Biotechnology Letters 22: 285–289, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Physiological aspects of hydrocarbon emulsification, metal resistance and DNA profile of biodegrading bacteria isolated from oil polluted sites Leonardo Colombo Fleck, Fl´avio Correa Bicca & Marco Antônio Zachia Ayub∗ Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090, Porto Alegre, RS, 91501-970; Brazil ∗ Author for correspondence (Fax: +55 51 3167048; E-mail:
[email protected]) Received 11 November 1999; Revisions requested 1 December 1999; Revisions received 23 December 1999; Accepted 24 December 1999
Key words: biodegradation, biosurfactant, heavy metal, hydrocarbon, Rhodococcus sp.
Abstract The production of biosurfactants was evaluated for seven bacterial strains isolated from different oil contaminated sites by the Emulsification Index using diesel oil as the hydrocarbon source. Minimum Inhibitory Concentrations of Mg2+ , Cr3+ and Cu2+ were determined to identify the less sensitive bacteria in order to select the best strains for bioremediation. Plasmid extraction was also performed in order to search for gene sequences involved with biosurfactant synthesis. All strains were able to emulsify diesel oil. Rhodococcus ruber AC239 presented the best index (58%), followed by other Rhodococcus strains. Pseudomonas aeruginosa, R. ruber AC239, AC87 and R. erytropolis AC272 presented smallest sensitivities to heavy metals used, being suitable for use in sites contaminated with high concentrations of them. No plasmid DNA was detected showing that biosurfactant coding genes should be in the chromosomal DNA.
Introduction There is global concern about the release of hydrocarbons to the environment, either from industrial activity or from accidental oil spills (Iqbal et al. 1994). Many of those hydrocarbons present hydrophobic structures, being insoluble in water and are toxic, handicapping their biodegradation and causing serious environmental impacts (Hunt et al. 1994, Melo & Azevedo 1997). One of the remediation alternatives is the addition of chemical surfactants (dispersants) that emulsify the hydrocarbons, allowing microbial action on those compounds. However, many synthetic surfactants have toxic effects and, usually, they are not biodegradable, contradictory to all current policies of environmental preservation techniques. An alternative technology is the use of biosurfactants produced by microorganisms, since these compounds are biodegradable and much less toxic (Banat 1995, Hunt et al. 1994, Lin 1996). Biosurfactants are amphipathic molecules that emulsify hydrophobic compounds, increasing the surface of action to the microorganisms,
being, in some cases, superior to the synthetic ones (Banat 1995, Bertrand et al. 1994, Lin 1996). This feature facilitates the microbial absorption of the hydrophobic compounds, which are subsequently degraded. The use of this remediation technique involves the direct use of the biosurfactant as well as the microbial producers of biosurfactants, which would produce this compound directly in situ (Harvey et al. 1990). Besides the recovery process of oil, biosurfactants can be used as substitutes for several synthetic surfactants, as in the petrochemical industry (in the cleaning of storage tanks and equipments), in remediation of soils or polluted effluents, in food processing, in agrochemical solubilization, in household and industrial cleaning, in phase dispersion for cosmetics and textiles (Banat 1995, Lin 1996). Heavy metals are found among hydrocarbons and increases the difficulty of biodegradation. Consequently, several isolated microorganisms of sites contaminated by hydrocarbons show low sensitivity to heavy metals existing at these sites, such as chromium, mercury and copper (Fredrickson et al. 1988). It is im-
286 portant, therefore, not to use sensitive microorganisms to the heavy metals present at the site that is being bioremediated. On the other hand, knowledge of the molecular basis of biosurfactants synthesis is of great interest since it could allow the enhancement of their production (Banat 1995, Lin 1996), through the isolation of the responsible genes for the synthesis of such molecules. It is known that several bacterial biodegrading enzymes and some biosurfactants are coded in plasmid DNA (Chakrabarty 1976, Lin 1996, Summers 1996). The discovery of plasmids coding for biosurfactants would reduce costs and time in isolating genes related to them. The aims of the present work are to study some bacteria strains isolated from oil polluted sites regarding their capability of producing biosurfactants, their minimum inhibitory concentration for some heavy metals and also the presence of plasmid DNA.
Fig. 1. Emulsification Index using diesel oil as carbon source in a culture medium at late exponential phase. The strains are Rhodococcus ruber (AC239, AC74, AC87), R. erytropolis (AC272, AC265), Bacillus subtilis B1, Pseudomonas aeruginosa P1 and, used as controls, Escherichia coli and B. cereus.
Material and methods Bacterial strains The strains used were obtained from three different sources. Five strains of Rhodococcus were obtained from the collection of alkanetrophic bacteria from the Institute of Ecology and Microbial Genetics of the Academy of Sciences of Ural, Russia, isolated from sites contaminated with hydrocarbons at different depths. These strains were: Rhodococcus ruber (AC74, AC87 and AC239) and Rhodococcus erythropolis (AC272 and AC265). A strain of Pseudomonas aeruginosa (P1) was obtained from the laboratory of Soil Microbiology of the Department of Soils of the Agronomy Institute of the Federal University of Rio Grande do Sul, Brazil, having been isolated from a soil sample in one of the ditches located at the Petrochemical Industrial Complex of Triunfo, RS, Brazil, used by landfarming as treatment for its discards, generated by petrochemical processing industries. The Bacillus subtilis (B1) strain B1 was isolated from sediments from the Porto de San Antonio Oeste, in Argentina, a site highly contaminated from oil tanker ships. Hydrocarbon used in the experiments The hydrocarbon used as carbon source was pure diesel oil, an alkane containing 9 to 20 carbons, sterilized by filtration through a Millipore membrane
Fig. 2. MIC of Mercury (Hg MIC, in black) and Copper (Cu MIC, in gray) for all strains (R. Rhodococcus ruber (AC239, AC74, AC87), R. erytropolis (AC272, AC265), Bacillus subtilis B1 and Pseudomonas aeruginosa P1.
0.22 mm, being obtained directly from the Petrobrás Alberto Pasqualini Oil Refinery, Canoas, Brazil. Media The media used were the following: Nutritious Agar (AN), Bushnell-Hass Mineral Medium (Bushnell & Hass 1941), Medium II (M2) (Bicca et al. 1999) (NaNO3 7.0 g l−1 ; K2 HPO4 1.0 g l−1 ; KH2 PO4 0.50 g l−1 ; KCl 0.10 g l−1 ; MgSO4 · 7H2 O 0.50 g l−1 ; CaCl2 0.01 g l−1 ; FeSO4 · 7H2 O 0.01 g l−1 ; yeast extract 0.10 g l−1 ; and agar 15.0 g l−1 , pH 7.0); Brain Heart Infusion (BHI)). All media were sterilized and all chemicals were of analytical grade. Diesel oil (1% (v/v)) was added to the media moments before solidification, when the temperature was between 50 and
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Fig. 3. Plasmid extraction in agarose gel 0.6%. The letters indicate the strains whose DNA was extracted. The left column of each letter represents the DNA digested with HindIII, whereas the right column represents undigested DNA. λ-lambda DNA used as pattern of molecular weight; A – B. subtilis B1; B – AC74; C – AC87; D – AC239; E – AC265; F – E. coli Yep352-PGK (cured); G – E. coli Yep352-PGK (digested in both columns); H – E. coli Yep352-PGK; I – AC272; J – P. aeruginosa P1.
55 ◦ C, being afterwards vigorously shaken and then poured into Petri plates (Bicca et al. 1999). Emulsification Index Determination (E24) E24 was the method used to quantify the emulsification caused by the produced biosurfactants. It was determined by the addition of 2 ml of diesel oil to the same amount of culture at late exponential phase, mixing with a vortex for 2 min, and leaving to stand for 24 h. The E24 index is given as percentage of height of emulsified layer (mm) divided by total height of the liquid column (mm) (Iqbal et al. 1994). As negative controls, strains of Escherichia coli and Bacillus cereus from our laboratory collection were used, since they were known to produce no biosurfactants. Metal MIC Minimum Inhibitory Concentrations for mercury, chromium and copper was determined according to the work of Fredrickson et al. (1988). Metal solutions were as follows: Cr3+ as CrCl3 · 6H2 O, Hg2+ as HgCl2 and Cu+2 as CuCl2 . The standard concentrations were: 25, 50, 100, 150, 200, 250 and 500 µg per disk. Plasmid extraction Plasmid extractions were performed by several methods (Assaf & Dick 1993, Sambrook et al. 1989 (lysis by alkali), Ashok et al. 1995 (Kado Liu Method), Frederickson et al. 1988 (Eckhart Method) and Concert Rapid Plasmid Purifucation Systems (Life Technologies – Gibco BRL)). Two E. coli strains bear-
ing Yep352-PGK and IprD2-LYS vectors were used as positive controls. E. coli strain Yep352-PGK was cured according to Ayub et al. (1992) and used as negative control. Digested and undigested samples were analysed through gel electrophoresis.
Results and discussion Emulsification index The emulsification index is a method extensively used to identify and quantify biosurfactants in microbial cultures. As shown in Figure 1, all strains isolated from oil contaminated sites presented varying degrees of emulsification, indicating production of biosurfactant compounds. Rhodococcus ruber strain AC239 showed the best result, reaching 58% of emulsification index. This value corresponds to very high emulsification when compared with values reported in the literature. For instance, Pruthi & Cameotra (1995) found that cultures of Pseudomonas aeruginosa growing on a similar medium presented a emulsification index value of 30%, and was considered an excellent producer of biosurfactant. Rhodococcus bacteria are mentioned as potential biodegradors of organic, toxic and xenobiotic compounds (Desai & Banat 1997, Finnerty 1992, Lin 1996). Their main characteristic is the high metabolic variability, which allows the use of several organic compounds as carbon source (Bicca et al. 1999). P. aeruginosa and B. subtilis strains presented lower values (21% and 18%). However, some strains of these species are cited as potencial biodegradors of hydrocarbons. P. aeruginosa strains are commonly
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Fig. 4. Plasmid extraction in agarose gel 0.8%. The letters indicate the strains whose DNA was extracted. The left column of each letter represent the DNA digested with HindIII, whereas the right column represent undigested DNA. λ-lambda DNA used as pattern of molecular weight.
isolated from sites contaminated with hydrocarbons around the world, and have already been used in bioremediation procedures (Exxon Valdez Petroleum spill in Alaska), increasing the speed of petroleum removal by two to three times (Harvey et al. 1990). Moreover, according to Makkar & Cameotra (1997), a Bacillus subtilis strain was one of the first species that had its biosurfactant (surfactin) used commercially. Results obtained in this work point to the use of Rhodococcus strains as alternative biosurfactants producers. Metal MIC The MIC analysis allowed the evaluation of which strains were less sensitive to toxic effects of heavy metals used. Heavy metals are, in certain cases, necessary for cellular metabolism. However, in concentrations slightly higher they may be highly toxic, interfering with bacterial metabolic processes. Mercury, for instance, alters proteins synthesis, disrupting enzymatic activity, translation and transcription (Chakrabarty 1976, Hurst 1997). As shown in Figure 2, all strains grew well in high concentration of copper, with the exception of R. erythropolis AC265, which also had a small growth in the presence of mercury. For all strains with the exception of P. aeruginosa, growth in the presence of mercury was highly inhibited, suggesting a low tolerance towards this metal. All strains grew without inhibition zone in the MIC analysis of chromium, even at the maximum concentration used, 500 µg ml−1 , showing that
these microorganisms are highly resistant to this heavy metal (results not shown). According to Bopp et al. (1983), studies demonstrated that bacteria isolated from polluted sites are 40 to 200 times more resistant to heavy metals than related strains isolated from unpolluted sites. Hurst et al. (1997) also considered that the presence of high concentrations of heavy metals is associated with an increase in the number of microorganisms resistant to them. Nevertheless, microorganisms with low sensitivity to heavy metals toxicity are also found in environments with small concentrations of them (Hurst et al. 1997). In spite of these results, it is incorrect to affirm that the sensitivity to the metals in the environment will be the same as in the laboratory tests. The association of the metals with the organic matter is complex. These elements are usually chelated or adsorbed, being unavailable to the cellular metabolism. Moreover, chemical factors, as the pH, influence deeply the availability of those toxic elements to bacteria (Hurst et al. 1997, Melo & Azevedo 1997). Although there are no standards concerning MIC (Hurst et al. 1997), our results show extraordinary high resistances towards the main heavy metals associated with pollution. Further research using these bacteria in bioremediation of metal-polluted areas is presently under progress by our group.
289 Plasmid DNA The best plasmid extractions were obtained using the method by Assaf & Dick (1993) (Figure 3) and the Concert Rapid Plasmid Purification Systems (Life Technologies – Gibco BRL) (Figure 4). The extractions of DNA allowed us to observe that, for the several techniques used, none of the strains presented plasmids, either of low or of high molecular weight (Figures 3 and 4). Thus, the strains tested by us might have been adapted for a long time to hydrocarbon contaminated environments and had the sequences responsible for biosurfactants production incorporated into their chromosomes.
Conclusions Although all strains showed emulsifying activity, the most efficient strains for hydrocarbon emulsification were R. ruber AC239 and R. erythropolis AC272. Concerning the MIC determination, P. aeruginosa, R. ruber AC239, AC87 and R. erytropolis AC272 presented the smallest sensitivities to heavy metals used, being suitable for use in sites contaminated with high concentrations of them. Plasmids were observed in none of the bacteria by the techniques used. This leads us to conclude that plasmids do not exist neither of high nor of low molecular weight in the studied strains, so that the coding genes are probably located in the chromosomal DNA. The genetic analysis of the biosurfactants production is still at the primary stages (Lin 1996). The cultivation of these microorganisms in bioreactors will allow the production of biosurfactants in an industrial scale (Banat 1995, Lin 1996), to be efficiently used in the bioremediation of polluted sites.
Acknowledgements The authors wish to thank the Brazilian Bureau of Science and Technology (CNPq) for funding this research.
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