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.

Corrigendum: Cross-Bioaugmentation Among Four Remote Soil Samples Contaminated With Oil Exerted Just Inconsistent Effects on Oil-Bioremediation.

[This corrects the article DOI: 10.3389/fmicb.2019.02827.].

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.

Cross-Bioaugmentation Among Four Remote Soil Samples Contaminated With Oil Exerted Just Inconsistent Effects on Oil-Bioremediation.

Soil samples were collected from Kuwait, Lebanon, Egypt, and Germany, and artificially polluted with 3% (w/w) crude oil. Cross-bioaugmentation was done among them, and the oil-consumption and the constituent communities of hydrocarbonoclastic bacteria were monitored periodically through 6 months. The results showed that cross-bioaugmentation did not bring about reproducible effects on oil-removal in the four soils. After 6 months, oil-removal values reached between 82 and 95% in most of the samples including the unbioaugmented controls. The numbers of hydrocarbonoclastic bacteria showed significant increases followed by significant decreases during the course of bioremediation also in the unbioaugmented controls. In most cases, the inoculated bacterial taxa failed to colonize the soils, and oil-removal was achieved mainly by the native (autochthonous) soil bacterial communities. those belonged to the genera Mycolicibacterium, Mycobacterium, Xanthobacter, Pseudoxanthomonas, Pseudomonas, Zavarzinia, and others. The microbial communities in the four soils also comprised nitrogen fixing bacteria belonging to the genera Gordonia, Rhizobium, Kocuria, and Azospirillum. Such diazotrophs are known to enrich the soils with fixed nitrogen and thus, contribute to enhancing the microbiological hydrocarbon-consumption. It was concluded that cross-bioaugmentation leads to unpredictable and inconsistent effects on oil removal. Therefore, it could not beregarded as the technology of choice for oil-bioremediation.

Bioaugmentation failed to enhance oil bioremediation in three soil samples from three different continents.

Soil samples from Kuwait, Lebanon, Egypt and Germany were polluted with 3% crude oil. Series of samples were left unbioaugmented, others were bioaugmented with Kuwaiti desert soil with a long history of oil pollution and still others with Kuwaiti marine biofouling material. In the samples from Kuwait, Egypt, and Germany, bioaugmentation did not enhance oil removal, whereas it did in the sample from Lebanon. Taxa from the desert-soil bioaugmented batches, but none of those from the biofouling-material bioaugmented ones, succeeded in colonizing the four studied soils. The dynamics of the hydrocarbonoclastic communities during bioremediation were monitored. Those communities differed in composition, not only according to the type of soil, but also for the same soil; at various phases of bioremediation. Although each soil seemed to have its characteristic microflora, they all were similar in harboring lower and higher actinomycetes and pseudomonads in addition to many other taxa. None of the taxa prevailed through all phases of bioremediation. The most powerful isolate in oil-removal; was Rhodococcus erythropolis (Germany), and the weakest was Arthrobacter phenanthrenivorans (Lebanon). The pure hydrocarbonoclastic isolates tolerated unusually high oil concentrations, up to 30%.

Ability of the So-Called Obligate Hydrocarbonoclastic Bacteria to Utilize Nonhydrocarbon Substrates Thus Enhancing Their Activities Despite their Misleading Name.

BACKGROUND: The group of the so-called obligate hydrocarbonoclastic bacteria (OHCB) are marine microorganisms affiliated with the genera Alcanivorax, Cycloclasticus, Oleiphilus and Thalassolituus. This small group plays a major role in oil-bioremediation in marine ecosystems. Marinobacter and Planomicrobium are considered related to this group. The OHCB are claimed to be obligate to hydrocarbon nutrition. This study argues against this claim. RESULTS: Four Alcanivorax species, three Marinobacter species and Planomicrobium okeanokoites from the Arabian/Persian Gulf proved to be not obligate to hydrocarbon nutrition. Although the eight strains grew on crude oil, n-octadecane and phenanthrene as sole carbon substrates, their growth was weaker than on certain nonhydrocarbon, organic compounds viz. peptone, glutamic acid, pyruvic acid, sucrose, mannose and others. Glucose and lactose failed to support the growth of seven of the eight tested strains. Mannose was utilized by five and sucrose by six strains. The well-known intermediate metabolite; pyruvic acid was utilized by all the eight strains, and lactic acid by five strains. In batch cultures, all the tested species consumed higher proportions of peptone than of n-alkanes and phenanthrene. When peptone and crude oil were provided together into the medium, the OHCB started to consume peptone first, and the enriched bacterial populations consumed oil effectively. Added nonhydrocarbon substrates biostimulated oil-consumption by all OHCB species. CONCLUSION: The tested OHCB species are not obligate hydrocarbon-utilizers. This provides them with the merit of survival, should their marine ecosystems become oil- or hydrocarbon-free. The fact that conventional, organic substrates biostimulated hydrocarbon-consumption by the tested bacterial species confirms the facultative nature of those organisms and is interesting from the practical point of view.

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.

Ferric Sulfate and Proline Enhance Heavy-Metal Tolerance of Halophilic/Halotolerant Soil Microorganisms and Their Bioremediation Potential for Spilled-Oil Under Multiple Stresses.

The aim of this study was to explore the heavy-metal resistance and hydrocarbonoclastic potential of microorganisms in a hypersaline soil. For this, hydrocarbonoclastic microorganisms were counted on a mineral medium with oil vapor as a sole carbon source in the presence of increasing concentrations of ZnSO4, HgCl2, CdSO4, PbNO3, CuSO4, and Na2HAsO4. The colony-forming units counted decreased in number from about 150 g-1 on the heavy-metal-free medium to zero units on media with 40-100 mg l-1 of HgCl2, CdSO4, PbNO3, or Na2HAsO4. On media with CuSO4 or ZnSO4 on the other hand, numbers increased first reaching maxima on media with 50 mg l-1 CuSO4 and 90 mg l-1 ZnSO4. Higher concentrations reduced the numbers, which however, still remained considerable. Pure microbial isolates in cultures tolerated 200-1600 mg l-1 of HgCl2, CdSO4, PbNO3, CuSO4, and Na2HAsO4 in the absence of crude oil. In the presence of oil vapor, the isolates tolerated much lower concentrations of the heavy metals, only 10-80 mg l-1. The addition of 10 Fe2(SO4)3 and 200 mg l-1 proline (by up to two- to threefold) enhanced the tolerance of several isolates to heavy metals, and consequently their potential for oil biodegradation in their presence. The results are useful in designing bioremediation technologies for oil spilled in hypersaline areas.

Coliform Bacteria for Bioremediation of Waste Hydrocarbons.

Raw, domestic sewage of Kuwait City contained about 106 ml-1 colony forming units of Enterobacter hormaechei subsp. oharae (56.6%), Klebsiella spp. (36%), and Escherichia coli (7.4%), as characterized by their 16S rRNA-gene sequences. The isolated coliforms grew successfully on a mineral medium with crude oil vapor as a sole source of carbon and energy. Those strains also grew, albeit to different degrees, on individual n-alkanes with carbon chains between C9 and C36 and on the individual aromatic hydrocarbons, toluene, naphthalene, phenanthrene, and biphenyl as sole sources of carbon and energy. These results imply that coliforms, like other hydrocarbonoclastic microorganisms, oxidize hydrocarbons to the corresponding alcohols and then to aldehydes and fatty acids which are biodegraded by ß-oxidation to acetyl CoA. The latter is a well-known key intermediate in cell material and energy production. E. coli cells grown in the presence of n-hexadecane (but not in its absence) exhibited typical intracellular hydrocarbon inclusions, as revealed by transmission electron microscopy. Raw sewage samples amended with crude oil, n-hexadecane, or phenanthrene lost these hydrocarbons gradually with time. Meanwhile, the numbers of total and individual coliforms, particularly Enterobacter, increased. It was concluded that coliform bacteria in domestic sewage, probably in other environmental materials too, are effective hydrocarbon-biodegrading microorganisms.

Gelatinizing oil in water and its removal via bacteria inhabiting the gels.

When crude oil samples were shaken (200 rpm) in seawater samples from the Arabian Gulf at 30 °C, usually oil-gels were produced spontaneously leaving the water quite clear. The gelators could probably be based on cholesteryl derivatives. Microscopic examination of the established gels revealed nanofibrellar structures similar to those described by earlier workers for artificially synthesized gelators. Communities of bacteria including prosthetic and stalked members as well as oil-degrading bacteria were recorded in such gels. Chemical analysis revealed that 88.5% of the oil entrapped by gelation was biodegraded after 40 days at 30 °C. Individual bacterial species isolated from the oil-gels biodegraded in batch cultures between 17.8 and 33.3% of the oil added at time zero in 12 days at 30 °C. Gelation is a promising approach, not only for clean, physical removal of oil spilled in aquatic habitats, as so far suggested, but also in its effective microbiological biodegradation, as the current study revealed.

Calcium (II) - and dipicolinic acid mediated-biostimulation of oil-bioremediation under multiple stresses by heat, oil and heavy metals.

The oil-producing Arabian Gulf states have hot summer seasons of about 7-month in length. Therefore, environmental oil spills should be bioremediated by the activity of indigenous, hydrocarbonoclastic (hydrocarbon-degrading) microorganisms with optimum growth at about 50 °C. Soils in such arid countries harbor thermophilic bacteria, whose oil-consumption potential is enhanced by calcium (II) - and dipicolinic acid (DPA)-supplement. Those organisms are, however, subjected to additional stresses including toxic effects of heavy metals that may be associated with the spilled oil. Our study highlighted the resistance of indigenous, thermophilic isolates to the heavy metals, mercury (II), cadmium (II), arsenic (II) and lead (II) at 50 °C. We also detected the uptake of heavy metals by 15 isolates at 50 °C, and identified the merA genes coding for Hg2+-resistance in 4 of the studied Hg2+-resistant isolates. Hg2+ was the most toxic metal and the metal toxicity was commonly higher in the presence of oil. The addition of Ca2+ and DPA enhanced the Hg2+-resistance among most of the isolates at 50 °C. Crude oil consumption at 50 °C by 4 selected isolates was inhibited by the tested heavy metals. However, Ca2+ and DPA limited this inhibition and enhanced oil-consumption, which exceeded by far the values in the control cultures.

Capabilities and limitations of DGGE for the analysis of hydrocarbonoclastic prokaryotic communities directly in environmental samples.

Prokaryotic communities in pristine and oil-contaminated desert soil, seawater, and hypersaline coastal soil were analyzed using culture-dependent and culture-independent approaches. The former technique was the dilution-plating method. For the latter, total genomic DNA was extracted and the 16S rRNA genes were amplified using a universal bacterial primer pair and primer pairs specific for Actinobacteria, Gammaproteobacteria, and Archaea. The amplicons were resolved using denaturing gradient gel electrophoresis (DGGE) and sequenced, and the sequences were compared to those in GenBank. The plating method offered the advantages of capturing the targeted hydrocarbonoclastic microorganisms, counting them and providing cultures for further study. However, this technique could not capture more than a total of 15 different prokaryotic taxa. Those taxa belonged predominantly to the genera Alcanivorax, Pseudoxanthomonas, Bosea, Halomonas, and Marinobacter. The individual isolates in culture consumed between 19 and 50% of the available crude oil in 10 days. Although the culture-independent approach revealed much more microbial diversity, it was not problem-free. The subdivision primers exhibited satisfactory specificity, but they failed to capture all the available taxa. The universal bacterial primer pair ignored Actinobacteria altogether, although the primer pair specific for Actinobacteria captured many of them, for example, the genera Geodermatophilus, Streptomyces, Mycobacterium, Pontimonas, Rhodococcus, Blastococcus, Kocuria, and many others. Because most researchers worldwide use universal primers for PCR, this finding should be considered critically to avoid misleading interpretations.

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.

Biostimulation of indigenous microorganisms for bioremediation of oily hypersaline microcosms from the Arabian Gulf Kuwaiti coasts.

Hypersaline soil and water samples were collected in summer and winter from the "sabkha" area at the Kuwaiti shore of the Arabian Gulf. Physicochemical parameters were analyzed, and found suitable for microbial oil-removal. Summer- and winter-microcosms were treated with individual cation (K+, Ca2+, Mg2+, Fe3+) salts, and with animal blood and commercial yeast, as cost-effective vitamin sources. Those microcosms were exposed to the open environment for six winter and six summer months, and analyzed for their hydrocarbonoclastic microorganisms at time zero and in two month intervals. The hydrocarbonoclastic microbial communities in the microcosms consisted of halophilic bacteria and haloarchaea. The constituent bacterial species varied according to the season. Three species, Dietzia kunjamensis, Marinobacter lacisalsi and Halomonas oxialensis consistently occurred both in summer- and winter-samples, but the remaining species were different. On the other hand, the haloarchaeal communities in summer and winter were quite similar, and consisted mainly of Haloferax spp and Halobacterium spp. Treating the microcosms with cations and with vitamin-containing natural products enhanced microbial numbers and oil-removal. The effectiveness of the cations in oil-removal was in the order; Fe3+ (94%) > Ca2+ (89%) > Mg2+ (85%) > K+ (82%). Thus, oily microcosms amended with trivalent and divalent cations lost most of the oil, and those amended with commercial yeast and with animal blood, as vitamin sources, lost 78% and 72% oil, respectively.

Consistent Occurrence of Hydrocarbonoclastic Marinobacter Strains in Various Cultures of Picocyanobacteria from the Arabian Gulf: Promising Associations for Biodegradation of Marine Oil Pollution.

Fifteen nonaxenic cultures of picocyanobacteria were isolated from the Arabian Gulf, from which 122 heterotrophic bacterial strains were obtained. Based on their 16S rRNA gene sequences, those strains were affiliated with 22 different species, 82.8% of which belonged to the genus Marinobacter, known to comprise hydrocarbonoclastic strains. The remaining species belonged to the genera Alcanivorax, Bacillus, Halomonas, Mesorhizobium, and Paenibacillus, and a Bacteriodetes bacterium also known to comprise hydrocarbonoclastic strains. All the picocyanobacterial cultures harbored one or more strains of Marinobacter. Marinobacter in addition to Alcanivorax and other genera isolated from those picocyanobacteria grew on Tween 80, crude oil, and pure hydrocarbons as sole sources of carbon and energy, i.e. they are related to the obligate hydrocarbonoclastic bacteria group. They consumed crude oil, n-octadecane, and phenanthrene in batch cultures. The results indicated that Marinobacter isolates seemed to grow better and consume more oil in the presence of their host picocyanobacteria than in their absence. Such natural microbial associations assumingly play a role in bioremediation of spilled hydrocarbons in the Arabian Gulf. Similar associations probably occur in other marine environments as well and are active in oil spill removal.

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.

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.

Bacterial communities associated with biofouling materials used in bench-scale hydrocarbon bioremediation.

Biofouling material samples from the Arabian (Persian) Gulf, used as inocula in batch cultures, brought about crude oil and pure-hydrocarbon removal in a mineral medium. Without any added nitrogen fertilizers, the hydrocarbon-removal values were between about 10 and 50 %. Fertilization with NaNO3 alone or together with a mixture of the vitamins thiamine, pyridoxine, vitamin B12, biotin, riboflavin, and folic acid increased the hydrocarbon-removal values, to reach 90 %. Biofouling material samples harbored total bacteria in the magnitude of 10(7) cells g(-1), about 25 % of which were hydrocarbonoclastic. These numbers were enhanced by NaNO3 and vitamin amendment. The culture-independent analysis of the total bacterioflora revealed the predominance of the gammaproteobacterial genera Marinobacter, Acinetobacter, and Alcanivorax, the Flavobacteriia, Flavobacterium, Gaetbulibacter, and Owenweeksia, and the Alphaproteobacteria Tistrella, Zavarzinia, and others. Most of those bacteria are hydrocarbonoclastic. Culture-dependent analysis of hydrocarbonoclastic bacteria revealed that Marinobacter hydrocarbonoclasticus, Dietzia maris, and Gordonia bronchialis predominated in the fouling materials. In addition, each material had several more-specific hydrocarbonoclastic species, whose frequencies were enhanced by NaNO3 and vitamin fertilization. The same samples of fouling materials were used in four successive crude-oil-removal cycles without any dramatic loss of their hydrocarbon-removal potential nor of their associated hydrocarbonoclastic bacteria. In the fifth cycle, the oil-removal value was reduced by about 50 % in only one of the studied samples. This highlights how firmly biofouling materials were immobilizing the hydrocarbonoclastic bacteria.