Selection and identification of biosurfactant-producing bacteria

A total of seven bacterial strains were characterized genetically and identified as the genus Pseudomonas. Four isolates were found in Zea mays rhizosphere (PAM7, PAM8, PAM9 and PAM 10) and the rest were isolated from compost (PAB11, PAB12 and PAB13). The strains showed 99% identity to P. aeruginosa PAO1 (reference strain); however, PAM79 and PAM810 isolates are genetically related to strains of Pseudomonas sp (Fig 1). All strains exhibited the ability to produce biosurfactants, which was confirmed by beta hemolysis in blood agar plates and dark halos in CTAB-Methylene blue agar.

Figure 1.
Phylogenic analysis by neighbor-joining method on 16S rDNA gene sequences. Scale bar = 0.5 substitutions per nucleotide position. Bacillus subtilis YS52 (HQ202539) was used as an outgroup sequence.
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The surface activity of biosurfactants present in the cultures was positive for the tests of foaming activity in PPGAS broth, drop collapse test and oil displacement test (Fig. 2).

Figure 2.
Evaluation of the production of biosurfactants. A) Blue halos formation in CTAB-Methylene blue agar. B) Hemolysis in blood agar. C) oil displacement assay. D) drop collapse test. E-F) Foaming activity in PPGAS broth.
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Emulsifying properties and cellular hydrophobicity associated with biosurfactants production

The biosurfactants produced can emulsify petroleum, diesel, toluene, xylene, cyclohexane and oils (mineral and vegetable) in different degrees, as shown on Table 1. The xylene hydrocarbon was mostly emulsified by the strains (average = 58%), while mineral oil did not exceed the 32% of average emulsification. P. aeruginosa strains with highest IE24 values were PAM8, PAM9, PAB11 and PAB13. The strain PAM8 has an average IE24 of 64% and PAM9 emulsifies cyclohexane up to 70% in vegetable oil. PAB11 emulsifies petroleum to 79% and PAB13 emulsifies xylene to 67%. The Zea mays rhizosphere strains showed better emulsification in diesel compared to the compost strains, and did not present significant differences in the other substrates. The strongly hydrophobic (% Cellular Hydrophobicity or BATH) P. aeruginosa strains were PAM8 and PAM9 with 38% and 30% of BATH values, respectively (Table 1).

Table 1.
Percent Index of emulsification (IE24) and cellular hydrophobicity by biosurfactants of P. aeruginosa.

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Quantification and physicochemical characterization of biosurfactants

The P. aeruginosa strains with the highest biosurfactant titers were PAM8 (187± 5 mg/L), from Zea mays rhizosphere, and PAB13 (162±7 mg/L), from compost (Fig.3). The strain PAB12 showed poor performance, with 125±4 mg/L of biosurfactant. All biosurfactants obtained from the isolated Pseudomonas strains were negatively charged and retained their capacity to disperse and emulsify oil after treatment at ≥ 100 °C, salinity at 10% and ≥ pH10 (Fig. 3). Significant differences were observed between compost and Zea mays rhizosphere strains in terms of amount of biosurfactant produced but not in their physicochemical properties.

Figure 3.
Comparison of production and physicochemical properties of rhamnolipids. *Statistically different between compost and rhizosphere strains accorded to unpaired Student’s t-test (p ≤ 0.05).
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Surface activity of biosurfactants in presence of pyocyanin pigment

The production of pigments was observed at the same time as formation of abundant foam in PPGAS culture so, due to its blue coloration, pyocyanin was quantified (Fig. 4). The highest production of pyocyanin was observed in Zea mays rhizosphere strains: PAM8 (11.9 ± 2.3 μL mL^-1), PAM9 (7.8 ± 1.3 μL mL^-1) and PAM10 (7.8 ± 1.0 μL mL^-1).

Figure 4.
Comparison of pyocyanim pigment between compost and rhizosphere strains. A) Quantification by colorimetric method. B) Intensity of pigment in PPGAS broth.
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Antibacterial activity of biosurfactants

The produced rhamnolipids have antimicrobial activity against two Grampositive and two Gramnegative bacteria evaluated (Table 2). Maize rhizosphere strains inhibit the bacterium Bacillus subtilis better than compost strains, and no significant differences in their antibacterial effect were observed for other bacteria. P. aeruginosa PAM8 showed the strongest activity against B. subtilis, S. aureus, S. paucimobilis and K. pneumoniae with inhibition halos of 26±6 mm, 22±4 mm, 32±5 mm and 24±3 mm, respectively.

Table 2.
Comparison of antibacterial activity of biosurfactants produced by Pseudomonas aeruginosa.

Authors* Statistically different between compost and rhizosphere strains accorded to unpaired Student's t-test (p ≤ 0.05).

ND=Not detected.

Heavy-metal resistance profile of P. aeruginosa strains

The petroleum and its derivatives contain traces of heavy metals and metalloids that give rise to the formation of organometallic compounds; therefore, we evaluated the heavy-metal resistance profile of biosurfactant-producing Pseudomonas. All the strains have similar MIC to heavy metals and metalloids in the following order: As⁵⁺ (>20 mM), As³⁺ (10 mM), Zn²⁺ (10 mM), Pb²⁺(6 mM), Fe³⁺(4 mM), Cd²⁺(4 mM), Cu²⁺(2 mM) and Cr⁶⁺(2mM). The colonies were hyper mucoid when grown in high concentrations of metals especially in Pb, Fe and Cu.

The bacterium P. aeruginosa is known for its ability to produce several bioactive compounds. In this study, report is made on seven strains isolated from the rhizosphere corn or compost; all isolates produced biosurfactants with emulsification indexes on the different hydrophobic substrates as shown on Table 1. Highlighted in the work by Pirôllo et al. (2008) , a P. aeruginosa LBI strain was isolated from places that were contaminated by hydrocarbons. The biosurfactant produced maintained the physicochemical properties after treatment at 120 °C, 10% salinity and only PAM8 biosurfactant is active under alkaline conditions (pH 12).

Strains synthetized both mono-rhamnolipids and di-rhamnolipids, except PAB12 that only produces mono-rhamnolipid, probably lacks some gene responsible for incorporating rhamnose during the biosynthetic pathway. Our results of IE₂₄ agree with the results obtained by Janek et al., (2013) , where it was reported that the rhamnolipid produced by a strain of Pseudomonas BD2 isolated in the Arctic, is capable of reducing the surface tension from 71 to 31 mN/m and emulsifies 70% olive oil, meanwhile for n-hexane, xylene, hexadecane and petroleum ether it emulsifies in a range from 51 to 65%.

It was observed that strains that synthesize more pigment also produce more biosurfactant and are better emulsifiers. Das and Ma (2013) report that the pyocyanin pigment enhances the biosurfactant-mediated hydrocarbon emulsification in clinical and environmental strains of P. aeruginosa. In the same way, the hydrocarbonoclastic activity (involved in the mineralization of hydrocarbon pollutants) is positively correlated with the pyocyanin production by P. aeruginosa strains isolated from gas stations, activated sludge and wastewater (Viana et al., 2018). Since the strains isolated in the present study are biosurfactant producers, they could potentially be used for Microbial Enhanced Oil Recovery (MEOR) in contaminated environments, in oil wells (Brown, 2010) or in agricultural soils (Sachdev and Cameotra, 2013) . The production and concentrations the biosurfactants vary depending on the culture conditions and nutrients, especially the carbon source, as reported by Vanavil et al. (2013). It was reported that the concentration of rhamnolipids obtained when growing the strain in glucose was 88 mg/L, and 15-fold higher when they added Mg²⁺ and Fe²⁺ to the media. In the case of this study, it is still needed to find conditions for the improvement of the biosurfactant production.

We propose PAM8, PAM9 and PAM13 strains as candidates for use in bioremediation of marine environments and other systems by biosurfactants that are stable in hypersaline and alkaline conditions. The strains in this study are similar to those that the microbial consortium reported by Darvishi et al. (2011). That consortium produces a biosurfactant capable of resisting temperatures of over 70°C, salt concentrations higher than 15%, and a range of pH from 4 to 10. It also breaks down hydrocarbons, a fact that points it as a good candidate for the remediation of contaminated sites with these compounds.

The strains PAM8 and PAM9 are capable to emulsify different hydrocarbons and have highest cell hydrophobicity, these are good features for the bioremediation of sites contaminated with oil. Kaczorek and Olszanowski (2010) evaluated the binding of hydrocarbons by Pseudomonas sp. when growing in different carbon sources. They observed that P. fluorescens PI growing in glucose had 8% hydrophobicity but when grown in ethylbenzene, its hydrophobicity increased by 30% similar to the hydrophobicity showed by the strains P. aeruginosa PAM8 (28%) and P. aeruginosa PAM9 (20%). PAB12 is the smallest emulsifier probably because it only produces mono-rhamnolipid in small quantities (Fig 3), so the critical micellar concentration is not easily achieved.

The crude extracts of strain PAM8 showed high activity against B. subtilis, S. aureus, S. paucimobilis and K. pneumoniae. These results are consistent with Bharali et al. (2013) where the antibacterial activity of the P. aeruginosa OBP1 rhamnolipids on S. aureus and K. pneumoniae was shown. The rhamnolipids produced by P. putida 21BN against B. subtillus (Tuleva et al., 2002) . Another study showed the effect of the biosurfactant produced by P. koreensis M9b on Grampositive bacteria (Toribio et al., 2011) . The antibacterial producers included in the CLSI (Clinical and Laboratory Standards Institute) to date are biosurfactants belonging to the class glycolipids and cyclic lipopeptides; for this reason, the biosurfactants produced by our isolates represent good candidates as antibacterial products.

The colonies showed fluorescence and were hyper-mucoid in presence of Pb, Zn and Fe. This was probably in response to the stress they underwent, since the exopolysaccharides have anionic functional groups as phosphates, sulfates, and carboxilic acids that bind to metals, sequestering them and avoiding the interactions of the metal inside the cell. Fluorescence was caused by the production of siderophores that could be related to the presence of specific metals in the soils that our isolates came from. Siderophores as the pioverdine and piochelin are able to bind Fe, Cd, Cu and Zn, reducing their toxicity for bacteria (Dao et al., 1999; Dimkpa et al., 2009) .

These data agree with Raja et al. (2006), who reported on a strain of P. aeruginosa FC1 being capable of growing in 800 ppm of Pb, 700 ppm of Ni, 500 ppm of Cd and 400 ppm of Cr. On the other hand, studies conducted by Hassan et al. (2008) showed bacterial resistance to heavy metals in contaminated soils in Egypt. However, 89% of the isolates were inhibited by 3.5 mM of the metal, which represents a lower concentration than our isolates could resist. Bodour et al. (2003) This shows the production of associated biosurfactants with resistance to heavy metals, which is similar to the yield of this study.