Hydrocarbon degradation capacity and population dynamics of a microbial consortium obtained using a sequencing batch reactor in the presence of molasses

A native microbial consortium capable of degrading hydrocarbons was employed as an inoculum source in a sequencing batch reactor (SBR) using molasses as a carbon source. The microbial biomass in the SBR was able to grow in the presence of molasses, degrading 88% of the reducing sugar. Moreover, the consortium produced in the SBR was capable of maintaining 75% of the capacity for biodegradation of oil with respect to the original capacity of the native microbial consortium. Monitoring of the microbial population structure was accomplished using PCR-DGGE. The results indicated that the microbial populations grown in molasses were stable during crude oil degradation, as judged by comparison to the population structure of the native microbial consortium. The results obtained demonstrated that molasses could be used as a carbon source to promote the growth of biomass with oildegrading capacity.     

Comparison of bioremediation strategies for soil impacted with petrochemical oily sludge

Different bioremediation techniques (natural attenuation, biostimulation and bioaugmentation) in contaminated soils with two oily sludge concentrations (1.5% and 6.0%) in open and closed microcosms systems were assessed during 90 days. The results showed that the highest biodegradation rates were obtained in contaminated soils with 6% in closed microcosms. Addition of microbial consortium and nutrients in different concentrations demonstrated higher biodegradation rate of total petroleum hydrocarbons (TPH) than those of the natural attenuation treatment. Soils treated in closed microcosms showed highest removal rate (84.1 ± 0.9%) when contaminated at 6% and bacterial consortium and nutrients in low amounts were added. In open microcosms, the soil contaminated at 6% using biostimulation with the highest amounts of nutrients (C:N:P of 100:10:1) presented the highest degradation rate (78.7 ± 1.3%). These results demonstrate that the application of microbial consortium and nutrients favored biodegradation of TPH present in oily sludge, indicating their potential applications for treatment of the soils impacted with this important hazardous waste.     

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.     

Response of 1,2-dichloroethane-adapted microbial communities to ex-situ biostimulation of polluted groundwater

The microbial community of a groundwater system contaminated by 1,2-dichloroethane (1,2-DCA), a toxic and persistent chlorinated hydrocarbon, has been investigated for its response to biostimulation finalized to 1,2-DCA removal by reductive dehalogenation. The microbial population profile of samples from different wells in the aquifer and from microcosms enriched in the laboratory with different organic electron donors was analyzed by ARISA (Amplified Ribosomal Intergenic Spacer Analysis) and DGGE (Denaturing Gradient Gel Electrophoresis) of 16S rRNA genes. 1,2-DCA was completely removed with release of ethene from most of the microcosms supplemented with lactate, acetate plus formate, while cheese whey supported 1,2-DCA dehalogenation only after a lag period. Microbial species richness deduced from ARISA profiles of the microbial community before and after electron donor amendments indicated that the response of the community to biostimulation was heterogeneous and depended on the well from which groundwater was sampled. Sequencing of 16S rRNA genes separated by DGGE indicated the presence of bacteria previously associated with soils and groundwater polluted by halogenated hydrocarbons or present in consortia active in the removal of these compounds. A PCR assay specific for Desulfitobacterium sp. showed the enrichment of this genus in some of the microcosms. The dehalogenation potential of the microbial community was confirmed by the amplification of dehalogenase-related sequences from the most active microcosms. Cloning and sequencing of PCR products indicated the presence in the metagenome of the bacterial community of a new dehalogenase potentially involved in 1,2-DCA reductive dechlorination.     

Kinetic and microbial community analysis of methyl ethyl ketone biodegradation in aquifer sediments

Methyl ethyl ketone (MEK) is a common groundwater contaminant often present with more toxic compounds of primary interest. Because of this, few studies have been performed to determine the effect of microbial community structure on MEK biodegradation rates in aquifer sediments. Here, microcosms were prepared with aquifer sediments containing MEK following a massive spill event and compared to laboratory-spiked sediments, with MEK biodegradation rates quantified under mixed aerobic/anaerobic conditions. Biodegradation was achieved in MEK-contaminated site sediment microcosms at about half of the solubility (356 mg/L) with largely Firmicutes population under iron-reducing conditions. MEK was biodegraded at a higher rate [4.0 ± 0.74 mg/(L days)] in previously exposed site samples compared to previously uncontaminated sediments [0.51 ± 0.14 mg/(L days)]. Amplicon sequencing and denaturing gradient gel electrophoresis of 16S rRNA genes were combined to understand the relationship between contamination levels, biodegradation, and community structure across the plume. More heavily contaminated sediments collected from an MEK-contaminated field site had the most similar communities than less contaminated sediments from the same site despite differences in sediment texture. The more diverse microbial community observed in the laboratory-spiked sediments reduced MEK concentration 47 % over 92 days. Results of this study suggest lower rates of MEK biodegradation in iron-reducing aquifer sediments than previously reported for methanogenic conditions and biodegradation rates comparable to previously reported nitrate- and sulfate-reducing conditions.     

Studies on Biodegradation of Kerosene in Soil under Different Bioremediation Strategies

The effectiveness of bioremediation is often a function of the microbial population and how they can be enriched and maintained in an environment. Strategies for inexpensive in situ bioremediation of soil contaminated with petroleum hydrocarbons include stimulation of the indigenous microorganisms by introduction of nutrients (biostimulation) and/or through inoculation of an enriched mixed microbial culture into soil (bioaugmentation). To demonstrate the potential use of bioremediation in soil contaminated with kerosene, a laboratory study with the objective of evaluating and comparing the effects of bioattenuation, biostimulation, bioaugmentation, and combined biostimulation and bioaugmentation was performed. The present study dealt with the biodegradation of kerosene in soil under different bioremediation treatment strategies: bioattenuation, biostimulation, bioaugmentation, and combined biostimulation and bioaugmentation, respectively. Each treatment strategy contained 10% (w/w) kerosene in soil as a sole source of carbon and energy. After 5 weeks of remediation, the results revealed that bioattenuation, bioaugmentation, biostimulation, and combined biostimulation and bioaugmentation exhibited 44.1%, 67.8%, 83.1%, and 87.3% kerosene degradation, respectively. Also, the total hydrocarbon-degrading bacteria (THDB) count in all the treatments increased with time up till the second week after which it decreased. The highest bacterial growth was observed for combined biostimulation and bioaugmentation treatment strategy. A first-order kinetic model equation was fitted to the biodegradation data to further evaluate the rate of biodegradation and the results showed that the specific degradation rate constant (k) value was comparatively higher for combined biostimulation and bioaugmentation treatment strategy than the values for other treatments. Therefore, value of the kinetic parameter showed that the degree of effectiveness of these bioremediation strategies in the clean up of soil contaminated with kerosene is in the following order: bioattenuation < bioaugmentation < biostimulation < combined biostimulation and bioaugmentation. Conclusively, the present work has defined combined biostimulation and bioaugmentation treatment strategy requirements for kerosene oil degradation and thus opened an avenue for its remediation from contaminated soil.     

Bioremediation strategies for diesel and biodiesel in oxisol from southern Brazil

Leaks and spillages during the extraction, transport and storage of petroleum and its derivatives may result in environmental contamination. Biodiesel is an alternative energy source that can contribute to a reduction in environmental pollution. The aim of the present work was to evaluate biodegradation of diesel, biodiesel, and a 20% biodiesel-diesel mixture in oxisols from southern Brazil, using two bioremediation strategies: natural attenuation and bioaugmentation/biostimulation. Fuel biodegradation was monitored over 60 days by dehydrogenase activity, CO₂ evolution and gas chromatography. The bacterial inoculum employed for bioaugmentation/biostimulation consisted of Bacillus megaterium, Bacillus pumilus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia and PCR-DGGE using 16S RNAr primers showed that some members of this consortium survived in the soil after 60 days. The biodegradation of pure biodiesel was higher for bioaugmentation/biostimulation than for natural attenuation, suggesting that the addition of the microbial consortium, together with adjustment of the macronutrient ratio, increased biodiesel degradation. The results of dehydrogenase and respiratory activity, together with GC analysis, suggested that the presence of biodiesel may, by stimulating general microbial degradative metabolism, increase the biodegradation of petroleum diesel. The microbial community was altered by both treatments, with natural attenuation producing a lower diversity index than the amended soil. The bioaugmentation/biostimulation strategy was showed to have a high potential for cleaning up soils contaminated with diesel and biodiesel blends.     

Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation

Bioremediation of diesel oil in soil can occur by natural attenuation, or treated by biostimulation or bioaugmentation. In this study we evaluated all three technologies on the degradation of total petroleum hydrocarbons (TPH) in soil. In addition, the number of diesel-degrading microorganisms present and microbial activity as indexed by the dehydrogenase assay were monitored. Soils contaminated with diesel oil in the field were collected from Long Beach, California, USA and Hong Kong, China. After 12 weeks of incubation, all three treatments showed differing effects on the degradation of light (C12-C23) and heavy (C23-C40) fractions of TPH in the soil samples. Bioaugmentation of the Long Beach soil showed the greatest degradation in the light (72.7%) and heavy (75.2%) fractions of TPH. Natural attenuation was more effective than biostimulation (addition of nutrients), most notably in the Hong Kong soil. The greatest microbial activity (dehydrogenase activity) was observed with bioaugmentation of the Long Beach soil (3.3-fold) and upon natural attenuation of the Hong Kong sample (4.0-fold). The number of diesel-degrading microorganisms and heterotrophic population was not influenced by the bioremediation treatments. Soil properties and the indigenous soil microbial population affect the degree of biodegradation; hence detailed site specific characterization studies are needed prior to deciding on the proper bioremediation method.     

Microbial Communities Associated with Zones of Elevated Magnetic Susceptibility in Hydrocarbon-Contaminated Sediments

Recent studies suggest that magnetic susceptibility (MS) measurements can play an important role in identifying zones where microbial-mediated iron mineral transformations are occurring. Here we investigated the microbial community variations within zones of elevated MS in a petroleum hydrocarbon-contaminated aquifer near Bemidji, Minnesota, USA. Our main objective was to 1) identify the key microbial populations that may play a role in hydrocarbon degradation, 2) analyze which microbial populations could be connected to the elevated MS and 3) explore the use of non-destructive geophysical techniques as a tool to guide microbial sampling. Clone libraries based on the 16S rRNA gene revealed the presence of iron-reducing β-Proteobacteria in the vadose zone, whereas the free petroleum phase on the water table was characterized by a methanogenic consortium, in which the syntrophic δ-proteobacterium Smithella and the hydrogenotrophic Methanoregula predominated. Nonmetric multidimensional scaling (NMDS) found a close relationship between elevated MS values and the methanogenic hydrocarbon-degrading consortium. Our results suggest that magnetic susceptibility measurements can guide microbiologists to zones of active microbial biodegradation in aged petroleum spills.     

Geochemical and physiological evidence for mixed aerobic and anaerobic field biodegradation of coal tar waste by subsurface microbial communities

We used geochemical analyses of groundwater and laboratory-incubated microcosms to investigate the physiological responses of naturally occurring microorganisms to coal-tar-waste constituents in a contaminated aquifer. Waters were sampled from wells along a natural hydrologic gradient extending from uncontaminated (1 well) into contaminated (3 wells) zones. Groundwater analyses determined the concentrations of carbon and energy sources (pollutants or total organic carbon), final electron acceptors (oxygen, nitrate, sulfate), and metabolic byproducts (dissolved inorganic carbon [DIC], alkalinity, methane, ferrous iron, sulfide, Mn2+). In the contaminated zone of the study site, concentrations of methane, hydrogen, alkalinity, and DIC were enhanced, while dissolved oxygen and nitrate were depleted. Field-initiated biodegradation assays using headspace-free serum bottle microcosms filled with groundwater examined metabolism of the ambient organic contaminants (naphthalene, 2-methylnaphthalene, benzothiophene, and indene) by the native microbial communities. Unamended microcosms from the contaminated zone demonstrated the simultaneous degradation of several coal-tar-waste constituents at the in situ temperature (10°C). Lag phases prior to the onset of biodegradation indicated the prevalence of both aerobic and anaerobic conditions in situ. Electron acceptor-amended microcosms from the most contaminated well waters demonstrated only aerobic naphthalene degradation. Collectively, the geochemical and microbial evidence show that biodegradation of coal-tar-waste constituents occurs via both aerobic and anaerobic terminal electron accepting processes at this site.     

Isolation and genetic identification of PAH degrading bacteria from a microbial consortium

Polycyclic aromatic hydrocarbons (PAH; naphthalene, anthracene and phenanthrene) degrading microbial consortium C2PL05 was obtained from a sandy soil chronically exposed to petroleum products, collected from a petrochemical complex in Puertollano (Ciudad Real, Spain). The consortium C2PL05 was highly efficient degrading completely naphthalene, phenanthrene and anthracene in around 18 days of cultivation. The toxicity (Microtox™ method) generated by the PAH and by the intermediate metabolites was reduced to levels close to non-toxic in almost 40 days of cultivation. The identified bacteria from the contaminated soil belonged to γ-proteobacteria and could be include in Enterobacter and Pseudomonas genus. DGGE analysis revealed uncultured Stenotrophomonas ribotypes as a possible PAH degrader in the microbial consortium. The present work shows the potential use of these microorganisms and the total consortium for the bioremediation of PAH polluted areas since the biodegradation of these chemicals takes place along with a significant decrease in toxicity.     

Characterization of the biodegradation,bioremediation and detoxification capacity of a bacterial consortium able to degrade the fungicide thiabendazole

Thiabendazole (TBZ) is a persistent fungicide used in the post-harvest treatment of fruits. Its application results in the production of contaminated effluents which should be treated before their environmental discharge. In the absence of efficient treatment methods in place, biological systems based on microbial inocula with specialized degrading capacities against TBZ could be a feasible treatment approach. Only recently the first bacterial consortium able to rapidly transform TBZ was isolated. This study aimed to characterize its biodegradation, bioremediation and detoxification potential. The capacity of the consortium to mineralize ¹⁴C-benzyl-ring labelled TBZ was initially assessed. Subsequent tests evaluated its degradation capacity under various conditions (range of pH, temperatures and TBZ concentration levels) and relevant practical scenarios (simultaneous presence of other postharvest compounds) and its bioaugmentation potential in soils contaminated with increasing TBZ levels. Finally cytotoxicity assays explored its detoxification potential. The consortium effectively mineralized the benzoyl ring of the benzimidazole moiety of TBZ and degraded spillage level concentrations of the fungicide in aqueous cultures (750 mg L^?1) and in soil (500 mg kg^?1). It maintained its high degradation capacity in a wide range of pH (4.5–7.5) and temperatures (15–37 °C) and in the presence of other pesticides (ortho-phenylphenol and diphenylamine). Toxicity assays using the human liver cancer cell line HepG2 showed a progressive decrease in cytotoxicity, concomitantly with the biodegradation of TBZ, pointing to a detoxification process. Overall, the bacterial consortium showed high potential for future implementation in bioremediation and biodepuration applications.     

Long-term simulation of in situ biostimulation of polycyclic aromatic hydrocarbon-contaminated soil

A continuous-flow column study was conducted to evaluate the long-term effects of in situ biostimulation on the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in soil from a manufactured gas plant site. Simulated groundwater amended with oxygen and inorganic nutrients was introduced into one column, while a second column receiving unamended groundwater served as a control. PAH and dissolved oxygen (DO) concentrations, as well as microbial community profiles, were monitored along the column length immediately before and at selected intervals up to 534?days after biostimulation commenced. Biostimulation resulted in significantly greater PAH removal than in the control condition (73% of total measured PAHs vs. 34%, respectively), with dissolution accounting for a minor amount of the total mass loss (~6%) in both columns. Dissolution was most significant for naphthalene, acenaphthene, and fluorene, accounting for >20% of the total mass removed for each. A known group of PAH-degrading bacteria, 'Pyrene Group 2' (PG2), was identified as a dominant member of the microbial community and responded favorably to biostimulation. Spatial and temporal variations in soil PAH concentration and PG2 abundance were strongly correlated to DO advancement, although there appeared to be transport of PG2 organisms ahead of the oxygen front. At an estimated oxygen demand of 6.2?mg O(2)/g dry soil and a porewater velocity of 0.8?m/day, it took between 374 and 466?days for oxygen breakthrough from the 1-m soil bed in the biostimulated column. This study demonstrated that the presence of oxygen was the limiting factor in PAH removal, as opposed to the abundance and/or activity of PAH-degrading bacteria once oxygen reached a previously anoxic zone.     

Bacterial community dynamics during biostimulation and bioaugmentation experiments aiming at chlorobenzene degradation in groundwater

A set of microcosm experiments was performed to assess different bioremediation strategies, i.e., biostimulation and bioaugmentation, for groundwater contaminated with chlorobenzenes. The biodegradative potential was stimulated either by the supply of electron acceptors (air, (NO ₃ ^– ), to increase the activity of the indigenous bacterial community, or by the addition of aerobic chlorobenzene-degrading bacteria (Pseudomonas putida GJ31, Pseudomonas aeruginosa RHO1, Pseudomonas putida F1CC). Experiments were performed with natural groundwater of the aquifer of Bitterfeld, which had been contaminated with 1,2-dichlorobenzene (1,2-DCB), 1,4-dichlorobenzene (1,4-DCB), and chlorobenzene (CB). The microcosms consisted of airtight glass bottles with 800 mL of natural groundwater and were incubated under in situ temperature (13°C). Behavior of the introduced strains within the indigenous bacterial community was monitored by fluorescent in situ hybridization (FISH) with species-specific oligonucleotides. Dynamics of the indigenous community and the introduced strains within the microcosms were followed by single-strand conformation polymorphism (SSCP) analysis of 16S rDNA amplicons obtained from total DNA of the microbial community. An indigenous biodegradation potential under aerobic as well as anaerobic denitrifying conditions was observed accompanied by fast and specific changes in the natural bacterial community composition. Augmentation with P. aeruginosa RHO1 did not enhance bio-degradation. In contrast, both P. putida GJ31 as well as P. putida F1CC were capable of growing in groundwater, even in the presence of the natural microbial community, and thereby stimulating chlorobenzene depletion. P. putida GJ31 disappeared when the xenobiotics were depleted and P. putida F1CC persisted even in the absence of CB. Detailed statistical analyses revealed that community dynamics of the groundwater microbiota were highly reproducible but specific to the introduced strain, its inoculum size, and the imposed physicochemical conditions. These findings could contribute to the design of better in situ bioremediation strategies for contaminated groundwater.     

Mixed aerobic and anaerobic microbial communities in benzene-contaminated groundwater

Aims:  To investigate the factors affecting benzene biodegradation and microbial community composition in a contaminated aquifer.
Methods and Results:  We identified the microbial community in groundwater samples from a benzene-contaminated aquifer situated below a petrochemical plant. Eleven out of twelve groundwater samples with in situ dissolved oxygen concentrations between 0 and 2·57 mg l⁻¹ showed benzene degradation in aerobic microcosm experiments, whereas no degradation in anaerobic microcosms was observed. The lack of aerobic degradation in the remaining microcosm could be attributed to a pH of 12·1. Three groundwaters, examined by 16S rRNA gene clone libraries, with low in situ oxygen concentrations and high benzene levels, each had a different dominant aerobic (or denitrifying) population, either Pseudomonas , Polaromonas or Acidovorax species. These groundwaters also had syntrophic organisms, and aceticlastic methanogens were detected in two samples. The alkaline groundwater was dominated by organisms closely related to Hydrogenophaga .
Conclusions:  Results show that pH 12·1 is inimical to benzene biodegradation, and that oxygen concentrations below 0·03 mg l⁻¹ can support aerobic benzene-degrading communities.
Significance and Impact of the Study:  These findings will help to guide the treatment of contaminated groundwaters, and raise questions about the extent to which aerobes and anaerobes may interact to effect benzene degradation.     

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) Bioremediation in Groundwater: Are Known RDX-Degrading Bacteria the Dominant Players?

Biodegradation of explosives in groundwater represents a promising remedial approach for these compounds. Although a range of bacteria capable of degrading the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in pure culture have been described, the role of these known strains (and the genera they represent) during RDX degradation in groundwater has not been established. RDX-contaminated groundwater was collected from the Pueblo Chemical Depot (CO, USA) and the Picatinny Arsenal (NJ, USA) where bioremediation technologies are being tested. Soil columns and enrichment cultures were derived from Picatinny Arsenal groundwater. Bacteria-specific primers were used to amplify the 16S rRNA genes that were used for phylogenetic analysis. The species detected ranged across multiple genera, many of which have not been previously associated with RDX biodegradation. None of the retrieved sequences were exact matches to previously described RDX-degrading strains, although multiple sequences that grouped with known explosive-degrading strains of Clostridium and Pseudomonas were recovered. Genes previously reported to be associated with RDX degradation, including xplA, hydA, onr, xenA, and xenB, were not detected in any of the groundwater samples. These preliminary results indicate that the previously described RDX-degrading bacteria likely do not capture the microbial diversity associated with RDX bioremediation in groundwater, especially under the general biostimulation approaches used during most remediation efforts.     

Polyvinyl alcohol biodegradation under denitrifying conditions

Polyvinyl alcohol was biodegraded under denitrifying conditions with a microbial community originated from a municipal wastewater treatment plant. The derived microbial consortium was capable of polyvinyl alcohol degradation under both denitrifying and aerobic conditions. The community dynamics was monitored by temperature gradient gel electrophoresis, and a principal utilizing organism was identified and assigned as Steroidobacter sp. PD. The possible role of Steroidobacter sp. PD was also investigated by sequencing the 16S rDNA clone library prepared from the degrading community. qPCR analysis showed that the fraction of the microorganism in the community was very low initially (0.02%) and had reached to about 16% by the end of the biodegradation experiment. The study revealed that polyvinyl alcohol can be biodegraded in a water environment not only under aerobic but also under denitrifying conditions.     

Intrinsic bioremediation in a solvent-contaminated alluvial groundwater

An industrial site contaminated with a mixture of volatile organic compounds in its subsurface differed from previously reported locations in that the contamination consisted of a mixture of chlorinated, brominated, and non-halogenated aromatic and aliphatic solvents in an alluvial aquifer. The source area was adjacent to a river. Of the contaminants present in the aquifer, benzene, toluene, and chlorobenzene (BTC) were of primary concern. Studies of the physical, chemical, and microbiological characteristics of site groundwater were conducted. The studies concentrated on BTC, but also addressed the fate of the other aquifer VOCs. Gas chromatographic analyses performed on laboratory microcosms demonstrated that subsurface microorganisms were capable of BTC degradation. Mineralization of BTC was demonstrated by the release of ¹⁴CO₂ from radiolabelled BTC. In the field, distribution patterns of nutrients and electron acceptors were consistent with expression of in situ microbial metabolic activity: methane, conductivity, salinity and o-phosphate concentrations were all positively correlated with contaminant concentration; while oxidation-reduction potential, nitrate, dissolved oxygen and sulfate concentrations were negatively correlated. Total aerobes, aerotolerant anaerobes, BTC-specific degraders, and acridine orange direct microscopic microorganism counts were strongly and positively correlated with field contaminant concentrations. The relative concentrations of benzene and toluene were lower away from the core of the plume compared to the less readily metabolized compound, chlorobenzene. Hydrodynamic modeling of electron-acceptor depletion conservatively estimated that 450 kg of contaminant have been removed from the subsurface yearly. Models lacking a biodegradation term predicted that 360 kg of contaminant would reach the river annually, which would result in measurable contaminant concentrations. River surveillance, however, has only rarely detected these compounds in the sediment and then only at trace concentrations. Thus, the combination of field modeling, laboratory studies, and site surveillance data confirm that significant in situ biodegradation of the contaminants has occurred. These studies establish the presence of intrinsic bioremediation of groundwater contaminants in this unusual industrial site subsurface habitat. Received 01 December 1995/ Accepted in revised form 27 July 1996     

Dynamic changes in microbial community structure and function in phenol-degrading microcosms inoculated with cells from a contaminated aquifer

Contamination of aquifers by organic pollutants threatens groundwater supplies and the environment. In situ biodegradation of organic pollutants by microbial communities is important for the remediation of contaminated sites, but our understanding of the relationship between microbial development and pollutant biodegradation is poor. A particular challenge is understanding the in situ status of microorganisms attached to solid surfaces, but not accessible via conventional sampling of groundwater. We have developed novel flow-through microcosms and examined dynamic changes in microbial community structure and function in a phenol-degrading system. Inoculation of these microcosms with a complex microbial community from a plume in a phenol-contaminated aquifer led to the initial establishment of a population dominated by a few species, most attached to the solid substratum. Initially, phenol biodegradation was incomplete, but as the microbial community structure became more complex, phenol biodegradation was more extensive and complete. These results were replicated between independent microcosms, indicating a deterministic succession of species. This work demonstrates the importance of examining community dynamics when assessing the potential for microbial biodegradation of organic pollutants. It provides a novel system in which such measurements can be made readily and reproducibly to study the temporal development and spatial succession of microbial communities during biodegradation of organic pollutants at interfaces within such environments.     

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.