On site bioremediation of hydrocarbon-contaminated Arctic tundra soils in inoculated biopiles

There is a need to develop technology to allow the remediation of soil in polar regions that have been contaminated by hydrocarbon fuel spills. Bioremediation is potentially useful for this purpose, but has not been well demonstrated in polar regions. We investigated biopiles for on-site bioremediation of soil contaminated with Arctic diesel fuel in two independent small-scale field experiments at different sites on the Arctic tundra. The results were highly consistent with one another. In biopiles at both sites, extensive hydrocarbon removal occurred after one summer. After 1 year in treatments with optimal conditions, total petroleum hydrocarbons were reduced from 196 to below 10 mg per kg of soil at one site, and from 2,109 to 195 mg per kg of soil at the other site. Addition of ammonium chloride and sodium phosphate greatly stimulated hydrocarbon removal and indicates that biodegradation was the primary mechanism by which this was achieved. Inoculation with cold-adapted, mixed microbial cultures further stimulated hydrocarbon removal during the summer immediately following inoculation. At one site, soil temperature was monitored during the summer season, and a clear plastic cover increased biopile soil temperature, measured as degree-day accumulation, by 30-49%. Our results show that on-site bioremediation of fuel-contaminated soil at Arctic tundra sites is feasible.     

Stimulation of Diesel Fuel Biodegradation by Indigenous Nitrogen Fixing Bacterial Consortia

Abstract Successful stimulation of N₂ fixation and petroleum hydrocarbon degradation in indigenous microbial consortia may decrease exogenous N requirements and reduce environmental impacts of bioremediation following petroleum pollution. This study explored the biodegradation of petroleum pollution by indigenous N₂ fixing marine microbial consortia. Particulate organic carbon (POC) in the form of ground, sterile corn-slash (post-harvest leaves and stems) was added to diesel fuel amended coastal water samples to stimulate biodegradation of petroleum hydrocarbons by native microorganisms capable of supplying a portion of their own N. It was hypothesized that addition of POC to petroleum amended water samples from N-limited coastal waters would promote the growth of N₂ fixing consortia and enhance biodegradation of petroleum. Manipulative experiments were conducted using samples from coastal waters (marinas and less polluted control site) to determine the effects of POC amendment on biodegradation of petroleum pollution by native microbial consortia. Structure and function of the microbial consortia were determined by measurement of N₂ fixation (acetylene reduction), hydrocarbon biodegradation (¹⁴C hexadecane mineralization), bacterial biomass (AODC), number of hydrocarbon degrading bacteria (MPN), and bacterial productivity (³H-thymidine incorporation). Throughout this study there was a consistent enhancement of petroleum hydrocarbon degradation in response to the addition of POC. Stimulation of diesel fuel biodegradation following the addition of POC was likely attributable to increases in bacterial N₂ fixation, diesel fuel bioavailability, bacterial biomass, and metabolic activity. Toxicity of the bulk phase water did not appear to be a factor affecting biodegradation of diesel fuel following POC addition. These results indicate that the addition of POC to diesel-fuel-polluted systems stimulated indigenous N₂ fixing microbial consortia to degrade petroleum hydrocarbons. Received: 29 December 1998; Accepted: 6 April 1999     

Release Of Petroleum Hydrocarbons From Bioremediated Soils

This article presents a qualitative evaluation of the extent to which the bioavailability (release) of a chemical is related to the biodegradation of hydrocarbons in a field bioremediation unit. The objectives of this research were to (1) quantify the rate of release of petroleum hydrocarbons from two soils that were bioremediated, (2) explore hydrocarbon release as a process affecting bioremediation; and (3) investigate the impact of bioremediation on chemical release in the two soils. An experimental protocol was used to quantify the rate of release of these hydrocarbons from two soils that had been bioremediated in a field-scale prepared bed land treatment unit. One soil showed little change in hydrocarbon concentration during 55 weeks of prepared bed bioremediation. The field study results indicated that, prior to the bioremediation, this soil had reached an environmentally acceptable endpoint. The second soil showed considerable hydrocarbon loss as a result of the bioremediation. The rate of hydrocarbon release was determined for the first soil and for the second soil at time zero and after 1, 2, and 7 months of prepared bed bioremediation. The results indicated: (1) the fraction (F) of the specific hydrocarbons that were released rapidly from the soil and the rates of release (k₂) of the residual hydrocarbons that were released slowly, (2) that the mass of each chemical of concern that was released from the first soil was very low; and (3) that the hydrocarbon released rapidly from the second soil decreased as treatment progressed. The experiments also verified, qualitatively, that some portion of each chemical evaluated was not able to be released, and thus was unavailable for bioremediation in the prepared bed land treatment unit.     

Prepared Bed Land Treatment of Soils Containing Diesel and Crude Oil Hydrocarbons

An ex situ, field-scale, prepared bed land treatment unit (LTU) was used to bio-remediate soils containing petroleum hydrocarbons. Two soils were treated in side-by-side units to compare performance: (1) a clayey silt containing crude oil hydrocarbons from releases 30 to 40 years ago and (2) a silty sand containing diesel fuel hydrocarbons from a leak about three years prior to the bioremediation. The effectiveness of the bioremediation in the LTU was evaluated over a period of 18 months. The results indicated that: (1) prepared bed bioremediation reduced the hydrocarbon concentration, mobility, and relative toxicity in the soil with the diesel fuel, and (2) chemical bioavailability appeared to limit bioremediation of the soil containing the crude oil hydrocarbons. Although the soils containing the crude oil hydrocarbons contained an average of 10,000?mg TPH/kg dry soil, these soils had limited hydrocarbon availability, nontoxic conditions, and low potential for chemical migration. For the soils containing the diesel fuel, active prepared bed bioremediation of about 15 weeks was adequate to reach an environmentally acceptable endpoint. At that time, there was little further TPH loss, no Microtox^TM toxicity, and limited hydrocarbon mobility.     

Surfactant enhances biodegradation of hydrocarbons: Microcosm and field study

The purpose of the present study was to provide new methods that would increase the rates of biodegradation of petroleum hydrocarbons in soil, thus reducing the time required to achieve a satisfactory level of residual hydrocarbon in an ex situ bioremediation. Results of laboratory studies on several techniques were used to guide our implementation of these methods in controlled field studies. Soils contaminated with nonvolatile hydrocarbons were treated with various combinations of (1) an anionic surfactant guanidinium cocoate (CGS), (2) a consortium of hydrocarbon‐degrading microorganisms, (3) a slow‐release form of nitrogen:urea, and (4) the bulking agent vermiculite. Laboratory results describing the activity of CGS have been presented previously (Jain et al., 1992). The amount and rate of hydrocarbon loss in treated soil was compared with hydrocarbon lost in soil that received no amendment other than water (water only). We also used a sheen screen method (Nelson et al., 1995), to assess the effectiveness of our field application of microorganisms.     

Biodegradation of Hopane Prevents Use as Conservative Biomarker During Bioremediation of PAHs in Petroleum Contaminated Soils

The pentacyclic triterpane C₃₀ 17α (H), 21β (H)-hopane, a biomarker commonly used in hydrocarbon bioremediation laboratory experiments and field studies, was found to be completely removed without the formation of the demethylated intermediate nor-hopane in a crude oil-contaminated soil undergoing slurry biotreatment, while PAHs such as benzo(e)pyrene were recalcitrant. The partial or complete biodegradation of hopane has also been previously reported in a few bioremediation studies and has been explored by petroleum geochemists in an effort to characterize crude oil deposits. It is currently not clear what conditions induce hopane biodegradation or biotransformation, although the use of microbial enrichment cultures appears to speed up the process. Considering that hopane is not necessarily conserved during a bioremediation study, the uncritical normalization of hydrocarbon concentrations using this biomarker can lead to incorrect estimates of biodegradation rates and extents. If hopane is found to be unstable in a particular case, other potential biomarkers such as pentahopane, oleanane, or vanadium may be used instead.     

Effect of scale-up and seasonal variation on biokinetics in the enhanced bioremediation of petroleum hydrocarbon-contaminated soil

The biodegradation rate of petroleum hydrocarbon-contaminated soil was evaluated by the effect of temperature variation through bioaugmentation and biostimulation. In this study, biokinetics of batch-, pilot-, and field-scale biodegradation were performed by the optimization of enhanced biodegradation, minimizing the inhibitory effects of seasonal variations such as the rainy and cold winter seasons. From the relationship between remedial timescale and initial concentration, the biokinetic isolines of the biodegradation were smaller in the winter than those in the other seasons. The scale-up of biodegradation process led to enhance its activation energy, and then the field-scale remedial action should be performed in the way to lower the activation energy from the chemical diffusion and microbial activation. Therefore, a remedial or field worker can obtain the remedial timescale from the given apparent data of biokinetics with respect to initial TPH concentration only after the simple remedial investigation.     

Microbial degradation of hydrocarbons in the environment.

The ecology of hydrocarbon degradation by microbial populations in the natural environment is reviewed, emphasizing the physical, chemical, and biological factors that contribute to the biodegradation of petroleum and individual hydrocarbons. Rates of biodegradation depend greatly on the composition, state, and concentration of the oil or hydrocarbons, with dispersion and emulsification enhancing rates in aquatic systems and absorption by soil particulates being the key feature of terrestrial ecosystems. Temperature and oxygen and nutrient concentrations are important variables in both types of environments. Salinity and pressure may also affect biodegradation rates in some aquatic environments, and moisture and pH may limit biodegradation in soils. Hydrocarbons are degraded primarily by bacteria and fungi. Adaptation by prior exposure of microbial communities to hydrocarbons increases hydrocarbon degradation rates. Adaptation is brought about by selective enrichment of hydrocarbon-utilizing microorganisms and amplification of the pool of hydrocarbon-catabolizing genes. The latter phenomenon can now be monitored through the use of DNA probes. Increases in plasmid frequency may also be associated with genetic adaptation. Seeding to accelerate rates of biodegradation has been shown to be effective in some cases, particularly when used under controlled conditions, such as in fermentors or chemostats.     

Biodegradation of aged diesel in diverse soil matrixes: impact of environmental conditions and bioavailability on microbial remediation capacity

While bioremediation of total petroleum hydrocarbons (TPH) is in general a robust technique, heterogeneity in terms of contaminant and environmental characteristics can impact the extent of biodegradation. The current study investigates the implications of different soil matrix types (anthropogenic fill layer, peat, clay, and sand) and bioavailability on bioremediation of an aged diesel contamination from a heterogeneous site. In addition to an uncontaminated sample for each soil type, samples representing two levels of contamination (high and low) were also used; initial TPH concentrations varied between 1.6 and 26.6 g TPH/kg and bioavailability between 36 and 100 %. While significant biodegradation occurred during 100 days of incubation under biostimulating conditions (64.4–100 % remediation efficiency), low bioavailability restricted full biodegradation, yielding a residual TPH concentration. Respiration levels, as well as the abundance of alkB, encoding mono-oxygenases pivotal for hydrocarbon metabolism, were positively correlated with TPH degradation, demonstrating their usefulness as a proxy for hydrocarbon biodegradation. However, absolute respiration and alkB presence were dependent on soil matrix type, indicating the sensitivity of results to initial environmental conditions. Through investigating biodegradation potential across a heterogeneous site, this research illuminates the interplay between soil matrix type, bioavailability, and bioremediation and the implications of these parameters for the effectiveness of an in situ treatment.     

Fenton's Reagent-Based In Situ Chemical Oxidation Treatment of Saturated and Unsaturated Soils at a Historic Railroad Site

A targeted treatment program utilizing in situ chemical oxidation was used to remediate diesel fuel-derived petroleum compounds in unsaturated and saturated soils at a historic railroad facility. This program consisted of multiple injections at varying depths within temporary Geoprobe® injection points. The actual treatment time was less than 3 months. Overall concentrations of volatile and semivolatile organic petroleum compounds were reduced by approximately 70%, while the total petroleum hydrocarbon concentration was reduced by nearly 50%. Treatment efficiency in unsaturated soil was similar to that in saturated soil. The results of the remedial program indicate that the effect of grain size of the subsurface materials on treatment efficacy is significant. The project has shown that the use of this technology can be as effective as other in situ treatment technologies used for treating subsurface diesel fuel contamination.     

Bioremediation of Soil Artificially Contaminated with Petroleum Hydrocarbon Oil Mixtures: Evaluation of the Use of Animal Manure and Chemical Fertilizer

The search for cheaper and environmentally friendly options of enhancing petroleum hydrocarbon degradation has continued to elicit research interest. One of such options is the use of animal manure as biostimulating agents. A combination of treatments consisting of the application of poultry manure, piggery manure, goat manure, and chemical fertilizer was evaluated in situ during a period of 4 weeks of remediation. Each treatment contained petroleum hydrocarbon mixture (kerosene, diesel oil, and gasoline mixtures) (10% w/w) in soil as a sole source of carbon and energy. After 4 weeks of remediation, the results showed that poultry manure, piggery manure, goat manure, and NPK (nitrogen, phosphorous, and potash [potassium]) fertilizer exhibited 73%, 63%, 50%, and 39% total petroleum hydrocarbon degradation, respectively. Thus, all the biostimulating treatment strategies showed the ability to enhance petroleum hydrocarbon microbial degradation. However, poultry manure, piggery manure, and goat manure treatments showed greater petroleum hydrocarbon reductions than NPK fertilizer treatment. A first-order kinetic equation was fitted to the biodegradation data and the specific degradation rate constant (k) values obtained showed that the order of effectiveness of these biostimulating strategies in the cleanup of soil contaminated with petroleum hydrocarbon mixtures (mixture of kerosene, diesel oil, and gasoline) is NPK fertilizer < goat manure < piggery manure < poultry manure. Therefore, this present work has indicated that the application of poultry manure, piggery manure, goat manure, and chemical fertilizer could enhance petroleum hydrocarbon degradation with poultry manure, showing a greater effectiveness and thus could be one of the severally sought environmentally friendly ways of remediating natural ecosystem contaminated with crude oil.     

Biostimulation and bioaugmentation for on-site treatment of weathered diesel fuel in Arctic soil

Bioremediation of weathered diesel fuel in Arctic soil at low temperature was studied both on-site in small-scale biopiles and in laboratory microcosms. The field study site was on Ellesmere Island (82°30'N, 62°20'W). Biostimulation was by fertilization with phosphorous and nitrogen. Bioaugmentation was with an enrichment culture originating from the field site. In biopiles, total petroleum hydrocarbons (TPH) were reduced from 2.9 to 0.5 mg/g of dry soil over a period of 65 days. In microcosms at 7 °C, TPH were reduced from 2.4 to 0.5 mg/g of dry soil over a period of 90 days. Inoculation had no effect on hydrocarbon removal in biopiles or in microcosms. Maximum TPH removal rates in the biopiles were approximately 90 μg of TPH g^–1 of soil day^–1, occurring during the first 14 days when ambient temperature ranged from 0 to 10 °C. The fate of three phylotypes present in the inoculum was monitored using most-probable-number PCR, targeting 16S rRNA genes. Populations of all three phylotypes increased more than 100-fold during incubation of both uninoculated and inoculated biopiles. The inoculum increased the initial populations of the phylotypes but did not significantly affect their final populations. Thus, biostimulation on site enriched populations that were also selected in laboratory enrichment cultures. Electronic Publication     

Bioventing soils contaminated with petroleum hydrocarbons

Summary Bioventing combines the capabilities of soil venting and enhanced bioremediation to cost-effectively remove light and middle distillate hydrocarbons from vadose zone soils and the groundwater table. Soil venting removes the more volatile fuel components from unsaturated soil and promotes aerobic biodegradation by driving large volumes of air into the subsurface. In theory, air is several thousand times more effective than water in penetrating and aerating fuel-saturated and low permeability soil horizons. Aerobic microbial degradation can mitigate both residual and vapor phase hydrocarbon concentrations. Soil venting is being evaluated at a number of U.S. military sites contaminated with middle distillate fuels to determine its potential to stimulate in situ aerobic biodegradation and to develop techniques to promote in situ vapor phase degradation. In situ respirometric evaluations and field pilot studies at sites with varying soil conditions indicate that bioventing is a cost-effective method to treat soils contaminated with jet fuels and diesel.     

Biodegradability of polar compounds formed from weathered diesel

Once released into the environment, petroleum is exposed to biological and physical weathering processes which can lead to the formation and accumulation of highly recalcitrant polar compounds. These polar compounds are often challenging to analyse and can be present as an “unresolved complex mixture” (UCM) in total petroleum hydrocarbon (TPH) analyses and can be mistaken for natural organic matter. Existing research on UCMs comprised of polar compounds is limited, with a majority of the compounds remaining unidentified and their long-term persistence unknown. Here, we investigated the potential biodegradation of these recalcitrant polar compounds isolated from weathered diesel contaminant, and the changes in the microbial community composition associated with the biodegradation process. Microcosms were used to study the biodegradability of the polar compounds under various aerobic and anaerobic conditions and the results compared against the biodegradation of fresh diesel. Under all conditions tested, the majority of the polar UCM contaminant remained recalcitrant to biodegradation. The degradation was limited to the TPH portion of the polar UCM, which represented a minor fraction of the total polar UCM concentration. Changes in microbial community composition were observed under different redox conditions and in the presence of different contaminants. This work furthers the understanding of the biodegradation and long-term recalcitrance of polar compounds formed through weathering at contaminated legacy sites.     

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.     

Functional genes reveal the intrinsic PAH biodegradation potential in creosote-contaminated groundwater following in situ biostimulation

A small-scale functional gene array containing 15 functional gene probes targeting aliphatic and aromatic hydrocarbon biodegradation pathways was used to investigate the effect of a pilot-scale air sparging and nutrient infiltration treatment on hydrocarbon biodegradation in creosote-contaminated groundwater. Genes involved in the different phases of polycyclic aromatic hydrocarbon (PAH) biodegradation were detected with the functional gene array in the contaminant plume, thus indicating the presence of intrinsic biodegradation potential. However, the low aerobic fluorescein diacetate hydrolysis, the polymerase chain reaction (PCR) amplification of 16S rRNA genes closely similar to sulphate-reducing and denitrifying bacteria and the negligible decrease in contaminant concentrations showed that aerobic PAH biodegradation was limited in the anoxic groundwater. Increased abundance of PAH biodegradation genes was detected by functional gene array in the monitoring well located at the rear end of the biostimulated area, which indicated that air sparging and nutrient infiltration enhanced the intrinsic, aerobic PAH biodegradation. Furthermore, ten times higher naphthalene dioxygenase gene copy numbers were detected by real-time PCR in the biostimulated area, which was in good agreement with the functional gene array data. As a result, functional gene array analysis was demonstrated to provide a potential tool for evaluating the efficiency of the bioremediation treatment for enhancing hydrocarbon biodegradation in field-scale applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assessing mineralization rates of petroleum hydrocarbons in soils in relation to environmental factors and experimental scale

Mineralization rates of non-volatile petroleum hydrocarbons (HCs) in five different oil-contaminated soils with initial HC contents ranging from 0.1 to 13 g kg^-1 are estimated as a function of environmental factors. The aim of the study is threefold, (i) to study the relevance of environmental factors that may influence the mineralization rate, (ii) to compare mineralization rates estimated in two experiments at different scales, after standardizing them to environmental reference conditions, (iii) to evaluate the CO₂ production rate as a measure for the mineralization rate of HCs. Experiments were performed at laboratory scale (30–50 cm³ soil volume) in closed-jars under constant environmental conditions and in lysimeters (0.81 m³ soil volume) under dynamic climatic and hydrological conditions. A biodegradation model, coupled to transport models for soil heat, water, and gas dynamics is employed for data interpretation. The transport models are used to simulate the environmental conditions that influence the mineralization rate in the non-steady lysimeter experiments. The results show that temperature, O₂ concentration and HC content have an effect on the mineralization rates. Water content could not be identified as a direct governing environmental factor. However, an indirect effect of water content is that it influences the effective gas diffusion coefficient in soils. The CO₂ production rate seems to be a good quantity to express the mineralization rate of HCs for HC contents>1 g kg^-1. Measured CO₂ production rates standardized to reference conditions are similar for the two different experimental scales. This demonstrates that the usage of biodegradation rates obtained in the laboratory to predict the biodegradation rates under field conditions is sound, as long as the differences in environmental conditions have been taken into account.     

Monitoring the Size and Metabolic Activity of the Bacterial Community during Biostimulation of Fuel-Contaminated Soil using Competitive PCR and RT-PCR

Efforts to understand and improve soil bioremediation are limited by our ability to determine how treatment variables affect microbial communities. A method was developed to monitor the density and metabolic activity of the total bacterial community in soil. This method was used to monitor the bacterial community in microcosms of Arctic soil after addition of N plus P to stimulate biodegradation of hydrocarbon contaminants. During 29 days of incubation, the total petroleum hydrocarbon level in the soil was reduced from 850 to 360 mg/g of soil. DNA and RNA were extracted from soil using a bead beating method, purified by ammonium acetate precipitation, and assayed by competitive PCR and RT-PCR assays with universal bacterial primers. The copy number of 16S rDNA in the soil microbial community was relatively stable and ranged from 1.7 × 109 to 4.5 × 109/g of soil throughout the incubation. The copy number of 16S rRNA changed substantially and ranged from 5.6 × 1010 to 1.0 × 1012/g of soil. The rRNA:rDNA ratio was highest during the phase of fastest hydrocarbon biodegradation. These results suggest that the treatment to stimulate hydrocarbon biodegradation did not substantially change the density of the bacterial community but did transiently increase its overall metabolic activity.     

石油烃和酚类物质在土中的生物降解与土壤酶活性

本文通过模拟实验,研究了不同条件下石油烃和酚类物质在土中的降解进程及其与土壤酶活性的关系,并在此基础上,对所述污染物的土地处理提出了若干建议。     

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.