Redalyc.BIODEGRADATION of CRUDE OIL by Pseudomonas

Tecnología Química ISSN: 0041-8420 [email protected] Universidad de Oriente Cuba

Pérez Silva, Rosa M.; Ábalos Rodríguez, Arelis; Gómez Montes de Oca, José M.; Cantero Moreno, Domingo BIODEGRADATION OF CRUDE OIL BY Pseudomonas aeruginosa AT18 STRAIN Tecnología Química, vol. XXVI, núm. 1, enero-abril, 2006, pp. 70-77 Universidad de Oriente Santiago de Cuba, Cuba

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative BIODEGRADATION OF CRUDE OIL BY Pseudomonas aeruginosa AT18 STRAIN Rosa M. Pérez Silva1; Arelis Ábalos Rodríguez2*; José M. Gómez Montes de Oca1, Domingo Cantero Moreno1 1Department of Chemical Engineering Technology, Food Technology and Environmental Technologies, Faculty of Sciences, University of Cadiz, Campus Rio San Pedro, 11510 Puerto Real (Cadiz), Spain

2Centro de Estudios de Biotecnología Industrial. Facultad de Ciencias Naturales. Universidad de Oriente. Santiago de Cuba, Cuba

The bioremediation of hydrocarbons in contaminated soils by Pseudomonas aeruginosa AT18 strain grow on crude oil, lubricant oil, naphthalene, toluene and, kerosene as unique carbon source and energy was isolated from samples soil contaminated with crude oil on Petroleum Refinery Hnos Díaz in Santiago de Cuba city. The biodegradative activity of the strain achieved 81% with the mixture Mesa 30/Puerto Escondido (8:2) crude oil. Key words: biodegradation, Pseudomonas aeruginosa. ______Pseudomonas aeruginosa AT18, es capaz de usar hidrocarburos del petróleo como fuente de carbono y energía para su metabolismo. En el presente trabajo se estudia la actividad biodegrativa de Pseudomonas aeruginosa AT18, aislada de suelos contaminados de la Refinería Hermanos Díaz (Santiago de Cuba), sobre petróleo y aceite lubricante. Los resultados obtenidos muestran un nivel de degradación del 81 % para el crudo de petróleo Mesa 30/Puerto Escondido (8:2). Palabras clave: biodegradación, Pseudomonas aeruginosa.

Introduction achieve extensive degradation. However, most of the crude oil degradation studies reported in the The hydrocarbons and petroleum constitute literature have been carried out with single or mixed bacterial strains isolated because of their one of the main environmental pollutants and ability to grow in mineral media with crude oil as their abundance and persistence in several only carbon source (Solanas, et al. 1984, polluted environmental compartments have Palittapongarnpim et al. 1998, Viñas et al., 2002, been reported (Casellas et al. 1995). The Gunn et al., 2003). In some studies the microbial petroleum contains thousands of individual activity has been increased with the use of hydrocarbons and related compounds. Their biosurfactants (Arino et. all 1998, Moran et al. main components are saturates (n- and 2000, Abalos, et al., 2004). branched-chain alkanes and cycloparafins rings) and aromatic and polynuclear compounds Pseudomonas aeruginosa is an opportunistic (PAHs) and resins and asphaltenes pathogen responsible of nosocomial infections in (heterocyclics, oxygenated hydrocarbons) (Vi- immunodeficiency patients (Noordman et al. 1998; ñas et al. 2002). The petroleum composition Anh, 1999) found ubiquitously in different determines their classification in heavy ecosystems (Holloway, 1996; Belhaj et. al., 2002). (aromatics and PHAs principally), half (the We studied the biodegradation of half crude oil Mesa 30 / Puerto Escondido (M30PP) by same proportion of saturates and aromatics) Pseudomonas aeruginosa AT18. The strain grow and light (saturated principally) oil. on kerosene (C12 – C14) lubricant oil (C18 – Many microorganisms capable of degrading C40), toluene (alkylbenzene), naphthalene petroleum components have been isolated. (polyaromatic) and evidence the presence of alk, Given the complexity of oil products, a tol, nah genes. It is unusual that a single strain combination of bacterial strains with broad contain three metabolic way to degrade enzymatic capabilities will be required to hydrocarbons.

70 TECNOLOGÍA QUÍMICA Vol. XXVI, No. 1, 2006 Material and Methods (1:1 v/v) with n-hexane (APHA, 1998). The amount residual of crude oil was determined by measuring the weight of the dry extract; and Bacterial Strain biodegradation was evaluated by comparison of its weight with a control sample. Pseudomonas aeruginosa AT18 was isolated from contaminated soil with crude oil at the Her- manos Díaz Refinery in Santiago of Cuba. The Results and Discussion strain is deposited in the Collection of Cultures at the Centro de Estudios de Biotecnología Indus- trial, Universidad de Oriente. Santiago de Cuba. Identification of Pseudomonas aeruginosa (Pérez et al. 2003). The strain was characterized AT18 strain by morphological and biochemical tests and was maintained on Triptone Soja Agar (TSA) tubes at A total of 44 bacterial strains from soil samples 4 oC with successive subcultures obtained every contaminated with crude oil were obtained. Thirty one month. eight (86 %) Gram-negative bacile, oxidase positive strains that grew on Kings A and B Culture Medium and Cultivation Conditions media, and produced siderophores pyoverdin and pyocyanin pigments were identified as P. The mineral medium used for Pseudomonas aeruginosa using classical test. These isolates -1 aeruginosa AT18 contained (gl ): NH4Cl, 0.1; were submitted to sequential enrichment during

K2HPO4, 0.1; KH2PO4, 0.05; CaCl2, 0.001; KCl, ten days with crude oil M30PP as carbon source

0.01; FeSO4.7H2O, 0.001; MgSO4.7H2O, 0.05 and in order to select the best strains of P. aeruginosa supplemented with 0.05 ml l-1 of a solution of oil-degraders. Finally five strains were obtained oligoelements, whose composition was: B (0.02 % v/ and Pseudomonas aeruginosa AT18 strain was v), Cu (0.05 % v/v), Mn (0.05 % v/v), Mo (0.006 % the best growing. The strain grew at 41 0C and v/v) and Zn (0.07 % v/v). The carbon source (0.2 % was capable of denitrification, showed haemolytic in all cases) used were: M30PP, lubricant oil, kero- and proteolytic properties and produced sene, toluene, and naphthalene. Controls without rhamnolipids (Pérez, et. all., 2003) and homoserine carbon sources were performed. The inoculum used lactone (HSL). In P. aeruginosa strains was 2 % (v/v) of a cellular suspension of concentration biosurfactants-producer had been studied that the 0.5 McFarland (Jorgensen, J. et al., 1999). The production of rhamnolipids is under quorum sensing strain was incubated in 500 –mL Erlenmeyer flasks mechanism trough an autoinducer homoserine- containing 100 mL of medium at 30 0C during 20 d type (Nakata et al., 1998). Several authors under shaking at 150 rpm. All the experiments were reported the rhamnolipids production by different carried out in triplicate. P. aeruginosa strains when cultivated on hydrocarbon or crude oil (Arino 1998, Syldatk et Cellular Growth Determination al., 1985; Déziel et al., 1999). The production of rhamnolipids becomes the carbon source Biomass concentration was measured by solubilisation and their assimilation by the cells. turbidymetric method at 580 nm and the obtained values converted to g cell dry/L using a previously Growth of Pseudomonas aeruginosa AT18 determined calibration curve (Madigan, 1999). on Hydrocarbons

Consumption of M30PP The growth kinetic for the strain P. aeruginosa AT18 was studied during ten days, and measuring The consumption of M30PP was measured by daily the absorbance and converting to g cell gravimetric analysis after liquid-liquid extraction dry L-1. The results are showed in Figure 1. The

TECNOLOGÍA QUÍMICA Vol. XXVI, No. 1, 2006 71 growth rate (µmax) was 0.20 d-1 approximately, which involve monooxygenase enzyme however the maximum growth was observed in (Vomberg and Klinner 2000). The complete -1 - kerosene (2.5 g L ) and lubricating oil (2 g L oxidation reaction can be represented as CnH2n 1 ). The lubricating oil and kerosene are mixture + 3/2N O2 → N CO2 + N H2O. The pathway for alkanes; the kerosene contains C12 – C14 degradation of n-alkanes has been extensively aliphatic chains, whereas lubricating oil contains studied in Pseudomonas putida and is codified heavy chains (C18 – C40). The alkanes are the by alk genes. These genes are expressed in n- most abundant compounds and more simples to alkanes presence. Alkanes are greatly oxide. Aliphatic hydrocarbons are degraded distribuited in the environment, and with greater speed, but the key step involves microorganisms are likely to utilize these highly oxidation of the molecule to increase their reduced compounds as carbon and energy sources. solubility. The first step of alkane degradation The alkanes degraders including strains of (Figure 2a) is the oxidation of the methyl group Pseudomonas, Acinetobacter, Alcaligenes and to the alcohol by the alkane hydroxylase system, Rhodoccocus (Whyte et. al., 1997).

2,5

1,5

dry weight (g/L) dry weight 1

0,5

0 012345678910

LO K T N Time (days)

Fig. 1 Growth kinetic for P. aeruginosa AT18 on lubricating oil (LO), kerosene (K), toluene (T) and naphtha lene (N) at 10 days of incubation. In all cases the carbon source was added at 0,2 % v/v.

72 TECNOLOGÍA QUÍMICA Vol. XXVI, No. 1, 2006 (a)

CH3-(CH2)n-CH3 monooxigenase

CH3-(CH2)n-CH2OH alcohol-deshydrogenase

CH3-(CH2)n-CHO aldehyde-deshydrogenase

CH3-(CH2)n-COOH

Beta-oxidation (c)

(b) CH3

toluene-dioxigenase naphtalene-dioxigenase

benzaldehyde-deshydrogenase salicylaldehyde-deshydrogenase COOH COOH

salicilate-hydroxilase benzoate-hydrocylase OH

OH catechol-dioxigenase meta orto

Piruvate Succinil-CoA + + Acetaldehyde Acetil-CoA

TCA

Fig. 2 Metabolic pathway of hydrocarbon degradation by Pseudomonas aeruginosa. (a) n-alkanes, (b) naphthalene and (c) toluene. The last step in the n-alkanes degradation involves beta-oxidation and the aromatics and polyaromatics ring degradation finish in TCA cycle. In both pathways the catechol is the intermediate.

TECNOLOGÍA QUÍMICA Vol. XXVI, No. 1, 2006 73 The growing of P. aeruginosa AT18 on toluene or water organisms. Pseudomonas has metabolic (1.2 g L-1) and naphthalene (1.73 g L-1) was 70 % versatility and this versatility is associated to lowest that growing on alkanes (Figure 1). The plasmids. It is know the degradative plasmids 60 % of soil bacteria strains (which the 50 % are ALK (alkanes), OCT (octane), XYL (xylene), Pseudomonas) can oxide the naphthalene. The CAM (alcanphour), NAH (naphthalene), TOL catabolic pathways of aromatics monoaromatics (toluene) and SAL (salicylic acid). and polynuclears including two oxygen-dependent enzymes: hydroxylase and diooxygenase (Casellas et al., 1998). The oxidative pathway of the Biodegradation of M30PP by Pseudomonas naphthalene (nah) and toluene (tol) involve the aeruginosa AT18 catechol molecule as intermediate (Figure 2b and 2c) which is converted in piruvate (meta break) or Considering that Pseudomonas aeruginosa succinic acid (orto break) that are incorporated AT18 grow on n-alkanes and aromatics into tricarboxilic acids (TCA) cycle. If the compounds we studied the degradation of crude incubation occurs under anaerobic conditions the oil. substrate is converted in CO2 and methane (Dunja and Vogel 1987). The anaerobic reaction is C7H8 M30PP is a mixture of crude oil Mesa 30 (from + 5H2O → 2.5CO2 + 4CH4. The complete oxidation Venezuela) with crude oil Puerto Escondido (from reaction can be represented as C7H8 + 9O2 → Cuban). The proportion in the mixture is 80:20 7CO2 + 4H2O for toluene and C10H8 + 12O2 → respectively. The crude oil M30PP has a density -1 10CO2 + 4H2O for naphthalene. The presence of 0.835 mg L , and 26,2 API gravity, their toluene pathway suggests the possibility of composition is n-parafines 23.9 %; iso-parafines degradation the alkylbenzenes toluene-related, 26.1 %; naphthenes 25.7 % and aromatics 24.3 %. for example xylenes and cresols. The M30PP crude oil is half crude because of the The presence of oxygenases in Pseudomonas proportion of the n-alkanes or parafines and iso- aeruginosa AT18 may reflect the double nature parafines is similar to the proportion of the naphthenes of this bacterium as pathogen and as common soil and aromatics.

0,8

0,6

0,4 cell growth (g/L) growth cell 0,2

0 02468101214

Time (days)

Fig. 3 Growth kinetic for P. aeruginosa AT18 on mineral media with M30PP crude oil as carbon source.

74 TECNOLOGÍA QUÍMICA Vol. XXVI, No. 1, 2006 The strain Pseudomonas aeruginosa AT18 molecules. The metabolic pathways are more is able to grow on petroleum as the only carbon complex, because of the hydroxylate source (Figure 3) and reaches 0.95 gL-1 of intermediate (catechol, gentisate and cellular biomass at 20 d of culture. The curve protocatecuate) are formed by different ways shows a diauxic growth, which is characteristic depending of the compound present in the crude of the assimilation of two or more carbon oil. In many cases is possible that some sources (Schlegel, 1997). The first stage of intermediate compound be toxic for the cells. growth begins approximately between first and We observed with naphthalene as carbon second day of incubation until fifth day where source, that P. aeruginosa AT18 have a poor was observed the stationary phase, reaching growth when the pH diminishes drastically by 0.31 g L-1 of biomass and the specific growth –2 -1 salicylic acid formation in the medium (Pérez rate (µ) was of 3.5 x 10 d . In the first stage, et al., 2003). The superficial tension not are degraded the n-alkanes and is possible the decreased which means that rhamnolipids degradation of some alkylic chains present in production not occurs during Pseudomonas other molecules (methylated aromatics) (Vi- aeruginosa AT18 growing on M30PP crude ñas et al., 2002). The aliphatic chains are oxide successively until fatty acid corresponding oil. Pseudomonas aeruginosa AT10 (CECT which is incorporated to the β-oxidation (Figu- 11769), strain isolated from contaminated soil re 2a). A second exponential phase was samples of the ERASOL vegetable oil refinery observed about ninth day of incubation. The in Santiago de Cuba, Cuba, can be produced specific growth rate of 2.4 x 10-3 d-1 and the until 16 g l-1 of rhamnolipids in mineral media biomass increased to 0.95 g L-1. The growth and waste free fatty acids as carbon source rate decreases with increase of the incubation (Ábalos et al., 2002); however when growth time for the different exponential growth phases on Casablanca crude oil, a light crude oil, not due to the cell have to adapt continuously to produce rhamnolipids. This strain degrades n- complex molecules. In the last stage are alkanes (34 %), but not degrades branches and degraded the mono and poly aromatics polyaromatics (Ábalos et al., 2004).

100

20 Biodegradation (%) 0 0 5 10 15 20

Time (days)

Fig. 4 Biodegradation of M30PP by P. aeruginosa AT18.

The biodegradative activity (Figure 4) increases the n-alkanes and aliphatic chains are first of 38 to 81 percentages in 20 d. When is calculated degraded. The yield biomass/substrate (Yx/s) was the consumption of petroleum after the different 46 % and for bioremediation purpose is very incubation times (dates not showed) we observed convenient a little biomass production with a high that the greater level of degradation takes place in utilization of the carbon source in order to no alter the first 5 days of culture, which means that the ecosystem.

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