Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.     

Change of oil-degrading activity in microorganisms stored under laboratory conditions

Change of the oil-degrading activity was studied in psychrophilic microbial strains Rhodococcus spp. DS-07, DS-21 and Pseudomonas spp. DS-09, DS-22 maintained on various media: rich and synthetic with a selective agent. After 2.5 years of storage on rich medium, the oil-degrading activity decreased by 50–60%, whereas this decrease was insignificant in the medium with oil. Passages to selective medium with oil after the storage partly restored the activity. It was found that storage of oil-degrading microorganisms caused loss of biodegradation plasmids. Their recovery and long-term preservation demand the presence of the selective agent in the medium.     

Soil lipase activity – a useful indicator of oil biodegradation

The evaluation of soil lipase activity as a tool to monitor the decontamination of a freshly oil-polluted soil was tested in a laboratory study. An arable soil was experimentally contaminated with diesel oil at 5 mg hydrocarbons g^–1 soil dry weight and incubated with and without fertilization (N-P-K) for 116 days at 20°C. Lipase activity and counts of oil-degrading microorganisms were measured at regular time intervals, and the correlations with the levels of hydrocarbon concentrations in soil were investigated. The residual soil hydrocarbon concentration correlated significantly negatively with soil lipase activity and with the number of oil-degrading microorganisms, independent of fertilization. The induction of soil lipase activity is a valuable indicator of oil biodegradation in naturally attenuated (unfertilized) and bioremediated (fertilized) soils.     

Composition and dynamics of biostimulated indigenous oil-degrading microbial consortia from the Irish, North and Mediterranean Seas: a mesocosm study

Diversity of indigenous microbial consortia and natural occurrence of obligate hydrocarbon-degrading bacteria (OHCB) are of central importance for efficient bioremediation techniques. To investigate the microbial population dynamics and composition of oil-degrading consortia, we have established a series of identical oil-degrading mesocosms at three different locations, Bangor (Menai Straits, Irish Sea), Helgoland (North Sea) and Messina (Messina Straits, Mediterranean Sea). Changes in microbial community composition in response to oil spiking, nutrient amendment and filtration were assessed by ARISA and DGGE fingerprinting and 16Sr RNA gene library analysis. Bacterial and protozoan cell numbers were quantified by fluorescence microscopy. Very similar microbial population sizes and dynamics, together with key oil-degrading microorganisms, for example, Alcanivorax borkumensis, were observed at all three sites; however, the composition of microbial communities was largely site specific and included variability in relative abundance of OHCB. Reduction in protozoan grazing had little effect on prokaryotic cell numbers but did lead to a decrease in the percentage of A.?borkumensis 16S rRNA genes detected in clone libraries. These results underline the complexity of marine oil-degrading microbial communities and cast further doubt on the feasibility of bioaugmentation practices for use in a broad range of geographical locations.     

Estimation of the hydrocarbon-oxidizing activity of microorganisms

A method allowing the microorganisms growing on substrates with a low water solubility (oil, fuel oil, resins, and asphalthenes) to be isolated and counted was developed. An advantage of the method is its ability to estimate the oil-utilizing activity of each strain visually according to the decolorized zones formed during its growth on oil products. The sizes of these zones indicate which oil-degrading strain is most active.     

Field evaluations of marine oil spill bioremediation.

Bioremediation is defined as the act of adding or improving the availability of materials (e.g., nutrients, microorganisms, or oxygen) to contaminated environments to cause an acceleration of natural biodegradative processes. The results of field experiments and trials following actual spill incidents have been reviewed to evaluate the feasibility of this approach as a treatment for oil contamination in the marine environment. The ubiquity of oil-degrading microorganisms in the marine environment is well established, and research has demonstrated the capability of the indigenous microflora to degrade many components of petroleum shortly after exposure. Studies have identified numerous factors which affect the natural biodegradation rates of oil, such as the origin and concentration of oil, the availability of oil-degrading microorganisms, nutrient concentrations, oxygen levels, climatic conditions, and sediment characteristics. Bioremediation strategies based on the application of fertilizers have been shown to stimulate the biodegradation rates of oil in aerobic intertidal sediments such as sand and cobble. The ratio of oil loading to nitrogen concentration within the interstitial water has been identified to be the principal controlling factor influencing the success of this bioremediation strategy. However, the need for the seeding of natural environments with hydrocarbon-degrading bacteria has not been clearly demonstrated under natural environmental conditions. It is suggested that bioremediation should now take its place among the many techniques available for the treatment of oil spills, although there is still a clear need to set operational limits for its use. On the basis of the available evidence, we have proposed preliminary operational guidelines for bioremediation on shoreline environments.     

Effects of temperature and biostimulation on oil-degrading microbial communities in temperate estuarine waters

Improved strategies for oil-spill remediation will follow a better understanding of the nature, activities and regulating parameters of petroleum hydrocarbon-degrading microbial communities in temperate marine environments. The addition of crude oil to estuarine water resulted in an immediate change in bacterial community structure, increased abundance of hydrocarbon-degrading microorganisms and a rapid rate of oil degradation, suggesting the presence of a pre-adapted oil-degrading microbial community and sufficient supply of nutrients. Relatively rapid degradation was found at 4°C, the lowest temperature tested; and it was temperature rather than nutrient addition that most influenced the community structure. A detailed phylogenetic analysis of oil-degrading microcosms showed that known hydrocarbonoclastic organisms like Thalassolituus and Cycloclasticus , as well as proposed oil degraders like Roseobacter , were present at both 4°C and 20°C, demonstrating the thermo-versatility of such organisms. Clones related to Oleispira antarctica (98% 16S rRNA similarity), a psychrophilic alkane degrader, were dominant in the 4°C oil-degrading community, whereas other clones constituting a different clade and showing 94% similarity 16S rRNA with O. antarctica were found in situ. These findings demonstrate the potential for intrinsic bioremediation throughout the course of the year in temperate estuarine waters, and highlight the importance of both versatile psychrotolerant and specialized psychrophilic hydrocarbon-degrading microbes in effecting this process at low temperatures.     

Estimation of the Hydrocarbon-Oxidizing Activity of Microorganisms

A method allowing microorganisms growing on substrates with low water solubility (oil, fuel oil, resins, and asphalthenes) to be isolated and counted was developed. The method makes it possible to estimate the oil-utilizing activity of each strain visually according to the decolorized zones formed during its growth on oil products. The sizes of these zones indicate which oil-degrading strains are most active.     

Continuous open flow-through system as a model for oil degradation in the arctic ocean

A continuous flow-through system incubated in situ was used to model oil biodegradation in Arctic coastal waters. High numbers of oil-degrading microorganisms were found in the Arctic coastal waters examined in this study. The microbial community underlying oil slicks increased and showed a population shift to a greater percentage of hydrocarbon-utilizing microorganisms. Microbial populations and oil biodegradation were increased by the addition of nitrogen and phosphorus. Both abiotic and biodegradative losses were lower than expected, perhaps due to the unusually harsh, ice-dominated Arctic summer, during which these tests were conducted. Chromatographic and spectrometric analyses showed that residual oils contained similar percentages of individual components and classes of hydrocarbons, regardless of the amount of degradation, indicating that most components of the oil were being degraded at similar rates.     

Oil degradation by basidiomycetes in soil and peat at low temperatures

A total of 17 basidiomycete strains causing white rot and growing on oil-contaminated substrates have been screened. Three strains with high (Steccherinum murashkinskyi), average (Trametes maxima), and low (Pleurotus ostreatus) capacities for the colonization of oil-contaminated substrates have been selected. The potential for degrading crude oil hydrocarbons has been assessed with the use of fungi grown on nonsterile soil and peat at low temperatures. Candida sp. and Rhodococcus sp. commercial strains have been used as reference organisms with oil-degrading ability. All microorganisms introduced in oil-contaminated soil have proved to be ineffective, whereas the inoculation of peat with basidiomycetes and oil-degrading microorganisms accelerated the destruction of oil hydrocarbons. The greatest degradation potential of oil-aliphatic hydrocarbons has been found in S. murashlinskyi. T. maxima turned out to be the most successful in degrading aromatic hydrocarbons. It has been suggested that aboriginal microflora contributes importantly to the effectiveness of oil-destructing microorganisms. T. maxima and S. murashkinskyi strains are promising for further study as oil-oxidizing agents during bioremediation of oil-contaminated peat soil under conditions of low temperatures.     

Study on the Lake Baikal microbial community in the areas of the natural oil seeps

We studied the composition of a natural microbial community, the distribution of different groups of microorganisms (including those able to degrade oil hydrocarbons) within the areas of natural oil seeps in the Lake Baikal. It was revealed that, in the bottom sediments, the oil-degrading microorganisms dominating the community have included the bacteria of g. Bacillus, while in the water column, dominating microbes are presented by species of genera Rhodococcus Pseudomonas, and Micrococcus. Under the conditions of the model experiment, the potential activity of Baikal microbes towards utilization of n-alcanes has been assessed. Under such conditions it was shown that the concentration of n-alcanes decreases to 60% during 20 days of the experiment (the initial oil concentration was 0.5 mg/l, i.e., ten maximal permissible concentrations, MPC).     

Degradation of machine oil by nocardiform bacteria]

Gas liquid chromatography (GLC) was used for the first time to screen for machine oil-degrading microorganisms. Oil degradation was evaluated from the microorganism respiratory activity during the utilization of oil as the sole carbon and energy source. The results are consistent with those obtained by the conventional weighing method. Substrate specificity of the active strains with respect to different machine oils was studied. Bacterial communities exhibited the highest activity, whereas a Rhodococcus erythropolis strain was the most active among pure cultures. Various stages of bacterial interaction with oil drops were followed by means of fluorescent microscopy.     

Hydrocarbon bioremediation potential of an unimpacted Kuwaiti oil-field environment

Seasonal variations in the hydrocarbon-degrading potential of soil samples from an unimpacted site in the Kuwaiti Burgan oil field environment were studied under mesophilic conditions. Hydrocarbon-degrading microorganisms occurred but varied all-year-round, and their numbers ranged from 1.3 x 10(7) to 9.3 x 10(7) CFU g(-1) dry soil, while hydrocarbon-degrading fungi ranged from 3.0 x 10(4) - 3.8 x 10(5) CFU g(-1) dry soil, depending on the sampling period. These hydrocarbon-degraders also comprised variable but generally high proportions of the total aerobic heterotrophic organisms (2 to > 98%) for bacteria and lower levels (7-9%) for fungi. The crude oil-degrading capacity of the oil-degrading populations (bacteria and fungi) ranged from 80-95% of the hexane-extractable fractions. Differential inhibition studies carried out on soil samples showed that bacteria were the greater contributors to hydrocarbon degradation (79-92%) than fungi. Pure hydrocarbon substrates, hexadecane and phenanthrene, were degraded to near completion after a 28-day incubation by both the bacterial and fungal portions of the soil flora.     

The characterization of oil-degrading microorganisms from lubricating oil contaminated (scale) soil

AIMS: To isolate and characterize oil-degrading microorganisms from contaminated (scale) soil. METHODS AND RESULTS: Oil-degrading microorganisms were isolated from enrichment cultures of scale soil. Each isolate was identified using 16S rDNA gene and oil degradability was determined on both unused and used lubricating oil. The weight of the extracted remaining oil revealed that most isolates degraded unused lubricating oil more than used lubricating oil. Chemical composition of oil analysed by TLC-FID and GC-MS demonstrated that Nocardia simplex W9 degraded used oil the best, and resulted in a decrease in saturates, aromatics and resins to 52.46, 38.13 and 18.81%, respectively. CONCLUSIONS:Nocardia simplex W9 is the best degrader, among all the isolates, on both used and unused lubricating oil. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Nocardia simplex W9 in scale soil enables iron to be recycled by biodegradation.     

Soybean oil-degrading bacterial cultures as a potential for control of green peach aphids (Myzus persicae)

Microorganisms capable of degrading crude oil were isolated and grown in soybean oil as a sole carbon source. The microbial cultures were used to control green peach aphids in vitro. Approximately 60% mortality of aphids was observed when the cultures were applied alone onto aphids. To examine the cultures as a pesticide formulation mixture, the cultures were combined with a low dose of the insecticide imidacloprid (one-fourth dose of recommended field-application rate) and applied onto aphids. The cultures enhanced significantly the insecticidal effectiveness of imidacloprid, which was higher than imidacloprid alone applied at the low dose. The isolated microorganisms exhibited high emulsifying index values and decreased surface tension values after being grown in soybean oil media. GC/MS analyses showed that microorganisms degraded soybean oil to fatty acids. The cultures were suggested to play the roles of wetting, spreading, and sticking agents to improve the effectiveness of imidacloprid. This is the first report on the control of aphids by using oil-degrading microbial cultures.     

Distribution and biodegradation potential of oil-degrading bacteria in North Eastern Japanese coastal waters

Abstract The distribution of oil-degrading microorganism in samples of surface water and sediment from North Eastern Japanese coastal waters was studied. Modified natural sea water (NSW) agar supplemented with emulsified crude oil (Arabian light, 5 g 1⁻¹) was used to enumerate oil-degrading bacteria. In addition, filtered samples were inoculated into NSW broth containing weathered crude oil. Incubation was carried out at 20°C for 7–10 days. Populations of oil-degrading microorganisms ranged from 3–230 CFU 100 ml⁻¹ in surface waters and 2.9 × 10³ to 1.2 × 10⁵ CFU g⁻ in sediment samples. Analysis of variance showed that oil-degraders were heterogenously distributed. Six mixed populations selected from 20 samples were studied to determine which of the constituent microflora were capable of crude oil biodegradation. Among 51 strains selected for identification, only 61% could be identified which formed 17 different bacterial species. Acinetobacter species (14 strains), Psychrobacter immobilis (9 strains) and Gram-positive cocci (10 strains) were the predominant types. Oil-degrading activity by various mixed populations (three each from water and sediment samples) was determined by using a conventional total weight reduction technique. Reduction in amount of various aliphatic and aromatic hydrocarbon substrates was verified using gas chromatography and high pressure liquid chromatography. Biodegradation of crude oil ranged from 35–58%. One mixed population of the sediment samples degraded more hydrocarbon (both aliphatic and aromatic) and the biodegradation of the aromatic hydrocarbon reached as high as 48%.     

大连湾原油降解菌的分离和多样性分析

[目的]研究大连湾原油污染海域可培养原油降解菌的多样性,并获得新的原油降解菌.[方法]通过大连湾海水、海泥和海绵样品采集,以原油作为唯一碳源,培养、富集、分离筛选原油降解菌,根据16S rRNA基因序列确定其系统进化地位.[结果]通过形态观察和16S rRNA基因分析,共获得22个属的50株菌.其中,有6株菌的16S rRNA序列与最相近的菌株序列一致性仅为95％-97％,可能是潜在的新菌.单菌实验表明,45株菌具有石油降解能力.[结论]揭示了大连湾可培养原油降解菌的多样性,并获得了新的原油降解菌,为海洋石油污染的生物治理提供新资源.     

Efficiency of Indigenous and Inoculated Cold-Adapted Soil Microorganisms for Biodegradation of Diesel Oil in Alpine Soils

Biodegradation of diesel oil (5 g(middot)kg [soil dry weight](sup-1)) was investigated in five alpine subsoils, differing in soil type and bedrock, in laboratory experiments during 20 days at 10(deg)C. The biodegradation activities of the indigenous soil microorganisms and of a psychrotrophic diesel oil-degrading inoculum and the effect of biostimulation by inorganic fertilization (C/N/P ratio = 100:10:2) were determined. Fertilization significantly enhanced diesel oil biodegradation activity of the indigenous soil microorganisms. Biostimulation by fertilization enhanced diesel oil biodegradation to a significantly greater degree than bioaugmentation with the psychrotrophic inoculum. In none of the five soils did fertilization plus inoculation result in a higher decontamination than fertilization alone. A total of 16 to 23% of the added diesel oil contamination was lost by abiotic processes. Total decontamination without and with fertilization was in the range of 16 to 31 and 27 to 53%, respectively.     

Enumeration of petroleum-degrading marine and estuarine microorganisms by the most probable number method.

Several media designed for use in a most probable number (MPN) determination of petroleum-degrading microorganisms were compared. The best results, i.e., largest numbers, were obtained using a buffered (32 mM PO4=) liquid medium containing 1% hydrocarbon substrate. Of 104 presumptive oil degraders tested, 20 grew on oil agar medium but did not utilize oil or a mixture of pure paraffinic hydrocarbons (C10 to C16 n-alkanes) in liquid (MPN) medium. Visible turbidity in the liquid medium was correlated with hydrocarbon utilization. Counts of petroleum degraders obtained using liquid medium (MPN) were in most cases higher than those obtained on an oil-amended silica gel medium. Both procedures yield an estimation of oil degraders, and the oil-amended agar permits growth of organisms which do not degrade crude oil. All strains of oil-degrading microorganisms examined in this study were lipolytic, but the converse was not always true.     

The genome sequence of Polymorphum gilvum SL003B-26A1(T) reveals its genetic basis for crude oil degradation and adaptation to the saline soil

Polymorphum gilvum SL003B-26A1(T) is the type strain of a novel species in the recently published novel genus Polymorphum isolated from saline soil contaminated with crude oil. It is capable of using crude oil as the sole carbon and energy source and can adapt to saline soil at a temperature of 45°C. The Polymorphum gilvum genome provides a genetic basis for understanding how the strain could degrade crude oil and adapt to a saline environment. Genome analysis revealed the versatility of the strain for emulsifying crude oil, metabolizing aromatic compounds (a characteristic specific to the Polymorphum gilvum genome in comparison with other known genomes of oil-degrading bacteria), as well as possibly metabolizing n-alkanes through the LadA pathway. In addition, COG analysis revealed Polymorphum gilvum SL003B-26A1(T) has significantly higher abundances of the proteins responsible for cell motility, lipid transport and metabolism, and secondary metabolite biosynthesis, transport and catabolism than the average levels found in all other genomes sequenced thus far, but lower abundances of the proteins responsible for carbohydrate transport and metabolism, defense mechanisms, and translation than the average levels. These traits support the adaptability of Polymorphum gilvum to a crude oil-contaminated saline environment. The Polymorphum gilvum genome could serve as a platform for further study of oil-degrading microorganisms for bioremediation and microbial-enhanced oil recovery in harsh saline environments.