**2. General characteristics of biosurfactants**

exploited commercially. This review article will describe microorganisms related to biosurfactant production, including yeasts, as well as their role in bioremediation.

**Keywords:** Biosurfactant, soil yeast, microbial communities in soil, bioremediation

Recently, there are many reports of soil and surface water locations that are contaminated with organic pollutants, with a great impact on soil and groundwater. Because of its low solubility in water and high interfacial tension, those contaminants cannot be easily removed. Bioreme‐ diation has become one of the methods used in the remediation of contaminated sites; bioremediation strategies are based on the use of different microorganisms: bacteria, yeasts, or fungi isolated from soil or from a place where there is a presence of contaminants such as hydrocarbons, which facilitate the cleaning of the contaminated sites. Bioremediation studies begin with the isolation and identification of microorganisms from soil and water that are able to degrade these contaminants. Some hydrocarbon-degrading microorganisms are also able to produce biosurfactants. Biosurfactants produced by the microorganisms in the environment help them to take the hydrocarbons as carbon source, either by making available the hydro‐ carbon by releasing biosurfactant into the environment or by changing its cell surface so that

Originally, biosurfactants attracted attention as hydrocarbon dissolving agents in the late 1960s as potential replacements for synthetic surfactants (carboxylates, sulfonates, and sulfate acid esters), especially in the food, pharmaceutical, and oil industries. Synthetic surfactants currently used are usually toxic and hardly degraded by microorganism, causing damage to the environment. Most of the biosurfactants are high molecular weight lipid complexes, which are normally produced under aerobic conditions. The classification of biosurfactants is based on their chemical composition, their mode of action, and the microorganisms that produce it. Biosurfactants can be of high or low molecular weight, and based on their composition, they can be glycolipids, phospholipids, lipopeptides, or a mixture of amphiphilic polysaccharides, proteins, lipoproteins, or lipopolysaccharides. Microorganisms also produce surfactants that are in some cases a combination of many chemical types referred to as the *polymeric microbial surfactants*. Because of their unique structures, biosurfactants may have a large range of

Regarding their mechanism of action, some compounds are better at decreasing the surface tension (biosurfactants), and others are able to produce stable emulsions (bioemulsifiers). Best known biosurfactants are produced by bacteria, and there are many studies on them, especially on *Pseudomonas* spp., strains that produce rhamnolipids. However, it is necessary to find new types of biosurfactants and bioemulsifiers, and the studies of other organisms are increasing recently. Yeast and fungi have demonstrated to produce biosurfactant and bioemulsifiers with very good results. The aim of this chapter is to describe microbial biosurfactant, especially

those produced by yeasts and to propose their use in bioremediation.

**1. Introduction**

94 Advances in Bioremediation of Wastewater and Polluted Soil

the contaminant can be absorbed.

properties that can be exploited commercially.

Recently, there are many reports of soil and surface water locations that are contaminated with organic pollutants, with a great impact on soil and groundwater. During this process, mole‐ cules of the pollutant have bound to the soil particles as it has moved toward the groundwater and are therefore difficult to remove it from soil because many of these pollutants have low solubility and high interfacial tensions with water [1].

To help on the solution of this problem, surfactants can be used to clean the contaminated soil and water. Surfactants are one of the most commonly used chemicals in everyday life. Since the beginning of the 20th century, the production of a wide spectrum of synthetic surfactants from petroleum resources has increased intensively. The amphiphiles that can improve surface–surface interactions by forming micelles are termed as surface active agents or *surfactants* [2]. Surfactants are amphiphilic molecules consisting of a hydrophobic and a hydrophilic portion [3]. Usually, the hydrophobic portion is a nonpolar long chain of fatty acids, whereas the hydrophilic domain can be nonionic, positively or negatively charged, or amphoteric, frequently a carbohydrate, an amino acid, or a phosphate [4–6].

Increasing concentrations of surfactant into an oil/water or water/air systems causes a reduction in surface tension up to a critical point where the surfactant can form structures like micelles, bilayers, or vesicles. This concentration defines the critical micelle concentration (CMC). To determine this value, the solution containing the surfactant is diluted severalfold; surface tension is measured for each dilution, and the CMC is calculated from this value. Surface tension can be easily measured with a tensiometer. There are surfactant molecules that are able to reduce the surface tension of water from 72 to around 27 mN m–1 [7]. When water, oil, and a surfactant are mixed, the surfactant rests at the water–oil interface; these systems are called *emulsions* or *microemulsions* depending on their stability [8, 9]. These characteristics confer excellent detergency and emulsifying, foaming, and dispersing capacities, which make surfactants one of the most versatile chemicals in industrial processes [10].

Current, worldwide surfactant market is around \$9.4 billion annually, while their production has been reported to be approximately 10 million tons, and their use is divided almost equally between household detergents and several industrial applications [10, 11]. Synthetic surfac‐ tants have increasingly been replaced by biotechnology-based compounds, derived either from enzymatic or microbial synthesis, because they can be produced using natural sources [12]. The group of surface active biomolecules produced by living organism is called *biosur‐ factants*.

Synthetic surfactants currently used are toxic and hardly degraded, causing damage to the environment. Initially, biosurfactants were considered to have applications in the food, pharmaceutical, and oil industries [13–15]. Biosurfactants have several advantages over chemical surfactants, including lower toxicity, higher biodegradability, effectiveness at extreme temperatures or pH values, biocompatibility, and digestibility. Also, biosurfactants can be produced using agroindustrial waste material; they can be economically produced and show better environmental compatibility. The microorganisms that produce the biosurfactant can be modified by genetic engineering or biological and biochemical techniques. Because the possibility of practical applications for biosurfactants depends on whether they can be produced economically, there have been many efforts to optimize its biological production [16–18]. To replace synthetic surfactants, biosurfactant production needs to be of low-cost, and up to now, there are few studies on the use of low-cost materials on the pilot plant or industrial scale [4].

Most of the biosurfactants are lipid-containing molecules, which are normally produced under aerobic conditions [16]. The classification of biosurfactants is based on their chemical compo‐ sition, their mode of action, and the microorganisms that produce it. Biosurfactants can be of high or low molecular weight, and based on their composition, they can be glycolipids, phospholipids, lipopeptides, or a mixture of amphipathic polysaccharides, proteins, lipopro‐ teins, or lipopolysaccharides. Biosurfactants with low molecular mass are efficient in lowering surface and interfacial tensions, whereas biosurfactants with high molecular mass are more effective at stabilizing oil-in-water emulsion (Figure 1) [19]. Microorganisms also produce surfactants that are in some cases a combination of many chemical types referred to as the *polymeric microbial surfactants* [8].

**Figure 1.** Stable emulsions (EI24) produced by mixing a cell-free supernatant from a biosurfactant-producing yeast and hexadecane. The tube on the left shows a clear emulsion characteristic of polymeric biosurfactants. The tube on the right shows a compact stable emulsion that is characteristic of low molecular weight biosurfactants.

Low molecular weight biosurfactants are usually glycolipids or lipopeptides; the later are usually produced by bacteria from the *Bacillus* genus and is composed of a cyclic peptide and a fatty acid residue. Among the glycolipids, the most studied is rhamnolipid, which is produced by *Pseudomonas aeruginosa* strains; it is composed of a backbone of two rhamnose moieties and two fatty acid residues. Other glycolipid biosurfactants include trehalolipids, produced by *Rhodococcus erytropolis* and other bacterial genera, and sophorolipids, produced by several yeast strains (Figure 2a and 2b). The physicochemical properties of low molecular weight biosurfactants are influenced by the fatty acid residues that contain, and those in fact depend on the bacterial strain used and on the growth conditions and nutrients present. High molecular weight biosurfactants are usually a complex mixture of macromolecules containing proteins, polysaccharides, and lipid residues. The most studied polymeric biosurfactant is emulsan, produced by *Acinetobacter calcoaceticus* (Figure 2c) [1, 10]. Because of their unique structures, biosurfactants may have a greater range of properties that can be exploited commercially [20].

can be modified by genetic engineering or biological and biochemical techniques. Because the possibility of practical applications for biosurfactants depends on whether they can be produced economically, there have been many efforts to optimize its biological production [16–18]. To replace synthetic surfactants, biosurfactant production needs to be of low-cost, and up to now, there are few studies on the use of low-cost materials on the pilot plant or industrial

Most of the biosurfactants are lipid-containing molecules, which are normally produced under aerobic conditions [16]. The classification of biosurfactants is based on their chemical compo‐ sition, their mode of action, and the microorganisms that produce it. Biosurfactants can be of high or low molecular weight, and based on their composition, they can be glycolipids, phospholipids, lipopeptides, or a mixture of amphipathic polysaccharides, proteins, lipopro‐ teins, or lipopolysaccharides. Biosurfactants with low molecular mass are efficient in lowering surface and interfacial tensions, whereas biosurfactants with high molecular mass are more effective at stabilizing oil-in-water emulsion (Figure 1) [19]. Microorganisms also produce surfactants that are in some cases a combination of many chemical types referred to as the

**Figure 1.** Stable emulsions (EI24) produced by mixing a cell-free supernatant from a biosurfactant-producing yeast and hexadecane. The tube on the left shows a clear emulsion characteristic of polymeric biosurfactants. The tube on the

Low molecular weight biosurfactants are usually glycolipids or lipopeptides; the later are usually produced by bacteria from the *Bacillus* genus and is composed of a cyclic peptide and a fatty acid residue. Among the glycolipids, the most studied is rhamnolipid, which is produced by *Pseudomonas aeruginosa* strains; it is composed of a backbone of two rhamnose moieties and two fatty acid residues. Other glycolipid biosurfactants include trehalolipids, produced by *Rhodococcus erytropolis* and other bacterial genera, and sophorolipids, produced by several yeast strains (Figure 2a and 2b). The physicochemical properties of low molecular weight biosurfactants are influenced by the fatty acid residues that contain, and those in fact

right shows a compact stable emulsion that is characteristic of low molecular weight biosurfactants.

scale [4].

*polymeric microbial surfactants* [8].

96 Advances in Bioremediation of Wastewater and Polluted Soil

**Figure 2.** Chemical structures of some of the most common biosurfactants. Low molecular weight glycolipids: (a) rhamnolipid and (b) sophorolipid; high molecular weight glycolipids: (c) emulsan.

## **3. Biosurfactant-producing microorganisms**

The ability of microorganisms to degrade hydrocarbons was first described in 1895 by Misyoshi, who reported the microbial degradation of paraffin. Many different microbial species of bacteria, yeast, and mold are capable of degrading hydrocarbons, and bacteria are the best described biosurfactant producer [21]. The exact reason why some microorganism can also produce biosurfactants is still not clear [22].

Bushnell and Hass (1941) were the first to demonstrate the bacterial production of biosurfac‐ tants, using a strain of *Corynebacterium simplex* and a strain of *Pseudomonas* grown in a mineral media containing kerosene, mineral oil, or paraffin. Since then, numerous studies on the structure and mechanisms involved in the production and action of biosurfactants have been reported [22]. It can be stated that biosurfactants are produced by a variety of microorganisms, and there is also a wide variety on the chemical composition and nature of the biosurfactant produced, as well as on the location (membrane-bound, extracellular) of the produced molecule [23]. The most reported genera of biosurfactant-producing bacteria include *Pseudo‐ monas* sp., *Acinetobacter* sp., *Bacillus* sp., and *Rhodococcus* sp., among others. Table 1 shows some of the most studied bacteria and the type of biosurfactant produced.


**Table 1.** Biosurfactant-producing bacteria

Microorganisms that produce biosurfactants are isolated mainly from sites that are or were contaminated with petroleum hydrocarbons: contaminated soils, effluents, and wastewater sites. Thus, these have an ability to grow on substrates considered potentially noxious for other nonbiosurfactant-producing microorganisms. Biosurfactants play a physiologic role in increasing bioavailability of hydrophobic molecules, which are involved in cellular signaling and differentiation processes, which facilitate the consumption of carbon sources present in soil [23, 28].

The physiological role of biosurfactants is not clear yet, but it might be related to an increase in the nutrient uptake of hydrophobic substrates, in enhancing the growth on hydrophobic surface, and in cellular motility and biofilm formation by reducing the surface tension at the phase boundary [10, 15]. The mechanism of uptake of liquid hydrocarbon substrates by microbial cells involves interfacial phenomena. The significant influence on the biodegrada‐ tion process is observed after the addition of surface active compounds [21]. Another physio‐ logical role of biosurfactants can be their observed antimicrobial activity [3].

Biosurfactants are produced predominantly when hydrophobic substrates provided as carbon source is used for microbial growth; they can be either secreted extracellularly or attached to the microbial cell wall. On the contrary, some microorganisms may produce biosurfactants in the presence of different types of substrates, including carbohydrates and other water soluble compounds. It has been reported that the carbon source used for biosurfactant production influences the structure of the compound produced by the microorganism. It is also affected by nitrogen sources as well as by the presence of minerals such as iron, magnesium, manga‐ nese, phosphorous, and sulfur [3, 23]. This capacity of modification of the biosurfactant molecule by the composition of the culture media can be used to produce compounds with specific applications. Industrial production of microbial metabolites is a very complex process, and for industrial production, many variables are needed to be considered; in the case of biosurfactants, media composition is a key element to control yield and specific productivity [29]. The success of biosurfactant production depends on the development of cheaper proc‐ esses and the use of low-cost raw materials, which account for 10% to 30% of the overall production cost. The literature shows that a wide range of carbon sources, including agricul‐ tural renewable resources, like sugars and oils, are suitable carbon sources for production of ecologically safe biosurfactants with good properties [30]. The use of agroindustrial waste products such as bagasses, molasses, and plant material residues can be good candidates for use in biosurfactant production.

Interest in microbially produced biosurfactants has led to a need for the further development of rapid and efficient qualitative and quantitative methods for screening and analyzing biosurfactant-producing microorganisms [20]. The development of rapid and reliable methods for screening and selection of microbes from thousands of potentially active organisms and the subsequent evaluation of surface activity holds the key for the discovery of new biosur‐ factants. Among the most important characteristics needed for rapid screening methods is the ability to identify microorganisms capable of biosurfactant production in large culture collections, as well as the use of reliable methods to quantify the compounds produced [31].
