**Abstract**

The *Actinobacteria* produce an array of valuable metabolites including biosurfactants which are gaining increased attention in the biotechnology industries as they are multifunctional, biorenewable and generally superior to chemically synthesized compounds. Biosurfactants are surface-active, amphipathic molecules present at the microbial cell-surface or released extracellularly and in a variety of chemical forms. The mycolic acid-containing actinobacteria (MACA), classified in the order *Corynebacteriales*, represent a potentially rich source of biosurfactants for novel applications and undiscovered biosurfactant compounds. Members of the mycolate genus *Rhodococcus* produce various well-characterised glycolipids. However, other mycolate genera including *Corynebacterium*, *Dietzia*, *Gordonia* and *Tsukamurella* although less extensively investigated also possess biosurfactant-producing strains. This chapter captures current knowledge on biosurfactant production amongst the MACA, including their chemical structures and producer organisms. It also provides an overview of approaches to the recovery of biosurfactant producing MACA from the environment and assays available to screen for biosurfactant production. Methodologies applied in the extraction, purification, and structural elucidation of the different types of biosurfactants are also summarised. Potential future applications of MACA-derived biosurfactants are highlighted with particular focus on biomedical and environmental possibilities. Further investigation of biosurfactant production by MACA will enable the discovery of both novel producing strains and compounds with the prospect of biotechnological exploitation.

**Keywords:** actinobacteria, antimicrobial, bioemulsifiers, bioremediation, biosurfactants, biotechnology, *Corynebacteriales*, mycolic acids, *Rhodococcus*

## **1. Introduction**

Members of the class *Actinobacteria* produce an impressive range of bioactive metabolites that are of commercial importance and many more that have the potential for future exploitation. This includes biosurfactants which are synthesised by many actinobacterial species. Microbial biosurfactants are gaining increased attention in the biotechnology industries as they are multifunctional, enabling diverse applications. Biosurfactants can also claim strong green credentials as not only are they biorenewable with the possibility of production on various substrates including wastes, but they may also be applied to environmental remediation [1]. Further, biosurfactants are generally considered superior to their chemically

synthesized counterparts. Amongst the most common biosurfactant producers are members of the mycolic acid-containing (mycolate) genus *Rhodococcus* which have received considerable attention. However, other related mycolate genera including *Corynebacterium*, *Dietzia*, *Gordonia* and *Tsukamurella* also possess biosurfactantproducing strains but have not been explored to the same extent. Additionally, there are several other mycolate genera that have received little or no investigation in this respect that may produce novel biosurfactant compounds.

Membership of the mycolic acid-containing actinobacterial (MACA) group has expanded considerably over the past 20 years with revisions to the classification of existing species and the publication of copious new mycolate species and genera [2]. This substantial and metabolically diverse group therefore warrants further attention in the search for valuable biosurfactants. This chapter provides an overview of the current knowledge on biosurfactants produced by members of this group and describes approaches to the recovery, screening and biosurfactant-producing strains from the environment and their growth requirements. Methodologies applied to screen for biosurfactant production and for extraction, purification, and structural elucidation of biosurfactant compounds are also described. Current and potential future applications of biosurfactants derived from MACA are examined with particular focus on potential biomedical and environmental possibilities.

#### **1.1 Biosurfactant properties**

Microbial biosurfactants are amphipathic compounds, with both hydrophilic (polar) and hydrophobic (non-polar) moieties. The hydrophobic portion has saturated, unsaturated, or hydroxylated long-chain fatty acids and the hydrophilic portion can contain amino acids, carbohydrate, carboxyl acid, peptides, phosphate, or alcohol [3]. Biosurfactants may be categorised according to molecular weight (low or high), ionic charge (anionic, cationic, neutral, or non-ionic) or according to chemical composition and structure. The main classes of biosurfactants include fatty acids, glycolipids, lipopeptides, lipoproteins, neutral lipids, phospholipids, and polymeric biosurfactants. Their amphipathic nature enables biosurfactants to partition at water-air, oil-air, or oil-water interfaces thereby reducing surface and/or interfacial tension. They exhibit many other useful properties including de-/emulsification, dispersion, foaming, lubrication, softening, stabilisation, viscosity reduction and wetting [4].

Biosurfactants may be located intracellularly, on the cell surface (cell-bound) or excreted extracellularly (free) [5] and are produced during growth on both hydrophilic and hydrophobic substrates, to reduce surface or interfacial properties of the microbial cell or the surrounding environment. Biosynthesis of these compounds is required for gliding, motility, swarming, and biofilm formation. Biosurfactants also mediate between cells and hydrophobic compounds, enabling enhanced solubilisation and uptake across the cell membrane for utilisation as a substrate for growth and energy (**Figure 1**).

Many microbially derived biosurfactants are already used in diverse industries including agriculture, bioremediation, cosmetics, food, healthcare and medicine, and the petrochemical industry (**Figure 2**). In addition to being multifunctional, biosurfactants have several advantages over chemically synthesised surfactants. They are less/non-toxic and biodegradable, have higher surface activity and lower critical micelle concentrations (CMC), greater biocompatibility and selectivity, they function over wide pH, salinity, and temperature ranges, and can be produced using renewable and waste substrates [6]. These unique eco-friendly features make biosurfactants particularly attractive options as industries focus on longer-term sustainability and working towards a circular economy.

**Figure 1.** *Emulsification of hydrocarbons by microbial biosurfactants to enhance bioavailability.*

**Figure 2.** *Various sectors of application for microbial biosurfactants.*

### **1.2 Mycolic acid-containing actinobacteria**

The MACA form a phylogenetically coherent group that resides in the order *Corynebacteriales* based on 16S rRNA gene sequence analysis. The members are Gram-positive with high guanine-plus-cytosine (G + C) content in their genomic DNA. They currently comprise more than 400 species classified in 15 genera, namely *Corynebacterium*, *Dietzia*, *Gordonia*, *Hoyosella*, *Lawsonella*, *Millisia*, *Mycobacterium*, *Nocardia*, *Rhodococcus*, *Segniliparus*, *Skermania*, *Smarigdococcus*, *Tomitella*,*Tsukamurella* and *Williamsia* [2]. The almost universal production of mycolic acids by members of this group is a synapomorphic trait that is unique to this phylogenetic lineage [7]. However, several members of this order appear to have lost the ability to produce mycolic acids over the course of evolution, including several species of the genus *Corynebacterium* and *Hoyosella*. It was recently proposed that the single species belonging to the genus *Turicella*, also characterised by the absence of mycolic acids, be reclassified in the genus *Corynebacterium* [8].

Mycolic acids, which are high molecular weight 3-hydroxy fatty acids with a long alkyl branch in the 2-position, represent the major lipid constituents of the cell envelope of these organisms. They show structural variations from relatively simple mixtures of saturated and unsaturated compounds in corynebacteria to highly complex mixtures in mycobacteria. Mycolic acids also vary in the number of carbons on the 2-alkyl-branch from C22–C38 in corynebacteria to C60–C90 in mycobacteria [9]. Mycolic acids play an essential role in the architecture and functions of the cell envelope, where attached to the cell wall arabinogalactan they help to form a barrier that contributes to impermeability and resilience and conveys hydrophobicity to the cell surface. Trehalose mycolates, also termed cord factors, play an important role in pathogenicity in mycobacterial species that cause infection [9]. The presence and carbon chain length of mycolic acids can be used as taxonomic markers for the identification and classification of actinobacteria to the order *Corynebacteriales* [2].

Members of order *Corynebacteriales* can usually be distinguished from one another and from corresponding taxa in the phylum Actinobacteria based on 16S rRNA phylogeny supported by phenotypic (cell wall chemistry and morphology) features. Cell morphology amongst the MACA varies from simple rods and cocci to branched filaments that fragment to pleomorphic forms (**Table 1**). Members of the species *Skermania piniformis* are micromorphologically unique in this group as they form pine tree-like acute-angle branched filaments [10]. Colonies growing on agar plates are normally visible within several days of inoculation (**Figure 3**) although slow-growing mycobacteria take considerably longer. Species vary widely in colony appearance and are often colourful however it is usually not possible to unambiguously assign strains to a genus based on this feature alone.

Chemotaxonomy is the study of the distribution of various cell wall components to classify and identify strains and is particularly useful to differentiate between the various mycolic acid-containing genera. Cell wall markers typically used to differentiate between MACA genera are summarised in **Table 2**. Some of the methods used to analyse these chemotaxonomic markers provide quantitative or semiquantitative data, as in the case of fatty acids, whereas other techniques provide only qualitative data as in the case of muramic acid type and phospholipid pattern.

Reliable identification of MACA strains to species level depends upon phylogenetic analysis of the gene encoding 16S rRNA and DNA:DNA homology determination provides definitive delineation of species with 70% homology and above signifying membership of same species [11]. Increasingly, whole-genome sequencing (WGS) is becoming a standard technique and comparative genomic analysis is providing useful insights to the relatedness and divergence of MACA species [11].


**Table 1.**

*General phenotypic features of mycolate genera classified in the order* Corynebacteriales.

Protein sequences from *Corynebacteriales* genomes have revealed many conserved signature indels (CSIs) conserved signature proteins (CSPs) that are specific for members of this order [12].
