Biodegradation of Trichloroethylene and Dichloromethane in Contaminated Soil and Groundwater

Soil column and serum bottle microcosm experiments were conducted to investigate the potential for in situ anaerobic bioremediation of trichloroethy lene (TCE) and dichloromethane (DCM) at the Pinellas site near Largo, Florida. Soil columns with continuous groundwater recycle were used to evaluate treatment with complex nutrients (casamino acids, methanol, lactate, sulfate); benzoate and sulfate; and methanol. The complex nutrients drove microbial dechlorination of TCE to ethene, whereas the benzoate/sulfate and methanol supported microbial dechlorination of TCE only to cis-1 ,2-dichloroethylene (cDCE). Microbial sulfate depletion in the benzoate/sulfate column allowed further dechlorination of cDCE to vinyl chloride. Serum bottle microcosms were used to investigate TCE dechlorination and DCM biodegradation in Pinellas soil slurries bioaugmented with liquid from the soil columns possessing TCE-dechlorinating activity and DCM biodegradation by indigenous microorganisms. Bioaugmented soil microcosms showed immediate TCE dechlorination in the microcosms with methanol or complex nutrients, but no dechlorination in the benzoate/sulfate microcosm. DCM biodegradation by indigenous microorganisms occurred in soil microcosms amended with either benzoate/sulfate or methanol, but not with complex nutrients. Bioaugmentation stimulated DCM biodegradation in both complex nutrient and methanol-amended microcosms, but appeared to inhibit DCM biodegradation in benzoate/sulfate-amended microcosms. TCE dechlorination occurred before DCM biodegradation in bioaugmented microcosms when both compounds were present.     

Kinetic Modelling and Half-Life Study on Bioremediation of Soil Co-Contaminated with Lubricating Motor Oil and Lead Using Different Bioremediation Strategies

The focus of this study was to investigate the effect of nutrient supplement (urea fertilizer) and microbial species augmentation (mixed culture of Aeromonas, Micrococcus, and Serratia sp.) on biodegradation of lubricating motor oil (LMO) and lead uptake by the autochthonous microorganism in LMO and lead-impacted soil were investigated. The potential inhibitory effects of lead on hydrocarbon utilization were investigated over a wide range of lead concentrations (25–200 mg/kg) owing to the complex co-contamination problem frequently encountered in most sites. Under aerobic conditions, total petroleum hydrocarbons (TPH) removal was 45.3% in the natural attenuation microcosm while a maximum of 72% and 68.2% TPH removal was obtained in biostimulation and bioaugmentation microcosms, respectively. Lead addition, as lead nitrate, to soil samples reduced the number of hydrocarbon degraders in all samples by a wide range (11–52%) depending on concentration and similarly, the metabolic activities were affected as observed in mineralization of LMO (3–60%) in soils amended with various lead concentrations. Moreover, the uptake of lead by the autochthonous microorganisms in the soil reduced with increase in the initial lead concentration. First-order kinetics described the biodegradation of LMO very well. The biodegradation rate constants were 0.015, 0.033, and 0.030 day^?1 for LMO degradation in natural attenuation, biostimulation and bioaugmentation treatment microcosms, respectively. The presence of varying initial lead concentration reduced the biodegradation rate constant of LMO degradation in the biostimulation treatment microcosm. Half-life times were 46.2, 21, and 23 days for LMO degradation in natural attenuation, biostimulation and bioaugmentation treatment microcosms, respectively. The half-life time in the biostimulation treatment microcosm was increased with a range between 10.7 and 39.2 days by the presence of different initial lead concentration. The results have promising potential for effective remediation of soils co-contaminated with hydrocarbons and heavy metals.     

Presence,diversity and enumeration of functional genes (bssA and bamA) relating to toluene degradation across a range of redox conditions and inoculum sources

The study investigates two functional genes for toluene degradation across three redox conditions (nitrate and sulfate amended and methanogenic). The genes targeted include benzylsuccinate synthase α-subunit (bssA) and a gene recently identified as being a strong indicator of anaerobic aromatic degradation, called 6-oxocylcohex-1-ene-1-carbonyl-CoA hydrolase (bamA). In all, sixteen different anaerobic toluene degrading microcosms were investigated using several primers sets targeting bssA and one primer set targeting bamA. One bssA primer set (7772f/8546r) was the most successful in producing a strong amplicon (eight from sixteen) with the other bssA primers sets producing strong amplicons in six or less samples. In contrast, the bamA primer set (bam-sp9 and bam-asp1) produced a strong amplicon in DNA extracted from all except one microcosm. Partial bssA and bamA sequences were obtained for a number of samples and compared to those available in GenBank. The partial bssA sequences (from nitrate amended and methanogenic microcosms) were most similar to Thauera sp. DNT-1, Thauera aromatica, Aromatoleum aromaticum EbN1 and bssA clones from a study involving sulfate reducing toluene degradation. The bamA sequences obtained could be placed into five previously defined clades (bamA-clade 1, Georgfuchsia/Azoarcus, Magnetospirillum/Thauera Syntrophus and Geobacter clades), with the placement generally depending on redox conditions. Gene numbers were also correlated with toluene degradation and the final gene number for both genes differed considerably between the range of redox conditions. The work is the first in depth investigation of bamA diversity over a range of redox conditions and inoculum sources.     

Anaerobic Benzene Biodegradation: A Microcosm Survey

Benzene-amended microcosms prepared with saturated soil or sediment from five hydrocarbon-contaminated sites and one pristine site were monitored for a year and a half to determine the rate of benzene biodegradation under a variety of electron-accepting conditions. Sustainable benzene degradation was observed under specific conditions in microcosms from four of the six sites. Significant differences were observed between sites with respect to lag times before the onset of degradation, rates of degradation, sustainability of the activity, and environmental conditions supporting degradation. Benzene degradation was observed under sulfate-reducing, nitrate-reducing, and iron(III)-reducing conditions, but not under methanogenic conditions. The presence of competing substrates such as toluene, xylenes, and ethylbenzene was found to inhibit anaerobic benzene degradation in microcosms where sulfate or possibly nitrate was the electron acceptor for benzene degradation, but not in microcosms from where iron(III) was the electron acceptor. The presence of organic matter, indicated by a high fraction organic carbon (f_oc), also appeared to inhibit the biodegradation of benzene; microcosms constructed with soils with the highest f_oc exhibited the longest lag times before the onset of benzene degradation. The initial extent of hydrocarbon contamination did not appear to correlate with anaerobic benzene-degrading activity.     

Bacterial community changes in TCE biodegradation detected in microcosm experiments

Laboratory microcosm experiments were set up to model biodegradation of trichloroethylene (TCE). Groundwater samples from two contaminated sites were taken, one of them with low (70 mg L⁻¹), the other with high sulfate (685 mg L⁻¹) concentration. In order to assess the biodegradative potential of natural microbiota, supplementary substrates (whey or molasses) were added to the bottles. At day 54, 98, 155, and 318, chemical and bacteriological parameters (i.e., Dehalococcoides test) were investigated. Terminal restriction fragment length polymorphism (T-RFLP) based diversity assessments were carried out to observe the bacterial community changes. Whey and molasses enhanced degradation at different rates. In the case of samples with high sulfate content and amended with whey, no ethylene, ethane, or methane was generated. Both ethylene and methane production was detected in samples of low sulfate content with added whey. The results of Dehalococcoides tests were positive for all control and amended samples. Based on T-RFLP analysis, the bacterial communities of high sulfate concentration groundwater microcosms amended with molasses or whey were very similar, while the communities of groundwater samples with low sulfate concentration were different when supplemented with whey or molasses. The rRNA and rDNA based investigations suggest that the proportions of the active microbes and the microbes present in the microcosms differ.     

Methyl tert-butyl ether (MTBE) degradation by a microbial consortium

The widespread use of methyl tert-butyl ether (MTBE) as a gasoline additive has resulted in a large number of cases of groundwater contamination. Bioremediation is often proposed as the most promising alternative after treatment. However, MTBE biodegradation appears to be quite different from the biodegradation of usual gasoline contaminants such as benzene, toluene, ethyl benzene and xylene (BTEX). In the present paper, the characteristics of a consortium degrading MTBE in liquid cultures are presented and discussed. MTBE degradation rate was fast and followed zero order kinetics when added at 100 mg l(-1). The residual MTBE concentration in batch degradation experiments ranged from below the detection limit (1 microg l(-1)) to 50 microg l(-1). The specific activity of the consortium ranged from 7 to 52 mgMTBE g(dw)(-1) h(-1) (i.e. 19-141 mgCOD g(dw) (-1) h(-1)). Radioisotope experiments showed that 79% of the carbon-MTBE was converted to carbon-carbon dioxide. The consortium was also capable of degrading a variety of hydrocarbons, including tert-butyl alcohol (TBA), tert-amyl methyl ether (TAME) and gasoline constituents such as benzene, toluene, ethylbenzene and xylene (BTEX). The consortium was also characterized by a very slow growth rate (0.1 d(-1)), a low overall biomass yield (0.11 gdw g(-1)MTBE; i.e. 0.040 gdw gCOD(-1)), a high affinity for MTBE and a low affinity for oxygen, which may be a reason for the slow or absence of MTBE biodegradation in situ. Still, the results presented here show promising perspectives for engineering the in situ bioremediation of MTBE.     

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.     

Anaerobic biodegradation of BTEX and gasoline in various aquatic sediments

We examined the extent of biodegradation of benzene, toluene, ethylbenzene and the three isomers of xylene (BTEX) as a mixture and from gasoline in four different sediments: the New York/New Jersey Harbor estuary (polluted); Tuckerton, N.J. (pristine); Onondaga Lake, N.Y. (polluted) and Blue Mtn. Lake, N.Y. (pristine). Enrichment cultures were established with each sediment using denitrifying, sulfidogenic, methanogenic and iron reducing media, as well as site water. BTEX loss, as measured by GC-FID, was extensive in the sediments which had a long history of pollution, with all compounds being utilized within 21–91 days in the most active cultures, and was very slight or non-existent in the pristine sediments. Also, the pattern of loss was different under the various reducing conditions within each sediment and between sediments. For example benzene loss was only observed in sulfidogenic cultures from the NY/NJ Harbor sediments while toluene was degraded under all redox conditions. The loss of BTEX was correlated to the reduction of the various electron acceptors. In cultures amended with gasoline the degradation was much slower and incomplete. These results show that the fate of the different BTEX components in anoxic sediments is dependent on the prevailing redox conditions as well as on the characteristics and pollution history of the sediment.     

Bacterial community changes in TCE biodegradation detected in microcosm experiments

Laboratory microcosm experiments were set up to model biodegradation of trichloroethylene (TCE). Groundwater samples from two contaminated sites were taken, one of them with low (70 mg L⁻¹), the other with high sulfate (685 mg L⁻¹) concentration. In order to assess the biodegradative potential of natural microbiota, supplementary substrates (whey or molasses) were added to the bottles. At day 54, 98, 155, and 318, chemical and bacteriological parameters (i.e., Dehalococcoides test) were investigated. Terminal restriction fragment length polymorphism (T-RFLP) based diversity assessments were carried out to observe the bacterial community changes. Whey and molasses enhanced degradation at different rates. In the case of samples with high sulfate content and amended with whey, no ethylene, ethane, or methane was generated. Both ethylene and methane production was detected in samples of low sulfate content with added whey. The results of Dehalococcoides tests were positive for all control and amended samples. Based on T-RFLP analysis, the bacterial communities of high sulfate concentration groundwater microcosms amended with molasses or whey were very similar, while the communities of groundwater samples with low sulfate concentration were different when supplemented with whey or molasses. The rRNA and rDNA based investigations suggest that the proportions of the active microbes and the microbes present in the microcosms differ.     

Effects of microcosm preparation on rates of toluene biodegradation under denitrifying conditions

Microcosms were prepared with subsurface material from two aquifers to examine the effects of preparation methods on rates of toluene biodegradation under denitrifying conditions. In both cases, the data fit a zero-order kinetics plot. However, rates of removal were generally proportional to initial toluene concentrations, resulting in similar half-lives. Increasing the solid/liquid mass ratio resulted in decreased lag times in one aquifer material, although in both cases the specific toluene mass removal rate (g toluene g sediment ⁻¹ day⁻¹) also decreased. Varying either the initial toluene concentrations or the solid/liquid ratios by two to three orders of magnitude resulted in a half-life variation of only a factor of two, with most of the differences occurring at the extreme ranges of the test variables. These data indicate that similar biodegradation rates might be expected from microcosms prepared with different contaminant concentrations and solid/liquid ratios, which is useful for design of microcosm studies to evaluate biodegradation at field sites. Received 14 March 1996/ Accepted in revised form 24 July 1996     

Microbial degradation of toluene under sulfate-reducing conditions and the influence of iron on the process.

Toluene degradation occurred concomitantly with sulfate reduction in anaerobic microcosms inoculated with contaminated subsurface soil from an aviation fuel storage facility near the Patuxent River (Md.). Similar results were obtained for enrichment cultures in which toluene was the sole carbon source. Several lines of evidence suggest that toluene degradation was directly coupled to sulfate reduction in Patuxent River microcosms and enrichment cultures: (i) the two processes were synchronous and highly correlated, (ii) the observed stoichiometric ratios of moles of sulfate consumed per mole of toluene consumed were consistent with the theoretical ratio for the oxidation of toluene to CO2 coupled with the reduction of sulfate to hydrogen sulfide, and (iii) toluene degradation ceased when sulfate was depleted, and conversely, sulfate reduction ceased when toluene was depleted. Mineralization of toluene was confirmed in experiments with [ring-U-14C]toluene. The addition of millimolar concentrations of amorphous Fe(OH)3 to Patuxent River microcosms and enrichment cultures either greatly facilitated the onset of toluene degradation or accelerated the rate once degradation had begun. In iron-amended microcosms and enrichment cultures, ferric iron reduction proceeded concurrently with toluene degradation and sulfate reduction. Stoichiometric data and other observations indicate that ferric iron reduction was not directly coupled to toluene oxidation but was a secondary, presumably abiotic, reaction between ferric iron and biogenic hydrogen sulfide.     

Microbial degradation of toluene under sulfate-reducing conditions and the influence of iron on the process.

Toluene degradation occurred concomitantly with sulfate reduction in anaerobic microcosms inoculated with contaminated subsurface soil from an aviation fuel storage facility near the Patuxent River (Md.). Similar results were obtained for enrichment cultures in which toluene was the sole carbon source. Several lines of evidence suggest that toluene degradation was directly coupled to sulfate reduction in Patuxent River microcosms and enrichment cultures: (i) the two processes were synchronous and highly correlated, (ii) the observed stoichiometric ratios of moles of sulfate consumed per mole of toluene consumed were consistent with the theoretical ratio for the oxidation of toluene to CO2 coupled with the reduction of sulfate to hydrogen sulfide, and (iii) toluene degradation ceased when sulfate was depleted, and conversely, sulfate reduction ceased when toluene was depleted. Mineralization of toluene was confirmed in experiments with [ring-U-14C]toluene. The addition of millimolar concentrations of amorphous Fe(OH)3 to Patuxent River microcosms and enrichment cultures either greatly facilitated the onset of toluene degradation or accelerated the rate once degradation had begun. In iron-amended microcosms and enrichment cultures, ferric iron reduction proceeded concurrently with toluene degradation and sulfate reduction. Stoichiometric data and other observations indicate that ferric iron reduction was not directly coupled to toluene oxidation but was a secondary, presumably abiotic, reaction between ferric iron and biogenic hydrogen sulfide.     

Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate,chelated ferric iron or nitrate

Flow-through aquifer columns were used to investigate the feasibility of adding sulfate, EDTA–Fe(III) or nitrate to enhance the biodegradation of BTEX and ethanol mixtures. The rapid biodegradation of ethanol near the inlet depleted the influent dissolved oxygen (8 mg l^-1), stimulated methanogenesis, and decreased BTEX biodegradation efficiencies from >99% in the absence of ethanol to an average of 32% for benzene, 49% for toluene, 77% for ethylbenzene, and about 30% for xylenes. The addition of sulfate, EDTA–Fe(III) or nitrate suppressed methanogenesis and significantly increased BTEX biodegradation efficiencies. Nevertheless, occasional clogging was experienced by the column augmented with EDTA–Fe(III) due to iron precipitation. Enhanced benzene biodegradation (>70% in all biostimulated columns) is noteworthy because benzene is often recalcitrant under anaerobic conditions. Influent dissolved oxygen apparently played a critical role because no significant benzene biotransformation was observed after oxygen was purged out of the influent media. The addition of anaerobic electron acceptors could enhance BTEX biodegradation not only by facilitating their anaerobic biodegradation but also by accelerating the mineralization of ethanol or other substrates that are labile under anaerobic conditions. This would alleviate the biochemical oxygen demand (BOD) and increase the likelihood that entraining oxygen would be used for the biotransformation of residual BTEX.     

The effects of carbon sources and micronutrients in fermented whey on the biodegradation of n-hexadecane in diesel fuel contaminated soil

Fermented whey has previously been shown to stimulate biodegradation of n-hexadecane in diesel contaminated soils. The proposed explanation for the stimulatory effect is that fermented whey provides easily accessible carbon and micronutrients, which give rise to an increased degrading biomass.The objective of this work has been to investigate the role of the different carbon sources and vitamins in fermented whey on the microbial degradation of n-hexadecane in soil.The effects of lactose, lactate, vitamins and free amino acids were tested in combinations according to a full factorial design experiment, at concentrations corresponding to those present in fermented whey. The target substance was ¹⁴C-labeled n-hexadecane in nutrient amended soil microcosms contaminated with 5000 mg diesel fuel kg⁻¹ dw. Biodegradation was monitored by determination of evolved ¹⁴CO₂.Significant effects on the biodegradation of n-hexadecane were observed for lactate and amino acids additions in a sandy soil. Lactate showed both an inhibitory effect in the early phase of the experiment and a stimulatory effect in the later phase. The effect of amino acids was slightly stimulatory, mainly evident as a shortening of the lag time.The degree of n-hexadecane degradation at the end of the experiment was correlated with the total concentration of organic compounds added to the soil.Scientific relevanceThere are a handful papers describing the potential of using organic amendments (often industrial by-products) with a content of both easily accessible carbon and micronutrients, to enhance the bioremediation of polluted soils. Enhanced biodegradation is often reported and the proposed explanations are that the combination of easily accessible carbon and micronutrients increases the degrading biomass.In this paper, we examine the effect of fermented whey on the degradation of n-hexadecane and correlate the observed effects on the biodegradation with the main components lactate, amino acids, lactose and B-vitamins. This has to our knowledge never been done before.     

The effects of carbon sources and micronutrients in fermented whey on the biodegradation of n-hexadecane in diesel fuel contaminated soil

Fermented whey has previously been shown to stimulate biodegradation of n-hexadecane in diesel contaminated soils. The proposed explanation for the stimulatory effect is that fermented whey provides easily accessible carbon and micronutrients, which give rise to an increased degrading biomass.The objective of this work has been to investigate the role of the different carbon sources and vitamins in fermented whey on the microbial degradation of n-hexadecane in soil.The effects of lactose, lactate, vitamins and free amino acids were tested in combinations according to a full factorial design experiment, at concentrations corresponding to those present in fermented whey. The target substance was ¹⁴C-labeled n-hexadecane in nutrient amended soil microcosms contaminated with 5000 mg diesel fuel kg⁻¹ dw. Biodegradation was monitored by determination of evolved ¹⁴CO₂.Significant effects on the biodegradation of n-hexadecane were observed for lactate and amino acids additions in a sandy soil. Lactate showed both an inhibitory effect in the early phase of the experiment and a stimulatory effect in the later phase. The effect of amino acids was slightly stimulatory, mainly evident as a shortening of the lag time.The degree of n-hexadecane degradation at the end of the experiment was correlated with the total concentration of organic compounds added to the soil.

Scientific relevance

There are a handful papers describing the potential of using organic amendments (often industrial by-products) with a content of both easily accessible carbon and micronutrients, to enhance the bioremediation of polluted soils. Enhanced biodegradation is often reported and the proposed explanations are that the combination of easily accessible carbon and micronutrients increases the degrading biomass.In this paper, we examine the effect of fermented whey on the degradation of n-hexadecane and correlate the observed effects on the biodegradation with the main components lactate, amino acids, lactose and B-vitamins. This has to our knowledge never been done before.     

Biodegradation of Trichloroethylene and Its Anaerobic Daughter Products in Freshwater Wetland Sediments

The wide range of redox conditions and diversity of microbial populations in organic-rich wetland sediments could enhance biodegradation of chlorinated solvents. To evaluate potential biodegradation rates of trichloroethylene (TCE) and its anaerobic daughter products (cis-1,2-dichloroethylene; trans-1,2-dichloroethylene; and vinyl chloride), laboratory microcosms were prepared under methanogenic, sulfate-reducing, and aerobic conditions using sediment and groundwater from a freshwater wetland that is a discharge area for a TCE contaminant plume. Under methanogenic conditions, biodegradation rates of TCE were extremely rapid at 0.30 to 0.37 d^-1 (half-life of about 2 days). Although the TCE biodegradation rate was slower under sulfate-reducing conditions (0.032 d^-1) than under methanogenic conditions, the rate was still two orders of magnitude higher than those reported in the literature for microcosms constructed with sandy aquifer sediments. In the aerobic microcosm experiments, biodegradation occurred only if methane consumption occurred, indicating that methanotrophs were involved. Comparison of laboratory-measured rates indicates that production of the 1,2-dichloroethylene isomers and vinyl chloride by anaerobic TCE biodegradation could be balanced by their consumption through aerobic degradation where methanotrophs are active in wetland sediment. TCE degradation rates estimated using field data (0.009 to 0.016 d^-1) agree with the laboratory-measured rates within a factor of 3 to 22, supporting the feasibility of natural attenuation as a remediation method for contaminated groundwater discharging in this wetland and other similar environments.     

Toluene biodegradation rates in unsaturated soil systems versus liquid batches and their relevance to field conditions

Contaminant biodegradation in unsaturated soils may reduce the risks of vapor intrusion. However, the reported rates show large variability and are often derived from slurry experiments that are not representative of unsaturated conditions. Here, different laboratory setups are used to derive the biodegradation capacity of an unsaturated soil layer through which gaseous toluene migrates from the water table upwards. Experiments in static unsaturated soil microcosms at 6–30 % water-filled porosity (WFP) and unsaturated soil columns at 9, 14, and 27 % WFP were compared with liquid batches containing the same culture of Alicycliphilus denitrificans. The biodegradation rates for the liquid batches were orders of magnitude lower than for the other setups. Hence, liquid batches do not necessarily reflect optimal conditions for bacteria; either oxygen or toluene mass transfer at the cell scale or the absence of soil–water–air interfaces seemed to be limiting bacterial activity. For the column setup, the rates were limited by mass supply. The microcosm results could be described by apparent first-order biodegradation constants that increased with WFP or through a numerical model that included biodegradation as a first-order process taking place in the liquid phase only. The model liquid phase first-order rates varied between 6.25 and 20 h^?1 and were not related to the water content. Substrate availability was the primary factor limiting bioactivity, with evidence for physiological stress at the lowest water-filled porosity. The presented approach is useful to derive liquid phase biodegradation rates from experimental data and to include biodegradation in vapor intrusion models.     

Novel aerobic benzene degrading microorganisms identified in three soils by stable isotope probing

The remediation of benzene contaminated groundwater often involves biodegradation and although the mechanisms of aerobic benzene biodegradation in laboratory cultures have been well studied, less is known about the microorganisms responsible for benzene degradation in mixed culture samples or at contaminated sites. To address this knowledge gap, DNA based stable isotope probing (SIP) was utilized to identify active benzene degraders in microcosms constructed with soil from three sources (a contaminated site and two agricultural sites). For this, replicate microcosms were amended with either labeled (¹³C) or unlabeled benzene and the extracted DNA samples were ultracentrifuged, fractioned and subject to terminal restriction fragment length polymorphism (TRFLP). The dominant benzene degraders (responsible for ¹³C uptake) were determined by comparing relative abundance of TRFLP phylotypes in heavy fractions of labeled benzene (¹³C) amended samples to the controls (from unlabeled benzene amended samples). Two phylotypes (a Polaromonas sp. and an Acidobacterium) were the major benzene degraders in the microcosms constructed from the contaminated site soil, whereas one phylotype incorporated the majority of the benzene-derived ¹³C in each of the agricultural soils (“candidate” phylum TM7 and an unclassified Sphingomonadaceae).     

Diversity of five anaerobic toluene-degrading microbial communities investigated using stable isotope probing

Time-series DNA-stable isotope probing (SIP) was used to identify the microbes assimilating carbon from [(13)C]toluene under nitrate- or sulfate-amended conditions in a range of inoculum sources, including uncontaminated and contaminated soil and wastewater treatment samples. In all, five different phylotypes were found to be responsible for toluene degradation, and these included previously identified toluene degraders as well as novel toluene-degrading microorganisms. In microcosms constructed from granular sludge and amended with nitrate, the putative toluene degraders were classified in the genus Thauera, whereas in nitrate-amended microcosms constructed from a different source (agricultural soil), microorganisms in the family Comamonadaceae (genus unclassified) were the key putative degraders. In one set of sulfate-amended microcosms (agricultural soil), the putative toluene degraders were identified as belonging to the class Clostridia (genus Desulfosporosinus), while in other sulfate-amended microcosms, the putative degraders were in the class Deltaproteobacteria, within the family Syntrophobacteraceae (digester sludge) or Desulfobulbaceae (contaminated soil) (genus unclassified for both). Partial benzylsuccinate synthase gene (bssA, the functional gene for anaerobic toluene degradation) sequences were obtained for some samples, and quantitative PCR targeting this gene, along with SIP, was further used to confirm anaerobic toluene degradation by the identified species. The study illustrates the diversity of toluene degraders across different environments and highlights the utility of ribosomal and functional gene-based SIP for linking function with identity in microbial communities.     

Is interspecies hydrogen transfer needed for toluene degradation under sulfate-reducing conditions?

Sediments from a hydrocarbon-contaminated aquifer, where periodic shifts between sulfate reduction and methanogenesis occurred, were examined to determine whether the degradation of toluene under sulfate-reducing conditions depended on interspecies hydrogen transfer. Toluene degradation under sulfate-reducing conditions was inhibited by the addition of 5 mM sodium molybdate, but the activity was not restored upon the addition of an actively growing, hydrogen-using methanogen. Toluene degradation was not inhibited in microcosms where hydrogen levels were maintained at a level theoretically sufficient to inhibit toluene degradation if the process proceeded via interspecies hydrogen transfer. Finally, the addition of carbon monoxide, a potent inhibitor of hydrogenase activity, inhibited hydrogen but not toluene consumption in sulfate-reducing microcosms. These results suggest that toluene is degraded directly by sulfate-reducing bacteria without the involvement of interspecies hydrogen transfer. The sequence of experiments used to reach this conclusion could be applied to determine the role of interspecies hydrogen transfer in the degradation of a variety of compounds in different environments or under different terminal electron-accepting conditions.