Moderately thermophilic, hydrocarbonoclastic bacterial communities in Kuwaiti desert soil: enhanced activity via Ca(2+) and dipicolinic acid amendment.

Pristine and oil-contaminated desert soil samples from Kuwait harbored between 10 and 100 cells g(-1) of hydrocarbonoclastic bacteria capable of growth at 50 °C. Enrichment by incubation of moistened soils for 6 months at 50 °C raised those numbers to the magnitude of 10(3) cells g(-1). Most of these organisms were moderately thermophilic and belonged to the genus Bacillus; they grew at 40-50 °C better than at 30 °C. Species belonging to the genera Amycolatopsis, Chelativorans, Isoptericola, Nocardia, Aeribacillus, Aneurinibacillus, Brevibacillus, Geobacillus, Kocuria, Marinobacter and Paenibacillus were also found. This microbial diversity indicates a good potential for hydrocarbon removal in soil at high temperature. Analysis of the same desert soil samples by a culture-independent method (combined, DGGE and 16S rDNA sequencing) revealed dramatically different lists of microorganisms, many of which had been recorded as hydrocarbonoclastic. Many species were more frequent in the oil contaminated than in the pristine soil samples, which may reflect their hydrocarbonoclastic activity in situ. The growth and hydrocarbon consumption potential of all tested isolates were dramatically enhanced by amendment of the cultures with Ca(2+) (up to 2.5 M CaSO4). This enhanced effect was even amplified when in addition 8 % w/v dipicolinic acid was amended. These novel findings are useful in suggesting biotechnologies for waste hydrocarbon remediation at moderately high temperature.

Hydrocarbonoclastic biofilms based on sewage microorganisms and their application in hydrocarbon removal in liquid wastes.

Attempts to establish hydrocarbonoclastic biofilms that could be applied in waste-hydrocarbon removal are still very rare. In this work, biofilms containing hydrocarbonoclastic bacteria were successfully established on glass slides by submerging them in oil-free and oil-containing sewage effluent for 1 month. Culture-dependent analysis of hydrocarbonoclastic bacterial communities in the biofilms revealed the occurrence of the genera Pseudomonas, Microvirga, Stenotrophomonas, Mycobacterium, Bosea, and Ancylobacter. Biofilms established in oil-containing effluent contained more hydrocarbonoclastic bacteria than those established in oil-free effluent, and both biofilms had dramatically different bacterial composition. Culture-independent analysis of the bacterial flora revealed a bacterial community structure totally different from that determined by the culture-dependent method. In microcosm experiments, these biofilms, when used as inocula, removed between 20% and 65% crude oil, n-hexadecane, and phenanthrene from the surrounding effluent in 2 weeks, depending on the biofilm type, the hydrocarbon identity, and the culture conditions. More of the hydrocarbons were removed by biofilms established in oil-containing effluent than by those established in oil-free effluent, and by cultures incubated in the light than by those incubated in the dark. Meanwhile, the bacterial numbers and diversities were enhanced in the biofilms that had been previously used in hydrocarbon bioremediation. These novel findings pave a new way for biofilm-based hydrocarbon bioremediation, both in sewage effluent and in other liquid wastes.

Bioremediation of oily hypersaline soil and water via potassium and magnesium amendment.

Ten hydrocarbonoclastic halobacterial species and 5 haloarchaeal species that had been isolated on a mineral medium with oil as the sole carbon source grew better and consumed more crude oil, as measured by gas-liquid chromatography, in media receiving between 0.50 and 0.75 mol/L KCl and between 1.50 and 2.25 mol/L MgSO4. Chemical analysis revealed that within a certain limit, the higher the KCl and MgSO4 concentrations in the medium, the more K⁺ and Mg²âº, respectively, was accumulated by cells of all the tested halobacteria and haloarchaea. Also, in experiments in which total natural microbial consortia in hypersaline soil and water samples were directly used as inocula, the consumption of hydrocarbons was enhanced in the presence of the above given concentrations of KCl and MgSO4. It was concluded that amendment with calculated concentrations of K⁺ and Mg²âº could be a promising practice for hydrocarbon bioremediation in hypersaline environments.

Oil-bioremediation potential of two hydrocarbonoclastic, diazotrophic Marinobacter strains from hypersaline areas along the Arabian Gulf coasts.

Two halophilic, hydrocarbonoclastics bacteria, Marinobacter sedimentarum and M. flavimaris, with diazotrophic potential occured in hypersaline waters and soils in southern and northern coasts of Kuwait. Their numbers were in the magnitude of 10(3) colony forming units g(-1). The ambient salinity in the hypersaline environments was between 3.2 and 3.5 M NaCl. The partial 16S rRNA gene sequences of the two strains showed, respectively, 99 and 100% similarities to the sequences in the GenBank. The two strains failed to grow in the absence of NaCl, exhibited best growth and hydrocarbon biodegradation in the presence of 1 to 1.5 M NaCl, and still grew and maintained their hydrocarbonoclastic activity at salinities up to 5 M NaCl. Both species utilized Tween 80, a wide range of individual aliphatic hydrocarbons (C9-C40) and the aromatics benzene, biphenyl, phenanthrene, anthracene and naphthalene as sole sources of carbon and energy. Experimental evidence was provided for their nitrogen-fixation potential. The two halophilic Marinobacter strains successfully mineralized crude oil in nutrient media as well as in hypersaline soil and water microcosms without the use of any nitrogen fertilizers.

Enhanced haloarchaeal oil removal in hypersaline environments via organic nitrogen fertilization and illumination.

Hypersaline soil and pond water samples were mixed with 3 % crude oil, some samples were autoclaved to serve as sterile controls; experimental samples were not sterilized. After 6-week incubation at 40 °C under light/dark cycles, the soil microflora consumed 66 %, and after 4 weeks the pond water microflora consumed 63 % of the crude oil. Soil samples treated with 3 % casaminoacids lost 89 % of their oil after 6 weeks and water samples lost 86 % after 4 weeks. Samples treated with casaminoacids and antibiotics that selectively inhibited bacteria, lost even more oil, up to 94 %. Soil-water mixtures incubated under continuous illumination lost double as much more oil than samples incubated in the dark. The soil-water mixture at time zero contained 1.3 × 10(4) CFU g(-1) of hydrocarbon-utilizing microorganisms which were affiliated to Halomonas aquamarina, Exiguobacterium aurantiacum, Haloferax sp., Salinococcus sp., Marinococcus sp. and Halomonas sp. After 6-week incubation with oil, these numbers were 8.7 × 10(7) CFU g(-1) and the Haloferax sp. proportion in the total microflora increased from 20 to 93 %. Experiments using the individual cultures and three other haloarchaea isolated earlier from the same site confirmed that casaminoacids and light enhanced their oil consumption potential in batch cultures.

Hydrocarbon-utilizing microorganisms naturally associated with sawdust.

Sawdust, one of the materials used as sorbent for removing spilled oil from polluted environments was naturally colonized by hydrocarbon-utilizing fungi, 1×10(5)-2×10(5) colony forming units (CFU) g(-1), depending on the hydrocarbon substrate. This sorbent was initially free of hydrocarbon-utilizing bacteria. Incubating wet sawdust at 30°C resulted in gradually increasing the fungal counts to reach after 6months between 5×10(6) and 7×10(6)CFUg(-1), and the appearance of hydrocarbon-utilizing bacteria in numbers between 8×10(4) and 3×10(5)cellsg(-1). The fungi belonged to the genera Candida (32% of the total), Penicillium (21%), Aspergillus (15%), Rhizopus (12%), Cladosporium (9%), Mucor (7%) and Fusarium (4%). Based on their 16S rRNA gene sequences the bacteria were affiliated to Actinobacterium sp. (38%), Micrococcus luteus (30%), Rhodococcus erythropolis, (19%) and Rhodococcus opacus (13%). Individual pure fungal and bacterial isolates grew on a wide range of individual pure aliphatic (n-alkanes with chain lengths between C(9) and C(40)) and aromatic (benzene, biphenyl, anthracene, naphthalene and phenanthrene) hydrocarbons as sole sources of carbon and energy. Quantitative determinations revealed that all fungal and bacterial isolates could consume considerable proportions of crude oil, phenanthrene (an aromatic hydrocarbon) and n-hexadecane (an aliphatic hydrocarbon) in batch cultures. It was concluded that when sawdust is used as a sorbent, the associated microorganisms probably contribute to the bioremediation of oil and hydrocarbon pollutants in the environment.

Mercury resistance and volatilization by oil utilizing haloarchaea under hypersaline conditions.

The hydrocarbon utilizing haloarchaea, Haloferax (two strains), Halobacterium and Halococcus from a hypersaline coastal area of the Arabian Gulf, had the potential for resistance and volatilization of Hg(2+). Individual haloarchaea resisted up to between 100 and 200 ppm HgCl2 in hydrocarbon free media with salinities between 1 and 4 M NaCl, but only up to between 20 and 30 ppm in a mineral medium containing 3 M NaCl, with 0.5% (w/v) crude oil, as a sole source of carbon and energy. Halococcus and Halobacterium volatilized more mercury than Haloferax. The individual haloarchaea consumed more crude oil in the presence of 3 M NaCl than in the presence of 2 M NaCl. At both salinities, increasing the HgCl2 concentration in the medium from 0 to 20 ppm resulted in decreasing the oil consumption values by the individual haloarchaea. However, satisfactory oil consumption still occurred in the presence of 10 ppm HgCl2. It was concluded that haloarchaea with the combined potential for mercury resistance and volatilization and hydrocarbon consumption could be useful in removing toxic mercury forms effectively from oil free, mercury contaminated, hypersaline environments, and mercury and oil, albeit less effectively, from oily hypersaline environments.

Phytoremediation of mercury in pristine and crude oil contaminated soils: Contributions of rhizobacteria and their host plants to mercury removal.

The rhizospheric soils of three tested legume crops: broad beans (Vicia faba), beans (Phaseolus vulgaris) and pea (Pisum sativum), and two nonlegume crops: cucumber (Cucumis sativus) and tomato, (Lycopersicon esculentum) contained considerable numbers (the magnitude of 10(5)g(-1) soil) of bacteria with the combined potential for hydrocarbon-utilization and mercury-resistance. Sequencing of the 16S rRNA coding genes of rhizobacteria associated with broad beans revealed that they were affiliated to Citrobacter freundii, Enterobacter aerogenes, Exiquobacterium aurantiacum, Pseudomonas veronii, Micrococcus luteus, Brevibacillus brevis, Arthrobacter sp. and Flavobacterium psychrophilum. These rhizobacteria were also diazotrophic, i.e. capable of N(2) fixation, which makes them self-sufficient regarding their nitrogen nutrition and thus suitable remediation agents in nitrogen-poor soils, such as the oily desert soil. The crude oil attenuation potential of the individual rhizobacteria was inhibited by HgCl(2), but about 50% or more of this potential was still maintained in the presence of up to 40 mgl(-1) HgCl(2). Rhizobacteria-free plants removed amounts of mercury from the surrounding media almost equivalent to those removed by the rhizospheric bacterial consortia in the absence of the plants. It was concluded that both the collector plants and their rhizospheric bacterial consortia contributed equivalently to mercury removal from soil.

Biodegradation of crude oil and pure hydrocarbons by extreme halophilic archaea from hypersaline coasts of the Arabian Gulf.

Two extreme halophilic Haloferax strains and one strain each of Halobacterium and Halococcus were isolated from a hypersaline coastal area of the Arabian Gulf on a mineral salt medium with crude oil vapor as a sole source of carbon and energy. These archaea needed at least 1 M NaCl for growth in culture, and grew best in the presence of 4 M NaCl or more. Optimum growth temperatures lied between 40 and 45 degrees C. The four archaea were resistant to the antibiotics chloramphenicol, cycloheximide, nalidixic acid, penicillin, streptomycin and tetracycline. The strains could grow on a wide scope of aliphatic and aromatic (both mono-and polynuclear) hydrocarbons, as sole sources of carbon and energy. Quantitative measurements revealed that these extreme halophilic prokaryotes could biodegrade crude oil (13-47%, depending on the strain and medium salinity), n-octadecane (28-67%) and phenanthrene (13-30%) in culture after 3 weeks of incubation. The rates of biodegradation by all strains were enhanced with increasing NaCl concentration in the medium. Optimal concentration was 3 M NaCl, but even with 4 M NaCl the hydrocarbon-biodegradation rates were higher than with 1 and 2 M NaCl. It was concluded that these archaea could contribute to self-cleaning and bioremediation of oil-polluted hypersaline environments.

Oil-utilizing bacteria associated with fish from the Arabian Gulf.

AIMS: The objectives were to count and identify the oil-utilizing bacteria associated with fish, and to study their hydrocarbon-degradation potential. METHODS AND RESULTS: The standard dilution-plate method using a medium with crude oil as a sole source of carbon and energy revealed that 10 different fish sorts from the Arabian Gulf and two from fish farms accommodated millions of oil-utilizing bacteria per square centimetre of fish surface and per gram of gills and guts. According to their 16S rRNA sequences, those bacteria were affiliated to Psychrobacter, Vibrio, Planococcus, Pseudomonas and Actinobacterium. Planktonic and benthic biomass samples from the Gulf were also rich in oil-utilizing bacteria, but with different composition. All isolates could grow on n-alkanes from C(8) to C(40) and three representative aromatics as individual sole sources of carbon and energy. Quantitative analysis of hydrocarbons by gas-liquid chromatography revealed that the biomass samples of the individual bacteria could consume crude oil, n-octadecane and phenanthrene in liquid media. CONCLUSIONS: The abundant oil-utilizing bacterial associated with fish have the potential for cleaning oily waters. SIGNIFICANCE AND IMPORTANCE OF THE STUDY: Aquatic fauna accommodates rich consortia of oil-utilizing bacteria.

Hydrocarbon accumulation by picocyanobacteria from the Arabian Gulf.

AIMS: The objective of this work was to study picocyanobacteria in the Arabian Gulf water in relation to oil pollution. METHODS AND RESULTS: Epifluorescent microscopic counting showed that offshore water samples along the Kuwaiti coast of the Arabian Gulf were rich in picocyanobacteria which ranged in numbers between about 1 x 10(5) and 6 x 10(5) ml(-1). Most dominant was the genus Synechococcus; less dominant genera were Synechocystis, Pleurocapsa and Dermocarpella. All isolates grew well in an inorganic medium containing up to 0.1% crude oil (w/v) and could survive in the presence of up to 1% crude oil. Hydrocarbon analysis by gas liquid chromatography (GLC) showed that representative strains of the four genera had the potential for the accumulation of hydrocarbons (the aliphatic n-hexadecane, aromatic phenanthrene and crude oil hydrocarbons) from aqueous media. Electron microscopy showed that the cells of these strains appeared to store hydrocarbons in their inter thylakoid spaces. Analysis by GLC of constituent fatty acids of total lipids and individual lipid classes from representative picoplankton strains grown in the absence and presence of hydrocarbons showed, however, that the fatty acid patterns were not markedly affected by the hydrocabon substrates, meaning that the test strains could not oxidize the accumulated hydrocarbons. CONCLUSION: The Arabian Gulf is among the water bodies of the world richest in picocyanobacteria. These micro-organisms accumulate hydrocarbons from the water body, but do not biodegrade these compounds. It is assumed that hydrocarbon-utilizing bacteria that were always found associated with all picocyanobacteria in nature may carry out the biodegradation of these compounds. SIGNIFICANCE AND IMPORTANCE OF THE STUDY: The results shed light on the potential role of picocyanobacteria in controlling marine oil pollution.

High-temperature hydrocarbon biodegradation activities in Kuwaiti desert soil samples.

Soil samples taken monthly from the Burgan South oil field of Kuwait for one year degraded crude oil, phenanthrene, and hexadecane. Bacteria were better degraders at high-temperature (55 degrees C) than fungi, especially in the drier, hotter months. Depending on the period of sampling, bacteria degraded hydrocarbons in the range of 46-86% (crude oil), 42-100% (hexadecane) and 5-58% (phenanthrene). Fungi alone accounted for degradation by 20-81% (crude oil), 30-95% (hexadecane) and less than 55% (phenanthrene).

Hydrocarbon uptake by Streptomyces.

Oligocarbophilic Streptomyces strains capable of hydrocarbon uptake and utilization were isolated from the polluted desert of Kuwait and used in this study. Transmission electron-microscopy of hyphae revealed that they become enriched with large less electron dense areas in the cytoplasm, when biomass samples were incubated with alkanes. The Streptomyces isolate could utilize n-hexadecane as sole carbon and energy source and their fatty acid content showed an increase in the fatty acids with chain length equivalent to those of the alkane substrates. Fluorescence measurements of diphenylhexatriene dissolved in the representative alkane, n-hexadecane, showed that the kinetics of hydrocarbon uptake are quite different in hydrocarbon-utilizer compared with non-utilizer Streptomyces strain. Microviscosity of the cellular membrane of the utilizer strain was also different from that of the non-utilizer control strain Streptomyces griseus after incubation in the presence of n-hexadecane. Very likely the hydrocarbon utilizer transported these compounds more efficiently across their membranes and accumulated them as inclusions in the cytoplasm.

Uptake and utilization of n-octacosane and n-nonacosane by Arthrobacter nicotianae KCC B35.

Arthrobacter nicotianae KCC B35 isolated from blue-green mats densely covering oil sediments along the Arabian Gulf coast grew well on C10 to C40 n-alkanes as sole sources of carbon and energy. Growth on C20 to C40 alkanes was even better than on C10 to C18 alkanes. Biomass samples incubated for 6 h with n-octacosane (C28) or n-nonacosane (C29) accumulated these compounds as the predominant constituent alkanes of the cell hydrocarbon fractions. The even chain hexadecane C16 and the odd chain pentadecane C15 were the second dominant constituent alkanes in C28 and C29 incubated cells, respectively. n-Hexadecane-incubated cells accumulated in their lipids higher proportions of C16-fatty acids than control cells not incubated with hydrocarbons. On the other hand, C28 and C29-incubated cells did not contain any fatty acids with the equivalent chain lengths, but the fatty acid patterns of the cell lipids suggest that there should have been mid-chain oxidation of these very long chain alkanes. This activity qualifies A. nicotianae KCC B35 to be used in cocktails for bioremediating environments polluted with heavy oil sediments.

Soil management enhancing hydrocarbon biodegradation in the polluted Kuwaiti desert.

Oil-polluted Kuwaiti desert samples, exposed to the open air, were subjected to specific types of management, once every 2 weeks, throughout a year; control samples were not treated. The total amounts of extractable alkanes from the control samples remained fairly constant during the dry hot months, but decreased during the rainy months reaching, after 1 year, slightly more than one-half of the amount at zero time. This result demonstrates the self-cleaning of the Kuwaiti desert and the essential role of moisture in this process. Out of the eight types of management studied, the repeated fertilization of the polluted sample with 3% KNO3 solution was most efficient, reducing the extractable alkanes after 1 year to about one-third of zero reading. Repeated fertilization with treated sewage effluent was inhibitory to alkane biodegradation, probably because of increasing soil acidity. The latter inhibitory effect was annulled by liming. Repeated irrigation with 3% NaCl solution was inhibitory, but 1% NaCl solution slightly promoted alkane biodegradation. The various samples contained 10(10)-10(11) oil-utilizing bacteria/g soil, predominantly Bacillus, Pseudomonas, Rhodococcus and Streptomyces. Oil-utilizing fungi were much less frequent and were predominantly Aspergillus and Penicillium species. The microbial numbers varied not only according to the type of soil management but also to the season.

Establishment of oil-degrading bacteria associated with cyanobacteria in oil-polluted soil.

A unique natural microbial cocktail with promising potential for remediating oil-polluted desert in the Gulf region is reported. Oil-degrading micro-organisms immobilized within dense cyanobacterial mats on oily coasts of the Arabian Gulf were successfully established in oil-contaminated sand. Those micro-organisms biodegraded 50% of the oil within 10-20 weeks. Nocardioforms belonging to the genus Rhodococcus predominated in the first few weeks, but after 22 weeks Pseudomonas spp. increased, sharing Rhodococcus in the predominance. Other oil-utilizing bacterial genera included Bacillus and Arthrobacter. Filamentous actinomycetes belonging to the genera Streptomyces and probably Thermoactinomyces, as well as fungi belonging mainly to Aspergillus and Penicillium increased in the contaminated sand during the experiment but declined later. Representative strains grew on spectra of the tested n-alkanes with chain lengths between C10 and C40, as sole sources of carbon and energy.

Wool-colonizing micro-organisms capable of utilizing wool-lipids and fatty acids as sole sources of carbon and energy.

Two keratinolytic fungi, Chrysosporium keratinophilum and Malbranchea anamorph of Uncinocarpus reesii, and another three wool-colonizing fungi not previously reported to be keratinolytic, Aspergillus fumigatus, A. Flavus and Scopulariopsis candida, were isolated from soil samples baited with animal wool. These fungi were tested for their ability to utilize wool-lipids as sole source of carbon and energy. The lipid contents of wool of various animals ranged between 2 and 5%. The different lipid extracts were similar in composition; they contained steryl esters, sterols, fatty acids, fatty alcohols and monoacylglycerols. The predominant acyl moiety in wool-lipids of different animals was linolenic acid (18:2). The five fungi tested grew well on an inorganic medium supplemented with total wool-lipids as sole sources of carbon and energy. Individual lipid classes fractionated by preparative thin layer chromatography and suspended into inorganic medium were able to support fungal growth as sole carbon sources. These fungi and another eight wool-colonizing strains, C. tropicum, C. zonatrum, C. anamorph of Arthroderma curryei, Microsporum canis, M. distortum, Trichophyton interdigitale, Emmonsia parva and Myceliophthora vellerae could also utilize standard lipids and fatty acids (cholesterol and palmitic and linoleic acids). Evidence fo the uptake and degradation of cholesterol by C. keratinophilum is presented.

Growth of Candida albicans on hydrocarbons: influence on lipids and sterols.

Candida albicans KTCC 89062 an isolate from a crude oil polluted soil sample in Kuwait grew adequately on n-alkanes with only 12 to 20 carbon chains but not on aromatic hydrocarbons. This isolate grew on glucose better than on any of the alkanes. Alkane-grown cells contained higher proportions of total lipids than glucose-grown cells, and the total lipid content was directly proportional to the alkane chain length. The sterol content also increased in alkane-grown cells; the highest level was with C12 as substrate and progressively lower sterol levels were obtained as the carbon chain length increased. The phospholipid:sterol ratio decreased when the cells were grown on alkanes as compared with glucose grown cells. The ratio of unsaturated:saturated fatty acids was higher in alkane than glucose grown cells and decreased progressively from C12 to C20 as substrates. Growth on alkanes but not on glucose was associated with pseudohyphal formation.