Modelling of cometabolic transformation ofortho-xylene in a denitrifying biofilm system

A model describing the cometabolic biotransformation ofo-xylene with toluene as primary carbon source in a continuously fed fixed biofilm reactor is presented. The model is based on the concept of competitive inhibition betweeno-xylene and toluene. The proposed model simulated successfully the transformation ofo-xylene and the associated by-products formation, as well as the toluene degradation. However, it appears that an accurate measurement of active biomass density and distribution in the biofilm is needed, since these factors dramatically affects the modelling. The modelling of various kinetic experiments indicates that the active biomass (or toluene degraders) is accumulated on the top of the biofilm, leading to the conclusion that only a minor part of the biofilm thickness was active. The calibrated model is able to predict the removal of toluene ando-xylene for concentrations ranging from 0 to 30 mg/L. For higher concentrations toxicity phenomena may decrease the accuracy of the model.     

Cometabolic transformation ofo-xylene in a biofilm system under nitrate reducing conditions

The purpose of this work was to investigate the anaerobic transformation ofo-xylene in a laboratory biofilm system with nitrate as an electron acceptor.o-Xylene was degraded cometabolically with toluene as primary carbon source. A mass balance showed thato-xylene was not mineralized but transformed.o-Methyl-benzalcohol ando-methyl-benzaldehyde were identified as intermediates ofo-xylene transformation which resulted in the formation ofo-methyl-benzoic acid as an end product. A cross inhibition phenomenon was observed between toluene ando-xylene. The presence of toluene was necessary for stimulation ofo-xylene transformation, but above a toluene concentration of 1–3 mg/L theo-xylene removal rate dramatically decreased. In returno-xylene inhibited the toluene degradation at concentrations above 2–3 mg/L.     

Transformation ofo-xylene too-methyl benzoic acid by a denitrifying enrichment culture using toluene as the primary substrate

A highly enriched denitrifying mixed culture transformedo-xylene cometabolically along with toluene by methyl group oxidation.o-Methyl benzaldehyde ando-methyl benzoic acid accumulated transiently as metabolic products ofo-xylene transformation. Transformation ofo-methyl benzyl alcohol ando-methyl benzaldehyde occurred independently of toluene degradation and resulted in the formation of a compound coeluting witho-methyl benzoic acid on a gas chromatograph. The cometabolic relationship between toluene ando-xylene could be attributed to a mechanism linked to the initial oxidation of the methyl group.     

Mesophilic and thermophilic BTEX substrate interactions for a toluene-acclimatized biofilter

Benzene, toluene, ethylbenzene and xylene (BTEX) substrate interactions for a mesophilic (25°C) and thermophilic (50°C) toluene-acclimatized composted pine bark biofilter were investigated. Toluene, benzene, ethylbenzene, o-xylene, m-xylene and p-xylene removal efficiencies, both individually and in paired mixtures with toluene (1:1 ratio), were determined at a total loading rate of 18.1 g m^–3 h^–1 and retention time ranges of 0.5–3.0 min and 0.6–3.8 min for mesophilic and thermophilic biofilters, respectively. Overall, toluene degradation rates under mesophilic conditions were superior to degradation rates of individual BEX compounds. With the exception of p-xylene, higher removal efficiencies were achieved for individual BEX compounds compared to toluene under thermophilic conditions. Overall BEX compound degradation under mesophilic conditions was ranked as ethylbenzene >benzene >o-xylene >m-xylene >p-xylene. Under thermophilic conditions overall BEX compound degradation was ranked as benzene >o-xylene >ethylbenzene >m-xylene >p-xylene. With the exception of o-xylene, the presence of toluene in paired mixtures with BEX compounds resulted in enhanced removal efficiencies of BEX compounds, under both mesophilic and thermophilic conditions. A substrate interaction index was calculated to compare removal efficiencies at a retention time of 0.8 min (50 s). A reduction in toluene removal efficiencies (negative interaction) in the presence of individual BEX compounds was observed under mesophilic conditions, while enhanced toluene removal efficiency was achieved in the presence of other BEX compounds, with the exception of p-xylene under thermophilic conditions.     

Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions forin situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, orp-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 10³ to 10⁵ bacteria ml^–1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain ofPseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.Abbreviations BTEX benzene, toluene, ethylbenzene, andp-xylene - HPLC high performance liquid chromatography - GAC granular activated carbon     

Isolation and characterization of acetonitrile utilizing bacteria

Summary Bacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified asChromobacterium sp. andPseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation andP. aeruginosa grew slightly faster thanChromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (¹⁴C)acetonitrile indicated 57% of acetonitrile was degraded byPseudomonas aeruginosa as compared to 43% byChromobacterium. The isolates utilized different nitrile compounds as carbon substrates.     

Tandem biodegradation of BTEX components by two Pseudomonas sp

A co-culture of two Pseudomonas putida isolates was enriched from sediment on a mixture of benzene, toluene, ethylbenzene, m-xylene, p-xylene, and o-xylene. The co-culture readily degraded each of the compounds present. Benzene, toluene, and ethylbenzene were used as growth substrates by one isolate, while toluene, m-xylene, and p-xylene were used as growth substrates by the other. Neither isolate could grow on o-xylene, but it was removed in the presence of the other compounds presumably by co-metabolism. The findings presented here support other reports in which constructed communities were effectively used to degrade blends of between two and four of the components of BTEX. However, here the co-culture of two P. putida isolates effectively degraded a complete BTEX stream containing all six of the components. Received: 4 September 2001 / Accepted: 19 October 2001     

Aerobic Biotransformation of Gasoline Aromatics in MultiComponent Mixtures

The primary objective of this study was to evaluate the impact of substrate interactions on the biotransformation rates and mineralization potentials of gasoline monoaromatics and methyl tert-butyl ether (MTBE), compounds that commonly co-exist in groundwater contaminant plumes. A mixed culture was derived from gasoline-contaminated aquifer material using toluene as the enrichment substrate. Two pure cultures, Rhodococcus sp. RR1 and RR2, were isolated from the mixed culture. The three toluene-grown cultures were shown to biotransform all of the six BTEX compounds (benzene, toluene, ethylbenzene, o-xylene, m-xylene, and p-xylene), both individually and in mixtures, over a broad range of concentrations. The mixed culture was shown to degrade all of the BTEX compounds to ¹⁴CO₂, while the two isolates mineralized BTE(m-/p-)X, but biotransformed o-xylene without production of carbon dioxide. Studies to evaluate substrate interactions caused by the concurrent presence of multiple BTEX compounds during their biodegradation revealed a number of patterns,including competitive inhibition and cometabolism. Ethylbenzene was shown to significantly inhibit BTX degradation in mixtures. MTBE was not biodegraded by any of the three toluene-grown cultures over a range of MTBE concentrations. Furthermore, the presence of MTBE at concentrations of 2 to 100?mg/L had no effect on BTEX biotransformation rates.     

Identification and characterization of <Emphasis Type="Italic">o</Emphasis>-xylene-degrading <Emphasis Type="Italic">Rhodococcus</Emphasis> spp. which were dominant species in the remediation of <Emphasis Type="Italic">o</Emphasis>-xylene-contaminated soils

Soils contaminated with o-xylene were more difficult to bioremediate than those contaminated with other BTEX hydrocarbons (benzene, toluene, ethylbenzene, m-xylene and p-xylene). In order to identify microorganisms responsible for o-xylene degradation in soil, microbial community structure analyses were carried out with two soil samples in the presence of o-xylene and mineral nutrients. In two different soil samples, Rhodococcus opacus became abundant. We were also able to isolate o-xylene degrading Rhodococcus species from these soil samples. A primer set was developed to specifically detect a cluster of this Rhodococcus group including isolated Rhodococcus strains, Rhodococcus opacus and Rhodococcus koreensis. The growth of this bacterial group in an o-xylene-contaminated soil was followed by competitive PCR (cPCR). The decrease in o-xylene clearly paralleled the growth of the Rhodococcus group.     

Metabolic pathways responsible for consumption of aromatic hydrocarbons by microbial associations: Molecular-genetic characterization

Genes for catechol 1,2- and 2,3-dioxygenases were cloned. These enzymes hold important positions in the ortho and meta pathways of the metabolism of aromatic carbons by microbial associations that consume the following volatile organic compounds in pilot minireactors: toluene, styrene, ethyl benzene, o-xylene, m-xylene, and naphthalene. Genes of both pathways were found in an association consuming m-xylene; only genes of the ortho pathway were found in associations consuming o-xylene, styrene, and ethyl benzene, and only genes of the meta pathway were found in associations consuming naphthalene and toluene. Genes of the ortho pathway (C12O) cloned from associations consuming o-xylene and ethyl benzene were similar to corresponding genes located on the pND6 plasmid of Pseudomonas putida. Genes of the ortho pathway from associations consuming o-xylene and m-xylene were similar to chromosomal genes of P. putida. Genes of the meta pathway (C23O) from associations consuming toluene and naphthalene were similar to corresponding genes formerly found in plasmids pWWO and pTOL.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 3, 2005, pp. 298–302.Original Russian Text Copyright © 2005 by Khomenkov, Shevelev, Zhukov, Kurlovich, Zagustina, Popov.     

Isopropylbenzene (cumene) — a new substrate for the isolation of trichloroethene-degrading bacteria

Various bacterial isolates from enrichments with isopropylbenzene (cumene), toluene or phenol as carbon and energy sources were tested as to their potential to oxidize trichloroethene (TCE). In contrast to toluene and phenol, all isolates enriched on isopropylbenzene were able to oxidize TCE. Two isolates, strain JR1 and strain BD1, were identified as Pseudomonas spec. and as Rhodococcus erythropolis, respectively. TCE oxidation was accompanied by the liberation of stoichiometric amounts of chloride. Initial TCE oxidation rate increased proportional to the substrate concentration from 25 to 200 M TCE. Maximal initial TCE-degradation rates found here were 4 to 5 nmol · min^-1 · mg protein^-1. The TCE degradation rate decreased with time. The two isolates showed a temperature optimum for TCE degradation between 10 and 20 °C. In addition to TCE, R. erythropolis BD1 degraded only cis- and trans-dichloroethene whereas Pseudomonas spec. JR1 was able to oxidize also 1,1-dichloroethene, vinyl chloride, trichloroethane, and 1,2-dichloroethane.Abbrevations DMF dimethylformamide - TCE trichloroethene     

Degradation of BTEX compounds in liquid media and in peat biofilters

A mixed culture, enriched from Sphagnum peat moss, contaminated with gasoline vapours, degraded individual and mixed components of BTEX (benzene, toluene, ethylbenzene, xylene). Complete degradation of radiolabelled toluene by the mixed culture was observed in mineralisation studies. Individual isolates from a mixed culture containingPseudomonas maltophilia, P. testosteroni andP. putida biotype A exhibited contrasting BTEX degradation patterns. WhileP. putida biotype A degraded all of the BTEX compounds,P. maltophilia andP. testosteroni, appeared unable to degrade benzene and xylenes, respectively. When the peat, inoculated with the mixed culture, was used as a biofilter (6.2 cm diameter ×93 cm length) for degradation of toluene and ethylbenzene vapours, percentage removal efficiencies were 99 and 85, respectively. When the capacity of the biofilter to degrade a combination of BTEX compounds was evaluated, percentage removal efficiencies for toluene, ethylbenzene,p-xylene,o-xylene and benzene were 99, 85, 82, 80 and 78, respectively. The importance of using the mixed culture as an inoculum in the biofilter was established and also the relationship between contaminated vapour flow rate and percentage removal efficiency.     

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.     

Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of study on the utilization of methyl-substituents of mono-cyclic aromatic hydrocarbons by Pseudomonas aeruginosa S668B2, some organic acids and phenolic compounds were found to be produced in culture broth.

Strain S668B2 was capable of producing ultraviolet absorbing and fluorescent substances from m-xylene. These substances were isolated in the form of crystal and identified as 3-methyl salicylic acid and m-toluic acid.

Strain S668B2 also produced ultraviolet absorbing and fluorescent substances from pseudocumene (1,2,4-trimethyl benzene). These substances were isolated in the crystalline form and identified as 3,4-dimethyl benzoic acid and 3,4-dimethyl phenol.

Strain S668B″ did not attack o-xylene. Under the similar conditions Pseudomonas desmolytica S449B3, which produced a large amount of cumic acid from p-cymene, did not oxidize o-xylene, but grew on p-xylene, m-xylene and 1,2,4-trimethyl benzene.

None out of 364 soil samples gave microorganisms which utilize o-xylene as a sole carbon source.     

Degradation of <Emphasis Type="Italic">o</Emphasis>-xylene and <Emphasis Type="Italic">m</Emphasis>-xylene by a novel sulfate-reducer belonging to the genus <Emphasis Type="Italic">Desulfotomaculum</Emphasis>

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO₂. Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbon-oxidizing bacterium in this genus.     

Alternative pathways foro-xylene orm-xylene andp-xylene degradation in aPseudomonas stutzeri strain

Pseudomonas stutzeri OX1 is able to grow ono-xylene but is unable to grow onm-xylene andp-xylene, which are partially metabolized through theo-xylene degradative pathway leading to the formation of dimethylphenols toxic to OX1.P. stutzeri spontaneous mutants able to grow onm-xylene andp-xylene have been isolated. These mutants soon lose the ability to grow ono-xylene. Data from HPLC analyses and from induction studies suggest that in these mutantsm-xylene andp-xylene could be metabolized through the oxidation of a methyl substituent.P. stutzeri chromosomal DNA is shown to share homology with pWW0 catabolic genes. In the mutant strains the region homologous to pWW0 upper pathway genes has undergone a genomic rearrangement.Abbreviations BADH benzylalcohol dehydrogenase - cat catechol - C23O catechol 2,3-dioxygenase - 2,3-,3,4-,2,4-,2,6-,3,5-2,5-DMP 2,3-,3,4-,2,4-,2,6-,3,5-,2,5-dimethylphenol - 2-MBOH 2-methylbenzyl alcohol - 3-MBOH 3-methylbenzyl alcohol - 4-MBOH 4-methylbenzyl alcohol - m-,p-tol m-,p-toluate - o-,m-,p-xyl o-,m-,p-xylene     

BTEX catabolism interactions in a toluene-acclimatized biofilter

BTEX substrate interactions for a toluene-acclimatized biofilter consortium were investigated. Benzene, ethylbenzene, o-xylene, m-xylene and p-xylene removal efficiencies were determined at a loading rate of 18.07 g m⁻³h⁻¹ and retention times of 0.5–3.0 min. This was also repeated for toluene in a 1:1 (m/m) ratio mixture (toluene: benzene, ethylbenzene, or xylene ) with each of the other compounds individually to obtain a final total loading of 18.07 g m⁻³h⁻¹. The results obtained were modelled using Michaelis–Menten kinetics and an explicit finite difference scheme to generate v _max and K _m parameters. The v _max/K _m ratio (a measure of the catalytic efficiency, or biodegradation capacity, of the reactor) was used to quantify substrate interactions occurring within the biofilter reactor without the need for free-cell suspended and monoculture experimentation. Toluene was found to enhance the catalytic efficiency of the reactor for p-xylene, while catabolism of all the other compounds was inhibited competitively by the presence of toluene. The toluene-acclimatized biofilter was also able to degrade all of the other BTEX compounds, even in the absence of toluene. The catalytic efficiency of the reactor for compounds other than toluene was in the order: ethylbenzene>benzene>o-xylene>m-xylene>p-xylene. The catalytic efficiency for toluene was reduced by the presence of all other tested BTEX compounds, with the greatest inhibitory effect being caused by the presence of benzene, while o-xylene and p-xylene caused the least inhibitory effect. This work illustrated that substrate interactions can be determined directly from biofilter reactor results without the need for free-cell and monoculture experimentation. Received: 13 April 2000 / Received revision: 20 July 2000 / Accepted: 27 July 2000     

Monocyclic Aromatic Hydrocarbon Degradation by Rhodococcus sp. Strain DK17

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.     

The influence of creosote compounds on the aerobic degradation of toluene

The inhibiting effect of 14 typical creosote compounds on the aerobic degradation of toluene was studied in batch experiments. Four NSO-compounds (pyrrole, 1-methylpyrrole, thiophene, and benzofuran) strongly inhibited the degradation of toluene. When the NSO-compounds were present together with toluene, little or no degradation of toluene was observed during 16 days of incubation, compared with a total removal of toluene within 4 days when the four compounds were absent. Indole (an N-compound) and three phenolic compounds (phenol, o-cresol, and 2,4-dimethylphenol) also inhibited the degradation of toluene, though the effect was much weaker that of the four NSO-compounds. O-xylene, p-xylene, naphthalene and 1-methylnaphthalene seemed to stimulate the degradation even though the influence was very weak. No effects of benzothiophene (an S-compound) and quinoline (an N-compound) were observed. Benzofuran (an O-compound) was identified as the compound that most inhibited the degradation of toluene. An effect could be detected even at low concentrations (40 g/l).Abbreviations bf benzofuran - bt benzothiophene - dmp 2,4-dimethylphenol - GC gas chromatograph - ind indole - mnap 1-methylnaphthalene - MAH monoaromatic hydrocarbons - mpyr 1-methylpyrrole - nap naphthalene - o-cre o-cresol - o-xyl o-xylene - phe phenol - pyr pyrrole - p-xyl p-xylene - tol toluene - thi thiophene - qui quinoline     

The roles of intermediates in biodegradation of benzene,toluene, and p-xylene by Pseudomonas putida F1

Several types of biodegradation experiments with benzene, toluene, or p-xylene show accumulation of intermediates by Pseudomonas putida F1. Under aerobic conditions, the major intermediates identified for benzene, toluene, and p-xylene are catechol, 3-methylcatechol, and 3,6-dimethylcatechol, respectively. Oxidations of catechol and 3-methylcatechol are linked to biomass synthesis. When oxygen is limited in the system, phenol (from benzene) and m-cresol and o-cresol (from toluene) accumulate.