BACTERIAS DEGRADADORAS DE HIDROCARBUROS AISLADAS EN SUELOS CONTAMINADOS DE PETROLEO Aislar cepas bacterianas y. Aislamiento de bacterias potencialmente degradadoras de petróleo en hábitats de ecosistemas costeros en la Bahía de Cartagena, Colombia. Download Citation on ResearchGate | SELECCIÓN DE BACTERIAS CON CAPACIDAD DEGRADADORA DE HIDROCARBUROS Estudio y selección de bacterias aerobias degradadoras de hidrocarburos del petróleo aisladas de costas.

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ABSTRACT The isolation of aerobic marine bacteria able to degrade hydrocarbons represents a promising alternative for the decontamination of oceanic and coastal environments. The obtained isolates were then subjected to selection in Bushnell-Haas medium supplemented with a heavy crude oil, selecting three strains able to degrade this hydrocarbon mixture within a period of seven days. Pure cultures of these strains were further used in crude oil biodegradability assays. Total petroleum hydrocarbon TPH degradation was evaluated through SARA analysis, employing gas chromatography with an FID detector and infrared spectroscopy to analyze the aliphatic and aromatic hydrocarbon fractions, respectively.

Two of the strains were also able to decrease C Two of these strains were phenotypically identified as sp. The degradation potential exhibited by these new isolates warrants further studies on their possible application to decontaminate coastal environments affected by oil spills. Las potencialidades biodegradadoras de estos microorganismos en la limpieza de costas marinas han generado nuevos estudios.

Usually found at low concentrations in non-contaminated areas, their populations bloom in chronically contaminated environments [1]. Anaerobic microorganisms, however, are less versatile regarding their growth substrate and often display increased sensitivity toward heavy metals, hence playing a smaller role in biodegradation [2]. Most research on bioremediation technology has focused therefore on aerobic heterotrophic bacteria, due not only to the taxonomic diversity of hydrocarbon-degrading representatives from this group, but to their ability to use xenobiotic compounds as carbon source in pure cultures [3].

Taxonomic and metabolic variety notwithstanding, individual species seldom have the complete enzymatic toolset required to completely degrade the main organic compounds contaminating the ecosystem at any given time, and thus biodegradation usually proceeds through the concerted action of mixed populations or microbial consortia. Viewed as a whole, the latter possess the necessary genetic information to produce all the enzymes required to completely degrade complex hydrocarbon mixtures in damaged areas [4, 5].

Environmental biotechnology techniques have been readily used to remediate ecological disasters caused by large oil spills, such as those caused by the collision of tanker Exxon Valdez with Bligh Reef at Prince William Sound, spilling some 50 tons of crude oil in March [6], and the sinking of Prestigewhich spilled 63 tons of crude and ended up affecting 1 km of French and Spanish coastlines in November [7].

Measures taken after the Exxon Valdez spill included the addition of nutrients in the form of fertilizers Inipol EAP 22 and Customblerwhich increased the rate of oil removal by three-fold [6]. They arrived to the laboratory for further processing within the first 24 h after their collection.

Strain isolation The primary selection process employed two different discrimination protocols, denominated here protocol A modified from [8] and B modified from [9]. Five replicates were used per sample. In protocol B the samples were stirred at rpm for 5 min, bottling mL from each one afterwards into a sterile mL threaded cap bottle, to which 6 mL of Mesa 30 crude and 0. These cultures were then further incubated for seven days under the same conditions, at the end of which the subculturing cycle was repeated once more.

After concluding the three subculture cycles, 0. In both protocols the marine bacteria isolation plates were periodically examined after 24 h of growth under a stereoscope, streaking onto separate plates all the colonies appearing during the seven day incubation period.

Pure stocks were obtained from colonies isolated by streaking, verifying their homogeneity by Gram staining and through the examination of culture characteristics. Phenotypic characterization of the strains The strains were characterized phenotypically using previously described morphological, physiological and biochemical tests [], using previously defined criteria to describe culture characteristics [13].

Morphological descriptions were based on bacterial shape, motility and pigmentation. Salt tolerance was estimated by seeding the test strains in marine bacteria isolation medium where seawater was replaced by distilled water and sodium chloride NaCl concentration was set at 0, 0. The cultures were discarded after 15 days. Biochemical characterization of the strains comprised tests for the fermentation of glucose, lactose, sucrose and mannitol, as well as for the production of indole, gas and hydrogen sulfide.

Three replicates were seeded per strain. The cultures were examined every 24 h, selecting all strains exhibiting vigorous growth within a seven day period for further study. The obtained bacterial suspensions constituted the inocula. Each strain was assayed in triplicate. Hydrocarbon determinations Hydrocarbon determinations were performed at day The organic phase of the samples was extracted with 45 mL of HPLC-grade dichloromethane three extractions with a volume of 15 mL each using the liquid-liquid method for 30 min in a separating funnel, filtering the obtained organic extract through anhydrous reagent grade sodium sulfate.


Asphaltenes were precipitated with n-pentane. All spectra were processed using the Omnic v5. This probably explains the high number of hydrocarbon-degrading microorganisms identified through isolation protocols A and B, which totaled 33 strains. Culturing these strains in the presence of hydrocarbons Mesa 30 crude favored the expression of enzyme systems degraddadoras in their degradation and bacteruas preferential isolation of hydrocarbon-tolerant clones. Finer taxonomic classification to the level of species was not pursued since, despite the existence of taxonomic identification schemes for microorganisms from marine ecosystems [12, 15], successfully identifying recently described genera and species of marine bacteria requires the application of molecular bacteria [16].

Two genera of Gram-positive bacilli were identified Bacillus and Kurthiaas well as five genera of Gram-negative bacilli Alcaligenes, Acinetobacter, Marinomonas, Pseudomonas and Azotobacter.

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Five strains belonging to the latter group could not be identified with the biochemical tests employed in this study. Many Bacillus species have been shown to be able to degrade hydrocarbons [], both in terrestrial [] and marine or aquatic environments []. A microbiological survey of the western Cuban continental shelf found this species in both northern and southern locations [27].

Some species of this genus have been isolated from marine environments contaminated with hydrocarbons [5, 17, 28]. It has been shown that strain WW1 of Alcaligenes denitrificans can degrade four-ring polyaromatic hydrocarbons [30].

These microorganisms can be found not only in soil and water petroleeo, but in clinical specimens, occasionally. Two of the isolated strains belonged to the Pseudomonas genus. Publications reporting the presence of this genus in hydrocarbon-contaminated ecosystems and describing its hydrocarbon-degrading abilities have appeared in the literature from degradadorxs early nineties [17], although its numbers have increased as of late [19, 24, 25, 31, 32].

Published reports indicate that they can also degrade phenanthrene in soils [38] as well as anthracene, phenol [41] and methyl methylbromide in marine environments [40].

Another two strains belonged to the genera Acinetobacter and Marinomonasrespectively. Recent studies on the degradation of hydrocarbons in water bodies have made reference to the former [26, 36, 37]; this genus has, in addition, been found in not only in terrestrial and marine environments, but in sewage as well.

Representatives of the Marinomonas genus were isolated for the first time from sediments contaminated with polycyclic aromatic hydrocarbons in [42]. Some authors place some species of this genus into Alteromonas instead [17].

Lastly, the genera Kurthia and Azotobacter degracadoras represented each by a single strain.

We were unable to find previous mentions in the literature of the presence of these microorganisms in hydrocarbon-contaminated environments. Kurthia is a genus of Gram-positive bacilli described as environmental bacteria, whereas Azotobacter is a typical inhabitant of water bodies and soils. Strain selection All 33 isolated strains were grouped according to culture, morphological, physiological and biochemical parameters.

At the end, 18 strains representing each unique combination of these parameters were chosen to be subjected to the selection process. Hydrocarbons are lipophilic compounds that inhibit growth when present at high concentrations pettroleo, inducing the bacterial stress response and a series of changes at the membrane, enzyme and protein levels [44, 46].

Characterization of the hydrocarbon degradation capacity of the selected strains The ability to degrade hydrocarbons from crude oil Mesa 30 crude was determined after 45 days of static culture. Strain F10S1 degraded Figure 3 shows the chromatographic profile of the saturated hydrocarbon fraction, in a signal intensity pico-amperes versus time chart for strains F9S, F10S1 and F1FLC.

Each of the images shown in the figure contains the profiles of the abiotic control and that of the strain under examination, for a number of different carbon chain lengths. Monocyclic and polycyclic aromatic hydrocarbon fractions were analyzed by FTIR spectroscopy, as described in Pettroleo and Methods.

Each chart depicts both the profile of the strain under examination and that of the standard sample. The concentration of associated hydroxyl groups OH -cm -1 increased for all treatments, as did that of carboxylic groups cm The present study has demonstrated that the bacterial strains selected are able to use hydrocarbons as sole carbon source when grown eptroleo pure cultures.

Since hydrocarbon determinations were performed solely at the end of the study day 45no data are available to evaluate the biodegradation process in earlier time points. Based on our results, however, together with the existing literature, it is possible to make some inferences. In general, biodegradation is expected to be more extensive in aliphatic hydrocarbons, which are far more amenable to this process than their aromatic counterparts [35, 47, 48]. The latter, in turn, are more susceptible to biodegradation than resins and asphaltenes [47, 48].


However, and despite the higher propensity of n-alkanes for oxidation [35, 49], no differences in biodegradation percentages were detected between saturated hydrocarbons, degrzdadoras and monocyclic aromatic hydrocarbons after 45 days. Aliphatic hydrocarbons decreased in comparison with those of the abiotic control regarding the non-resolved background cycloalkanes, resins and asphaltenes. This decrease in isoprenoids is a telltale sign of effective biodegradation; whereas strain F1FLC exhibits values above those of the abiotic control.

The spectra in figure 4 reveal the presence of aromatic compounds in the crudes treated with bacterial strains.

No statistically significant differences were found for this parameter among the examined strains, although the increased proportion of carboxyl and hydroxyl groups demonstrates the presence of biological oxidation processes.

The increased levels of phenols and phenoxides may be directly se to the accumulation of end compounds produced by the degradation of resins and asphaltenes. All three strains under examination produced a notable decrease in the concentration of asphaltenes, compared to the control. This phenomenon was more pronounced for strains F10S and F9S, which did not exhibit statistically significant differences when compared in this regard, but did so when compared to strain F1FLC.

Strain F10S1 lowered the concentration of all fractions in comparison with the control. No alkanes with backbones shorter than 12 carbon atoms were detected in these samples. Several authors have pointed out that short chain aliphatic hydrocarbons usually volatilize during the first hours after a spill.

Since their physico-chemical properties make them toxic compounds for the growth of most bacteria [44, 50], it is assumed dr the degradation of aromatic compounds does not start until saturated hydrocarbons have been used up. However, direct experimental observation has revealed that low molecular weight aromatic compounds may start to be degraded much earlier, sooner, in fact, than many degradadoars molecules [51]. An analysis of these results leads us to suppose that linear chains up to 30 carbon petdoleo long and some low molecular weight aromatic compounds were degraded during the first 10 to 15 days.

Had hydrocarbon composition been determined at that point, we would have most likely found practically identical levels of asphaltenes and resins in crudes treated with the strains under examination and in their controls, significantly decreased levels of saturated hydrocarbons in the former, and only a small drop in the concentration of aromatic hydrocarbons, since the bulk of their degradation takes place after 21 days.

When comparing the degradation of asphaltenes with that of saturated and aromatic hydrocarbons regarding the time point at which they first become detectable as well as their kinetics, it is useful to take into account the environmental characteristics of the habitat from which the microorganisms under examination have been isolated; that is, their environmental adaptations. In addition, it must be noted that synthesis of the enzyme complexes required to degrade the heavier fractions is not induced until lighter fractions have been exhausted, following the principle of maximum cellular ddgradadoras metabolic regulation.

Regardless, Joseph et al. These authors attributed such a phenomenon to adaptations of these microorganisms to the chronic contamination of their original habitats. Varadero crude is higher in asphaltenes, whereas the main constituents of Pina crude are saturated hydrocarbons with chain lengths smaller than 18 carbon atoms.

BACTERIAS DEGRADADORAS DE HIDROCARBUROS by Mirelly Katherine Diaz Gamarra on Prezi

In addition, other authors have shown that the biotransformation of asphaltenes and resins leads to the accumulation of simpler saturated and aromatic derivates, increasing the concentration of these fractions [53, 54].

Bacterial metabolism is readjusted as culture ages and less complex substrates saturated linear alkanes and low molecular weight aromatic compounds are exhausted, shifting towards the synthesis of enzyme complexes geared towards the degradation of more complex molecules. These compounds did not mineralize completely, accumulating instead as intermediary metabolites consisting of linear chains and aromatic compounds. The host enzyme machinery must, therefore, have adapted to fluctuations in the levels of these different compounds.

They were taxonomically identified as members of the Bacillus, Alcaligenes, Pseudomonas, Acinetobacter, Marinomonas, Kurthia and Azotobacter genera. The strains Alcaligenes sp. F9S and Bacillus sp. Asphaltenes, monocyclic aromatic compounds and saturated hydrocarbons were the fractions undergoing degradation to the largest extent.