Methyl Tert-butyl Ether Biodegradation Research Articles

A multicomponent propane monooxygenase catalyzes the initial degradation of methyl tert-butyl ether in Mycobacterium vaccae JOB5.

Methyl tert-butyl ether (MTBE) has been recognized as a groundwater contaminant due to its widespread distribution and potential threat to human health. The limited understanding of the enzymes catalyzing MTBE degradation restricts their application in MTBE bioremediation. In this study, an MTBE-degrading soluble di-iron monooxygenase that clusters phylogenetically with a known propane monooxygenase (PRM) encoded by the prmABCD gene cluster was identified and functionally characterized, revealing their role in MTBE metabolism by Mycobacterium vaccae JOB5. Transcriptome analysis demonstrated that the expression of prmABCD was upregulated when JOB5 was induced by MTBE. Escherichia coli Rosetta heterologously expressing prmABCD from JOB5 could transform MTBE, indicating that the PRM of JOB5 is capable of the initial degradation of MTBE. The loss of the gene encoding the oxygenase α-subunit or β-subunit, the coupling protein, or the reductase disrupted MTBE transformation by the recombinant E. coli Rosetta. In addition, the catalytic capacity of PRM is likely affected by residue G95 in the active site pocket and residues I84, P165, A269, and V270 in the substrate tunnel structure. Mutation of amino acids in the active site and substrate tunnel resulted in inefficiency or inactivation of MTBE degradation, and the activity in 1,4-dioxane (1,4-D) degradation was diminished less than that in MTBE degradation.IMPORTANCEMulticomponent monooxygenases catalyzing the initial hydroxylation of MTBE are important in MTBE biodegradation. Previous studies of MTBE degradation enzymes have focused on P450s, alkane monooxygenase and MTBE monooxygenase, but the vital role of soluble di-iron monooxygenases has rarely been reported. In this study, we deciphered the essential catalytic role of a PRM and revealed the key residues of the PRM in MTBE metabolism. Our findings provide new insight into the MTBE-degrading gene cluster and enzymes in bacteria. This characterization of the PRM associated with MTBE degradation expands our understanding of MTBE-degrading gene diversity and provides a novel candidate enzyme for the bioremediation of MTBE-contaminated sites.

Enhanced Methyl Tert-Butyl Ether Removal by Mixed Consortium: Performance and Adaptability

Methyl tert-butyl ether (MTBE) is widely used as a gasoline additive with toxicity and carcinogenicity, and has caused environmental pollution worldwide. Biodegradation is a promising method for the removal of MTBE from contaminated sites. In this paper, three strains with high adaptability and different degrading characteristics to MTBE were cultured. The kinetic models were established to systematically simulate the biodegradation of MTBE by various strains at different concentrations. Moreover, the removal of MTBE in a synergistic system containing a mixture of three pure strains was studied. The mixed consortium enhanced MTBE removal at high concentrations (30–50 mg/L), and the degradation efficiency was increased by about 20% compared to pure strains at a concentration of 30 mg/L. Further, the mixed consortium degraded MTBE nearly three times faster than any of the individual, indicating that the co-cultures of three pure cultures improved both efficiency and the rate of MTBE biodegradation. In addition, it was found that the mixed consortium effectively removed MTBE in the presence of other gasoline components, and exhibited stronger adaptability, especially at low or high temperatures. This study supports the cultivation of a mixed consortium to remediate MTBE-contaminated environments, either as a single substrate or in environments containing other gasoline components.

A Survey of Gasoline Ameliorator, Methyl-Tert-Butyl Ether (MTBE) on Bovine Serum Albumin: A Spectroscopy and Molecular Dynamic Simulation Study.

Methyl-Tert-Butyl Ether (MTBE) as a gasoline modifier is frequently added to fuels and used in plenty of worldwide applications. MTBE biodegradation in groundwater occurs slowly and produces water miscibility; therefore, it causes diverse environmental and human health concerns. The interaction of MTBE with bovine serum albumin (BSA) as a model protein at physiological conditions is investigated to illustrate the possible interactions of MTBE with the body's proteins. Uv-visible, fluorescence, circular dichroism (CD) spectroscopy methods, and molecular modeling were used to analyze the MTBE's effect on BSA structure and dynamics. The constant protein concentration and various MTBE contents were used for possible interactions. The protein structural analysis shows that MTBE binds to BSA via positive enthalpy and entropy via hydrophobic interactions. Molecular docking shows the participation of several amino acids in the MTBE-BSA interaction. The CD spectroscopy results show that the BSA structure was not changed in the MTBE concentrations utilized in the study. Molecular dynamics (MD) simulation results suggest that MTBE can slightly change protein structure in the last 50ns. Comparing experimental and MD simulation results demonstrated that the BSA secondary structure was maintained in the low concentration of the MTBE. The entropy and enthalpy parameters asserted the hydrophobic interaction was the major force in the interaction between the BSA and MTBE.

Interaction of tetrahydrofuran and methyl tert-butyl ether in waste gas treatment by a biotrickling filter bioaugmented with Piscinibacter caeni MQ-18 and Pseudomonas oleovorans DT4

Bioaugmented biotrickling filter (BTF) seeded with Piscinibacter caeni MQ-18, Pseudomonas oleovorans DT4, and activated sludge was established to investigate the treatment performance and biodegradation kinetics of the gaseous mixtures of tetrahydrofuran (THF) and methyl tert-butyl ether (MTBE). Experimental results showed an enhanced startup performance with a startup period of 9 d in bioaugmented BTF (25 d in control BTF seeded with activated sludge). The interaction parameter I2,1 of control (7.462) and bioaugmented BTF (3.267) obtained by the elimination capacity–sum kinetics with interaction parameter (EC–SKIP) model indicated that THF has a stronger inhibition of MTBE biodegradation in the control BTF than in the bioaugmented BTF. Similarly, the self-inhibition EC–SKIP model quantified the positive effects of MTBE on THF biodegradation, as well as the negative effects of THF on MTBE biodegradation and the self-inhibition of MTBE and THF. Metabolic intermediate analysis, real-time quantitative polymerase chain reaction, biofilm-biomass determination, and high-throughput sequencing revealed the possible mechanism of the enhanced treatment performance and biodegradation interactions of MTBE and THF.

Kinetic modeling of Methyl Tert-Butyl Ether (MTBE) removal by bacterial isolated from contaminated soils

ABSTRACT Methyl Tert-Butyl Ether(MTBE) is among oxygenised additives to gasoline, which has high solubility and leads to pollution of water resources. So far, the microbial species affecting the of high MTBEconcentrationsremoval have been less investigated. This study aimed to remove high concentrations of MTBEusing isolated bacteriafrom contaminatedsoil with petroleum compounds in southern regions of Iran and determine their kinetics. After isolating the bacteria and determining their growth in different concentrations, the effects of the following parameters on the removal percentage were examined: incubation time = 1–7 days, pH = 5.0–7.0, and 9.0, initial MTBE concentration = 1000–5000 mg/L, and C: N:P. Then, the four strains with high efficiency were selected, and their kinetic were analysed by zero-order, first-order, and second-order models. The results showed that the highest growth in MTBE concentration (50,000 mg/L) was observed for Acinetobacter sp, Bacillus sp, Ochrobactrum sp, and Sphingomonas sp. after four days. Then, Ochrobactrum sp.was used as a pure strain as well as a microbial consortium for the next steps, including the effects of nutrient ratio and initial MTBE concentration. In this study, the optimal conditions for incubation time and the initial MTBE concentration were 72 hours and 5000 mg/L, respectively. The kinetic modeling indicated that the second-order model (R2 = 0.95, Root Mean Squared Error = 0.012) and the Micheal-Menten model (R2 = 0.99) were the best modelfor MTBE biodegradation. Overall, the results indicated that pure Ochrobactrum sp. and a microbial consortium consisting of resistant bacteria at high MTBEconcentrations could remove MTBE> 90%.

Characterization of co-metabolic biodegradation of methyl tert-butyl ether by a Acinetobacter sp. strain.

Co-metabolic bioremediation is a promising approach for the elimination of methyl tert-butyl ether (MTBE), which is a common pollutant found worldwide in ground water. In this paper, a bacterial strain able to co-metabolically degrade MTBE was isolated and named as Acinetobacter sp. SL3 based on 16S rRNA gene sequencing analysis. Strain SL3 could grow on n-alkanes (C5–C8) accompanied with the co-metabolic degradation of MTBE. The number of carbons present in the n-alkane substrate significantly influenced the degradation rate of MTBE and accumulation of tert-butyl alcohol (TBA), with n-octane resulting in a higher MTBE degradation rate (Vmax = 36.7 nmol min−1 mgprotein−1, Ks = 6.4 mmol L−1) and lower TBA accumulation rate. A degradation experiment in a fed-batch reactor revealed that the efficiency of MTBE degradation by Acinetobacter sp. strain SL3 did not show an obvious decrease after nine rounds of MTBE replenishment ranging from 0.1–0.5 mmol L−1. The results of this paper reveal the preferable properties of Acinetobacter sp. SL3 for the bioremediation of MTBE via co-metabolism and leads towards the development of new MTBE elimination technologies.

Biodegradation of Methyl tert-Butyl Ether by Co-Metabolism with a Pseudomonas sp. Strain.

Co-metabolic bioremediation is supposed to be an impressive and promising approach in the elimination technology of methyl tert-butyl ether (MTBE), which was found to be a common pollutant worldwide in the ground or underground water in recent years. In this paper, bacterial strain DZ13 (which can co-metabolically degrade MTBE) was isolated and named as Pseudomonas sp. DZ13 based on the result of 16S rRNA gene sequencing analysis. Strain DZ13 could grow on n-alkanes (C5-C8), accompanied with the co-metabolic degradation of MTBE. Diverse n-alkanes with different carbon number showed a significant influence on the degradation rate of MTBE and accumulation of tert-butyl alcohol (TBA). When Pseudomonas sp. DZ13 co-metabolically degraded MTBE with n-pentane as the growth substrate, a higher MTBE-degrading rate (Vmax = 38.1 nmol/min/mgprotein, Ks = 6.8 mmol/L) and lower TBA-accumulation was observed. In the continuous degradation experiment, the removal efficiency of MTBE by Pseudomonas sp. Strain DZ13 did not show an obvious decrease after five times of continuous addition.

Anaerobic biodegradation of MTBE in a field site above the Israeli Coastal Aquifer: evidence from δ13C compound‐specific isotope analysis

AbstractBACKGROUNDThe fuel additive, methyl tert‐butyl ether (MTBE), is one of the most frequently occurring groundwater pollutants worldwide. The present study focused on assessing microbial degradation of MTBE in groundwater below a gasoline station situated above the Israeli Coastal Aquifer. Groundwater was monitored in six monitoring wells over a time period of 2.5 years.RESULTSThe site water geochemistry indicated that anaerobic conditions prevailed in two of the most contaminated wells. During a year MTBE concentrations declined from 81 to 4.2 mg L−1, concurrently, with a significant change in δ13C from −31.4 to −11.8 ‰ in a source area monitoring well. MTBE biodegradation during microcosm experiments under sulfate reducing and methanogenic conditions resulted in carbon isotopic enrichment factors (ε) of −17.2 ± 1.1 ‰.CONCLUSIONSignificant decrease in MTBE concentrations, along with pronounced changes in carbon isotope composition, indicates in situ degradation of MTBE occurring at the site. This evidence was supported by a microcosm experiment with indigenous microbial cultures. The estimated rate of biodegradation is 0.7 year−1, which is about 70% of the total natural attenuation (NA) processes. © 2016 Society of Chemical Industry

Deep RNA-Seq profile reveals biodiversity, plant-microbe interactions and a large family of NBS-LRR resistance genes in walnut (Juglans regia) tissues.

Deep RNA-Seq profiling, a revolutionary method used for quantifying transcriptional levels, often includes non-specific transcripts from other co-existing organisms in spite of stringent protocols. Using the recently published walnut genome sequence as a filter, we present a broad analysis of the RNA-Seq derived transcriptome profiles obtained from twenty different tissues to extract the biodiversity and possible plant–microbe interactions in the walnut ecosystem in California. Since the residual nature of the transcripts being analyzed does not provide sufficient information to identify the exact strain, inferences made are constrained to the genus level. The presence of the pathogenic oomycete Phytophthora was detected in the root through the presence of a glyceraldehyde-3-phosphate dehydrogenase. Cryptococcus, the causal agent of cryptococcosis, was found in the catkins and vegetative buds, corroborating previous work indicating that the plant surface supported the sexual cycle of this human pathogen. The RNA-Seq profile revealed several species of the endophytic nitrogen fixing Actinobacteria. Another bacterial species implicated in aerobic biodegradation of methyl tert-butyl ether (Methylibium petroleiphilum) is also found in the root. RNA encoding proteins from the pea aphid were found in the leaves and vegetative buds, while a serine protease from mosquito with significant homology to a female reproductive tract protease from Drosophila mojavensis in the vegetative bud suggests egg-laying activities. The comprehensive analysis of RNA-seq data present also unraveled detailed, tissue-specific information of ~400 transcripts encoded by the largest family of resistance (R) genes (NBS-LRR), which possibly rationalizes the resistance of the specific walnut plant to the pathogens detected. Thus, we elucidate the biodiversity and possible plant–microbe interactions in several walnut (Juglans regia) tissues in California using deep RNA-Seq profiling.

Monitoring In Situ Biodegradation of MTBE Using Multiple Rounds of Compound‐Specific Stable Carbon Isotope Analysis

AbstractAt a large industrial facility, methyl tert‐butyl ether (MTBE) was released to the subsurface and dispersed into the light, non‐aqueous phase liquids (LNAPL), in the first aquifer, with the LNAPL serving as a continuous source of MTBE in groundwater. Compound‐specific isotope analysis was conducted on both MTBE and tert‐butyl alcohol (TBA) in groundwater samples collected in 2008, 2011, and 2013 from wells located along and off the center line of the MTBE plume. The study demonstrated the onset and progress of biodegradation of MTBE between 2008 and 2013. The TBA observed in 2008 appears to be derived only in part from MTBE transformation while a significant portion of TBA might be contributed directly from LNAPL sources. In 2011 to 2013, the dominant source of TBA in the mid‐gradient plume was MTBE transformation. A contribution of an offsite LNAPL source, in particular to the down‐gradient area of the plume, is possible but could not be unequivocally confirmed. The time series provided direct evidence for MTBE biodegradation, but also a valuable insight on the sources of TBA.

Microbial Community from MTBE-Contaminated Soil for Aerobic Biodegradation of MTBE

This Methyl tert-butyl ether (MTBE) is one of the main additives in gasoline to increase octane rating and consequently reduce air pollution. The physico-chemical properties of this substance (high water solubility, low sorption in soil) result in high mobility and considerable concentrations in aquifers. In this survey, Isfehan Refinery that was encountered with MTBE contamination problem was selected as a case study and the MTBE degradation ability of this contaminated area by its indigenous microorganisms was investigated. In the first step of this survey, the influence of various factors on the aerobic degradation of MTBE such as mixed culture type, incubation time, microbial culture and optimal concentration of MTBE were investigated in shaking flasks and the most important factors were specified by means of fractional factorial design 1/2. In the second stage by using optimal values which obtained from the first stage, the effects of co-substare parameter and inoculum parameter were assayed by means of response surface method. The results of the experiments showed that the mixed culture type and initial concentration of MTBE were the most significant factors. The results of the experiments showed that the mixed indigenous culture acted better than activated sludge. The initial concentration of MTBE was also one of the most significant factors. At the best condition about 31 percent of MTBE was treated by co-substrating with n-hexane in a ratio of 0.2.

Cometabolism of methyl tert-butyl ether by a new microbial consortium ERS.

The release of methyl tert-butyl-ether (MTBE) into the environment has increased the worldwide concern about the pollution of MTBE. In this paper, a microbial consortium was isolated from the soil sample near an oil station, which can degrade MTBE directly with a low biomass yield and MTBE degrading efficiency. Further research has indicated that this consortium can degrade MTBE efficiently when grown on n-octane as the cometabolic substrate. The results of 16S rDNA based on phylogenetic analysis of the selected operating taxonomic units (OTUs) involved in the consortium revealed that one OTU was related to Pseudomonas putida GPo1, which could cometabolically degrade MTBE on the growth of n-octane. This may help explain why n-octane could be the optimal cometabolic substrate of the consortium for MTBE degradation. Furthermore, the degradation of MTBE was observed along with the consumption of n-octane. Different K s values for MTBE were observed for cells grown with or without n-octane, suggesting that different enzymes are responsible for the oxidation of MTBE in cells grown on n-octane or MTBE. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism.

Combined toxicities of methyl tert-butyl ether and its metabolite tert-butyl alcohol on earthworms via different exposure routes

Methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) are among the major soil contaminants that threaten the health of soil ecosystems. Many MTBE-contaminated sites accumulate TBA, because TBA is the intermediate of MTBE biodegradation. To access the risk of MTBE and TBA in soil, we investigated the combined toxicities of MTBE and TBA using two earthworm species, Perionyx excavatus and Eisenia andrei, as well as the toxic effects via different exposure routes. The combined toxicity showed weak antagonistic effects (LC50mix values were slightly greater than 1.0), and sensitivity toward same pollutants differed in the two earthworm species. Moreover, the toxicity of MTBE and TBA was also affected by the exposure route; both filter paper and artificial soil tests showed that dermal-only exposure to MTBE had an even greater toxic effect than combined dermal and oral exposure. Thus, we suggest that diverse environmental factors including organic materials, the physicochemical properties of the contact media, and the exposure routes of the organism, should be taken into consideration when assessing the effects of pollutants on organisms in diverse environmental systems.

Lab-scale tests and numerical simulations for in situ treatment of polluted groundwater

Methyl tert-butyl ether (MTBE) is used at significant percentages as an additive of unleaded gasoline. The physical–chemical properties of the substance (water solubility, soil organic carbon–water partition coefficient) cause high mobility and high concentrations in groundwater. Laboratory scale batch and column tests and mathematical modeling were performed to study the feasibility of a biobarrier (BB), that is an in situ permeable biological barrier with or without inoculation, for the remediation of MTBE and other gasoline-derived pollutants (benzene, toluene, ethylbenzene, o-xylene and m+p-xylenes, BTEXs) polluted groundwater and to estimate kinetic constants. The experimental results showed simultaneous biodegradation of MTBE and BTEXs, with similar removals in the uninoculated and the inoculated systems. Ranges for the first order kinetic removal were obtained for MTBE ((0.18±0.02)/(0.28±0.11d−1)), B ((0.39±0.12)/(0.56±0.12d−1)), T ((0.51±0.03)/(0.78±0.15d−1)), E ((0.46±0.18)/(1.57±0.21d−1)), o-X ((0.24±0.08)/(0.64±0.09d−1)) and m+p-X ((0.20±0.04)/(1.21±0.04d−1)). The results of the laboratory tests allowed to improve mathematical modeling in order to design a full-scale BB at a gasoline-contaminated site.

Rapid biodegradation of methyl tert-butyl ether (MTBE) by pure bacterial cultures

Two pure bacterial strains capable of rapid degrading methyl tert-butyl ether (MTBE) were isolated from an industrial wastewater treatment plant, identified and characterized. These strains are able to grow on MTBE as the sole carbon and energy sources and completely mineralize it to the biomass and carbon dioxide. The strains were identified as Bacillus cereus and Klebsiella terrigena. Both strains are able to grow in the presence of 48 gl -1 MTBE in water, which is almost the maximum concentration of MTBE in the water. They were able to completely degrade 10 gl -1 MTBE in less than a day. The specific degradation rate of MTBE at optimum conditions were 5.89 and 5.78 g(MTBE) g(cells) -1 h -1 for B. cereus and K. terrigena, respectively. The biomass yield was 0.085 and 0.076 gg -1 , respectively. The cultivations were carried out successfully at 25, 30 and 37 °C, while they showed the best performance at 37 °C. Neither of the strains was able to grow and degrade MTBE anaerobically.

Evaluation of Intrinsic Bioremediation of Methyl Tert-butyl Ether (MTBE) Contaminated Groundwater

This paper reported the use of real-time polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and the culture-based method in the intrinsic bioremediation study at a petroleum contaminated site. The study showed that phenol hydroxylase gene was detected in groundwater contaminated with benzene, toluene, ethylbenzene, xylene isomers (BTEX) and methyl tert-butyl ether (MTBE). This indicated that intrinsic bioremediation occurred at the site. DGGE analyses revealed that the petroleum-hydrocarbon plume caused the variation in microbial communities. MTBE degraders including Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01, and Enterobacter sp. NKNU02 were isolated from the contaminated groundwater using the cultured-based method. Among these five strains, Enterobacter sp. NKNU02 is the most effective stain at degrading MTBE without the addition of pentane. The MTBE biodegradation experiment indicated that the isolated bacteria were affected by propane. Biodegradation of MTBE was decreased but not totally inhibited in the mixtures of BTEX. Enterobacter sp. NKNU02 degraded about 60% of MTBE in the bioreactor study. Tert-butyl alcohol (TBA), acetic acid, 2-propanol, and propenoic acid were detected using gas chromatography/mass spectrometry during MTBE degraded by the rest cells of Enterobacter sp. NKNU02. The effectiveness of bioremediation of MTBE was assessed for potential field-scale application.

호기/혐기성 조건에서의 BTEX 및 MTBE 동시 분해특성

호기/혐기성 조건에서의 BTEX 및 MTBE 동시 분해특성

Using DNA‐Stable Isotope Probing to Identify MTBE‐ and TBA‐Degrading Microorganisms in Contaminated Groundwater

Although the anaerobic biodegradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) has been documented in the laboratory and the field, knowledge of the microorganisms and mechanisms involved is still lacking. In this study, DNA-stable isotope probing (SIP) was used to identify microorganisms involved in anaerobic fuel oxygenate biodegradation in a sulfate-reducing MTBE and TBA plume. Microorganisms were collected in the field using Bio-Sep® beads amended with 13C5-MTBE, 13C1-MTBE (only methoxy carbon labeled), or13C4-TBA. 13C-DNA and 12C-DNA extracted from the Bio-Sep beads were cloned and 16S rRNA gene sequences were used to identify the indigenous microorganisms involved in degrading the methoxy group of MTBE and the tert-butyl group of MTBE and TBA. Results indicated that microorganisms were actively degrading 13C-labeled MTBE and TBA in situ and the 13C was incorporated into their DNA. Several sequences related to known MTBE- and TBA-degraders in the Burkholderiales and the Sphingomonadales orders were detected in all three13C clone libraries and were likely to be primary degraders at the site. Sequences related to sulfate-reducing bacteria and iron-reducers, such as Geobacter and Geothrix, were only detected in the clone libraries where MTBE and TBA were fully labeled with 13C, suggesting that they were involved in processing carbon from the tert-butyl group. Sequences similar to the Pseudomonas genus predominated in the clone library where only the methoxy carbon of MTBE was labeled with 13C. It is likely that members of this genus were secondary degraders cross-feeding on 13C-labeled metabolites such as acetate.

The Biodegradation of Methyl Tert-Butyl Ether (MTBE) by Indigenous Bacillus cereus Strain RJ1 Isolated From Soil

Methyl Tert-Butyl Ether (MTBE) is an oxygenated organic compound extensively substituted for lead in gasoline worldwide. MTBE can affect human and environment. In this research, biodegradation capability of MTBE by identified indigenous Bacillus cereus strain RJ1 was studied. Obtained results showed that biological removal of MTBE with 200 mg/L samples in 28°C is 27.5% while in 37°C within 120 days reaches to 34%. In addition, biodegradation of Bacillus cereus RJ1 in 500 mg/L and 1,000 mg/L is 28% and 23.9% in 28°C, respectively. Therefore, this bacterium could clean up MTBE from the environment.

Anaerobic biodegradation of methyl tert-butyl ether and tert-butyl alcohol in petrochemical wastewater

A laboratory-scale anaerobic sequencing batch reactor was used to evaluate treatment of a synthetic substrate mixture representing petrochemical wastewater containing methyl tert-butyl ether (MTBE), ethanol and acetic acid. Influent MTBE concentrations were 5, 10 and 50 mg/l (corresponding to MTBE loading rates of 0.2, 0.4 and 2 mg/l.d) with overall organic loading rates (OLRs) of 1.51, 3.23 and 3.25 g COD/l.d, respectively. These OLRs resulted in removal efficiencies for MTBE of 78%, 98% and 88%. Removal efficiencies for chemical oxygen demand were 85% and 90% with influent MTBE concentrations of 5 and 10 mg/l, but were significantly reduced to 72% with influent MTBE concentrations of 50 mg/l. During all reactor runs, effluent concentrations of tert-butyl alcohol (TBA) were below the detection limit. Batch degradation of the organic substrate mixture demonstrated initial inhibitory effects when exposed to MTBE concentrations of 50 mg/l and complete inhibition with MTBE concentrations above 2000 mg/l. It is interesting to note that in batch tests using MTBE as the sole organic substrate (initial MTBE concentrations of 50, 100 and 200 mg/l), the specific methanogenic activity decreased to below detection within the first 96 hours, but following a 72-hour lag phase the methane production increased again. Based on low volatile fatty acid (VFA) concentration, disappearance of TBA peaks and no findings of any other intermediate via gas chromatography/mass spectrometry, while the MTBE concentration is still high, it can be suggested that during the batch tests the breakdown of gas production and the following lag phase were the direct effect of higher MTBE concentrations (more than 50 mg/l) and not because of the TBA or VFA accumulations.