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Selection of Biosurfactan/Bioemulsifier-Producing Bacteria From Hydrocarbon-Contaminated Soil

Petroleum-derived hydrocarbons are among the most persistent soil contaminants, and some hydrocarbon-degrading microorganisms can produce biosurfactants to increase bioavailability and degradation. The aim of this work was to identify biosurfactant-producing bacterial strains isolated from hydrocarbon-contaminated sites, and to evaluate their biosurfactant properties. The drop-collapse method and minimal agar added with a layer of combustoleo were used for screening, and positive strains were grown in liquid medium, and surface tension and emulsification index were determined in cell-free supernantant and cell suspension. A total of 324 bacterial strains were tested, and 17 were positive for the drop-collapse and hydrocarbon-layer agar methods. Most of the strains were Pseudomonas, except for three strains (Acinetobacter, Bacillus, Rhodococcus). Surface tension was similar in cell-free and cell suspension measurements, with values in the range of 58 to 26 (mN/m), and all formed stable emulsions with motor oil (76-93% E24). Considering the variety of molecular structures among microbial biosurfactants, they have different chemical properties that can be exploited commercially, for applications as diverse as bioremediation or degradable detergents.


INTRODUCTION
The release of contaminants to the environment, including petroleum and petroleum-derived products, is one of the main causes of global contamination (29). It is also a risk for human and animal health, since many of these contaminants have demonstrated to be toxic and carcinogenic (27). Hydrocarbon molecules that are released into the environment are hard to remove, since they adsorb to surfaces and are trapped by capillarity in a water-immiscible phase. Bioremediation has proven to be an alternative to diminish the effects caused by hydrocarbon contamination of soil and water, using the metabolic capacities of microorganisms that can use hydrocarbons as source of carbon and energy, or that can modify them by cometabolism. The efficiency of removal is directly related to the compounds' chemical structure, to its bioavailabity (concentration, toxicity, mobility and access) and to the physicochemical conditions present in the environment (10).
Biosurfactants are produced by many organisms, in order Selection of biosurfactan/bioemulsifier-producing bacteria to metabolize water-immiscible substrates, allowing its adsorption, emulsification or dispersion. For the microorganisms, production of biosurfactants is an advantage in soil, giving them advantages in specific conditions (30).
Microbial biosurfactants are mainly produced by aerobic microorganisms, using as carbon sources carbohydrates, hydrocarbons, animal or vegetal oils or a mixture of them (6,14 (5), and it has been described that the carbon source used to grow the microorganisms is important on the identification of biosurfactat producing strains (6). Therefore, the aim of this work was to identify biosurfactant and/or bioemulsifier producing bacteria from a collection of soil bacterial isolates obtained from hydrocarbon-contaminated sites.

Bacterial strains
Bacterial strains were isolated from soil contaminated with either combustoleo ( (4). After growth, biosurfactant production was tested. Cells were recovered by centrifugation (12,000 x g, 5 min) and resuspended in 1 ml of M9 broth. Cell suspension and supernatant were used in the biosurfactant activity assay.

Qualitative evaluation of biosurfactant production
The drop-collapse method was used for initial identification of biosurfactant-producing bacteria (4). Briefly,

Determination of Surface Tension
Bacterial strains that were positive in the drop-collapse test were also evaluated for surface tension and for stable emulsion formation. Strains were grown in M9 broth added with glucose (2% w/v for Pseudomonas) or sucrose (1% w/v for Bacillus, Acinetobacter, Rhodococcus) and incubated at 120 rpm for five days. For surface tension measurements, 5 ml of broth supernatant were transferred to a glass tube that was submerged in a water bath at a constant temperature (28°C).
Surface tension was calculated by measuring the height reached by the liquid when freely ascended trough a capillary tube (23). As control, non-inoculated broth was used, and the surface tension was calculated according to the following formula: 2 g rh = γ (1) γ = Surface tension (mN/m); δ = Density (g/mL); g = gravity (980 cm/s 2 ); r = capillary radius (0.05 cm); h = height of the liquid column (cm).

Determination of emulsification index
For determination of the emulsification index, 2 ml of supernatant or cell suspension and 3 ml of a selected hydrocarbon were mixed in a test tube and vortexed for 2 min.
The test tubes were maintained at 25°C and the height of emulsion layer was measured after 24 h to determine the emulsification index (11). The equation used to determine the emulsification index (E 24 ) is as follows:

Statistical analysis
The data analysis was carried out with Minitab Inc. software (version14.13). One-way ANOVA was used to determine whether significant (p < 0.05) variation occurred among emulsification index and surface tension measurements of bacterial isolates. Tukey's-test was used to perform multiple comparisons between means.

Screening of biosurfactant producing bacteria
Bacterial strains were isolated from two different soils contaminated with hydrocarbons (fuel oil No. 6 and used motor oil) that were subjected to different bioremediation procedures in laboratory-scale bioreactors and were isolated during the period that the bioreactors were followed (24) reported as hydrocarbon degraders (7,29).

One of the factors that is important in biosurfactant
production is the carbon source present, since some microorganisms produce biosurfactants in water insoluble substrates, such as vegetable or mineral oils, and other microorganisms can produce this metabolites when carbohydrates are present as carbon sources (6). Therefore, all 324 bacterial strains were grown in M9 added with glucose, sucrose or olive oil, and the supernatant was used to select for biosurfactant-production bacteria by the drop-collapse test.
Seventeen isolates were positive and of those, 11 strains were growing in glucose, 6 in olive oil and only 3 in sucrose; of those, 3 strains produced biosurfactant in olive oil as well as in glucose as substrates ( Table 1). The substrates used have been reported to promote biosurfactant production (4,26,32).
Although the biodegradability capacity of the 324 strains has not been tested by reduction of hydrocarbons in pure culture, many are likely to degrade hydrocarbons since they were isolated in M9 Agar plates, incubated in a hydrocarbonsaturated atmosphere as carbon source. However, only a small proportion were biosurfactant producers, which support the concept of bacterial community, whereas the biosurfactant producers will liberate the surface-tension-active molecules, that will help other microorganisms to degrade the wide variety of hydrocarbons found at the site (28, 30).   has also been reported that the most known biosurfactant that they produce, is a rhamnolipid (22,33). Although most of the reports on rhamnolipid production are attributed to
When the 17 isolates described in Table 1 Table 2).
Even though lypopeptides produced by Bacillus are known as one of the most powerful microbial biosurfactants (20), Pseudomonas rhamnolipids are also effective, and both of them are extracellular (21). Isolates that liberate biosurfactants into the culture medium are interesting from an industrial point of view, because the product can be easily removed from the culture media (19).  (14).
The debate is if a surfactant that reduces the surface tension of water, can form stable emulsions (3,9). As described in Table   2, most of the strains that had the lower surface tension values were also the ones that formed the largest and more stable