Dominance of Geobacteraceae in BTX-degrading enrichments from an iron-reducing aquifer

Microbial community structure was linked to degradation potential in benzene-, toluene- or xylene- (BTX) degrading, iron-reducing enrichments derived from an iron-reducing aquifer polluted with landfill leachate. Enrichments were characterized using 16S rRNA gene-based analysis, targeting of the benzylsuccinate synthase-encoding bssA gene and phospholipid fatty acid (PLFA) profiling in combination with tracking of labelled substrate. 16S rRNA gene analysis indicated the dominance of Geobacteraceae, and one phylotype in particular, in all enrichments inoculated with polluted aquifer material. Upon cultivation, progressively higher degradation rates with a concomitant decrease in species richness occurred in all primary incubations and successive enrichments. Yet, the same Geobacteraceae phylotype remained common and dominant, indicating its involvement in BTX degradation. However, the bssA gene sequences in BTX degrading enrichments differed considerably from those of Geobacter isolates, suggesting that the first steps of toluene, but also benzene and xylene oxidation, are carried out by another member of the enrichments. Therefore, BTX would be synthrophically degraded by a bacterial consortium in which Geobacteraceae utilized intermediate metabolites. PLFA analysis in combination with (13)C-toluene indicated that the enriched Geobacteraceae were assimilating carbon originally present in toluene. Combined with previous studies, this research suggests that Geobacteraceae play a key role in the natural attenuation of each BTX compound in situ.     

Isolation,gene detection and solvent tolerance of benzene,toluene and xylene degrading bacteria from nearshore surface water and Pacific Ocean sediment

BTX (benzene, toluene and xylene) degrading bacteria were isolated from Pacific Ocean sediment and nearshore surface water. In the seawater near a ferry dock, degrading bacteria of a relatively wide diversity were detected, including species of Pseudomonas, Rhodococcus, Exiguobacterium and Bacillus; while species of Bacillus only have been detected from the deep-sea sediment. Most of the isolates showed degradation to more than one compound. Generally better growth was obtained with p-xylene and ethylbenzene than with the other two. All the bacteria could tolerate and grow with the compounds at 5–20% (v/v). Both benzene and toluene degradation related genes had been successfully PCR cloned from the isolates of nearshore water, the detected benzene dioxygenase gene was identical among all the species and close to its soil counterpart. However, they were not detected in all the isolates from deep sea. Results in this report suggested that BTX degrading bacteria widely spread in marine environments and they might be of potentials in biotreatment of BTEX in saline environments.     

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

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

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO2. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and o-Xylene in Sediment-Free Iron-Reducing Enrichment Cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO₂. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Enrichment of dissimilatory Fe(III)-reducing bacteria from groundwater of the Siklós BTEX-contaminated site (Hungary)

Dissimilatory iron-reducing bacteria are commonly found in microbial communities of aromatic hydrocarbon-contaminated subsurface environments where they often play key role in the degradation of the contaminants. The Siklós benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated area is one of the best characterized petroleum hydrocarbon-contaminated sites of Hungary. Continuous monitoring of the microbial community in the center of the contaminant plume indicated the presence of an emerging Geobacter population and a Rhodoferax phylotype highly associated with aromatic hydrocarbon-contaminated subsurface environments. The aim of the present study was to make an initial effort to enrich Rhodoferax-related and other dissimilatory iron-reducing bacteria from this environment. Accordingly, four slightly different freshwater media were used to enrich Fe(III) reducers, differing only in the form of nitrogen source (organic, inorganic nitrogen or gaseous headspace nitrogen). Although enrichment of the desired Rhodoferax phylotype was not succeeded, Geobacter-related bacteria were readily enriched. Moreover, the different nitrogen sources caused the enrichment of different Geobacter species. Investigation of the diversity of benzylsuccinate synthase gene both in the enrichments and in the initial groundwater sample indicated that the Geobacter population in the center of the contaminant plume may not play a significant role in the anaerobic degradation of toluene.     

Effects of co-substrates and inhibitors on the anaerobic O-demethylation of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether (MTBE)

Methyl tert-butyl ether (MTBE) contamination is widespread in aquifers near urban areas around the world. Since this synthetic fuel oxygenate is resistant to most physical methods of treating fuel-contaminated water, biodegradation may be a useful means of remediation. Currently, information on anaerobic MTBE degradation is scarce. Depletion has been observed in soil and sediment microcosms from a variety of locations and under several redox conditions, but the responsible organisms are unknown. We are studying anaerobic consortia, enriched from contaminated sediments for MTBE-utilizing microorganisms for over a decade. MTBE degradation occurred in the presence of other fuel components and was not affected by toluene, benzene, ethanol, methanol, or gasoline. Many aryl O-methyl ethers, such as syringic acid, that are O-demethylated by acetogenic bacteria, were also O-demethylated by the MTBE-utilizing enrichment cultures. The addition of these compounds as co-substrates increased the rate of MTBE degradation, offering a potentially useful method of stimulating the MTBE degradation rate in situ. Propyl iodide caused light-reversible inhibition of MTBE degradation, suggesting that the MTBE degradation process is corrinoid dependent. The anaerobic MTBE degradation process was not directly coupled to methanogenesis or sulfidogenesis and was inhibited by the bactericidal antibiotic, rifampicin. These results suggest that MTBE degradation is mediated by acetogenic bacteria.     

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.     

Composition of soil microbial communities enriched on a mixture of aromatic hydrocarbons

Soil contaminated with C5+, which contained benzene (45%, wt/wt), dicyclopentadiene (DCPD) plus cyclopentadiene (together 20%), toluene (6%), styrene (3%), xylenes (2%), naphthalene (2%), and smaller quantities of other compounds, served as the source for isolation of 55 genomically distinct bacteria (standards). Use of benzene as a substrate by these bacteria was most widespread (31 of 44 standards tested), followed by toluene (23 of 44), xylenes (14 of 44), styrene (10 of 44), and naphthalene (10 of 44). Master filters containing denatured genomic DNAs of all 55 standards were used to analyze the community compositions of C5+ enrichment cultures by reverse sample genome probing (RSGP). The communities enriched from three contaminated soils were similar to those enriched from three uncontaminated soils from the same site. The compositions of these communities were time dependent and showed a succession of Pseudomonas and Rhodococcus spp. before convergence on a composition dominated by Alcaligenes spp. The dominant community members detected by RSGP were capable of benzene degradation at all stages of succession. The enrichments effectively degraded all C5+ components except DCPD. Overall, degradation of individual C5+ hydrocarbons followed first-order kinetics, with the highest rates of removal for benzene.     

Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria

Despite its high chemical stability, benzene is known to be biodegradable with various electron acceptors under anaerobic conditions. However, our understanding of the initial activation reaction and the responsible prokaryotes is limited. In the present study, we enriched a bacterial culture that oxidizes benzene to carbon dioxide under sulfate-reducing conditions. Community analysis using terminal restriction fragment length polymorphism, 16S rRNA gene sequencing and FISH revealed 95% dominance of one phylotype that is affiliated to the Gram-positive bacterial genus Pelotomaculum showing that sulfate-reducing Gram-positive bacteria are involved in anaerobic benzene degradation. In order to get indications of the initial activation mechanism, we tested the substrate utilization, performed cometabolism tests and screened for putative metabolites. Phenol, toluene, and benzoate could not be utilized as alternative carbon sources by the benzene-degrading culture. Cometabolic degradation experiments resulted in retarded rates of benzene degradation in the presence of phenol whereas toluene had no effect on benzene metabolism. Phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, and benzoate were identified as putative metabolites in the enrichment culture. However, hydroxylated aromatics were shown to be formed abiotically. Thus, the finding of benzoate as an intermediate compound supports a direct carboxylation of benzene as the initial activation mechanism but additional reactions leading to its formation cannot be excluded definitely.     

Anaerobic Biodegradation of Alkylbenzenes in Laboratory Microcosms Representing Ambient Conditions

A microcosm study was performed to document the anaerobic biodegradation of benzene, toluene, ethylbenzene, m- xylene, and/or o-xylene in petroleum-contaminated aquifer sediment from sites in Michigan (MI) and North Carolina (NC) and relate the results to previous field investigations of intrinsic bioremediation. Laboratory microcosms, designed to simulate ambient conditions, were constructed under anaerobic conditions with sediment and groundwater from source, mid-plume, and end-plume locations at each site. The general patterns of biodegradation and electron acceptor utilization in the microcosms were consistent with field data. At the MI site, methane was produced after a moderate lag period, followed by toluene degradation in all sets of microcosms. At the NC site, biodegradation of the target compounds was not evident in the source area microcosms. In the mid-plume microcosms, toluene and o-xylene biodegraded first, followed by m-xylene and benzene, a pattern consistent with contaminant decay along the plume length. Chemical extraction of microcosm sediment at the beginning and end of me incubation indicated that iron-reducing conditions were dominant and iron reduction occurred on a sediment fraction not extracted by 0.5N HC1. In the end-plume microcosms, degradation of benzene, toluene, and xylene isomers occurred but was variable between replicates. Consistent with field data, dissolved concentrations of the target contaminant(s) persisted at low but detectable levels (0.05 to 0.25 μM) in microcosms from both sites where biodegradation was measured.     

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.     

Composition of Soil Microbial Communities Enriched on a Mixture of Aromatic Hydrocarbons

Soil contaminated with C5+, which contained benzene (45%, wt/wt), dicyclopentadiene (DCPD) plus cyclopentadiene (together 20%), toluene (6%), styrene (3%), xylenes (2%), naphthalene (2%), and smaller quantities of other compounds, served as the source for isolation of 55 genomically distinct bacteria (standards). Use of benzene as a substrate by these bacteria was most widespread (31 of 44 standards tested), followed by toluene (23 of 44), xylenes (14 of 44), styrene (10 of 44), and naphthalene (10 of 44). Master filters containing denatured genomic DNAs of all 55 standards were used to analyze the community compositions of C5+ enrichment cultures by reverse sample genome probing (RSGP). The communities enriched from three contaminated soils were similar to those enriched from three uncontaminated soils from the same site. The compositions of these communities were time dependent and showed a succession of Pseudomonas and Rhodococcus spp. before convergence on a composition dominated by Alcaligenes spp. The dominant community members detected by RSGP were capable of benzene degradation at all stages of succession. The enrichments effectively degraded all C5+ components except DCPD. Overall, degradation of individual C5+ hydrocarbons followed first-order kinetics, with the highest rates of removal for benzene.     

The effect of BTX compounds on the biodegradation of ETBE by an ETBE degrading bacterial consortium

Ethyl tert-butyl ether (ETBE) is a fuel oxygenate that is commonly used in Europe to achieve complete combustion of automobile fuels and to control air pollution. It is potentially toxic and can enter the human system via contaminated water bodies. In the present study, we have identified an enriched bacterial consortium from a gasoline-contaminated site that can degrade ETBE. Bacterial consortium A was able to degrade 47% of the added ETBE in 4 days and it continued to degrade up to 51% in 9 days. Consortium A consisted of Xanthomonas sp., Methylibium sp., Methylobacillus sp., and Methylovorus sp. which were identified as the participating bacteria during ETBE degradation by DGGE — 16S rDNA analysis. In addition to ETBE, this consortium degraded benzene, toluene and xylene isomers (BTX) when they were present as the sole carbon source. The degradation efficiency increased predominantly when ETBE was included as an additional carbon source. Interestingly, the degradation of ETBE decreased to 14% in 9 days when present with BTX compounds. We report that ETBE degradation is slowed down or inhibited when BTX compounds are present. This is a crucial observation for ETBE degradation in the natural environment.     

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

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

Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures.

Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene was correlated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures. However, m-xylene was degraded by a subculture that was incubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.     

Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures

Seven different sources of inocula that included sediments, contaminated soils, groundwater, process effluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, and xylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion of toluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene was correlated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene were observed in these initial enrichment cultures. However, m-xylene was degraded by a subculture that was incubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for the degradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greater than 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of these compounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradative capabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.     

Anaerobic reductive dechlorination of chlorinated dioxins in estuarine sediments

The biotransformation of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-tetraCDD) under anaerobic sulfate-reducing, methanogenic, and iron-reducing conditions was examined with anaerobic enrichment cultures established with sediment from an estuarine intertidal strait in the New York/New Jersey harbor. In addition, the effect of prior enrichment on 2-bromophenol or a mixture of 2-, 3-, and 4-bromophenol on dioxin dechlorination was examined. All enrichments were spiked with 1 ppm 1,2,3,4-tetraCDD and monitored by gas chromatography-mass spectrometry for up to a 3-year period. Reductive dechlorination was initially observed only under methanogenic conditions in the cultures enriched on all three bromophenol isomers. 1,2,3,4-TetraCDD was dechlorinated in the lateral position to 1,2,4-triCDD. The initial appearance of 1,2,4-triCDD was observed after 2 months, with further dechlorination to 1,3-diCDD within 17 months.