Alginate beads as a storage, delivery and containment system for genetically modified PCB degrader and PCB biosensor derivatives of Pseudomonas fluorescens F113

Aims: Pseudomonas fluorescens F113Rifpcb is a genetically engineered rhizosphere bacterium with the potential to degrade polychlorinated biphenyls (PCBs). F113Rifpcbgfp and F113L::1180gfp are biosensor strains capable of detecting PCB bioavailability and biodegradation. The aim of this paper is to evaluate the use of alginate beads as a storage, delivery and containment system for use of these strains in PCB contaminated soils. Methods and Results: The survival and release of Ps. fluorescens F113Rifpcb from alginate beads were evaluated. Two Ps. fluorescens F113‐based biosensor strains were encapsulated, and their ability to detect 3‐chlorobenzoate (3‐CBA) and 3‐chlorobiphenyl (3‐CBP) degradation in soil was assessed. After 250 days of storage, 100% recovery of viable F113Rifpcb cells was possible. Amendments to the alginate formulation allowed for the timed release of the inoculant. Encapsulation of the F113Rifpcb cells provided a more targeted approach for the inoculation of plants and resulted in lower inoculum populations in the bulk soil, which may reduce the risk of unintentional spread of these genetically modified micro‐organisms in the environment. Encapsulation of the biosensor strains in alginate beads did not interfere with their ability to detect either 3‐CBA or 3‐CBP degradation. In fact, detection of 3‐CBP degradation was enhanced in encapsulated biosensors. Conclusions: Alginate beads are an effective storage and delivery system for PCB degrading inocula and biosensors. Significance and Impact of the Study: Pseudomonas fluorescens F113Rifpcb and the F113 derivative PCB biosensor strains have excellent potential for detecting and bioremediation of PCB contaminated soils. The alginate bead delivery system could facilitate the application of these strains as biosensors.     

Genetically modified Pseudomonas biosensing biodegraders to detect PCB and chlorobenzoate bioavailability and biodegradation in contaminated soils

Whole cell microbial biosensors offer excellent possibilities for assaying the complex nature of the bioavailable and bioaccessible fraction of pollutants in contaminated soils, which currently cannot be easily addressed. This paper describes the application and evaluation of three microbial biosensor strains designed to detect the bioavailability and biodegradation of PCBs (and end-products) in contaminated soils and sediments. Polychlorinated biphenyls (PCBs) are considered to be one of the most wide spread, hazardous and persistent pollutants. Herein we describe that there was a positive correlation between the PCB levels within the samples and the percentage of biosensor cells that were expressing their reporter gene; gfp. Immobilisation of the biosensors in calcium alginate beads allowed easy and accurate detection of the biosensor strains in contaminated soil and sludge samples. The biosensors also showed that PCB degradation activity was occurring at a much greater level in Pea inoculated planted soil compared to inoculated unplanted soil indicating rhizoremediation (the removal of pollutants by plant root associated microbes) shows considerable promise as a solution for removing organic xenobiotics from the environment.     

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.     

Biodegradation of disperse textile dye Brown 3REL by newly isolated Bacillus sp. VUS

Aims:  To isolate the potential micro-organism for the degradation of textile disperse dye Brown 3 REL and to find out the reaction mechanism.
Methods and Results:  16S rDNA analysis revealed an isolate from textile effluent contaminated soil as Bacillus sp. VUS and was able to degrade (100%) dye Brown 3REL within 8 h at static anoxic condition. A significant increase in the activities of lignin peroxidase, laccase and NADH-DCIP reductase was observed up to complete decolourization of Brown 3REL. The optimum temperature required for degradation was 40°C and pH 6·5–12·0. Phyto-toxicity and chemical oxygen demand revealed nontoxic products of dye degradation. The biodegradation was monitored by UV–VIS, FTIR spectroscopy and HPLC. The final products 6,8-dichloro-quinazoline-4-ol and cyclopentanone were characterized by gas chromatography-mass spectrometry. This Bacillus sp. VUS also decolourized (80%) textile dye effluent within 12 h.
Conclusions:  This study suggests that Bacillus sp. VUS could be a useful tool for textile effluent treatment.
Significance and Impact of the Study:  The newly isolated Bacillus sp. VUS decolourized 16 textile dyes and textile dye effluent also. It achieved complete biodegradation of Brown 3REL. Phytotoxicity study demonstrated no toxicity of the biodegraded products for plants with respect to Triticum aestivum and Sorghum bicolor .     

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.     

Soil biosensor for the detection of PAH toxicity using an immobilized recombinant bacterium and a biosurfactant

A biosensor for detecting the toxicity of polycylic aromatic hydrocarbons (PAHs) contaminated soil has been successfully constructed using an immobilized recombinant bioluminescent bacterium, GC2 (lac::luxCDABE), which constitutively produces bioluminescence. The biosurfactant, rhamnolipids, was used to extract a model PAH, phenanthrene, and was found to enhance the bioavailability of phenanthrene via an increase in its rate of mass transfer from sorbed soil to the aqueous phase. The monitoring of phenanthrene toxicity was achieved through the measurement of the decrease in bioluminescence when a sample extracted with the biosurfactant was injected into the minibioreactor. The concentrations of phenanthrene in the aqueous phase were found to correlate well with the corresponding toxicity data obtained by using this toxicity biosensor. In addition, it was also found that the addition of glass beads to the agar media enhanced the stability of the immobilized cells. This biosensor system using a biosurfactant may be applied as an in-situ biosensor to detect the toxicity of hydrophobic contaminants in soils and for performance evaluation of PAH degradation in soils.     

Basic examinations on chemical pre-oxidation by ozone for enhancing bioremediation of phenanthrene contaminated soils

Biological treatment of polycyclic aromatic hydrocarbons (PAH) has been demonstrated to be a feasible and common remediation technology which has been successfully applied to the clean-up of contaminated soils. Because bioavailability of the contaminants is of great importance for a successful bioremediation, a chemical pre-oxidation step by ozone was tested to enhance the subsequent biodegradation steps. Oxidation of PAH by ozone should result in reaction products that have a better solubility in water and thus a better bioavailability. A major part of this work was done by examinations of the model substance phenanthrene as a typical compound of PAH. After initial ozonation of phenanthrene, analysis by GC-MS showed at least seven identified conversion-products of phenanthrene. In comparison with phenanthrene these conversion products were more efficiently biodegraded by Sphingomonas yanoikuyae or mixed cultures when the ozonation process resulted in monoaromatic compounds. Primary ozonation products with biphenylic structures were found not to be biodegradable. Investigations into the toxicity of contaminated and ozonated soils were carried out by well-established toxicity assays using Bacillus subtilis and garden cress. The ozonated soils surprisingly showed higher toxic or inhibitory effects towards different organisms than the phenanthrene or PAH itself. The microbial degradation of phenanthrene in slurry reactors by S. yanoikuyae was not enhanced significantly by preozonation of the contaminated soil.     

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.     

Rhamnolipid biosurfactants decrease the toxicity of chlorinated phenols to Pseudomonas putida DOT-T1E

Aims:  To investigate the effect of a mixture of rhamnolipid R1 and R2 biosurfactants produced by a Pseudomonas aeruginosa strain on the toxicity of phenol and chlorophenols to Pseudomonas putida DOT-T1E.
Methods and Results:  Toxicity was quantified by the effective concentration 50% (EC50), that is the concentration that causes a 50% inhibition of bacterial growth. The presence of 300 mg l⁻¹ rhamnolipids, that is at about twice their critical micelle concentration (CMC), increased the EC50 of phenol, 4-chlorophenol, 2,4-dichlorophenol and 2,4,5-trichlorophenol by about 12, 19, 32 and 40%, respectively, and consequently reduced the bioavailability and the freely dissolved concentration of the toxic phenolic compounds. The reduction was related to the phenols' octanol–water partition coefficients ( K _ow).
Conclusions:  The reduction in toxicity of the phenols can be explained by a combination of toxin accumulation in biosurfactant micelles and hydrophobic interactions of the phenols with rhamnolipid-based dissolved organic carbon.
Significance and Impact of the Study:  Results provide evidence that next to the effect of the micelle formation also hydrophobic interactions with rhamnolipid-based dissolved organic carbon affects the bioavailability of the phenols. Quantifying the effect of biosurfactants on the toxicity of hydrophobic compounds such as phenols thus appears to be a useful approach to assess their bioavailable equilibrium concentration.     

Broad substrate specificity of naphthalene- and biphenyl-utilizing bacteria

 Although aromatic compounds are most often present in the environment as components of complex mixtures, biodegradation studies commonly focus on the degradation of individual compounds. The present study was performed to investigate the range of aromatic substrates utilized by biphenyl- and naphthalene-degrading environmental isolates and to ascertain the effects of co-occurring substrates during the degradation of mono-aromatic compounds. Bacterial strains were isolated on the basis of their ability to utilize either biphenyl or naphthalene as a sole source of carbon. Growth and transformation assays were conducted on each isolate to determine the range of substrates degraded. One isolate, Pseudomonas putida BP18, was tested for the ability to biodegrade benzene, toluene, ethylbenzene and xylene isomers (BTEX) individually and as components of mixtures. Overall, the results indicate that organisms capable of growth on multi-ring aromatic compounds may be particularly versatile in terms of aromatic hydrocarbon biodegradation. Furthermore, growth and transformation assays performed with strain BP18 suggest that the biodegradation of BTEX and biphenyl by this strain is linked to a catabolic pathway with overlapping specificities. The broad substrate specificity of these environmental isolates has important implications for bioremediation efforts in the field. Received: 4 August 1999 / Received revision: 25 October 1999 / Accepted: 5 November 1999     

Populations of heavy fuel oil-degrading marine microbial community in presence of oil sorbent materials

Aims:  To investigate the feasibility of applying sorbent material X-Oil^® in marine oil spill mitigation and to survey the interactions of oil, bacteria and sorbent.
Methods and Results:  In a series of microcosms, 25 different treatments including nutrient amendment, bioaugmentation with Alcanivorax borkumensis and application of sorbent were tested. Microbial community dynamics were analysed by DNA fingerprinting methods, RISA and DGGE. Results of this study showed that the microbial communities in microcosms with highly active biodegradation were strongly selected in favour of A. borkumensis . Oxygen consumption measurements in microcosms and gas chromatography of oil samples indicated the fast and intense depletion of linear alkanes as well as high oxygen consumption within 1 week followed by consequent slower degradation of branched and polyaromatic hydrocarbons.
Conclusion:  Under given conditions, A. borkumensis was an essential organism for biodegradation, dominating the biofilm microbial community formation and was the reason of emulsification.
Significance and Impact of the Study:  This study strongly emphasizes the pivotal importance of A. borkumensis as an essential organism in the initial steps of marine hydrocarbon degradation. Interaction with the sorbent material X-Oil^® proved to be neutral to beneficial for biodegradation and also promoted the growth of yet unknown micro-organisms.     

Development of a biological test to evaluate the bioavailability of iron in culture media

Aims:  To develop an easy-to-use and pathogen-free protocol giving reliable information on the bioavailability of iron in a medium.
Methods and Results:  In aerobic conditions, iron bioavailability is very low, and most of its forms cannot be assimilated by micro-organisms. Media with similar iron contents can differ considerably in iron bioavailability, something that is not easily achieved using conventional physicochemical methods. The assay developed in the present work is based on a pyoverdin siderophore release by fluorescent Pseudomonas in response to iron stress.
Conclusions:  The test was applied to a complex medium used for the production of diphtheria toxin (DT). A significant difference between the bioavailable iron level and the total chemical concentrations contributed by the various compounds used to make the medium could thus be detected. This can be explained by the formation of salt complexes trapping the iron, which thus cannot be used directly by the micro-organism for its metabolism.
Significance and Impact of the Study:  The assay can easily be applied to any medium designed for the production of iron-regulated compounds. This is particularly useful when dealing with processes that use pathogenic strains as was shown in the case based on DT production.     

Isolation and characterization of a phorate degrading bacterium

Aims:  To study the degradation of phorate by a bacterium isolated from phorate-contaminated sites.
Methods and Results:  Ralstonia eutropha strain AAJ1 isolated from soil was found to degrade phorate (supplied as sole carbon source) upto 85% in 10 days in liquid medium. Half-life ( t _½) of phorate in the liquid medium in control (uninoculated) and in experimental (inoculated with R. eutropha , strain AAJ1) samples was recorded as 36·49 and 6·29 days, respectively. Kinetics revealed that phorate degradation depends on time and the reaction follows the first order kinetics. Diethyl dithiophosphate was one of the degradation products, which is markedly less toxic than the parent compound; other degradation products included phorate sulfoxide and phorate sulfone. Release of inorganic phosphates and sulfates indicated the potential of the isolate to further degrade the above-mentioned metabolites to simpler forms. The strain was also found to posses phosphomonoesterase and phosphodiesterase enzymatic activity, which are involved in biodegradation of organophosphorus compounds.
Conclusions:  Ralstonia eutropha AAJ1 could degrade and detoxify phorate upto 85% in 10 days in laboratory conditions.
Significance and Impact of the Study:  The isolate has the potential to be utilized for remediation of phorate-contaminated water and soil.     

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.     

Isolation of alkali-tolerant benzene-degrading bacteria from a contaminated aquifer

Aims:  To isolate benzene-degrading strains from neutral and alkaline groundwaters contaminated by benzene, toluene, ethylbenzene, xylenes (BTEX) from the SIReN aquifer, UK, and to test their effective pH range and ability to degrade TEX.
Methods and Results:  The 14 isolates studied had an optimum pH for growth of 8, and could degrade benzene to below detection level (1  μ g l⁻¹). Five Rhodococcus erythropolis strains were able to metabolize benzene up to pH 9, two distinct R. erythropolis strains to pH 10, and one Arthrobacter strain to pH 8·5. These Actinobacteria also degraded benzene at least down to pH 5·5. Six other isolates, a Hydrogenophaga and five Pseudomonas strains, had a narrower pH tolerance for benzene degradation (pH 6 to 8·5), and could metabolize toluene; in addition, the Hydrogenophaga and two Pseudomonas strains utilized o- , m- or p- xylenes. None of these strains degraded ethylbenzene.
Conclusions:  Phylogenetically distinct isolates, able to degrade BTX compounds, were obtained, and some degraded benzene at high pH.
Significance and Impact of the Study:  High pH has previously been found to inhibit in situ degradation of benzene, a widespread, carcinogenic groundwater contaminant. These benzene-degrading organisms therefore have potential applications in the remediation or natural attenuation of alkaline waters.     

Degradation of BTEX by anaerobic bacteria: physiology and application

Pollution of the environment with aromatic hydrocarbons, such as benzene, toluene, ethylbenzene and xylene (so-called BTEX) is often observed. The cleanup of these toxic compounds has gained much attention in the last decades. In situ bioremediation of aromatic hydrocarbons contaminated soils and groundwater by naturally occurring microorganisms or microorganisms that are introduced is possible. Anaerobic bioremediation is an attractive technology as these compounds are often present in the anoxic zones of the environment. The bottleneck in the application of anaerobic techniques is the lack of knowledge about the anaerobic biodegradation of benzene and the bacteria involved in anaerobic benzene degradation. Here, we review the existing knowledge on the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria, in particular the physiology and application, including results on the (per)chlorate stimulated degradation of these compounds, which is an interesting new alternative option for bioremediation.     

PHYTOREMEDIATION OF BTEX HYDROCARBONS: POTENTIAL IMPACTS OF DIURNAL GROUNDWATER FLUCTUATION ON MICROBIAL DEGRADATION

Volatile hydrocarbons have multiple potential fates in phytoremediation. This research investigated the relationship between biodegradation and plant uptake of BTEX compounds in laboratory and field settings. At a phytoremediation site, preliminary studies revealed minimal uptake into trees and enhanced degradation potential in the rhizosphere and in the bulk soil. Increased oxygen transport to the vadose zone caused by diurnal rise and fall of the water table was hypothesized to enhance degradation in the bulk soil. A detailed greenhouse study was then conducted to investigate potential bioremediation impacts using field-site soil and DN34 hybrid poplar trees.

In rhizosphere soils, the contaminated-planted reactor had significantly higher BTEX degrader populations versus the uncontaminated-planted reactor, as was anticipated. The bulk soil in the planted-contaminated reactor had increased degrader populations than the unplanted-contaminated soil or planted-uncontaminated soil, and planting increased degradation throughout the soil profile, not just in the limited volume of rhizosphere soils. Oxygen diffusive and advective transport into reactors was modeled and calculated. Oxygen input in planted reactors was at least 3 to 5 times higher than in unplanted reactors, and increasing oxygen input lead to increased degrader populations in a linear manner. These results combined with the knowledge that high-transpiration trees draw the contaminated groundwater to the capillary fringe and the rhizosphere indicate that phytoremediation can aid microbial degradation via multiple mechanisms: increasing degrader populations, increasing oxygen input via groundwater diurnal fluctuations, and transporting contaminants to the biologically-enriched soil profile.     

Selection and characterization of aerobic bacteria capable of degrading commercial mixtures of low-ethoxylated nonylphenols

Aims:  Isolation and characterization of new bacterial strains capable of degrading nonylphenol ethoxylates (NP n EO) with a low ethoxylation degree, which are particularly recalcitrant to biodegradation.
Methods and Results:  Seven aerobic bacterial strains were isolated from activated sludges derived from an Italian plant receiving NP n EO-contaminated wastewaters after enrichment with a low-ethoxylated NP n EO mixture. On the basis of 16S rDNA sequence, the strains were positioned into five genera: Ochrobactrum , Castellaniella , Variovorax , Pseudomonas and Psychrobacter . Their degradation capabilities have been evaluated on two commercial mixtures, i.e. Igepal CO-210 and Igepal CO-520, the former rich in low ethoxylated congeners and the latter containing a broader spectrum of NP n EO, and on 4- n -nonylphenol (NP). The strains degraded Igepal CO-210, Igepal CO-520 and 4- n -NP all applied at the initial concentration of 100 mg l⁻¹, by 35–75%, 35–90% and 15–25%, respectively, after 25 days of incubation.
Conclusions:  Some of the isolated strains, in particular the Pseudomonas strains BCb12/1 and BCb12/3, showed interesting degradation capabilities towards low ethoxylated NP n EO congeners maintaining high cell vitality.
Significance and Impact of the Study:  Increased knowledge of bacteria involved in NP n EO degradation and the possibility of using the isolated strains in tailored process for a tertiary biological treatment of effluents of wastewater treatment plants.     

Assessment of Factors Governing Biodegradability of PAHs in Three Soils Aged Under Field Conditions

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants of great environmental concern due to their toxic, mutagenic and carcinogenic properties. This study correlates soil characteristics (i.e. soil organic matter, particle- and pore-size distribution) with extractability and toxicity data (LUMIStox, Ostracod) to investigate factors that govern biodegradability of PAHs in three historically contaminated soils. Desorption of PAHs occurred most readily from soil TA1 (82%), followed by soil AS3.7 (69%) and soil WG2 (20%). This is in line with toxicity data, as the soil in which the greatest contaminant desorption (SFE) was observed exhibited the highest toxicity (TA1). Of the three soils, pronounced biodegradation of 2-4-ring, and slight biodegradation of 5-ring PAHs was observed only in AS3.7, while no decrease of PAHs was reported for soils WG2 and TA1 during the degradation experiment. Strong sorption reduced pollutant bioavailability in WG2 and hence hampered biodegradation. By contrast, pollutant sorption was weak in TA1 and microbial activity was most likely inhibited due to high toxicity in this soil. Based on our results we conclude that biodegradation of PAHs in soils is determined by a number of phenomena with complex interactions between them. Consideration of a single factor will be misleading and may result in false prediction of the biodegradation potential.     

Multi-substrate biodegradation kinetics of MTBE and BTEX mixtures by Pseudomonas aeruginosa

Biodegradation of MTBE under various multi-substrate conditions by Pseudomonas aeruginosa was investigated in this research. The addition of BTEX in various combinations significantly inhibited MTBE biodegradation. This result was mainly due to the non-competitive inhibition between MTBE and BTEX compounds. The rate of MTBE biodegradation decreased with the increasing substrate number for multi-substrate conditions. Additionally, the kinetic models developed in this research successfully simulate the degradation of MTBE under various multi-substrate conditions. However, the accumulation of TBA during MTBE biodegradation revealed that P. aeruginosa was unable to degrade TBA during the period of time tested.