Indigenous oil-degrading bacteria more efficient in soil bioremediation than microbial consortium and active even in super oil-saturated soils.

A microbial consortium of the hydrocarbonoclastic bacterial species, comprising Actinotalea ferrariae, Arthrobacter ginsengisoli, Dietzia cinnamea, Dietzia papillomatosis, and Pseudomonas songnenensis, isolated from oil-saturated desert soil did not consume more oil in batch cultures than the individual species with the maximum oil consumption. In oil-polluted desert soil microcosms, the rate of oil removal in the soil samples bioaugmented with the microbial consortium was similar to the rate of oil removal in the unbioaugmented ones through a 6-month bioremediation experiment. Although the composition of hydrocarbonoclastic bacterial communities in the unbioaugmented and bioaugmented soil samples was different, the predominant bacterial species during most of the months were the same. Toward the end of the bioremediation experiment, Ar. ginsengisoli prevailed in both soil samples, suggesting its important role in oil removal. Self-cleaning proceeded in desert soil samples artificially polluted with 1, 10, 20, and 30% of crude oil and incubated at 30 °C for 6 months. Oil was removed effectively at rates reaching 73.6 and 69.3% in the soils polluted with 1 and 10% oil concentrations, respectively, and reached 50% in desert soils polluted with 20 and 30% oil concentrations. The bacterial numbers increased in all soil samples from hundreds of thousands per gram of soil samples at time zero to millions and tens of millions per gram of soil samples after 6 months. It was concluded that bioaugmenting oil-polluted soil samples with microbial consortium of hydrocarbonoclastic bacterial species with high oil removal potential did not drastically enhance oil bioremediation and that even in super oil-saturated soils, indigenous oil-degrading bacteria will prevail and effectively contribute to oil removal from the surrounding environment.

Self-cleaning of very heavily oil-polluted sites proceeds even under heavy-metal stress while involved bacteria exhibit bizarre pleomorphism.

Two substrates saturated with crude oil, a desert soil sample (17.3% oil) and an olive-pomace (plant-based oil sorbent) sample (41% oil) showed effective self-cleaning via their own native microorganisms. The oil in such systems did not gather in one compact layer as it may be expected, but became dispensed as vesicles of varying dimensions connected together with narrow tunnels. Bacteria colonized the oil vesicles but only at the borders between the oil and the watery substrates. Through this architectural arrangement, the cells were capable of absorbing oil through their oil-contact surfaces and oxygen, water and water soluble nutrients through their substrate-contact surfaces. The cells involved were those of indigenous hydrocarbonoclastic bacterial communities. Many of those bacteria also tolerated and removed the amended heavy-metals, Hg2+, Cd2+, Pb2+, AsO43- and AsO33-. In the presence of heavy-metals, some of the bacterial species particularly of the pseudomonads exhibited bizarre pleomorphic cell-forms. It was concluded that even environments toxified with extremely high oil concentrations and heavy-metals can be remediated rather effectively via their already existing native microorganisms.

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."

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.