N.A. Sorkhoh

Rhizospheric hydrocarbon-utilizing microorganisms as potential contributors to phytoremediation for the oily Kuwaiti desert.

Roots of the wild desert plants, Senecio glaucus, Cyperus conglomeratus, Launaea mucronata, Picris babylonica and Salsola imbricata and the crop plants Vicia faba and Lupinus albus grown in oil polluted and clean soils were densely associated with hydrocarbon utilizing bacteria. The most dominant were Cellulomonas flavigena, Rhodococcus erythropolis and Arthrobacter sp. The rhizosphere soils of all plants contained more hydrocarbon-utilizers than the soils apart. This rhizosphere effect was much more pronounced for plants growing in oil-polluted than clean soils. C. flavigena predominant in the rhizosphere of Vicia faba took up representative test aliphatic and aromatic hydrocarbons. Thus, these bacteria could be contributing in nature to detoxifying and bioremediating the soil around the roots. It was concluded that vegetation may be a feasible approach for cleaning oil-polluted soil, including the polluted Kuwaiti desert areas.

Soil management enhancing hydrocarbon biodegradation in the polluted Kuwaiti desert

Oil-polluted Kuwaiti desert samples, exposed to the open air, were subjected to specific types of management, once every 2 weeks, throughout a year; control samples were not treated. The total amounts of extractable alkanes from the control samples remained fairly constant during the dry hot months, but decreased during the rainy months reaching, after 1 year, slightly more than one-half of the amount at zero time. This result demonstrates the self-cleaning of the Kuwaiti desert and the essential role of moisture in this process. Out of the eight types of management studied, the repeated fertilization of the polluted sample with 3% KNO3 solution was most efficient, reducing the extractable alkanes after 1 year to about one-third of zero reading. Repeated fertilization with treated sewage effluent was inhibitory to alkane biodegradation, probably because of increasing soil acidity. The latter inhibitory effect was annulled by liming. Repeated irrigation with 3% NaCl solution was inhibitory, but 1% NaCl solution slightly promoted alkane biodegradation. The various samples contained 1010–1011 oil-utilizing bacteria/g soil, predominantly Bacillus, Pseudomonas, Rhodococcus and Streptomyces. Oil-utilizing fungi were much less frequent and were predominantly Aspergillus and Penicillium species. The microbial numbers varied not only according to the type of soil management but also to the season.

Lipids of n-Alkane-Utilizing Microorganisms and Their Application Potential

Publisher Summary This chapter describes the lipids of n -Alkane-utilizing microorganisms and their application potential. The chapter refers the potential commercial values of various lipid classes and fatty acids. In addition, reference is made to environmental considerations associated with the proposed application of microorganisms in controlling oil pollution and in enhanced oil recovery. That lipids of n -alkaline-utilizing microorganisms should be expected to differ from the lipids of the same organisms grown on conventional substrates is apparent from the following arguments. (1) n -Alkanes, being water insoluble, expectedly induce in cell membranes alterations that allow for their enhanced active transport. Such alterations may involve the membrane lipids which contribute to about 50% of the membrane weight. )2) Alkanes, themselves lipids, are taken up, chemically unchanged, and thus directly contribute to the total cell lipids. (3) Initial phases of n -alkane metabolism involve the oxidation of these substrates to fatty alcohols and fatty acids that become, in part, incorporated into complex cell lipid compounds. (4) Several n -alkane-utilizing microorganisms reveal cytological entities that are associated with their growth on n -alkanes as substrates. Thus, certain bacteria produce intracytoplasmic membranes, and yeasts produce peroxisomes. Like other biological membranes and organelles, these cytological entities are expected to be rich in lipids.

The potential of oil-utilizing bacterial consortia associated with legume root nodules for cleaning oily soils

The surfaces of root nodules of Vicia faba and Lupinus albus (legume crops), were colonized with bacterial consortia which utilized oil and fixed nitrogen. Such combined activities apparently make those periphytic consortia efficient contributors to bioremediation of oily nitrogen-poor desert soils. This was confirmed experimentally in this study. Thus, cultivating V. faba, L. albus and, for comparison, Solanum melongena, a nonlegume crop, separately in oily sand samples resulted in more oil attenuation than in an uncultivated sample. This effect was more pronounced with the legume crops than with the nonlegume crop. Furthermore, in flask cultures, V. faba plants with nodulated roots exhibited a higher potential for oil attenuation in the surrounding water than plants with nodule-free roots. Denaturation gradient gel electrophoresis (DGGE) of polymerase chain reaction amplified 16S rRNA coding genes revealed that periphytic bacteria had DGGE bands not matching those of the oil-utilizing rhizospheric bacteria. Legume nodules also contained endophytic bacteria whose 16S rDNA bands did not match those of Rhizobium nor those of all other individual periphytic and rhizospheric strains. It was concluded that legume crops host on their roots bacterial consortia with a satisfactory potential for oil phytoremediation.

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.

Oil phytoremediation potential of hypersaline coasts of the Arabian Gulf using rhizosphere technology.

The rhizosphere and phyllosphere of the halophyte Halonemum strobilaceum naturally inhabiting hypersaline coastal areas of the Arabian Gulf harbor up to 8.1 x 10(4)g(-1) and 3 x 10(2)g(-1), respectively, of extremely halophilic oil-utilizing microorganisms. Such organisms were 14- to 38-fold more frequent in the rhizosphere than in the plant-free soil. Frequent genera in the rhizosphere were affiliated to the archaea Halobacterium sp. and Halococcus sp., the firmicute Brevibacillus borstenlensis, and the proteobacteria Pseudoalteromonas ruthenica and Halomonas sinaensis. The phyllospheric microflora consisted of the dimorphic yeast Candida utilis and the two proteobacteria Ochrobactrum sp. and Desulfovibrio sp. Individual strains grew on a range of pure aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy. All the strains, except C. utilis which could not tolerate salinities >2M NaCl, grew also in media with salinities ranging between 1 and 4M NaCl, with optimum growth between 1 and 2M NaCl. With the exception of the two archaeal genera, all isolates could grow in a nitrogen-free medium. The total rhizospheric and phyllospheric microbial consortia could attenuate crude oil in complete (nitrogen-containing) medium, but also equally well in a nitrogen-free medium. It was concluded that H. strobilaceum could be a valuable halophyte for phytoremediation of oil-polluted hypersaline environments via rhizosphere technology.

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.

PlANT-ASSOCIATED BACTERIA AS TOOLS FOR THE PHYTOREMEDIATION OF OILY NITROGEN-POOR SOILS

The rhizospheres and phyllospheres of peas, beans, tomatoes, and squash raised in a desert sand soil mixed with 0.5% crude oil were rich in oil-utilizing bacteria and accommodated large numbers of free-living diazotrophic bacteria, with potential for hydrocarbon utilization. According to their 16S rRNA-sequences, the cultivable oil-utilizing bacteria were affiliated with the following genera, arranged in decreasing frequency: Bacillus, Ochrobactrum, Enterobacter, Rhodococcus, Arthrobacter, Pontola, Nocardia, and Pseudoxanthomonas. Diazotrophic isolates were affiliated with Rhizobium, Bacillus, Rhodococcus, Leifsonia, Cellulosimicrobium, Stenotrophomonas, Kocuria, Arthrobacter, and Brevibacillus. The crude oil–utilizing and diazotrophic isolates grew, with varying growth intensities, on individual aliphatic (C8 to C40) and aromatic hydrocarbons, as sole sources of carbon and energy. Quantitative gas liquid chromatographic measurements showed that representative bacterial isolates eliminated pure n-hexadecane, n-decosane, phenanthrene, and crude oil from the surrounding liquid media. Cultivation of oily sand–soil samples with any of the four tested crops led to enhanced oil degradation in that soil, as compared with the degradation in uncultivated oily sand–soil samples.

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.

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.