Bioremediation of Atmospheric Hydrocarbons via Bacteria Naturally Associated with Leaves of Higher Plants.

Bacteria associated with leaves of sixteen cultivated and wild plant species from all over Kuwait were analyzed by a culture-independent approach. This technique depended on partial sequencing of 16S rDNA regions in total genomic DNA from the bacterial consortia and comparing the resulting sequences with those in the GenBank database. To release bacterial cells from leaves, tough methods such as sonication co-released too much leaf chloroplasts whose DNA interfered with the bacterial DNA. A more satisfactory bacterial release with a minimum of chloroplast co-release was done by gently rubbing the leaf surfaces with soft tooth brushes in phosphate buffer. The leaves of all plant species harbored on their surfaces bacterial communities predominated by hydrocarbonoclastic (hydrocarbon-utilizing) bacterial genera. Leaves of 6 representative plants brought about in the laboratory effective removal of volatile hydrocarbons in sealed microcosms. Each individual plant species had a unique bacterial community structure. Collectively, the phyllospheric microflora on the studied plants comprised the genera Flavobacterium, Halomonas, Arthrobacter, Marinobacter, Neisseria, Ralstonia, Ochrobactrum. Exiguobacterium, Planomicrobium, Propionibacterium, Kocuria, Rhodococcus and Stenotrophomonas. This community structure was dramatically different from the structure we determined earlier for the same plants using the culture-dependent approach, although in both cases, hydrocarbonoclastic bacteria were frequent.

The potential of established turf cover for cleaning oily desert soil using rhizosphere technology.

The rhizosphere of two turf cover sorts; Bermuda grass and American grass contained high numbers, 8.1 to 16.8 x 10(6) g(-1) of cultivable oil-utilizing and diazotrophic bacteria belonging predominantly to the genera Agrobacterium, Arthrobacter, Pseudomonas, Gordonia, and Rhodococcus. Those bacteria also grew on a nitrogen-free medium and demonstrated the ability to reduce acetylene to ethylene. These isolates grew on a wide range of n-alkanes (C9 to C40) and aromatic hydrocarbons, as sole sources of carbon. Quantitative determinations revealed that predominant bacteria consumed crude oil and representative aliphatic (n-octadecane) and aromatic (phenanthrene) hydrocarbons efficiently. The fact that those organisms had the combined activities of hydrocarbon-utilization and nitrogen-fixation makes them suitable tools for bioremediating oily desert areas that are normally poor in nitrogenous compounds. Phytoremediation experiments showed that spreading turf cover on oily desert soil inhibited oil volatilization and enhanced oil loss in soil by about 15%. Oil loss was also enhanced in turf free soil samples fertilized with NH4NO3. In conclusion, covering this oil-polluted soil with turf cover minimized atmospheric pollution, increased the numbers of the oil-utilizing/nitrogen-fixing bacteria by about 20 to 46% thus, encouraging oil attenuation.

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 phytoremediation potential of hypersaline coasts of the Arabian Gulf using rhizosphere technology.

The rhizosphere and phyllosphere of the halophyte Halonemum strobilaceum naturally inhabiting hypersaline coastal areas of the Arabian Gulf harbor up to 8.1 x 10(4)g(-1) and 3 x 10(2)g(-1), respectively, of extremely halophilic oil-utilizing microorganisms. Such organisms were 14- to 38-fold more frequent in the rhizosphere than in the plant-free soil. Frequent genera in the rhizosphere were affiliated to the archaea Halobacterium sp. and Halococcus sp., the firmicute Brevibacillus borstenlensis, and the proteobacteria Pseudoalteromonas ruthenica and Halomonas sinaensis. The phyllospheric microflora consisted of the dimorphic yeast Candida utilis and the two proteobacteria Ochrobactrum sp. and Desulfovibrio sp. Individual strains grew on a range of pure aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy. All the strains, except C. utilis which could not tolerate salinities >2M NaCl, grew also in media with salinities ranging between 1 and 4M NaCl, with optimum growth between 1 and 2M NaCl. With the exception of the two archaeal genera, all isolates could grow in a nitrogen-free medium. The total rhizospheric and phyllospheric microbial consortia could attenuate crude oil in complete (nitrogen-containing) medium, but also equally well in a nitrogen-free medium. It was concluded that H. strobilaceum could be a valuable halophyte for phytoremediation of oil-polluted hypersaline environments via rhizosphere technology.

The potential of oil-utilizing bacterial consortia associated with legume root nodules for cleaning oily soils.

The surfaces of root nodules of Vicia faba and Lupinus albus (legume crops), were colonized with bacterial consortia which utilized oil and fixed nitrogen. Such combined activities apparently make those periphytic consortia efficient contributors to bioremediation of oily nitrogen-poor desert soils. This was confirmed experimentally in this study. Thus, cultivating V. faba, L. albus and, for comparison, Solanum melongena, a nonlegume crop, separately in oily sand samples resulted in more oil attenuation than in an uncultivated sample. This effect was more pronounced with the legume crops than with the nonlegume crop. Furthermore, in flask cultures, V. faba plants with nodulated roots exhibited a higher potential for oil attenuation in the surrounding water than plants with nodule-free roots. Denaturation gradient gel electrophoresis (DGGE) of polymerase chain reaction amplified 16S rRNA coding genes revealed that periphytic bacteria had DGGE bands not matching those of the oil-utilizing rhizospheric bacteria. Legume nodules also contained endophytic bacteria whose 16S rDNA bands did not match those of Rhizobium nor those of all other individual periphytic and rhizospheric strains. It was concluded that legume crops host on their roots bacterial consortia with a satisfactory potential for oil phytoremediation.

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.

Crude oil and hydrocarbon-degrading strains of Rhodococcus rhodochrous isolated from soil and marine environments in Kuwait.

Soil and marine samples collected from different localities in Kuwait were screened for microorganisms capable of oil degradation. Both fungi and bacteria were isolated. The fungal flora consisted of Aspergillus terreus, A. sulphureus, Mucor globosus, Fusarium sp. and Penicillum citrinum. Mucor globosus was the most active oil degrading fungus isolated. Bacterial isolates included Bacillus spp. Enterobacteriaceae, Pseudomonas spp., Nocardia spp., Streptomyces spp.,and Rhodococcus spp. Among these Rhodococcus strains were the most efficient in oil degradation and, relatively speaking, the most abundant. Bacterial and fungal isolates differed in their ability to degrade crude oil, with Rhodococcus isolates being more active that fungin in n-alkane biodegradation, particularly in the case of R. rhodochrous. In addition to medium chain n-alkanes, fungi utilized one or more of the aromatic hydrocarbons studied, while bacteria failed to do so. R. rhodochorous KUCC 8801 was shown by GLC and post-growth studies to be more efficient in oil degradation than isolates known to be active oil degraders.

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.