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Biosurfactants (BS) are amphiphilic molecules that, as any surfactant, consist in two contrasting moieties: (i) one hydrophilic part showing affinity to polar substances like water, and (ii) one lipophilic displaying attraction to non‐polar media such as oil and fats (Figure 2.1) [1]. The prefix “bio” refers to the fact that these surfactants are exclusively produced through biochemical transformations within microorganisms under certain conditions; this is why BS are also called microbial surfactants [2]. The adjustable binary hydrophilic–lipophilic feature confers surfactants appealing properties for applications in all types of industrial sectors.

Surfactants can be categorized according to their (i) origin, (ii) electrostatic status, and (iii) the ratio of their hydrophilic and lipophilic components (Table 2.1). In the first category, surfactants can be seen as synthetic, semi‐synthetic, and BS. Synthetic surfactants are those that have been manufactured on a large scale for many years from raw materials of petrochemical origin. Semi‐synthetic surfactants were introduced to the market to offer less hazardous alternatives to conventional surfactants. These are constituted by lipophilic fragments coming from biocompatible motifs like fatty esters, acids, or alcohols extracted from natural oils (oleum), ergo these surfactants are also called oleochemical surfactants. Nevertheless, further steps of conventional synthesis are always required to afford finished products (semi‐synthetic = half‐synthetic). Currently, it is estimated that >27% of consumed surfactants are oleochemicals. Europe and North America have dominated this particular market, and the increased consumption in recent years can be associated with the interest in products for personal care, home care, and agriculture.

In an attempt to solve relevant drawbacks related to low biodegradability profiles of synthetic surfactants, pollution of traditional chemical processes, and sacrifice of natural resources, new sustainable approaches have recently emerged, implying biotechnological means for surfactant obtention. In this context, BS are known to be secondary metabolites of living organisms just as bacteria and yeast and to be involved in some microbial physiological functions, e.g. cell development, biofilm formation, and osmotic pressure regulation. Furthermore, these play a key role in microbial survival in front of adverse situations such as lack of nutrients and the need to incorporate non‐soluble substances like hydrocarbons as the only carbon source [2]. These natural phenomena have been exploited by bioengineering in productive microbial fermentation, and since BS are achieved through complex enzymatic reactions, such biomolecules are endowed of robust chemical architectures, which are tunable from elegant processes of molecular biology, genetic engineering, or careful selection of culture conditions.

In the second category, surfactants are normally grouped according to the presence or absence of formal electrostatic charges in the hydrophilic moiety of the molecule. Thus, cationic surfactants contain formal positive charges (+) on the polar head of the surfactant molecule and are systematically escorted by negative counterions that neutralize the charges. Anionic surfactants have negative formal charges (−), an example of this latter is represented in Figure 2.1. The third class of ionic surfactants is those species comprising both positive and negative charges in the same body (±). These species are known as inner salts or zwitterions (from German zwitter = hybrid). The last case of this classification is the absence of formal charges in the surfactant molecule, so these are called nonionic surfactants.

Figure 2.1 3D chemical structure of lauryl sulfate as an example of a surfactant molecule. The hydrophilic head of sulfate (SO₄²⁻) is surrounded by water molecules, while the lipophilic tail avoids any contact with water.

Source: Based on [1].

Table 2.1 Classification of surfactants by origin, ionic status, and hydrophilic–lipophilic balance.

Category	Example
I. Origin	Synthetic	Nonylphenol ethoxylates
Oleochemical	Lauryl alcohol ethoxylates
Biosurfactants	Sophorolipids
II. Electrostatic status	Oonic	Cationic	Ammonium salts
Anionic	Lauryl sulfates
Zwitterionic	Betaines
Nonionic	Oxirane and 2‐methyoxirane copolymers
III. Hydrophilic–lipophilic balance	01–03	Antifoaming agents
03–08	w/o emulsifiers
07–10	Wetting agents
08–16	o/w emulsifiers
13–16	Detergents
16–19	Solubilizing agent

Finally, surfactants can be ranked according to their amphiphilic nature, otherwise by their hydrophilic–lipophilic balance (HLB). Although there are a significant number of theoretical and experimental approaches, the most used HLB system is the Griffin’s [3]. In a simplistic way, HLB values are estimated from the division of the mass of the hydrophilic fragment by the mass of the entire molecule, and the resulting quotient is then multiplied by a conventional value of 20. HLB can predict whether a surfactant will behave as an emulsifier, solubilizer, dispersant, or other; therefore, this system has been a useful guide to formulate products containing conventional surfactants for specific applications, and certainly, will be just as convenient for the case of BS. The hydrophilic part of BS is normally constituted by carbohydrates, amino acids, proteins, phosphates, carboxylic acids, or alcohol motifs; and these can be ionic or nonionic. The lipophilic part commonly is long chains of carbon atoms, just as in fatty acids. Both molecular components, hydrophilic and lipophilic, are assembled via linking biochemical functionalities, e.g. ethers (C−O−C), amides (N−C=O), and esters (O−C=O). According to the nature of each moiety hydrophilic and lipophilic, BS are commonly classified in the following groups: (i) glycolipids, (ii) lipopolysaccharides, (iii) lipopeptides, (iv) phospholipids, and (v) fatty acids; each one with specific physicochemical characteristics and physiological roles [4, 5]; for example, Emulsan and other complex lipopolysaccharides are polymeric BS with known emulsification capabilities.

From all the types of BS, glycolipids have the greatest opportunity to be manufactured on a large scale due to the high yield of obtention compared to other BS such as lipoproteins. It is clear that BS produced in higher yields will represent a lower cost for production [6]. This is why glycolipids have captured our attention for this chapter. Glycolipids result from the condensation of aliphatic fatty acids (lipids) and carbohydrates. Their names are taken from the nature of the carbohydrate moiety. Consequently, glycolipids containing sophorose in the hydrophilic segment are called sophorolipids (SL); those containing rhamnose are named rhamnolipids; those with trehalose, trehalose‐lipids, and so on. Of all the glycolipid types, SL and rhamnolipids have been among the most studied [7, 8].

SL (Figure 2.2) contain the disaccharide sophorose linked to a fatty acid via an ether function. The fatty acid must be previously hydroxylated somewhere in the carbon chain, usually at the other end of the carboxylic acid function. SL can be found as different chemical structures. The most evident and well known is the divergence between open and cyclic arrangements [9]. Open SL are those that have the chemical functionality of carboxylic acid (COOH) at the end of the lipophilic chain, while cyclic arrangements are those having an ester functionality as a result of the condensation between the fatty acid and one of the hydroxyl motifs of the sophorose.

Figure 2.2 Chemical structures for two species of sophorolipids as a result of the condensation of sophorose with oleic acid. On the left side, it is shown the open form of carboxylic acid and on the right side, the cyclic form of the lactone.

Cyclic esters are called lactones. Hence, there are two types of SL: the acidic forms and the lactone forms. Other less notable molecular variables are (i) the presence or absence of acetyl groups attached to the hydroxyl moieties on the carbohydrate periphery, (ii) the length of the alkyl chain, (iii) the degree of unsaturation (unsaturation = double or triple bonds), (iv) the position of the hydroxyl group in the alkyl chain, (v) the position of the hydroxyl group of the sophorose that serves to build the ether bond with the fatty alcohol, and (vi) the position of the hydroxyl group of sophorose serves to construct the ester bond with the fatty acid in the lactone forms, inter alia.

Figure 2.2 shows the chemical structures of two SL: one in the acid form (left) and one in the lactone form (right). Both species do not contain any acetyl groups but have a fatty acid moiety of 18 carbon atoms with only one unsaturation in position C‐9 with Z geometry. This fatty acid (oleic acid) must have been previously hydroxylated inside the cell by some biochemical β‐oxidation at position C‐17. This latter allows its association with the free hydroxyl moiety of the anomeric carbon of sophorose, which produces the ether bridge of sophorose‐lipid equally found in both acid and lactone arrangements. Thus, the chemical name for the acid form should be (S,Z)‐17‐{[(2S,3R,4S,5S,6R)‐4,5‐dihydroxy‐6‐hydroxymethyl‐3‐{[(2S,3R,4S,5S,6R)‐3,4,5‐trihydroxy‐6‐hydroxymethyltetrahydro‐2H‐pyran‐2‐yl]oxy}tetrahydro‐2H‐pyran‐2‐yl]oxy}octadec‐9‐enoic acid.

In the case of lactone, the carboxylic acid was condensed with the hydroxyl group in C‐5 of the other monosaccharide fragment of sophorose. After imagining the number of possible combinations of structural variations, one would not expect microorganisms to produce unique and pure compounds, but rather a wide variety of many different species. Therefore, the selection of microorganism strain, culture conditions, culture media, and substrates are the first fundamental factors playing a decisive role in the complexity (or simplicity) of obtained mixtures.

The other type of interesting glycolipids is rhamnolipids. Like SL, there are abounding varied possible structures. In the case of rhamnolipids, there are even two more features: (i) the number of rhamnose monomers and (ii) the number of condensed fatty acids. So, rhamnolipids can be constituted by one or two units of rhamnose linked to one or two molecules of fatty acids (Figure 2.3). To our knowledge, lactone rhamnolipid is not yet reported.

The manufacture of surfactants has been an exclusive task for industrial organic chemistry. However, just as microorganisms have been used in industrial processes to afford enzymes, vaccines, antibiotics, wine, and beer, the production of surfactants can also be carried out in this way. Furthermore, the rapid and recent advance on bioprocesses envisions the feasibility of producing BS on a large scale. Research and technological developments have tried to look for cost competitiveness proposing cheaper raw materials and more affordable downstream processes. For example, it has been demonstrated that some agro‐industrial wastes such as molasses from sugar industry [10] or whey from dairy industry [11] can be useful. Other aspect on BS is the possibility to improve physicochemical properties or the productivity of microorganisms via biochemical or genetic engineering techniques.

BS have very attractive properties such as low toxicity and high biodegradability (degraded by other microorganisms); which is very well aligned to current needs imposed by international regulations. It now is considered that biocompatibility of a product is a parameter as important as its cost and performance, so the chemical industry is compelled to implement new strategies for manufacturing more sustainable materials without scarifying efficiency. The numerous advantages of versatile BS compared to synthetic surfactants explain the increasing attention on the topic from the early twenty‐first century (Figure 2.4). Although publishable activity on BS dates back 57 years, the boom has emerged for the last 20 years. Only in the last eight years, more than half of the known references on BS have been reported. BS are recognized for resisting a wide range of pH, salt concentration, and temperature [12] and have the ability to reduce surface tension in exactly the same way as chemical and oleochemical surfactants, so these can find the same application niches, i.e. as the key components of countless formulations for almost any sector of the contemporary industry [13]. Hence, BS are excellent candidates for different industrial applications like oil recovery, detergents, cleaning products, degreasers, fertilizers, agrochemicals, textile products, paints, mining, inter alia. Moreover, BS can attend applications where strong eco‐friendly features are demanded, e.g. bioremediation of soil and water due to hydrocarbon spills [14, 15], water treatment, food processing [16], health, sanitizers, cosmetics, and pharmaceuticals [17].

Figure 2.3 General chemical structures for four types of rhamnolipids: monorhamnose‐monolipid, monorhamnose‐dilipid, dirhamnose‐monolipid, and dirhamnose‐dilipid.

As it has been observed, classification for a surfactant molecule comprises all the chemical species that share a binary amphiphilic feature. The production of microbial surfactants involves a strong character of sustainability and circular economy. Its production seems to be the right choice that will revolutionize the way the chemical industry, applications, and markets work. Glycolipids such as SL and rhamnolipids appear to be the species with the greatest potential to be developed at larger scales in the coming years. In addition, there are some synergies with other chemical compounds that can enhance surface activities and performances, which makes ipso facto possible the introduction of BS in the market via innovative formulations.

Figure 2.4 Number of publications on biosurfactants from 1963 to April 2020.

Among all primary and secondary metabolites, BS play interesting roles for microbial life. Some authors suggest that their emulsifying properties help microorganisms to adapt to environments, enhancing nutrient availability in soil or water and allowing cell adherence for water‐insoluble substrate transport [18]. Since BS can inhibit microbial growth, those microorganisms producing BS can become predominant in their environment [19].

As mentioned above, BS are biodegradable and suitable for different industrial applications; for this reason, there is abundant research about their production and natural sources. Biosynthesis of BS are distributed among archaea, bacteria, yeasts, and molds, but depending on the group, genus, and species of microorganisms, BS structures become significantly different.