Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction

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Abstract

In the present study, we proposed methyl tertiary-butyl ether (MTBE) as a solvent for extraction of biosurfactants from Rhodococcus bacterial cultures. After comparison with other well known solvent systems used for biosurfactant extraction, it was found that MTBE was able to extract crude surfactant material with high product recovery (10 g/l), efficiency (critical micelle concentration (CMC), 130–170 mg/l) and good functional surfactant characteristics (surface and interfacial tensions, 29 and 0.9 mN/m, respectively). The isolated surfactant complex contained 10% polar lipids, mostly glycolipids possessing maximal surface activity. Ultrasonic treatment of the extraction mixture increased the proportion of polar lipids in crude extract, resulting in increasing surfactant efficiency. Due to certain characteristics of MTBE, such as relatively low toxicity, biodegradability, ease of downstream recovery, low flammability and explosion safety, the use of this solvent as an extraction agent in industrial scale biosurfactant production is feasible.

Introduction

Surfactants of biological origin are of increasing interest for many industries due to their chemical diversity, multifunctional characteristics and low toxicity in comparison to synthetic, petrochemical-derived surfactants. Their production from renewable resources is an attraction receiving increasing attention. Fields of application or potential use of biosurfactants are diverse. In the near term, the most promising applications of biosurfactants are the environmental remediation technologies, since product purity is of less concern. At present, however, the industrial use of biosurfactants is not generally competitive with synthetics because of the higher production cost for biosurfactants, mainly due to downstream processing.

The recovery and concentration of biosurfactants from fermentation broth largely determines the production cost. Often, low concentration and the amphiphilic nature of microbial surfactants limit their recovery (Georgeou et al., 1992). Different methods used for biosurfactant isolation include high-speed centrifugation, dia- and ultrafiltration, acid and salt precipitation, solvent extraction and adsorption chromatography Syldatk and Wagner, 1987, Bryant, 1990, Mulligan, 1990, Kurane et al., 1994. Bacteria of the genus Rhodococcus synthesise considerable amounts of glycolipid surfactants, e.g. total product yield reached 20–30 g/l for Rhodococcus erythropolis (Kim et al., 1990), and 9–11 g/l for R. ruber (present study). However, surfactants produced by rhodococci under unrestricted growth conditions are predominantly cell-associated trehalose mycolates, which can be effectively isolated only by organic solvent extraction Rapp et al., 1979, Lang and Philp, 1998. By comparison, the rhamnolipids of Pseudomonas aeruginosa are excreted.

A wide variety of organic solvents (e.g. methanol, ethanol, diethyl ether, pentane, acetone, chloroform, dichloromethane) have been used, either singly or in combination, for biosurfactant extraction (Desai and Banat, 1997). Most effective are the mixtures of chloroform and methanol in various ratios, which facilitates adjustment of the polarity of extraction agent to the target extractable material. However, the use of chloroform for preparative extractions demanding large volumes of solvent is not economically warranted. Moreover, chloroform is a highly toxic chloro-organic compound regarded as harmful for the environment and for human health Heitmann et al., 1996, Agency for Toxic Substances and Disease Registry (ATSDR), 1997, Lilly et al., 1997, Mills et al., 1998. Thus, there is a need for inexpensive and low toxicity solvents for biosurfactant extraction suitable for industrial applications.

To isolate rhodococcal non-ionic glycolipid surfactants having low hydrophilic–lipophilic balance (HLB) requires a solvent with low polarity. We suggested that methyl tertiary-butyl ether (MTBE), the commonly used octane-enhancing gasoline additive, might be a promising candidate for this application (Ivshina et al., 1996). It is relatively non-toxic, less likely to form peroxides and less explosive than other solvents Rosenkranz and Klopman, 1991, Gupta and Lin, 1995. Alkyl ethers, the chemical class to which MTBE belongs, are more polar than hydrocarbons, but less polar than alcohols, ketones, esters and chlorinated hydrocarbons (Reichardt, 1988).

In this work, we compared MTBE with other well known solvent systems used for biosurfactant extraction. The surface-active properties and chemical composition of the biosurfactant isolated from the culture of R. ruber by different extraction methods were evaluated.

Section snippets

Bacterial strain and culture conditions

The strain R. ruber IEGM 231 was from the Regional Specialised Alkanotrophic Microorganisms Collection of the Institute of Ecology and Genetics of Microorganisms, Perm, Russia.

The organism was maintained on nutrient agar slants. A mineral salts medium contained (per litre of distilled water): KH2PO4, 2.0 g; K2HPO4, 2.0 g; KNO3, 1.0 g; (NH4)2SO4, 2.0 g; NaCl, 1.0 g; MgSO4·7H2O, 0.2 g; CaCl2·2H2O, 0.02 g; FeCl3·7H2O, 0.01 g; trace elements solution, 1.0 ml; thiamine, 4 mg. n-Hexadecane was used

Extraction of crude biosurfactant

The ability of solvent systems to isolate surface-active components from 60-h cultures of R. ruber differed under various extraction conditions. Although differences in the amount of biosurfactant extracted by tested solvent systems were small, use of MTBE and chloroform–methanol resulted in greater crude extract yield than the use of dichloromethane (Table 1). When extraction was performed with MTBE or dichloromethane, the solvent and water fractions produced two clearly separate layers after

Acknowledgments

This work was supported by the Royal Society grant (638072 P750/LJH) and the grant from the Ministry for Science and Technology of the Russian Federation (No. 991F).

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