Characterization of Methyl tert-Butyl Ether-Degrading Bacteria from a Gasoline-Contaminated Aquifer

Molecular microbial community analysis was combined with traditional cultivation strategies to investigate the presence of methyl tert-butyl ether (MTBE)-degrading bacteria in a gasoline-contaminated aquifer (Ronan, MT). A bacterial consortium, RS24, which is capable of complete mineralization of MTBE as a sole carbon and energy source was enriched from soil and aquifer materials taken from the contaminated site. The consortium was capable of degrading MTBE at rates up to 0.66 mg d^-1, with corresponding gross biomass yields of 0.25±0.02 mg dry biomass (mg MTBE)^-1. Two MTBE-degrading isolates identified as Pseudomonas Ant9 and Rhodococcus koreensis were obtained from the consortium. However, both isolates required the presence of 2-propanol as a cosubstrate for MTBE degradation. Denaturing gradient gel electrophoresis (DGGE) of Poly-merase Chain Reaction (PCR)-amplified 16S rDNA confirmed the presence of both isolates in the initial consortium and indicated their disappearance with transfer and subculturing. MTBE degradation and cell growth by the consortium was stimulated by the presence of spent culture medium, suggesting the production of a growth factor during MTBE degradation. These results indicate the presence of naturally occurring MTBE-degrading bacteria in a contaminated aquifer and suggest the potential for natural attenuation or enhanced aerobic oxidation.     

Bioremediation of gasoline-contaminated groundwater in a pilot-scale packed-bed anaerobic reactor

This work reports on the anaerobic treatment of gasoline-contaminated groundwater in a pilot-scale horizontal-flow anaerobic immobilized biomass reactor inoculated with a methanogenic consortium. BTEX removal rates varied from 59 to 80%, with a COD removal efficiency of 95% during the 70 days of in situ trial. BTEX removal was presumably carried out by microbial syntrophic interactions, and at the observed concentrations, the interactions among the aromatic compounds may have enhanced overall biodegradation rates by allowing microbial growth instead of co-inhibiting biodegradation. There is enough evidence to support the conclusion that the pilot-scale reactor responded similarly to the lab-scale experiments previously reported for this design.     

Microbial Diversity in a Hydrocarbon- and Chlorinated-Solvent-Contaminated Aquifer Undergoing Intrinsic Bioremediation

A culture-independent molecular phylogenetic approach was used to survey constituents of microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents undergoing intrinsic bioremediation. Samples were obtained from three redox zones: methanogenic, methanogenic-sulfate reducing, and iron or sulfate reducing. Small-subunit rRNA genes were amplified directly from aquifer material DNA by PCR with universally conserved or Bacteria- or Archaea-specific primers and were cloned. A total of 812 clones were screened by restriction fragment length polymorphisms (RFLP), approximately 50% of which were unique. All RFLP types that occurred more than once in the libraries, as well as many of the unique types, were sequenced. A total of 104 (94 bacterial and 10 archaeal) sequence types were determined. Of the 94 bacterial sequence types, 10 have no phylogenetic association with known taxonomic divisions and are phylogenetically grouped in six novel division level groups (candidate divisions WS1 to WS6); 21 belong to four recently described candidate divisions with no cultivated representatives (OP5, OP8, OP10, and OP11); and 63 are phylogenetically associated with 10 well-recognized divisions. The physiology of two particularly abundant sequence types obtained from the methanogenic zone could be inferred from their phylogenetic association with groups of microorganisms with a consistent phenotype. One of these sequence types is associated with the genus Syntrophus; Syntrophus spp. produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen. The organism represented by the other sequence type is closely related to Methanosaeta spp., which are known to be capable of energy generation only through aceticlastic methanogenesis. We hypothesize, therefore, that the terminal step of hydrocarbon degradation in the methanogenic zone of the aquifer is aceticlastic methanogenesis and that the microorganisms represented by these two sequence types occur in syntrophic association.     

Bioremediation of MTBE: a review from a practical perspective

The addition of methyl tert-butyl ether (MTBE) to gasoline has resulted in public uncertainty regarding the continued reliance on biological processes for gasoline remediation. Despite this concern, researchers have shown that MTBE can be effectively degraded in the laboratory under aerobic conditions using pure and mixed cultures with half-lives ranging from 0.04 to 29 days. Ex-situ aerobic fixed-film and aerobic suspended growth bioreactor studies have demonstrated decreases in MTBE concentrations of 83% and 96% with hydraulic residence times of 0.3 hrs and 3 days, respectively. In microcosm and field studies, aerobic biodegradation half-lives range from 2 to 693 days. These half-lives have been shown to decrease with increasing dissolved oxygen concentrations and, in some cases, with the addition of exogenous MTBE-degraders. MTBE concentrations have also been observed to decrease under anaerobic conditions; however, these rates are not as well defined. Several detailed field case studies describing the use of ex-situ reactors, natural attenuation, and bioaugmentation are presented in this paper and demonstrate the potential for successful remediation of MTBE-contaminated aquifers. In conclusion, a substantial amount of literature is available which demonstratesthat the in-situ biodegradation of MTBE is contingent on achieving aerobic conditions in the contaminated aquifer.     

Effect of Benzene, Toluene, Ethylbenzene, and p-Xylene (BTEX) Mixture on Biodegradation of Methyl tert-Butyl Ether (MTBE) and tert-Butyl Alcohol (TBA) by Pure Culture UC1

The effect of a BTEX mixture on the biodegradation of methyl tert-butyl ether (MTBE) and its degradation intermediate, tert-butyl alcohol (TBA) was investigated in the pure bacterial culture UC1, which has been identified to be a strain of the known MTBE-degrader PM1 based on greater than 99% 16S rDNA similarity. Several degradation studies were carried out on UC1 at three initial concentration levels of MTBE or TBA: 6-7; 15-17; and 40-45 mg/l, both with and without BTEX present cumulatively at about half of the MTBE or TBA molar mass in the system. The BTEX mixture was observed not to affect either the rate or the degradation lag period of MTBE or TBA degradation, except that the TBA degradation rate actually increased when BTEX was present initially in the highest concentration studies. When serving as the sole substrate, the MTBE degradation rate ranged from 48 +/- 1.2 to 200 +/- 7.0 mg(MTBE)/g(dw) h, and the TBA degradation rate from 140 +/- 18 to 530 +/- 70 mg(TBA)/g(dw) h. When present with BTEX, MTBE and TBA rates ranged from 46 +/- 2.2 to 210 +/- 14 and 170 +/- 28 to 780 +/- 43 mg(TBA)/g(dw) h, respectively. In studies where varying concentrations of TBA were present with 5 mg/l MTBE, both compounds were degraded simultaneously with no obvious preference for either substrate. In the highest concentration study of TBA with 5 mg/l MTBE, BTEX was also observed to increase the ultimate rate of TBA degradation. In addition to exploring the affect of BTEX, this study also provides general insight into the metabolism of MTBE and TBA by pure culture UC1.     

Bioremediation of benzene,toluene, ethylbenzene,xylenes-contaminated soil: a biopile pilot experiment

Aims: In this study, we evaluated the removal efficiency of fuel hydrocarbons from a jet fuel contaminated area using bioaugmentation treatment in biopile. Methods and Results: The hydrocarbon analysis of the sample revealed total hydrocarbons mainly constituted by benzene, toluene, ethylbenzene, xylenes (BTEX) and heavy aliphatic hydrocarbons. Enrichments of soil sample were performed with BTEX, pristane and fuel JP-5, respectively, selected hydrocarbon-degrading strains, namely Acinetobacter sp., Pseudomonas sp. and Rhodococcus sp. Three hundred litres of culture containing 10⁸ cell ml⁻¹ of each strain and nutrients sprayed on the biopile allowed a removal of 90% of total hydrocarbons in 15 days. Bioremediation process was monitored by observation of the respiration rate and the bacterial abundance and GC-MS analysis. Conclusions: The efficiency of the treatment in the biopile was considerable. The assessment of microbial activity during the experiment is necessary for interventions targeted to improve environmental parameters such as humidity, temperature, pH and nutrients for optimization of the bioremediation process. Significance and Impact of the Study: A better knowledge of microbial successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in biopile study improve our understanding of processes occurring during oil pollution.     

Monitoring Soil Ecosystem Recovery Following Bioremediation of a Terrestrial Crude Oil Spill With and Without a Fertilizer Amendment

The effect of fertilizer as an amendment in the bioremediation of a terrestrial crude oil spill has been investigated in terms of the subsequent recovery of the soil ecosystem following bioremediation. Two different spills in the same area with different initial hydrocarbon concentrations (33,500 mg kg^-1 and 4,800 mg kg^-1) were compared. At the higher initial hydrocarbon concentration fertilizer addition increased the rate of bioremediation (first-order rate constant of 0.0033 days-1 with fertilizer amendment vs. 0.0020 days-1 without) and resulted in more rapid recovery of soil bacteria (numbers, community structure, diversity) and nematodes (trophic diversity and community structure). The effect of the fertilizer amendment was more significant at the higher initial concentration of crude oil hydrocarbons, presumably due to greater depletion of soil nutrient pools in the absence of the amendment. A second objective of this work was to identify sensitive and cost-effective ecological indicators useful for monitoring the recovery of soil ecosystems impacted by crude oil. Ecological indicators used included: microbial numbers, community structure, and activity as revealed by biomarker analysis (phospholipid fatty acids); nitrogen availability; nematode numbers and community structure (trophic groups and colonizer-persister classes); and ultimately, plant cover and diversity. All ecological indicators investigated were sensitive to disturbances in the soil food web in a hydrocarbon-impacted site. However, nematode community structure analysis offered the greatest sensitivity coupled with low cost and readily available sources for the analysis.     

Limitations to Natural Bioremediation of Perchlorate in a Contaminated Site

Perchlorate (ClO₄ ^?) has been detected in many drinking water supplies in the United States, including the Las Vegas Wash and Lake Mead, Nevada. These locations are highly contaminated and contribute perchlorate to Lake Mead and the Colorado River system. Essential elements for perchlorate bioremediation at these locations were examined, including the presence of perchlorate-reducing bacteria (PRB), sufficient electron donors, occurrence of competing electron acceptors, and ability of PRB to utilize a variety of electron donors. Enumeration of PRB was performed anoxically using most probable number (MPN). Values ranged from ≤20 to 230 PRB/100 ml or ≤20 to ≥ 1.6× 10⁵ PRB/g for Lake Mead water samples and Las Vegas Wash sediments, respectively. 16S rRNA sequences revealed that isolates were γ -proteobacteria, Aeromonas, Dechlorosoma, Rahnella and Shewanella. A screening of potential electron donors using BIOLOG^TM demonstrated that all isolates were capable of metabolic versatility. Measurements of total organic carbon (TOC), nitrate and dissolved oxygen (DO) indicated limited presence of electron donor at all sites, whereas the electron acceptors varied throughout the Wash and Lake Mead. The persistence of perchlorate in the sites is attributed to lack of available electron donor and/or the presence of competing electron acceptors. A location has been identified where perchlorate biodegradation could be implemented thereby halting the transport of perchlorate to Lake Mead and the Colorado River.     

Limitations to Natural Bioremediation of Perchlorate in a Contaminated Site

Perchlorate (ClO₄^-) has been detected in many drinking water supplies in the United States, including the Las Vegas Wash and Lake Mead, Nevada. These locations are highly contaminated and contribute perchlorate to Lake Mead and the Colorado River system. Essential elements for perchlorate bioremediation at these locations were examined, including the presence of perchlorate-reducing bacteria (PRB), sufficient electron donors, occurrence of competing electron acceptors, and ability of PRB to utilize a variety of electron donors. Enumeration of PRB was performed anoxically using most probable number (MPN). Values ranged from ≤20 to 230 PRB/100 ml or ≤20 to ≥ 1.6× 10⁵ PRB/g for Lake Mead water samples and Las Vegas Wash sediments, respectively. 16S rRNA sequences revealed that isolates were γ -proteobacteria, Aeromonas, Dechlorosoma, Rahnella and Shewanella. A screening of potential electron donors using BIOLOG^TM demonstrated that all isolates were capable of metabolic versatility. Measurements of total organic carbon (TOC), nitrate and dissolved oxygen (DO) indicated limited presence of electron donor at all sites, whereas the electron acceptors varied throughout the Wash and Lake Mead. The persistence of perchlorate in the sites is attributed to lack of available electron donor and/or the presence of competing electron acceptors. A location has been identified where perchlorate biodegradation could be implemented thereby halting the transport of perchlorate to Lake Mead and the Colorado River.     

Characterizing Intrinsic Bioremediation in a Petroleum Hydrocarbon-Contaminated Aquifer by Combined Chemical,Isotopic, and Biological Analyses

Chemical, isotopic, and biological parameters were evaluated over a 1-year period to characterize microbial processes associated with intrinsic bioremediation in a petroleum hydrocarbon-contaminated aquifer located in Studen, Switzerland. Chemical parameters measured included oxidants such as O₂, NO₃ ^?, and SO₄ ^2?, reduced species such as Fe²⁺ and CH₄, and dissolved inorganic carbon (DIC). Stable carbon isotope analyses of DIC were used to differentiate between different processes that contribute to DIC production. Microbial populations were identified by sequence analysis of archaeal 16S rDNA and in situ hybridization using a general DNA binding dye (DAPI) and specific probes targeting the domain Archaea (Arch915) and Bacteria (Eub338), as well as the species Methanosaeta concilii (Rotcl1) and Methanospirillum sp. (Rotcl2). Groundwater exhibited reduced conditions and elevated concentrations of DIC within the contaminated zone. Spatially distinct values of δ¹³C ranging from ?16.5l%c to ?4.44%o were found, indicating the presence of different ongoing microbial processes. Detected microbial populations (% of DAPI-stained cells) within the contaminated zone belonged to Archaea (9±2% to 31±13%) and Bacteria (13±3% to 32±13%). In wells with methanogenic activity, Methanosaeta concilii accounted for up to 26% of all DAPI-detected microorganisms. These results demonstrate that this novel combination of chemical, isotopic, and biological analysis provides valuable insights that can be used for the characterization of microbial processes in contaminated aquifers.     

Implementation of natural attenuation at a JP-4 jet fuel release after active remediation

After eighteen months of active remediation at a JP-4 jet-fuel spill, aresidual of unremediated hydrocarbon remained. Further site characterizationwas conducted to evaluate the contribution of natural attenuation to controlexposure to hazards associated with the residual contamination in thesubsurface. Activities included the detailed characterization ofground-water flow through the spill; the distribution of fuel contaminantsin groundwater; and the analysis of soluble electron acceptors moving intothe spill from upgradient. These activities allowed a rigorous evaluation ofthe transport of contaminants from the spill to the receptor of groundwater,the Pasquotank River. The transport of dissolved contaminants of concern,that is benzene, toluene, ethyl benzene, xylene isomers (BTEX) andmethyl-tertiary-butyl ether (MTBE), into the river from the source area wascontrolled by equilibrium dissolution from the fuel spill to the adjacentgroundwater, diffusion in groundwater from the spill to permeable layers inthe aquifer, and advective transport in the permeable layers. The estimatedyearly loading of BTEX compounds and MTBE into the receptor was trivial evenwithout considering biological degradation. The biodegradation ofhydrocarbon dissolved in groundwater through aerobic respiration,denitrification, sulfate reduction, and iron reduction was estimated fromchanges in ground-water chemistry along the flow path. The concentrations oftarget components in permanent monitoring wells continue to decline overtime. Long term monitoring will ensure that the plume is under control, andno further active remediation is required.     

Aerobic MTBE biodegradation in the presence of BTEX by two consortia under batch and semi-batch conditions

This study explores the effect of microbial consortium composition and reactor configuration on methyl tert-butyl ether (MTBE) biodegradation in the presence of benzene, toluene, ethylbenzene and p-xylenes(BTEX). MTBE biodegradation was monitored in the presence and absence of BTEX in duplicate batch reactors inoculated with distinct enrichment cultures: MTBE only (MO—originally enriched on MTBE) and/or MTBE BTEX (MB—originally enriched on MTBE and BTEX). The MO culture was also applied in a semi-batch reactor which received both MTBE and BTEX periodically in fresh medium after allowing cells to settle. The composition of the microbial consortia was explored using a combination of 16S rRNA gene cloning and quantitative polymerase chain reaction targeting the known MTBE-degrading strain PM1^T. MTBE biodegradation was completely inhibited by BTEX in the batch reactors inoculated with the MB culture, and severely retarded in those inoculated with the MO culture (0.18 ± 0.04 mg/L-day). In the semi-batch reactor, however, the MTBE biodegradation rate in the presence of BTEX was almost three times as high as in the batch reactors (0.48 ± 0.2 mg/L-day), but still slower than MTBE biodegradation in the absence of BTEX in the MO-inoculated batch reactors (1.47 ± 0.47 mg/L-day). A long lag phase in MTBE biodegradation was observed in batch reactors inoculated with the MB culture (20 days), but the ultimate rate was comparable to the MO culture (0.95 ± 0.44 mg/L-day). Analysis of the cultures revealed that strain PM1^T concentrations were lower in cultures that successfully biodegraded MTBE in the presence of BTEX. Also, other MTBE degraders, such as Leptothrix sp. and Hydrogenophaga sp. were found in these cultures. These results demonstrate that MTBE bioremediation in the presence of BTEX is feasible, and that culture composition and reactor configuration are key factors.     

Microbiological Comparison of Core and Groundwater Samples Collected from a Fractured Basalt Aquifer with that of Dialysis Chambers Incubated In Situ

Microorganisms associated with basalt core were compared to those suspended in groundwater pumped from the same well in the eastern Snake River Plain Aquifer (Idaho, USA). Two wells located at different distances from the source of a mixed-waste plume in the fractured basalt aquifer were examined. In the well more distal from the plume source, an array of dialysis chambers filled with either deionized water or crushed basalt was equilibrated to compare the microorganisms collected in this fashion with those from core and groundwater samples collected in a traditional manner from the same well. The samples were characterized to determine the total amount of biomass, presence of specific populations or physiological groups, and potential community functions. Microorganisms and their activities were nearly undetectable in core and groundwater collected from the well farthest from the plume source and substantially enriched in both core and groundwater from the well closest to the plume source. In both wells, differences (statistically significant for some measures) were found between bacteria associated with the cores and those suspended in the groundwater. Significantly higher populations were found in the basalt- and water-filled dialysis chambers incubated in the open well compared with core and groundwater samples, respectively. For a given parameter, the variation among dialysis chambers incubated at different depths was much less than the high variation observed among core samples. Analyses on selected basalt- and water-filled dialysis chamber samples suggested that these two communities were compositionally similar but exhibited different potential functions. Documented knowledge of cell physiological changes associated with attachment and potential differences between attached and unattached communities in aquifers indicate that careful consideration should be given to the type of sample media (i.e., core, groundwater, substrata incubated in a well) used to represent a subsurface environment.     

Geochemical, Microbiological, and Geophysical Assessments of Anaerobic Immobilization of Heavy Metals

Bioremediation methods that precipitate contaminants in situ as solid (mineral) phases can provide cost-effective options for removing dissolved metals in contaminated groundwater. The current field-scale experiments demonstrate that indigenous bacteria can be stimulated to remove metals by injection of electron-donating substrates and nutrients into a contaminated aquifer. Groundwater at the investigation site is aerobic and contains high levels of lead, cadmium, zinc, copper, and sulfuric acid (pH = 3.1) derived from a car-battery recycling plant. During the experiments, lead, cadmium, zinc, and copper were almost completely removed by precipitation of solid sulfide phases, as pH increased from 3 to ∼ 5 and Eh dropped from +400 mV to -150 mV. X-ray and transmission electron microscopy (TEM) analyses of filtered material from the treated groundwater indicated the presence of newly formed nanocrystalline metal sulfides. Genetic sequencing indicated that the principal species of sulfate-reducing bacteria involved in the bioremediation process was Desulfosporosinus orientis. Geochemical modeling shows that oxidation of added substrates and subsequent bacterial sulfate reduction produced desired geochemical conditions (i.e., decreasing Eh and increasing pH) for the precipitation and sorption of metal sulfides. Geophysical survey results suggest that bioremediation lowers electrical conductance of groundwater and possibly increases the magnetic susceptibility of porous media. This study demonstrates that integrated geochemical, geophysical, and microbiological analyses, combined with theoretical modeling, can successfully track and predict the progress of subsurface bioremediation.     

Bioremediation by Composting of Heavy Oil Refinery Sludge in Semiarid Conditions

The present work attempts to ascertain the efficacy of low cost technology (in our case, composting) as a bioremediation technique for reducing the hydrocarbon content of oil refinery sludge with a large total hydrocarbon content (250–300 g kg⁻¹), in semiarid conditions. The oil sludge was produced in a refinery sited in SE Spain The composting system designed, which involved open air piles turned periodically over a period of 3 months, proved to be inexpensive and reliable. The influence on hydrocarbon biodegradation of adding a bulking agent (wood shavings) and inoculation of the composting piles with pig slurry (a liquid organic fertiliser which adds nutrients and microbial biomass to the pile) was also studied. The most difficult part during the composting process was maintaining a suitable level of humidity in the piles. The most effective treatment was the one in which the bulking agent was added, where the initial hydrocarbon content was reduced by 60% in 3 months, compared with the 32% reduction achieved without the bulking agent. The introduction of the organic fertiliser did not significantly improve the degree of hydrocarbon degradation (56% hydrocarbon degraded). The composting process undoubtedly led to the biodegradation of toxic compounds, as was demonstrated by ecotoxicity tests using luminescent bacteria and tests on plants in Petri dishes.     

Aerobic Cr(VI) Reduction by Bacteria in Culture and Soil Conditions

We compared the performance of aerobic Cr(VI)-reducing bacteria isolated from Cr(VI)-contaminated soil in pure and mixed cultures of five isolated strains. The mixed culture had increased reduction rates compared to individual cultures. Cr(VI) reduction was observed in sterile soil inoculated with Pseudomonas fluorescens and in non-sterile soil with and without inoculation with P. fluorescens at initial pore water concentrations up to 1,600 mg Cr(VI)/L, whereas in culture the maximum inhibitory concentration was 500 mg Cr(VI)/L. Linear rates of Cr(VI) reduction in non-sterile soil amended with peptone were ～5 to 8 times higher than those observed in the mixed culture. Inoculation of non-sterile soil with P. fluorescens did not further enhance Cr(VI) reduction rates. Our results indicate that evaluation of Cr(VI) reduction capacity in Cr(VI)-contaminated soil for in-situ bioremediation purposes should not be done solely in pure culture. Although the latter may be used initially to assess the effects of process parameters (e.g., pH, temperature), the rate and extent of Cr(VI) reduction should be determined in soil for bioremediation design purposes.     

Effect of carbon addition and predation on acetate-assimilating bacterial cells in groundwater

Groundwater microbial community dynamics are poorly understood due to the challenges associated with accessing subsurface environments. In particular, microbial interactions and their impact on the subsurface carbon cycle remain unclear. In the present project, stable isotope probing with uniformly labeled [¹³C]-acetate was used to identify metabolically active and inactive bacterial populations based on their ability to assimilate acetate and/or its metabolites. Furthermore, we assessed whether substrate availability (bottom–up control) or grazing mortality (top–down control) played a greater role in shaping bacterial community composition by separately manipulating the organic carbon supply and the protozoan grazer population. A community fingerprinting technique, terminal restriction fragment length polymorphism, revealed that the bacterial community was not affected by changes in acetate availability but was significantly altered by the removal of protozoan grazers. In silico identification of terminal restriction fragments and 16S rRNA gene sequences from clone libraries revealed a bacterial community dominated by Proteobacteria, Firmicutes , and Bacteroidetes . Elucidation of the factors that structure the bacterial community will improve our understanding of the bacterial role in the carbon cycle of this important subterranean environment.