Samar Salamah

Bioremediation of oily sea water by bacteria immobilized in biofilms coating macroalgae

Abstract Using the standard plate method and a solid mineral medium containing crude oil as a sole source of carbon and energy, 10 different macroalgae from the Arabian Gulf were found associated with large numbers of oil-utilizing bacteria. Each gram fresh alga was associated with about two to about 30 million cells of bacteria predominantly belonging to the nocardioforms and the genus Acinetobacter . Shaking macroalgal samples in sea water batches containing known amounts of individual hydrocarbons led to considerable attenuation of these compounds as measured by GLC. Thus, bacteria associated with macroalgae consumed about 64–98% of n- octadecane and about 38–56% phenanthrene from medium aliquots containing 0.03% of the test hydrocarbon after 2 weeks. Meanwhile, the oil-utilizing bacteria, especially the nocardioforms, associated with the macroalgae increased in number by about 32–490 fold, depending on the macroalgae and hydrocarbons studied. On the other hand, relatively negligible numbers of bacteria were released into the sea water compared with the numbers immobilized on the macroalgal surfaces. Individual bacterial isolates could grow on a wide range of pure alkanes and aromatic hydrocarbons as sole sources of carbon and energy. It was concluded that macroalgae submerged in the sea waters are coated with biofilms rich in oil-utilizing bacteria, that contribute to hydrocarbon attenuation in water. These natural biological consortia represent valuable tools that could be of high potential for phytoremediation of oily sea water.

Enhanced remediation of hydrocarbon contaminated desert soil fertilized with organic carbons

Fertilizing an oily desert soil sample with a mixture of glucose and peptone resulted in enhancing hydrocarbon disappearance in that soil. The magnitude of hydrocarbon attenuation was too high to be attributed to the nitrogen content of the added peptone alone. Fertilization with KNO3 containing the same amount of nitrogen as in peptone, brought about an enhanced hydrocarbon attenuation aect, but lower in magnitude than fertilization with glucose=peptone. Addition of glucose=peptone to a clean desert soil with hydrocarbon utilizing microorganisms resulted in dramatic increase in their numbers. After 13 days, the microorganisms had depleted all the added organic matter and their numbers decreased. In the oily desert soil, glucose=peptone addition also increased microbial numbers, but after utilization of the glucose=peptone, microbial numbers remained high and enhanced attenuation of hydrocarbons was found. The use of sea water instead of fresh water in these experiments blocked the hydrocarbon attenuation eect. c 2000 Elsevier Science Ltd. All rights reserved.

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.

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.

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 × 106 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.

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.

Culture-independent analysis of hydrocarbonoclastic bacterial communities in environmental samples during oil-bioremediation

To analyze microbial communities in environmental samples, this study combined Denaturing Gradient Gel Electrophoresis of amplified 16S rRNA‐genes in total genomic DNA extracts from those samples with gene sequencing. The environmental samples studied were oily seawater and soil samples, that had been bioaugmented with natural materials rich in hydrocarbonoclastic bacteria. This molecular approach revealed much more diverse bacterial taxa than the culture‐dependent method we had used in an earlier study for the analysis of the same samples. The study described the dynamics of bacterial communities during bioremediation. The main limitation associated with this molecular approach, namely of not distinguishing hydrocarbonoclastic taxa from others, was overcome by consulting the literature for the hydrocarbonoclastic potential of taxa related to those identified in this study. By doing so, it was concluded that the hydrocarbonoclastic bacterial taxa were much more diverse than those captured by the culture‐dependent approach. The molecular analysis also revealed the frequent occurrence of nifH‐genes in the total genomic DNA extracts of all the studied environmental samples, which reflects a nitrogen‐fixation potential. Nitrogen fertilization is long known to enhance microbial oil‐bioremediation. The study revealed that bioaugmentation using plant rhizospheres or soil with long history of oil‐pollution was more effective in oil‐removal in the desert soil than in seawater microcosms.