by Pseudomonas aeruginosa JC2 in Different

Rhamnolipid Production by Pseudomonas aeruginosa JC2 in Different N-Heterocylic Aromatic Hydrocarbons Aparna P * Sasikala Ch ** and Ramana Ch V *** An isolate from garden soil, Pseudomonas aeruginosa JC2 produced rhamnolipid whose structure was determined by mass spectral analysis and was found to be L-rhamanosyl L-rhamnolsyl-hydroxydecanoyl--hydroxydecanoate. The concentration of rhamnolipid produced was checked in the presence of various nitrogen containing heterocyclic aromatic hydrocarbons (1 mM) with glucose (1%) as carbon source. Pyrazine-2-carboxylate served as best source as it contributed to the production of 240 mg/L concentration of rhamnolipid when compared with other compounds. Highest amounts of rhamnolipid (360 mg/L) were produced when 6.4 mM concentration of pyrazine-2-carboxylate was used. Ke yw or ds : N-heterocyclic aromatic hydrocarbons, Pseudomonas aeruginosa JC2, Rhamnolipid

Introduction Surfactants constitute an important class of industrial chemicals widely used in almost every sector of modern industry. Biosurfactants are amphiphilic compounds of microbial origin having advantages over their chemical counterparts in biodegradability and effectiveness at extreme conditions (Banat et al., 2000). Biosurfactants are produced by various microbes like Pseudomonas aeruginosa, Bacillus subtilis and also some Yeast species (Cooper and Goldenberg 1987; and Johnson et al., 1992). Rhamnolipid, a biosurfactant containing rhamnose and  -hydroxydecanoic acid is produced by Pseudomonas aeruginosa. A mixture of rhamnolipids containing either one or two residues and two lipid chains are produced by various Pseudomonas spp. with different strains producing varying concentrations of rhamnolipids when grown on various substrates (Lang and Wulbrandt, 1999). Rhamnolipids from Pseudomonas spp are most commonly isolated type of biosurfactants, having potential applications in agriculture, cosmetics, pharmaceuticals, personal care products, food processing, textile manufacturing, Author pl p r o v i d e designatio ns and email ids of the authors

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xxxxxxxxxxxxxxxxx, Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, JNT University, Kukatpally, Hyderabad 500072, India; and is the corresponding author. E-mail: [email protected]

**

xxxxxxxxxxxxxxxxx, Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, JNT University, Kukatpally, Hyderabad 500072, India. E-mail: xxxxxxxxxxxxxxxxxxx

*** xxxxxxxxxxxxxxxxx, Department of Plant Sciences, School of Life Sciences, University of Hyderabad, PO Central University, Hyderabad 500046, India. E-mail: xxxxxxxxxxxxxxxx Rhamnolipid Pseudomonas aeruginosa JC2 © 2010 IUPProduction . All Rightsby Reserved. in Different N-Heterocylic Aromatic Hydrocarbons

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etc. The most important of it is their role in bioremediation which is attributed to their capability of increasing the bioavailability of poorly soluble organic compounds, such as polycyclic aromatics (Banat et al., 2000; and Maier and Soberón-Chávez, 2000). Though polycyclic hydrocarbons (Déziel et al., 1996) and carbohydrates (Guerra-Santos et al., 1984) were used as substrates for the production of rhamnolipids, their production in the presence of n-heterocyclic aromatic hydrocarbons is studied very little. An attempt was made to study the levels of rhamnolipids produced in the presence of heterocyclic aromatic hydrocarbons.

Materials and Methods Organism and Culture Conditions Pseudomonas aeruginosa JC2 was isolated from rhizosphere soil and was grown aerobically. The mineral medium contained (per liter of deionized water), 0.5 g KH2PO4, 0.2 g MgSO4.7H20, 0.05 g CaCl2.2H20, 0.001 g FeSO4.7H2O, 10 g glucose and N-heterocyclic aromatic hydrocarbons (1 mM). Different n-heterocyclic aromatic compounds used in the study were pyrazine, pyrazinamide, pyridine, picoline, nicotinic acid, dipicolinic acid, indole, imidazole. The final pH of the liquid medium was adjusted to 7.0. 25 mL of mineral medium was inoculated with 10% inoculum of Pseudomonas aeruginosa JC2 in 100 mL conical flasks which were incubated at 37 °C with 125 rpm agitation for 2 days and the concentration of rhamnolipids produced was checked at regular intervals. Growth of the culture was measured in terms of absorbance at an optical density (OD) 540 nm.

Rhamnolipid Assay and Purification Rhamnolipid concentration in the samples was determined by measuring the concentration of rhamnose by the orcinol method (Chandrasekaran and Bemiller, 1980) after acid hydrolysis of the sample. 2 L of the mineral medium was used for rhamnolipid extraction from the culture supernatant as per the procedure described earlier (Wei et al., 2005).

Characterization of Rhamnolipid Mass spectral analysis of rhamnolipid was done in Shimadzu LC-MS (LCMS-2010A). The analysis was performed at 40 °C (LC column oven) and 85 °C (MS ionization chamber) with Luna 5  C18 (2) 100 A column (250  4.6 mm). Acetonitrile and water (1:1) were used as solvent at 0.2 mL min–1. The column effluent from the LC was nebulized into an Electron Spray Ionization (ESI) region under N2 gas for generating molecular masses ranging from 200 to 800 m/z, which were detected in negative mode.

Results and Discussion Rhamnolipid production in the presence of various nitrogen containing heterocyclic aromatic hydrocarbons was studied and among all the aromatics used, pyrazine-2-carboxylate gave the high amounts of rhamnolipid followed by pyrazinamide, nicotinic acid, dipicolinic acid, indole and imidazole when used in 1 mM concentration (Figure 1). 2

The IUP Journal of Life Sciences, Vol. IV, No. 2, 2010

Figure 1: Production of Rhamnolipid in the Presence of Various N-Heterocyclic Aromatic Compounds 300

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Rhamnolipid production was very minimal when no heterocyclic compound was present. Compared to the commercial production of rhamnolipids that reached 100 g/L (Lang and Wullbrandt, 1999) and the production up to 3,600 mg/L, when olive oil was used as substrate (Wei et al., 2005), the production in the presence of n-heterocyclic aromatic hydrocarbons is very low. But the conditions can be optimized for increasing the levels of rhamnolipid. Figure 2: Increase in the Production of Rhamnoplipid Corresponding to the Growth of Pseudomonas aeruginosa JC2 0.8

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When pyrazine-2-carboxylic acid was supplemented in the medium at different concentrations, there was an increase in the rhamnolipid production up to 6.4 mM and it decreased subsequently with increasing concentrations. The increase in the production of rhamnolipid was corresponding to the growth of the culture at that particular time interval, as shown in Figure 2. Mass spectral analysis of the purified product gave a mass of 673.0124 which corresponds to the mass of L-rhamanosyl L-rhamnolsyl--hydroxydecanoyl--hydroxydecanoate, a di-rhamnolipid produced by this particular strain of Pseudomonas aeruginosa JC2 (Figure 3). The purified product from the culture supernatant contained only RL2, possibly playing a major role in the degradation of n-heterocyclic aromatic hydrocarbons. Figure 3: LC-Mass Spectral Analysis of Rhamnolipid OH CH3 OH

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4292276 3108207 3692266

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Biosurfactants, that are environmentally compatible and nontoxic are having potential applications in industry ranging from biotechnology to environmental cleanup. Studies were conducted on effect of rhamnolipids, produced by Pseudomonas aeruginosa in the degradation of long chain hydrocarbons, polycyclic aromatic hydrocarbons and also in solubilization of pesticides (Zhang and Miller, 1992; Arino et al., 1996; Mata-Sandoval et al., 2000; Mohammed et al., 2007; and Chang-Zheng et al., 2008). Biosurfactant production by Pseudomonas growing on polycyclic aromatic hydrocarbons was also reported 4


(Déziel et al., 1996), but their production in the presence of n-heterocyclic aromatic hydrocarbons were not reported till now up to our knowledge. In this present study, some n-heterocyclic aromatic hydrocarbons were introduced into the mineral medium and the production of rhamnolipids was monitored. The production of rhamnolipids can be attributed to its degradation capability as Pseudomonas has immense capacity to degrade n-heterocyclic aromatic hydrocarbons (Mattey and Harley, 1976; and Kaiser et al., 1996). High concentrations of rhamnolipid in pyrazine-2-carboxylic acid and pyrazinamide suggest that pyrazines contribute the most for their production compared to other n-heterocyclic aromatic hydrocarbons.

Conclusion LC-Mass metabolome profile of the purified rhamnolipid has given the structure of the RL2, probably the predominant rhamnolipid produced during the degradation of the n-heterocyclic aromatic compounds. The condition for higher rhamnolipid production by the strain has to be optimized and this study can be further continued to employ this strain in bioremediation studies. 

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8. Guerra-Santos L, Käppeli O and Fiechter A (1984), “Pseudomonas aeruginosa Biosurfactant Production in Continuous Culture with Glucose as Carbon Source”, Applied Environmental Microbiology, Vol. 48, pp. 301-305. 9. Johnson V, Singh M, Saini V et al. (1992), “Bioemulsifier Production by an Oleaginous Yeast Rhodotorula glutinis IIP-30”, Biotechnology Letters, Vol. 24, pp. 225-229. 10. Kaiser J P, Feng Y and Bollag J M (1996), “Microbial Metabolism of Pyridine, Quinoline, Acridine and Their Derivatives Under Aerobic and Anaerobic Conditions”, Microbiol. Reviews, Vol. 60, pp. 483-498. 11. Lang S and Wulbrandt D (1999), “Rhamnose Lipids-Biosynthesis, Microbial Production and Application Potential”, Applied Microbiology and Biotechnology , Vol. 51, pp. 22-32. 12. Maier R M and Soberón-Chávez G (2000), “Pseudomonas aeruginosa Rhamnolipids: Biosynthesis and Potential Applications”, Applied Microbiology and Biotechnology, Vol. 54, pp. 625-633. 13. Mata-Sandoval J C, Karns J and Torrents A (2000), “Effect of Rhamnolipids Produced by Pseudomonas aeruginosa UG2 on the Solubilization of Pesticides”, Environ. Sci. Technol., Vol. 34, pp. 4923-4930. 14. Mattey M and Harley E M (1976), “Aerobic Metabolism of Pyrazine Compounds by Pseudomonas Species”, Biochem. Soc. Trans., Vol. 4, No. 3, pp. 492-494. 15. Mohammad H, Akio U and Hitoshi I et al. (2007), “Degradation of Long-Chain NAlkanes (C36 and C40) by Pseudomonas aeruginosa Strain WatG”, International Biodeterioration & Biodegradation, Vol. 59, No. 1, pp. 40-43. 16. Wei Y, Chou C and Chang J (2005), “Rhamnolipid Production by Indigenous Pseudomonas aeruginosa J4 Originating from Petrochemical Wastewater”, Biochemical Engineering Journal, Vol. 27, pp. 146-154. 17. Zhang Y and Miller R M (1992), “Enhanced Octadecane Dispersion and Biodegradation by Pseudomonas Rhamnolipid Surfactant (Biosurfactant)”, Applied Environmental Microbiology, Vol. 58, pp. 3276-3282. Reference # 54J-2010-05-xx-01

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