On-Site Treatment of Contaminated Soils: An Approach to Bioremediation of Weathered Petroleum Compounds

A bench-scale investigation was conducted prior to on-site bioremediation of 52,000 cubic yards of contaminated soil containing weathered, structurally complex petroleum compounds from an inactive oil refinery. Addition of bulking agents was required to improve soil physical properties. A supplemental study was also conducted to evaluate the effectiveness of bio-enhancement products. Loss of n-alkanes was rapid in soil mixtures containing a high nitrogen sludge compost, but very slow in mixtures containing wood products as bulking agents. By completion of the study at day 110, the isoprenoids pristane and phytane had nearly disappeared from mixtures containing sludge compost. Clearly, pristane and phytane are inadequate biomarkers when conditions favor an advanced stage of biodegradation. Nearly half the complex branched and cyclic alkanes in the unresolved complex mixture also degraded. After 70 days, depletion of dibenzo-thiophenes and phenan-threnes was 75 and 90%, respectively. The most stable PAHs within each group were the highly methylated homologues. Because of their complex structures, both steranes and hopanes were stable in all soil mixtures. Data were normalized to hopanes as a conserved internal standard or biomarker. Use of hopane-normalized data successfully eliminated much of the data variability and permitted a more accurate assessment of biodegradation. A relatively slow decline in total hydrocarbons occurred later in the study. This slowing tendency of microbial utilization is caused not only by substrate depletion, but also because remaining hydrocarbons are structurally more complex and persistent. Because of this, it is important to avoid using kinetic data from early stages of bioremediation to predict later hydrocarbon losses, such as the time required to attain a cleanup standard. In the supplemental study, an oleophilic fertilizer product accelerated hydrocarbon degradation when compared with a conventional fertilizer. This product will be tested in combination with organic bulking agents under field conditions to determine its cost effectiveness.     

石油污染土壤生物降解生态条件研究

生物治理是石油污染土壤主要的有效的治理方法。微生物、污染物及环境等方面的因素都影响着生物降解效率。通过最佳生物降解条件的研究,提出了提高生物降解效率的措施。     

石油污染土壤的生物修复技术

1　前　言在石油生产、贮运、炼制加工及使用过程中 ,由于事故 ,不正常操作及检修等原因 ,都会有石油烃类的溢出和排放。例如 ,油田开发过程中的井喷事故 ;输油管线和贮油罐的泄漏事故 ;油槽车和油轮的泄漏事故 ;油井清蜡和油田地面设备检修 ;炼油和石油化工生产装置检修等。石油烃类大量溢出 ,应当尽可能予以回收 ,但有的情况下回收很困难 ,即使尽力回收 ,仍会残留一部分 ,对环境 (土壤、地面和地下水 )造成污染。其进入土壤后 ,会破坏土壤结构 ,分散土粒 ,使土壤的透水性降低。其富含的反应基能与无机氮、磷结合并限制硝化作用和脱磷酸作…     

Microbial Factors Rather Than Bioavailability Limit the Rate and Extent of PAH Biodegradation in Aged Crude Oil Contaminated Model Soils

The rate and extent of polynuclear aromatic hydrocarbons (PAH) biodegradation in a set of aged model soils that had been contaminated with crude oil at the high concentrations (i.e.,>20,000?mg/kg) normally found in the environment were measured in 90-week slurry bioremediation experiments. Soil properties such as organic matter content, mineral type, particle diameter, surface area, and porosity did not significantly influence the PAH biodegradation kinetics among the 10 different model soils. A comparison of aged and freshly spiked soils indicates that aging affects the biodegradation rate and extent only for higher-molecular-weight PAHs, while the effects of aging are insignificant for 4-ring PAHs and total PAHs. In all model soils with the exception of kaolinite clay, the rate of abiotic desorption was faster than the rate of biodegradation during the initial phase of bioremediation treatment, indicating that PAH biodegradation was limited by microbial factors. Similarly, any of the higher-molecular-weight PAHs that were still present after 90 weeks of treatment were released rapidly during abiotic desorption tests, which demonstrates that bioavailability limitations were not responsible for the recalcitrance of these hydrocarbons. Indeed, an analysis of microbial counts indicates that a severe reduction in hydrocarbon degrader populations may be responsible for the observed incomplete PAH biodegradation. Therefore, it can be concluded that the recalcitrance of PAHs during bioremediation is not necessarily due to bioavailability limitations and that these residual contaminants therefore might pose a greater risk to environmental receptors than previously thought.     

Prediction of Petroleum Hydrocarbon Bioavailability in Contaminated Soils and Sediments

Recently, several laboratory methods have been developed for the prediction of contaminant bioavailability. So far, none of these methods has been extensively tested for petroleum hydrocarbons. In the present study we investigated solid-phase extraction and persulfate oxidation for the prediction of total petroleum hydrocarbon (TPH) bioavailability. One sediment and two soil samples were subjected to solid-phase extraction, persulfate oxidation, and biodegradation, after which hydrocarbon removal was compared. It was demonstrated that a short solid-phase extraction (168?h) provided a good method for the prediction of the extent of TPH degradation in an optimized slurry reactor (84?d). Solid-phase extraction slightly underestimated the degradation of readily biodegradable hydrocarbons, whereas it slightly overestimated the degradation of poorly biodegradable hydrocarbons. Persulfate oxidation appeared to be unfit for the prediction of TPH bioavailability as persulfate was unable to oxidize hydrocarbons with a high ionization potential. Hydrocarbons that were affected were likely to be transformed rather than completely oxidized. Nevertheless, persulfate oxidation provided a good method for the prediction of polycyclic aromatic hydrocarbon (PAH) bioavailability.     

Bioremediation of Soils Contaminated with Nigerian Petroleum Products Using Composted Municipal Wastes

The efficiency of ready-to-use, source-separated, composted municipal organic wastes of Nigerian origin on degradation of soil total petroleum hydrocarbons (TPHs) in soils polluted with petroleum products (crude oil, diesel, and spent engine oil) was assessed in screen house experiments. The effect of compost:soil ratios and combined effect of compost-phytoremediation technique were also studied. TPH was determined spectrophotometrically, after extraction with 1:1 acetone-dichloromethane mixture at 425 nm. Soil pH, electrical conductivity, and phytotoxicity to seed germination and growth of maize (Zea mays L.) served as risk assessments on soil quality and evidence of recovery for the oil-impacted soil. Results showed that the treatments increased soil pH and electrical conductivity but reduced TPH. Reductions in TPH by compost technology ranged from 40% to 75.87%. Toxicity to seed germination reduced from 100% to 16.12%. Positive correlations were obtained for plant agronomical parameters and growth period, for all treatments, with coefficients in the range of .905 to .996, p < .05. This study revealed that ready-to-use composted waste has the potential for bioremediation of soils polluted with petroleum and petroleum products. This study is a contribution to the data bank of relatively simple bioremediation methods, suitable for workers in the developing countries, where there is no easy access to high-technology facilities. However, further development of this technique to achieve zero residual TPH is recommended.     

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.     

Aerobic Biodegradation of DDT by Advenella Kashmirensis and Its Potential Use in Soil Bioremediation

The aim of this study was to select a bacterial strain able to degrade 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT), and to use it for bioaugmentation in order to decontamination soil. Advenella Kashmirensis MB-PR (A. Kashmirensis MB-PR) was isolated from DDT contaminated soil, and the degradation ability of DDT by this strain in the mineral salt medium was screened by gas chromatography. The efficiency of degradation was 81% after 30 days of bacterial growth. The study of intermediary products during the degradation of DDT showed the appearance and accumulation of DDD and DDE, which emerged from the first days of the experiment. Other metabolites were detected at a lower number of chlorine atoms, such as DBH. DNA samples were isolated and screened for the linA gene, encoding dehydrochlorinase. The bioaugmentation by A. Kashmirensis MB-PR of polluted sterile soil showed that 98% of DDT disappeared after 20 days of experience. This study demonstrates the significant potential use of A. Kashmirensis MB-PR for the bioremediation of DDT in the environment.     

Incorporation of Petroleum Carbon Into Soil Organic Matter During Biodegradation

A procedure, based on measurement of the stable carbon isotope ¹³C, has been developed for determining the extent to which petroleum carbon is incorporated into soil organic matter (SOM) by humification of biomass produced during biodegradation of the petroleum in soil. We have shown that a crude oil having a δ¹³C of-27.4%, when biodegraded in a soil containing SOM with a δ¹³C of-15.7%, resulted in a change of the δ¹³C of the bound SOM reflecting that of petroleum carbon. Comparison of five soil biodegradation tests using different amounts and types of fertilizer to stimulate biodegradation of the oil in this soil showed that the extent of the δ¹³C change in the bound SOM varied with the extent of oil biodegradation observed. To obtain ¹³C data on the SOM, the residual petroleum was first removed by rigorous extraction with dichloromethane using a Soxhlet apparatus. The extracted soil was then combusted to release bound carbon as CO₂, which was analyzed for ¹³C. Where the SOM has a δ¹³C similar to that of petroleum, ¹⁴C measurements of SOM would give similar results. This type of data, referred to as the petroleum “footprint” in the SOM, could be useful in identifying or confirming intrinsic biodegradation of petroleum in contaminated soil.     

生物反应器法处理油泥污染土壤的研究

采油过程产生的油泥是整个石油烃污染源的重点。在陆地生态环境中 ,烃类的大量存在往往对植物的生物学质量产生不利影响 ,更重要的是石油中的一些多环芳烃是致癌和致突变物质 ,这些致癌和致突变的有机污染物进入农田生态系统后 ,在动植物体内逐渐富集 ,进而威胁人类的生存和健康[1 ,1 1 ] 。大量的废弃油泥 ,不仅污染农田 ,同时也给石油行业带来巨大的经济损失。污染土壤的治理主要有物理、化学和生物 (生物修复 )方法 ,生物修复方法被认为最有生命力。污染土壤生物修复技术主要有 3种 ,即原位处理、挖掘堆置处理和反应器处理。反应器处理是…     

Enhanced Biodegradation of Petroleum Hydrocarbons in Contaminated Soil

Soil samples taken from a contaminated site in Northern Quebec, Canada, exhibited a low capacity for biodegradation of total petroleum hydrocarbons (TPH), despite a high capacity for the mineralization of aromatic hydrocarbons and a low toxicity of soil leachates as measured by Microtox assay. Toxicity assays directly performed on surface soil, including earthworm mortality and barley seedling emergence, indicated moderate to high levels of toxicity. Soil biostimulation did not improve the removal of petroleum hydrocarbons, while bioaugmentation of soil with a developed enrichment culture increased the efficiency of hydrocarbon removal from 20.4% to 49.2%. A considerable increase in the removal of TPH was obtained in a bioslurry process, enhancing the mass transfer of hydrocarbons from soil to the aqueous phase and increasing the efficiency of hydrocarbon removal to over 70% after 45 days of incubation. The addition of ionic or nonionic surfactants did not have a significant impact on biodegradation of hydrocarbons. The extent of hydrocarbon mineralization during the bioslurry process after 45 days of incubation ranged from 41.3% to 58.9%, indicating that 62.7% to 83.1% of the eliminated TPH were transformed into CO₂ and water.     

Enhanced Biodegradation of Petroleum Hydrocarbons in Contaminated Soil

Soil samples taken from a contaminated site in Northern Quebec, Canada, exhibited a low capacity for biodegradation of total petroleum hydrocarbons (TPH), despite a high capacity for the mineralization of aromatic hydrocarbons and a low toxicity of soil leachates as measured by Microtox assay. Toxicity assays directly performed on surface soil, including earthworm mortality and barley seedling emergence, indicated moderate to high levels of toxicity. Soil biostimulation did not improve the removal of petroleum hydrocarbons, while bioaugmentation of soil with a developed enrichment culture increased the efficiency of hydrocarbon removal from 20.4% to 49.2%. A considerable increase in the removal of TPH was obtained in a bioslurry process, enhancing the mass transfer of hydrocarbons from soil to the aqueous phase and increasing the efficiency of hydrocarbon removal to over 70% after 45 days of incubation. The addition of ionic or nonionic surfactants did not have a significant impact on biodegradation of hydrocarbons. The extent of hydrocarbon mineralization during the bioslurry process after 45 days of incubation ranged from 41.3% to 58.9%, indicating that 62.7% to 83.1% of the eliminated TPH were transformed into CO₂ and water.     

Improving Mathematical Model in Biodegradation of PAHs Contaminated Soil Using Gram-Positive Bacteria

In published literature there are limited studies on the estimation of kinetic parameters of polycyclic aromatic hydrocarbons (PAHs) in soil. In addition, neither the kinetic studies were performed with Gram-positive bacteria nor conducted under non-indigenous condition in order to understand their removal performance. Thus, a mathematical model describing biodegradation of phenanthrene-contaminated soil by Corynebacterium urealyticum, bacterium isolated from municipal sludge, was developed in this study. The model includes three kinetic parameters that were determined using TableCurve 2D software, namely qmax (maximum substrate utilization rate per unit mass of bacteria), X (biomass concentration) and Ks (substrate concentration at one half the maximum substrate utilization rate). These parameters were evaluated and verified in five different initial phenanthrene concentrations. Highest degradation rate was determined to be 79.24 mg kg^?1 day^?1 at 500 mg kg^?1 initial phenanthrene concentrations. This high concentration shows that bacteria perform better in contaminated sand compared to liquid media. High r² values, ranging from 0.92 to 0.99, were obtained excluding 1000 mg/kg phenanthrene. The kinetic parameters, i.e., qmax and Ks, increased with the phenanthrene concentration and thus suggest that bacteria degrade at a higher degradation rate. This model successfully described the biodegradation profiles observed at different initial phenanthrene concentrations. The established model can be used to simulate the duration of phenanthrene degradation using only the value of the initial PAHs concentration.     

Use of hopane as a conservative biomarker for monitoring the bioremediation effectiveness of crude oil contaminating a sandy beach

Much of the variability inherent in crude oil bioremediation field studies can be eliminated by normalizing analyte concentrations to the concentration of a nonbiodegradable biomarker such as hopane. This was demonstrated with data from a field study in which crude oil was intentionally released onto experimental plots on the Delaware shoreline. Five independent replicates of three treatments were examined: no nutrient addition, addition of inorganic mineral nutrients alone, and nutrient addition plus indigenous oil-degrading microorganisms from the site. Samples collected biweekly were analyzed for the Most Probable Numbers (MPNs) of alkane and aromatic degraders and oil component analysis by GC/MS. The data were normalized to either the mass of sand that was extracted or to the concentration of hopane that was measured. Hopane normalization enabled detection of significant treatment differences in hydrocarbon biodegradation that were not detected when the data were normalized to sand mass. First-order loss rates for the hopane-normalized data were lower than those for the sand-normalized data because hopane normalization accounts only for loss due to biodegradation whereas sand normalization includes all loss mechanisms. Plots amended with nutrients alone and nutrients plus the inoculum showed enhanced removal of hydrocarbons compared to unamended control plots. However, no differences were detected between the nutrient-amended plots and the nutrient/inoculum-amended plots. Received 06 November 1995/ Accepted in revised form 26 June 1996     

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.     

An Investigation of the Ability of a 14C-Labeled Hydrocarbon Mineralization Test to Predict Bioremediation of Soils Contaminated with Petroleum Hydrocarbons

Bioremediation is a widely accepted technology for the remediation of hydrocarbon-contaminated soil. Treatability studies are usually carried out to assess the biodegradation potential of the contaminants and to design optimal treatments. Laboratory studies measuring soil respiration are often used. One method consists of monitoring the mineralization of a ¹⁴C-labeled hydrocarbon surrogate added to the contaminated soil. This study investigates the ability of this method to properly predict the removal of the hydrocarbon contaminants initially found in soils. Mineralization of ¹⁴C-labeled hexadecane was monitored in seven soils contaminated with various hydrocarbon mixtures, both fresh and weathered, in microcosm experiments. Reduction of total petroleum hydrocarbon (TPH) concentrations was measured simultaneously in separate microcosms. Both types of microcosms were subjected to the same amendment regimes. For all soils, poor correlation was observed between the mineralization and TPH reduction data sets. Mineralization data supported contaminants removal data in only one soil. Findings indicate that the radioactive surrogate method does not reliably predict the extent of, and the effect of amendments on, the removal of the hydrocarbons initially present in soil, and may therefore predict suboptimal treatment regimes. Recommendations for soil treatability protocols are provided.     

Site-Specific Limitations of Using Urea as a Nitrogen Source in Biodegradation of Petroleum Wastes in Soil

Nutrient addition is important to achieving the carbon/nitrogen balance and successful biodegradation of petroleum contaminants. Urea has been considered as a preferred nitrogen source in enhancing biodegradation, because of its high nitrogen content and commercial availability. This study investigated urea in the biodegradation of petroleum-contaminated soils collected from an arid and sandy area in Egypt. Ammonium nitrate served as the nitrogen amendment control in this study. Biodegradation of petroleum-contaminated soils from Wyoming was monitored as a comparison. Addition of urea failed to improve the enhancement of biodegradation of petroleum-impacted soil from the Egyptian site; in addition, urea demonstrates an adverse effect on the biodegradation rates. Results indicate that urea or its intermediates may inhibit the microorganisms involved in petroleum degradation. Data from this study suggest that the application of urea in the enhancement of biodegradation of petroleum compounds should consider site specificity, and may not be applicable in geological areas or soils structures similar to those in this study.     

Biodegradation of Chlorpyrifos by Enterobacter Strain B-14 and Its Use in Bioremediation of Contaminated Soils

Six chlorpyrifos-degrading bacteria were isolated from an Australian soil and compared by biochemical and molecular methods. The isolates were indistinguishable, and one (strain B-14) was selected for further analysis. This strain showed greatest similarity to members of the order Enterobacteriales and was closest to members of the Enterobacter asburiae group. The ability of the strain to mineralize chlorpyrifos was investigated under different culture conditions, and the strain utilized chlorpyrifos as the sole source of carbon and phosphorus. Studies with ring or uniformly labeled [¹⁴C]chlorpyrifos in liquid culture demonstrated that the isolate hydrolyzed chlorpyrifos to diethylthiophospshate (DETP) and 3, 5, 6-trichloro-2-pyridinol, and utilized DETP for growth and energy. The isolate was found to possess mono- and diphosphatase activities along with a phosphotriesterase activity. Addition of other sources of carbon (glucose and succinate) resulted in slowing down of the initial rate of degradation of chlorpyrifos. The isolate degraded the DETP-containing organophosphates parathion, diazinon, coumaphos, and isazofos when provided as the sole source of carbon and phosphorus, but not fenamiphos, fonofos, ethoprop, and cadusafos, which have different side chains. Studies of the molecular basis of degradation suggested that the degrading ability could be polygenic and chromosome based. Further studies revealed that the strain possessed a novel phosphotriesterase enzyme system, as the gene coding for this enzyme had a different sequence from the widely studied organophosphate-degrading gene (opd). The addition of strain B-14 (10⁶ cells g⁻¹) to soil with a low indigenous population of chlorpyrifos-degrading bacteria treated with 35 mg of chlorpyrifos kg⁻¹ resulted in a higher degradation rate than was observed in noninoculated soils. These results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment.     

Biodegradation of Pyrene in Marine Beach Sediments

The potential of chitosan (0.1% dry weight equivalent) as a bioremediation additive for removal of the recalcitrant polycyclic aromatic hydrocarbon (PAH) pyrene in marine beach sediments was investigated using an open irrigation system over a 63-day period. Osmocote, a slow release fertilizer, was used as the key nutrient supplement at a concentration of 1% in sediment (dry weight equivalent). Osmocote significantly (p < .05) enhanced nutrient levels, and the metabolic activity of the indigenous microbial biomass. Both additives were comparable in stimulating pyrene biodegradation rates; with chitosan (0.062 day^?1) being slightly more effective as an amendment than Osmocote (0.051 day^?1). Loss of pyrene in a control sediment (i.e., pyrene, without additives) was 66.6% over a 63-day period. The concurrent application of additives yielded the greatest biodegradation rates (0.072day^?1), resulting in a 98.2% loss of pyrene over 63 days. The treatment of oil contaminated beach sediments with both osmocote (1%) and chitosan (0.1%) is therefore recommended as an effective treatment for the intrinsic biodegradation of recalcitrant PAHs in oil-contaminated beach sediments.     

低温微生物修复石油烃类污染土壤研究进展

耐冷菌、嗜冷菌等低温微生物广泛存在于极地、高山以及高纬度等土壤环境中,是石油烃类污染物在低温条件下降解与转化的重要微生物资源.利用低温微生物的独特优势,石油污染土壤的低温生物修复技术的研究成为当前热点领域.本文系统综述了低温石油烃降解菌的分类及冷适机制,低温微生物对不同类型石油烃组分的降解特征和降解机理,低温环境中接种降解菌、添加营养物质和表面活性剂等强化技术在石油污染土壤中生物修复的应用.以及微生物分子生物学技术在低温微生物降解石油烃的研究现状,为拓展我国石油污染土壤生物修复技术提供参考.