacter sp. (strain MG) and A. calcoaceticus (strain M10) consumed 100 (cometabolic medium) and 82 mg/L (mineral medium) of oxygenate TAME in 21 d, respectively, under aerobic conditions. Rhodococcus ruber (strain E10) was observed to use MTBE and ETBE as the sole carbon and energy source, whereas G. amicalis (strain T3) used TAME as the sole carbon and energy source for growth. All the bacterial strains transformed oxygenates better in the presence of an alternative carbon source (ethanol) with the exception of A. calcoaceticus (strain M10). The capacity of the selected strains to remove MTBE, ETBE, and TAME looks promising for application in bioremediation technologies.

Application of microbial fuel cell technology for wastewater treatment and electricity generation under Nordic countries climate conditions: Study of performance and microbial communities.

Two microbial fuel cells were inoculated with activated sludge from Finland and operated under moderate (25 °C) and low (8 °C) temperatures. Operation under real urban wastewater showed similarities in chemical oxygen demand removal and voltage generated, although moderate temperature supported higher ammonium oxidation. Fungi disappeared in the microbial fuel cell operated at temperature of 25 °C. Archaea domain was dominated by methanogenic archaea at both temperature scenarios. Important differences were observed in bacterial communities between both temperatures, however generating similar voltage. The results supported that the implementation of microbial fuel cells in Nordic countries operating under real conditions could be successful, as well as suggested the flexibility of cold-adapted inoculum for starting-up microbial fuel cells, regardless of the operating temperature of the system, obtaining higher COD removal and voltage generation performances at low temperature than at moderate temperature.

Removal of organic load from olive washing water by an aerated submerged biofilter and profiling of the bacterial community involved in the process.

The present work aims to use a biofilter technology (aerated submerged filters) for the aerobic transformation at laboratory-scale of olive washing water (OWW) generated in the first steps of olive oil processing, as well as the genetic profiling and identification to the species level of the bacteria involved in the formation of the biofilm, by means of TGGE. Chemical parameters, such as biological oxygen demand at five days (BOD5) and chemical oxygen demand (COD), decreased markedly (up to 90 and 85%, respectively) by the biological treatment, and the efficiency of the process was significantly affected by aeration and inlet flow rates. The total polyphenol content of inlet OWW was only moderately reduced (around 50% decrease of the inlet content) after the biofilter treatment, under the conditions tested. Partial 16S rRNA genes were amplified using total DNA extracted from the biofilm and separated by TGGE. Sequences of isolated bands were mostly affiliated to the alpha-subclass of Proteobacteria, and often branched in the periphery of bacterial genera commonly present in soil (Rhizobium, Reichenowia, Agrobacterium, and Sphingomonas). The data obtained by the experimentation at laboratory scale provided results that support the suitability of the submerged filter technology for the treatment of olive washing waters with the purpose of its reutilization.

BSocial: Deciphering Social Behaviors within Mixed Microbial Populations.

Ecosystem functionality depends on interactions among populations, of the same or different taxa, and these are not just the sum of pairwise interactions. Thus, know-how of the social interactions occurring in mixed-populations are of high interest, however they are commonly unknown due to the limitations posed in tagging each population. The limitations include costs/time in tediously fluorescent tagging, and the number of different fluorescent tags. Tag-free strategies exist, such as high-throughput sequencing, but ultimately both strategies require the use of expensive machinery. Our work appoints social behaviors on individual strains in mixed-populations, offering a web-tool (BSocial http://m4m.ugr.es/BSocial.html) for analyzing the community framework. Our quick and cheap approach includes the periodic monitoring of optical density (OD) from a full combinatorial testing of individual strains, where number of generations and growth rate are determined. The BSocial analyses then enable us to determine how the addition/absence of a particular species affects the net productivity of a microbial community and use this to select productive combinations, i.e., designate their social effect on a general community. Positive, neutral, or negative assignations are applied to describe the social behavior within the community by comparing fitness effects of the community against the individual strain. The usefulness of this tool for selection of optimal inoculum in biofilm-based methyl tert-butyl ether (MTBE) bioremediation was demonstrated. The studied model uses seven bacterial strains with diverse MTBE degradation/growth capacities. Full combinatorial testing of seven individual strains (triplicate tests of 127 combinations) were implemented, along with MTBE degradation as the desired function. Sole observation of highest species fitness did not render the best functional outcome, and only when strains with positive and neutral social assignations were mixed (Rhodococcus ruber EE6, Agrobacterium sp. MS2 and Paenibacillus etheri SH7), was this obtained. Furthermore, the use of positive and neutral strains in all its combinations had a significant higher degradation mean (x1.75) than exclusive negative strain combinations. Thus, social microbial processes benefit bioremediation more than negative social microbial combinations. The BSocial webtool is a great contributor to the study of social interactions in bioremediation processes, and may be used in other natural or synthetic habitat studies.

Draft Genome Sequence of Paenibacillus etheri sp. nov. SH7T, a Methyl Tert-Butyl Ether Degrader.

We report here the draft genome sequence of Paenibacillus etheri sp. nov. SH7(T) (= CECT 8558(T) = DSM 29760(T)), isolated from a hydrocarbon-contaminated soil pilot plant in Granada, Spain. The bacterium was isolated and sequenced due to its methyl tert-butyl ether (MTBE)-degrading properties.

Growth and nitrite and nitrous oxide accumulation of Paracoccus denitrificans ATCC 19367 in the presence of selected pesticides.

The effects of the application of eight pesticides (aldrin, lindane, dimetoate, methylparathion, methidation, atrazine, simazine, and captan) on growth, respiratory activity (as CO2 production), denitrifying activity (as N2O released), and nitrite accumulation in the culture medium by Paracoccus denitrificans strain ATCC 19367 were studied. The fungicide captan totally inhibited growth and biological activity of P. denitrificans, while the rest of the tested pesticides delayed the growth and CO2 release of P. denitrificans but did not drastically affect the bacterial growth or respiratory capacity after 96 h of culture. The denitrifying activity of P. denitrificans ATCC 19367 (as N2O released) was negatively affected by all tested pesticides. The release of N2O was strongly inhibited by several organochlorinated and organophosphorated insecticides (aldrin, lindane, dimetoate, and methidation), which led to high accumulation of nitrite in the surrounding medium. Atrazine decreased N2O release after 48 h of culture because of negative effects on growth, and methylparathion and simazine delayed the onset of N2O release by P. denitrificans. These three pesticides reduced the accumulation of NO2- compared to unamended control cultures.

Biofilm formation and microbial activity in a biofilter system in the presence of MTBE, ETBE and TAME.

Emerging water contaminants derived from unleaded gasoline such as methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), are in need of effective bioremediation technologies for restoring water resources. In order to design the conditions of a future groundwater bioremediating biofilter, this work assesses the potential use of Acinetobacter calcoaceticus M10, Rhodococcus ruber E10 and Gordonia amicalis T3 for the removal of MTBE, ETBE and TAME in consortia or as individual strains. Biofilm formation on an inert polyethylene support material was assessed with scanning electron microscopy, and consortia were also analysed with fluorescent in situ hybridisation to examine the relation between the strains. A. calcoaceticus M10 was the best coloniser, followed by G. amicalis T3, however, biofilm formation of pair consortia favoured consortium M10-E10 both in formation and activity. However, degradation batch studies determined that neither consortium exhibited higher degradation than individual strain degradation. The physiological state of the three strains was also determined through flow cytometry using propidium iodide and 3'-dihexylocarbocyanine iodide thus gathering information on their viability and activity with the three oxygenates since previous microbial counts revealed slow growth. Strain E10 was observed to have the highest physiological activity in the presence of MTBE, and strain M10 activity with TAME was only maintained for 24 h, thus we believe that biotransformation of MTBE occurs within the active periods established by the cytometry analyses. Viable cell counts and oxygenate removal were determined in the presence of the metabolites tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA), resulting in TBA biotransformation by M10 and E10, and TAA by M10. Our results show that A. calcoaceticus M10 and the consortium M10-E10 could be adequate inocula in MTBE and TAME bioremediating technologies.