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.

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.

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.