Effects of humic substances and soya lecithin on the aerobic bioremediation of a soil historically contaminated by polycyclic aromatic hydrocarbons (PAHs)

The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) strongly reduces their bioavailability in aged contaminated soils, thus limiting their bioremediation. The biodegradation of PAHs in soils can be enhanced by employing surface-active agents. However, chemical surfactants are often recalcitrant and exert toxic effects in the amended soils. The effects of two biogenic materials as pollutant-mobilizing agents on the aerobic bioremediation of an aged-contaminated soil were investigated here. A soil historically contaminated by about 13 g kg(-1) of a large variety of PAHs, was amended with soya lecithin (SL) or humic substances (HS) at 1.5% w/w and incubated in aerobic solid-phase and slurry-phase reactors for 150 days. A slow and only partial biodegradation of low-molecular weight PAHs, along with a moderate depletion of the initial soil ecotoxicity, was observed in the control reactors. The overall removal of PAHs in the presence of SL or HS was faster and more extensive and accompanied by a larger soil detoxification, especially under slurry-phase conditions. The SL and HS could be metabolized by soil aerobic microorganisms and enhanced the occurrence of both soil PAHs and indigenous aerobic PAH-degrading bacteria in the reactor water phase. These results indicate that SL and HS are biodegradable and efficiently enhance PAH bioavailability in soil. These natural surfactants significantly intensified the aerobic bioremediation of a historically PAH-contaminated soil under treatment conditions similar to those commonly employed in large-scale soil bioremediation.     

Activation of an indigenous microbial consortium for bioaugmentation of pentachlorophenol/creosate contaminated soils

Soil activation, a concept based on the cultivation of biomass from a fraction of a comtaminated soil for subsequent use as an inoculum for bioaugmentation of the same soil, was studied as a method for the aerobic biodegradation of pentachlorophenol (PCP) and polycyclic hydrocarbons (PAH) in contaminated soils. A microbial consortium able to degrade PCP and PAH in contaminated soil from wood-preserving facilities was isolated and characterized for PCP degradation and resistance. To obtain an active consortium from the contaminated soil in a fed-batch bioreactor, the presence of soil as a support or source of nutrients was found to be essential. During the 35 days of bioreactor operation, residual PCP in solution remained near zero up to a loading rate of 700mg/l per day. The PCP meneralization rate increased from 70 mg/l per day when no PCP was added to the bioreactor to 700 mg/l per day at the maximum loading rate. The consortium tolerated a PCP concentration of 400 mg/l in batch experiments. Production of a PCP-degrading consortium in a fed-batch slurry bioreactor enhanced the activity of PCP biodegradation by a factor of ten. PAH biodegradation increased, during the same time period, by a factor of 30 and 81 for phenanthrene and pyrene, respectively. Preliminary laboratory-scale results indicated that a significant reduction in the time required for degradation of PCP and PAH in contaminated soil could be achieved using activated soil as an inoculum.Issued as NRC 33861 correspondence to: R. Samson     

Adsorption of polycyclic aromatic hydrocarbons (PAHs) by soil particles: influence on biodegradability and biotoxicity

Summary Polycyclic aromatic hydrocarbon (PAH) biodegradation was investigated in contaminated soils from two different industrial sites under simulated land treatment conditions. Soil samples from a former impregnation plant (soil A) showed high degradation rates of PAHs by the autochthonous microorganisms, whereas PAHs in material of a closed-down coking plant (soil B) were not degraded even after inoculation with bacteria known to effectively degrade PAHs. As rapid PAH biodegradation in soil B was observed after PAHs were extracted and restored into the extracted soil material, the kind of PAH binding in soil B appears to completely prevent biodegradation. Sorption of PAHs onto extracted material of soil B follows a two-phase process (fast and slow); the latter is discussed in terms of migration of PAHs into soil organic matter, representing less accessible sites within the soil matrix. Such sorbed PAHs are suggested to be non-bioavailable and thus non-biodegradable. By eluting soil B with water, no biotoxicity, assayed as inhibition of bioluminescence, was detected in the aqueous phase. When treating soil A analogously, a distinct toxicity was observed, which was reduced relative to the amount of activated carbon added to the soil material. The data suggest that sorption of organic pollutants onto soil organic matter significantly affects biodegradability as well as biotoxicity.     

Aerobic and Anaerobic Biodegradation of Aged Pentachlorophenol by Indigenous Microorganisms

Pentachlorophenol (PCP) use as a general biocide, particularly for treating wood, has led to widespread environmental contamination. Biodegradation has emerged as the main mechanism for PCP degradation in soil and groundwater and a key strategy for remediation. Examining the microbial biodegrading potential for PCP at a contaminated site is crucial in determining its fate. Hundreds of studies have been published on PCP microbial degradation, but few have described the biodegradation of PCP that has been in contact with soils for many years. The bioavailability of “aged” hydrophobic organics is a significant concern. PCP- and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)-contaminated soil samples from several depths at a former wood treatment site were placed under varying conditions in the laboratory to determine the anaerobic and aerobic potential for biodegradation of chlorophenols at the site. PCP biodegradation occurred in both anaerobic and aerobic soil samples. Rapid aerobic degradation occurred in samples spiked with 2- and 4-chlorophenol, but not with 3-chlorophenol. Reductive dechlorination of PCP in anaerobic samples resulted in the accumulation of 3-chlorophenol. In most anaerobic replicates, 3-chlorophenol was degraded with the appearance of detectable, but not quantifiable amounts of phenol. These results indicate excellent potential for remediation at the site using the indigenous microorganisms under both aerobic and anaerobic conditions. However, a fraction of the PCP was unavailable for degradation.     

Chemical Vapor Intrusion from Soil or Groundwater to Indoor Air: Significance of Unsaturated Zone Biodegradation of Aromatic Hydrocarbons

The soil vapor to indoor air exposure pathway is considered in a wide number of risk-based site management programs. In screening-level assessments of this exposure pathway, models are typically used to estimate the transport of vapors from either subsurface soils or groundwater to indoor air. Published studies indicate that the simple models used to evaluate this exposure pathway often over estimate the impact for aromatic hydrocarbons (e.g., benzene, toluene, ethylbenzene, and xy-lene or BTEX), while showing reasonable agreement for estimates of chlorinated hydrocarbon impacts (e.g., PCE, TCE, DCE). Aerobic biodegradation of the petroleum hydrocarbons is most often attributed as the source of this disparity in the model/ data comparisons. This paper looks at the significance of aerobic biodegradation of aromatic hydrocarbons as part of the assessment of chemical vapor intrusion from soil or groundwater to indoor air. A review of relevant literature summarizing the available field data as well as various modeling approaches that include biodegradation is presented. This is followed by a simple modeling analysis that demonstrates the potential importance of biodegradation in the assessment of the soil vapor to indoor air exposure pathway. The paper concludes with brief discussions of other model considerations that are often not included in simple models but may have a significant impact on the intrusion of vapors into indoor air.     

Soil biotransformation of thiodiglycol, the hydrolysis product of mustard gas: understanding the factors governing remediation of mustard gas contaminated soil

Thiodiglycol (TDG) is both the precursor for chemical synthesis of mustard gas and the product of mustard gas hydrolysis. TDG can also react with intermediates of mustard gas degradation to form more toxic and/or persistent aggregates, or reverse the pathway of mustard gas degradation. The persistence of TDG have been observed in soils and in the groundwater at sites contaminated by mustard gas 60 years ago. The biotransformation of TDG has been demonstrated in three soils not previously exposed to the chemical. TDG biotransformation occurred via the oxidative pathway with an optimum rate at pH 8.25. In contrast with bacteria isolated from historically contaminated soil, which could degrade TDG individually, a consortium of three bacterial strains isolated from the soil never contaminated by mustard gas was able to grow on TDG in minimal medium and in hydrolysate derived from an historical mustard gas bomb. Exposure to TDG had little impacts on the soil microbial physiology or on community structure. Therefore, the persistency of TDG in soils historically contaminated by mustard gas might be attributed to the toxicity of mustard gas to microorganisms and the impact to soil chemistry during the hydrolysis. TDG biodegradation may form part of a remediation strategy for mustard gas contaminated sites, and may be enhanced by pH adjustment and aeration.     

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.     

Biodegradation of RDX in Unsaturated Soil

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a military explosive that is a common soil and groundwater contaminant at facilities that manufacture, handle, and dispose of munitions. One such facility is the U.S. Department of Energy Pantex Plant, the focus of this research in which the feasibility of in situ bioremediation of contaminated soil in the vadose zone was assessed. A batch technique using ¹⁴C-RDX was developed to investigate the degradation of RDX under aerobic, microaerobic, and anaerobic conditions. In addition, the effect of nutrients (organic carbon and phosphorus) on biodegradation rates was studied. The extent of mineralization was quantified by monitoring the production of ¹⁴CO₂, and RDX biodegradation rates were estimated for each environmental condition. The results showed that RDX degraders were indigenous to the contaminated soil and degraded RDX to a significant extent under anaerobic conditions. Little biotransformation was observed under aerobic conditions. The addition of a biodegradable organic carbon source significantly increased the RDX biodegradation rate. Under appropriate environmental conditions, significant mineralization of RDX also was observed. The half-lives for the degradation of RDX under anaerobic conditions were approximately 60 days and decreased to approximately 40 days with nutrient addition. In contrast, the half-life for aerobic degradation was on the order of 1000 days, with an upper 95% confidence interval approaching infinity.     

Bioremediation of experimental petroleum spills on mineral soils in the Vestfold Hills,Antarctica

The effect of nutrient and water enhancement on the biodegradation of petroleum was tested in Antarctic mineral soils. Nitrogen, phosphorus and potassium were applied in solution, with or without gum xanthan or plastic covers, to sites artificially contaminated with distillate. The effectiveness of these procedures was assessed by measuring changes in total petroleum hydrocarbons; heptadecane/pristane and octadecane/phytane ratios; in concentrations of major hydrocarbon components and in microbial numbers and activity.Significantly lower hydrocarbon concentrations were recorded after one year in soils treated with fertilizer solutions, but only in the surface 3 cm. These soils also showed lowered heptadecane/pristane and octadecane/ phytane ratios and had the highest levels of microbial activity relative to other plots. Soils treated with gum xanthan. or covered with plastic had the highest residual hydrocarbon levels. Both treatments inhibited evaporative loss of hydrocarbon, and there were indications that gum xanthan was utilized by the microbiota as an alternative carbon source to distillate. Higher temperatures were recordecd under the plastic but no stimulation of biodegradation was detected.Estimated numbers of metabolically active bacteria were in the range 10⁷ to 10⁸ g^–1 dry weight of soil, with an estimated biomass of 0.03 to 0.26 mg g^–1 soil. Estimated numbers of amoebae were in the range 10⁶ to 10⁷ g^–1 soil (biomass of 2 to 4 mgg^–1). The highest populations were recorded in fertilized, contaminated soils, the only soils where petroleum degradation was demonstrated.     

The abundance of nahAc genes correlates with the 14C-naphthalene mineralization potential in petroleum hydrocarbon-contaminated oxic soil layers

In this study, we evaluated whether the abundance of the functional gene nahAc reflects aerobic naphthalene degradation potential in subsurface and surface samples taken from three petroleum hydrocarbon contaminated sites in southern Finland. The type of the contamination at the sites varied from lightweight diesel oil to high molecular weight residuals of crude oil. Samples were collected from both oxic and anoxic soil layers. The naphthalene dioxygenase gene nahAc was quantified using a replicate limiting dilution-polymerase chain reaction (RLD-PCR) method with a degenerate primer pair. In the non-contaminated samples nahAc genes were not detected. In the petroleum hydrocarbon-contaminated oxic soil samples nahAc gene abundance [range 3 x 10(1)-9 x 10(4) copies (g dry wt soil)(-1)] was correlated (Kendall non-parametric correlation r2=0.459, p<0.01) with the aerobic 14C-naphthalene mineralization potential (range 1 x 10(-5)-0.1 d(-1)) measured in microcosms at in situ temperatures (8 degrees C for subsurface and 20 degrees C for surface soil samples). In these samples nahAc gene abundance was also correlated with total microbial cell counts (r2=0.471, p<0.01), respiration rate (r2=0.401, p<0.01) and organic matter content (r2=0.341, p<0.05). NahAc genes were amplified from anoxic soil layers indicating that, although involved in aerobic biodegradation of naphthalene, these genes or related sequences were also present in the anoxic subsurface. In the samples taken from the anoxic layers, the aerobic 14C-naphthalene mineralization rates were not correlated with nahAc gene abundance. In conclusion, current sequence information provides the basis for a robust tool to estimate the naphthalene degradation potential at oxic zones of different petroleum hydrocarbon-contaminated sites undergoing in situ bioremediation.     

Anaerobic biodegradation of no. 2 diesel fuel in soil: a soil column study

Soil and sediments are contaminated with petroleum hydrocarbons in many parts of the world. Anaerobic degradation of petroleum hydrocarbon is very relevant in removing oil spills in the anaerobic zones of soil and sediments. This research investigates the possibility of degrading no. diesel fuel under anaerobic conditions. Anaerobic packed soil columns were used to simulate and study in situ bioremediation of soil contaminated with diesel fuel. Several anaerobic conditions were evaluated in soil columns, including sulfate reducing, nitrate reducing, methanogenic, and mixed electron acceptor conditions. The objectives were to determine the extent of diesel fuel degradation in soil columns under various anaerobic conditions and identify the best conditions for efficient removal of diesel fuel. Diesel fuels were degraded significantly under all conditions compared to no electron supplemented soil column (natural attenuation). However, the rate of diesel degradation was the highest under mixed electron acceptor conditions followed in order by sulfate reducing, nitrate reducing, and methanogenic conditions. Under mixed electron acceptor condition 81% of diesel fuel was degraded within 310 days. While under sulfate reducing condition 54.5% degradation of diesel fuel was observed for the same period. This study showed evidence for diesel fuel metabolism in a mixed microbial population system similar to any contaminated field sites, where heterogeneous microbial population exists.     

Genotoxicity Assessment of Wood-Preserving Waste-Contaminated Soil Undergoing Bioremediation

Bioremediation represents one of the most cost-effective technologies for treatment of petroleum hydrocarbons in contaminated surface soils. A major concern for regulatory agencies when evaluating bioremediation is how to determine acceptable levels for residual organics in soil. Although guidelines have been developed for some organics in soil, limited information is available to define acceptable levels of the metabolites of biological degradation. The products of oxidative degradation are likely to be more water soluble and may also be more toxic. The purpose of the current study was to monitor changes in compound concentration and genotoxicity in soils undergoing bioremediation. The site selected for this study was a former wood-preserving site in the northwestern United States. Soil samples were collected over a 4-year period from two 6075-m² land treatment units. Conditions for biodegradation were enhanced by the addition of water and nutrients, as well as by frequent tilling to add oxygen. Due to the location of the facility, the temperature was conducive to a more rapid rate of biodegradation for approximately 6 months per year. Soil samples were collected using a grid system and solvent extracted. Polycyclic aromatic hydrocarbons were quantified in soil extracts using gas chromatography-mass spectrometry (GC/MS), and genotoxicity measured using the Salmonella/microsome assay. After 2 years of treatment, concentrations of total and carcinogenic polycyclic aromatic hydrocarbons (PAHs) were reduced to approximately 10% the concentration in the untreated soil. However, the mean weighted activity of the untreated soil was 293 net revertants per g soil, whereas the extracts of soil collected after 2 years induced a mean weighted activity of 325 net revertants per g soil. Thus, although biodegradation clearly reduced the concentration of total and carcinogenic PAHs in the surface soils, the results from the genotoxicity bioassay indicate that there was a lag in the reduction of mutagenicity in treated soils.     

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).     

Bioremediation of soils contaminated with the herbicide 2-sec-butyl-4,6-dinitrophenol (dinoseb).

A novel soil treatment method for achieving the removal of dinoseb (2-sec-butyl-4,6-dinitrophenol) from contaminated soils was investigated. One soil contained dinoseb as the major contaminant, although several other hazardous compounds were also present. A second soil was highly contaminated with dinoseb. Dinoseb was not degraded in these soils under the aerobic conditions at each site. Pretreatment of the soils by the addition of a starchy potato-processing by-product and flooding with phosphate buffer stimulated the consumption of oxygen and nitrate from the soils, thereby lowering the redox potential and creating anaerobic conditions. Anaerobiosis (Eh less than -200 mV) promoted the establishment of an anaerobic microbial consortium that degraded dinoseb completely, without the formation of the polymerization products seen under aerobic or microaerophilic conditions. When dinoseb was present at low concentrations in a chronically contaminated soil, the natural microflora was capable of establishing anaerobic conditions and degrading dinoseb as a result of starch degradation. Inoculation of this soil with an aerobic starch-degrading microorganism and then an acclimated, anaerobic, dinoseb-degrading consortium did not improve dinoseb degradation. In a second acutely contaminated soil, these inoculations improved dinoseb degradation rates over those of uninoculated controls.     

Bioremediation of soils contaminated with the herbicide 2-sec-butyl-4,6-dinitrophenol (dinoseb).

A novel soil treatment method for achieving the removal of dinoseb (2-sec-butyl-4,6-dinitrophenol) from contaminated soils was investigated. One soil contained dinoseb as the major contaminant, although several other hazardous compounds were also present. A second soil was highly contaminated with dinoseb. Dinoseb was not degraded in these soils under the aerobic conditions at each site. Pretreatment of the soils by the addition of a starchy potato-processing by-product and flooding with phosphate buffer stimulated the consumption of oxygen and nitrate from the soils, thereby lowering the redox potential and creating anaerobic conditions. Anaerobiosis (Eh less than -200 mV) promoted the establishment of an anaerobic microbial consortium that degraded dinoseb completely, without the formation of the polymerization products seen under aerobic or microaerophilic conditions. When dinoseb was present at low concentrations in a chronically contaminated soil, the natural microflora was capable of establishing anaerobic conditions and degrading dinoseb as a result of starch degradation. Inoculation of this soil with an aerobic starch-degrading microorganism and then an acclimated, anaerobic, dinoseb-degrading consortium did not improve dinoseb degradation. In a second acutely contaminated soil, these inoculations improved dinoseb degradation rates over those of uninoculated controls.     

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.     

Aerobic biomineralization of alpha-hexachlorocyclohexane in contaminated soil

The factors identified to be important for the aerobic biodegradation of alpha-hexachlorocyclohexane (alpha-HCH) in a soil slurry are temperature, auxiliary carbon source, substrate concentration, and soil inhomogeneities. Temperatures in the range of 20 to 30 degrees C were determined to be most favorable for biodegradation of alpha-HCH. No alpha-HCH biodegradation was detected at temperatures below 4 degrees C and above 40 degrees C. The addition of auxiliary organic carbon compounds showed repressive effects on alpha-HCH biomineralization. Increased oxygen partial pressures reduced the repressive effects of added auxiliary organic carbon compounds. A linear relationship between alpha-HCH concentration and its conversion rate was found in a Lineweaver-Burk plot. Inhomogeneities such as clumping of alpha-HCH significantly affected its biodegradation. Inhomogeneity as an influence on biodegradation has not drawn sufficient attention in the past, even though it certainly has affected both laboratory studies and the application of biotechnological methods to clean up contaminated sites. On the basis of metabolites detected during degradation experiments, the initial steps of aerobic alpha-HCH bioconversion in a soil slurry are proposed.     

Aerobic biomineralization of alpha-hexachlorocyclohexane in contaminated soil.

The factors identified to be important for the aerobic biodegradation of alpha-hexachlorocyclohexane (alpha-HCH) in a soil slurry are temperature, auxiliary carbon source, substrate concentration, and soil inhomogeneities. Temperatures in the range of 20 to 30 degrees C were determined to be most favorable for biodegradation of alpha-HCH. No alpha-HCH biodegradation was detected at temperatures below 4 degrees C and above 40 degrees C. The addition of auxiliary organic carbon compounds showed repressive effects on alpha-HCH biomineralization. Increased oxygen partial pressures reduced the repressive effects of added auxiliary organic carbon compounds. A linear relationship between alpha-HCH concentration and its conversion rate was found in a Lineweaver-Burk plot. Inhomogeneities such as clumping of alpha-HCH significantly affected its biodegradation. Inhomogeneity as an influence on biodegradation has not drawn sufficient attention in the past, even though it certainly has affected both laboratory studies and the application of biotechnological methods to clean up contaminated sites. On the basis of metabolites detected during degradation experiments, the initial steps of aerobic alpha-HCH bioconversion in a soil slurry are proposed.     

Degradation of polycyclic aromatic hydrocarbons at low temperature under aerobic and nitrate-reducing conditions in enrichment cultures from northern soils

The potential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not well understood, but such biodegradation would be very useful for remediation of polluted sites. Biodegradation of a mixture of 11 different PAHs with two to five aromatic rings, each at a concentration of 10 micro g/ml, was studied in enrichment cultures inoculated with samples of four northern soils. Under aerobic conditions, low temperature severely limited PAH biodegradation. After 90 days, aerobic cultures at 20 degrees C removed 52 to 88% of the PAHs. The most extensive PAH degradation under aerobic conditions at 7 degrees C,53% removal, occurred in a culture from creosote-contaminated soil. Low temperature did not substantially limit PAH biodegradation under nitrate-reducing conditions. Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene were degraded. The most extensive PAH degradation under nitrate-reducing conditions at 7 degrees C, 39% removal, occurred in a culture from fuel-contaminated Arctic soil. In separate transfer cultures from the above Arctic soil, incubated anaerobically at 7 degrees C, removal of 2-methylnaphthalene and fluorene was stoichiometrically coupled to nitrate removal. Ribosomal intergenic spacer analysis suggested that enrichment resulted in a few predominant bacterial populations,including members of the genera Acidovorax,Bordetella, Pseudomonas, Sphingomonas, and Variovorax. Predominant populations from different soils often included phylotypes with nearly identical partial 16S rRNA gene sequences (i.e., same genus) but never included phylotypes with identical ribosomal intergenic spacers (i.e., different species or subspecies). The composition of the enriched communities appeared to be more affected by presence of oxygen, than by temperature or source of the inoculum.     

Benzene Degradation at a Site Amended with Nitrate or Chlorate

Aromatic hydrocarbons are widespread in nature and often contribute to the pollution of soils, sediments, and groundwater. The contamination of soil with mobile aromatic compounds, generally termed BTEX (benzene, toluene, ethylbenzene, xylene) is observed at many industrial sites, especially those associated with the petrochemical industry. In situ bioremediation of sites that are contaminated with BTEX can be applied both aerobically and anaerobically. The use of anaerobic in situ bioremediation is advantageous because supply of oxygen is not needed. Nevertheless, anaerobic in situ bioremediation is less commonly used for BTEX contaminated sites. This paper describes push-pull experiments in order to stimulate the degradation of benzene by the addition of nitrate or chlorate. Deuterated benzene was subjected with nitrate-amended groundwater to the aquifer, and the mineralization was traced by the enrichment of deuterium in the groundwater. Nitrate can be used as electron acceptor, and the addition of nitrate at a site in The Netherlands resulted in partial degradation of benzene. This was demonstrated by comparing various push-pull experiments, benzene concentration measurements, stable isotope analyses of benzene and water, and modeling. Chlorate can be used for the in situ production of oxygen, followed by degradation of benzene with oxygen as electron acceptor. The addition of chlorate at the site resulted in the complete removal of benzene demonstrating a complete degradation within 4 weeks. A pull phase was not needed during this run.