Effect of carbon source during enrichment on BTEX degradation by anaerobic mixed bacterial cultures

A comprehensive study on the effects of different carbon sources during the bacterial enrichment on the removal performances of benzene, toluene, ethylbenzene, and xylenes (BTEX) compounds when present as a mixture was conducted. Batch BTEX removal kinetic experiments were performed using cultures enriched with individual BTEX compounds or BTEX as a mixture or benzoate alone or benzoate–BTEX mixture. An integrated Monod-type non-linear model was developed and a ratio between maximum growth rate (μ _max) and half saturation constant (K_s) was used to fit the non-linear model. A higher μ _max/K_s indicates a higher affinity to degrade BTEX compounds. Complete removal of BTEX mixture was observed by all the enriched cultures; however, the removal rates for individual compounds varied. Degradation rate and the type of removal kinetics were found to be dependent on the type of carbon source during the enrichment. Cultures enriched on toluene and those enriched on BTEX mixture were found to have the greatest μ _max/K_s and cultures enriched on benzoate had the least μ _max/K_s. Removal performances of the cultures enriched on all different carbon sources, including the ones enriched on benzoate or benzoate–BTEX mixture were also improved during a second exposure to BTEX. A molecular analysis showed that after each exposure to the BTEX mixture, the cultures enriched on benzoate and those enriched on benzoate–BTEX mixture had increased similarities to the culture enriched on BTEX mixture.     

Detection of benzene, toluene, ethyl benzene, and xylenes (BTEX) using toluene dioxygenase-peroxidase coupling reactions

We have developed a simple, whole-cell bioassay for the detection of bioavailable benzene, toluene, ethyl benzene, and xylenes (BTEX) and similar compounds. A genetically engineered E. coli strain expressing toluene dioxygenase (TDO) and toluene dihydrodiol dehydrogenase (TodD) was constructed, enabling the conversion of BTEX into their respective catechols, which were quickly converted into colored products by a horseradish peroxidase (HRP)-coupled reaction. The intensity of the color formation was correlated to concentrations of the BTEX compounds. Under the optimized conditions, a detection limit (defined as three times the standard deviation of the response obtained from the blank) of 10, 10, 20, and 50 microM was observed for benzene, toluene, ethyl benzene, and xylene, respectively. The bioassay was selective toward BTEX-related compounds with no interference observed with commonly used pesticides, herbicides, and organic solvent. The bioassay was very stable with little change in response over a 10-week period. The excellent stability suggests that the reported bioassay may be suitable for field monitoring of BTEX to identify and track contaminated water and follow the bioremediation progress.     

Degradation of benzene, toluene, ethylbenzene, and xylenes (BTEX) by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Degradation of the BTEX (benzene, toluene, ethylbenzene, and o-, m-, and p-xylenes) group of organopollutants by the white-rot fungus Phanerochaete chrysosporium was studied. Our results show that the organism efficiently degrades all the BTEX components when these compounds are added either individually or as a composite mixture. Degradation was favored under nonligninolytic culture conditions in malt extract medium, in which extracellular lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) are not produced. The noninvolvement of LIPs and MNPs in BTEX degradation was also evident from in vitro studies using concentrated extracellular fluid containing LIPs and MNPs and from a comparison of the extents of BTEX degradation by the wild type and the per mutant, which lacks LIPs and MNPs. A substantially greater extent of degradation of all the BTEX compounds was observed in static than in shaken liquid cultures. Furthermore, the level of degradation was relatively higher at 25 than at 37 degrees C, but pH variations between 4.5 and 7.0 had little effect on the extent of degradation. Studies with uniformly ring-labeled [14C]benzene and [14C]toluene showed substantial mineralization of these compounds to 14CO2.     

Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO2. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Thermophilic biodegradation of BTEX by two consortia of anaerobic bacteria

Two thermophilic anaerobic bacterial consortia (ALK-1 and LLNL-1), capable of degrading the aromatic fuel hydrocarbons, benzene, toluene, ethylbenzene, and the xylenes (BTEX compounds), were developed at 60 °C from the produced water of ARCO'S Kuparuk oil field at Alaska and the subsurface water at the Lawrence Livermore National Laboratory gasoline-spill site, respectively. Both consortia were found to grow at 45–75 °C on BTEX compounds as their sole carbon and energy sources with 50 °C being the optimal temperature. With 3.5 mg total BTEX added to sealed 50-ml serum bottles, which contained 30 ml mineral salts medium and the consortium, benzene, toluene, ethylbenze, m-xylene, and an unresolved mixture of o- and p-xylenes were biodegraded by 22%, 38%, 42%, 40%, and 38%, respectively, by ALK-1 after 14 days of incubation at 50 °C. Somewhat lower, but significant, percentages of the BTEX compounds also were biodegraded at 60 °C and 70 °C. The extent of biodegradation of these BTEX compounds by LLNL-1 at each of these three temperatures was slightly less than that achieved by ALK-1. Use of [ring-¹⁴C]toluene in the BTEX mixture incubated at 50 °C verified that 41% and 31% of the biodegraded toluene was metabolized within 14 days to water-soluble products by ALK-1 and LLNL-1, respectively. A small fraction of it was mineralized to ¹⁴CO₂. The use of [U-¹⁴C]benzene revealed that 2.6%–4.3% of the biodegraded benzene was metabolized at 50 °C to water-soluble products by the two consortia; however, no mineralization of the degraded [U-¹⁴C]benzene to ¹⁴CO₂ was observed. The biodegradation of BTEX at all three temperatures by both consortia was tightly coupled to sulfate reduction as well as H₂S generation. None was observed when sulfate was omitted from the serum bottles. This suggests that sulfate-reducing bacteria are most likely responsible for the observed thermophilic biodegradation of BTEX in both consortial cultures. Received: 12 July 1996 / Received revision: 31 December 1996 / Accepted: 31 January 1997     

BTEX-Contaminated Gas Treatment in a Shallow, Sparged, Suspended-Growth Bioreactor

Treatment of a gas contaminated with a mixture of benzene, toluene, ethylbenzene, and o-xylene (BTEX) compounds in a 40-cm-deep laboratory-scale bioreactor containing suspended biomass was investigated. Gas treatment efficiency was not significantly impacted by different BTEX mixtures, and approximately 99% removal was achieved for volumetric loadings of 11 to 18 mg-BTEX/L-reactor volume/hr (specific biomass loadings of 0.27 to 0.83 g-BTEX/g-VSS/d; inlet concentrations of total BTEX of 2.3 to 4.3 mg/L) and operational solids retention times (SRTs) of 1.7, 2.7, and 9.2 days. Maximum specific biodegradation rates of the reactor biomass increased as the reactor SRTs decreased. Under specific loadings greater than 1 g-BTEX/g-VSS/d the gas treatment became biokinetically limited, such that BTEX and unidentified BTEX metabolites accumulated in the bioreactor liquid over time. BTEX gas-liquid mass transfer was sufficient in the 40-cm-deep sparged liquid reactor to provide high BTEX treatment efficiency.     

Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and o-Xylene in Sediment-Free Iron-Reducing Enrichment Cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO₂. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Kinetics of BTEX degradation in a packed-bed anaerobic reactor

The ever-increasing diversity of industrial activity is responsible for the discharge of compounds that are toxic or difficult to degrade into the environment. Some of the compounds found in surface and ground waters, usually deriving from the contamination of oil-based products, are benzene, toluene, ethylbenzene and xylenes (BTEX). To remove these compounds from contaminated water, a bench-scale horizontal-flow anaerobic immobilized biomass reactor, containing anaerobic biomass from various sources immobilized in polyurethane foam matrices, was employed to treat a synthetic substrate composed of protein, carbohydrates and BTEX solution in ethanol, as well as a BTEX solution in ethanol as the sole carbon source. The reactor removed up to 15.0 mg/l of each BTEX compound over a hydraulic detention time of 11.4 h. A first-order kinetic model fitted the experimental data well, showing correlation coefficients higher than 0.994. The apparent first-order coefficient values, , ranged from 8.4±1.5 day⁻¹ for benzene to 10.7±1.4 day⁻¹ for o-xylene in the presence of ethanol, protein and carbohydrates, and from 10.0±2.0 day⁻¹ for benzene to 13.0±1.7 day⁻¹ for o-xylene in the presence of ethanol. The BTEX degradation rates estimated here were 10- to 94-fold higher than those found in reports on microcosm studies.     

Effects of Oxygenation on Hydrocarbon Biodegradation in a Hypoxic Environment

In 1984, an underground storage tank leaked approximately 41,000 L of gasoline into the ground water at the Naval Construction Battalion Command in Port Hueneme, CA (USA). Benzene, toluene, ethylbenzene, and xylenes (BTEX) contamination stimulated remedial action. In 1995, a ground water circulation well (GCW) and network of surrounding monitoring wells were installed. After year of operation, dissolved oxygen and nitrate concentrations remained low in all monitoring wells. Benzene utilization (the sum of respiration, uptake, and conversion to polar compounds) ranged from 0.03 to 4.6 µg L^-1 h^-1, and toluene utilization ranged from 0.01 to 5.2 µg L^-1 h^-1. Heterotrophic bacterial productivity (total carbon assimilation) increased dramatically in the GCW, although benzene and toluene utilization decreased markedly relative to surrounding wells. Benzene and toluene uptake accounted for a significant proportion (mean=22%) of the heterotrophic bacterial productivity except within the GCW, indicating other fuel contaminant or indigenous organic carbon and not BTEX compounds served as primary carbon source. The GCW effectively air-stripped BTEX compounds, but failed to stimulate benzene and toluene biodegradation and thus would not be effective for stimulating BTEX bioremediation under current deployment parameters. Air stripping was three orders of magnitude more effective than biodegradation for removing benzene and toluene in the GCW.     

Utilization of monocyclic aromatic hydrocarbons individually and in mixture by bacteria isolated from petroleum-contaminated soil

The fate of benzene, ethylbenzene, toluene, xylenes (BTEX) compounds through biodegradation was investigated using two different bacteria, Ralstonia picketti (BP-20) and Alcaligenes piechaudii (CZOR L-1B). These bacteria were isolated from extremely polluted soils contaminated with petroleum hydrocarbons. PCR and Fatty Acid Methyl Ester (FAME) were used to identify the isolates. In this study, BTEX biodegradation, applied as a mixture or as individual compounds by the bacteria was evaluated. Both bacteria were shown to degrade each of the BTEX compounds individually and in mixture. However, Alcaligenes piechaudii was a better degrader of BTEXs both in the mixture and individually. Differences between BTEX biodegradation in the mixture and individually were observed, especially in the case of benzene. The degradation of all BTEXs in the mixture was lower than the degradation of individual compounds for both bacteria tested. In the all experiments, toluene and m + p- xylenes were better removed than the other BTEXs. No intermediates of biodegradation were detected. Biosurfactant production was observed by culture techniques. In addition, 3-hydroxy fatty acids, important in biosurfactant production, were observed by FAME analysis. The test results indicate that the bacteria could contribute to bioremediation of aromatic hydrocarbon pollution.     

Iron (II) Activated Persulfate Oxidation of MGP Contaminated Soil

The persulfate anion (S₂O₈ ^2?) is a strong oxidant with a redox potential of 2.01 V. However, when mixed with iron (II), it is capable of forming the sulfate radical (SO₄ ^?.) that has an even higher redox potential (E ^o = 2.6 V). In these studies the sulfate radical was investigated to determine if it was a feasible oxidant for the destruction of BTEX and PAH compounds found in MGP contaminated soil. The sulfate radical was generated by either the sequential addition of iron (II) solutions or by a single addition of a citric acid chelated iron (II) solution. The sequentially added iron destroyed 86% of the total BTEX concentration and 56% of the total PAH concentration in the soil. The citric acid chelated iron destroyed 95% of the total BTEX concentration and 85% of the total PAH concentration. A second dose of persulfate and citric acid chelated iron (II) resulted in the destruction of 99% of the total BTEX concentration and 92% of the total PAH concentration. In both the sequential and chelated iron studies the lower molecular weight BTEX compounds were oxidized to a greater extent than the higher molecular weight BTEX compounds, whereas the oxidation of PAH compounds showed no preference to molecular weight.     

Lab-Scale Biodegradation Study of BTEX under the Unsaturated Condition and Its Effect on Soil Matric Potential

Benzene, toluene, ethylbenzene, and xylene are collectively known as BTEX which contributes to volatile environmental contaminants. This present study investigates the microbial degradation of BTEX in batch and continuous soil column experiments and its effects on soil matric potential. Batch degradation experiments were performed with different initial concentrations of BTEX using the BTEX tolerant culture isolated from petroleum-contaminated soil. In batch study, the degradation pattern for single substrate showed that xylene was degraded much faster than other compounds followed by ethylbenzene, toluene, and benzene with the highest μ_max = 0.140 h^?1 during initial substrate concentration of 100 mg L^?1. Continuous degradation experiments were performed in a soil column with an inlet concentration of BTEX of about 2000 mg L^?1 under unsaturated flow in anaerobic condition. BTEX degradation pattern was studied with time and the matric potential of the soil at different parts along the length of the column were determined at the end of the experiment. In continuous degradation study, BTEX compounds were degraded with different degradation pattern and an increase in soil matric potential was observed with an increase in depth from top to bottom in the column with applied suction head. It was found that column biodegradation contributed to 69.5% of BTEX reduction and the bacterial growth increased the soil matric potential of about 34% on an average along the column height. Therefore, this study proves that it is significant to consider soil matric potential in modeling fate and transport of BTEX in unsaturated soils.     

Plants as Bio-Indicators of Subsurface Conditions: Impact of Groundwater Level on BTEX Concentrations in Trees

Numerous studies have demonstrated trees’ ability to extract and translocate moderately hydrophobic contaminants, and sampling trees for compounds such as BTEX can help delineate plumes in the field. However, when BTEX is detected in the groundwater, detection in nearby trees is not as reliable an indicator of subsurface contamination as other compounds such as chlorinated solvents. Aerobic rhizospheric and bulk soil degradation is a potential explanation for the observed variability of BTEX in trees as compared to groundwater concentrations. The goal of this study was to determine the effect of groundwater level on BTEX concentrations in tree tissue. The central hypothesis was increased vadose zone thickness promotes biodegradation of BTEX leading to lower BTEX concentrations in overlying trees. Storage methods for tree core samples were also investigated as a possible reason for tree cores revealing lower than expected BTEX levels in some sampling efforts. The water level hypothesis was supported in a greenhouse study, where water table level was found to significantly affect tree BTEX concentrations, indicating that the influx of oxygen coupled with the presence of the tree facilitates aerobic biodegradation of BTEX in the vadose zone.     

Distribution of Petroleum and Aromatic Hydrocarbons at a Former Crude Oil and Natural Gas Production Facility

Site characterization and remediation activities were performed at a former crude oil and natural gas production facility prior to redevelopment of the site. Field activities included delineation, excavation and segregation of approximately 1,250,000 m³ of soil impacted by total petroleum hydrocarbons (TPH) and the aromatic volatile organic compounds (VOCs) benzene, toluene, ethylbenzene, and xylenes (hereafter, collectively referred to as BTEX). Petroleum hydrocarbon chain length information was used to determine whether remediation was required in impacted areas, because the site-specific cleanup values for TPH compounds, established by the California State Regional Water Quality Control Board (RWQCB), were based on hydrocarbon chain length. Site-specific cleanup levels were also established by the RWQCB for BTEX. Subsurface investigation activities performed at the site indicated that the mean percentage of condensate and TPH compounds in the gasoline range was significantly greater at depths ranging from 4.6 to 18 m than in shallower samples. There was no significant difference in the mean concentration of BTEX compounds and mean percentage of diesel range and heavier hydrocarbons with depth. The occurrence of BTEX, diesel range, and heavier hydrocarbons at depth may result from preferential pathways for downward migration of contaminants, including blown out wells, abandoned wellbores, and the presence of faults. Vapor phase diffusion may also be a major transport mechanism controlling movement of BTEX compounds beneath the site.     

Plants as Bio-Indicators of Subsurface Conditions: Impact of Groundwater Level on Btex Concentrations in Trees

Numerous studies have demonstrated trees’ ability to extract and translocate moderately hydrophobic contaminants, and sampling trees for compounds such as BTEX can help delineate plumes in the field. However, when BTEX is detected in the groundwater, detection in nearby trees is not as reliable an indicator of subsurface contamination as other compounds such as chlorinated solvents. Aerobic rhizospheric and bulk soil degradation is a potential explanation for the observed variability of BTEX in trees as compared to groundwater concentrations. The goal of this study was to determine the effect of groundwater level on BTEX concentrations in tree tissue. The central hypothesis was increased vadose zone thickness promotes biodegradation of BTEX leading to lower BTEX concentrations in overlying trees. Storage methods for tree core samples were also investigated as a possible reason for tree cores revealing lower than expected BTEX levels in some sampling efforts. The water level hypothesis was supported in a greenhouse study, where water table level was found to significantly affect tree BTEX concentrations, indicating that the influx of oxygen coupled with the presence of the tree facilitates aerobic biodegradation of BTEX in the vadose zone.     

Aerobic Biotransformation of Gasoline Aromatics in MultiComponent Mixtures

The primary objective of this study was to evaluate the impact of substrate interactions on the biotransformation rates and mineralization potentials of gasoline monoaromatics and methyl tert-butyl ether (MTBE), compounds that commonly co-exist in groundwater contaminant plumes. A mixed culture was derived from gasoline-contaminated aquifer material using toluene as the enrichment substrate. Two pure cultures, Rhodococcus sp. RR1 and RR2, were isolated from the mixed culture. The three toluene-grown cultures were shown to biotransform all of the six BTEX compounds (benzene, toluene, ethylbenzene, o-xylene, m-xylene, and p-xylene), both individually and in mixtures, over a broad range of concentrations. The mixed culture was shown to degrade all of the BTEX compounds to ¹⁴CO₂, while the two isolates mineralized BTE(m-/p-)X, but biotransformed o-xylene without production of carbon dioxide. Studies to evaluate substrate interactions caused by the concurrent presence of multiple BTEX compounds during their biodegradation revealed a number of patterns,including competitive inhibition and cometabolism. Ethylbenzene was shown to significantly inhibit BTX degradation in mixtures. MTBE was not biodegraded by any of the three toluene-grown cultures over a range of MTBE concentrations. Furthermore, the presence of MTBE at concentrations of 2 to 100?mg/L had no effect on BTEX biotransformation rates.     

Effect of Benzene, Toluene, Ethylbenzene, and p-Xylene (BTEX) Mixture on Biodegradation of Methyl tert-Butyl Ether (MTBE) and tert-Butyl Alcohol (TBA) by Pure Culture UC1

The effect of a BTEX mixture on the biodegradation of methyl tert-butyl ether (MTBE) and its degradation intermediate, tert-butyl alcohol (TBA) was investigated in the pure bacterial culture UC1, which has been identified to be a strain of the known MTBE-degrader PM1 based on greater than 99% 16S rDNA similarity. Several degradation studies were carried out on UC1 at three initial concentration levels of MTBE or TBA: 6-7; 15-17; and 40-45 mg/l, both with and without BTEX present cumulatively at about half of the MTBE or TBA molar mass in the system. The BTEX mixture was observed not to affect either the rate or the degradation lag period of MTBE or TBA degradation, except that the TBA degradation rate actually increased when BTEX was present initially in the highest concentration studies. When serving as the sole substrate, the MTBE degradation rate ranged from 48 +/- 1.2 to 200 +/- 7.0 mg(MTBE)/g(dw) h, and the TBA degradation rate from 140 +/- 18 to 530 +/- 70 mg(TBA)/g(dw) h. When present with BTEX, MTBE and TBA rates ranged from 46 +/- 2.2 to 210 +/- 14 and 170 +/- 28 to 780 +/- 43 mg(TBA)/g(dw) h, respectively. In studies where varying concentrations of TBA were present with 5 mg/l MTBE, both compounds were degraded simultaneously with no obvious preference for either substrate. In the highest concentration study of TBA with 5 mg/l MTBE, BTEX was also observed to increase the ultimate rate of TBA degradation. In addition to exploring the affect of BTEX, this study also provides general insight into the metabolism of MTBE and TBA by pure culture UC1.     

SEQUENCE Visualization of Natural Attenuation Trends at Hill Air Force Base, Utah

For monitored natural attenuation to be considered as an acceptable remedial approach, the proponent must clearly document converging lines of evidence that illustrate the effectiveness of this measure. SEQUENCE, a visualization tool based on a modified radial diagram approach, is ideally suited for evaluating spatial and temporal trends that provide supporting evidence for the efficacy of monitored natural attenuation. SEQUENCE was applied to evaluate the natural attenuation of benzene, toluene, ethylbenzene, and total xylene (BTEX) concentrations observed in groundwater at Hill Air Force Base, Utah. SEQUENCE-BTEX maps provided an efficient means of documenting the declining BTEX concentrations downgradient from the source area. SE-QUENCE-Redox maps were used to facilitate a correlation between elevated BTEX concentrations; decreasing electron acceptor concentrations (oxygen, nitrate, and sulfate); and elevated metabolic byproduct concentrations (iron(II) and methane) providing a second line of evidence that suggests BTEX compounds are being destroyed through biodegradation processes downgradient from the source area. Site-specific guidelines for interpolating representative data sets for use with the SEQUENCE approach are discussed.     

Degradation of BTEX compounds in liquid media and in peat biofilters

A mixed culture, enriched from Sphagnum peat moss, contaminated with gasoline vapours, degraded individual and mixed components of BTEX (benzene, toluene, ethylbenzene, xylene). Complete degradation of radiolabelled toluene by the mixed culture was observed in mineralisation studies. Individual isolates from a mixed culture containingPseudomonas maltophilia, P. testosteroni andP. putida biotype A exhibited contrasting BTEX degradation patterns. WhileP. putida biotype A degraded all of the BTEX compounds,P. maltophilia andP. testosteroni, appeared unable to degrade benzene and xylenes, respectively. When the peat, inoculated with the mixed culture, was used as a biofilter (6.2 cm diameter ×93 cm length) for degradation of toluene and ethylbenzene vapours, percentage removal efficiencies were 99 and 85, respectively. When the capacity of the biofilter to degrade a combination of BTEX compounds was evaluated, percentage removal efficiencies for toluene, ethylbenzene,p-xylene,o-xylene and benzene were 99, 85, 82, 80 and 78, respectively. The importance of using the mixed culture as an inoculum in the biofilter was established and also the relationship between contaminated vapour flow rate and percentage removal efficiency.