Production of Current by Syntrophy Between Exoelectrogenic and Fermentative Hyperthermophilic Microorganisms in Heterotrophic Biofilm from a Deep-Sea Hydrothermal Chimney.

To study the role of exoelectrogens within the trophic network of deep-sea hydrothermal vents, we performed successive subcultures of a hyperthermophilic community from a hydrothermal chimney sample on a mix of electron donors in a microbial fuel cell system. Electrode (the electron acceptor) was swapped every week to enable fresh development from spent media as inoculum. The MFC at 80 °C yielded maximum current production increasing from 159 to 247 mA m-2 over the subcultures. The experiments demonstrated direct production of electric current from acetate, pyruvate, and H2 and indirect production from yeast extract and peptone through the production of H2 and acetate from fermentation. The microorganisms found in on-electrode communities were mainly affiliated to exoelectrogenic Archaeoglobales and Thermococcales species, whereas in liquid media, the communities were mainly affiliated to fermentative Bacillales and Thermococcales species. The work shows interactions between fermentative microorganisms degrading complex organic matter into fermentation products that are then used by exoelectrogenic microorganisms oxidizing these reduced compounds while respiring on a conductive support. The results confirmed that with carbon cycling, the syntrophic relations between fermentative microorganisms and exoelectrogens could enable some microbes to survive as biofilm in extremely unstable conditions. Graphical Abstract Schematic representation of cross-feeding between fermentative and exoelectrogenic microbes on the surface of the conductive support. B, Bacillus/Geobacillus spp.; Tc, Thermococcales; Gg, Geoglobus spp.; Py, pyruvate; Ac, acetate.

Biohydrogen production from hyperthermophilic anaerobic digestion of fruit and vegetable wastes in seawater: Simplification of the culture medium of Thermotoga maritima.

Biohydrogen production by the hyperthermophilic and halophilic bacterium T. maritima, using fruit and vegetable wastes as the carbon and energy sources was studied. Batch fermentation cultures showed that the use of a culture medium containing natural seawater and fruit and vegetable wastes can replace certain components (CaCl2, MgCl2, Balch's oligo-elements, yeast extract, KH2PO4 and K2HPO4) present in basal medium. However, a source of nitrogen and sulfur remained necessary for biohydrogen production. When fruit and vegetable waste collected from a wholesale market landfill was used, no decreases in total H2 production (139 mmol L-1) or H2 yield (3.46 mol mol-1) was observed.

Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima Part II: modeling and experimental approaches for hydrogen production.

BACKGROUND: Thermotoga maritima is a hyperthermophilic bacterium known to produce hydrogen from a large variety of substrates. The aim of the present study is to propose a mathematical model incorporating kinetics of growth, consumption of substrates, product formations, and inhibition by hydrogen in order to predict hydrogen production depending on defined culture conditions. RESULTS: Our mathematical model, incorporating data concerning growth, substrates, and products, was developed to predict hydrogen production from batch fermentations of the hyperthermophilic bacterium, T. maritima. It includes the inhibition by hydrogen and the liquid-to-gas mass transfer of H2, CO2, and H2S. Most kinetic parameters of the model were obtained from batch experiments without any fitting. The mathematical model is adequate for glucose, yeast extract, and thiosulfate concentrations ranging from 2.5 to 20 mmol/L, 0.2-0.5 g/L, or 0.01-0.06 mmol/L, respectively, corresponding to one of these compounds being the growth-limiting factor of T. maritima. When glucose, yeast extract, and thiosulfate concentrations are all higher than these ranges, the model overestimates all the variables. In the window of the model validity, predictions of the model show that the combination of both variables (increase in limiting factor concentration and in inlet gas stream) leads up to a twofold increase of the maximum H2-specific productivity with the lowest inhibition. CONCLUSIONS: A mathematical model predicting H2 production in T. maritima was successfully designed and confirmed in this study. However, it shows the limit of validity of such mathematical models. Their limit of applicability must take into account the range of validity in which the parameters were established.

Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima part I: effects of sulfured nutriments, with thiosulfate as model, on hydrogen production and growth.

BACKGROUND: Thermotoga maritima and T. neapolitana are hyperthermophile bacteria chosen by many research teams to produce bio-hydrogen because of their potential to ferment a wide variety of sugars with the highest theoretical H2/glucose yields. However, to develop economically sustainable bio-processes, the culture medium formulation remained to be optimized. The main aim of this study was to quantify accurately and specifically the effect of thiosulfate, used as sulfured nutriment model, on T. maritima growth, yields and productivities of hydrogen. The results were obtained from batch cultures, performed into a bioreactor, carefully controlled, and specifically designed to prevent the back-inhibition by hydrogen. RESULTS: Among sulfured nutriments tested, thiosulfate, cysteine, and sulfide were found to be the most efficient to stimulate T. maritima growth and hydrogen production. In particular, under our experimental conditions (glucose 60 mmol L-1 and yeast extract 1 g L-1), the cellular growth was limited by thiosulfate concentrations lower than 0.06 mmol L-1. Under these conditions, the cellular yield on thiosulfate (Y X/Thio) could be determined at 3617 mg mmol-1. In addition, it has been shown that the limitations of T. maritima growth by thiosulfate lead to metabolic stress marked by a significant metabolic shift of glucose towards the production of extracellular polysaccharides (EPS). Finally, it has been estimated that the presence of thiosulfate in the T. maritima culture medium significantly increased the cellular and hydrogen productivities by a factor 6 without detectable sulfide production. CONCLUSIONS: The stimulant effects of thiosulfate at very low concentrations on T. maritima growth have forced us to reconsider its role in this species and more probably also in all thiosulfato-reducer hyperthermophiles. Henceforth, thiosulfate should be considered in T. maritima as (1) an essential sulfur source for cellular materials when it is present at low concentrations (about 0.3 mmol g-1 of cells), and (2) as both sulfur source and detoxifying agent for H2 when thiosulfate is present at higher concentrations and, when, simultaneously, the pH2 is high. Finally, to improve the hydrogen production in bio-processes using Thermotoga species, it should be recommended to incorporate thiosulfate in the culture medium.

Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1)) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

Characterization of artificially dried biofilms for air biofiltration studies.

One of the main problems associated with the operation of air biofilters is the loss of performance caused by drying of the bioactive support, as the removal capacity of contaminants by the microorganisms is dependent on their water content. In this work, biofilms from a microbial consortium adapted to toluene were grown on stainless steel slides. The biofilms were dried in stoppered flasks with saturated saline solutions to obtain final water activities of 97.4 %, 83.9 %, 74.8 % and 32 %. The biofilms were characterized by a sorption isotherm Type III with toluene; the water desorption isotherm was fitted to the BET model and the biofilm hydrophobicity was also determined. Specific oxygen consumption rates decreased at lower Aw from 60 µg O(2)/mg protein/h to zero activity. Biofilm activity, represented by a toluene consumption rate, and others physical properties presented a critical point between Aw 0.84 and 0.97. Biological activity of dried biofilms was restored either partially or completely, depending on the extent of drying and rewetting method.

Oxygen uptake rates in the hyperthermophilic anaerobe Thermotoga maritima grown in a bioreactor under controlled oxygen exposure: clues to its defence strategy against oxidative stress.

A 2.3-L bioreactor was specially adapted to grow hyperthermophilic microorganisms under controlled conditions of temperature, pH, redox potential and dissolved O(2). Using this bioreactor regulated at 80°C and pH 7.0, we demonstrated that Thermotoga maritima recovered its growth despite being exposed to oxygen for a short time (30 min with a maximum concentration of 23 µM of dissolved oxygen). Under these conditions, we demonstrated that O(2) uptake rate, estimated at 73.6 µmoles O(2) min(-1) g proteins(-1) for dissolved oxygen, was optimal and constant, when dissolved oxygen was present in a range of 22-5 µM. Transcription analyses revealed that during short oxygen exposure, T. maritima expressed genes coding for enzymes to deal with O(2) and reactive oxygen species (ROS) such as peroxides. Thus, genes encoding ROS-scavenging systems, alkyl hydroperoxide reductase (ahp), thioredoxin-dependent thiol peroxidase (bcp 2) and to a lesser extent neelaredoxin (nlr) and rubrerythrin (rbr), were found to be upregulated during oxygen exposure. The oxygen reductase FprA, homologous to the rubredoxin-oxygen oxidoreductase (ROO) found in Desulfovibrio species, is proposed as a primary consumer of O(2) in T. maritima. Moreover, the expression of frpA was shown to depend on the redox (Eh) level of the culture medium.

Phenol biodegradation by the thermoacidophilic archaeon Sulfolobus solfataricus 98/2 in a fed-batch bioreactor.

Toxic at low concentrations, phenol is one of the most common organic pollutants in air and water. In this work, phenol biodegradation was studied in extreme conditions (80°C, pH = 3.2) in a 2.7 l bioreactor with the thermoacidophilic archaeon Sulfolobus solfataricus 98/2. The strain was first acclimatized to phenol on a mixture of glucose (2000 mg l(-1)) and phenol (94 mg l(-1)) at a constant dissolved oxygen concentration of 1.5 mg l(-1). After a short lag-phase, only glucose was consumed. Phenol degradation then began while glucose was still present in the reactor. When glucose was exhausted, phenol was used for respiration and then for biomass build-up. After several batch runs (phenol < 365 mg l(-1)), specific growth rate (µ(X)) was 0.034 ± 0.001 h(-1), specific phenol degradation rate (q(P)) was 57.5 ± 2 mg g(-1) h(-1), biomass yield (Y(X/P)) was 52.2 ± 1.1 g mol(-1), and oxygen yield factor (Y(X/O2)) was 9.2 ± 0.2 g mol(-1). A carbon recovery close to 100% suggested that phenol was exclusively transformed into biomass (35%) and CO(2) (65%). Molar phenol oxidation constant (Y(O2/P)) was calculated from stoichiometry of phenol oxidation and introducing experimental biomass and CO(2) conversion yields on phenol, leading to values varying between 4.78 and 5.22 mol mol(-1). Respiratory quotient was about 0.84 mol mol(-1), very close to theoretical value (0.87 mol mol(-1)). Carbon dioxide production, oxygen demand and redox potential, monitored on-line, were good indicators of growth, substrate consumption and exhaustion, and can therefore be usefully employed for industrial phenol bioremediation in extreme environments.

New cultural approaches for microaerophilic hyperthermophiles.

This article reports on a new culture system designed for studying the effects of nutritional factors on the growth of hyperthermophilic and chemolithotrophic microorganisms. The system comprises 5-l stainless steel jars, an automatic gas dispenser, propylene microplates, and a robotic platform. The culture system was validated using Aquifex aeolicus, a hyperthermophilic, chemolithotrophic, and microaerophilic bacterium, which requires hydrogen, oxygen, CO2, and minerals for growth. We demonstrated that the cell densities measured on 147 cultures of A. aeolicus microplated in jar at 80°C under partial pressures (in kPa) of water vapor (47), H2 (117.7), O2 (28.1), CO2 (31.4), and N2 (3.9), followed a normal distribution, with a mean of 0.72 and a standard deviation of 0.04 (variation coefficient: 5.7%). In addition, cross-comparison of the growth kinetics of A. aeolicus in serum bottles and in a jar system highlighted similar kinetics patterns (both mean growth rates were 0.18 and 0.17 h-1, respectively), whereas the maximum cell densities reached were slightly lower in jar than in bottle (0.73 vs. 0.88 OD units, respectively). Furthermore, these results showed that, contrary to bottles, the total pressure of gas in jars remained constant throughout the biotic experiments, even with seven microplates completely filled with grown cultures. In addition, this system has been validated also for hyperthermophilic strictly anaerobes such as Thermotoga maritima or aerobes such as Sulfolobus solfataricus. This new culture system offers an interesting alternative for cultivating hyperthermophiles, using gas as substrate under constant pressure, thus making it possible to miniaturize experiments and study a large number of nutritional factors in one experimental run.

Effect of Oxygen and Redox Potential on Glucose Fermentation in Thermotoga maritima under Controlled Physicochemical Conditions.

Batch cultures of Thermotoga maritima were performed in a bioreactor equipped with instruments adapted for experiments performed at 80°C to mimic the fluctuating oxidative conditions in the hot ecosystems it inhabits. When grown anaerobically on glucose, T. maritima was shown to significantly decrease the redox potential (Eh) of the culture medium down to about -480 mV, as long as glucose was available. Addition of oxygen into T. maritima cultures during the stationary growth phase led to a drastic reduction in glucose consumption rate. However, although oxygen was toxic, our experiment unambiguously proved that T. maritima was able to consume it during a 12-hour exposure period. Furthermore, a shift in glucose metabolism towards lactate production was observed under oxidative conditions.

First evidence of aerobic biodegradation of BTEX compounds by pure cultures of Marinobacter.

Marinobacter vinifirmus was shown to degrade toluene as sole carbon and energy source under aerobiosis and at NaCl concentrations in the range 30-150 g/L. Maximum toluene consumption rate, total CO2, and biomass productions were measured in the presence of 60 g/L of NaCl. Under these conditions, 90% of the carbon from toluene was recovered as CO2 and biomass. Maximum specific toluene consumption rate was about 0.12 mgC toluene mgC biomass(-1) h(-1) at NaCl concentrations between 30 and 60 g/L. It decreased to 0.03 mgC toluene mgC biomass(-1) h(-1) at 150 g/L. Besides toluene, M. vinifirmus degraded benzene, ethylbenzene, and p-xylene. Benzene and toluene were utilized to a lesser extent by another Marinobacter sp., Marinobacter hydrocarbonoclasticus.

Effect of O2 concentrations on Sulfolobus solfataricus P2.

Sulfolobus solfataricus P2 was grown aerobically at various O(2) concentrations. Based on growth parameters in microcosms, four types of behavior could be distinguished. At 35% O(2) (v/v; gas phase), the cultures did not grow, indicating a lethal dose of oxygen. For 26-32% O(2), the growth was significantly affected compared with the reference (21%), suggesting a moderate toxicity by O(2). For 16-24% O(2), standard growth was observed. For 1.5-15% O(2), growth was comparable with the reference, but the yield on O(2) indicated a more efficient use of oxygen. These results indicate that S. solfataricus P2 grows optimally in the range of 1.5-24% O(2), most likely by adjusting its energy-transducing machinery. To gain some insight into control of the respiratory system, transcriptomes of the strain cultivated at different O(2) concentrations, corresponding to each behavior (1.5%, 21% and 26%), were compared using a DNA microarray approach. It showed differential expression of several genes encoding terminal oxidases, indicating an adaptation of the strain's respiratory system in response to fluctuating oxygen concentrations.

New process for fungal delignification of sugar-cane bagasse and simultaneous production of laccase in a vapor phase bioreactor.

We propose a new process using a vapor phase bioreactor (VPB) to simultaneously (i) delignify sugar-cane bagasse, a residue of sugar production that can be recycled in paper industry, and (ii) produce laccase, an enzyme usable to bleach paper pulp. Ethanol vapor, used as laccase inducer, was blown up through a VPB packed with bagasse and inoculated with Pycnoporus cinnabarinusss3, a laccase-hyperproducing fungal strain. After 28 days, the laccase activity in the ethanol-treated bagasse was 80-fold higher (80 U g(ds)(-)(1)) and the bagasse delignification percentage was 12-fold (12%) higher than in the reference samples produced in the absence of ethanol, corresponding to a high overall pulp yield of 96.1%. In the presence of ethanol, the total soluble phenols amount was 2.5-fold (3 mg FA g(ds)(-)(1)) higher than that without ethanol. Six monomeric phenols were detected: p-coumaric (4-hydroxyphenyl-2-propenoic), ferulic (4-hydroxy-3-methoxyphenyl-2-propenoic), syringic (4-hydroxy-3,5-dimethoxybenzoic), vanillic (4-hydroxy-3-methoxybenzoic) and 4-hydroxybenzoic acids, and 2-methoxyhydroquinone. Higher concentrations of phenolic compounds were observed when ethanol vapor was added, confirming a more efficient bagasse delignification. After 28 days, the fungal-treated bagasse (with ethanol addition) was pulped and refined. For a freeness of 81 mL CSF, this processing required 50% less energy than with untreated bagasse (without inoculation and ethanol addition), which indicated a significant potential economy for the pulp and paper industry. Handsheets were made from pulp obtained after fungal-treated and untreated bagasse. Comparison of bagasse-pulp characteristics for freeness of 35 and 181 mL CSF showed an average increment by 35% for tensile index and breaking strength and length. VPB allowed a simultaneous production of laccase (90 U g(ds)(-)(1), after pressing of the bagasse) that improved the overall profitability of the process.

Study of a hexane-degrading consortium in a biofilter and in liquid culture: biodiversity, kinetics and characterization of degrading strains.

A gasoline-degrading consortium, originating from a Mexican soil, was used to study its hexane-degradation kinetics in liquid culture and in a biofilter with mineral support. The biodiversity of the consortium depending on the culture conditions and electron and energy source (gasoline, hexane in liquid or hexane in the biofilter) was analyzed using a 16S rRNA-based approach. Significant differences between the populations were observed, indicating a probable adaptation to the substrate. Two strains, named SP2B and SP72-3, isolated from the consortium, belonged to Actinomycetes and demonstrated a high metabolic potential in hexane degradation. Even though the SP2B strain was related to Rhodococcus ruber DSM 43338(T) by phylogenetic studies, it displayed enlarged metabolic properties in hexane and other short-alkane degradation compared with the collection strain.

Correlation of biological activity and reactor performance in biofiltration of toluene with the fungus Paecilomyces variotii CBS115145.

A biofiltration system inoculated with the mold Paecilomyces variotii CBS115145 showed a toluene elimination capacity (EC) of around 250 g/m3 of biofilter/h, which was higher than the values usually reported for bacteria. P. variotii assimilated m- and p-cresols but not the o isomer. Initial toluene hydroxylation occurred both on the methyl group and through the p-cresol pathway. These results were corroborated by detecting benzyl alcohol, benzaldehyde, and p-cresol as volatile intermediates. In liquid cultures with toluene as a substrate, the activity of toluene oxygenase (TO) was 5.6 nmol of O2/min/mg of biomass, and that of benzyl alcohol dehydrogenase was 16.2 nmol of NADH/min/mg of protein. Toluene biodegradation determined from the TO activity in the biofilter depended on the biomass distribution and the substrate concentration. The specific enzymatic activity decreased from 6.3 to 1.9 nmol of O2/min/mg of biomass along the reactor. Good agreement was found between the EC calculated from the TO activity and the EC measured on the biofilter. The results were confirmed by short-time biofiltration experiments. Average EC measured in different biofiltration experiments and EC calculated from the TO activity showed a linear relation, suggesting that in the biofilters, EC was limited by biological reaction. As the enzymatic activities of P. variotii were similar to those reported for bacteria, the high performance of the fungal biofilters can possibly be explained by the increased transfer of the hydrophobic compounds, including oxygen, from the gas phase to the mycelia, overcoming the transfer problems associated with the flat bacterial biofilms.

Effect of drying on biofilter performance: modeling and experimental approach.

The moisture content of biofilter media is a key parameter for its adequate performance. Control of moisture requires a better understanding of the drying of the support due to changes in inlet air temperature and relative humidity and from metabolic heat production by pollutant oxidation. A dynamic one-dimensional model was developed to describe drying and its effect on biofilter performance. Mass and energy balances were established on an elementary representative volume. The biological reaction term incorporated temperature, water content, and pollutant concentration effects. The model describes the variations in pollutant concentration, air relative humidity, temperature, and water content of the media. It predicts (1) water evaporation from the packing material as a consequence of metabolic heat generation and variations of the relative humidity of the inlet air stream, and (2) the resulting decrease in biofilter performance. The model was validated with biofiltration experiments treating gaseous toluene using peat as support. Various ranges of inlet air relative humidity, temperature, air velocity, and inlet pollutant concentration were assayed.

Enhancing phenanthrene biomineralization in a polluted soil using gaseous toluene as a cosubstrate.

Laboratory experiments were conducted to study the potential of adding gaseous toluene, as a readily degradable carbon source, to enhance phenanthrene mineralization in polluted soil (1,000 mg/kg(dry soil)) aged for 400 days. Experiments were conducted in 0.5-L column reactors packed with a mixture of (80:20 w(wet)/w(wet)) spiked soil and vermiculite and fed with 1 g m(-3)reactor h(-1) toluene load in air. Removal efficiencies of 100% for toluene and greater than 95% for phenanthrene were obtained in 190 h. Evolved CO2 showed that phenanthrene mineralization increased from 39% to 86% in columns treated with gaseous toluene. Phthalic acid was identified as the principal soluble intermediate, which accumulated when no toluene was added. Increased phenanthrene uptake and mineralization with toluene can be attributed to increased biomass and the induction of enzymes involved in the intermediate mineralization. In microcosm experiments, phthalic acid mineralization increased from 19% to 81% within 50 h in the presence of toluene. Experiments with 14C-labeled phenanthrene confirmed the enhancement of phenanthrene mineralization from 45% to 83% in 385 h with toluene as a second carbon source. The results indicate thatthe addition of an appropriate gaseous cosubstrate could be an adequate strategy to enhance mineralization of PAHs in soil.

Biofiltration of methyl tert-butyl ether vapors by cometabolism with pentane: modeling and experimental approach.

Degradation of methyl tert-butyl ether (MTBE) vapors by cometabolism with pentane using a culture of pentane-oxidizing bacteria (Pseudomonas aeruginosa) was studied in a 2.4-L biofilter packed with vermiculite, an inert mineral support. Experimental pentane elimination capacity (EC) of approximately 12 g m(-3) h(-1) was obtained for an empty bed residence time (EBRT) of 1.1 h and inlet concentration of 18.6 g m(-3). For these experimental conditions, EC of MTBE between 0.3 and 1.8 g m(-3) h(-1) were measured with inlet MTBE concentration ranging from 1.1 to 12.3 g m(-3). The process was modeled with general mass balance equations that consider a kinetic model describing cross-competitive inhibition between MTBE (cosubstrate) and pentane (substrate). The experimental data of pentane and MTBE removal efficiencies were compared to the theoretical predictions of the model. The predicted pentane and MTBE concentration profiles agreed with the experimental data for steady-state operation. Inhibition by MTBE of the pentane EC was demonstrated. Increasing the inlet pentane concentration improved the EC of MTBE but did not significantly change the EC of pentane. MTBE degradation rates obtained in this study were much lower than those using consortia or pure strains that can mineralize MTBE. Nevertheless, the system can be improved by increasing the active biomass.

Cometabolic biodegradation of methyl tert-butyl ether by a soil consortium: Effect of components present in gasoline.

A soil consortium was tested for its ability to degrade reformulated gasoline, containing methyl tert-butyl ether (MTBE). Reformulated gasoline was rapidly degraded to completion. However, MTBE tested alone was not degraded. A screening was carried out to identify compounds in gasoline that participate in cometabolism with MTBE. Aromatic compounds (benzene, toluene, xylenes) and compounds structurally similar to MTBE (tert-butanol, 2,2-dimethylbutane, 2,2,4-trimethylpentane) were unable to cometabolize MTBE. Cyclohexane was resistant to degradation. However, all n-alkanes tested for cometabolic activity (pentane, hexane, heptane) did enable the biodegradation of MTBE. Among the alkanes tested, pentane was the most efficient (200 &mgr;g/day). Upon the depletion of pentane, the consortium stopped degrading MTBE. When the consortium was spiked with pentane, MTBE degradation continued. When the ratio of MTBE to pentane was increased, the amount of MTBE degraded by the consortium was higher. Finally, diethylether was tested for cometabolic degradation with MTBE. Both compounds were degraded, but the process differed from that observed with pentane.