Microbial Surfactants: Current Perspectives and Role in Bioremediation

Pooja Shivanand1*, Nur Bazilah Afifah Binti Matussin1 and Lee Hoon Lim2

■‘Environmental and Life Sciences, Faculty of Science, Universiti Brunei Darussalam, Jalan

Tungku Link, Gadong BE1410, Negara Brunei Darussalam 2Chemical Sciences, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link,

Gadong BE1410, Negara Brunei Darussalam.

INTRODUCTION

Surfactants are molecules consisting of both hydrophilic and hydrophobic moieties, which enable them to reduce interfacial tension between the molecules themselves and their interface (Ron and Rosenberg 2001, Chandran and Das 2010, Fracchia et al. 2012, Bhardwaj 2013). These surface- active agents produced from microorganisms and plants possess biological properties such as antimicrobial, antiviral, and anticancer activities and find suitable applications in pharmaceutical, cosmetic and textile industries, oil recovery, wastewater treatment and bioremediation (Randhawa and Rahman 2014, Bouassida et al. 2018).

A wide variety of biosurfactants produced by various microorganisms have been reported, among which surfactin, a bacterial cyclic lipopeptide, was first reported and named in 1968 (Alima et al. 1968). In the late 1960s, a biosurfactant study attracted the attention of researchers due to its properties as a hydrocarbon dissolution agent and its associated applications, which could reduce dependence on chemical surfactants such as carboxylates, sulphonates and sulphate esters (De et al. 2008). These biomolecules have multi-functionality and low toxicity compared to chemical surfactants (Anna et al. 2002, Rufino et al. 2013). Rapid growth in industry and ♦Corresponding author: This email address is being protected from spam bots, you need Javascript enabled to view it ; This email address is being protected from spam bots, you need Javascript enabled to view it ; Tel: +673 246 0923; ORCID ID: 0000-0002-5740-6234

transportation sectors in the past decades have led to prevailing environmental hazards like petroleum products. It is imperative to understand the mechanism of degradation of these hydrocarbons in order to devise effective remediation tools. Biosurfactants from microorganisms are excreted extracellularly, either totally or partially, assisting in the adhesion of oil droplets and subsequent degradation of hydrocarbons, thereby offering an eco-friendly alternative to chemical remediation technology. Hence, biosurfactant production by various microorganisms has been studied extensively over the past few years (Parthipan et al. 2017, Keikha 2018).

CLASSIFICATION OF BIOSURFACTANTS

Biosurfactants are classified according to their composition and source of production (Rikalovic et al. 2015). The structure of a biosurfactant includes a hydrophilic domain consisting of amino acids or peptides, mono-, di- or polysaccharides and a hydrophobic domain consisting of unsaturated or saturated fatty acids (Chen et al. 2015). Based on their chemical structure, biosurfactants are broadly classified into five classes: glycolipids, phospholipids, lipopeptides, polymeric and particulate biosurfactants (Randhawa and Rahman 2014, Chen et al. 2015).

Glycolipids

As one of the most common biosurfactants, glycolipids comprise long chain fatty acids or hydroxyl fatty acids in combination with mono- or disaccharide moieties (Fracchia et al. 2012, Mnif and Ghribi 2016). These compounds are further divided into several groups depending on the nature of their carbohydrate moieties, such as rhamnolipids, trehalose lipids, sophorolipids, cellobiose lipids, and mannosylerythritol lipids (Fracchia et al. 2012, Mnif and Ghribi 2016). Table 1 summarizes the general structures and producer microorganisms of different biosurfactants (Kulakovskaya et al. 2009, Perfumo et al. 2010, Fracchia et al. 2012, Hoffmann et al. 2012, Mongkolthauaruk 2012, Morita et al. 2015).

Rhamnolipids are composed of either one or two rhamnose sugar molecules linked to /)-hydroxy fatty acids (Uzoigwe et al. 2015, Mnif and Ghribi 2016). They are exclusively produced by Pseudomonas sp. (Peter and Singh 2014, Li et al. 2016). These rhamnolipids enhance the cell-surface hydrophobicity of P. aeruginosa, which aids in bacterial growth and increases the rate of bioremediation. They are also easily biodegradable and considered to be nontoxic (Mohan et al. 2006, Mnif and Ghribi 2016).

Trehalose lipids are biosurfactants commonly found in Rhodococcus and other actinomycetes. They are composed of two glucose units connected by an a,a-l,l-glycosidic linkage. These lipids are mostly produced when hydrocarbons are used as a source of nutrients for microbes to grow on. The lipids help overcome the low solubility of hydrocarbons (Franzetti et al. 2010) and have potential research applications as antitumour agents (Fracchia et al. 2012, Duarte et al. 2014).

Sophorolipids are produced by different strains of Torulopsis and consist of two glucose units connected by a (1,2)-/) linkage and a 1-hydroxyoleic acid glycone (Hoffmann et al. 2012). Sophorolipids have the ability to increase surfactant-organic interactions, which can be utilized in the flushing of hydrocarbons from soil (Kang et al. 2010). Cellobiose lipids excreted by Candida, Geotrichum, Kurtzmanomyces and Pseudozyma were studied for their ability to dissolve organic compounds for consumption by microbes (Kulakovskaya et al. 2009). These lipids consist of two glucose units linked by a 1,4'-//-glycoside bond and a fatty acid aglycone (Kulakovskaya and Kulakovskaya 2014). They serve as natural biocontrol agents against fungal spoilage (Kulakovskaya et al. 2009, Morita et al. 2011). Mannosylerythritol lipids are commonly excreted by yeasts and consist of 4-0-/j-D-mannopyranosylerythntol or l-0-/)-D-mannopyranosylerythiitol linked to fatty acids. These lipids have antioxidant and healing agent properties, which make them suitable candidates for pharmaceutical use (Morita et al. 2015, Yu et al. 2015).

Table 1 General structures of different types of glycolipids and lipopeptides with their producers (Kulakovskaya et al. 2009, Perfumo et al. 2010, Fracchia et al. 2012, Hoffmann et al. 2012, Mongkolthanaruk 2012, Morita et al. 2015)

Type of glycolipid

Genera1 structure Producer

Rhamnolipid

Pseudomonas sp.

Trehalose lipid

Actinomycete sp. Rhodococcus sp.

Sophorolipid

Tondopsis bombicola, Candida sp., Trichosporon asahii

Cellobiose lipid

Cvyptococcus humicola, Saccharomyces cerevisiae

Mannosylerythritol

lipid

Basidiomycetous yeast (Pseudozyma)

Type of lipopeptide

Surfactin

Bacillus subtilis

Iturin

Bacillus subtilis, Bacillus methylotrophicus

Fengycin

Bacillus subtilis

Lipopeptides

Lipopeptides are classified as cyclic and linear based on their chemical structure. Cyclic lipopeptides contain a cyclic component formed with a carboxyl group in the C-terminus bonded to the amino group of the peptide chain or a hydroxyl group of the fatty acid chain. The best known cyclic lipopeptides are surfactin, iturins, fengycins, lichenysins, viscosins, amphisin and putisolvins (Vandana and Singh 2018). Linear lipopeptides contain a linear chain of amino acids and fatty acids bonded to the a-amino group or other hydroxyl groups. Lipopeptides are mainly produced by Bacillus sp. (such as B. subtilis, B. licheniformis and B. polymyxa), Pseudomonas, Streptomyces, Aspergillus, Serratia and Actinoplanes species (Biniarz et al. 2017).

Surfactin is composed of a peptide chain formed by seven a-amino acids bonded to a hydroxyl fatty acid by a lactone bond to form a cyclic lipopeptide. The typical sequence of amino acids in the peptide ring is L-Glu1-L-Leu2-D-Leu3-L-Val4-L-Asps-D-Leu6-L-Leu7 (Dliiman et al. 2016). The cyclic lipopeptide surfactin is mainly produced by Bacillus sp. (Vandana and Singh 2018). It acts as an inhibitor of fibrin clot formation and has antibacterial and antitumour properties (Walia 2015).

Iturin is a cyclic lipo-heptapeptide that contains a fi-amino fatty acid in its extended chain. The molecular mass of iturin is ~1.1 kDa, consisting of 7 amino acids linked from C14-C17. The lipopeptide profile and bacterial hydrophobicity vary with strain, with iturin A being the only lipopeptide produced by all B. subtilis strains (Dhiman et al. 2016). Iturin was found to be a strong antifungal agent with constrained antibacterial activity against Micrococcus and Sarcina strains (Walia 2015).

Fengycin is a deca-peptide with an internal lactone ring in the peptidic moiety and a /3-hydroxy fatty acid chain. Fengycins are classified into fengycin A and B. These are also known as plipastatin (Walia 2015, Dhiman et al. 2016). Fengycin is produced by Bacillus subtilis, and plipastatin is produced from Bacillus cereus. Fengycins are found to have strong antifungal activities (Walia 2015).

Lichenysin is produced by Bacillus licheniformis and acts synergistically, exhibiting excellent stability towards temperature, pH and salt. Furthermore, this molecule has a similar structure and physiochemical properties as that of surfactin (Walia 2015, Dhiman et al. 2016).

HIGH-MOLECULAR-WEIGHT BIOSURFACTANTS

Biosurfactants with high molecular weights are generally grouped together as polymeric biosurfactants. They are produced by a number of bacteria and include lipoproteins, proteins, polysaccharides, lipopolysaccharides or complexes containing several of these structural types. Polymeric biosurfactants include alasan, liposan, lipomann, emulsan and other polysaccharide- protein complexes (Siiieriz et al. 2001). The most commonly studied biopolysaccharides are emulsan and alasan. Emulsan isolated from Acinetobacter calcoaceticus RAG-1 ATCC 31012 was reported to be composed of hydrophobic fatty acid chains and an anionic polysaccharide backbone. It was found that alasan isolated from Acinetobacter radioresistens has a molecular weight of 1000 kDa (Walzer et al. 2006, Perfumo et al. 2010). Emulsan is a valuable emulsifying agent for hydrocarbons in water, even at a concentration as low as 0.01 to 0.1%. Moreover, emulsan and biodispersants produced by Acinetobacter calcoaceticus comprise a heteropolysaccharide backbone where fatty acids are covalently linked. Liposan is an extracellular water-soluble emulsifier commonly produced by Candida sp. and Yarroma sp. and is composed of carbohydrate and protein complexes (Shekhar et al. 2015).

PROPERTIES OF BIOSURFACTANTS

Critical Micelle Concentration

Critical micelle concentration (CMC) is the amount of surfactant molecule in the bulk stage beyond which aggregates of surface-active agents are formed (Anna et al. 2002). Amphiphilic molecules can disperse on the surface of the aqueous phase so that the polar moiety interacts with the aqueous phase (Shekhar et al. 2015). The CMCs for microbial surfactants generally range from 5-100 mg/1. A low concentration of surfactants is sufficient to reduce the surface tension because these surface-active agents have low CMC values. At low concentrations, biosurfactants reside on the surface of water. As the surface becomes crowded, excess biosurfactant molecules assemble as micelles (Roy 2017). Biosurfactants with lower CMCs can attain higher emulsification potential, offering industrial and biotechnological applications (Sharma et al. 2016).

Increase in Surface Area of Hydrophobic Water-insoluble Substrates

At the phase boundary, biosurfactants reduce surface tension, which allows microorganisms to grow on water-immiscible substrates, thus making substrates more readily available for uptake and metabolism (Suryanti et al. 2009). The number of fatty acid chains can significantly affect the hydrophilic-lipophilic balance (HLB) of biosurfactants, and it is likely that cells interact with different substrates by modulating the overall properties of their surface to shift from hydrophilic to hydrophobic and vice versa (Banat et al. 2000, Perfumo et al. 2010).

Increase in Bioavailability of Hydrophobic Water-insoluble Substrates

Microbial growth on bound substrates is improved by biosurfactants by desorbing them from surfaces or by increasing their apparent water solubility. Low-molecular-weight biosurfactants that have low CMCs increase the apparent solubility of hydrocarbons by incorporating them into the hydrophobic cavities of micelles (Ron and Rosenberg 2001).

Emulsifier Production

The growth of microbes on hydrocarbons induces emulsifier production. Microorganisms that cannot produce emulsifiers grow poorly on hydrocarbons. The emulsifier (lipid-carbohydrate- lipid) complex is associated with the bacterial cell wall during the log phase of growth and generally shows its extracellular emulsification activity in the stationary phase (Muller-Hurtig et al. 1993, Beal and Betts 2000).

Biosurfactants form stable emulsions with a lifespan of several months to years. They can also stabilize or destabilize emulsions. High molecular mass biosurfactants, such as liposan, have the ability to emulsify edible oil but do not reduce surface tension, whereas sophorolipids produced by T. bombicola have been shown to reduce surface tension but are not good emulsifiers (De et al. 2008, Sharma et al. 2016).

Reduction of Surface Tension

Biosurfactants can reduce the surface tension of water from 72 to 35 mN/m and lower the interfacial tension of water against n-hexadecane from 40 to 1 mN/m. It has been reported that the CMC value of biosurfactants is 10-40 times lower than that of synthetic surfactants, which makes biosurfactants more efficient and effective (De et al. 2008). Surfactin produced by Bacillus subtilis can lower the surface tension of water to 25 mN/m and the interfacial tension of water against и-hexadecane to less than 1 mN/m (Liu et al. 2015).

Toxicity

Although biosurfactants are thought to be environmentally friendly, some experiments have shown that under certain circumstances, microbial surfactants can exhibit certain toxicity. For example, lipopeptides synthesized by Bacillus subtilis ATCC 6633 were reported to cause rupture of erythrocytes (Gudina et al. 2013). However, biosurfactants do not pose harmful effects to organs, nor do they interfere with blood coagulation in normal clotting time (De et al. 2008).

In a study by Sharma et al. (2016) on biosurfactant produced by Enterococcus, faecium MRTL9, it was found that 6.25 mg ml-1 of biosurfactant resulted in 90% viability of mouse fibroblast cells. Furthermore, L.jensenii and I. rhamnosus producing biosurfactant were observed to have low toxicity on eukaryotic cells, and biosurfactant concentrations ranging from 25 to 100 mg/ml revealed no toxicity (Sharma et al. 2016).

Different concentrations of biosurfactants synthesized by E. faecium MRTL9 ranging from 1.25 to 5 mg/ml were tested on different plants, such as Brassica nigra and Triticum aestivum. It was found that vital growth parameters such as root elongation, vigour index and germination index were excellent when treated with biosurfactant compared to sodium dodecyl sulphate. Moreover, the vital growth parameter increased with increasing concentration of biosurfactant (Saharan et al. 2012, Sharma et al. 2016).

 
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