M. Eliyas

The potential of epiphytic hydrocarbon-utilizing bacteria on legume leaves for attenuation of atmospheric hydrocarbon pollutants.

The leaves of two legumes, peas and beans, harbored on their surfaces up to 9×10⁷ cells g⁻¹ of oil-utilizing bacteria. Less numbers, up to 5×10⁵ cells g⁻¹ inhabited leaves of two nonlegume crops, namely tomato and sunflower. Older leaves accommodated more of such bacteria than younger ones. Plants raised in oily environments were colonized by much more oil-utilizing bacteria than those raised in pristine (oil-free) environments. Similar numbers were counted on the same media in which nitrogen salt was deleted, indicating that most phyllospheric bacteria were probably diazotrophic. Most dominant were Microbacterium spp. followed by Rhodococcus spp., Citrobacter freundii, in addition to several other minor species. The pure bacterial isolates could utilize leaf tissue hydrocarbons, and consume considerable proportions of crude oil, phenanthrene (an aromatic hydrocarbon) and n-octadecane (an alkane) in batch cultures. Bacterial consortia on fresh (but not on previously autoclaved) leaves of peas and beans could also consume substantial proportions of the surrounding volatile oil hydrocarbons in closed microcosms. It was concluded that phytoremediation through phyllosphere technology could be useful in remediating atmospheric hydrocarbon pollutants.

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.

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.

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.

Most hydrocarbonoclastic bacteria in the total environment are diazotrophic, which highlights their value in the bioremediation of hydrocarbon contaminants.

Eighty-two out of the 100 hydrocarbonoclastic bacterial species that have been already isolated from oil-contaminated Kuwaiti sites, characterized by 16S rRNA nucleotide sequencing, and preserved in our private culture collection, grew successfully in a mineral medium free of any nitrogenous compounds with oil vapor as the sole carbon source. Fifteen out of these 82 species were selected for further study based on the predominance of most of the isolates in their specific sites. All of these species tested positive for nitrogenase using the acetylene reduction reaction. They belonged to the genera Agrobacterium, Sphingomonas, and Pseudomonas from oily desert soil and Nesiotobacter, Nitratireductor, Acinetobacter, Alcanivorax, Arthrobacter, Marinobacter, Pseudoalteromonas, Vibrio, Diatzia, Mycobacterium, and Microbacterium from the Arabian/Persian Gulf water body. A PCR-DGGE-based sequencing analysis of nifH genes revealed the common occurrence of the corresponding genes among all the strains tested. The tested species also grew well and consumed crude oil effectively in NaNO3 -containing medium with and without nitrogen gas in the top space. On the other hand, these bacteria only grew and consumed crude oil in the NaNO3 -free medium when the top space gas contained nitrogen. We concluded that most hydrocarbonoclastic bacteria are diazotrophic, which allows for their wide distribution in the total environment. Therefore, these bacteria are useful for the cost-effective, environmentally friendly bioremediation of hydrocarbon contaminants.

Culture-Dependent and Culture-Independent Analysis of Hydrocarbonoclastic Microorganisms Indigenous to Hypersaline Environments in Kuwait

The halophilic, hydrocarbonoclastic bacteria and archaea inhabiting two hypersaline coastal areas in Kuwait, one in the north and the other in the south, were counted and characterized. Environmental parameters in both areas were similar, with the exception of the soil organic carbon content, which was in the north higher than in the south. The hydrocarbonoclastic bacterial and haloarchaeal numbers and identities as analyzed using nutrient media of various salinities were similar in soil and pond water samples from both areas. The bacterial species recorded by this culture-dependent method belonged to the genera Halomonas, Chromohalobacter, Marinobacter, Exiguobacterium, Stenotrophomonas, Pseudomonas, Salinivibrio, and Bacillus. The haloarchaeal species belonged to the genera Haloferax and Halobacterium. When analyzed by fingerprinting of their amplified genomic DNA followed by sequencing of the electrophoresis-resolved bands, the same environmental samples revealed a different microbial composition. Bacterial phylotypes recorded by this culture-independent method were affiliated with the genera Ochrobactrum, Stenotrophomonas, Rhodococcus, and “Halomicrobium,” whereas the archaeal phylotypes were affiliated with Halorussus, Halomicrobium, and Halorientalis. The observed diversity and composition similarity of the hydrocarbonocalastic microflora in both hypersaline areas suggest an effective potential for oil mineralization therein. This potential has been confirmed experimentally.

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.

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.

Ferric Sulfate and Proline Enhance Heavy-Metal Tolerance of Halophilic/Halotolerant Soil Microorganisms and Their Bioremediation Potential for Spilled-Oil Under Multiple Stresses

The aim of this study was to explore the heavy-metal resistance and hydrocarbonoclastic potential of microorganisms in a hypersaline soil. For this, hydrocarbonoclastic microorganisms were counted on a mineral medium with oil vapor as a sole carbon source in the presence of increasing concentrations of ZnSO4, HgCl2, CdSO4, PbNO3, CuSO4, and Na2HAsO4. The colony-forming units counted decreased in number from about 150 g-1 on the heavy-metal-free medium to zero units on media with 40–100 mg l-1 of HgCl2, CdSO4, PbNO3, or Na2HAsO4. On media with CuSO4 or ZnSO4 on the other hand, numbers increased first reaching maxima on media with 50 mg l-1 CuSO4 and 90 mg l-1 ZnSO4. Higher concentrations reduced the numbers, which however, still remained considerable. Pure microbial isolates in cultures tolerated 200–1600 mg l-1 of HgCl2, CdSO4, PbNO3, CuSO4, and Na2HAsO4 in the absence of crude oil. In the presence of oil vapor, the isolates tolerated much lower concentrations of the heavy metals, only 10–80 mg l-1. The addition of 10 Fe2(SO4)3 and 200 mg l-1 proline (by up to two- to threefold) enhanced the tolerance of several isolates to heavy metals, and consequently their potential for oil biodegradation in their presence. The results are useful in designing bioremediation technologies for oil spilled in hypersaline areas.

Consistent Occurrence of Hydrocarbonoclastic Marinobacter Strains in Various Cultures of Picocyanobacteria from the Arabian Gulf: Promising Associations for Biodegradation of Marine Oil Pollution

Fifteen nonaxenic cultures of picocyanobacteria were isolated from the Arabian Gulf, from which 122 heterotrophic bacterial strains were obtained. Based on their 16S rRNA gene sequences, those strains were affiliated with 22 different species, 82.8% of which belonged to the genus Marinobacter, known to comprise hydrocarbonoclastic strains. The remaining species belonged to the genera Alcanivorax, Bacillus, Halomonas, Mesorhizobium, and Paenibacillus, and a Bacteriodetes bacterium also known to comprise hydrocarbonoclastic strains. All the picocyanobacterial cultures harbored one or more strains of Marinobacter. Marinobacter in addition to Alcanivorax and other genera isolated from those picocyanobacteria grew on Tween 80, crude oil, and pure hydrocarbons as sole sources of carbon and energy, i.e. they are related to the obligate hydrocarbonoclastic bacteria group. They consumed crude oil, n-octadecane, and phenanthrene in batch cultures. The results indicated that Marinobacter isolates seemed to grow better and consume more oil in the presence of their host picocyanobacteria than in their absence. Such natural microbial associations assumingly play a role in bioremediation of spilled hydrocarbons in the Arabian Gulf. Similar associations probably occur in other marine environments as well and are active in oil spill removal.