Benzene, Toluene, Ethylbenzene And Xylene Biodegradation Research Articles

Anoxic biodegradation of BTEX in a biotrickling filter

Emissions of BTEX (benzene, toluene, ethylbenzene and xylene) from the petrochemical industry are characterized by a low pollutants concentration and the absence of oxygen. Biodegradation of these pollutants using nitrate as the electron acceptor is of key interest to reuse the residual gas for inertization purposes. However, the biological mineralization of BTEX is often limited by their recalcitrant nature and the toxicity of the secondary metabolites produced. The potential of an anoxic biotrickling filter for the treatment of a model O2-free BTEX-laden emission at inlet individual concentrations of ~700mgm−3 was here evaluated. A UV oxidation step was also tested both in the recycling liquid and in the inlet gas emission prior to biofiltration. Removal efficiencies >90% were achieved for both toluene and ethylbenzene, corresponding to elimination capacities (ECs) of 1.4±0.2gm−3h−1 and 1.5±0.3gm−3h−1, respectively, while ~45% of xylene (EC=0.6±0.1g m−3h−1) was removed at a liquid recycling rate of 2mh−1. Benzene biodegradation was however limited by the accumulation of toxic metabolites in the liquid phase. The oxidation of these intermediates in the recycling liquid by UV photolysis boosted benzene abatement, achieving an average EC of 0.5±0.2gm−3h−1 and removals of ~40%. However, the implementation of UV oxidation as a pretreatment step in the inlet gas emission resulted in the deterioration of the BTEX biodegradation capacity of the biotrickling filter. Finally, a high bacterial diversity was observed throughout the entire experiment, the predominant phyla being Proteobacteria and Deinococcus-thermus.

Gene Expression during BTEX Biodegradation by a Microbial Consortium Acclimatized to Unleaded Gasoline and a Pseudomonas putida Strain (HM346961) Isolated from It.

Pseudomonas putida strain (HM346961) was isolated from a consortium of bacteria acclimatized to unleaded gasoline-contaminated water. The consortium can efficiently remove benzene, toluene, ethylbenzene and xylene (BTEX) isomers, and a similar capability was observed with the P. putida strain. Proteome of this strain showed certain similarities with that of other strains exposed to the hydrocarbon compounds. Furthermore, the toluene di-oxygenase (tod) gene was up-regulated in P. putida strain when exposed to toluene, ethylbenzene, xylene, and BTEX. In contrast, the tod gene of P. putida F1 (ATCC 700007) was up-regulated only in the presence of toluene and BTEX. Several differences in the nucleotide and protein sequences of these two tod genes were observed. This suggests that tod up-regulation in P. putida strain may partially explain their great capacity to remove aromatic compounds, relative to P. putida F1. Therefore, new tod and P. putida strain are promising for various environmental applications.

BTEX biodegradation in contaminated groundwater using a novel strain (Pseudomonas sp. BTEX-30)

BTEX (benzene, toluene, ethylbenzene and xylenes) compounds are the most frequently encountered subsurface contaminants among the various petroleum hydrocarbons. In this study, a new strain, able to degrade BTEX compounds, was isolated from oil contaminated groundwater. Phylogenetic analysis revealed that the isolated strain was most closely related to Pseudomonas zhaodongensis with 98% 16SrRNA gene sequence similarity. A series of batch experiments were carried out to investigate the ability of the new strain for removing BTEX compounds using single and mixed substrates. The optimized values of pH, temperature, and inorganic nutrients (as percent of BTEX concentration) were 7.6, 28.9 °C, and 200%, respectively which were obtained by using the response surface methodology for the biodegradation of BTEX. Results showed that the identified strain was able to completely degrade benzene, toluene, and ethylbenzene in the single substrate batch experiments while m-xylene remained non-degradable. Results of dual-substrate experiments revealed co-metabolism of m-xylene in the presence of benzene and toluene. Dioxygenase was found to be the key enzyme incorporating in the co-metabolism of m-xylene. Within the mixed substrate batch experiments all BTEX compounds can be degraded. An increase in the production of cell growth due to the degradation of benzene and toluene accelerated the degradation process of m-xylene in the mixed substrate experiments.

Biodiesel presence in the source zone hinders aromatic hydrocarbons attenuation in a B20-contaminated groundwater

The behavior of biodiesel blend spills have received limited attention in spite of the increasing and widespread introduction of biodiesel to the transportation fuel matrix. In this work, a controlled field release of biodiesel B20 (100L of 20:80 v/v soybean biodiesel and diesel) was monitored over 6.2years to assess the behavior and natural attenuation of constituents of major concern (e.g., BTEX (benzene, toluene, ethyl-benzene and xylenes) and PAHs (polycyclic aromatic hydrocarbons)) in a sandy aquifer material. Biodiesel was preferentially biodegraded compared to diesel aromatic compounds with a concomitant increase in acetate, methane (near saturation limit (≈22mgL−1)) and dissolved BTEX and PAH concentrations in the source zone during the first 1.5 to 2.0years after the release. Benzene and benzo(a)pyrene concentrations remained above regulatory limits in the source zone until the end of the experiment (6.2years after the release). Compared to a previous adjacent 100-L release of ethanol-amended gasoline, biodiesel/diesel blend release resulted in a shorter BTEX plume, but with higher residual dissolved hydrocarbon concentrations near the source zone. This was attributed to greater persistence of viscous (and less mobile) biodiesel than the highly-soluble and mobile ethanol in the source zone. This persistence of biodiesel/diesel NAPL at the source zone slowed BTEX and PAH biodegradation (by the establishment of an anaerobic zone) but reduced the plume length by reducing mobility. This is the first field study to assess biodiesel/diesel blend (B20) behavior in groundwater and its effects on the biodegradation and plume length of priority groundwater pollutants.

Anaerobic BTEX degradation in oil sands tailings ponds: Impact of labile organic carbon and sulfate-reducing bacteria

The extraction of bitumen from oil sands in Alberta (Canada) produces volumes of tailings that are pumped into large anaerobic settling-basins. Beside bitumen, tailings comprise fractions of benzene, toluene, ethylbenzene and xylenes (BTEX) that derive from the application of industrial solvents. Due to their toxicity and volatility, BTEX pose a strong concern for gas- and water-phase environments in the vicinity of the ponds. The examination of two pond profiles showed that concentrations of indigenous BTEX decreased with depth, pointing at BTEX transformation in situ. With depth, the relative contribution of ethylbenzene and xylenes to total BTEX significantly decreased, while benzene increased relatively from 44% to 69%, indicating preferential hydrocarbon degradation. To predict BTEX turnover and residence time, we determined BTEX degradation rates in tailings of different depths in a 180-days microcosm study. In addition, we evaluated the impact of labile organic substrates (e.g. acetate) generally considered to stimulate hydrocarbon degradation and the contribution of sulfate-reducing bacteria (SRB) to BTEX turnover. In all depths, BTEX concentrations significantly decreased due to microbial activity, with degradation rates ranging between 4 and 9μgkg−1d−1. BTEX biodegradation decreased linearly in correlation with initial concentrations, suggesting a concentration-dependent BTEX transformation. SRB were not significantly involved in BTEX consumption, indicating the importance of methanogenic degradation. BTEX removal decreased to 70–90% in presence of organic substrates presumptively due to an accumulation of acetate that lowered BTEX turnover due to product inhibition. In those assays SRB slightly stimulated BTEX transformation by reducing inhibitory acetate levels.

Kinetics of BTEX biodegradation coupled with Fe(III) reduction by indigenous microorganisms in simulated underground environment

Pressure on widespread contamination of BTEX (benzene, toluene, ethylbenzene, and xylene) in sediments and groundwater requires better understanding of biogeochemical removal process of the pollutant. Oxidation of BTEX coupled with reduction of Fe(III) is one of the most efficient ways. BTEX degradation dynamics was investigated by observing BTEX oxidation rates at different concentrations (from 50 to 200 mg/L) at the presence of Fe(III) (from 106 to 212 mg/L). The dual-substrate Michaelis–Menten model was derived ( mg/L d, mg/L, mg/L). Experiment data agreed well with the model when the initial BTEX concentration was under 100 mg/L, which indicated that microorganisms in the system could keep balance. With the increasing of initial BTEX concentration, oxidation rates did not fit the model, indicating that high BTEX concentration would be toxic to the function microorganisms and would dampened the BTEX oxidation reaction. The dynamics of BTEX with different compositions (the B:T:E:X ratios) coupled to the dissimilatory Fe(III) reduction in contaminated underground environment was also modified. The proposal is to remedy contaminated groundwater by using Fe(III) in aquifer material instead of other substances, so that the operating conditions do not destruct ecological environment of groundwater. These findings provide important parameters for the remediation of BTEX in sediments and groundwater coupled with Fe(III) reduction.

Application of natural attenuation for the control of petroleum hydrocarbon plume: Mechanisms and effectiveness evaluation

Summary The objective of this study was to evaluate the effectiveness and mechanisms of natural attenuation (NA) on the remediation of petroleum-hydrocarbon contaminated groundwater caused by gasoline and diesel fuel leakage. During the effectiveness investigation, site groundwater samples were collected and analyzed for the variations in geochemical indicators, microbial diversity, dominant bacteria, biodegradation rates, biodegradation capacity, and the trend of contaminant degradation. Results show that the total BTEX (benzene, toluene, ethylbenzene, and xylenes) concentrations dropped from 9.5 mg/L at the source zone to 0.06 mg/L at a 120-m downgradient line. The calculated BTEX biodegradation capacity (20 mg/L) was much higher than the BTEX concentrations (9.5 mg/L) within the most contaminated area. This indicates that the spilled BTEX could be completely biodegraded through different intrinsic microbial processes using available electron acceptors. Results from polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and nucleotide sequence analyses reveal that the petroleum-hydrocarbon plume caused the bacterial variation in microbial communities, and more than 27 different petroleum-hydrocarbon degrading bacteria were observed in site groundwater indicating that BTEX could be biodegraded by the intrinsic bacterial consortium. Results also imply that the occurrence of intrinsic biodegradation was the major cause of the limited spreading of the BETX plume and reduction of contaminant concentrations. Results from Mann–Kendall test reveal that BTEX concentrations were decreasing in all monitor wells, and the BTEX first-order NA rate was 5.4 × 10−4 1/d along the transport path. The observed NA processes within the plume included the following: (1) depletion of electron acceptors (e.g., dissolved oxygen, nitrate, sulfate), (2) production of the degradation products (e.g., ferrous iron, sulfide, methane, CO2), (3) decreased BTEX concentrations along the groundwater flow direction, (4) a decreased trend of BTEX concentrations in monitor wells, (5) increased alkalinity, (6) increased microbial populations, (7) deceased pH in the spill source area, and (8) preferential removal of certain BTEX components along the transport path. Results indicate that NA can effectively contain the plume, and intrinsic biodegradation mechanisms were the major causes for contaminant removal. Results will be useful in applying NA as an alternative option for the management of petroleum-hydrocarbon contaminated sites.

Application of micronucleus test and comet assay to evaluate BTEX biodegradation

The BTEX (benzene, toluene, ethylbenzene and xylene) mixture is an environmental pollutant that has a high potential to contaminate water resources, especially groundwater. The bioremediation process by microorganisms has often been used as a tool for removing BTEX from contaminated sites. The application of biological assays is useful in evaluating the efficiency of bioremediation processes, besides identifying the toxicity of the original contaminants. It also allows identifying the effects of possible metabolites formed during the biodegradation process on test organisms. In this study, we evaluated the genotoxic and mutagenic potential of five different BTEX concentrations in rat hepatoma tissue culture (HTC) cells, using comet and micronucleus assays, before and after biodegradation. A mutagenic effect was observed for the highest concentration tested and for its respective non-biodegraded concentration. Genotoxicity was significant for all non-biodegraded concentrations and not significant for the biodegraded ones. According to our results, we can state that BTEX is mutagenic at concentrations close to its water solubility, and genotoxic even at lower concentrations, differing from some described results reported for the mixture components, when tested individually. Our results suggest a synergistic effect for the mixture and that the biodegradation process is a safe and efficient methodology to be applied at BTEX-contaminated sites.

Biodegradation of BTEX mixture by Pseudomonas putidaYNS1 isolated from oil‐contaminated soil

The presence of mixed contaminants, such as BTEX (benzene, toluene, ethylbenzene and xylene isomers) can affect the biodegradation, fate and environmental impacts of each compound. To understand the influence of interactions among BTEX compounds on their biodegradation, four bacteria were isolated from oil-contaminated soil and assayed for BTEX biodegradation in vitro. The isolate exhibiting maximum biodegradation was identified as Pseudomonas putida based on the 16S rDNA sequence. The biodegradation of the BTEX compounds was greatly influenced by pH, temperature, and salinity. Substrate mixture studies (binary, tertiary and quaternary) revealed that the presence of toluene increased the biodegradation rate of benzene, ethylbenzene, and xylene.

Remediation of BTEX contaminated groundwater: best technology assessment between pump&treat and bioremediation by oxygen injection

The presence of benzene, toluene, ethylbenzene and xylene (BTEX) dissolved in the groundwater and migrated from a light non-aqueous phase liquid (LNAPL) source in an alluvial aquifer required a remedial action to be taken by the responsible party as established by the Italian regulation (Legislative Decree 152/06 and subsequent amendments). For such purpose, field investigations were conducted on site in order to define the site conceptual model and to identify the appropriate remediation technology to be applied. The remediation design was developed by means of a flow and reactive transport mathematical model, applied to saturated media, using the numerical codes MODFLOW and RT3D. Groundwater field observations showed evidence of occurring BTEX biodegradation processes by bacteria naturally present in the aquifer. Since such specific bacterial activity would be significantly enhanced by the injection of free oxygen in the aquifer, the performance of traditional pump and treat systems (P&T) was assessed and compared with cost/efficiency of reactive oxygen bio-barrier technology (OD). The results showed a clear advantage in terms of cost/efficiency with the application of the OD. This presents an overall cost of about 30% of the P&T installation and maintenance, and it reaches remedial target in a shorter timeframe. Moreover, the system is also applicable as a bioremediation technology in case of Environmental Emergency Measures (MISE). The site examined is part of an industrial plant located in Central Italy.

Groundwater microbial analysis to assess enhanced BTEX biodegradation by nitrate injection at a gasohol-contaminated site

Groundwater samples from a gasohol contaminated aquifer were analyzed to investigate the effects of nitrate injection on microbial communities associated with benzene, toluene, ethylbenzene and xylenes (BTEX) biodegradation. Real-time quantitative PCR was used to quantify total bacteria (16S rDNA), nitrate-, iron-, sulfate-reducing bacteria and methanogens. Anaerobic BTEX degradation potential was assessed by targeting the bssA gene which encodes for benzylsuccinate synthase (BSS), an enzyme that initiates the biodegradation of toluene and xylenes. Aerobic BTEX biodegradation potential was assessed by targeting the catabolic genes: toluene dioxygenase (TOD), naphthalene dioxygenase (NAH), ring hydroxylating monooxygenase (RMO), phenol hydroxylase (PHE), and/or biphenyl dioxygenase (BPH). 16S rDNA gene copies were higher (∼4 × 10 5 cells ml −1) at the plume centerline coinciding with the highest concentrations of BTEX (∼26 mg-total l −1) and ethanol (∼3 mg l −1). Regions with high nitrate consumption coincided with the increased nitrate-reducing bacteria population. The establishment of Fe(III)-reducing zones were unlikely associated with bacteria belonging to Geobacter genus. Sulfate-reducing bacteria and methanogens were not detected corroborating with the geochemical footprints. Nitrate did not stimulate the fortuitous growth of anaerobic BTEX degraders as indicated by the absence of bssA amplification. Nitrate alleviated the high biological oxygen demand (BOD) associated with BTEX and ethanol biodegradation thus maintaining microaerophilic niche that supported the growth of aerobic BTEX degraders as indicated by the presence of PHE gene copy numbers (∼2 × 10 5 PHE gene copies ml −1). Among the genes tested, the non-functional 16S rDNA showed significant correlation ( r 2 = 0.94; p < 0.05) with BTEX (but not ethanol) first-order ( k′) biodegradation rates. Further investigations are required, however, to validate qPCR accuracy and reliability to estimate contaminants removal rates for a wide range of contaminated sites.

Aerobic biodegradation of iso-butanol and ethanol and their relative effects on BTEX biodegradation in aquifer materials

The aerobic biodegradability of iso-butanol, a new biofuel, and its impact on benzene, toluene, ethylbenzene and xylenes (BTEX) degradation was investigated in aerobic microcosms consisting of groundwater and sediment from a California site with a history of gasoline contamination. To the best of our knowledge this is the first study directly examining the effects of iso-butanol on BTEX degradation. Microcosms that received either low (68 μM) or high (3400 μM) concentrations of iso-butanol showed complete biodegradation of iso-butanol within 7 and 23 d, respectively, of incubation at 15 °C under aerobic conditions. A maximum utilization rate coefficient of 2.3 ± 0.1 × 10 −7 μmol cell −1 h −1 and a half saturation constant of 610 ± 54 μM were regressed from the iso-butanol data. Iso-butanol biodegradation resulted in transient formation of the degradation intermediate products iso-butylaldehyde and iso-butyric acid, and both compounds were subsequently degraded within the timeframe of the experiments. Ethanol was biodegraded more slowly than iso-butanol. Ethanol also exhibited greater adverse impacts on BTEX biodegradation than iso-butanol. Results of the study suggest that iso-butanol added to fuels will be readily biodegraded in the environment under aerobic conditions without the accumulation of major intermediate products ( iso-butylaldehyde and iso-butyric acid), and that it will pose less impacts on BTEX biodegradation than ethanol.

BTEX biodegradation by bacteria from effluents of petroleum refinery

Groundwater contamination with benzene, toluene, ethylbenzene and xylene (BTEX) has been increasing, thus requiring an urgent development of methodologies that are able to remove or minimize the damages these compounds can cause to the environment. The biodegradation process using microorganisms has been regarded as an efficient technology to treat places contaminated with hydrocarbons, since they are able to biotransform and/or biodegrade target pollutants. To prove the efficiency of this process, besides chemical analysis, the use of biological assessments has been indicated. This work identified and selected BTEX-biodegrading microorganisms present in effluents from petroleum refinery, and evaluated the efficiency of microorganism biodegradation process for reducing genotoxic and mutagenic BTEX damage through two test-systems: Allium cepa and hepatoma tissue culture (HTC) cells. Five different non-biodegraded BTEX concentrations were evaluated in relation to biodegraded concentrations. The biodegradation process was performed in a BOD Trak Apparatus (HACH) for 20 days, using microorganisms pre-selected through enrichment. Although the biodegradation usually occurs by a consortium of different microorganisms, the consortium in this study was composed exclusively of five bacteria species and the bacteria Pseudomonas putida was held responsible for the BTEX biodegradation. The chemical analyses showed that BTEX was reduced in the biodegraded concentrations. The results obtained with genotoxicity assays, carried out with both A. cepa and HTC cells, showed that the biodegradation process was able to decrease the genotoxic damages of BTEX. By mutagenic tests, we observed a decrease in damage only to the A. cepa organism. Although no decrease in mutagenicity was observed for HTC cells, no increase of this effect after the biodegradation process was observed either. The application of pre-selected bacteria in biodegradation processes can represent a reliable and effective tool in the treatment of water contaminated with BTEX mixture. Therefore, the raw petroleum refinery effluent might be a source of hydrocarbon-biodegrading microorganisms.

Control of petroleum-hydrocarbon contaminated groundwater by intrinsic and enhanced bioremediation

In the first phase of this study, the effectiveness of intrinsic bioremediation on the containment of petroleum hydrocarbons was evaluated at a gasoline spill site. Evidences of the occurrence of intrinsic bioremediation within the BTEX (benzene, toluene, ethylbenzene, and xylenes) plume included (1) decreased BTEX concentrations; (2) depletion of dissolved oxygen (DO), nitrate, and sulfate; (3) production of dissolved ferrous iron, methane, and CO 2; (4) deceased pH and redox potential; and (5) increased methanogens, total heterotrophs, and total anaerobes, especially within the highly contaminated areas. In the second phase of this study, enhanced aerobic bioremediation process was applied at site to enhance the BTEX decay rates. Air was injected into the subsurface near the mid-plume area to biostimulate the naturally occurring microorganisms for BTEX biodegradation. Field results showed that enhanced bioremediation process caused the change of BTEX removal mechanisms from anaerobic biodegradation inside the plume to aerobic biodegradation. This variation could be confirmed by the following field observations inside the plume due to the enhanced aerobic bioremediation process: (1) increased in DO, CO 2, redox potential, nitrate, and sulfate, (2) decreased in dissolved ferrous iron, sulfide, and methane, (3) increased total heterotrophs and decreased total anaerobes. Field results also showed that the percentage of total BTEX removal increased from 92% to 99%, and the calculated total BTEX first-order natural attenuation rates increased from 0.0092% to 0.0188% per day, respectively, after the application of enhanced bioremediation system from the spill area to the downgradient area (located approximately 300 m from the source area).

Determination of Biodegradation Products from Benzene, Toluene, Ethylbenzene and Xylenes in Seawater by Purge and Trap Gas Chromatography

Benzene, toluene, ethylbenzene and xylenes (BTEX) are commonly found in crud oil and used as the direct indicating substance in the Geochemical Investigation for Oil and Gas. BTEX are easily volatile and can be degraded by microorganism, which affects the precise measurement seriously. A method for the determination of the biodegradation process of BTEX in seawater is described. The determination is performed by dynamic headspace (purge and trap) gas chromatography with a photoionization detector (PID). The features of the described method are: detection limits of 7.3–13.2 ng l −1, no sample preparation required, and recovery rate of 92.84%–100.92%. The results of the BTEX biodegradation process are of great significance in the collection, transportation, preservation, and measurement of samples in the Geochemical Investigation for Oil and Gas.

Analysis of benzylsuccinates in groundwater by liquid chromatography/tandem mass spectrometry and its use for monitoring in situ BTEX biodegradaton.

Benzylsuccinic acid (BS) and methylbenzylsuccinic acid (MeBS) isomers have been proposed as distinctive indicators of anaerobic toluene and xylene metabolism in fuel-contaminated aquifers; however, labor-intensive analytical procedures have limited their analysis at field sites. In this article, a rapid electrospray LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry) method for benzylsuccinates is described that involves selected reaction monitoring, internal standard quantification with [ring-2H5]BS, small sample size (<1 mL), and no extraction/concentration steps. The highly selective LC/MS/ MS method was shown to be sensitive (detection limits ca. 0.3 microg/L), accurate, and precise. The method was used to characterize geographic and temporal distributions of BS and MeBS isomers in an anaerobic, hydrocarbon-contaminated aquifer. BS was never detected, and MeBS isomers were detected in the three wells with the highest concentrations of BTEX (benzene, toluene, ethylbenzene, and xylenes); MeBS concentrations ranged from <0.3 to 205 microg/L. A strong linear correlation (r2 = 0.94; n = 12) was found between concentrations of total MeBS isomers and their parent compounds, xylenes, which suggests that anaerobic xylene metabolism at this site was probably first-order rather than zero-order. The novel LC/MS/MS method for BS and MeBS isomers is a promising technique for rapid and reliable monitoring of in situ bioremediation of gasoline-contaminated groundwater.

Thermophilic biodegradation of BTEX by two Thermus Species

Two thermophilic bacteria, Thermus aquaticus ATCC 25104 and Thermus species ATCC 27978, were investigated for their abilities to degrade BTEX (benzene, toluene, ethylbenzene, and xylenes). Thermus aquaticus and the Thermus sp. were grown in a nominal medium at 70 degrees C and 60 degrees C, respectively, and resting cell suspensions were used to study BTEX biodegradation at the same corresponding temperatures. The degradation of BTEX by these cell suspensions was measured in sealed serum bottles against controls that also displayed significant abiotic removals of BTEX under such high-temperature conditions. For T. aquaticus at a suspension density of only 1.3 x 10(7) cells/mL and an aqueous total BTEX concentration of 2.04 mg/L (0.022 mM), benzene, toluene, ethylbenzene, m-xylene, and an unresolved mixture of o-and p-xylenes were biodegraded by 10, 12, 18, 20, and 20%, respectively, after 45 days of incubation at 70 degrees C. For the Thermus sp. at a suspension density of 1.1 x 10(7) cells/mL and an aqueous total BTEX concentration of 6.98 mg/L (0.079 mM), benzene, toluene, ethylbenzene, m-xylene, and the unresolved mixture of o-and p-xylenes were biodegraded by 40, 35, 32, 33, and 33%, respectively, after 45 days of incubation at 60 degrees C. Raising the BTEX concentrations lowered the extents of biodegradation. The biodegradations of both benzene and toluene were enhanced when T. aquaticus and the Thermus sp. were pregrown on catechol and o-cresol, respectively, as carbon sources. Use of [U-(14)C]benzene and [ring-(14)C]toluene verified that a small fraction of these two compounds was metabolized within 7 days to water-soluble products and CO(2) by these nongrowing cell suspensions. Our investigation also revealed that the nominal medium can be simplified by eliminating the yeast extract and using a higher tryptone concentration (0.2%) without affecting the growth and BTEX degrading activities of these cells. (c) 1995 John Wiley & Sons, Inc.