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.     

Bioremediation of volatile oil hydrocarbons by epiphytic bacteria associated with American grass (Cynodon sp.) and broad bean (Vicia faba) leaves

Fresh leaves of American grass and broad beans grown in pristine soil were naturally colonized with cultivable volatile oil hydrocarbon-utilizing bacteria, whose numbers increased significantly in plants grown in oily soil. According to their 16S rRNA gene sequences those bacteria were affiliated to various species of the genera Rhodococcus and Pseudomonas. Qualitative growth studies revealed that pure cultures of these phyllospheric bacteria could grow successfully on a solid mineral medium containing individual alkanes with chain lengths of C₉ through C₄₀ and the aromatics phenanthrene, naphthalene, and biphenyl as sole sources of carbon and energy. Quantitative measurements showed that the individual pure bacterial isolates degraded between about 20 and 30% of crude oil, n-hexadecane, or phenanthrene in batch culture after a one-week incubation. These results reflect the high hydrocarbon degradation potential of those bacteria. The isolates were diazotrophic (nitrogen fixers), meaning that they were self-dependent in covering their nitrogen requirements. Incubating fresh leaves in closed microcosms containing volatile oil hydrocarbons resulted in up to more than 80% attenuation of these compounds after two weeks. Experimental evidence was provided that the leaf tissues did not contribute to this attenuation, which was exclusively due to the bacterial activity.     

Alkaliphilic and halophilic hydrocarbon-utilizing bacteria from Kuwaiti coasts of the Arabian Gulf

Green animate materials from the intertidal zone of the Arabian Gulf coast accommodated more alkaliphilic and halophilic bacteria than inanimate materials. The alkaliphilic oil-utilizing bacteria, as identified by their 16S ribonucleic acid sequences, belonged to the following genera arranged in decreasing frequences: Marinobacter, Micrococcus, Dietzia, Bacillus, Oceanobacillus, and Citricoccus. The halophilic oil-utilizing bacteria belonged to the genera: Marinobacter, Georgenia, Microbacterium, Stappia, Bacillus, Isoptericola, and Cellulomonas. Most isolates could grow on a wide range of pure n-alkanes and aromatic compounds, as sole sources of carbon and energy. Quantitative gas liquid chromatographic analysis showed that individual isolates attenuated crude oil and representative pure hydrocarbons in culture. The optimum pH for most of the alkaliphilic genera was pH 10, and the optimum salinity for the halophiles ranged between 2.5 and 5% NaCl (w/v). It was concluded that as far as their microbial makeup is concerned, oily alkaline and saline intertidal areas of the Kuwaiti coasts have a self-cleaning potential.     

Biodegradation of Crude Oil by Thermophilic Bacteria Isolated from a Volcano Island

One-hundred and fifty different thermophilic bacteria isolated from a volcanic island were screened for detection of an alkane hydroxylase gene using degenerated primers developed to amplify genes related to the Pseudomonas putida and Pseudomonas oleovorans alkane hydroxylases. Ten isolates carrying the alkJ gene were further characterized by 16s rDNA gene sequencing. Nine out of ten isolates were phylogenetically affiliated with Geobacillus species and one isolate with Bacillus species. These isolates were able to grow in liquid cultures with crude oil as the sole carbon source and were found to degrade long chain crude oil alkanes in a range between 46.64% and 87.68%. Results indicated that indigenous thermophilic hydrocarbon degraders of Bacillus and Geobacillus species are of special significance as they could be efficiently used for bioremediation of oil-polluted soil and composting processes.     

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.     

Identities of Epilithic Hydrocarbon-Utilizing Diazotrophic Bacteria from the Arabian Gulf Coasts, and Their Potential for Oil Bioremediation Without Nitrogen Supplementation

Gravel particles from four sites along the Arabian Gulf coast in autumn, winter, and spring were naturally colonized with microbial consortia containing between 7 and 400 × 10² cm⁻² of cultivable oil-utilizing bacteria. The 16S rRNA gene sequences of 70 representatives of oil-utilizing bacteria revealed that they were predominantly affiliated with the Gammaproteobacteria and the Actinobacteria. The Gammaproteobacteria comprised among others, the genera Pseudomonas, Pseudoalteromonas, Shewanella, Marinobacter, Psychrobacter, Idiomarina, Alcanivorax, Cobetia, and others. Actinobacteria comprised the genera Dietzia, Kocuria, Isoptericola, Rhodococcus, Microbacterium, and others. In autumn, Firmicutes members were isolated from bay and nonbay stations while Alphaproteobacteria were detected only during winter from Anjefa bay station. Fingerprinting by denaturing gradient gel electrophoresis of amplified 16S rRNA genes of whole microbial consortia confirmed the culture-based bacterial diversities in the various epilithons in various sites and seasons. Most of the representative oil-utilizing bacteria isolated from the epilithons were diazotrophic and could attenuate oil also in nitrogen-rich (7.9–62%) and nitrogen-free (4–54%) cultures, which, makes the microbial consortia suitable for oil bioremediation in situ, without need for nitrogen supplementation. This was confirmed in bench-scale experiments in which unfertilized oily seawater was bioremediated by epilithon-coated gravel particles.     

Enhancing the growth of Vicia faba plants by microbial inoculation to improve their phytoremediation potential for oily desert areas

Two experiments were conducted to investigate the effect of inoculating Vicia faba plants (broad beens) raised in clean and oily sand with nodule-forming rhizobia and plant-growth-promoting rhizobacteria (PGPR) on growth of these plants in sand and to test whether this can improve the phytoremediation potential of this crop for oily desert areas. It was found that crude oil in sand at concentrations < 1.0% (w/w) enhanced the plant heights, their fresh and dry weights, the total nodule weights per plant, and the nitrogen contents of shoots and fruits. Similar enhancing effects were recorded when roots of the young plants were inoculated with nodule bacteria alone, PGPR alone, or a mixture of one strain of nodule bacteria and one of the PGPR. Such plant growth effects were associated with a better phytoremediation potential of V. faba plants for oily sand. The total numbers of oil-utilizing bacteria increased in the rhizosphere and more hydrocarbons were eliminated in sand close to the roots. The nodule bacteria tested were two strains of Rhizobium leguminosarum and the PGPR were Pseudomonas aeruginosa and Serratia liquefaciens. The four strains were found to use crude oil, n-octadecane, and phenanthrene as sole sources of carbon and energy. It was concluded that coinoculation of V. faba plant roots in oily sand with nodule bacteria and PGPR enhances the phytoremediation potential of this plant for oily desert sand through improving plant growth and nitrogen fixation.     

Inoculation of 1-aminocyclopropane-1-carboxylate deaminase–producing bacteria along with biosurfactant application enhances the phytoremediation efficiency of Medicago sativa in hydrocarbon-contaminated soils

Phytoremediation efficiency of Alfa alfa (Medicago sativa) was evaluated in hydrocarbon-contaminated soil with the combined application of 1-aminocyclopropane-1-carboxylate (ACC) deaminase–producing Bacillus sp. PVMX4 and an isolated biosurfactant from this strain. Results on the plant growth–promoting (PGP) traits of Bacillus sp. PVMX4 revealed that phosphate (P) solubilization, indole-3-acetic acid (IAA) production, and ACC deaminase activity were not affected by low-concentration hydrocarbon amendment in the form of crude oil. Bacillus sp. PVMX4 was able to utilize crude oil as a sole carbon source in mineral salt medium (MSM), and this strain synthesized significant quantities of biosurfactant in growth medium quantified by an emulsification index of 69.2 EI_24% and surface tension reduction of 26.2 mN/m at the end of the experimental period. Biosurfactant, when partially purified and characterized by thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR), revealed it to be a lipopeptide-type biosurfactant. Pilot-scale phytoremediation studies conducted under growth chamber conditions in hydrocarbon-contaminated soil using Medicago sativa along with combined application of ACC deaminase–containing bacteria and biosurfactant recorded 76.4% hydrocarbon degradation.     

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.     

Plant and fertiliser effects on rhizodegradation of crude oil in two soils with different nutrient status

Soils and sediments polluted with crude oil are of major environmental concern on various contaminated sites. Outdoors pot experiments were conducted to test the phytodegradation potential of common reed (Phragmites australis) and poplar (Populus nigra × maximowiczii) in fertilised and non-fertilised control treatments. Two topsoils (E, G) of different texture were mixed with crude oil. Soil analysis included hydrocarbon (HC) measurements, detection of labile phosphorus and mineralised nitrogen as well as dehydrogenase activity. Increased HC degradation by native soil biota was clearly related to higher P availability in soil G and to fertilisation in soil E. Except of the non-fertilised common reed treatment, plants did not enhance crude oil degradation. We found even inhibited degradation of high molecular weight HC in the presence of plants together with declining labile phosphorous concentrations due to planting on soil E. Native soil biota were able to use the whole range of crude oil compounds (C₁₀ to C₆₀) as a carbon source in the presence of sufficient nutrient concentrations in soil. This study is the first to show that reduced HC degradation in the higher molecular weight crude oil fraction (C₂₀ to C₄₀) is likely to be a consequence of decreased phosphorus availability for microorganisms in the plant rhizosphere.     

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

N.A. SORKHOH, R.H. AL-HASAN, M. KHANAFER AND S.S. RADWAN. 1995. 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 Rhodcoccus 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.     

Hydrocarbon degradation in relation to cell-surface hydrophobicity among bacterial hydrocarbon degraders from petroleum-contaminated Kuwait desert environment

Forty six bacterial isolates able to grow on crude oil were isolated from various hydrocarbon-contaminated sites in Kuwait. The extent of crude oil degradation varied over a wide range (1–87%) among the isolates. Isolates were predominantly Gram-positive bacteria (79% of total isolates) belonging to the genera Bacillus (93%) and Paenibacillus (7%). Among the few Gram-negative isolates were from the genera Acinetobacter, Alcaligenes, Klebsiella, Burkholderia, Pseudomonas, and Williamsia. Analyses of their cell-surface hydrophobicity (CSH) by various methods equally showed a wide variation among the isolates. About 74% of isolates that degraded significant amounts of crude oil (>40% degradation) possessed high level of CSH, while 58% of all the isolates exhibited high levels of CSH. Statistical analyses showed significantly high correlation between the ability to degrade crude oil and CSH. The ability of the isolates to bind to polystyrene and salt-aggregation test as measures of CSH were more strongly correlated with hydrocarbon-degrading ability than adherence to hydrocarbons.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

A refinery sludge deposition site: presence of nahH and alkJ genes and crude oil biodegradation ability of bacterial isolates

204 bacterial isolates from four Greek refinery sludge deposition sites were investigated for the presence of nahH and alkJ genes encoding key enzymes of both aromatic and aliphatic hydrocarbon degradation pathways by PCR and DNA hybridisation. Members of Pseudomonas, Acinetobacter, Bacillus, Rhodococcus and Arthrobacter play important role in bioremediation processes in sandy/loam soil contaminated with oil and nahH and alkJ genes were present in the 73% of the isolates. Consortia of bacterial isolates that were used for biodegradation of aliphatic and aromatic hydrocarbons in crude oil using liquid cultures exhibited rates from 35% to 48% within 10 days of incubation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of bacteria-immobilized cotton fibers to absorb and degrade crude oil

Using enrichment culture technique, two isolates that brought a significant degradation and dispersion of crude oil were obtained from contaminated sediments of the Bohai Bay, China. 16S rRNA gene sequencing and phylogenetic analysis indicated that the two bacterial strains affiliated with the genera Vibrio and Acinetobacter. Subsequently, the bacterial cells were immobilized on the surface of cotton fibers. Cotton fibers were used as crude oil sorbent as well as a biocarrier for bacteria immobilization. Among the two isolates, the marine bacteria Acinetobacter sp. HC8-3S showed a strong binding to the cotton fibers, possibly enhanced through extracellular dispersant excreted by Acinetobacter sp. HC8-3S. Both planktonic and immobilized bacteria showed relatively high biodegradation (>60%) of saturated hydrocarbons fraction of crude oil, in the pH range of 5.6–8.6. The degradation activities of planktonic and immobilized bacteria were not affected significantly when the NaCl concentration reached 70 g/L. The immobilized bacterial cells exhibited an enhanced biodegradation of crude oil. The efficiency of saturated hydrocarbons degradation by the immobilized bacterial cells increased about 30% compared to the planktonic bacterial cells.     

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

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.     

Microbial changes during oil decomposition in soil

An examination has been made of the changes in bacterial and fungal populations during the decomposition of oil in contaminated soil. The number of aerobic and anaerobic bacteria and the length of mycelium increased in the oily soil whereas the number of CFU (= colony forming units) of fungi was highest in a control soil. The percentage of oil-utilizing fungi increased from 60% to 82%, while the bacterial utilization figure increased from 3% to 50%. The important oil-utilizing fungus Scolecobasidium appeared only in the oily soil, but otherwise the composition of the fungal flora changed only little after addition of oil. In laboratory experiments the chemical Pajab FI was shown to increase microbial activity.     

Development of a method for the application of solid-phase microextraction to monitor biodegradation of volatile hydrocarbons during bacterial growth on crude oil

A quantitative solid-phase microextraction, gas chromatography, flame ionization detector (SPME-GC-FID) method for low-molecular-weight hydrocarbons from crude oil was developed and applied to live biodegradation samples. Repeated sampling was achieved through headspace extractions at 30°C for 45 min from flasks sealed with Teflon Mininert. Quantification without detailed knowledge of oil–water–air partition coefficients required the preparation of standard curves. An inverse relationship between retention time and mass accumulated on the SPME fibre was noted. Hydrocarbons from C₅ to C₁₆ were dated and those up to C₁₁ were quantified. Total volatiles were quantified using six calibration curves. Biodegradation of volatile hydrocarbons during growth on crude oil was faster and more complete with a mixed culture than pure isolates derived therefrom. The mixed culture degraded 55% of the compounds by weight in 4 days versus 30–35% by pure cultures of Pseudomonas aeruginosa, Rhodococcus globerulus or a co-culture of the two. The initial degradation rate was threefold higher for the mixed culture, reaching 45% degradation after 48 h. For the mixed culture, the degradation rate of individual alkanes was proportional to the initial concentration, decreasing from hexane to undecane. P. fluorescens was unable to degrade any of the low-molecular-weight hydrocarbons and methylcyclohexane was recalcitrant in all cases. Overall, the method was found to be reliable and cost-effective. Journal of Industrial Microbiology & Biotechnology (2000) 25, 155–162. Received 04 March 2000/ Accepted in revised form 25 June 2000     

Bench-Scale Production of Biosurfactants and their Potential in Ex-Situ MEOR Application

The fermentative production of biosurfactants by five Bacillus strains in a bench-scale bioreactor and evaluation of biosurfactant-based enhanced oil recovery using sand pack columns were investigated. Adjusting the initial dissolved oxygen to 100% saturation, without any further control and with collection of foam and recycling of biomass, gave higher biosurfactant production. The microorganisms were able to produce biosurfactants, thus reducing the surface tension and interfacial tension to 28 mN/m and 5.8–0.5 mN/m, respectively, in less than 10 hours. The crude surfactant concentration of 0.08–1.1 g/L, and critical micelle concentration (CMC) values of 19.4–39 mg/L, corresponding to the biosurfactants produced by the different Bacillus strains, were observed. The efficiency of crude biosurfactant preparation obtained from Bacillus strains for enhanced oil recovery, by sand pack column studies, revealed it to vary from 30.22–34.19% of the water flood residual oil saturation. The results are indicative of the potential of the strains for the development of ex-situ, microbial-enhanced, oil recovery processes.     

Vitamin requirements of hydrocarbon-utilizing soil bacteria

The numbers of oil-utilizing bacteria in several samples of clean and oil-polluted soils counted on vitamin-containing media were severalfold higher than the numbers counted on vitamin-free media. Colonies that grew on a medium containing a vitamin mixture were tested for growth on the same medium lacking any vitamins. More than 90% of the total colonies failed to grow. The remaining 10% grew, yet their growth was enhanced, when vitamins were added. The predominant oil-utilizing bacteria in one of the test desert soil samples were various strains of Cellulomonas flavigena and Rhodococcus erythropolis. Minor organisms belonged to the genera Pseudomonas, Bacillus and Arthrobacter. Two vitamin-requiring biovars of C. flavigena and R. erythropolis were selected for further study. Their growth on n-octadecane and phenanthrene as sole sources of carbon and energy as well as their potential for hydrocarbon consumption were enhanced by added vitamins, e.g. folic acid, pyridoxine, vitamin B12, biotin and others. In a field experiment, it was confirmed that vitamin fertilization of an oil-polluted sand sample enhanced the biodegradation of constituent hydrocarbons of that sample.