Selection of Microorganisms Capable of Degrading Petroleum and Its Products at Low Temperatures

Of 150 cultures capable of degrading petroleum at +6°C, 40 strains growing in a liquid mineral nutrient medium containing petroleum (2%) as the sole source of carbon were selected. Of them, 13 cultures displaying a petroleum degradation rate exceeding 25% were selected. Abilities of these cultures and their associations to utilize fuel oil and its components—oils and benzene resins—were studied. A culture exhibiting degradation rates of fuel oil, its oils, benzene resins, and petroleum amounting to 17, 26, 10, and 51%, respectively, was selected. This culture can be used for cleanup of petroleum pollution under cold climatic conditions.     

Effect of photosynthetic bacteria and compost on degradation of petroleum products in soil

Addition of diesel fuel and waste engine oil to soil was found to cause biostimulation of hydrocarbon-oxidizing microorganisms. Corynebacteria constitute a large group of hydrocarbon-oxidizing microorganisms. Addition of a liquid culture of photosynthetic bacteria to soil not only facilitates degradation of petroleum products, but also stimulates growth of hydrocarbon-oxidizing microorganisms. Combined addition of photosynthetic bacteria and compost to soil polluted with petroleum products causes even a more significant increase in the count of hydrocarbon-oxidizing bacteria and substantially increases the rate of pollutant degradation.     

Influence of UV irradiation on microbiological degradation of petroleum products

Changes in the rates of microbiological degradation of kerosene, diesel fuel, and fuel oil under the effect of UV irradiation were estimated by testing the respiratory activity of microbial communities. The strongest inhibitory effect was observed upon simultaneous UV irradiation of both natural water and petroleum products. Concentrations of CO₂ in the microbial communities (microcosms) decreased from 6.7 to 3.6 vol. % upon oxidation of kerosene, from 5.9 to 0.8 vol. % upon oxidation of diesel fuel, and from 5.7 to 0.05 vol. % upon oxidation of fuel oil.     

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.     

Low-temperature microbial degradation of crude oil products differing in the extent of condensation

Out of the 30 strains capable of oil degradation at 4-6 degrees C, four were selected by the ability to degrade 40% of the oil substrate present in the growth medium: Rhodococcus spp. DS-07 and DS-21 and Pseudomonas spp. DS-09 and DS-22. We studied the activity of these strains as degraders of oil products of various condensation degrees (crude oil, masut, petroleum oils, benzene resins and ethanol-benzene resins) at 4-6 degrees C. The maximum degrees of degradation of masut and ethanol-benzene resins were observed in Pseudomonas spp. DS-22 (17.2% and 5.2%, respectively). The maximum degradation of petroleum oils and benzene resins was observed in Rhodococcus spp. DS-07 (40% and 16.6%, respectively). The strains provide a basis for developing biodegrader preparations applicable to bioremediation of oil-polluted sites under the conditions of cold climate.     

Microbial degradation of petroleum hydrocarbons in a polluted tropical stream

Summary Crude oil degradation was observed in water samples from three sites along the course of a polluted stream in Lagos, Nigeria. Consistent increase and decrease in the total viable counts (TVCs) of indigenous organisms occurred in the test and control experiments, respectively. Enrichments of the water samples with crude oil resulted in the isolation of nine bacteria belonging to seven genera. A mixed culture was developed from the assemblage of the nine species. The defined microbial consortium utilized a wide range of pure HCs including cycloalkane and aromatic HCs. Utilization of crude oil and petroleum cuts, i.e., kerosene and diesel resulted in an increase in TVC (till day 10) concomitant with decreases in pH and residual oil concentration. Crude oil, diesel and kerosene were degraded by 88, 85 and 78%, respectively, in 14 days. Substrate uptake studies with axenic cultures showed that growth was not sustainable on either cyclohexane or aromatics while degradation of the petroleum fractions fell below 67% in spite of extended incubation period (20 day). From the GC analysis of recovered oil, while reductions in peaks of n-alkane fractions and in biomarkers namely n-C₁₇/pristane and n-C₁₈/phytane ratios were observed in culture fluids of pure strains, complete removal of all the HC components of kerosene, diesel and crude oil including the isoprenoids was obtained with the consortium within 14 days.     

Studies on the effect of inoculation of activated sludge with bacteria actively degrading hydrocarbons on the biodegradation of petroleum products

Eighteen strains of bacteria were isolated from activated sludge purifying petroleum-refining wastewaters. These strains were plated on solidified mineral medium supplemented with oil fraction in concentration 1000 mg/l. Four of the strains that grew best in the presence of oil were selected for further studies. The strains were identified based on Bonde's scheme and microscopic observations. Three of them belonged to the genus Arthrobacter and one to the genus Micrococcus. Stationary cultures of single strains and their mixtures were set up in mineral medium containing oil (sterile and non-sterile) as sole carbon source in concentration 1000 mg/l. The oils were found to be removed the most efficiently by a mixture of the strains. After 14 days of culture the amount of oil was utilized by from 63 to 95%. In the next stage of the studies the bacteria were used to inoculate activated sludge. Stationary cultures of the activated sludge were set up in mineral medium with oil. The utilisation of petroleum products by non-inoculated activated sludge (control), activated sludge inoculated with a single strain or a mixture of all four strains was examined. In both inoculated activated sludge cultures approximately 80% of the oils were removed, compared to 60% in the control activated sludge. Therefore, inoculated activated sludge showed 20% higher effectiveness of removal of petroleum derivatives.     

Low-Temperature Microbial Degradation of Oil Products Differing in the Extent of Condensation

Out of the 30 strains capable of oil degradation at 4–6°C, four were selected by their ability to degrade 40% of the oil substrate present in the growth medium: Rhodococcus spp. DS-07 and DS-21 and Pseudomonas spp. DS-09 and DS-22. We studied the activity of these strains as degraders of oil products of various condensation degrees (crude oil, masut, petroleum oils, benzene resins and ethanol–benzene resins) at 4–6°C. The maximum degrees of degradation of masut and ethanol–benzene resins were observed in Pseudomonas spp. DS-22 (17.2% and 5.2%, respectively). The maximum degradation of petroleum oils and benzene resins was observed in Rhodococcus spp. DS-07 (40% and 16.6%, respectively). These strains provide a basis for developing biodegrader preparations applicable to the bioremediation of oil-polluted sites under the conditions of a cold climate.     

Comparison of autochthonous bacteria and commercially available cultures with respect to their effectiveness in fuel oil degradation

Summary This study examined the microbial degradation of fuel oil by nine highly adapted different commercially available mixed bacterial cultures (DBC-plus, Flow Laboratories, Meckenheim, F.R.G.) and a bacterial community from a domestic sewage sludge sample. All mixed cultures were cultivated under aerobic batch conditions shaking (110 rpm) at 20°C in a mineral base medium containing 1 or 5% (v/v) fuel oil as the sole carbon source. Percent degradation of fuel oil and the n-alkane fraction was recorded for the nine DBC-plus cultures and the mixed population of the activated sludge sample. The increase in colony counts, protein, and optical density was studied during a 31-day incubation period for DBC-plus culture A, DBC-plus culture A2 and the activated sludge sample. The activated sludge mixed culture was most effective in degrading fuel oil, but various isolated bacterial strains from this bacterial community were not able to grow on fuel oil as the sole carbon source. In contrast, the n-alkane degradation rates of the DBC-cultures were lower, but single strains from the commercially available mixed cultures were able to mineralize fuel oil hydrocarbons. Strains ofPseudomonas aeruginosa were isolated most frequently and these organisms were able to grow very rapidly on fuel oil as a complex sole carbon source. The results indicate that fuel oil degradation in domestic sewage sludge is performed by mixed populations of naturally occurring bacteria and does not depend on the application of highly adapted commercially available cultures.     

Temperature-dependent differences in community structure of bacteria involved in degradation of petroleum hydrocarbons under sulfate-reducing conditions

Aim: The aim of this study was to characterize the microbial community involved in anaerobic degradation of petroleum hydrocarbon under low‐ and moderate‐temperature conditions. Methods and Results: Sulfate‐reducing enrichment cultures growing on crude oil and p‐xylene were established at low and moderate temperatures. Bacterial community structures of the cultures were characterized by 16S rRNA gene‐based analysis and organisms responsible for degradation of p‐xylene were investigated by analysis of the bamA gene, involved in anaerobic degradation of aromatic compounds. The PCR‐denaturing gradient gel electrophoresis analysis indicated significant differences in microbial community structures among the cultures, depending on the temperatures of incubation. Difference depending on the temperatures was also observed in the cloning analysis of the bamA gene performed on the p‐xylene‐degrading enrichment cultures. Majority of clones detected in the culture of moderate temperature were related to Desulfosarcina ovata, whereas more diverse bamA gene sequences were obtained from the culture incubated at low temperature. Conclusions: Temperature‐dependent differences in microbial community were demonstrated by the analyses of two genes. It was suggested that sulfate‐reducing bacteria of phylogenetically different groups might be involved in the degradation of petroleum hydrocarbons in different temperature environments. Significance and Impact of the Study: This study is the first report of p‐xylene‐degrading sulfate‐reducing enrichment culture at low temperature. The results of the experiments at low temperature were distinctly different from those reported in previous studies performed at moderate temperatures.     

一株不动杆菌降解石油烃的特性及关键烷烃降解基因分析

【背景】Acinetobacter sp. Tust-DM21 (GenBank登录号KX390866)是本实验室前期从渤海湾海洋石油勘探船废油收集区采集的水油混合样中分离出的一株高效石油降解菌,其对短、中、长链烷烃均表现出很强的降解能力,有较好的应用前景。【目的】从应用层面探究其最佳降解条件,同时从生物信息层面探究其降解基因的作用。【方法】将其在不同温度、pH下培养144h,通过GC-MS内标法测定石油烃各组分的变化情况,计算出其最佳降解条件;同时,通过生物信息学手段确定基因组中的降解基因,每个基因分别选择7个同源基因,对它们的蛋白序列进行比较;最后对2个降解基因在0-144 h的表达情况进行了Real-time PCR分析。【结果】Acinetobacter sp. Tust-DM21最佳降解条件为35°C、pH 8.5,该条件下对石油降解率可达97.5%,其中,对长链烷烃降解率达98.5%,对环烃为81%,对芳香烃为87%;同时,研究发现基因组中含有常见烷烃降解基因alk B(GenBank登录号MH368539)和长链烷烃降解基因alm A (GenBank登录号MH357335),2个降解基因的蛋白经比较均与其同源蛋白表现出一定的相似性,同属菌的相似性最高;通过Real-timePCR发现这2个基因在0-144 h的相对表达量随时间逐步提高。【结论】Acinetobacter sp. Tust-DM21在最佳降解条件下对石油各组分都显示出了优良的降解能力,特别对长链烷烃的降解能力尤为突出;将2个降解基因的相对表达量结合该时间段的生长趋势,证明了菌株Acinetobacter sp. Tust-DM21的生长和降解与alk B和alm A基因的上调表达存在关联。     

Accelerated biodegradation of petroleum hydrocarbon waste

Conventional landfarming approaches to bioremediation of refinery and other petroleum sludges are not acceptable environmentally and are banned in most North American jurisdictions. While initial bioreactor-based systems for treatment of these sludges required batch-cycle process-times of 1–3 months, an accelerated process has now been developed which can be completed in 10–12 days. In this process, up to 99% of total petroleum hydrocarbons are degraded and the sludges are converted from hazardous to non-hazardous according to the United States EPA's toxicity characteristic leachate procedure criteria. Understanding and exploiting mechanisms to improve hydrocarbon accession to the degrading microorganisms was a key development component of the process. Contrasting physiological mechanisms were observed for different component organisms of the mixed culture with respect to their associations with the hydrocarbon substrate; and the beneficial effects of using surfactants were demonstrated. The mixed culture used in the process exhibited a capacity for high-rate degradation of volatile organic carbons and the potential use of the culture as a liquid biofilter was demonstrated. The culture was also effective as an inoculant for the bioaugmentation of total petroleum hydrocarbon-contaminated soil and as a de-emulsifier of oilfield emulsions and could transform some other environmental contaminants which are not predominant components of crude oil.     

Combustion characteristics of a turbocharged DI compression ignition engine fueled with petroleum diesel fuels and biodiesel

In this study, the combustion characteristics and emissions of two different petroleum diesel fuels (No. 1 and No. 2) and biodiesel from soybean oil were compared. The tests were performed at steady state conditions in a four-cylinder turbocharged DI diesel engine at full load at 1400-rpm engine speed. The experimental results compared with No. 2 diesel fuel showed that biodiesel provided significant reductions in PM, CO, and unburned HC, the NO(x) increased by 11.2%. Biodiesel had a 13.8% increase in brake-specific fuel consumption due to its lower heating value. However, using No. 1 diesel fuel gave better emission results, NO(x) and brake-specific fuel consumption reduced by 16.1% and 1.2%, respectively. The values of the principal combustion characteristics of the biodiesel were obtained between two petroleum diesel fuels. The results indicated that biodiesel may be blended with No. 1 diesel fuel to be used without any modification on the engine.     

Long-chain n-alkanes occurring during microbial degradation of petroleum.

Five axenic cultures and a mixed culture were examined for ability to degrade South Louisiana, Brass River Nigerian, Anaco Venezuelan, and Altamont crude oils. A wax was observed during microbial degradation of Altamont crude oil, but not during weathering of the oil. The high-boiling n-alkanes in the wax were associated with microbial degradation of the oil and appeared to be similar to components of tarballs found in the open ocean.     

Biodegradation of petroleum sludge and petroleum polluted soil by a bacterial consortium: a laboratory study

This article presents a study of the efficiency and degradation pattern of samples of petroleum sludge and polluted sandy soil from an oil refinery. A bacterial consortium, consisting of strains from the genera Pseudomonas, Achromobacter, Bacillus and Micromonospora, was isolated from a petroleum sludge sample and characterized. The addition of nitrogen and phosphorus nutrients and a chemical surfactant to both the samples and bioaugmentation to the soil sample were applied under laboratory conditions. The extent of biodegradation was monitored by the gravimetric method and analysis of the residual oil by gas chromatography. Over a 12-week experiment, the achieved degree of TPH (total petroleum hydrocarbon) degradation amounted to 82–88% in the petroleum sludge and 86–91% in the polluted soil. Gas chromatography–mass spectrometry was utilized to determine the biodegradability and degradation rates of n-alkanes, isoprenoids, steranes, diasteranes and terpanes. Complete degradation of the n-alkanes and isoprenoids fractions occurred in both the samples. In addition, the intensities of the peaks corresponding to tricyclic terpenes and homohopanes were decreased, while significant changes were also observed in the distribution of diasteranes and steranes.     

Community dynamics of a mixed-bacterial culture growing on petroleum hydrocarbons in batch culture

The effects of various hydrocarbon substrates, and a chemical surfactant capable of enhancing crude-oil biodegradation, on the community structure of a mixed-bacterial inoculum were examined in batch culture. Of 1000 TSA-culturable isolates, 68.6% were identified at the genus level or better by phospholipid fatty acid analysis over 7-day time course experiments. Cultures were exposed to 20 g/L Bow River crude oil with and without 0.625 g/L Igepal CO-630 (a nonylphenol ethoxylate surfactant), 5 g/L saturates, 5 g/L aromatics, or 125 g/L refinery sludge. A group of six genera dominated the cultures: Acinetobacter, Alcaligenes, Ochrobactrum, Pseudomonas/Flavimonas, Stenotrophomonas, and Yersinia. Species from four of the genera were shown to be capable of hydrocarbon degradation, and counts of hydrocarbon degrading and total heterotrophic bacteria over time were nearly identical. Pseudomonas/Flavimonas and Stenotrophomonas normally dominated during the early portions of cultures, although the lag phase of Stenotrophomonas appears to have been increased by surfactant addition. Acinetobacter calcoaceticus was the most frequently isolated microorganism during exposure to the saturate fraction of crude oil. Regardless of substrate, the culture medium supported a greater variety of organisms during the latter portions of cultures. Understanding the community structure and dynamics of mixed bacterial cultures involved in treatment of heterogeneous waste substrates may assist in process development and optimization studies.     

Microbial petroleum degradation: application of computerized mass spectrometry.

An analytical procedure is presented for obtaining detailed characterization of petroleum hydrocarbons which undergo microbial degradation. The procedure includes column chromatographic separation and characterization of the resulting fractions by mass spectrometry and gas chromatography. The use of computerized low-resolution mass spectrometry is offered as a method for assessing microbial degradation of petroleum. This method provides information which cannot, at the present time, be obtained by other available analytical methods. Use of this method to evaluate degradation of a South Louisiana crude oil by a mixed culture of estuarine bacteria revealed that asphaltenes and resins increased by 28% after degradation, while saturates and aromatics decreased by 83.4% and 70.5%, respectively. Most of the normal and branched-chain alkanes were degraded (96.4%), but an increase in long-chain alkanes (C28-C32) after degradation was observed by gas-liquid chromatography. Susceptibility of cycloalkanes to degradation was less as the structure varied, i.e., 6-ring greater than 1-ring greater than 2-ring greater than 3-ring greater than 5-ring greater than 4-ring. Susceptibility of aromatic components to degradation decreased with increase in the number of rings, viz., monoaromatics greater than diaromatics greater than triaromatics greater than tetraaromatics greater than pentaaromatics. Aromatic nuclei containing sulfur were twice as refractory as non-sulfur analogs.     

Biodegradation of Petroleum Tar by Pseudomonas Spp. From Oil Field of Assam,India

ABSTRACT

Petroleum tar produced during the processing of crude oil is one of the earth's major pollutants. The potential of certain soil bacteria in the biodegradation of petroleum tar was assessed to develop an active indigenous bacterial consortium for bioremediation of petroleum tar–polluted sites of Assam, India. In vitro enrichment cultures of five Pseudomonas spp. were found to metabolize petroleum tar. The Fourier transform infrared (FTIR) analyses of the enrichment cultures revealed the presence of the functional groups, viz., –OH, –CHO, C?O, and –COOH, which provided evidence for the biodegradation of petroleum tar. Further, gas chromatography–flame ionization detection (GC-FID) analyses revealed complete degradation of low-molecular-weight hydrocarbons, and the subsequent appearance of some additional peaks reflected the formation of intermediate metabolites during the degradation of petroleum tar. A mixed culture with 0.1% Tween 80 as a surfactant exhibited almost complete degradation in contrast to the degradation by the mixed culture without Tween 80. This confirmed the effect of a surfactant for acceleration of the biodegradation process of petroleum tar.     

Gas chromatographic determination for forensic purposes of petroleum fuel inhaled just before fatal burning

The determination of petroleum fuel in the blood of burned bodies was carried out by three different gas chromatographic procedures. Seven components of gasoline (isopentane, n-pentane, 2-methylpentane, benzene, 2-methylhexane, 3-methylhexane and toluene) and five of kerosene (xylene, C₉H₂₀, mesitylene, pseudocumene and C₁₁H₂₄) were chosen as indicators with a coefficient of variation of 5–24%. The methods were applied to four autopsy cases with a relatively low carboxyhaemoglobin (HbCO) content. When gasoline exposure had occurred, the blood concentrations determined were almost identical whatever the components selected. Great variations in the components determined were found after kerosene exposure, and hydrocarbons ≥C₁₄ were hardly inhaled by the victims. A higher content of fuel in the left than in the right ventricular blood observed in the autopsy cases suggests fuel inhalation just before death. The same phenomenon was also observed in the content of blood HbCO. Determinations of petroleum fuel and HbCO in both the right and left ventricular blood would be useful for the forensic diagnosis on burned bodies with a low HbCO content.     

Advance in the toxic effects of petroleum water accommodated fraction on marine plankton

Recently, the impact of petroleum pollution on marine plankton has been complemented by a great concern. This review summarizes the reports about toxic effects of oil water accommodated fraction (WAF) on marine phytoplankton, zooplankton and early life stage of animal. For the oil WAF, toxicants are mainly composed of the aromatic hydrocarbons, such as the benzene hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) with 2–5 rings. The oil WAF, especially the PAHs, can be accumulated in plankton due to their great lipophilic abilities, and thus elicites various deleterious effects. Toxicological tests show that marine plankton is very sensitive to the petroleum WAF, as the order of median effective/lethal concentration is merely μg/L or mg/L. There are species and developmental stages differences of plankton tolerance to petroleum WAF, and the toxicity of different oil WAF is various. Generally, its toxicity enhances with increasing carbonic chain length and benzene ring number. Many studies on the acute and sub-acute toxic effects of oil WAF have been done, however few researches on its chronic toxic effects has been carried out till now. Besides, most reports focused on the levels from molecule to individual, though very little work of petroleum toxic effects has ever been performed on the marine plankton population or community levels. Therefore, it is necessary to continue these studies in future.