Biodegradation and biotransformation of groundwater pollutant mixtures by Mycobacterium vaccae.

Mycobacterium vaccae can catabolize a number of major groundwater pollutants. When added singly, acetone, cyclohexane, styrene, benzene, ethylbenzene, propylbenzene, dioxane, and 1,2-dichloroethylene can be catabolized by M. vaccae. Catabolism of a number of these chemicals was monitored by gas-chromatographic analysis. Gas-chromatographic analysis indicated that the products of benzene degradation are phenol and hydroquinone. The products of chlorobenzene and ethylbenzene degradation are 4-chlorophenol and 4-ethylphenol. The extent that some compounds were catabolized when present as mixtures was also investigated. When toluene and benzene were present concomitantly, toluene was catabolized and benzene oxidation was delayed. Although toluene promoted the degradation of styrene, a lower rate of toluene degradation occurred when styrene was present. Both 4-chlorophenol and 4-ethylphenol had an antagonistic effect on the ability of M. vaccae to degrade other aromatic compounds. Studies with [14C]benzene indicated that M. vaccae can mineralize small amounts of this compound. These results suggest that components in mixtures may have a positive or a negative effect on the rates of biodegradation of other pollutants.     

Evidence for diverse oxidations in the catabolism of toluene by Rhodococcus rhodochrous strain OFS

Rhodococcus rhodochrous strain OFS grew on toluene as a sole source of carbon and energy with a maximum growth rate of 0.011 h⁻¹. Initial reaction products were extracted, derivatized and identified by GC-MS. Oxygen consumption studies indicated that OFS grown on an aliphatic substrate required an induction period before oxidizing toluene. OFS grown on toluene transformed an array of aromatic ground water pollutants including styrene, ethylbenzene and chlorobenzene. Products of these transformations were identified. The sole product of chlorobenzene biotransformation was 4-chlorophenol. Products from toluene oxidation included 3- and 4-methylcatechol as well as benzyl alcohol, p-cresol and cis-toluene dihydrodiol. The identification of these and the products of other aromatic substrate conversions affirm that oxidation occurred on the functional group as well as directly on the aromatic nucleus. Received: 23 July 1999 / Received revision: 4 October 1999 / Accepted: 16 October 1999     

Catabolism of 2,4,6-trinitrotoluene byMycobacterium vaccae

Mycobacterium vaccae strain JOB-5 cometabolized 2,4,6-trinitrotoluene (TNT) in the presence of propane as a carbon and energy source. Two novel oxidized metabolites, as well as several known reduced products, were generated during catabolism of TNT byM. vaccae. During the cometabolic process, there was transient production of a brown chromophore. This compound was identified as 4-amino-2,6-dinitrobenzoic acid. WhenM. vaccae was incubated with [^14CTNT and propane, 50% of the added radiolabel was incorporated into the cellular lipid fraction. These results suggest that ring cleavage occurred prior to the incorporation of radiolabelled carbon into phosphatidyl-l-serine, phosphatidylethanolamine, cardiolipin, and other polar lipids.     

Degradation of volatile hydrocarbons from steam-classified solid waste by a mixture of aromatic hydrocarbon-degrading bacteria

Steam classification is a process for treatment of solid waste that allows recovery of volatile organic compounds from the waste via steam condensate and off-gases. A mixed culture of aromatic hydrocarbon-degrading bacteria was used to degrade the contaminants in the condensate, which contained approx. 60 hydrocarbons, of which 38 were degraded within 4 d. Many of the hydrocarbons, including styrene, 1,2,4-trimethylbenzene, naphthalene, ethylbenzene, m-/p-xylene, chloroform, 1,3-dichloropropene, were completely or nearly completely degraded within one day, while trichloroethylene and 1,2,3-trichloropropane were degraded more slowly.     

Regioselective hydroxylation of chlorobenzene and chlorophenols by a Pseudomonas putida

Summary Pseudomonas putida MST, previously isolated in the presence of -methylstyrene, has been shown to transform several substituted aromatic compounds. It was able to modify halogenated aromatic compounds by co-oxidation. It regiospecifically hydroxylates chlorobenzene and 2-chlorophenol to 3-chlorocatechol, and 4-chlorophenol to 4-chlorocatechol; both metabolites were identified in the cultures.     

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

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

Morpho-functional identification of abdominal olfactory receptors in the midge Culicoides imicola

The aim of the present study was to examine the presence and the possible role of abdominal olfactory sensilla in Culicoides imicola mediating the search for potential hosts and oviposition sites, by means of a morphological, electrophysiological and behavioural approach. The results reported here show that in the midge C. imicola the whole abdomen, comprising the ovipositor, are endowed with three morphotypes of multiporous sensilla that display olfactory sensitivity towards kairomones related to the host-animal skin such as l-(+)-lactic acid and 1-octen-3-ol, to the host-animal urine such as 3-ethylphenol and 4-propylphenol, and to the potent attractant sesame seed oil. Electrophysiological and behavioural data for the first time suggest in the midge the involvement of abdominal olfactory structures in the choice of the oviposition sites and allow in discussing their possible role in the host-animal localisation. Field experiments showed that light traps baited with the aforementioned compounds elicited a stronger degree of attractiveness on midges with respect to the unbaited traps (control), although to a different extent. Our results, while implying a number of considerations concerning the role of molecules tested as kairomones, also suggest their use in the control of the midge C. imicola population.     

Anaerobic biodegradability of alkylphenols and fuel oxygenates in the presence of alternative electron acceptors

Alkylphenols and fuel oxygenates are important environmental pollutants produced by the petrochemical industry. A batch biodegradability test was conducted with selected ortho-substituted alkylphenols (2-cresol, 2,6-dimethylphenol and 2-ethylphenol), fuel oxygenates (methyl tert-butyl ether, ethyl tert-butyl ether and tert-amylmethyl ether) and tert-butyl alcohol (TBA) as model compounds. The ortho-substituted alkylphenols were not biodegraded after 100 days of incubation under methanogenic, sulfate-, or nitrate-reducing conditions. However, biodegradation of 2-cresol and 2-ethylphenol (150 mg l⁻¹) was observed in the presence of Mn (IV) as electron acceptor. The biodegradation of these two compounds took place in less than 15 days and more than 90% removal was observed for both compounds. Mineralization was indicated since no UV-absorbing metabolites accumulated after 23 days of incubation. These alkylphenols were also slowly chemically oxidized by Mn (IV). No biodegradation of fuel oxygenates or TBA (1 g l⁻¹) was observed after 80 or more days of incubation under methanogenic, Fe (III)-, or Mn (IV)-reducing conditions, suggesting that these compounds are recalcitrant under anaerobic conditions. The fuel oxygenates caused no toxicity towards acetoclastic methanogens activity in anaerobic granular sludge. Received: 8 February 2000 / Received revision: 15 May 2000 / Accepted: 19 May 2000     

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

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

Roles of ring-hydroxylating dioxygenases in styrene and benzene catabolism in Rhodococcus jostii RHA1

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ΔbphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the β-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.     

Trichloroethylene degradation by butane-oxidizing bacteria causes a spectrum of toxic effects

The physiological consequences of trichloroethylene (TCE) transformation by three butane oxidizers were examined. Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 utilize distinctly different butane monooxygenases (BMOs) to initiate degradation of the recalcitrant TCE molecule. Although the primary toxic event resulting from TCE cometabolism by these three strains was loss of BMO activity, species differences were observed. P. butanovora and Nocardioides sp. CF8 maintained only 4% residual BMO activity following exposure to 165 μM TCE for 90 min and 180 min, respectively. In contrast, M. vaccae maintained 34% residual activity even after exposure to 165 μM TCE for 300 min. Culture viability was reduced 83% in P. butanovora, but was unaffected in the other two species. Transformation of 530 nmol of TCE by P. butanovora (1.0 mg total protein) did not affect the viability of BMO-deficient P. butanovora cells, whereas transformation of 482 nmol of TCE by toluene-grown Burkholderia cepacia G4 caused 87% of BMO-deficient P. butanovora cells to lose viability. Together, these results contrast with those previously reported for other bacteria carrying out TCE cometabolism and demonstrate the range of cellular toxicities associated with TCE cometabolism.     

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

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

Anaerobic co-metabolic oxidation of 4-alkylphenols with medium-length or long alkyl chains by <Emphasis Type="Italic">Thauera</Emphasis> sp., strain R5

A 4-alkylphenol-degrading facultative anaerobic bacterium, strain R5, was isolated from paddy soil after enrichment with 4-n-propylphenol, 4-n-butylphenol and 4-hydroxybenzoate (4-HBA) under nitrate-reducing conditions. Strain R5 is a Gram-negative rod bacillus grown on phenolic compounds with short alkyl chains (≤C2), organic acids and ethanol. The sequence of the 16S ribosomal RNA gene revealed that the strain is affiliated with Thauera sp. In the presence of 4-HBA as a carbon source, the strain transformed 4-n-alkylphenols with a medium or long-length alkyl chain (C3–C8) to the corresponding oxidised products as follows: 1-(4-hydroxyphenyl)-1-alkenes, -(4-hydroxyphenyl)-1-alkanones and/or 1-(4-hydroxyphenyl)-1-alcohols. The strain also transformed 4-i-propylphenol and 4-sec-butylphenol to (4-hydroxyphenyl)-i-propene and (4-hydroxyphenyl)-sec-butene but not 4-alkylphenols with tertiary alkyl chains (4-t-butylphenol or 4-t-octylphenol). The biotransformation did not proceed without another carbon source and was coupled with nitrate reduction. Biotransformation activity was high in the presence of p-cresol, 4-ethylphenol, 4′-hydroxyacetophenone and 4-HBA as carbon sources and low in the presence of organic acids and ethanol. We suggest that strain R5 co-metabolically transforms alkylphenols to the corresponding metabolites with oxidised alpha carbon in the alkyl chain during coupling with nitrate reduction.     

Degradation of mixtures of monochlorophenols and phenol as substrates for free and immobilized cells of Alcaligenes sp. A7-2

Summary The biodegradation of the three isomeric monochlorophenols 2-(2CP), 3- (3CP) and 4-chlorophenol (4CP) and phenol by the constructed strain Alcaligenes sp. A7-2 was investigated. Mineralization took place in the order: phenol >4CP >2CP >3CP, whereas 3CP was mineralized only co-metabolically. In substrate mixtures with phenol, degradation of 4CP was decelerated but degradation of 2CP was accelerated. Free cells in batch culture showed biphasic growth with an equimolar mixture of 2CP and 4CP as substrates, perhaps due to diauxie. Degradation patterns obtained with free cells in batch culture were confirmed with immobilized cells in continuous culture. Immobilized cells of Alcaligenes sp. A7-2 built up a biofilm on the lava that was used as filling material in the packed-bed reactors. The continuous cultures remained stable despite increasing input rates of chlorophenol and phenol mixtures up to 1.16 mMo1.1^–1.h^–1 for several weeks. Correspondence to: H.-J. Rehm     

Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319

A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of R. ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using polymerase chain reaction. The primary protein structure suggests a natural self-sufficient fusion protein consisting of ferredoxin, flavin-containing reductase, and P450 monooxygenase. The only flavin found within the enzyme was riboflavin 5′-monophosphate. The enzyme was successfully expressed in Escherichia coli, purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene, and ethyl benzene. The conversion of naphthalene, acenaphthene, and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene, and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.     

Bioremediation Assessment of a BTEX‐Contaminated Soil

The elimination of BTEX (benzene, toluene, ethylbenzene, o‐xylene) compounds from soil was studied. After 18 days at 20 °C, 21% of the initial total BTEX contamination (400 mg/kg soil) was lost due to sorption onto soil. Biodegradation decreased in the order ethylbenzene > toluene > benzene > o‐xylene. NPK fertilisation stimulated biodegradation, particularly that of benzene and toluene, significantly, and oleophilic fertilisation inhibited biodegradation. After 18 days, the residual contamination in the NPK‐fertilised, unfertilised and with oleophilic nutrients amended soil was 96, 166 and 196 mg total BTEX/kg soil, respectively. The presence of BTEX initially inhibited the biological activity of the soil (fluorescein diacetate hydrolysis) considerably. This short‐term, reversible inhibition was significantly higher in the unfertilised soil than in the fertilised soil.     

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     

Isolation and Characterization of a New Benzene,Toluene, and Ethylbenzene Degrading Bacterium, <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. B113

A bacterium designated strain B113, able to degrade benzene, toluene, and ethylbenzene compounds (BTE), was isolated from gasoline-contaminated sediment at a gas station in Geoje, Korea. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Acinetobacter. The biodegradation rates of benzene, toluene, and ethylbenzene were relatively low in MSB broth, but the addition of yeast extract had a substantial impact on the biodegradation of BTE compounds, which suggested that yeast extract might provide a factor that was necessary for its growth or BTE biodegradation activity. However, interestingly, the biodegradation of BTE compounds occurred very quickly in slurry systems amended with sterile soil. Moreover, if soil was combusted first to remove organic matters, the enhancement effect on BTE biodegradation was lost, indicating that some insoluble organic compounds were probably beneficial for BTE degradation in contaminated sediment. This study suggests that strain B113 may play an important role for biodegradation of BTE in the contaminated site.     

Propylphenols are metabolites in the anaerobic biodegradation of propylbenzene under iron-reducing conditions

The metabolism of monoaromatic hydrocarbons by an iron-reducing bacterial enrichment culture originating from diesel-contaminated groundwater was examined using d₇-propylbenzene as a model hydrocarbon. Sequence analysis of the 16S rDNA gene showed that the dominant part (10 of 10 clones) of the enrichment culture consisted of a bacterium closely related to clones found in benzene-contaminated groundwater and to the iron-reducing -proteobacterium, Rhodoferax ferrireducens (similarity values were 99.5% and 98.3%, respectively). In degradation studies conducted over 18 weeks, d₇-propylphenols were detected by gas chromatography–mass spectrometry (GC/MS) as intra-cellular metabolites concomitant with cell growth in the cultures. The amount of propylphenols increased during the exponential growth phase, and by the end of this phase 4 × 10^–14 moles of ferric iron were reduced and 3 × 10^–15 moles propylphenol produced for every cell formed. During the stationary growth phase the cell density was approximately 10⁷ ml^–1, with significantly correlated amounts of propylphenols. Succinate derivates of propylbenzene or phenylpropanol previously shown to be the initial metabolites in the anaerobic degradation of alkylbenzenes could not be identified. This study is the first to report that oxidation of propylbenzene to propylphenols can initiate anaerobic propylbenzene degradation and that iron-reducing bacteria are responsible for this process. In addition, the study shows the importance of taking account of the metabolites adhering to solid phases when determining the extent of biodegradation, so as not to underestimate the extent of the process.     

Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase

Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol, and 2-methoxy-4-propylphenol into 1-(4'-hydroxyphenyl)ethanol, 1-(4'-hydroxyphenyl)propanol, and 1-(4'-hydroxy-3'-methoxyphenyl)propanol, respectively, with an ee of 94% for the R enantiomer. The stereochemical outcome of the reactions was established by comparing the chiral GC retention times of the products to those of chiral alcohols obtained by the action of the lipases from Candida antarctica and Pseudomonas cepacia. Isotope labeling experiments revealed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO-mediated conversion of 4-ethylphenol/4-propylphenol, 4-vinylphenol/4-propenylphenol are formed as side products. With 2-methoxy-4-propylphenol as a substrate, this competing side reaction is nearly abolished, resulting in less than 1% of the vinylic product, isoeugenol. The VAO-mediated conversion of 4-alkylphenols also results in small amounts of phenolic ketones indicative for a consecutive oxidation step. Copyright 1998 John Wiley & Sons, Inc.