Evaluation of growth in diesel fuel and surfactants production ability by bacteria isolated from fuels in Costa Rica

Carlos E. Rodríguez-Rodríguez*, Carolina Zúñiga-Chacón, Carolina Barboza-Solano

Sección de Microbiología de Alimentos y Aguas, Facultad de Microbiología, Universidad de Costa Rica, Costa Rica.

* Correspondencia: E-mail: carlos.rodriguezrodriguez@ucr.ac.cr

Abstract: A total of 149 bacterial strains previously isolated from fuels stored in Costa Rica were selected in terms of their ability to grow aerobically in diesel and produce bioemulsifier active compounds. The diesel growth was evaluated by a redox-indicator based test, and surfactant production was estimated indirectly by both the emulsification index determination (E₂₄) and hemolytic activity. Twenty-six strains (16.8%) were considered as capable of growing in diesel, while surfactant production was detected in 22 (14.8%), estimated according to E₂₄. Seven strains showed high production of biosurfactants (E₂₄ ≥ 50%), headed by Pseudomonas aeruginosa 148D-O, P. aeruginosa 87R-B and Bacillus pumilus 133S-B. No significant correlation was observed between hemolytic patterns and growth outcomes in diesel or E₂₄. Surfactant producing strains should be studied further to assess its potential applications.

Keywords: bioremediation, biosurfactant, emulsification index, fuel, diesel.

Evaluación de crecimiento en combustible diesel y capacidad de producción de surfactantes en bacterias aisladas de combustibles en Costa Rica

Resumen: Un total de 149 cepas bacterianas previamente aisladas de combustibles almacenados en Costa Rica fueron seleccionadas en términos de sus habilidades para crecer aeróbicamente en diesel y producir compuestos con actividad bioemulsificante. El crecimiento en diesel fue evaluado por medio de un test basado en un indicador redox, y la producción de surfactantes fue estimada indirectamente con las determinaciones del índice de emulsificación (E₂₄) y la actividad hemolítica. Veintiseis cepas (16,8%) fueron consideradas como capaces de crecer en diesel, mientras que la producción de surfactantes fue detectada en 22 (14,8%), estimado de acuerdo con el E₂₄. Siete cepas mostraron alta producción de biosurfactantes (E₂₄ ≥ 50%), encabezadas por Pseudomonas aeruginosa 148D-O, P. aeruginosa 87R-B y Bacillus pumilus 133S-B. No se observó correlación significativa entre los patrones de hemólisis y los resultados de crecimiento en diesel o E₂₄. Las cepas productoras de surfactantes deben ser estudiadas más a fondo para evaluar sus potenciales aplicaciones.

Palabras clave: biorremediación, biosurfactante, índice de emulsificación, combustible, diesel.

Recibido 29 de enero de 2012; aceptado 31 de mayo de 2012

Introduction

Bacterial microbiota from regular gas, premium gas and diesel was recently determined for the first time in Costa Rican fuels [1], although the ability of these isolates to grow in the hydrocarbons was not evaluated. To assess the importance of particular microorganisms in fuel deterioration, it is essential to determine their ability to grow, rather than merely exist in fuel systems [2]. The presence of microorganisms in fuel storage tanks and transportation systems may cause problems such as acid formation, increase in viscosity, suspended solids (sludge) and corrosion residues, which translate in filtration problems, deterioration of equipment and loss of fuel quality [3].

In addition, growth on fuels may be correlated to the production of biosurfactants, interesting compounds with potential industrial and biotechnological applications. Biosurfactants are microbial molecules which exhibit high surface and emulsifying activities, and their production is closely related to the ability to grow in hydrocarbon contaminated environments, since they are predominantly produced during growth on water-immiscible substrates [4-6]. Most surfactants produced today are synthetic; however, the use of biosurfactants has advantages such as biodegradability, low toxicity and economically susteinable production cost [7], which makes them an attractive option for industrial purposes. The biosurfactants most commonly isolated are glycolipids containing sugars as rhamnose and trehalose [8], although other different kinds, produced by a wide variety of microorganisms, have been described [6]. Some biosurfactant applications include: oil recovery, additives in food industry, health care and cosmetic industry, microbial remediation of hydrocarbons and crude oil contaminated soils [6-9].

This work aimed to evaluate the ability to grow in diesel oil of bacteria isolated from fuels, and to find indigenous microorganisms from Costa Rican environments capable to produce emulsifier compounds.

Materials and methods

Growth on diesel test: Diesel degradation ability was evaluated in 149 bacterial strains previously isolated from storage tanks of automotive fuels from Costa Rican oil distribution facilities, identified by miniaturized biochemical-test galleries as described by Rodríguez-Rodríguez et al. [1]. Diesel oil is an excellent model for hydrocarbon biodegradation studies [5] and also it may act as a substrate for biosurfactant production. A modified version of a methodology based on a redox-indicator change, developed by Hanson et al. [10] was used; briefly, 125 µL suspension of each strain (30% T, λ = 600 nm) was added separately onto duplicate test tubes containing 7.5 mL of sterile Bushnell-Hass medium (which lacks of carbon sources) [11] supplemented with triphenyl tetrazolium chloride (TTC, 0.225 gL^-1) and 50 µL of diesel-oil. Tubes were incubated at 25 °C for 21 days, and daily monitored for the production of a reddish color, considered as a positive result. Tubes without bacterial inoculum were used as negative controls. This metod is based on the use of TTC as an electron acceptor, which receives electrons from microbial oxidation of hydrocarbons and changes from the colorless (oxidized-form) to the red-reddish (reduced-form).

Hemolyitic activity: Hemolytic activity was also assessed on bacterial strains by plating cells (a loop) onto duplicate blood agar plates and subsequent incubation (25 °C and 35 °C respectively). Hemolytic activity is considered by some authors as indicative of biosurfactant production and used as a rapid method for bacterial screening [5,12,13].

Surfactant production screening: Potential diesel-degraders were screened for surfactant production by determination of the Emulsification Index (E₂₄) [4,5,14-16]. Previously, Correa Bicca et al. [5] designed a yeast extract-containing medium called M2, and demonstrated its improved performance for biosurfactant production. Inocula for E₂₄ tests were prepared by adding a loop of each strain into flasks containing 50 mL of M2 medium supplemented with 1% of diesel and subsequent incubation in agitation (200 rpm) at 28 °C for 48 h; then O.D. was adjusted at 0.650±0.030 (λ=600 nm). Cultures for E₂₄ tests were performed in triplicate, adding 5 mL of the inoculainoculum into flasks containing M2 medium, to obtain a final volume of 50 mL and a diesel concentration of 1%; flasks were incubated at 28 °C in agitation at 200 rpm. Negative controls without bacteria were used by triplicate. At times 0, 1, 2, 3, 4, 7, 9 and 10 days, aliquots of 2 mL were withdrawn from the cultures and mixed in vortex for 2 minutes with 2 mL of sterile diesel in flat bottom tubes (diameter 1.5 cm); the tubes were allowed to stand for exactly 24 h. E₂₄ index was given as the percentage of emulsified layer height (mm) divided by the total height of the liquid column (mm) [17].

Results and discussion

A set of 149 bacterial strains, previously obtained from storage tanks of the main automobile fuels (gasoline, premium gasoline and diesel), was evaluated for a fuel degradation test. A total of 25 strains (17%) showed a positive result in the diesel-degrading screening test. The time necessary to produce a color change in TTC differed among the strains, as it is observed on table 1, ranging from one day for Pseudomonas aeruginosa 149B-D, to 8 days for Bacillus pumilus 133S-B, with a mean of 5 days. These results suggest that only a fraction of the microorganisms previously isolated from stored Costa Rican fuels are able to exert some degree of diesel degradation and consequently reduce the quality. It is remarkable that all the strains of P. aeruginosa were able to grow on diesel, as well as P. pseudoalcaligenes, but not the remaining strains of this genus. Pseudomonas metabolic diversity related to hydrocarbon assimilation has been described, and the use of some strains for the removal of fuel from polluted sites has been documented [6,8,18,19]. Additionally Gram-negative diesel-degraders were only represented by Burkholderia cepacia, best known for its ability to degrade polycyclic aromatic hydrocarbons [20]. Among Gram-positive bacteria, the most frequently diesel-degrading genera were Bacillus (5 strains) and Micrococcus (4 strains), representing 18% and 15% respectively. The ability to degrade some fuel components has been reported for species such as Micrococcus sp., Bacillus sp., B. cereus, B. sphaericus, B. pumilus and B. subtilis [21-23]. On the other hand, only two out of 32 Staphylococcus strains showed positive results in the screening test. Arthrobacter stood out among the remaining isolates, since it is considered as one of the main fuel degraders in soil [21]; Rhodococcus, a metabolic-diverse nocardioform microorganism [5] and Deinococcus or Kurthia, are unusual hydrocarbon-degraders too.

Table 1. Emulsification index E₂₄ and hemolytic activity in potential diesel oil degrading strains isolated from stored Costa Rican fuels.

^a A: alpha-hemolysis; B: beta-hemolysis; G: gamma-hemolysis (no hemolysis); N.G.: no growth. ^b Non-identified strains; both correspond to Gram-positive aerobic bacilli.

Regarding the production of bioemulsifier compounds, tested by monitoring E₂₄ in 10 day-cultures supplemented with diesel, 22 strains from the 25 potential diesel-degraders (88%) yielded positive results. This is not surprising, since biosurfactant production is correlated to the ability to grow in polluted environments with highly hydrophobic substrates [6]. However, E₂₄ results for most of the strains were below 50% (Table 1). Therefore, and since most published data reported E₂₄>50% for potential biosurfactant producing microorganims in the present work we have identified seven promising microbial candidates for biosurfactant production. Nonetheless, it should be taken into account that the maximum E₂₄ value was obtained in most cases after 10 days of culture (Table 1, figure. 1), which indicates that higher values might be accomplished by longer periods than those tested here. Those promising strains are headed by P. aeruginosa 148D-O (E₂₄=77%, 9 d), P. aeruginosa 87R-B (E₂₄=65%, 7 d) and B. pumilus 133S-B (E₂₄=62%, 10 d). E₂₄ values ranging from 70 to 78% have been reported for hexadecane and diesel oil in surfactant-producing P. aeruginosa strains [14,15,17]. Similarly, values of 59% for diesel and values ranging from 66 to 76% for other hydrocarbons, have been obtained with B. pumilus [4,5]. Reports of surfactant-producing strains of B. megaterium have been published [13], although no results of E₂₄ are available; in this work, B. megaterium 122R-G yielded an E₂₄ of 57%. Several studies report the ability of both bacterial genus, Pseudomonas [24-26] and Bacillus [24,27-30], to produce diverse biosurfactants as well.

In the case of Micrococcus sp., values up to 50% were obtained, while Adebusoye et al. [14] reported 49% with diesel and Toledo et al. [16]values ranging from 65 to 73% with other hydrocarbons. The finding of E₂₄=56% for D. radiodurans 54R-M is highly remarkable, since it is, as far as the authors know, the first report of surfactant-production evidence by this specie. Members of the genus Deinococcus are known for their remarkable tolerance towards ionizing radiation and desiccation, and have been isolated from very diverse environments, including exposed to extreme physical conditions [31].

As previously mentioned, hemolytic activity was included in this work as it is widely used to screen biosurfactant production [13]. Moreover, a possible association between hemolytic activity and bioemulsifier-compounds has been proposed [12]. Nonetheless, as it is shown in table 1, a correlation between hemolytic patterns and the ability to grow on diesel oil or E₂₄ values could not be determined, given that only 40% of the strains that were able to grow on diesel showed either beta or alpha hemolysis at 25 °C and/or 35 °C. Similarly, only 45% of the strains with positive results in the E₂₄ test presented some kind of hemolysis, and only half of the strains with E₂₄>40% were able to produce lysis on blood agar plates. These results suggest that hemolytic activity assay may lead to misrecognize many good biosurfactant producers and therefore it should not be used as the only screening method for this purpose.

Given the origin of the strains analyzed, the findings of this study suggest the presence of potential risks caused by bacterial growth in stored fuels. However, for a more accurate assessment of such risks, further studies should be conducted in situations that resemble storage conditions. Results also led to the identification of potential candidates for the production of bioemulsifier compounds, with potential biotechnological applications such as the bioremediation of polluted sites.

Acknowledgements

The authors would like to thank ML Arias for the review of the manuscript. This work was partially supported by Vicerrectoría de Investigación de la Universidad de Costa Rica.

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