Petroleum-degrading microbial numbers in rhizosphere and non-rhizosphere crude oil-contaminated soil

Phytoremediation can be a cost-effective and environmentally acceptable method to clean up crude oil-contaminated soils in situ. Our research objective was to determine the effects of nitrogen (N) additions and plant growth on the number of total hydrocarbon (TH)-, alkane-, and polycyclic aromatic hydrocarbon (PAH)-degrading microorganisms in weathered crude oil-contaminated soil. A warm-season grass, sudangrass (Sorghum sudanense (Piper) Stapf), was grown for 7 wk in soil with a total petroleum hydrocarbon (TPH) level of 16.6 g TPH/kg soil. Nitrogen was added based upon TPH-C:added total N (TPH-C:TN) ratios ranging from 44:1 to 11:1. Unvegetated and unamended controls were also evaluated. The TH-, alkane-, and PAH-degrading microbial numbers per gram of dry soil were enumerated from rhizosphere and non-rhizosphere soil for vegetated pots and non-rhizosphere soil populations were enumerated from non-vegetated pots. Total petroleum-degrading microbial numbers were also calculated for each pot. The TH-, alkane-, and PAH-degrading microbial numbers per gram of dry soil in the sudangrass rhizosphere were 3.4, 2.6, and 4.8 times larger, respectively, than those in non-rhizosphere soil across all N rates. The presence of sudangrass resulted in significantly more TH-degrading microorganisms per pot when grown in soil with a TPH-C:TN ratio of 11:1 as compared to the control. Increased plant root growth in a crude oil-contaminated soil and a concomitant increase in petroleum-degrading microbial numbers in the rhizosphere have the potential to enhance phytoremediation.     

Effects of oil spills on microbial heterotrophs in Antarctic soils

Oil spillage on the moist coastal soils of the Ross Sea region of Antarctica can impact on populations of microbial heterotrophs in these soils, as determined by viable plate counts and a most probable number technique. Elevated numbers of culturable hydrocarbon degraders, bacteria and fungi were detected in surface and subsurface soils from oil-contaminated sites, compared with nearby control sites. Culturable yeasts were not detected in soil from coastal control sites, yet reached >10⁵ organisms g^-1 dry weight in contaminated soils. The presence of hydrocarbons in soils resulted in a shift in the genera of culturable filamentous fungi. Chrysosporium dominated control soils, yet Phialophora was more abundant in oil-contaminated soils. Hydrocarbon degraders are most likely bacteria; however, fungi could play a role in degradation of hydrocarbons or their metabolites. Depleted levels of nitrate detected in some contaminated soils and decreased pH may be the result of growth of hydrocarbon degraders. Numbers and diversity of culturable microbes from Antarctic soil varied depending on whether a pristine site or a human-impacted (in this case, by fuel spills) site is studied.     

Microbial Degradation of Organic Pollutants in Soil in a Cold Climate

Cold-adapted microorganisms are potentially interesting for use in environmental biotechnology applications since a large part of the biosphere has low temperatures during at least parts of the year. Many studies have shown that both oil-contaminated and uncontaminated soils in the Arctic, the Antarctic and the Alps contain microbes that can degrade different hydrocarbons deriving from oils. A few studies have also been conducted on degradation of herbicides in soils at low temperatures. Furthermore, phenols and some polychlorinated biphenyl (PCB) congeners have proved to be degradable at low temperatures, using microorganisms isolated from sediments or soils. Additions of nitrogen and phosphorous to polluted soils have been shown to enhance the degradation of hydrocarbons in many cases. Bioaugmentation with hydrocarbon degrading cold-adapted microorganisms has given varying results. The inoculated microorganisms have probably been out-competed by the indigenous microorganisms in some cases. Different ways to increase the efficiency of microbial degradation of organic pollutants in soil in a cold climate is discussed.     

Screening of plants for phytoremediation of oil-contaminated soil

Several species of ornamental flowering plants were evaluated regarding their phytoremediation ability for the cleanup of oil-contaminated soil in Japanese environmental conditions. Thirty-three species of plants were grown in oil-contaminated soil, and Mimosa, Zinnia, Gazania, and cypress vine were selected for further assessment on the basis of their favorable initial growth. No significant difference was observed in the above-ground and under-ground dry matter weight of Gazania 180 days after sowing between contaminated and non-contaminated plots. However, the other 3 species of plants died by the 180^th day, indicating that Gazania has an especially strong tolerance for oil-contaminated soil. The total petroleum hydrocarbon concentration of the soils in which the 4 species of plants were grown decreased by 45–49% by the 180^th day. Compared to an irrigated plot, the dehydrogenase activity of the contaminated soil also increased significantly, indicating a phytoremediation effect by the 4 tested plants. Mimosa, Zinnia, and cypress vine all died by the 180^th day after seeding, but the roots themselves became a source of nutrients for the soil microorganisms, which led to a phytoremediation effect by increase in the oil degradation activity. It has been indicated that Gazania is most appropriate for phytoremediation of oil-contaminated soil.     

Synergistic plant-microbes interactions in the rhizosphere: a potential headway for the remediation of hydrocarbon polluted soils

Soil pollution is an unavoidable evil; many crude-oil exploring communities have been identified to be the most ecologically impacted regions around the world due to hydrocarbon pollution and their concurrent health risks. Several clean-up technologies have been reported on the removal of hydrocarbons in polluted soils but most of them are either very expensive, require the integration of advanced mechanization and/or cannot be implemented in small scale. However, “Bioremediation” has been reported as an efficient, cost-effective and environment-friendly technology for clean-up of hydrocarbon”s contaminated soils. Here, we suggest the implementation of synergistic mechanism of bioremediation such as the use of rhizosphere mechanism which involves the actions of plant and microorganisms, which involves the exploitation of plant and microorganisms for effective and speedy remediation of hydrocarbon”s contaminated soils. In this mechanism, plant”s action is synergized with the soil microorganisms through the root rhizosphere to promote soil remediation. The microorganisms benefit from the root metabolites (exudates) and the plant in turn benefits from the microbial recycling/solubilizing of mineral nutrients. Harnessing the abilities of plants and microorganisms is a potential headway for cost-effective clean-up of hydrocarbon”s polluted sites; such technology could be very important in countries with great oil producing activities/records over many years but still developing.     

Functional gene diversity of soil microbial communities from five oil-contaminated fields in China

To compare microbial functional diversity in different oil-contaminated fields and to know the effects of oil contaminant and environmental factors, soil samples were taken from typical oil-contaminated fields located in five geographic regions of China. GeoChip, a high-throughput functional gene array, was used to evaluate the microbial functional genes involved in contaminant degradation and in other major biogeochemical/metabolic processes. Our results indicated that the overall microbial community structures were distinct in each oil-contaminated field, and samples were clustered by geographic locations. The organic contaminant degradation genes were most abundant in all samples and presented a similar pattern under oil contaminant stress among the five fields. In addition, alkane and aromatic hydrocarbon degradation genes such as monooxygenase and dioxygenase were detected in high abundance in the oil-contaminated fields. Canonical correspondence analysis indicated that the microbial functional patterns were highly correlated to the local environmental variables, such as oil contaminant concentration, nitrogen and phosphorus contents, salt and pH. Finally, a total of 59% of microbial community variation from GeoChip data can be explained by oil contamination, geographic location and soil geochemical parameters. This study provided insights into the in situ microbial functional structures in oil-contaminated fields and discerned the linkages between microbial communities and environmental variables, which is important to the application of bioremediation in oil-contaminated sites.     

A halotolerant Alcanivorax sp. strain with potential application in saline soil remediation

Biodegradation of petroleum compounds in saline environments seems intricate and needs more attention. In this study, tetracosane was used to enrich alkane-degrading bacteria from oil-contaminated saline soils. Among the isolates, strain Qtet3, with the highest 16s rRNA gene sequence similarity to Alcanivorax dieselolei B-5^T, was able to grow at a wide range of NaCl concentrations and was shown by GC analysis to degrade more than 90% of tetracosane in 10 days. This strain has at least two alkB genes and could grow on crude oil and diesel fuel, and utilize various pure aliphatic hydrocarbon substrates (from C₁₂ to C₃₄). Highly hydrophobic cell surfaces and lack of significant surface tension reduction in the media suggest that the main mechanism of the cells for accessing substrate is to attach directly to hydrocarbon particles. Application of this strain for remediating crude oil-contaminated soils irrigated with defined saline water demonstrated that this halotolerant bacterium could survive and grow in saline soils irrigated with NaCl solutions up to 5% w/v, with the highest hydrocarbon degradation of 26.1% observed at 2.5% NaCl. This strain is promising for future industrial applications especially in bioremediation of saline soils and wastes.     

Microbial population dynamics associated with crude-oil biodegradation in diverse soils

Soil bacterial population dynamics were examined in several crude-oil-contaminated soils to identify those organisms associated with alkane degradation and to assess patterns in microbial response across disparate soils. Seven soil types obtained from six geographically distinct areas of the United States (Arizona, Oregon, Indiana, Virginia, Oklahoma, and Montana) were used in controlled contamination experiments containing 2% (wt/wt) crude oil spiked with [1-(14)C]hexadecane. Microbial populations present during hydrocarbon degradation were analyzed using both 16S rRNA gene sequence analysis and by traditional methods for cultivating hydrocarbon-oxidizing bacteria. After a 50-day incubation, all seven soils showed comparable hydrocarbon depletion, where >80% of added crude oil was depleted and approximately 40 to 70% of added [(14)C]hexadecane was converted to (14)CO(2). However, the initial rates of hydrocarbon depletion differed up to 10-fold, and preferential utilization of shorter-chain-length n-alkanes relative to longer-chain-length n-alkanes was observed in some soils. Distinct microbial populations developed, concomitant with crude-oil depletion. Phylogenetically diverse bacterial populations were selected across different soils, many of which were identical to hydrocarbon-degrading isolates obtained from the same systems (e.g., Nocardioides albus, Collimonas sp., and Rhodococcus coprophilus). In several cases, soil type was shown to be an important determinant, defining specific microorganisms responding to hydrocarbon contamination. However, similar Rhodococcus erythropolis-like populations were observed in four of the seven soils and were the most common hydrocarbon-degrading organisms identified via cultivation.     

Metagenomics of petroleum muck: revealing microbial diversity and depicting microbial syntrophy

Present study attempts in revealing taxonomic and functional diversity of microorganism from petroleum muck using metagenomics approach. Using Ion Torrent Personal Genome Machine, total of 249 Mb raw data were obtained which was analysed using MG-RAST platform. The taxonomic analysis revealed predominance of Proteobacteria with Gammaproteobacteria as major class and Pseudomonas stutzeri as most abundant organism. Several enzymes involved in aliphatic and aromatic hydrocarbon degradation through both aerobic and anaerobic routes and proteins related to stress response were also present. Comparison of our metagenome with the existing metagenomes from oil-contaminated sites and wastewater treatment plant indicated uniqueness of this metagenome taxonomically and functionally. Based on these results a hypothetical community model showing survival and syntrophy of microorganisms in hydrocarbon-rich environment is proposed. Validation of the metagenome data was done in three tiers by validating major OTUs by isolating oil-degrading microbes, confirmation of key genes responsible for hydrocarbon degradation by Sanger sequencing and studying functional dynamics for degradation of the hydrocarbons by the muck meta-community using GC–MS.     

Preface

Biological properties of soil are not only essential for the maintenance of soil fertility and the sustainability of the plant-soil ecosystems, but also indicators of land reclamation of contaminated or disturbed soils. This experiment involves two plants (barley and field pea) growing in four soils with different hydrocarbon contents. The objective was to study the effect of hydrocarbons on plant growth and microbial activity, and to evaluate the acid phosphatase activity as an indicator of reclamation of hydrocarbon-contaminated soils. Barley root mass decreased with the increase of the hydrocarbon content but field pea roots were not sensitive to the hydrocarbon content in this experiment. The hydrocarbon contamination reduced the plant growth but increased the microbial activity. The acid phosphatase activity was controlled by both plant root production and microbial activity, therefore it was not a good indicator of the reclamation of oil-contaminated soils.     

Potential for biodegradation of hydrocarbons in soil from the Ross Dependency, Antarctica

Oil spills occur in the Antarctic when fuel oils such as JP8 jet fuel are moved or stored. Hydrocarbons, both n-alkanes and aromatic compounds, have been detected in oil-contaminated soils of the Ross Dependency. In such areas hydrocarbon-degrading microbes, if naturally occurring, could be used for clean-up. Soil samples from oil-impacted and control sites were analysed for hydrocarbon-degrading microbes and for a range of parameters known to limit biodegradative activity. Soils were analysed for water content, pH, concentrations of nutrients (N and P) and electrical conductivity. Numbers of culturable heterotrophic bacteria and hydrocarbon degraders were greater in some of the oil-contaminated samples. Mineralisation studies with ¹⁴C-radiolabelled hexadecane and naphthalene demonstrated that nitrogen amendments significantly enhanced hydrocarbon mineralisation rates in an oil-impacted soil. Received: 30 June 1997 / Received revision: 15 September 1997 / Accepted: 19 September 1997     

Assimilation of liquid hydrocarbon by microorganisms. I. Mechanism of hydrocarbon uptake.

The uptake mechanism of liquid hydrocarbons of low solubility in water was investigated, using microorganisms with different affinities for liquid hydrocarbon. Microorganisms which could utilize hydrocarbon were much more adherent to hydrocarbon than those which could not. The adhesive force between Candida intermedia IFO 0761 and hydrocarbon was higher than that of Candida tropicalis ATCC 20336, though both could utilize hydrocarbon; The total hydrocarbon uptake from the drop and accommodation forms of hydrocarbons was much higher than that from dissolved hydrocarbon. The uptake rate of drop-form hydrocarbon was nearly equal to that of accommodation-form hydrocarbon for C. intermedia, but was lower for C. tropicalis which shows lower adhesion to hydrocarbon.     

Effect of Butyric Acid on the Physiological Activity of Hydrocarbon-Oxidizing Rhodococci

Laboratory experiments showed that butyric acid not only fails to meet the trophic requirements of hydrocarbon-oxidizing microorganisms, but even specifically inhibits their assimilatory and dissimilatory activity. Therefore, butyric acid can be referred to as growth inhibitors. The combined mineralization of carbohydrates and hydrocarbons can be described as follows. Plants polymers are converted to monosugars by heterotrophic soil microorganisms. As the concentration of the monosugars grows and oxygen becomes deficient, the monosugars are no longer oxidized completely but are fermented. As a result, glucose transforms to butyric acid, which inhibits hydrocarbon-oxidizing bacteria. It is concluded that, to be efficient, the cleanup of oil-contaminated soils must include measures to intensify the mineralization of carbohydrates and to inhibit their fermentation.     

石油污染土壤自然衰减过程多环芳烃降解基因动态特征

新疆石油污染土壤中微生物多环芳烃(polycyclic aromatic hydrocarbons,PAHs)降解功能基因研究甚少,且环境因子和功能基因之间相关性仍不清楚。【目的】揭示新疆石油污染砂质土壤自然衰减过程中多环芳烃降解关键基因结构和变化规律。【方法】以新疆准东油田为研究区,分析同一采油区不同石油污染年限土壤理化因子和多环芳烃含量变化,采用扩增子测序研究石油自然衰减过程中多环芳烃降解酶基因结构变化规律,利用Mental检验探讨其环境驱动因子。【结果】石油污染时间1年和3年的土壤中有多项理化指标与背景土存在显著性差异,而污染5年土壤与背景土之间仅2项指标具有显著性差异,随石油自然衰减逐渐恢复至正常。石油污染1年的土壤中16种多环芳烃除苊烯和?以外,其余14种多环芳烃均高于石油污染3年和5年土壤,多环芳烃总量和含油率污染1年土壤均显著高于污染3年和5年的土壤,多环芳烃会在污染后短时间内迅速被降解。扩增子测序结果显示,萘双加氧酶基因分类操作单元(operational taxonomic units,OTUs)序列随污染年限延长逐渐增多;芳环羟化双加氧酶基因OTUs序列BLAST(...     

The Use of Conocarpus lancifolius Trees for the Remediation of Oil-Contaminated Soils

The role of the Conocarpus lancifolius tree in remediaitng oil-contaminated soil, which was bioremediated using conventional methods, was investigated. The selected tree was used to phytoremediate bioremediated oil-contaminated soil for three successive growing seasons. At the end of the phytoremediation experiment, 85.7% of measurable total petroleum hydrocarbon (TPH) was degraded in Conocarpus lancifolius rhizosphere, and the detectable concentrations of some poly aromatic hydrocarbons (PAHs) were less than 0.02 ppm. A number of hydrocarbon degrading microorganisms (HDMs) were isolated at 35°C under aerobic conditions, and were identified using 16S rRNA gene sequencing and fatty acid methyl ester (FAME) analysis. The efficiency of the isolated HDMs in degrading a mixture of hydrocarbon compounds (HC) was assessed. Among the bacterial isolates, Rhodococcus equi was distinguished from the other isolates because of its efficient degradation of some compounds in the HC mixture.

Samples were also collected from Conocarpus lancifolius vegetative parts and were analyzed for heavy metal and mineral accumulation. The results demonstrated that the Conocarpus lancifolius tree was able to uptake high levels of chromium (Cr), vanadium (V), and nickel (Ni) and accumulate them in the tree's roots. Additionally, Conocarpus trees tolerated high concentration and accumulated several metals in all plant tissues. These metals included aluminum (Al), calcium (Ca) and iron (Fe).     

Activation of indigenous micorflora in oil-contaminated soils using photoluminescent films

A stimulating effect of sunlight transformed by a photoluminescent polymer film on the abundance dynamics and fermentation and respiration of indigenous microflora in oil-contaminated soils was investigated. Polymer film doped with photoluminophores based on inorganic Eu-complexes and common glasshouse film was used as a cover material for oil-contaminated soils at experimental and control sites. The application of photoluminescent film has been reported to stimulate a hundredfold growth of the microflora population, with the soil respiration intensity and catalase activity being increased by a factor of 2.5–3, respectively. The extents of biodegradation of petroleum hydrocarbons within 60 days were up to 70 and 30% of the overall background pollution level for the experimental and control site, respectively. Residual hydrocarbons extracted from samples of the contaminated soils were analyzed by infrared spectroscopy to show the appearance of additional absorption bands at 3350, 1600, and 1710 cm^?1, thus indicating the formation of metabolites during enzymatic oxidation of oil. Chromatographic data corroborated the occurrence of intense oxidation. The hydrocarbon biodegradation factor increases sixfold when the photoluminescent films are used.     

Microbial Population Dynamics Associated with Crude-Oil Biodegradation in Diverse Soils

Soil bacterial population dynamics were examined in several crude-oil-contaminated soils to identify those organisms associated with alkane degradation and to assess patterns in microbial response across disparate soils. Seven soil types obtained from six geographically distinct areas of the United States (Arizona, Oregon, Indiana, Virginia, Oklahoma, and Montana) were used in controlled contamination experiments containing 2% (wt/wt) crude oil spiked with [1-¹⁴C]hexadecane. Microbial populations present during hydrocarbon degradation were analyzed using both 16S rRNA gene sequence analysis and by traditional methods for cultivating hydrocarbon-oxidizing bacteria. After a 50-day incubation, all seven soils showed comparable hydrocarbon depletion, where >80% of added crude oil was depleted and approximately 40 to 70% of added [¹⁴C]hexadecane was converted to ¹⁴CO₂. However, the initial rates of hydrocarbon depletion differed up to 10-fold, and preferential utilization of shorter-chain-length n-alkanes relative to longer-chain-length n-alkanes was observed in some soils. Distinct microbial populations developed, concomitant with crude-oil depletion. Phylogenetically diverse bacterial populations were selected across different soils, many of which were identical to hydrocarbon-degrading isolates obtained from the same systems (e.g., Nocardioides albus, Collimonas sp., and Rhodococcus coprophilus). In several cases, soil type was shown to be an important determinant, defining specific microorganisms responding to hydrocarbon contamination. However, similar Rhodococcus erythropolis-like populations were observed in four of the seven soils and were the most common hydrocarbon-degrading organisms identified via cultivation.     

Relationships between root growth of Zinnia hybrid “profusion orange” flowers and phytoremediation of oil-contaminated soil

Relationships exist between plant root growth and the phytoremediation of oil-contaminated soils. In a previous study, we demonstrated that zinnia flowers are well suited for the remediation of oil-contaminated soil. In this study, our goal was to quantify the relationship between zinnia root growth and purification of oil-contaminated soils. Three treatments were used: (1) cultivation of zinnia in oil-contaminated soil (contaminated pots), (2) cultivation in non-contaminated soil (non-contaminated pots), and (3) contaminated soil with no cultivation and only irrigation (irrigated pots). Growth of the Zinnia plants, including their roots, was significantly reduced in the contaminated pots compared with the noncontaminated pots. The soil dehydrogenase activity increased between 45 and 90?days after planting in all parts of the contaminated pots, especially the upper parts. The soil total petroleum hydrocarbon (TPH) concentrations in the contaminated pots decreased throughout the study period. Interestingly, the soil dehydrogenase activity increased, and the soil TPH concentration decreased even in lower parts of the pots where there was very little root growth. Therefore, the cultivation of plants can have a remediative effect on oil-contaminated soil even below the depth reached by the plant roots.     

Biodegradation of crude oil by soil microorganisms in the tropic

Five microorganisms, three bacteria and two yeasts, capable of degrading Tapis light crude oil were isolated from oil-contaminated soil in Bangkok, Thailand. Soil enrichment culture was done by inoculating the soil in mineral salt medium with 0.5% v/v Tapis crude oil as the sole carbon source. Crude oil biodegradation was measured by gas chromatography method. Five strains of pure microorganisms with petroleum degrading ability were isolated: three were bacteria and the other two were yeasts. Candida tropicalis strains 7Y and 15Y were identified as efficient oil degraders. Strain 15Y was more efficient, it was able to reduce 87.3% of the total petroleum or 99.6% of n-alkanes within the 7-day incubation period at room temperature of 25 ± 2 °C.     

Influence of petroleum contamination and biostimulation treatment on the diversity of Pseudomonas spp. in soil microcosms as evaluated by 16S rRNA based-PCR and DGGE

AIMS: The aim of this study was to apply a group specific PCR system followed by denaturing gradient gel electrophoresis (DGGE) analysis to evaluate the effect of oil contamination and the biostimulation process on the diversity of Pseudomonas populations in soil ecosystems. METHODS AND RESULTS: Direct DNA extraction from biostimulated- and oil-contaminated soil samples was performed. Primers specific for the genus Pseudomonas spp. were used to amplify 16S rRNA genes and then a semi-nested PCR reaction was applied to obtain smaller fragments for comparing the PCR products by DGGE. Whether in bulk, oil-contaminated or biostimulated soils, the DGGE profiles revealed little change in Pseudomonas community throughout the 270 days of experiment. The presence of a few additional bands observed only in treated samples indicated that a bacterial shift occurred with the addition of nutrients and with oil contamination. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of semi-nested PCR and DGGE was found to be a rapid and sensitive technique to study the diversity within the genus Pseudomonas and may be suitable for further studies concerning the role of this bacterial group in large-scale oil-contaminated areas.