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.     

Amplification of toluene dioxygenase genes in a hybrid Pseudomonas strain to enhance the biodegradation of benzene, toluene, and p-xylene mixture

A hybrid metabolic pathway through which benzene, toluene, and p-xylene (BTX) mixture could be simultaneously mineralized was previously constructed in Pseudomonas putida TB101 (Lee, Roh, Kim, Biotechnol. Bioeng 43: 1146-1152, 1994). In this work, we improved the performance of the hybrid pathway by cloning the todC1C2BA genes in the broad-host-range multicopy vector RSF1010 and by introducing the resulting plasmid pTOL037 into P. putida mt-2 which harbors the archetypal TOL plasmid. As a result, a new hybrid strain, P. putida TB103, possessing the enhanced activity of toluene dioxygenase in the hybrid pathway was constructed. The degradation rates of benzene, toluene, and p-xylene by P. putida TB103 were increased by about 9.3-, 3.7-, and 1.4-fold, respectively, compared with those by previously constructed P. putida TB101. Apparently, this improved capability of P. putida TB103 for the degradation of BTX mixture resulted from the amplification of the todC1C2BA genes. Furthermore, a relatively long lag period for benzene degradation observed when P. putida TB101 was used for the degradation of BTX mixture at low dissolved oxygen (DO) tension disappeared when P. putida TB103 was employed. (c) 1995 John Wiley & Sons, Inc.     

Kinetics of aerobic biodegradation of benzene and toluene in sandy aquifer material

Monod's equation adequately described aerobic biodegradation rates of benzene and toluene by the microbial population of a sandy aquifer when these compounds were initially present at concentrations lower than 100 mg/l each. Concentrations higher than 100 mg/l were inhibitory, and no benzene or toluene degradation was observed when these compounds were initially present at 250 mg/l each. The Monod coefficients were calculated as k = 8.3 g-benzene/g-cells/day and Ks = 12.2 mg/l for benzene, and k = 9.9 g-toluene/g-cells/day and Ks = 17.4 mg/l for toluene. Specific first-order coefficients would be 0.68 l/mg.day for benzene and 0.57 l.mg.day for toluene.     

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.     

Regiospecific oxidation of naphthalene and fluorene by toluene monooxygenases and engineered toluene 4-monooxygenases of Pseudomonas mendocina KR1

The regiospecific oxidation of the polycyclic aromatic hydrocarbons naphthalene and fluorene was examined with Escherichia coli strains expressing wildtype toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1, toluene para-monooxygenase (TpMO) from Ralstonia pickettii PKO1, toluene ortho-monooxygenase (TOM) from Burkholderia cepacia G4, and toluene/ortho-xylene monooxygenase (ToMO) from P. stutzeri OX1. T4MO oxidized toluene (12.1+/-0.8 nmol/min/mg protein at 109 microM), naphthalene (7.7+/-1.5 nmol/min/mg protein at 5 mM), and fluorene (0.68+/-0.04 nmol/min/mg protein at 0.2 mM) faster than the other wildtype enzymes (2-22-fold) and produced a mixture of 1-naphthol (52%) and 2-naphthol (48%) from naphthalene, which was successively transformed to a mixture of 2,3-, 2,7-, 1,7-, and 2,6-dihydroxynaphthalenes (7%, 10%, 20%, and 63%, respectively). TOM and ToMO made 1,7-dihydroxynaphthalene from 1-naphthol, and ToMO made a mixture of 2,3-, 2,6-, 2,7-, and 1,7-dihydroxynaphthalene (26%, 22%, 1%, and 44%, respectively) from 2-naphthol. TOM had no activity on 2-naphthol, and T4MO had no activity on 1-naphthol. To take advantage of the high activity of wildtype T4MO but to increase its regiospecificity on naphthalene, seven engineered enzymes containing mutations in T4MO alpha hydroxylase TmoA were examined; the selectivity for 2-naphthol by T4MO I100A, I100S, and I100G was enhanced to 88-95%, and the selectivity for 1-naphthol was enhanced to 87% and 99% by T4MO I100L and G103S/A107G, respectively, while high oxidation rates were maintained except for G103S/A107G. Therefore, the regiospecificity for naphthalene oxidation was altered to practically pure 1-naphthol or 2-naphthol. All four wildtype monooxygenases were able to oxidize fluorene to different monohydroxylated products; T4MO oxidized fluorene successively to 3-hydroxyfluorene and 3,6-dihydroxyfluorene, which was confirmed by gas chromatography-mass spectrometry and 1H nuclear magnetic resonance analysis. TOM and its variant TomA3 V106A oxidize fluorene to a mixture of 1-, 2-, 3-, and 4-hydroxyfluorene. This is the first report of using enzymes to synthesize 1-, 3-, and 4-hydroxyfluorene, and 3,6-dihydroxyfluorene from fluorene as well as 2-naphthol and 2,6-dihydroxynaphthalene from naphthalene.     

Peptide conjugates of benzene and toluene metabolites in English ryegrass

Biotransformation of [1-6-¹⁴C]benzene and [1-¹⁴C]toluene in English ryegrass (Lolium perenne L.) seedlings was investigated. Vapors of these compounds were absorbed by the leaves of this plant. Benzene and toluene were oxidized, forming phenol and benzoic acid, respectively. A portion of phenol and benzoic acid was bound by low-molecular-weight peptides forming conjugates. A qualitative amino acid composition of the peptides involved in the conjugation was determined. After removing plants from the atmosphere containing [1-6-¹⁴C]benzene and [1-¹⁴C]toluene, the radioactivity of the conjugates gradually decreased. This process was accompanied by the evolution of ¹⁴CO₂, indicating the breakdown of these conjugates. Radioactive compounds thus formed were oxidized, yielding carbon dioxide. A portion of phenol and benzoic acid, along with peptide conjugation, was subjected to further oxidative transformations up to disruption of the aromatic ring. By this pathway, nonvolatile carboxylic acids, such as muconic, fumaric, succinic, malic, malonic, glycolic, and glyoxylic, were formed. Using electron microscopy, a damaging effect of benzene on the cell ultrastructure of English ryegrass leaves was shown, and this toxic effect depended on the benzene concentration.     

Quantitative analysis of experiments on bacterial chemotaxis to naphthalene

A mathematical model was developed to quantify chemotaxis to naphthalene by Pseudomonas putida G7 (PpG7) and its influence on naphthalene degradation. The model was first used to estimate the three transport parameters (coefficients for naphthalene diffusion, random motility, and chemotactic sensitivity) by fitting it to experimental data on naphthalene removal from a discrete source in an aqueous system. The best-fit value of naphthalene diffusivity was close to the value estimated from molecular properties with the Wilke-Chang equation. Simulations applied to a non-chemotactic mutant strain only fit the experimental data well if random motility was negligible, suggesting that motility may be lost rapidly in the absence of substrate or that gravity may influence net random motion in a vertically oriented experimental system. For the chemotactic wild-type strain, random motility and gravity were predicted to have a negligible impact on naphthalene removal relative to the impact of chemotaxis. Based on simulations using the best-fit value of the chemotactic sensitivity coefficient, initial cell concentrations for a non-chemotactic strain would have to be several orders of magnitude higher than for a chemotactic strain to achieve similar rates of naphthalene removal under the experimental conditions we evaluated. The model was also applied to an experimental system representing an adaptation of the conventional capillary assay to evaluate chemotaxis in porous media. Our analysis suggests that it may be possible to quantify chemotaxis in porous media systems by simply adjusting the model's transport parameters to account for tortuosity, as has been suggested by others.     

Dynamic response of naphthalene biodegradation in a continuous flow soil slurry reactor

Periodic perturbations were used to evaluate the system stability and robustness of naphthalene biodegradation in a continuous flow stirred tank reactor (CSTR) containing a soil slurry. The experimental design involved perturbing the test system using a sinusoidal input either of naphthalene or non-naphthalene organic carbon at different frequencies during steady state operation of the reactors. The response of the test system was determined by using time series off-gas analysis for naphthalene liquid phase concentration and degradation, total viable cell counts, and gene probe analysis of naphthalene degradative genotype, and by batch mineralization assays.Naphthalene biodegradation rates were very high throughout the experimental run (95 to >99% removed) resulting in very low or undetectable levels of naphthalene in the off-gas and reactor effluent. Attempts to reduce the rate of naphthalene biotransformation by either reducing the reactor temperature from 20°C to 10°C or the dissolved oxygen level (>1 mg/L) were unsuccessful. Significant naphthalene biodegradation was observed at 4°C. While variable, the microbial community as measured by population densities was not significantly affected by temperature changes. In terms of naphthalene biotransformation, the system was able to adapt readily to all perturbations in the reactor.Department of Chemical EngineeringDepartment of Microbiology and The Graduate Program in EcologyDepartment of Civil Engineering, New Orleans University     

Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review

Bioremediation of aromatic hydrocarbons in groundwater and sediments is often limited by dissolved oxygen. Many aromatic hydrocarbons degrade very slowly or not at all under anaerobic conditions. Nitrate is a good alternative electron acceptor to oxygen, and denitrifying bacteria are commonly found in the subsurface and in association with contaminated aquifer materials. Providing both nitrate and microaerophilic levels of oxygen may result in oxidation of the stable benzene rings in aromatic contaminants and allow for the intermediates of this oxidation to degrade via denitrification. The effects of using mixed electron acceptors on biodegradation of subsurface contaminants is unclear. Below some critical oxygen threshold, aerobic biodegradation is inhibited, however high levels of oxygen inhibit denitrification. The mechanisms which regulate electron transfer to oxygen and nitrate are complex. This review: 1) describes the factors which may affect the utilization of oxygen and nitrate as dual electron acceptors during biodegradation; 2) summarizes the incidence of dual use of nitrate and oxygen (aerobic denitrification); and 3) presents evidence of the effectiveness of bioremediation under mixed oxygen/nitrate conditions. Received 08 November 1995/ Accepted in revised form 09 June 1996     

Cloning and expression of a pathway for benzene and toluene fromBacillus stearothermophilus

Bacillus stearothermophilus strain BR325 demonstrating broad aromatic substrate capability was isolated from petroleum-contaminated soil. The chromosomally-located aromatic pathway from this isolate was cloned intoEscherichia coli as a 32 kb insert in cosmid pHC79, conferring growth on benzene, phenol, and toluene as sole carbon sources.     

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.     

生物降解萘的研究进展*

萘的生物降解具有低成本,效果佳,无二次污染等优势,受到全世界的广泛关注。本文从萘降解菌的种类、降解质粒与降解基因的开发与研究、三种降解途径的发展以及表面活性剂、菌体固定化技术和有机溶剂在萘降解和环境污染治理当中的应用情况等方面综述了生物降解萘的发展历程。从降解菌株的研究和表面活性剂以及生物技术手段的应用情况等角度分析了目前生物降解萘还存在的问题,并为解决这些问题提出了合理的方案。论述了两相体系技术在生物降解萘上的可应性,对其强大的发展潜力进行了展望。     

Genetics of naphthalene and phenanthrene degradation by Comamonas testosteroni

Naphthalene and phenanthrene have long been used as model compounds to investigate the ability of bacteria to degrade polycyclic aromatic hydrocarbons. The catabolic pathways have been determined, several of the enzymes have been purified to homogeneity, and genes have been cloned and sequenced. However, the majority of this work has been performed with fast growing Pseudomonas strains related to the archetypal naphthalene-degrading P. putida strains G7 and NCIB 9816-4. Recently Comamonas testosteroni strains able to degrade naphthalene and phenanthrene have been isolated and shown to possess genes for polycyclic aromatic hydrocarbon degradation that are different from the canonical genes found in Pseudomonas species. For instance, C. testosteroni GZ39 has genes for naphthalene and phenanthrene degradation which are not only different from those found in Pseudomonas species but are also arranged in a different configuration. C. testosteroni GZ42, on the other hand, has genes for naphthalene and phenanthrene degradation which are arranged almost the same as those found in Pseudomonas species but show significant divergence in their sequences. Received 10 August 1997/ Accepted in revised form 15 August 1997     

Behaviour of toluene, benzene and naphthalene under anaerobic conditions in sediment columns

The biotransformation of toluene, benzene and naphthalene was examined in anaerobic sediment columns. Five columns filled with a mixture of sediments were operated in the presence of bicarbonate, sulfate, iron, manganese, or nitrate as electron acceptor. The columns were continuously percolated with a mixture of the three organic compounds (individual concentrations 25–200 M) at 20°C.Toluene was transformed readily (within 1 to 2 months) under all redox conditions tested. Benzene was recalcitrant over the test period of 375–525 days in all five columns. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect was observed after adding and omitting an easier degradable substrate. [¹⁴C]naphthalene was used to confirm this disappearance to be the result of degradation; two third of the naphthalene was converted to CO₂.     

Functional analysis of genes involved in biphenyl, naphthalene, phenanthrene, and m-xylene degradation by Sphingomonas yanoikuyae B1

Sphingomonas yanoikuyae B1 is able to utilize toluene, m-xylene, p-xylene, biphenyl, naphthalene, phenanthrene, and anthracene as sole sources of carbon and energy for growth. A forty kilobase region of DNA containing most of the genes for the degradation of these aromatic compounds was previously cloned and sequenced. Insertional inactivation of bphC results in the inability of B1 to grow on both polycyclic and monocyclic compounds. Complementation experiments indicate that the metabolic block is actually due to a polar effect on the expression of bphA3, coding for a ferredoxin component of a dioxygenase. Lack of the ferredoxin results in a nonfunctional polycyclic aromatic hydrocarbon dioxygenase and a nonfunctional toluate dioxygenase indicating that the electron transfer components are capable of interacting with multiple oxygenase components. Insertional inactivation of a gene for a dioxygenase oxygenase component downstream of bphA3 had no apparent effect on growth besides a polar effect on nahD which is only needed for growth of B1 on naphthalene. Insertional inactivation of either xylE or xylG in the meta-cleavage operon results in a polar effect on bphB, the last gene in the operon. However, insertional inactivation of xylX at the beginning of this cluster of genes does not result in a polar effect suggesting that the genes for the meta-cleavage pathway, although colinear, are organized in at least two operons. These experiments confirm the biological role of several genes involved in metabolism of aromatic compounds by S. yanoikuyae B1 and demonstrate the interdependency of the metabolic pathways for polycyclic and monocyclic aromatic hydrocarbon degradation. Received 13 May 1999/ Accepted in revised form 05 July 1999     

Behaviour of toluene, benzene and naphthalene under anaerobic conditions in sediment columns

The biotransformation of toluene, benzene and naphthalene was examined in anaerobic sediment columns. Five columns filled with a mixture of sediments were operated in the presence of bicarbonate, sulfate, iron, manganese, or nitrate as electron acceptor. The columns were continuously percolated with a mixture of the three organic compounds (individual concentrations 25–200 μM) at 20°C. Toluene was transformed readily (within 1 to 2 months) under all redox conditions tested. Benzene was recalcitrant over the test period of 375–525 days in all five columns. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect was observed after adding and omitting an easier degradable substrate. [¹⁴C]naphthalene was used to confirm this disappearance to be the result of degradation; two third of the naphthalene was converted to CO₂.     

Substrate inhibition kinetics for toluene and benzene degrading pure cultures and a method for collection and analysis of respirometric data for strongly inhibited cultures

We present an evaluation of the qualitative and quantitative effects that high concentrations of benzene and toluene have on the growth rate of several pure cultures that use these compounds as their sole carbon and energy source. The cultures employed were five widely studied environmental isolates: Pseudomonas putida F1, P. putida mt2, P. mendocina KR, Ralstonia pickettii PKO1, and Burkholderia cepacia G4. Three cultures degraded toluene following a pattern consistent with the kinetic model of Wayman and Tseng (1976) while the other two followed a modification of this model introduced by Alagappan and Cowan (2001). The pattern followed for benzene degradation was different than that for toluene degradation for all four capable pure cultures and consistent with that described by the model of Luong (1987). Mechanisms of substrate inhibition and solvent toxicity are discussed, used to conceptually evaluate the reasons for the differences in inhibition behavior, and used to support a call for more widespread use of the empirical, terminal substrate concentration inhibition models employed here. We also present the methodology developed to overcome a limitation commonly encountered when attempting to collect oxygen uptake data for use in quantifying substrate inhibition kinetics. The experimental method was effective for use in the collection of high quality data and the substrate inhibition models most useful in representing the growth of bacteria on these solvents are those that show a complete loss of activity at high concentration rather than the more popular asymptotic inhibition models.     

Modeling of DNAPL-Dissolution, Rate-Limited Sorption and Biodegradation Reactions in Groundwater Systems

This article presents an approach for modeling the dissolution process of single component dense non-aqueous phase liquids (DNAPL), such as tetrachloroethene and trichloroethene, in a biologically reactive porous medium. In the proposed approach, the overall transport processes are conceptualized as three distinct reactions. Firstly, the dissolution (or dissolving) process of a residual DNAPL source zone is conceptualized as a mass-transfer limited reaction. Secondly, the contaminants dissolved from the DNAPL source are allowed to partition between sediment and water phases through a rate-limited sorption reaction. Finally, the contaminants in the solid and liquid phases are allowed to degrade by a set of kinetic-limited biological reactions. Although all of these three reaction processes have been researched in the past, little progress has been made towards understanding the combined effects of these processes. This work provides a rigorous mathematical model for describing the coupled effects of these three fundamental reactive transport mechanisms. The model equations are then solved using the general-purpose reactive transport code RT3D (Clement, 1997).     

Construction of a bacterial consortium for the biofiltration of benzene,toluene and xylene emissions

On equal parts of benzene, toluene and p-xylene (BTX), a stable bacterial consortium was enriched for removal of BTX vapours from air. As demonstrated by gas chromatographic monitoring, this consortium removed all three BTX components but was able to grow only on benzene and/or toluene. A Pseudomonas putida strain, PPO1, isolated from this consortium behaved in an identical manner. When immobilized on a porous peat/perlite column, both the consortium and the PPO1 isolated removed all three BTX components from metered air streams. However, due to the accumulation of products from the incompletely metabolized p-xylene, the removal rates were unsatisfactory and declined further with time. P. putida ATCC 33015 bearing the TOL plasmid was capable of growing on toluene, on para- and on meta- xylene isomers, but not on benzene. When the PPO1 and ATCC 33015 strains were immobilized, in equal parts, on peat/perlite columns a much improved and sustainable removal of all three BTX components was observed at the rate of 40–50 g/h. m3 filter bed. Due to the dominance of the ring-hydroxylating pathways over the TOL pathway, the classical enrichment approach did not result in a consortium capable of the sustained removal of all BTX components. However, a rationally formulated consortium consisting of members with complementary metabolic abilities was capable of this task and should be of use both in industrial emission control and in soil venting operations.