Bioremediation of soils saturated with spilled crude oil.

A desert soil sample was saturated with crude oil (17.3%, w/w) and aliquots were diluted to different extents with either pristine desert or garden soils. Heaps of all samples were exposed to outdoor conditions through six months, and were repeatedly irrigated with water and mixed thoroughly. Quantitative determination of the residual oil in the samples revealed that oil-bioremediation in the undiluted heaps was nearly as equally effective as in the diluted ones. One month after starting the experiment. 53 to 63% of oil was removed. During the subsequent five months, 14 to 24% of the oil continued to be consumed. The dynamics of the hydrocarbonoclastic bacterial communities in the heaps was monitored. The highest numbers of those organisms coordinated chronologically with the maximum oil-removal. Out of the identified bacterial species, those affiliated with the genera Nocardioides (especially N. deserti), Dietzia (especially D. papillomatosis), Microbacterium, Micrococcus, Arthrobacter, Pseudomonas, Cellulomonas, Gordonia and others were main contributors to the oil-consumption. Some species, e.g. D. papillomatosis were minor community constituents at time zero but they prevailed at later phases. Most isolates tolerated up to 20% oil, and D. papillomatosis showed the maximum tolerance compared with all the other studied isolates. It was concluded that even in oil-saturated soil, self-cleaning proceeds at a normal rate. When pristine soil receives spilled oil, indigenous microorganisms suitable for dealing with the prevailing oil-concentrations become enriched and involved in oil-biodegradation.

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.

Oil uptake by plant-based sorbents and its biodegradation by their naturally associated microorganisms.

The plant waste-products, wheat straw, corn-cobs and sugarcane bagasse took up respectively, 190, 110 and 250% of their own weights crude oil. The same materials harbored respectively, 3.6 × 105, 8.5 × 103 and 2.3 × 106 g-1 cells of hydrocarbonoclastic microorganisms, as determined by a culture-dependent method. The molecular, culture-independent analysis revealed that the three materials were associated with microbial communities comprising genera known for their hydrocarbonoclastic activity. In bench-scale experiments, inoculating oily media with samples of the individual waste products led to the biodegradation of 34.0-44.9% of the available oil after 8 months. Also plant-product samples, which had been used as oil sorbents lost 24.3-47.7% of their oil via their associated microorganisms, when kept moist for 8 months. In this way, it is easy to see that those waste products are capable of remediating spilled oil physically, and that their associated microbial communities can degrade it biologically.

Autochthonous bioaugmentation with environmental samples rich in hydrocarbonoclastic bacteria for bench-scale bioremediation of oily seawater and desert soil.

Oil-contaminated seawater and desert soil batches were bioaugmented with suspensions of pea (Pisum sativum) rhizosphere and soil with long history of oil pollution. Oil consumption was measured by gas-liquid chromatography. Hydrocarbonoclastic bacteria in the bioremediation batches were counted using a mineral medium with oil vapor as a sole carbon source and characterized by their 16S ribosomal RNA (rRNA)-gene sequences. Most of the oil was consumed during the first 2-4 months, and the oil-removal rate decreased or ceased thereafter due to nutrient and oxygen depletion. Supplying the batches with NaNO3 (nitrogen fertilization) at a late phase of bioremediation resulted in reenhanced oil consumption and bacterial growth. In the seawater batches bioaugmented with rhizospheric suspension, the autochthonous rhizospheric bacterial species Microbacterium oxidans and Rhodococcus spp. were established and contributed to oil-removal. The rhizosphere-bioaugmented soil batches selectively favored Arthrobacter nitroguajacolicus, Caulobacter segnis, and Ensifer adherens. In seawater batches bioaugmented with long-contaminated soil, the predominant oil-removing bacterium was the marine species Marinobacter hydrocarbonoclasticus. In soil batches on the other hand, the autochthonous inhabitants of the long-contaminated soil, Pseudomonas and Massilia species were established and contributed to oil removal. It was concluded that the use of rhizospheric bacteria for inoculating seawater and desert soil and of bacteria in long-contaminated soil for inoculating desert soil follows the concept of "autochthonous bioaugmentation." Inoculating seawater with bacteria in long-contaminated soil, on the other hand, merits the designation "allochthonous bioaugmentation."

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.

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.

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.

Indigenous soil bacteria with the combined potential for hydrocarbon consumption and heavy metal resistance.

INTRODUCTION: Transconjugant bacteria with combined potential for hydrocarbon utilization and heavy metal resistance were suggested by earlier investigators for bioremediation of soils co-contaminated with hydrocarbons and heavy metals. The purpose of this study was to offer evidence that such microorganisms are already part of the indigenous soil microflora. METHODS: Microorganisms in pristine and oily soils were counted on nutrient agar and a mineral medium with oil as a sole carbon source, in the absence and presence of either sodium arsenate (As V), sodium arsenite (As III) or cadmium sulfate, and characterized via 16S rRNA gene sequencing. The hydrocarbon-consumption potential of individual strains in the presence and absence of heavy metal salts was measured. RESULTS: Pristine and oil-contaminated soil samples harbored indigenous bacteria with the combined potential for hydrocarbon utilization and As and Cd resistance in numbers up to 4 × 105 CFU g⁻¹. Unicellular bacteria were affiliated to the following species arranged in decreasing order of predominance: Bacillus subtilis, Corynebacterium pseudotuberculosis, Brevibacterium linens, Alcaligenes faecalis, Enterobacter aerogenes, and Chromobacterium orangum. Filamentous forms were affiliated to Nocardia corallina, Streptomyces flavovirens, Micromonospora chalcea, and Nocardia paraffinea. All these isolates could grow on a wide range of pure aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy, and could consume oil and pure hydrocarbons in batch cultures. Low As concentrations, and to a lesser extent Cd concentrations, enhanced the hydrocarbon-consumption potential by the individual isolates. CONCLUSION: There is no need for molecularly designing microorganisms with the combined potential for hydrocarbon utilization and heavy metal resistance, because they are already a part of the indigenous soil microflora.

Hydrocarbon utilization by nodule bacteria and plant growth-promoting rhizobacteria.

Standard and locally isolated nodule bacteria and plant growth-promoting rhizobacteria (PGPR) were grown on crude oil and individual pure hydrocarbons as sole sources of carbon and energy. The nodule bacteria included two standard Rhizobium leguminosarum strains, two standard Bradyrhizobium japonicum strains, and one unknown nodule bacterial strain that was locally isolated from Vicia faba nodules. The PGPR included one standard Serratia liquefaciens strain and two locally isolated strains of Pseudomonas aeruginosa and Flavobacterium sp. The pure hydrocarbons tested included n-alkanes with chain lengths from C9 to C40 and the aromatic hydrocarbons benzene, biphenyle, naphthalene, phenanthrene, and toluene. Quantitative gas liquid chromatographic analyses confirmed that pure cultures of representative nodule bacteria and PGPR could attenuate n-octadecane and phenanthrene in the surrounding nutrient medium. Further, intact nodules of V. faba containing bacteria immobilized on and within those nodules reduced hydrocarbon levels in a medium in which those nodules were shaken. It was concluded that legume crops are suitable phytoremediation tools for oily soil, since they enrich such soils not only with fixed nitrogen, but also with hydrocarbon-utilizing microorganisms. Further, legume nodules may have biotechnological value as materials for cleaning oily liquid wastes.

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.