Efficiencies of introduction of an oil-oxidizing <Emphasis Type="Italic">Dietzia maris</Emphasis> strain and stimulation of natural microbial communities in remediation of polluted soil

Two approaches to bioremediation of oil-polluted soils are compared: use of active degrader strain Dietzia maris AM3 and stimulation of natural microflora. Introduction of D. maris AM3 to soil freshly polluted with oil accelerated its remediation twofold within the first month in comparison with the stimulation. After three months, the purification degrees were approximately equal. By the end of bioremediation, the soil with the introduced strain had higher dehydrogenase and catalase activities. In soil with aged pollution, introduced strain D. maris AM3 did not affect the rate of oil product degradation, and no significant differences between the two bioremediation methods were detected in purification degree and biological activity of soil after three months.     

Effect of the media salinity on destruction of petroleum oils by nocardioform bacteria]

Oil degradation by cultures of Rhodococcus erythropolis and Dietzia maris was found to depend on the NaCl concentration in the medium. Optimal utilization of turbine oil by R. erythropolis and D. maris was observed at 0.5 and 2 to 5% NaCl concentration, respectively. Mineral oil and a mixture of paraffins (C14-C18) were utilized within a broader range of the medium salinity. As shown by fluorescent microscopy, D. maris colonies formed on the oil drop surface, whereas R. erythropolis cells penetrated the drops. The strains studied may populate various ecological niches in oil-containing ecosystems. They are promising for the development of microbial preparations for cleaning the environment from oil pollution.     

Bioremediation of Crude Oil-Contaminated Soil Using Slurry-Phase Biological Treatment and Land Farming Techniques

Field-scale experiments on bioremediation of soil heavily contaminated with crude oil were undertaken on the territory of the Kokuyskoye oil field (Perm region, West Urals, Russia) owned by the LUKOIL Company. The pollution consisted of the contents of a oil waste storage pit, which mostly received soils contaminated after accidental oil spills and also the solid n-alkane (paraffin) wastes removed from the surface of drilling equipment. Laboratory analyses of soil samples indicated contamination levels up to 200?g/kg of total recoverable petroleum hydrocarbons (TRPH). Average oil composition consisted of 64% aliphatics, 25% aromatics, 8% heterocyclics, and 3% of tars/asphaltenes. Ex situ bioremediation techniques involved the successive treatment of contaminated soil using a bioslurry reactor and land farming cells. An oleophilic biofertilizer based on Rhodococcus surfactant complexes was used in both treatment systems. An aerobic slurry bioreactor was designed, and the biofertilizer applied weekly. Slurry-phase biotreatment of the contaminated soil resulted in an 88% reduction in oil concentration after 2 months. The resulting reactor product, containing approximately 25?g/kg of TRPH, was then loaded into land farming cells for further decontamination. To enhance bioremediation, different treatments (e.g., soil tilling, bulking with woodchips, watering, and biofertilizer addition) were used. The rates of oil biodegradation were 300 to 600?ppm/day. As a result, contamination levels dropped to 1.0 to 1.5?g/kg of TRPH after 5 to 7 weeks. Tertiary soil management involved phytoremediation where land farming cells were seeded with a mixture of three species of perennial grass. The effect of phytoremediation on the residual decontamination and rehabilitation of soil fertility is being evaluated.     

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.     

AM真菌对重金属污染土壤生物修复的应用与机理

土壤重金属污染威胁人类健康和整个生态系统,而高效、低耗、安全的生物修复技术显示出了极大的应用潜力,特别是利用植物-微生物共生体增强生物修复效应的应用.丛枝菌根(Arbuscular Mycorrhizae,AM)真菌是一类广泛分布于土壤生态系统中的有益微生物,能与90％以上的陆生高等植物形成共生体.研究发现,AM真菌能够增强宿主植物对土壤中重金属胁迫的耐受性.当前,利用AM真菌开展重金属污染土壤的生物修复已经引起环境学家和生态学家的广泛关注.基于此,围绕AM真菌在重金属污染土壤生物修复作用中的最新研究进展,从物理性防御体系的形成、对植物生理代谢的调控、生化拮抗物质的产生、基因表达的调控等角度探究AM真菌在重金属污染土壤生物修复中的作用机理,以期为利用AM真菌开展重金属污染的生物修复提供理论依据,并对本领域未来的发展和应用前景进行了展望.     

石油污染土壤的生物刺激和生物强化修复

随着工业化的发展,土壤的石油污染问题日益严重,石油污染物的处理已成为各国亟待解决的环境问题。由于生物修复具有高效、安全、成本低、无二次污染等诸多优点而成为近些年来主要的石油污染处理方法。概述了生物刺激和生物强化处理方法的基本原理,以及近年来国内外相关的研究进展。生物刺激和生物强化是生物修复法中最常用的两类方法,而且生物强化通常比生物刺激的效率高,但两者处理效率的高低均与多方面的因素有关,因此在实际应用中应注意考虑多方面的效益,制定出较合理的处理方案。     

Identification and biodegradation potential of a novel strain of Dietzia cinnamea isolated from a petroleum-contaminated tropical soil

A bacterial strain, named P4, isolated previously from microcosms containing oil-contaminated soil collected from an environmentally protected area of a tropical Atlantic forest (Biological Reserve of Poço das Antas) located in Brazil was identified as Dietzia cinnamea by morphological, biochemical and genotypic tests. Arabian Light and Marlin oils were both degraded when strain P4 was tested for oil degradation ability in microplates. Total Petroleum Hydrocarbons (TPH) analysis, determined by gas chromatography, showed that strain P4 degraded a wide range of n-alkanes, and also pristane and phytane. Furthermore, this strain was also able to grow in mineral liquid media amended with carbazole, quinoline, naphthalene, toluene, gasoline and diesel as the sole carbon sources. The species D. cinnamea has been previously described with only one representative strain isolated from a perianal swab of a patient with a bone marrow transplant. With the results presented here this species is implicated not only as a human pathogen but also as a potential strain for further studies concerning its role for bioremediation of oil contaminated soil.     

Liposomes enhance bioremediation of oil-contaminated soil

Liposomes (composed of soy phosphatides) in the form of small unilamellar vesicles (SUV), when added to soil contaminated by crude oil, accelerate bioremediation. After three weeks incubation at 30 degrees C, using soil experimentally contaminated (with 10,000 ppm crude oil), level of bioremediation increased from 40% without SUV to 75% with SUV (0.1 wt% phospholipids per dry weight soil). Similarly, for accidentally contaminated soil (with approximately 17,000 ppm crude oil), addition of 0.1 wt% SUV to the soil increased the bioremediation level from 55 to 80%. The enhancing effect of liposomes is explained by two interrelated phenomena: a large increase both in total bacteria number and in diversity of bacterial species in the soil. Comparison after four weeks revealed 21 bacterial species in the presence of liposomes (many being oil-degrading bacterial species) and only nine species in the absence of liposomes. Both effects may be related to the physical effects of liposome phospholipids, which modify the crude oil by wetting it, thereby making it more accessible to the microorganisms. In addition, liposome phospholipids serve as phosphate and nitrogen sources for the bacteria.     

Low temperature bioremediation of oil-contaminated soil using biostimulation and bioaugmentation with a Pseudomonas sp. from maritime Antarctica

AIMS: To identify native Antarctic bacteria capable of oil degradation at low temperatures. METHODS AND RESULTS: Oil contaminated and pristine soils from Signy Island (South Orkney Islands, Antarctica) were examined for bacteria capable of oil degradation at low temperatures. Of the 300 isolates cultured, Pseudomonas strain ST41 grew on the widest range of hydrocarbons at 4 degrees C. ST41 was used in microcosm studies of low temperature bioremediation of oil-contaminated soils. Microcosm experiments showed that at 4 degrees C the levels of oil degradation increased, relative to the controls, with (i) the addition of ST41 to the existing soil microbial population (bioaugmentation), (ii) the addition of nutrients (biostimulation) and to the greatest extent with (iii) a combination of both treatments (bioaugmentation and biostimulation). Addition of water to oil contaminated soil (hydration) also enhanced oil degradation, although less than the other treatments. Analysis of the dominant species in the microcosms after 12 weeks, using temporal temperature gradient gel electrophoresis, showed Pseudomonas species to be the dominant soil bacteria in both bioaugmented and biostimulated microcosms. CONCLUSIONS: Addition of water and nutrients may enhance oil degradation through the biostimulation of indigenous oil-degrading microbial populations within the soil. However, bioaugmentation with Antarctic bacteria capable of efficient low temperature hydrocarbon degradation may enhance the rate of bioremediation if applied soon after the spill. SIGNIFICANCE AND IMPACT OF THE STUDY: In the future, native soil bacteria could be of use in bioremediation technologies in Antarctica.     

Degradation of 3-phenoxybenzoic acid in soil by Pseudomonas pseudoalcaligenes POB310(pPOB) and two modified Pseudomonas strains.

Pseudomonas pseudoalcaligenes POB310(pPOB) and Pseudomonas sp. strains B13-D5(pD30.9) and B13-ST1(pPOB) were introduced into soil microcosms containing 3-phenoxybenzoic acid (3-POB) in order to evaluate and compare bacterial survival, degradation of 3-POB, and transfer of plasmids to a recipient bacterium. Strain POB310 was isolated for its ability to use 3-POB as a growth substrate; degradation is initiated by POB-dioxygenase, an enzyme encoded on pPOB. Strain B13-D5 contains pD30.9, a cloning vector harboring the genes encoding POB-dioxygenase; strain B13-ST1 contains pPOB. Degradation of 3-POB in soil by strain POB310 was incomplete, and bacterial densities decreased even under the most favorable conditions (100 ppm of 3-POB, supplementation with P and N, and soil water-holding capacity of 90%). Strains B13-D5 and B13-ST1 degraded 3-POB (10 to 100 ppm) to concentrations of <50 ppb with concomitant increases in density from 10(6) to 10(8) CFU/g (dry weight) of soil. Thus, in contrast to strain POB310, the modified strains had the following two features that are important for in situ bioremediation: survival in soil and growth concurrent with removal of an environmental contaminant. Strains B13-D5 and B13-ST1 also completely degraded 3-POB when the inoculum was only 30 CFU/g (dry weight) of soil. This suggests that in situ bioremediation may be effected, in some cases, with low densities of introduced bacteria. In pure culture, transfer of pPOB from strains POB310 and B13-ST1 to Pseudomonas sp. strain B13 occurred at frequencies of 5 x 10(-7) and 10(-1) transconjugant per donor, respectively. Transfer of pPOB from strain B13-ST1 to strain B13 was observed in autoclaved soil but not in nonautoclaved soil; formation of transconjugant bacteria was more rapid in soil containing clay and organic matter than in sandy soil. Transfer of pPOB from strain POB310 to strain B13 in soil was never observed.     

Bioremediation of oil-polluted soil with an association including the fungus Pleurotus ostreatus and soil microflora

The possibility of application of the Pleurotus ostreatus D1-soil microflora to bioremediation of oil-polluted soils was studied. The fungus degraded mainly the aromatic fraction, whereas soil microflora intensely degraded paraffin and naphthene oil fractions. Introduction of the fungus Pleurotus ostreatus D to soil induces degradation of a wider range of oil hydrocarbons. It is reasonable to further investigate the discovered phenomenon in order to improve procedures of remediation of oil-polluted soils.     

Multi-Species Ecotoxicity Assessment of Petroleum-Contaminated Soil

In 1992, a study was begun to compare the effect of landfarming vs. natural attenuation on the restoration of soil that had been contaminated with crude oil. Each of three lysimeters was filled with a sandy loam topsoil, and crude oil was applied to two of the lysimeters. One of the contaminated lysimeters was tilled, watered, and received a one-time application of fertilizer (N, P, K). No amendments were added to the second contaminated lysimeter, and the third was left uncontaminated. The lysimeters were monitored for 6 months and then left unattended. In 1995 and again in 1997 we sampled these lysimeters to evaluate the long-term effects of contamination and bioremediation. In 1995 we found marked effects on soil chemistry, bacterial, fungal, nematode, and plant populations and a higher rate of bioremediation in the fertilized-contaminated lysimeter (Lawlor et al., 1997). Data from 1997 and previously unreported data from 1995 are the subject of the current report. In 1997, low densities of hydrocarbon-degrading bacteria were found in all the lysimeters and little loss of TPH from the two contaminated lysimeters, suggesting a decreased rate of bioremediation. Nevertheless, there were increases in diversity and number of functional groups of bacteria, nematodes, and native plant species. However, molecular analyses revealed marked differences remained in the composition of dominant eubacterial species, and tests of soybeans indicated field conditions remained unsuitable for these plants.     

Liposomes Enhance Bioremediation of Oil-Contaminated Soil

Abstract

Liposomes (composed of soy phosphatides) in the form of small unilamellar vesicles (SUV), when added to soil contaminated by crude oil, accelerate bioremediation. After three weeks incubation at 30°C, using soil experimentally contaminated (with 10,000 ppm crude oil), level of bioremediation increased from 40% without SUV to 75% with SUV (0.1 wt% phospholipids per dry weight soil). Similarly, for accidentally contaminated soil (with ～17,000 ppm crude oil), addition of 0.1 wt% SUV to the soil increased the bioremediation level from 55 to 80%. The enhancing effect of liposomes is explained by two interrelated phenomena: a large increase both in total bacteria number and in diversity of bacterial species in the soil. Comparison after four weeks revealed 21 bacterial species in the presence of liposomes (many being oil-degrading bacterial species) and only nine species in the absence of liposomes. Both effects may be related to the physical effects of liposome phospholipids, which modify the crude oil by wetting it, thereby making it more accessible to the microorganisms. In addition, liposome phospholipids serve as phosphate and nitrogen sources for the bacteria.     

Bioremediation of soil polluted with fuels by sequential multiple injection of native microorganisms: Field-scale processes in Poland

Research was conducted to estimate impact of the multiple bioaugmentation on the treatment of soil contaminated by fuels - diesel oil and aircraft fuel. The bacteria used to inoculate the remediation plots were isolated from the polluted soil and proliferated in field conditions. The amount of biomass applied to the polluted soil was set to ensure the total number of bacteria in soil 10⁷-10⁸ cfu/g d.w. The multiple inoculation of soil with indigenous bacteria active in diesel oil and engine oil (plot A) degradation increased bioremediation effectiveness by 50% in comparison to the non-inoculated control soil and by 30% in comparison to the soil that was inoculated only once. The multiple inoculation of soil with indigenous microorganisms was then applied in bioremediation of the soil polluted with double high concentration of diesel oil (soil B) and in bioremediation of the soil polluted with aircraft fuel (soil C). The process efficiency was 80% and 98% removal of TPH for soil B and C, respectively.     

The effect of the bioremediation of soil contaminated with petroleum derivatives on the occurrence of epigeic and edaphic fauna

This field study investigated the colonization process of soil contaminated with different petroleum products (petrol, diesel fuel, spent engine oil; dose: 6000 mg of fuel·kg^?1 dry mass [d.m.] of soil) by epigeic and edaphic invertebrates during the progress of natural bioremediation and bioremediation enhanced using selected microorganisms (ZB-01 biopreparation). Epigeic fauna was captured using pitfall traps. Occurrence of edaphic fauna in soil samples as well as total petroleum hydrocarbon contents (TPH) were also investigated. Results showed that inoculation with ZB-01 biocenosis allowed the degradation of petroleum derivatives in the soil contaminated with diesel fuel and engine oil, with 82.3% and 75.4% efficiency, respectively. Applying bioremediation to all contaminated soils accelerated the process of recolonization by edaphic invertebrates. However, the 28-month period was too short to observe full population recovery in soils contaminated with diesel fuel and engine oil. Microbe-enhanced bioremediation accelerated recolonization by epigeic invertebrates on soil contaminated with diesel fuel, whereas it exerted inhibitory effect on recolonization of soil contaminated with engine oil (especially by Collembola). The observed discrepancies in the rates of recolonization for soils contaminated with petrol and diesel fuel that were still noted at the stage of no longer different TPH levels justify the idea to include the survey of edaphic faunal density as one of the parameters in the ecological risk assessment of various bioremediation techniques.     

Bioremediation of Crude Oil–Contaminated Soil By Immobilized Bacteria on an Agroindustrial Waste—Sunflower Seed Husks

This study reports the immobilization and performance of a hydrocarbon-degrading Rhodococcus sp. strain (designated as QBTo) on sunflower seed husks (SH) for the bioremediation of soils polluted with crude oil. The SH performance as inoculants carrier was compared with peat, which is a vegetal material traditionally used in carrier-based inoculants production. The stability of the immobilized culture under storage conditions was assessed by viability at different times when stored at 25°C and 10°C. The catabolic activity of immobilized and free QTBo cells introduced into sandy loam soil, freshly contaminated with crude oil, was studied in microcosms. A higher number of viable QTBo cells were recovered from the inoculants formulated with SH (QTBo-SH) after prolonged storage at 10°C and 25°C. The microcosms amended with QTBo-SH inoculants showed a removal of about 66% of total petroleum hydrocarbons (TPH), whereas in those inoculated with QTBo-peat inoculants, the decrease was of about 47%. In the control microcosms (noninoculated) and liquid culture–amended soils, the TPH removal was about 28%. SH is a waste of edible oil industry, nontoxic, and biodegradable and has demonstrated to confer to the immobilized cultures greater potential to survive not only during storage but also in the soil environment, improving bioremediation process.     

Nitrification and utilization of ammonium and nitrate during oil bioremediation at different soil water potentials

Bioremediation of petroleum spills requires aerobic soil conditions and readily available N, which may be susceptible to leaching. Our objectives were to determine the influence of soil water potential on nitrification in the presence of crude oil, the toxicity of oil to NHj‐oxidizing bacteria, and the preferences of microorganisms for NH⁺ ₄ or NO^? ₃. A Weswood clay loam was amended with crude oil to contain 0, 5, and 10% by soil dry weight, and N was added to achieve C:N ratios of 90:1 and 120:1. Soil water potentials were maintained at ‐0.02, ‐0.1, and ‐1.0 kJ/kg or allowed to fluctuate between ‐0.02 and ‐3 kJ/kg. Concentrations of NH⁺ ₄ and NO₃ ^?were measured during an incubation period of 40 d. Nitrification in soil not amended with oil was rapid at water potentials of ‐0.02 and ‐0.1 kJ/kg but inactive at a water potential of ‐1.0 kJ/kg. Oil reduced nitrification rates and populations of NH⁺ ₄‐oxidizing bacteria. Little NO^? ₃ accumulated when the C:N ratio was 120:1, but when the C:N ratio was 90:1, up to 150 μg of NO₃‐N/g of soil accumulated at a soil water potential of ‐0.02 kJ/kg. Soil water potential in the range used did not greatly influence the extent of oil bioremediation but significantly influenced nitrification. Ammonium was preferentially used over NO^? ₃ by microorganisms during oil bioremediation. Nitrate accumulation from urea applied to stimulate oil bioremediation was low when N application matched requirements for oil bioremediation, and nitrification was restricted by controlling soil water content.     

Enhanced biodegradation of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in the presence of mycolic acid

AIMS: The aim of the present study was to isolate and characterize a bacterium, strain EN3, capable of using diesel oil as a major carbon and energy source, and to analyse the enhancement of diesel oil degradation by this organism using synthetic mycolic acid (2-hexyl-3-hydroxyldecanoic acid). METHOD AND RESULTS: An actinomycete with the ability to degrade diesel oil was isolated from oil contaminated soil and characterized. The strain had phenotypic properties consistent with its classification in the genus Rhodococcus showing a 16S rRNA gene similarity of 99.7% with Rhodococcus baikonurensis DSM 44587(T). The ability of the characterized strain to degrade diesel oil at various concentrations (1000, 5000, 10 000 and 20 000 mg l(-1)) was determined. The effect of synthetic mycolic acid on the biodegradation of diesel oil was investigated at the 20 000 mg l(-1) concentration; the surfactant was added to the flask cultures at three different concentrations (10, 50 and 100 mg l(-1)) and degradation followed over 7 days. Enhanced degradation was found at all three concentrations of the surfactant. In addition, the enhancement of diesel oil degradation by other surfactants was observed. CONCLUSIONS: The synthetic mycolic acid has potential for the remediation of petroleum-contaminated sites from both an economic and applied perspective as it can stimulate biodegradation at low concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that the synthesized mycolic acid can be used for potential applications in the bioremediation industries, for example, in oil spill clean-up, diesel fuel remediation and biostimulation.     

Biosurfactant synthesis by Pseudomonas aeruginosa LBI isolated from a hydrocarbon-contaminated site

Aims: Pseudomonas aeruginosa LBI (Industrial Biotechnology Laboratory) was isolated from hydrocarbon-contaminated soil as a potential producer of biosurfactant and evaluated for hydrocarbon biodegradation. The emulsifying power and stability of the product was assessed in the laboratory, simulating water contamination with benzene, toluene, kerosene, diesel oil and crude oil at various concentrations. Methods and Results: Bacteria were grown at 30°C and shaken at 200 rpm for 168 h, with three repetitions. Surface tension, pH and biosurfactant stability were observed in the cell-free broth after 168 h of incubation. The strain was able to produce biosurfactant and grow in all the carbon sources under study, except benzene and toluene. When cultivated in 30% (w/v) diesel oil, the strain produced the highest quantities (9·9 g l⁻¹) of biosurfactant. The biosurfactant was capable of emulsifying all the hydrocarbons tested. Conclusion: The results from the present study demonstrate that Ps. aeruginosa LBI can grow in diesel oil, kerosene, crude oil and oil sludge and the biosurfactant produced has potential applications in the bioremediation of hydrocarbon-contaminated sites. Significance and Impact of the Study: Pseudomonas aeruginosa LBI or the biosurfactant it produces can be used in the bioremediation of environmental pollution induced by industrial discharge or accidental hydrocarbon spills.     

Comparison of bio-augmentation and composting for remediation of oily sludge: A field-scale study in China

Two bioremediation technologies were performed in order to explore a better treatment process for an oily sludge restoration in China during 2004. The bioremediation by augmentation of biopreparation was compared with a conventional composting. The oily sludge and oil-polluted soil were received from an oil production plant. The total hydrocarbon content (THC) varied from 327.7 to 371.2 g kg⁻¹ of dry sludge and the THC in contaminated soil was 151.0 g kg⁻¹. Before application of preparation, straw, sawdust, top sand and pure soil were added in different proportions to the sludge and soil and mixed thoroughly. Such sludge and soil composites were used for negative controls and for activation of indigenous oil degrading microorganisms with addition of fertilizer (positive controls). For composting, crude manure and straw were added to the oily sludge and the THC was 101.4 g kg⁻¹. The biopreparation was applied every 2 weeks and experiment lasted 56 days under the ambient temperature. The sludge was mixed and watered every 3 days. After three times of biopreparation application, the THC decreased by 46–53% in the oily sludge and soil, while in the positive controls (activation of indigenous microorganisms) the THC decreased by 13–23%, and there was no oil degradation in negative controls After composting, the THC decreased by 31% in the oily sludge. The planting of Tall Fescue (Festuca arundinace) revealed a decrease of sludge toxicity after application of both bioremediation technologies and additionally decreased the THC by 5–7%.