H. Al-Awadhi

Rhizospheric hydrocarbon-utilizing microorganisms as potential contributors to phytoremediation for the oily Kuwaiti desert.

Roots of the wild desert plants, Senecio glaucus, Cyperus conglomeratus, Launaea mucronata, Picris babylonica and Salsola imbricata and the crop plants Vicia faba and Lupinus albus grown in oil polluted and clean soils were densely associated with hydrocarbon utilizing bacteria. The most dominant were Cellulomonas flavigena, Rhodococcus erythropolis and Arthrobacter sp. The rhizosphere soils of all plants contained more hydrocarbon-utilizers than the soils apart. This rhizosphere effect was much more pronounced for plants growing in oil-polluted than clean soils. C. flavigena predominant in the rhizosphere of Vicia faba took up representative test aliphatic and aromatic hydrocarbons. Thus, these bacteria could be contributing in nature to detoxifying and bioremediating the soil around the roots. It was concluded that vegetation may be a feasible approach for cleaning oil-polluted soil, including the polluted Kuwaiti desert areas.

Bias problems in culture-independent analysis of environmental bacterial communities: a representative study on hydrocarbonoclastic bacteria

Culture-dependent methods for bacterial community analysis are currently considered obsolete; therefore, molecular techniques are usually used instead. The results of the current study on hydrocarbonoclastic bacteria in various oily habitats in Kuwait showed however, that the bacterial identities varied dramatically according to the analytical approach used. For six desert and six seawater samples used in this study, the culture-independent and culture-dependent techniques each led to a unique bacterial composition. Problems related to the culture-dependent technique are well known. The results of the current study highlighted bias problems other than those already recorded in the literature for the molecular approaches. Thus, for example, in contrast to the culture-dependent technique, the primers used in the molecular approach preferentially amplified the 16S rDNAs of hydrocarbonoclastic bacteria in total genomic DNAs of all the studied environmental samples, and in addition, failed to reveal in any environmental sample members of the Actinobacteria. The primers used in the molecular approach also amplified certain “pure” 16S rDNAs, but failed to do so when these DNAs were in mixture. In view of these results, it is recommended that the two analytical approaches should be used simultaneously because their combined results would reflect the bacterial community composition more precisely than either of them can do alone.

Indigenous hydrocarbon-utilizing bacterioflora in oil-polluted habitats in Kuwait, two decades after the greatest man-made oil spill

Kuwaiti habitats with two-decade history of oil pollution were surveyed for their inhabitant oil-utilizing bacterioflora. Seawater samples from six sites along the Kuwaiti coasts of the Arabian Gulf and desert soil samples collected from seven sites all over the country harbored oil-utilizing bacteria whose numbers made up 0.0001–0.01% of the total, direct, microscopic counts. The indigenous bacterioflora in various sites were affiliated to many species. This was true when counting was made on nitrogen-containing and nitrogen-free media. Seawater samples harbored species belonging predominantly to the Gammaproteobacteria and desert soil samples contained predominantly Actinobacteria. Bacterial species that grew on the nitrogen-free medium and that represented a considerable proportion of the total in all individual bacterial consortia were diazotrophic. They gave positive acetylene-reduction test and possessed the nifH genes in their genomes. Individual representative species could utilize a wide range of aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy. Quantitative determination showed that the individual species consumed crude oil, n-octadecane and phenanthrene, in batch cultures. It was concluded that the indigenous microflora could be involved in bioremediation programs without bioaugmentation or nitrogen fertilization. Irrigation would be the most important practice in bioremediation of the polluted soil desert areas.

Cropping as a Phytoremediation Practice for Oily Desert Soil with Reference to Crop Safety as Food

ABSTRACT Broad beans (Vicia faba)could tolerate up to 10% (w)w) crude oil in desert soil (sand), therefore, the potential of this crop for cleaning oily desert soil via rhizosphere technology was investigated. The amounts of hydrocarbons recovered from oily desert soil samples supporting V. faba plants were less than the amounts extracted from uncultivated oily soil samples. Excised fresh V. faba roots with their intact rhizospheres resulted in the attenuation of n-octadecane, phenanthrene, and crude oil when shaken into sterile desert soil extract containing these hydrocarbons. The amounts of hydrocarbons eliminated were greater with roots of plants previously raised in oily soil than with roots of plants raised in clean soil. Similar hydrocarbon attenuation effects were recorded when, instead of excised roots, whole plants were used with their roots submerged in the hydrocarbon containing soil extract. The various parts of plants raised in oily desert soil contained more linolenic acid in their total lipids than did the same parts of plants raised in clean desert soil. This was much more pronounced for the roots than for shoots and seeds. The hydrocarbons of roots and shoots of V. faba plants were not as affected by oil pollution as were those of seeds, in which the proportions of very long chain hydrocarbons increased with increasing oil concentration in the soil. Those hydrocarbons are not recommended for human and animal nutrition.

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.

Establishing oil-degrading biofilms on gravel particles and glass plates

Abstract A method is described for “artificially” establishing biofilms rich in hydrocarbon degrading bacteria on gravel particles and glass plates. The microbial consortia in the biofilms included in additions, filamentous cyanobacteria, picoplankton and diatoms. Phototrophic microorganisms were pioneer colonizers. Hydrocarbon utilizing bacteria, namely Acinetobacter calcoaceticus and nocardioforms were in part attached to filaments of cyanobacteria. In batch cultures, it was shown that those artificial biofilms had an attenuation effect on crude-oil in contaminated sea water samples. The potential use of these biofilms for preparing trickling filters (gravel particles), and in bioreactors (glass plates) for attenuating hydrocarbons in oily liquid wastes before their disposal in the open environment is suggested and discussed.

PlANT-ASSOCIATED BACTERIA AS TOOLS FOR THE PHYTOREMEDIATION OF OILY NITROGEN-POOR SOILS

The rhizospheres and phyllospheres of peas, beans, tomatoes, and squash raised in a desert sand soil mixed with 0.5% crude oil were rich in oil-utilizing bacteria and accommodated large numbers of free-living diazotrophic bacteria, with potential for hydrocarbon utilization. According to their 16S rRNA-sequences, the cultivable oil-utilizing bacteria were affiliated with the following genera, arranged in decreasing frequency: Bacillus, Ochrobactrum, Enterobacter, Rhodococcus, Arthrobacter, Pontola, Nocardia, and Pseudoxanthomonas. Diazotrophic isolates were affiliated with Rhizobium, Bacillus, Rhodococcus, Leifsonia, Cellulosimicrobium, Stenotrophomonas, Kocuria, Arthrobacter, and Brevibacillus. The crude oil–utilizing and diazotrophic isolates grew, with varying growth intensities, on individual aliphatic (C8 to C40) and aromatic hydrocarbons, as sole sources of carbon and energy. Quantitative gas liquid chromatographic measurements showed that representative bacterial isolates eliminated pure n-hexadecane, n-decosane, phenanthrene, and crude oil from the surrounding liquid media. Cultivation of oily sand–soil samples with any of the four tested crops led to enhanced oil degradation in that soil, as compared with the degradation in uncultivated oily sand–soil samples.

Olive-pomace harbors bacteria with the potential for hydrocarbon-biodegradation, nitrogen-fixation and mercury-resistance: Promising material for waste-oil-bioremediation

Olive-pomace, a waste by-product of olive oil industry, took up >40% of its weight crude oil. Meanwhile, this material harbored a rich and diverse hydrocarbonoclastic bacterial population in the magnitude of 10(6) to 10(7) cells g(-1). Using this material for bioaugmentation of batch cultures in crude oil-containing mineral medium, resulted in the consumption of 12.9, 21.5, 28.3, and 43% oil after 2, 4, 6 and 8 months, respectively. Similar oil-consumption values, namely 11.0, 29.3, 34.7 and 43.9%, respectively, were recorded when a NaNO3-free medium was used instead of the complete medium. Hydrocarbonoclastic bacteria involved in those bioremediation processes, as characterized by their 16S rRNA-gene sequences, belonged to the genera Agrococcus, Pseudomonas, Cellulosimicrobium, Streptococcus, Sinorhizobium, Olivibacter, Ochrobactrum, Rhizobium, Pleomorphomonas, Azoarcus, Starkeya and others. Many of the bacterial species belonging to those genera were diazotrophic; they proved to contain the nifH-genes in their genomes. Still other bacterial species could tolerate the heavy metal mercury. The dynamic changes of the proportions of various species during 8 months of incubation were recorded. The culture-independent, phylogenetic analysis of the bacterioflora gave lists different from those recorded by the culture-dependent method. Nevertheless, those lists comprised among others, several genera known for their hydrocarbonoclastic potential, e.g. Pseudomonas, Mycobacterium, Sphingobium, and Citrobacter. It was concluded that olive-pomace could be applied in oil-remediation, not only as a physical sorbent, but also for bioaugmentation purposes as a biological source of hydrocarbonoclastic bacteria.

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.

Dynamics of bacterial populations during bench-scale bioremediation of oily seawater and desert soil bioaugmented with coastal microbial mats.

This study describes a bench‐scale attempt to bioremediate Kuwaiti, oily water and soil samples through bioaugmentation with coastal microbial mats rich in hydrocarbonoclastic bacterioflora. Seawater and desert soil samples were artificially polluted with 1% weathered oil, and bioaugmented with microbial mat suspensions. Oil removal and microbial community dynamics were monitored. In batch cultures, oil removal was more effective in soil than in seawater. Hydrocarbonoclastic bacteria associated with mat samples colonized soil more readily than seawater. The predominant oil degrading bacterium in seawater batches was the autochthonous seawater species Marinobacter hydrocarbonoclasticus. The main oil degraders in the inoculated soil samples, on the other hand, were a mixture of the autochthonous mat and desert soil bacteria; Xanthobacter tagetidis, Pseudomonas geniculata, Olivibacter ginsengisoli and others. More bacterial diversity prevailed in seawater during continuous than batch bioremediation. Out of seven hydrocarbonoclastic bacterial species isolated from those cultures, only one, Mycobacterium chlorophenolicum, was of mat origin. This result too confirms that most of the autochthonous mat bacteria failed to colonize seawater. Also culture‐independent analysis of seawater from continuous cultures revealed high‐bacterial diversity. Many of the bacteria belonged to the Alphaproteobacteria, Flavobacteria and Gammaproteobacteria, and were hydrocarbonoclastic. Optimal biostimulation practices for continuous culture bioremediation of seawater via mat bioaugmentation were adding the highest possible oil concentration as one lot in the beginning of bioremediation, addition of vitamins, and slowing down the seawater flow rate.