Carbon spectrum utilization by an indigenous strain of Pseudomonas aeruginosa NCIM 5514: Production, characterization and surface active properties of biosurfactant

Highlights

• P. aeruginosa NCIM 5514 showed unique properties of carbon spectrum utilization.

• It produced 3.178 ± 0.071 g/l biosurfactant with glucose as the best carbon substrate.

• P. aeruginosa NCIM 5514 biosurfactant showed desired surface active properties.

• Biosurfactant was composed of Rha-Rha-C₁₀-C_14:1 and Rha-C₈-C₁₀.

• Biosurfactant is a promising product for application in petroleum and other industries.

Abstract

The present research work was undertaken with a mandate to study carbon spectrum utilization and structural characterization of biosurfactant produced by indigenous Pseudomonas aeruginosa NCIM 5514, which showed unique properties to utilize a large number of carbon sources effectively for production of biosurfactant, although glucose was the best carbon substrate. In Bushnell-Hass medium supplemented with glucose (1%, w/v), 3.178 ± 0.071 g/l biosurfactant was produced by this isolate in 96 h. The biosurfactant produced showed surface tension and emulsification activity values from 29.14 ± 0.05 to 62.29 ± 0.13 mN/m and 88.50 ± 1.96 to 15.40 ± 0.91%, respectively. Toluene showed highest emulsification activity followed by kerosene. However, kerosene exhibited emulsion stability for 30 days. Biosurfactant was characterized as a mixture of di-rhamnolipid (Rha-Rha-C₁₀-C_14:1) and mono-rhamnolipid (Rha-C₈-C₁₀) by FTIR, ESI-MS and LC–MS techniques. High biosurfactant yield opens up doors for the isolate to find utility in various industries.

Introduction

The demand for bio-based processes and materials in petroleum industry has significantly increased during last decade (Muller et al., 2012, Varjani and Upasani, 2016). Microorganisms present in oil polluted environments produce various bioactive compounds. Biosurfactant, a secondary metabolite is one of the several bioactive compounds that have attracted major interest and attention due to their structural and functional diversity (Soberon-Chavez et al., 2005, Janek et al., 2010, Muller et al., 2012). These natural compounds have many advantages over synthetic surfactants, such as lower toxicity, enhanced biodegradability, enhanced foaming capacity, high specificity, better environmental compatibility and ability to be synthesized from renewable feed-stocks (Desai and Banat, 1997, Janek et al., 2010, Gudina et al., 2016). Bacterial species belonging to the genera Bacillus, Halomonas and Pseudomonas can grow exclusively on hydrocarbons and produce biosurfactant(s) (Satpute et al., 2008). Pseudomonas is one of the most prominent hydrocarbon utilizers and biosurfactant producer reported from hydrocarbon polluted sites (Deziel et al., 1996, Das and Mukherjee, 2005, Batista et al., 2006, Thavasi et al., 2011, Phan et al., 2013, Varjani and Upasani, 2016). Biosurfactants have extensive applications in many industries such as petroleum, food, beverage, cosmetics, detergents, pharmaceuticals, lubricant formulation, mining and metallurgy and environmental protection as well as management, etc. (Benincasa et al., 2004, Rahman et al., 2003). They can be used as emulsifiers, de-emulsifiers, wetting, dispersing and foaming agents (Moya et al., 2015, Gudina et al., 2016).

Effective screening methodologies and improved purification techniques plays critical role in achieving desired quality and quantity of biosurfactant (Satpute et al., 2008). Screening methods used for biosurfactant producers are hemolytic activity, cetyltrimethyl ammonium bromide (CTAB) agar plate method, drop collapse, oil spread, emulsification activity, surface tension and interfacial tension (Satpute et al., 2008, Varjani et al., 2014).

The structure and production of this secondary metabolite strictly depends on medium composition, e.g. carbon and nitrogen sources (Rahman et al., 2002, Gudina et al., 2011). Microbial surfactants encompass a wide spectrum of molecules, such as lipopeptides, glycolipids, phospholipids, fatty acids, neutral lipids and polymeric biosurfactants (Rahman et al., 2003, Janek et al., 2010, Moya et al., 2015). Glycolipids are one of the most promising biosurfactants. They consist of carbohydrates and long-chain aliphatic or hydroxyaliphatic acids (Gudina et al., 2016). The most effective glycolipids with reference to surface active properties are trehalolipids of Rhodococcus erythropolis, sophorolipids of Candida bombicola, and rhamnolipids of Pseudomonas sp. (Wei et al., 2005, Raza et al., 2009, Varjani and Upasani, 2016). Rhamnolipids (RLs) produced by Pseudomonas strains have a glycosyl head group (a rhamnose moiety) and a 3-(hydroxyalkanoyloxy) alkanoic acid (HAA) (Soberon-Chavez et al., 2005, Gudina et al., 2016). Muller et al. (2012), reviewed that shake flask experiments are more useful for studying optimization of rhamnolipid production. They have critically reviewed factors influencing rhamnolipid production in Pseudomonas sp., and reported it to be capable of using both water soluble carbon substrates, such as glucose, glycerol, mannitol and fructose (Gudina et al., 2011, Hamzah et al., 2013, Varjani et al., 2014) as well as water immiscible n-alkanes to produce rhamnolipid (Varjani et al., 2013, Varjani et al., 2015). Not only the type of carbon and nitrogen source but also the respective C/N ratio strongly influence total rhamnolipid production (Wu et al., 2008, Muller et al., 2012).

Most methods for recovery of rhamnolipids have been very well reviewed (Desai and Banat, 1997, Heyd et al., 2008, Pornsunthorntawee et al., 2008). One of the easiest methods for recovery was precipitation by acid (Zhang and Miller, 1992; Deziel et al., 1996; Varjani and Upasani, 2016) or aluminium sulphate (Schenk et al., 1995), the latter precipitates the rhamnolipid by salting out process which can be recovered by centrifugation. However, solvent extraction is the most adopted method (Schenk et al., 1995, Varjani and Upasani, 2016). Extracted biosurfactant can be characterized by chromatographic and/or spectrophotometric techniques, viz. Thin layer chromatography (TLC), Fourier transform infrared spectroscopy (FTIR), Electrospray ionization mass spectrometry (ESI-MS), High-performance thin layer chromatography (HP-TLC), High performance liquid chromatography – mass spectrometry (HPLC-MS), Liquid chromatography – mass spectrometry (LC–MS) and Nuclear magnetic resonance (NMR), etc. (Rahman et al., 2002, Benincasa et al., 2004, Abdel-Mawgoud et al., 2009, Raza et al., 2009; Lotfabad et al., 2010, Varjani and Upasani, 2016).

In the present investigation, carbon spectrum utilization and biosurfactant production by P. aeruginosa NCIM 5514 has been studied. Physico-chemical properties of the biosurfactant such as surface activity (minimum surface tension and critical micelle dilution) and emulsification activity of the cell free supernatant have also been reported.