Dynamics of an oligotrophic bacterial aquifer community during contact with a groundwater plume contaminated with benzene, toluene, ethylbenzene, and xylenes: an in situ mesocosm study

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.     

Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions forin situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, orp-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 10³ to 10⁵ bacteria ml^–1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain ofPseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.Abbreviations BTEX benzene, toluene, ethylbenzene, andp-xylene - HPLC high performance liquid chromatography - GAC granular activated carbon     

Dynamics of an Oligotrophic Bacterial Aquifer Community during Contact with a Groundwater Plume Contaminated with Benzene, Toluene, Ethylbenzene, and Xylenes: an In Situ Mesocosm Study

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.     

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.     

Indole-based assay to assess the effect of ethanol on <Emphasis Type="Italic">Pseudomonas putida</Emphasis> F1 dioxygenase activity

Toluene dioxygenase (TDO) is ubiquitous in nature and has a broad substrate range, including benzene, toluene, ethylbenzene and xylenes (BTEX). Pseudomonas putida F1 (PpF1) induced on toluene is known to produce indigo from indole through the activity of TDO. In this work, a spectrophotometric assay previously developed to measure indole to indigo production rates was modified to characterize the effects of various ethanol concentrations on toluene aerobic biodegradation activity and assess catabolite repression of TDO. Indigo production rate by cells induced on toluene alone was 0.0012 ± 0.0006 OD₆₁₀ min⁻¹. The presence of ethanol did not fully repress TDO activity when toluene was also available as a carbon source. However, indigo production rates by PpF1 grown on ethanol:toluene mixtures (3:1 w/w) decreased by approximately 50%. Overall, the proposed spectrophotometric assay is a simple approach to quantify TDO activity, and demonstrates how the presence of ethanol in groundwater contaminated with reformulated gasoline is likely to interfere with naturally occurring microorganisms from fully expressing their aerobic catabolic potential towards hydrocarbons bioremediation.     

Kinetics of BTEX biodegradation by a coculture of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> and<Emphasis Type="Italic"> Pseudomonas fluorescens</Emphasis> under hypoxic conditions

Pseudomonas putida and Pseudomonas fluorescens present as a coculture were studied for their abilities to degrade benzene, toluene, ethylbenzene, and xylenes (collectively known as BTEX) under various growth conditions. The coculture effectively degraded various concentrations of BTEX as sole carbon sources. However, all BTEX compounds showed substrate inhibition to the bacteria, in terms of specific growth, degradation rate, and cell net yield. Cell growth was completely inhibited at 500mgl^–1 of benzene, 600mgl^–1 of o-xylene, and 1000mgl^–1 of toluene. Without aeration, aerobic biodegradation of BTEX required additional oxygen provided as hydrogen peroxide in the medium. Under hypoxic conditions, however, nitrate could be used as an alternative electron acceptor for BTEX biodegradation when oxygen was limited and denitrification took place in the culture. The carbon mass balance study confirmed that benzene and toluene were completely mineralized to CO₂ and H₂O without producing any identifiable intermediate metabolites.     

Correcting for Sample Contamination in Genotype Calling of DNA Sequence Data

DNA sample contamination is a frequent problem in DNA sequencing studies and can result in genotyping errors and reduced power for association testing. We recently described methods to identify within-species DNA sample contamination based on sequencing read data, showed that our methods can reliably detect and estimate contamination levels as low as 1%, and suggested strategies to identify and remove contaminated samples from sequencing studies. Here we propose methods to model contamination during genotype calling as an alternative to removal of contaminated samples from further analyses. We compare our contamination-adjusted calls to calls that ignore contamination and to calls based on uncontaminated data. We demonstrate that, for moderate contamination levels (5%–20%), contamination-adjusted calls eliminate 48%–77% of the genotyping errors. For lower levels of contamination, our contamination correction methods produce genotypes nearly as accurate as those based on uncontaminated data. Our contamination correction methods are useful generally, but are particularly helpful for sample contamination levels from 2% to 20%.     

Phospholipid Fatty Acid Composition, Biomass, and Activity of Microbial Communities from Two Soil Types Experimentally Exposed to Different Heavy Metals

The phospholipid fatty acid (PLFA) pattern was analyzed in a forest humus and in an arable soil experimentally polluted with Cd, Cu, Ni, Pb, or Zn at different concentrations. In both soil types, there were gradual changes in the PLFA patterns for the different levels of metal contamination. The changes in the forest soil were similar irrespective of which metal was used, while in the arable soil the changes due to Cu contamination differed from those due to the other metals. Several PLFAs reacted similarly to the metal amendments in the two soil types, while others showed different responses. In both soils, the metal pollution resulted in a decrease in the iso-branched PLFAs i15:0 and i17:0 and in the monounsaturated 16:1ω5 and 16:1ω7c fatty acids, while increases were found for i16:0, the branched br17:0 and br18:0, and the cyclopropane cy17:0 fatty acids. In the forest soil, the methyl branched PLFAs 10Me16:0, 10Me17:0, and 10Me18:0 increased in metal-polluted soils, indicating an increase in actinomycetes, while in the arable soil a decrease was found for 10Me16:0 and 10Me18:0 in response to most metals. The bacterial PLFAs 15:0 and 17:0 increased in all metal-contaminated samples in the arable soil, while they were unaffected in the forest soil. Fatty acid 18:2ω6, which is considered to be predominantly of fungal origin, increased in the arable soil, except in the Cu-amended samples, in which it decreased instead. Effects on the PLFA patterns were found at levels of metal contamination similar to or lower than those at which effects on ATP content, soil respiration, or total amount of PLFAs had occurred.     

Substrate interactions during the biodegradation of BTEX and THF mixtures by Pseudomonas oleovorans DT4

The efficient tetrahydrofuran (THF)-degrading bacterium, Pseudomonas oleovorans DT4 was used to investigate the substrate interactions during the aerobic biotransformation of THF and BTEX mixtures. Benzene and toluene could be utilized as growth substrates by DT4, whereas cometabolism of m-xylene, p-xylene and ethylbenzene occurred with THF. In binary mixtures, THF degradation was delayed by xylene, ethylbenzene, toluene and benzene in descending order of inhibitory effects. Conversely, benzene (or toluene) degradation was greatly enhanced by THF leading to a higher degradation rate of 39.68 mg/(h g dry weight) and a shorter complete degradation time about 21 h, possibly because THF acted as an “energy generator”. Additionally, the induction experiments suggested that BTEX and THF degradation was initiated by independent and inducible enzymes. The transient intermediate hydroquinone was detected in benzene biodegradation with THF while catechol in the process without THF, suggesting that P. oleovorans DT4 possessed two distinguished benzene pathways.     

Effects of long-term benzene pollution on bacterial diversity and community structure in groundwater

In this study we analysed the relationship between bacterial community structures and geochemistry of groundwater in a sandstone aquifer (SIReN site) impacted mainly by BTEX hydrocarbons (benzene, toluene, ethylbenzene and xylenes), of which benzene is most abundant. The long-term presence of benzene reduced bacterial diversity: in groundwaters contaminated with more than 1.8 x 10(4) microg l(-1) of benzene, bacterial diversity was half of that in clean groundwaters. Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rDNA revealed that the community structures were very similar in uncontaminated groundwaters, whereas communities subjected to long-term benzene contamination were different, not only from uncontaminated groundwater communities, but also from each other. Canonical correspondence analysis of the community profiles and the geochemical data showed that this divergence in community structure was not primarily caused by the direct toxic or stressful effects of benzene, but by the environmental changes brought about by benzene metabolism, in particular a decrease in redox potential.     

Isolation and characterization of ethylbenzene degrading <Emphasis Type="Italic">Pseudomonas putida</Emphasis> E41

Pseudomonas putida E41 was isolated from oil-contaminated soil and showed its ability to grow on ethyl-benzene as the sole carbon and energy source. Moreover, P. putida E41 show the activity of biodegradation of ethylbenzene in the batch culture. E41 showed high efficiency of biodegradation of ethylbenzene with the optimum conditions (a cell concentration of 0.1 g wet cell weight/L, pH 7.0, 25°C, and ethylbenzene concentration of 50 mg/L) from the results of the batch culture. The maximum degradation rate and specific growth rate (μ_max) under the optimum conditions were 0.19+0.03 mg/mg-DCW (Dry Cell Weight)/h and 0.87+0.13 h⁻¹, respectively. Benzene, toluene and ethylbenzene were degraded when these compounds were provided together; however, xylene isomers persisted during degradation by P. putida E41. When using a bioreactor batch system with a binary culture with P. putida BJ10, which was isolated previously in our lab, the degradation rate for benzene and toluene was improved in BTE mixed medium (each initial concentration: 50 mg/L). Almost all of the BTE was degraded within 4 h and 70–80% of m-, p-, and o-xylenes within 11 h in a BTEX mixture (initial concentration: 50 mg/L each). In summary, we found a valuable new strain of P. putida, determined the optimal degradation conditions for this isolate and tested a mixed culture of E41 and BJ10 for its ability to degrade a common sample of mixed contaminants containing benzene, toluene, and xylene.     

Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site

Eight new primer sets were designed for PCR detection of (i) mono-oxygenase and dioxygenase gene sequences involved in initial attack of bacterial aerobic BTEX degradation and of (ii) catechol 2,3-dioxygenase gene sequences responsible for meta-cleavage of the aromatic ring. The new primer sets allowed detection of the corresponding genotypes in soil with a detection limit of 10(3)-10(4) or 10(5)-10(6) gene copies g(-1) soil, assuming one copy of the gene per cell. The primer sets were used in PCR to assess the distribution of the catabolic genes in BTEX degrading bacterial strains and DNA extracts isolated from soils sampled from different locations and depths (vadose, capillary fringe and saturated zone) within a BTEX contaminated site. In both soil DNA and the isolates, tmoA-, xylM- and xylE1-like genes were the most frequently recovered BTEX catabolic genes. xylM and xylE1 were only recovered from material from the contaminated samples while tmoA was detected in material from both the contaminated and non-contaminated samples. The isolates, mainly obtained from the contaminated locations, belonged to the Actinobacteria or Proteobacteria (mainly Pseudomonas). The ability to degrade benzene was the most common BTEX degradation phenotype among them and its distribution was largely congruent with the distribution of the tmoA-like genotype. The presence of tmoA and xylM genes in phylogenetically distant strains indicated the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer. Overall, these results show spatial variation in the composition of the BTEX degradation genes and hence in the type of BTEX degradation activity and pathway, at the examined site. They indicate that bacteria carrying specific pathways and primarily carrying tmoA/xylM/xylE1 genotypes, are being selected upon BTEX contamination.     

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.     

The biofiltration permeable reactive barrier: Practical experience from Synthesia

The paper refers to utilization of biological elements within permeable reactive barriers. The concept of a biofiltration permeable barrier has been tested in the laboratory and in pilot-scale. Oxyhumolite (oxidized young lignite) was examined as an absorption material and a biofilm carrier. Laboratory tests performed before the pilot verification confirmed that oxyhumolite adsorbs organic pollutants at a minimum value, but that it can be used for biofilm attachment. An experimental barrier was built on premises of a chemical factory contaminated mainly by various organic pollutants [benzene, toluene, ethylbenzene, and xylenes (BTEX), chlorobenzenes, naphthalene, nitro-derivatives, phenols, trichloroethylene (TCE), and total petroleum hydrocarbon (TPH)]. Before the barrier was installed, a preliminary survey of the unsaturated zone, hydrogeological investigation, and a microbiological survey had been performed. The barrier was designed as a trench-and-gate system with an in situ bioreactor. During the year 2004, measurements of groundwater flux and retention time under current hydrological conditions, together with chemical and microbiological monitoring, were carried out on the site. The results showed high effectiveness of organic contamination removal. Average elimination varied from 57.3% (naphthalene) to 99.9% (nitro-derivatives, BTEX); microbial density in the bioreactor was approx. 10⁵ CFU mL⁻¹.     

A Shallow BTEX and MTBE Contaminated Aquifer Supports a Diverse Microbial Community

Microbial communities in subsurface environments are poorly characterized and the impacts of anthropogenic contamination on their structure and function have not been adequately addressed. The release of contaminant(s) to a previously unexposed environment is often hypothesized to decrease the diversity of the affected community. We characterized the structure of microbial communities along a gradient of benzene, toluene, ethylbenzene, and xylene (BTEX) and methyl-tert-butyl-ether (MTBE) contamination, resulting from a petroleum spill, within a shallow sandy aquifer at Vandenberg Air Force Base (VAFB) in Lompoc, CA. Differences in microbial community composition along the contaminant plume were assessed via a combinatorial approach utilizing denaturing gradient gel electrophoresis (DGGE), cloning and sequencing, intergenic transcribed spacer analysis (ITS), and comparative phylogenetic analysis of partial 16S rDNA sequences. Substantial bacterial sequence diversity, similar levels of species richness, and similar phylo-groups (including the Cytophaga–Flavobacterium–Bacteroidetes group and numerous members of the -, -, -, -, and -groups of the proteobacteria) were observed in both uncontaminated and contaminated regions of the aquifer. High-resolution measures (ITS fingerprinting and phylogenetic inference) readily separated communities impacted by the original petroleum spill (in source zone) from those in other parts of the aquifer and indicated that communities exposed to MTBE only were similar to communities in uncontaminated regions. Collectively, these data suggest that petroleum contamination alters microbial community structure at the species and subspecies level. Further study is required to determine whether these changes have an impact on the functioning of this subsurface ecosystem.     

Radioactive Fingerprinting of Microorganisms That Oxidize Atmospheric Methane in Different Soils

Microorganisms that oxidize atmospheric methane in soils were characterized by radioactive labelling with ¹⁴CH₄ followed by analysis of radiolabelled phospholipid ester-linked fatty acids (¹⁴C-PLFAs). The radioactive fingerprinting technique was used to compare active methanotrophs in soil samples from Greenland, Denmark, the United States, and Brazil. The ¹⁴C-PLFA fingerprints indicated that closely related methanotrophic bacteria were responsible for the oxidation of atmospheric methane in the soils. Significant amounts of labelled PLFAs produced by the unknown soil methanotrophs coeluted with a group of fatty acids that included i17:0, a17:0, and 17:1ω8c (up to 9.0% of the total ¹⁴C-PLFAs). These PLFAs are not known to be significant constituents of methanotrophic bacteria. The major PLFAs of the soil methanotrophs (73.5 to 89.0% of the total PLFAs) coeluted with 18:1 and 18:0 fatty acids (e.g., 18:1ω9, 18:1ω7, and 18:0). The ¹⁴C-PLFAs fingerprints of the soil methanotrophs that oxidized atmospheric methane did not change after long-term methane enrichment at 170 ppm CH₄. The ¹⁴C-PLFA fingerprints of the soil methanotrophs were different from the PLFA profiles of type I and type II methanotrophic bacteria described previously. Some similarity at the PLFA level was observed between the unknown soil methanotrophs and the PLFA phenotype of the type II methanotrophs. Methanotrophs in Arctic, temperate, and tropical regions assimilated between 20 and 54% of the atmospheric methane that was metabolized. The lowest relative assimilation (percent) was observed for methanotrophs in agricultural soil, whereas the highest assimilation was observed for methanotrophs in rain forest soil. The results suggest that methanotrophs with relatively high carbon conversion efficiencies and very similar PLFA compositions dominate atmospheric methane metabolism in different soils. The characteristics of the methane metabolism and the ¹⁴C-PLFA fingerprints excluded any significant role of autotrophic ammonia oxidizers in the metabolism of atmospheric methane.     

Depth-Resolved Quantification of Anaerobic Toluene Degraders and Aquifer Microbial Community Patterns in Distinct Redox Zones of a Tar Oil Contaminant Plume

Microbial degradation is the only sustainable component of natural attenuation in contaminated groundwater environments, yet its controls, especially in anaerobic aquifers, are still poorly understood. Hence, putative spatial correlations between specific populations of key microbial players and the occurrence of respective degradation processes remain to be unraveled. We therefore characterized microbial community distribution across a high-resolution depth profile of a tar oil-impacted aquifer where benzene, toluene, ethylbenzene, and xylene (BTEX) degradation depends mainly on sulfate reduction. We conducted depth-resolved terminal restriction fragment length polymorphism fingerprinting and quantitative PCR of bacterial 16S rRNA and benzylsuccinate synthase genes (bssA) to quantify the distribution of total microbiota and specific anaerobic toluene degraders. We show that a highly specialized degrader community of microbes related to known deltaproteobacterial iron and sulfate reducers (Geobacter and Desulfocapsa spp.), as well as clostridial fermenters (Sedimentibacter spp.), resides within the biogeochemical gradient zone underneath the highly contaminated plume core. This zone, where BTEX compounds and sulfate—an important electron acceptor—meet, also harbors a surprisingly high abundance of the yet-unidentified anaerobic toluene degraders carrying the previously detected F1-cluster bssA genes (C. Winderl, S. Schaefer, and T. Lueders, Environ. Microbiol. 9:1035-1046, 2007). Our data suggest that this biogeochemical gradient zone is a hot spot of anaerobic toluene degradation. These findings show that the distribution of specific aquifer microbiota and degradation processes in contaminated aquifers are tightly coupled, which may be of value for the assessment and prediction of natural attenuation based on intrinsic aquifer microbiota.     

Temperature effects and substrate interactions during the aerobic biotransformation of BTEX mixtures by toluene-enriched consortia and Rhodococcus rhodochrous

A microbial consortium derived from a gasoline-contaminated aquifer was enriched on toluene (T) in a chemostat at 20 degrees C and was found to degrade benzene (B), ethylbenzene (E), and xylenes (X). Studies conducted to determine the optimal temperature for microbial activity revealed that cell growth and toluene degradation were maximized at 35 degrees C. A consortium enriched at 35 degrees C exhibited increased degradation rates of benzene, toluene, ethylbenzene, and xylenes in single-substrate experiments; in BTEX mixtures, enhanced benzene, toluene, and xylene degradation rates were observed, but ethylbenzene degradation rates decreased. Substrate degradation patterns over a range of BTEX concentrations (0 to 80 mg/L) for individual aromatics were found to differ significantly from patterns for aromatics in mixtures. Individually, toluene was degraded fastest, followed by benzene, ethylbenzene, and the xylenes. In BTEX mixtures, degradation followed the order of ethylbenzene, toluene, and benzene, with the xylenes degraded last. A pure culture isolated from the 35 degrees C-enriched consortium was identified as Rhodococcus rhodochrous. This culture was shown to degrade each of the BTEX compounds, individually and in mixtures, following the same degradation patterns as the mixed cultures. Additionally, R. rhodochrous was shown to utilize benzene, toluene, and ethylbenzene as primary carbon and energy sources. Studies conducted with the 35 degrees C-enriched consortium and R. rhodochrous to evaluate potential substrate interactions caused by the concurrent presence of multiple BTEX compounds revealed a range of substrate interaction patterns including no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism. In the case of the consortium, benzene and toluene degradation rates were slightly enhanced by the presence of o-xylene, whereas the presence of toluene, benzene, or ethylbenzene had a negative effect on xylene degradation rates. Ethylbenzene was shown to be the most potent inhibitor of BTEX degradation by both the mixed and pure cultures. Attempted quantification of these inhibition effects in the case of the consortium suggested a mixture of competitive and noncompetitive inhibition kinetics. Benzene, toluene, and the xylenes had a negligible effect on the biodegradation of ethylbenzene by both cultures. Cometabolism of o-, m-, and p-xylene was shown to be a positive substrate interaction.     

Biodegradation of benzene,toluene, ethylbenzene,and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (>1000 mg l⁻¹) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l⁻¹. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate,chelated ferric iron or nitrate

Flow-through aquifer columns were used to investigate the feasibility of adding sulfate, EDTA–Fe(III) or nitrate to enhance the biodegradation of BTEX and ethanol mixtures. The rapid biodegradation of ethanol near the inlet depleted the influent dissolved oxygen (8 mg l^-1), stimulated methanogenesis, and decreased BTEX biodegradation efficiencies from >99% in the absence of ethanol to an average of 32% for benzene, 49% for toluene, 77% for ethylbenzene, and about 30% for xylenes. The addition of sulfate, EDTA–Fe(III) or nitrate suppressed methanogenesis and significantly increased BTEX biodegradation efficiencies. Nevertheless, occasional clogging was experienced by the column augmented with EDTA–Fe(III) due to iron precipitation. Enhanced benzene biodegradation (>70% in all biostimulated columns) is noteworthy because benzene is often recalcitrant under anaerobic conditions. Influent dissolved oxygen apparently played a critical role because no significant benzene biotransformation was observed after oxygen was purged out of the influent media. The addition of anaerobic electron acceptors could enhance BTEX biodegradation not only by facilitating their anaerobic biodegradation but also by accelerating the mineralization of ethanol or other substrates that are labile under anaerobic conditions. This would alleviate the biochemical oxygen demand (BOD) and increase the likelihood that entraining oxygen would be used for the biotransformation of residual BTEX.