Ralstonia Pickettii PKO1 Research Articles

The effects of substrate exposure history and carbon starvation-induced stress on the EPS synthesis of TCE degrading toluene oxidizing soil bacteria

A series of batch experiments was undertaken to explore the influence of growth conditions on the extracellular polymeric substances (EPS) production of five representative strains of trichloroethylene (TCE) degrading toluene oxidizing bacteria of importance to in situ bioremediation. EPS production for three distinct carbon sources (i.e., lactate, glucose, and toluene) was examined under starvation and non-starvation conditions. EPS extraction was accomplished using DOWEX™ (cation exchange resin; CER), and total carbohydrate, protein, and uronic acid were quantified as the main EPS constituents. Experimental results reveal that most bacteria grown on toluene synthesized more EPS per colony forming unit (CFU) than those grown on other substrates and that EPS production is enhanced with increasing length of starvation. These observations suggest that EPS may be produced under conditions of environmental stress and may be linked to enzyme deactivation. The results from this work additionally indicate that the mutant strain of Ralstonia pickettii PKO1 with the lack of gene encoding TbuX (an outer membrane protein) is more hydrophobic due to less EPS synthesis ability. The relatively lower amount of EPS synthesis in the mutant strain of R. pickettii PKO1 suggests that TbuX, an outer membrane protein encoded a part of the toluene catabolic regulation of R. pickettii PKO1, has a role in bacterial EPS production.

Benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate

The regulation of benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate was investigated in batch and continuous culture. In batch culture, R. pickettii PKO1 achieved a maximum specific growth rate with benzene of 0.18 h(-1), while succinate allowed much faster growth (mu(max) = 0.5 h(-1)). Under carbon excess conditions succinate repressed benzene consumption resulting in diauxic growth whereas under carbon-limited conditions in the chemostat both substrates were used simultaneously. Moreover, the effect of succinate on the adaptation towards growth with benzene was investigated in carbon-limited continuous culture at a dilution rate of 0.1 h(-1) by changing the inflowing carbon substrate from succinate to different mixtures of benzene and succinate. The adaptation process towards utilisation of benzene was rather complex. Three to seven hours after the medium shift biomass production from benzene started. Higher proportions of succinate in the mixture had a positive effect on both the onset of biomass production and on the time required for induction of benzene utilisation. Strikingly, after the initial increase in biomass and benzene-catabolising activities, the culture collapsed regularly and wash-out of biomass was observed. After a transient phase of low biomass concentrations growth on benzene resumed so that finally rather stable and high biomass concentrations were reached. The decrease in biomass and degradative activities cannot be explained so far, but the possibilities of either intoxication of the cells by benzene itself, or of inhibition by degradation intermediates were ruled out.

Induction kinetics of aerobic toluene degradation as a function of carbon starvation history

In this study, the effects of carbon starvation history on the induction kinetics of aerobic toluene degradation by Ralstonia pickettii PKO1 were quantitatively explored. Experimental results suggested that (i) the durations of the lag and transient phases depended upon both the duration of prior carbon starvation ( T stv) and the inducer concentration, and (ii) that post-transient-phase (PTP) induction levels were independent of T stv but dependent upon inducer concentration. The observed relationships among toluene degradation, carbon starvation history, and inducer concentration were incorporated into a modified Michaelis–Menten equation in order to refine the traditional model of enzymatic biodegradation. The refined model was able to predict toluene breakthrough in a porous medium in which previously carbon-starved cells were reactivated with a continuous supply of toluene. These findings suggested a means of improving the accuracy of existing models of biodegradation in groundwater under conditions of fluctuating substrate/inducer concentration.

Degradation of toluene by a mixed population of archetypal aerobes, microaerophiles, and denitrifiers: Laboratory sand column experiment and multispecies biofilm model formulation

An experiment was conducted in a saturated sand column with three bacterial strains that have different growth characteristics on toluene, Pseudomonas putida F1 which degrades toluene only under aerobic conditions, Thauera aromatica T1 which degrades toluene only under denitrifying conditions, and Ralstonia pickettii PKO1 has a facultative nature and can perform nitrate-enhanced biodegradation of toluene under hypoxic conditions (DO <2 mg/L). Steady-state concentration profiles showed that oxygen and nitrate appeared to be utilized simultaneously, regardless of the dissolved oxygen concentration and the results from fluorescent in-situ hybridization (FISH) indicated that PKO1 maintained stable cells numbers throughout the column, even when the pore water oxygen concentration was high. Since PKO1's growth rate under aerobic condition is much lower than that of F1, except under hypoxic conditions, these observations were not anticipated. Therefore these observations require a mechanistic explanation that can account for localized low oxygen concentrations under aerobic conditions. To simulate the observed dynamics, a multispecies biofilm model was implemented. This model formulation assumes the formation of a thin biofilm that is composed of the three bacterial strains. The individual strains grow in response to the substrate and electron acceptor flux from bulk fluid into the biofilm. The model was implemented such that internal changes in bacterial composition and substrate concentration can be simulated over time and space. The model simulations from oxic to denitrifying conditions compared well to the experimental profiles of the chemical species and the bacterial strains, indicating the importance of accounting for the biological activity of individual strains in biofilms that span different redox conditions.

Spatial distribution and physiological state of bacteria in a sand column experiment during the biodegradation of toluene

Toxic organic contaminants frequently serve as growth substrates for bacteria. However, long-term exposure to the organic contaminants can result in significant stress or “injury” to bacterial cells such that bacteria may lose, either temporarily or permanently, their capacity to degrade a specific toxic organic contaminant. In order to understand the relationship between biodegradability and physiological conditions of bacteria after a prolonged exposure to a contaminant, biomass samples collected from a sand column experiment, with toluene as the carbon source, were analyzed for bacterial physiology and spatial population distribution in the porous media. The column was seeded with three bacterial isolates that perform aerobic ( Pseudomonas putida F1), denitrifying (Thauera aromatica T1), and facultative ( Ralstonia pickettii PKO1) degradation of toluene were analyzed. Total, viable but not culturable with toluene, and toluene-culturable cells were enumerated using 4′6-diamidino-2-phenylindole (DAPI) staining and plate counting methods. Comparison of three types of cell counts showed that toluene-culturable cells were less than 40% of the total cell numbers. However, viable colonies transferred to a toluene media after cultivation on rich media regained their ability to degrade toluene. This implies that the temporary loss of their toluene degradation capacity is either due to an intracellular accumulation of degradation by-products, which have to be consumed in order for the cells to degrade toluene, or it is possible that cells have shifted to degrade other substrates such as toluene degradation intermediates or organic materials resulting from cell turnover. Comparison of cell counts with toluene concentration showed no exponential increase in total and viable cell numbers, as reported for flat bed biofilm reactor experiments. The overall fraction of toluene-culturable cells was highest at the highest toluene concentration near the column inlet, which indicates that the observed temporary loss of toluene culturability was not solely caused by a direct toxic effect from the long-term exposure to toluene.

Kinetics of 1,4-Dioxane Biodegradation by Monooxygenase-Expressing Bacteria

1,4-Dioxane is a probable human carcinogen, and an important emerging water contaminant. In this study, the biodegradation of dioxane by 20 bacterial isolates was evaluated, and 13 were found to be capable of transforming dioxane. Dioxane served as a growth substrate for Pseudonocardia dioxanivorans CB1190 and Pseudonocardia benzenivorans B5, with yields of 0.09 g protein g dioxane(-1) and 0.03 g protein g dioxane(-1), respectively. Cometabolic transformation of dioxane was observed for monooxygenase-expressing strains that were induced with methane, propane, tetrahydrofuran, or toluene including Methylosinus trichosporium OB3b, Mycobacterium vaccae JOB5, Pseudonocardia K1, Pseudomonas mendocina KR1, Ralstonia pickettii PKO1, Burkholderia cepacia G4, and Rhodococcus RR1. Product toxicity resulted in incomplete dioxane degradation for many of the cometabolic reactions. Brief exposure to acetylene, a known monooxygenase inhibitor, prevented oxidation of dioxane in all cases, supporting the hypothesis that monooxygenase enzymes participated in the transformation of dioxane by these strains. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene-2- and toluene-4-monooxygenases of G4, KR1 and PKO1 were also capable of cometabolic dioxane transformation. Dioxane oxidation rates measured at 50 mg/L ranged from 0.01 to 0.19 mg hr(-1) mg protein(-1) for the metabolic processes, 0.1-0.38 mg hr(-1) mg protein(-1) for cometabolism by the monooxygenase-induced strains, and 0.17-0.60 mg hr(-1) mg protein(-1) for the recombinant strains. Dioxane was not degraded by M. trichosporium OB3b expressing particulate methane monooxygenase, Pseudomonas putida mt-2 expressing a toluene side-chain monooxygenase, and PseudomonasJS150 and Pseudomonas putida F1 expressing toluene-2,3-dioxygenases. This is the first study to definitively show the role of monooxygenases in dioxane degradation using several independent lines of evidence and to describe the kinetics of metabolic and cometabolic dioxane degradation.

Aerobic biodegradation of N‐nitrosodimethylamine (NDMA) by axenic bacterial strains

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen whose appearance in the environment is related to the release of rocket fuel and to chlorine-based disinfection of water and wastewater. Although this compound has been shown to be biodegradable, there is minimal information about the organisms capable of this degradation, and little is understood of the mechanisms or biochemistry involved. This study shows that bacteria expressing monooxygenase enzymes functionally similar to those demonstrated to degrade NDMA in eukaryotes have the capability to degrade NDMA. Specifically, induction of the soluble methane monooxygenase (sMMO) expressed by Methylosinus trichosporium OB3b, the propane monooxygenase (PMO) enzyme of Mycobacterium vaccae JOB-5, and the toluene 4-monooxygenases found in Ralstonia pickettii PKO1 and Pseudomonas mendocina KR1 resulted in NDMA degradation by these strains. In each of these cases, brief exposure to acetylene gas, a suicide substrate for certain monooxygenases, inhibited the degradation of NDMA. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene monooxygenases found in strains PKO1 and KR1 mimicked the behavior of the parent strains. In contrast, M. trichosporium OB3b expressing the particulate form of MMO, Burkholderia cepacia G4 expressing the toluene 2-monooxygenase, and Pseudomonas putida mt-2 expressing the toluene sidechain monooxygenase were not capable of NDMA degradation. In addition, bacteria expressing aromatic dioxygenases were not capable of NDMA degradation. Finally, Rhodococcus sp. RR1 exhibited the ability to degrade NDMA by an unidentified, constitutively expressed enzyme that, unlike the confirmed monooxygenases, was not inhibited by acetylene exposure.

Physiological relevance of successive hydroxylations of toluene by toluene para-monooxygenase of Ralstonia pickettii PKO1

We have recently found that toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1 (encoded by tbuA1UBVA2C) performs successive hydroxylations of benzene (Appl. Environ. Microbiol. 70: 3814, 2004) as well as hydroxylates toluene to a mixture of 90% p-cresol and 10% m-cresol which are then further oxidized to 100% 4-methylcatechol (J. Bacteriol. 186: 3117, 2004) whereas it was thought previously that TpMO forms 100% m-cresol and is not capable of successive hydroxylations. Here we propose a modification of the degradation pathway originally described by Olsen et al. (J. Bacteriol. 176: 3749, 1994) that now relies primarily on TpMO for conversion of toluene to 4-methylcatechol (instead of m-cresol) since both m-cresol and p-cresol are shown here to be good substrates for Escherichia coli expressing TpMO (Vmax/Km=0.046, 0.036, and 0.055 mL min−1 mg−1 protein for the oxidation of toluene, m-cresol, and p-cresol, respectively). In light of the broader activity of TpMO, phenol hydroxylase (encoded by tbuD) appears to facilitate conversion of any m-cresol or p-cresol formed from toluene oxidation by TpMO to 4-methylcatechol; hence, the cell has a redundant method for making this important intermediate 4-methylcatechol. Further, it is suggested that the physiological relevance of the 10% m-cresol formed from toluene oxidation by TpMO is needed for induction of the meta cleavage operon tbuWEFGKIHJ to enable full metabolism of toluene since p-cresol (and o-cresol) do not induce the meta-cleavage pathway. Therefore both the successive hydroxylation of toluene by TpMO and the product distribution are of physiological relevance to the cell.

Trichloroethylene degradation by toluene-oxidizing bacteria grown on non-aromatic substrates.

The potential of trichloroethylene (TCE) to induce and non-aromatic growth substrates to support TCE degradation in five strains (Pseudomonas mendocina KR1, Ralstonia pickettii PKO1, Pseudomonas putida F1, Burkholderia cepacia G4, B. cepacia PR1) of toluene-oxidizing bacteria was examined. LB broth and acetate did not support TCE degradation in any of the wild-type strains. In contrast, fructose supported the highest specific levels of TCE oxidation observed in each of the strains tested, except B. cepacia G4. We discuss the potential mechanisms and implications of this observation. In particular, cells of P. mendocina KR1 degraded significant amounts of TCE during cell growth on non-aromatic substrates. Apparently, TCE degradation was not completely constrained by any given factor in this microorganism, as was observed with P. putida F1 (TCE was an extremely poor substrate) or B. cepacia G4 (lack of oxygenase induction by TCE). Our results indicate that multiple physiological traits are required to enable useful TCE degradation by toluene-oxidizing bacteria in the absence of aromatic cosubstrates. These traits include oxygenase induction, effective TCE turnover, and some level of resistance to TCE mediated toxicity.

Degradation of mixtures of aromatic and chloroaliphatic hydrocarbons by aromatic hydrocarbon-degrading bacteria

The well-characterized toluene-oxidizing bacteria Pseudomonas putida PaW1, P. putida F1, P. mendocina KR1, Burkholderia cepacia G4, B. cepacia JS150, and Ralstonia pickettii PKO1, as well as several strains of bacteria isolated from activated sludge, were examined for their ability to degrade mixtures of aromatic and chloroaliphatic hydrocarbons. Collectively, the strains tested were able to transform all of the 14 aromatic hydrocarbons included in the mixtures, with most strains degrading five or more. Few strains degraded trichloroethylene well under these conditions, only one degraded methylene chloride, and none were able to transform chloroform. G4, PKO1, KR1, F1, and JS150 exhibited generally broad but oxygenase-specific degradation profiles, with the three monooxygenase strains degrading significantly more o-xylene and trichloroethylene, and F1 and JS150 degrading greater quantities of isopropylbenzene and 1,4-dichlorobenzene. PaW1 degraded only the methylaromatic hydrocarbons and styrene. Sludge isolates enriched on benzene, toluene, styrene and the xylenes exhibited degradation profiles similar to F1 or PaW1, while the pattern of hydrocarbon degradation for the ethylbenzene, isopropylbenzene, 1,3,5-trimethylbenzene, and 1,3-dichlorobenzene isolates were distinct from the other strains and from each other. Overall, our results showed that many of the bacteria which utilize aromatic compounds are capable of degrading a diverse array of aromatic hydrocarbons in mixtures, but that chloroaliphatics such as methylene chloride and chloroform may be recalcitrant to co-oxidation in the presence of aromatics or trichloroethylene.

Degradation of mixtures of aromatic and chloroaliphatic hydrocarbons by aromatic hydrocarbon-degrading bacteria

The well-characterized toluene-oxidizing bacteria Pseudomonas putida PaW1, P. putida F1, P. mendocina KR1, Burkholderia cepacia G4, B. cepacia JS150, and Ralstonia pickettii PKO1, as well as several strains of bacteria isolated from activated sludge, were examined for their ability to degrade mixtures of aromatic and chloroaliphatic hydrocarbons. Collectively, the strains tested were able to transform all of the 14 aromatic hydrocarbons included in the mixtures, with most strains degrading five or more. Few strains degraded trichloroethylene well under these conditions, only one degraded methylene chloride, and none were able to transform chloroform. G4, PKO1, KR1, F1, and JS150 exhibited generally broad but oxygenase-specific degradation profiles, with the three monooxygenase strains degrading significantly more o-xylene and trichloroethylene, and F1 and JS150 degrading greater quantities of isopropylbenzene and 1,4-dichlorobenzene. PaW1 degraded only the methylaromatic hydrocarbons and styrene. Sludge isolates enriched on benzene, toluene, styrene and the xylenes exhibited degradation profiles similar to F1 or PaW1, while the pattern of hydrocarbon degradation for the ethylbenzene, isopropylbenzene, 1,3,5-trimethylbenzene, and 1,3-dichlorobenzene isolates were distinct from the other strains and from each other. Overall, our results showed that many of the bacteria which utilize aromatic compounds are capable of degrading a diverse array of aromatic hydrocarbons in mixtures, but that chloroaliphatics such as methylene chloride and chloroform may be recalcitrant to co-oxidation in the presence of aromatics or trichloroethylene.

Characterization of the adaptive response to trichloroethylene-mediated stresses in Ralstonia pickettii PKO1.

In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression.

Influence of substrate exposure history on biodegradation in a porous medium

This study investigates the influence of fluctuating toluene concentrations on aerobic toluene degradation in a sandy porous medium colonized with Ralstonia pickettii PKO1. Column effluent toluene concentrations were found to increase after a temporary decrease in influent toluene concentration. Subsequent examination of the spatial gradient of toluene degradative activity in the column suggested that the observed increase in effluent toluene concentrations was attributable to an adverse effect of toluene limitation on the biodegradative activity of attached cells. The traditional Michaelis–Menten-type biodegradation equation associated with batch-measured V max (2.26 mg toluene/mg living cell/day) and K S (1.20 mg toluene/l) of nonstarved cells was unable to predict the observed toluene breakthrough behavior when the column had been previously exposed to no-toluene conditions. An alternative modeling approach was developed based upon the assumptions that (i) degradative activity was completely deactivated within the no-toluene exposure period (53.5 h) and (ii) a lag-phase was present prior to the subsequent reactivation of degradative activity in previously toluene-starved cells. These assumptions were independently verified by batch microbial investigations, and the modified model provided a good fit to the same observed toluene breakthrough curve. Application of single lag-time and threshold concentration values, however, failed to predict observed toluene breakthrough under different toluene exposure conditions. Results of this experimental and modeling investigation suggested that substrate exposure history, including the length of the starvation period and the level of substrate concentration, affected the induction of biodegradation in the porous medium.

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.