Modeling substrate interactions during the biodegradation of mixtures of toluene and phenol by Burkholderia species JS150

The biodegradation kinetics of toluene, phenol, and a mixture of toluene and phenol by Burkholderia species JS150 was measured and modeled. Both of these compounds can serve as the sole source of carbon and energy for this microorganism. The single-substrate biodegradation kinetics was described well using the Monod model, with model constants of mu(max,T) = 0.39 h(-1) and K(S,T) = 0.011 mM for growth on toluene and mu(max,P) = 0.309 h(-1) and K(S,P) = 0.0054 mM for growth on phenol. Degradation of the mixture of toluene and phenol followed simultaneous utilization kinetics with toluene being the preferred substrate. Toluene was found to inhibit the rate of utilization of phenol while the presence of phenol had little effect on the rate of degradation of toluene. Of the kinetic models that were tested, one developed for microbial degradation of multiple substrates was able to describe substrate interactions and to model the mixture utilization by strain JS150. Simple competitive, noncompetitive, or uncompetitive substrate kinetics were not sufficient to describe the observed inhibitory interactions.     

Use of 16S-rRNA to investigate microbial population dynamics during biodegradation of toluene and phenol by a binary culture

Interspecies interactions and changes in the rate and extent of biodegradation in mixed culture-mixed substrate studies were investigated. A binary mixed culture of Pseudomonas putida F1 and Burkholderia sp. JS150 degraded toluene, phenol, and their mixture. Both toluene and phenol can serve as sole sources of carbon and energy for both P. putida F1 and strain JS150. To investigate the population dynamics of this system, a fluorescent in-situ hybridization method was chosen because of its ability to produce quantitative data, its low standard error, and the ease of use of this method. When the binary mixed culture was grown on toluene or phenol alone, significant interactions between the species were observed. These interactions could not be explained by a pure-and-simple competition model and were substrate dependent. Strain JS150 growth was slightly inhibited when grown with P. putida F1 on phenol, and P. putida F1 grew more rapidly than expected. Conversely, when the two species were grown together on toluene alone, P. putida F1 was inhibited while strain JS150 was unaffected. During growth of the mixed culture on a combination of toluene and phenol, the interactions were similar to that observed during growth on phenol alone; P. putida F1 growth was enhanced while strain JS150 was unaffected. Because of the observed interspecies interactions, monoculture kinetic parameters were not sufficient to describe the mixed culture kinetics in any experiment. This is one of the first reports of microbial population dynamics in which molecular microbial ecology and mathematical modeling have been combined. The use of the 16S-rRNA-based method allowed for observation and understanding of interspecies interactions that were not observable with standard culture-based methods. These results suggest the need for more investigations that account for both substrate and microbial interactions when predicting the fate of organic pollutants in real systems.     

Multisubstrate biodegradation kinetics of naphthalene, phenanthrene, and pyrene mixtures.

Biodegradation kinetics of naphthalene, phenanthrene and pyrene were studied in sole-substrate systems, and in binary and ternary mixtures to examine substrate interactions. The experiments were conducted in aerobic batch aqueous systems inoculated with a mixed culture that had been isolated from soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Monod kinetic parameters and yield coefficients for the individual compounds were estimated from substrate depletion and CO(2) evolution rate data in sole-substrate experiments. In all three binary mixture experiments, biodegradation kinetics were comparable to the sole-substrate kinetics. In the ternary mixture, biodegradation of naphthalene was inhibited and the biodegradation rates of phenanthrene and pyrene were enhanced. A multisubstrate form of the Monod kinetic model was found to adequately predict substrate interactions in the binary and ternary mixtures using only the parameters derived from sole-substrate experiments. Numerical simulations of biomass growth kinetics explain the observed range of behaviors in PAH mixtures. In general, the biodegradation rates of the more degradable and abundant compounds are reduced due to competitive inhibition, but enhanced biodegradation of the more recalcitrant PAHs occurs due to simultaneous biomass growth on multiple substrates. In PAH-contaminated environments, substrate interactions may be very large due to additive effects from the large number of compounds present.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.     

Interactions between benzene, toluene, and p-xylene (BTX) during their biodegradation

A microbial consortium and Pseudomonas strain (PPO1) were used in studying biodegradation of benzene, toluene, and p-xylene under aeorbic conditions. Studies involved removal of each compound individually as well as in mixture with the others. Both cultures exhibited a qualitatively similar behavior toward each compound. Both the pure culture and the consortium grew on benzene following Monod kinetics, on toluene following inhibitory (Andrews) kinetics, whereas neither could grow on P-xylene. Benzene and toluene mixtures were removed under cross-inhibitory (competitive inhibition) kinetics. In the presence of benzene and/or toluene, p-xylene was cometabolically utilized by both cultures, but was not completely mineralized. Metabolic intermediates of p-xylene accumulated in the medium and were identified. Benzene and toluene were completely mineralized. Cometabolic removal of p-xylene reduced the yields on both benzene and toluene. Except for cometabolism, kinetic constants were determined from data analysis and are compared with values published recently by other researchers. (c) 1994 John Wiley & Sons, Inc.     

Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates

Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot-scale fluidized bed reactor treating groundwater containing benzene, toluene, and p-xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p-xylene in the presence of toluene. Strain X1 grew on toluene and p-xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth-rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p-xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p-xylene. (c) 1993 Wiley & Sons, Inc.     

Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.     

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.     

Metabolic engineering of Pseudomonas putida for the simultaneous biodegradation of benzene, toluene, and p-xylene mixture

For the complete biodegradation of a mixture of benzene, toluene, and p-xylene (BTX), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. Toluate-cis-glycol dehydrogenase in the tol pathway was found to attack benzene-cis-glycol, toluene-cis-glycol, and p-xylene-cis-glycol, which are metabolic intermediates of the tod pathway. Based on this observation, a hybrid strain, Pseudomonase putida TB101, was constructed by introduction of the TOL plasmid pWW0 into P. putida F39/D, a derivative of P. putida F1, which is unable to transform cis-glycol compounds to corresponding catechols. The metabolic flux of BTX into the tod pathway was redirected to the tol pathway at the level of cis-glycol compounds by the action of toluate-cis-glycol dehydrogenase in P. putida TB101, resulting in the simultaneous mineralization of BTX mixture without accumulation of any metabolic intermediates. The profile of specific degradation rates showed a similar pattern as that of the specific growth rate of the microorganism, and the maximum specific degradation rates of benzene, toluene, and p-xylene were determined to be about 0.27, 0.86, and 2.89 mg/mg biomass/h, respectively. P. putida TB101 is the first reported microorganism that mineralizes BTX mixture simultaneously. (c) 1994 John Wiley & Sons, Inc.     

Anaerobic degradation of toluene and o-xylene by a methanogenic consortium.

Toluene and o-xylene were completely mineralized to stoichiometric amounts of carbon dioxide, methane, and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was creosote-contaminated sediment from Pensacola, Fla. The adaptation periods before the onset of degradation were long (100 to 120 days for toluene degradation and 200 to 255 days for o-xylene). Successive transfers of the toluene- and o-xylene-degrading cultures remained active. Cell density in the cultures progressively increased over 2 to 3 years to stabilize at approximately 10(9) cells per ml. Degradation of toluene and o-xylene in stable mixed methanogenic cultures followed Monod kinetics, with inhibition noted at substrate concentrations above about 700 microM for o-xylene and 1,800 microM for toluene. The cultures degraded toluene or o-xylene but did not degrade m-xylene, p-xylene, benzene, ethylbenzene, or naphthalene. The degradative activity was retained after pasteurization or after starvation for 1 year. Degradation of toluene and o-xylene was inhibited by the alternate electron acceptors oxygen, nitrate, and sulfate. Degradation was also inhibited by the addition of preferred substrates such as acetate, H2, propionate, methanol, acetone, glucose, amino acids, fatty acids, peptone, and yeast extract. These data suggest that the presence of natural organic substrates or contaminants may inhibit anaerobic degradation of pollutants such as toluene and o-xylene at contaminated sites.     

Growth kinetics of Pseudomonas putida G7 on naphthalene and occurrence of naphthalene toxicity during nutrient deprivation

The objectives of this work were (1) to demonstrate how the chemostat approach could be modified to allow determination of kinetic parameters for a sparingly soluble, volatile substrate such as naphthalene and (2) to examine the influence of the interactions of various nutrients on possible growth-inhibitory effects of naphthalene. Pseudomonas putida G7 was used as a model naphthalene-degrading microorganism. Naphthalene was found to be toxic to P. putida G7 in the absence of a nitrogen source or oxygen. The death rate of cells grown on minimal medium plus naphthalene and then exposed to naphthalene under anoxic conditions was higher than that observed under oxic conditions in the absence of a nitrogen source. The presence of necessary nutrients for the biodegradation of PAH compounds is indicated to be important for the survival of microorganisms that are capable of PAH degradation. The amounts of ammonia and oxygen necessary for naphthalene biodegradation and for suppression of naphthalene toxicity were calculated from growth yield coefficients. A chemostat culture of P. putida G7 using naphthalene as a carbon and energy source was accomplished by using a feed augmented with a methanol solution of naphthalene so as to provide sufficient growth to allow accurate evaluation of kinetic parameters. When naphthalene was the growth-limiting substrate, the degradation of naphthalene followed Monod kinetics. Maximum specific growth rate (micrometer) and Monod constant (Ks) were 0.627 +/- 0.007 h-1 and 0.234 +/- 0.0185 mg/L, respectively. The evaluation of biodegradation parameters will allow a mathematical model to be applied to predict the long-term behavior of PAH compounds in soil when combined with PAH transport parameters.     

Biodegradation kinetics of select polycyclic aromatic hydrocarbon (PAH) mixtures by <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> EPA505

Many contaminated sites commonly have complex mixtures of polycyclic aromatic hydrocarbons (PAHs) whose individual microbial biodegradation may be altered in mixtures. Biodegradation kinetics for fluorene, naphthalene, 1,5-dimethylnaphthalene and 1-methylfluorene were evaluated in sole substrate, binary and ternary systems using Sphingomonas paucimobilis EPA505. The first order rate constants for fluorene, naphthalene, 1,5-dimethylnaphthalene, and 1-methylfluorene were comparable; yet Monod parameters were significantly different for the tested PAHs. S. paucimobilis completely degraded all the components in binary and ternary mixtures; however, the initial degradation rates of individual components decreased in the presence of competitive PAHs. Results from the mixture experiments indicate competitive interactions, demonstrated mathematically. The generated model appropriately predicted the biodegradation kinetics in mixtures using parameter estimates from the sole substrate experiments, validating the hypothesis of a common rate-determining step. Biodegradation kinetics in mixtures were affected by the affinity coefficients of the co-occurring PAHs and mixture composition. Experiments with equal concentrations of substrates demonstrated the effect of concentration on competitive inhibition. Ternary experiments with naphthalene, 1,5-dimethylnaphthalene and 1-methylfluorene revealed delayed degradation, where depletion of naphthalene and 1,5-dimethylnapthalene occurred rapidly only after the complete removal of 1-methylfluorene. The substrate interactions observed in mixtures require a multisubstrate model to account for simultaneous degradation of substrates. PAH contaminated sites are far more complex than even ternary mixtures; however these studies clearly demonstrate the effect that interactions can have on individual chemical kinetics. Consequently, predicting natural or enhanced degradation of PAHs cannot be based on single compound kinetics as this assumption would likely overestimate the rate of disappearance.     

Effect of ethanol, acetate, and phenol on toluene degradation activity and tod-lux expression in Pseudomonas putida TOD102: evaluation of the metabolic flux dilution model

The reporter strain Pseudomonas putida TOD102 (with a tod-lux fusion) was used in chemostat experiments with binary substrate mixtures to investigate the effect of potentially occurring cosubstrates on toluene degradation activity. Although toluene was simultaneously utilized with other cosubstrates, its metabolic flux (defined as the toluene utilization rate per cell) decreased with increasing influent concentrations of ethanol, acetate, or phenol. Three inhibitory mechanisms were considered to explain these trends: (1) repression of the tod gene (coding for toluene dioxygenase) by acetate and ethanol, which was quantified by a decrease in specific bioluminescence; (2) competitive inhibition of toluene dioxygenase by phenol; and (3) metabolic flux dilution (MFD) by all three cosubstrates. Based on experimental observations, MFD was modeled without any fitting parameters by assuming that the metabolic flux of a substrate in a mixture is proportional to its relative availability (expressed as a fraction of the influent total organic carbon). Thus, increasing concentrations of alternative carbon sources "dilute" the metabolic flux of toluene without necessarily repressing tod, as observed with phenol (a known tod inducer). For all cosubstrates, the MFD model slightly overpredicted the measured toluene metabolic flux. Incorporating catabolite repression (for experiments with acetate or ethanol) or competitive inhibition (for experiments with phenol) with independently obtained parameters resulted in more accurate fits of the observed decrease in toluene metabolic flux with increasing cosubstrate concentration. These results imply that alternative carbon sources (including inducers) are likely to hinder toluene utilization per unit cell, and that these effects can be accurately predicted with simple mathematical models.     

Benzene, toluene, and o-xylene degradation by free and immobilized P. putida F1 of postconsumer agave-fiber/polymer foamed composites

Benzene, toluene, and o-xylene (BTX) degradation by immobilized Pseudomonas putida F1 of postconsumer agave-fiber/polymer foamed-composites (AFPFC) and suspended cultures was studied under controlled conditions. Analyses using FTIR-ATR and SEM showed that P. putida F1 adhered onto the composite surface and developed a biofilm. In this sense, the AFPFC were successfully used as a support for bacterial immobilization. Both systems, immobilized and suspended cells of P. putida F1, were able to completely degrade benzene and toluene from initial concentrations of 15, 30, 60, and 90 mg l⁻¹. An inhibitory effect of the intermediary catechol from benzene degradation was observed in suspended cultures but it was not presented in the immobilized system. The degradation of o-xylene was partially accomplished in both systems. The Monod equation was used to model the experimental data obtained from the biodegradation kinetics, and they were adequately described with this model.     

Genetic engineering of Pseudomonas putida KT2442 for biotransformation of aromatic compounds to chiral cis-diols

Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylations of aromatic compounds. Pseudomonas putida KT2442 (pSPM01) harboring TDO genes could effectively biotransform a wide-range of aromatic substrates into their cis-diols products. In shake-flask culture, approximately 2.7gl(-1) benzene cis-diols, 8.8gl(-1) toluene cis-diols and 6.0gl(-1) chlorobenzene cis-diols were obtained from the biotransformation process. Furthermore, vgb gene encoding Vitreoscilla hemoglobin protein (VHb) which enhances oxygen microbial utilization rate under low dissolved oxygen concentration was integrated into P. putida KT2442 genome. The oxidation ability of the mutant strain P. putida KTOY02 (pSPM01) harboring TDO gene was increased in the presence of VHb protein. As a result, approximately 3.8, 15.1 or 6.8gl(-1) different cis-diols production was achieved in P. putida KTOY02 (pSPM01) grown in shake-flasks when benzene, toluene or chlorobenzene was used as the substrate. The above results indicate that P. putida KT2442 could be used as a cell factory to biotransform aromatic compounds.     

Kinetics of biodegradation of gasoline and its hydrocarbon constituents

Aerobic biodegradation of gasoline and its constituents, benzene, toluene and ethylbenzene were studied by an enrichment from soil indigenous microbial population. The enrichment culture completely degraded 16.1–660 mg/l gasoline in 2.5–16 days respectively, without accumulation of any by-products. The kinetics of gasoline as well as benzene, toluene and ethylbenzene biodegradation was investigated with initial gasoline concentrations of 16.1–62.6 mg/l. The maximum specific rates of biodegradation of benzene, toluene and ethylbenzene were 0.12, 0.38 and 0.19 mg mg biomass⁻¹ day⁻¹ respectively. When benzene and toluene were used as sole substrate, the maximum specific rates of their biodegradation were 62.9 and 16.4 times greater than the corresponding values for a mixture (gasoline). The microbial culture was able to mineralize up to 200 mg/l pure toluene and benzene. Maximum mineralization efficiencies of benzene and toluene were 76.7 ± 5.1% and 76.8 ± 1.3% respectively. Self-inhibition and competitive inhibition patterns were observed during the biodegradation of benzene and toluene alone and in the mixture respectively. The observed kinetics was modeled according to Andrews' inhibition model. Received: 6 August 1997 / Received revision: 18 November 1997 / Accepted: 29 November 1997     

Anaerobic degradation of benzene by a marine sulfate-reducing enrichment culture, and cell hybridization of the dominant phylotype

The anaerobic biodegradation of benzene, a common constituent of petroleum and one of the least reactive aromatic hydrocarbons, is insufficiently understood with respect to the involved microorganisms and their metabolism. To study these aspects, sulfate-reducing bacteria were enriched with benzene as sole organic substrate using marine sediment as inoculum. Repeated subcultivation yielded a sediment-free enrichment culture constituted of mostly oval-shaped cells and showing benzene-dependent sulfate reduction and growth under strictly anoxic conditions. Amplification and sequencing of 16S rRNA genes from progressively diluted culture samples revealed an abundant phylotype; this was closely related to a clade of Deltaproteobacteria that includes sulfate-reducing bacteria able to degrade naphthalene or other aromatic hydrocarbons. Cell hybridization with two specifically designed 16S rRNA-targeted fluorescent oligonucleotide probes showed that the retrieved phylotype accounted for more than 85% of the cells detectable via DAPI staining (general cell staining) in the enrichment culture. The result suggests that the detected dominant phylotype is the 'candidate species' responsible for the anaerobic degradation of benzene. Quantitative growth experiments revealed complete oxidation of benzene with stoichiometric coupling to the reduction of sulfate to sulfide. Suspensions of benzene-grown cells did not show metabolic activity towards phenol or toluene. This observation suggests that benzene degradation by the enriched sulfate-reducing bacteria does not proceed via anaerobic hydroxylation (mediated through dehydrogenation) to free phenol or methylation to toluene, respectively, which are formerly proposed alternative mechanisms for benzene activation.     

Studies on biodegradation of a mixture of toxic and nontoxic pollutant using Arthrobacter species

The effect of a nontoxic easily degradable substrate, glucose, on the biodegradation of toxic pollutant, phenol, was studied in batch reactors using a phenol degrading culture (Arthrobacter species). The effect of glucose on phenol degradation was determined at different glucose concentrations. The effect of different inoculum on substrate removal in a phenol and glucose mixture was also studied. Results indicated that when a mixed substrate (phenol and glucose) was used, phenol acclimated population showed an initial preference for phenol and utilised glucose after phenol removal. However phenol degradation rate was reduced in the presence of glucose. It was also observed that phenol degradation was completely inhibited when the glucose concentration exceeds 2 g/l. The substrate removal pattern changed completely when inoculum was drawn from mixed substrate acclimatised culture. The glucose utilisation started immediately and the rate of glucose utilisation was not affected by the presence of phenol. The phenol degradation also started simultaneously. In presence of phenol only, the rate of phenol degradation for the culture acclimatised to mixed substrates was lower than that of phenol acclimatised culture. These results indicate that nontoxic substrate can affect the biodegradation of toxic pollutants is suitable and acclimatisation may be necessary for biodegradation of mixed substrate.