Biosurfactant Production by Mycolic Acid-Containing Actinobacteria

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.

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 biosurfactant-producing 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).

Figure 1.

Emulsification of hydrocarbons by microbial biosurfactants to enhance bioavailability.

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 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.

Genus	Micro-morphology	Acid-fastness	Aerial hyphae	Visible colonies (days)	Strictly aerobic
Corynebacterium	Pleomorphic rods, often club-shaped in palisade or angular arrangements	Some weakly acid-fast	Absent	1–2	No
Dietzia	Short rods and cocci	No	Absent	1–3	Yes
Gordonia	Rods, cocci and/or moderately branching hyphae	Partially acid-alcohol fast	Absent	1–3	Yes
Hoyosella	Cocci occur singly, in pairs, tetrads or in groups	Slightly acid–alcohol-fast	Absent	2	Yes
Lawsonella	Pleomorphic bacilli and cocci	Partially acid-fast	Absent	5–7	No
Millisia	Short rods	Acid-alcohol fast	Absent	1–3	Yes
Mycobacterium	Rods, occasionally branched filaments that fragment to rods and cocci	Strongly acid-fast	Rare	2–40	Yes
Nocardia	Mycelia that fragment into rods and cocci	Partially acid-fast	Present	1–5	Yes
Rhodococcus	Rods to extensive substrate mycelia that fragment to irregular rods and cocci	Partially acid-fast	Absent	1–3	Yes
Segniliparus	Rods	Acid-alcohol fast	Absent	3–4	Yes
Skermania	Acute angled branched mycelia	No	Only visible under the microscope	10–21	No
Smaragdicoccus	Coccoid	ND	Absent	7–14	Yes
Tomitella	Irregular rods	ND	Absent	ND	Yes
Tsukamurella	Single rods or in pairs or masses, sometimes rudimentary filaments and coccobacillary forms	Partially alcohol-acid fast	Absent	1–3	Yes
Williamsia	Thin rods or cocci in pairs or clusters	ND	Present	1–4	Yes

Table 1.

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

ND, not determined.

Adapted from [2].

Figure 3.

The appearance of (a) Gordonia amarae, (b) Rhodococcus erythropolis and (c) Tsukamurella spumae on glucose yeast-extract agar after 7 days incubation at 30°C.

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 semi-quantitative 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.

Genus	Mycolic acids (chain length)	Fatty acids*	Phospholipid type	Major menaquinone(s)	Muramic acid type	gDNA G + C (mol%)
Corynebacterium	22–38	S,U	I	MK-8(H2)	Acetylated	51–67
Dietzia	34–38	S,U,T	II	MK-8(H2)	Acetylated	65.5–73
Gordonia	46–66	S,U,T	II	MK-9(H2)	Glycolated	63–69
Hoyosella	30–38		II	MK-8	Acetylated	49.3–61.8
Lawsonella	α+-mycolate	S,U	I	MK-9	Acetylated	58.6
Millisia	44–52	S,U, T	II	MK-8(H2)	Glycolated	64.7
Mycobacterium	60–90	S,U,T	II	MK-9(H2)	Glycolated	57–73
Nocardia	48–60	S,U,T	II	MK-8(H4, Ѡ-cycl)	Glycolated	63–72
Rhodococcus	30–54	S,U,T	II	MK-8(H2)	Glycolated	63–73
Segniliparus	α+-mycolate	T	ND	ND	ND	68–72
Skermania	58–64	S,U,T	II	MK-8(H4, Ѡ-cycl)	Glycolated	67.5
Smaragdicoccus	43–49	S,U	II	SQA-8(H4, Ѡ-cycl); SQB(H4, dicycl)	Glycolated	63.7
Tomitella	42–52	S,U	II	MK-9(H2)	Glycolated	67.5–71.6
Tsukamurella	64–78	S,U,T	II	MK-9	Glycolated	67–78
Williamsia	50–56	S,U,T	II	MK-9(H2)	Glycolated	64–65

Table 2.

Chemotaxonomic features of mycolate genera classified in the order Corynebacteriales.

*

S, straight-chain saturated fatty acids; U, straight-chain unsaturated fatty acids; T, tuberculostearic acid.

Adapted from [2].

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]. 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].

2. Biosurfactants produced by MACA

In addition to Rhodococcus, diverse members of the order Corynebacteriales have been reported to synthesise extra-cellular and cell-bound biosurfactants, including members of the genera Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocardia, and Tsukamurella. Species belonging to the genus Rhodococcus have been most extensively investigated and are known to produce different chemical types, including a variety of glycolipids. However, an interesting array of biosurfactant structures are synthesized by MACA including lipopeptides, oligosaccharide lipids, polymeric glycolipids, terpenoid glycosides, trehalose corynemycolates, trehalose mycolates and dimycolates, and trehalose lipid (THL) esters [13]. Example structures of the different types of biosurfactants produced by MACA are shown in Figure 4. The chemical structure of trehalose-containing glycolipids have perhaps been studied in most detail. Several structural types have been reported including mono-, di- and tri-corynemycolates which have been characterised for species such as Rhodococcus erythropolis, Rhodococcus ruber and Rhodococcus wratislaviensis [14] and trehalose di-nocardiomycolates which have been characterised for Rhodococcus opacus [13]. The mycobacterial trehalose mycolates or di-mycolates (cord factors) are also thoroughly investigated given their role as modulators of mycobacterial pathogenesis and host immune response.

Figure 4.

Types and key structural features of various biosurfactants produced by MACA. (Adapted from [13]).

3. Habitats, recovery, and growth requirements of MACA

MACA are widely distributed in the environment including natural habitats such as mangroves, soil, freshwater, and deep ocean sediments as well as man-made sites such as activated sludge foams, biofilters, industrial wastewater and indoor building materials. Although predominantly saprophytic, many species are opportunistic pathogens forming parasitic associations with plants and animals, including humans, notably immunocompromised individuals. Several members of the genus Mycobacterium cause a plethora of diseases most notably tuberculosis caused by Mycobacterium bovis and Mycobacterium tuberculosis.

MACA capable of producing various biosurfactants have been isolated from environments (Table 3) including oil-contaminated soils [24, 25], water from oil wells [26], wastewater from the rubber industry [21], activated sludge, and effluent and sediment from pesticide manufacturing facilities [23]. The ability of MACA to produce biosurfactants in these habitats appears to be driven by the environmental conditions to which they are exposed whereby the biosurfactants act as mediators for the biodegradation of hydrophobic carbon substrates. Genes involved in biosynthesis of rhamnolipids by Dietzia maris for example have been shown to be upregulated in the presence of hydrophobic substrates including n-hexadecane, n-tetradecane and pristane [15]. However, the true distribution of biosurfactant-producing MACA in the environment may not solely depend on the presence of hydrophobic substrates.

MACA species	Source of isolation	Biosurfactant type	References
D. maris As-13-3	Deep-sea hydrothermal field	Di-rhamnolipid (DRL)	[15]
Dietzia cinnamea KA-1	Water and sediments collected from oil-polluted seasonal ponds	Methylated ester	[16]
Nocardia otitidiscaviarum MTCC 6471	Oil contaminated seawater	Rhamnolipid	[17]
T. spumae DSM44113, T. spumae DSM44114 and T. pseudospumae DSM44117	Activated sludge foam	THL	[18]
Gordonia amicalis; HS-11	Oil contaminated soil	Glycolipid	[19]
Gordonia westfalica GY40	Agricultural soil	Glycolipid	[20]
R. erythropolis 16 LM. USTHB	Water polluted by rejections of 2-mercaptobenzothiazole and its derivatives used in the rubber industry	Fatty acid methyl esters	[21]
Rhodococcus fascians strain A-3	Fell field soil	Rhamnose-containing glycolipid	[22]
Rhodococcus pyridinivorans NT2	Effluent-sediment collected from a pesticide manufacturing facility	THLs	[23]

Table 3.

Various environmental sources of biosurfactant-producing MACA.

Isolation of biosurfactant producers largely relies on selective isolation strategies, utilising hydrophobic compounds as sole carbon sources for energy and growth. Typically, strains are isolated and cultivated using mineral salt medium containing essential trace elements supplemented with a hydrocarbon substrate such as crude oil, diesel, n-alkanes, n-hexadecane, paraffin, polyaromatic hydrocarbons (PAHs), or vegetable oils such as olive oil and rapeseed oil, as the sole carbon source. These may be incorporated into the liquid or solid medium, spread across the agar surface or soaked onto a filter in the lid of petri dishes. Besides the selectivity of the culture medium, pre-enrichment techniques utilising hydrophobic compounds as the sole carbon source, can be used [27]. The principle of enrichment is to provide growth conditions that are favourable for the organisms of interest but not for competing organisms. This selective advantage allows target populations to expand through a series of passages, maximising the chances of successful recovery at the isolation stage. Incorporating antibiotics into the isolation media may provide a useful additional selective pressure to eliminate or reduce unwanted fungi and bacteria.

The ability of an organism to grow on hydrophobic compounds is a good indicator of biosurfactant production but is not a guarantee. It is therefore important that isolates of interest are tested in pure culture for biosurfactant production using further screening assays. It is also possible that biosurfactant-producing organisms may be present in an environment but not enriched by in the conditions provided or indeed producers may be recovered from the environment but not synthesize biosurfactants under the culture conditions imposed. Mining genomes for cryptic biosurfactant biosynthesis pathways, and metagenomic screening of DNA from environmental samples promise an alternative approach to biosurfactant discovery that may circumvent some of the issues associated with culture-dependent strategies [28].

4. Detection and characterisation of biosurfactants

4.1 Biosurfactant screening methods

A variety of methods, both qualitative and quantitative, have been applied to screen microbial cultures and cell-free media for total (intracellular, surface-bound, and freely released) and freely released biosurfactants, respectively. As biosurfactants are structurally diverse, complex molecules, most of these methods are indirect, reliant on physico-chemical properties such as emulsification, surface activity or hydrophobicity. Commonly reported screening methods used to detect biosurfactant production amongst MACA strains are listed in Table 4. Besides the bacterial adhesion to hydrocarbons (BATH) assay [37] other tests based on cell surface hydrophobicity include salt aggregation [38] and hydrocarbon overlay [39] assays. The atomized oil assay [40] may be used to directly screen colonies growing on primary isolation plates and is therefore useful as an initial screen for novel-producing strains recovered from the environment. The microplate assay [41] which relies on the wetting properties of biosurfactants and the penetration assay [42], which relies on the reduction of interfacial tension are also considered useful for screening large numbers of strains. Recently, a rapid, high throughput assay that utilises Victoria pure blue BO dye, and is based on surface-active properties, has been developed for quantitative screening, but has not yet been applied to MACA [43].

Detection property	Screening method	MACA species	Reference
Surface activity	Oil spreading	R. erythropolis	[29]
		Gordonia spp.	[30]
		Dietzia spp.	[31]
	Drop collapse/; modified drop collapse	G. westfalica	[20]
		R. erythropolis	[29]
	Surface and interfacial tension measurement	Rhodococcus spp.	[14, 26, 29]
		Gordonia spp.	[20, 30, 32]
		Tsukamurella spp.	[18]
		Dietzia sp.	[31, 33]
		Nocardia sp.	[17]
Emulsification	Emulsification assay	G. amicalis	[34]
	Emulsification index	Rhodococcus sp.	[14, 22, 35]
		Gordonia spp.	[30, 32]
		Tsukamurella spp.	[18]
		Dietzia spp.	[31, 33]
		Nocardia spp.	[17]
Cell-surface hydrophobicity	Microbial adhesion to hydrocarbons (MATH)/BATH assay	R. fascians	[22]
		Gordonia alkanivorans	[36]
		D. maris	[33]
		N. otitidiscaviarum	[17]

Table 4.

Examples of screening methods used to detect biosurfactant production by MACA.

These assays are simpler and more rapid than chemical analytical procedures, and most enable larger-scale screening for biosurfactant production. However, perhaps owing to the general and indirect nature of these assays and various limitations associated with some, test results between assays are not always congruent and no one assay is considered definitive for biosurfactant production. It is thus advisable to use several methods in combination, adopting simple methods to undertake preliminary screening of large strains collections prior to further investigation of those found to be most promising. The development of high-throughput screening, metabolic profiling technologies, and whole-genome analysis promise a more thorough investigation of potential biosurfactant producing strain in the future [28].

4.2 Extraction and structural analysis of biosurfactants

Crude biosurfactant extracts may be obtained from cell cultures (cell-associated and free surfactants) or cell-free broth (free surfactant only) by acidification and solidification followed by solvent extraction of the precipitate. In the case of MACA commonly used solvents include MTBE, dichloromethane, or varying ratios of chloroform–methanol or MTBE–chloroform [44]. Various analytical techniques are used in combination to detect, quantify, and characterise biosurfactants. Thin layer chromatography (TLC) is a straightforward method to separate biosurfactant fractions present in crude extracts. Samples are spotted at the base of a silica plate before development in a solvent system, then air-dried and sprayed with a particular reagent to detect certain chemical groups based on spot colour and/or R_f values. Orcinol, for example, allows detection and differentiation of glycolipids and can distinguish mono-rhamnolipid (MRL) and DRL congeners [45]. However, TLC provides little further detail on congener structure, and it is not generally considered suitable for quantitative analysis although densiometry has been used for this purpose [46]. Biosurfactants may be further separated by silica gel column chromatography.

High-performance liquid chromatography-mass spectrometry (HPLC-MS) allows more precise and accurate characterisation and quantitation of biosurfactant compounds. Isocratic HPLC-UV has been reported for structural and yield determination of THLs produced by R. erythropolis strain MTCC 2794 from semi-purified extractions of whole-cell broth [47]. Nuclear magnetic resonance spectroscopy (NMR) is considered the gold standard method to characterise the chemical structure of novel biosurfactants. This has been used in combination with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF/MS) to elucidate the structure of two novel extracellular THLs TL A and TL B from Tsukamurella spp. [18].

A combination of Fourier transform infrared spectroscopy (FTIR), NMR, and liquid chromatography-mass spectrometry (LC-MS) enabled structural characterisation of a novel cyclic lipopeptide, Coryxin, produced by Corynebacterium xerosis NS5 [48]. Multiple-Stage Linear Ion-Trap Mass Spectrometry with Electrospray Ionization has been used to determine the structure of trehalose monomycolate (TMM) and trehalose dimycolate (TDM) in the cell wall of Rhodococcus equi and R. opacus [49]. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been utilised successfully for the purification and characterisation of sophorolipids and rhamnolipids in Pseudomonas aeruginosa [50] and could be applied to similar compounds produced by mycolate species. Gas chromatography-mass spectrometry (GC-MS) is used to characterisation of the fatty acid and mycolic acid components and for the carbohydrate portion of THLs.

5. Potential applications of biosurfactants from MACA

Biosurfactants produced by rhodococci and related MACA have been investigated primarily for their potential application in oil remediation but are otherwise under-studied and under-exploited. However, research studies reveal various potential applications for these molecules, including in environmental and medical fields as summarised in Figure 5.

Figure 5.

Promising medical and environmental applications for biosurfactants produced by MACA.

5.1 Biomedical applications

Biosurfactants produced by microorganisms are reported to have various potential biomedical and pharmaceutical applications which have been reviewed widely [1, 51, 52]. This stems from an array of biological properties including anti-adhesion and antibiofilm, anti-inflammatory, antimicrobial (anti-bacterial, anti-fungal and anti-viral), antioxidant, anti-tumour, and wound healing activities. Other potential applications include adjuvants for antigens in vaccines, pulmonary surfactants, drug delivery systems, enhanced vehicles for gene therapy and in dermatological care. Biosurfactants also have several applications in therapeutic dentistry [53]. Daptomycin, a cyclic lipopeptide produced by the actinobacterium Streptomyces filamentosus, is used as an antibiotic to treat serious blood and skin infections caused by Gram-positive pathogens [54] and there are other examples of actinobacteria that produce surfactants with potential biomedical applications, such as Nocardiopsis strains [55]. Only limited investigation has focused on the biomedical potential of biosurfactants from MACA, except for TDM or cord factors synthesised by intracellular pathogens of the genera Mycobacterium. Nevertheless, as shown in Table 5, studies over the past two decades reveal that various biosurfactants produced by members of the genera Corynebacterium, Nocardia, Rhodococcus, and Tsukamurella demonstrate a range of promising properties.

Strain (origin)	Biosurfactant	Biomedical properties	Reference
C. xerosis NS5 (human axilla)	Purified Coryxin (lipopeptide)	Antibacterial activity, biofilm inhibition and disruption of pre-formed biofilms of Gram-positive S. aureus and Streptococcus mutans and Gram-negative E. coli and P. aeruginosa strains	[48]
Nocardia brasiliensis IFM- 0406 (patient with lung nocardiosis)	Aliphatic macrolide (Brasilinolide)	Moderately antifungal against Aspergillis, Candida, Cryptococcus and Paecilomyces species	[56]
N. farcinica BN26 (hydrocarbon polluted soil)	THL	Anti-tumour activity: cytotoxic effects on human tumour cell lines BV-173 and SKW-3, and to a lesser extent, HL-60. Mediated cell death by the induction of partial apoptotic DNA laddering	[57]
N. vaccinii IMB B7405 (K-8) (oil-contaminated soil)	Complex of amino lipids; neutral lipids (mycolic and n-alkanic acids); trehalose di-acelates and di-mycolates (surfactant solution and supernatant)	Anti-adhesive activity against Gram-negative bacteria E. coli, Proteus vulgaris, P. aeruginosa and Enterobacter cloaceae and the yeast Candida albicans on silicon urogenital catheters. Anti-adhesive activity against fungus C. albicans and bacterium E. coli on treated acrylic dental material and against Gram-positive Bacillus subtilis and micromycete Aspergillis niger when coated on various abiotic substrates	[58]
R. erythropolis SD-74 (alkaline soil)	Purified STL-1	In vitro induction of human monocytoid leukemic cell line U937 differentiation and cytotoxicity against human lung carcinoma cell line A549	[59, 60]
		In vitro induction of human promyelocytic leukaemia (HL60) cell line differentiation into monocytes and inhibition of protein kinase C
R. erythropolis IMВ Ac-5017 (EK-1) (oil-contaminated soil)	Complex of trehalose mono- and di-mycolates; neutral lipids (cetyl alcohol, palmitic acid, methyl ether of n-pentadecanoic acid, mycolic acids); phospholipids (phosphatidylglycerol, phosphatidylethanol-amine) (surfactant solution and supernatant)	Antibacterial activity against Gram-positive bacteria B. subtilis and S. aureus and Gram-negative E. coli and Pseudomonas sp., and anti-fungal activity against C. albicans, C. utilis and C. tropicalis	[58]
		Anti-adhesive activity against Gram-negative bacteria and fungus C. albicans on silicon urogenital catheters. Anti-adhesive activity against B. subtilis on various abiotic substrates, against C. albicans and E. coli on acrylic dental material and S. aureus and P. aeruginosa on plastic and steel
R. fascians BD8 (Arctic soil polluted with hydrocarbons	THL	Antibacterial activity against Vibrio harveyi and P. vulgaris, and partial inhibition of other Gram-positive and negative bacteria and fungus C. albicans. Anti-adhesion properties on polystyrene against various Gram-positive and negative strains and fungal strains of C. albicans. Biofilm inhibition on glass, polystyrene, and silicone urethral catheters against Gram-positive Enterococcus hirae and E. faecalis, Gram-negative E. coli, and fungus C. albicans	[61]
Rhodococcus sp. I2R (marine sediment)	Extracellular complex of glycolipids (crude extracts and purified fractions)	Antiviral activity against HSV-1 and human coronavirus HCoV-OC43. Antiproliferation activity against human prostatic carcinoma cell line PC3	[62]
R. ruber IEGM 231 (spring water, oil-extracting enterprise)	Crude trehalolipids	Anti-adhesive activity against exponentially growing Gram-positive bacteria Arthrobacter simplex, B. subtilis, Brevibacterium linens, Corynebacterium glutamicum, and Micrococcus luteus and against Gram-negative bacteria E. coli and P. fluorescens on polystyrene.	[63]
	Mixture of TDM, diacyltrehalose and monoacyltrehalose isolated by column chromatography	In vitro induction of IL-1β, IL-6, and TNF-α cytokine secretion by human monocytes	[64]
		In vitro induction of Th1-polarizing factors IL-12 and IL-18 by human mononuclear cells and monocytes and reactive oxygen species (ROS) by peripheral blood leukocytes	[65]
	Glycolipid	In vivo induction of IL-1β by mouse peritoneal macrophages	[66]
	Monoacyltrehalose fraction (MAT)	In vivo suppression of bactericidal activity and proinflammatory cytokine IL-1β of mouse peritoneal macrophages, antibody production by splenocytes and stimulates the production of IL-10	[67]
Rhodococcus sp. TB-42 (soil)	Analogues of STL-3	Inhibited growth and induced the differentiation of human HL-60 promyelocytic leukaemia cell line	[66]
T. tyrosinosolvens DSM 44370 (oil containing soil)	Purified oligosaccharide lipids	Antimicrobial activity against Gram-positive bacterial strain of Bacillus megaterium, Gram-negative E. coli and fungal strain Ustilago violacea	[67]

Table 5.

Biomedical research on biosurfactants produced by MACA.

The amphipathic nature of biosurfactants makes them suitable for anti-adhesion and anti-biofilm applications such as the development of anti-adhesive coatings for intra-urinary devices that are prone to the formation of intractable biofilms, to prevent or delay the onset of biofilm growth by pathogens such as Escherichia coli and Proteus mirabilis. C. xerosis strain NS5, Nocardia vaccinii K-8 and various Rhodococcus strains demonstrate anti-adhesion, biofilm inhibition and/or biofilm disruption effects against various clinically significant pathogens (Table 5). Some also exhibit antimicrobial properties although in the case of R. ruber strain IEGM 231 the trehalolipids had no effect on cell viability despite preventing adhesion of various bacteria to polystyrene [63]. Oligosaccharides produced by Tsukamurella tyrosinosolvens (DSM 44370) showed some activity against Gram-positive bacteria, although the pathogenic strain Staphylococcus aureus was not affected. Rhodococcus strain I2R shows anti-viral activity against herpes simplex virus 1 (HSV-1) and human coronavirus HcoV-OC43 [62].

Nocardia farcinica BN26 produces a THL with anti-cancer effects, showing cytotoxicity against human tumour and promyelocytic leukaemia (HL60) cell lines [57]. Rhodococcus erythropolis SD-74 and Rhodococcus sp. TB-43 also cause the induction of HL60 cells [59, 60]. R. ruber has been studied in some detailed and reported to show immunomodulatory effects, including both in vitro induction of Th1-polarizing factors IL-12 and IL-18 by human mononuclear cells and monocytes and in vivo induction of IL-1β by mouse peritoneal macrophages [64, 65, 68, 69]. Two succinoyl trehalose lipids, STL-1 and STL-3, produced by R. erythropolis SD-74 inhibit growth and induce cell differentiation into monocytes instead of cell proliferation when tested on the HL60 cell line.

Glycolipid bearing mycolic acids, such as trehalose dimycolate (TDM) have attracted extensive investigation as they play a central role in pathogenesis during infection by intracellular pathogens such as M. tuberculosis and R. equi. TDM’s have been researched as a possible tuberculosis vaccine and as an adjuvant. In addition, modification of mycobacterial TDM has been shown to reduce virulence and suppress the host immune response [9]. Interestingly, TDM also possesses biological activities that point towards medical and pharmaceutical applications, such as antitumor activity and immunomodulating functions. Despite this, the potential for TDM is perhaps limited by relatively high toxicity and the pathogenic nature of the species that produce them.

Although biologics including surfactants are generally regarded as less toxic than synthesized pharmaceuticals not much work has focussed on this with respect to MACA surfactants. However, a THL from R. erythropolis strain 51T7 has been reported to be suitable for use in cosmetic preparations as it was less irritating than SDS when tested on mouse fibroblast and human keratinocyte lines [70]. Further investigation into the potential biomedical and pharmaceutical applications of biosurfactants produced by members of the MACA, including toxicity testing, is certainly warranted. The high costs and technical challenges associated with production and downstream extraction of biosurfactants may not be a barrier to their commercial application in biomedical fields given that smaller-scale productions would likely be required.

5.2 Environmental applications

Biosurfactants have a range of promising, and increasingly important, applications in the environmental, industrial, and agricultural sectors (Table 6). These include bioremediation of both organic pollutants (especially hydrocarbons) and metals, microbial enhanced oil recovery (MEOR), cleaning and maintenance of tanks and pipelines in the petroleum industry, wastewater treatment, and agricultural applications such as promotion of plant growth/health and inhibition of phytopathogenic fungi [1, 78]. MACA-derived surfactants have been investigated in some of these contexts, although the focus is on well-known species such as R. ruber and R. erythropolis. Members of Gordonia, Corynebacterium, Nocardia and Dietzia have also been investigated but there is likely to be much unexplored potential within the group [79]. This is supported by the promising results obtained with rhamnolipids produced by other bacteria, most notably P. aeruginosa, and their commercialisation [80]. It is not unreasonable to expect that rhamnolipids produced by MACA may also exhibit such properties. Indeed, the search for non-pathogenic producers is important for further development of biosurfactant production at industrial scale [81].

Pollution of soils with organic and inorganic chemical compounds is a major environmental issue. Biosurfactants are used to improve the solubility of hydrocarbon organic compounds, either to make them available for subsequent biodegradation or to facilitate removal by soil washing. A remediation agent called JE1058BS containing biosurfactant from Gordonia sp. strain JE-1058 was evaluated as an oil spill dispersant using the baffled flask test recommended by the US Environmental Protection Agency and performed better than commercially available dispersants. It also enhanced the bioremediation of crude oil by indigenous marine bacteria and significantly improved removal of crude oil from contaminated sea sand by washing compared with the use of seawater alone [73]. Various Dietzia, Gordonia and Rhodococcus strains have been shown to degrade hydrocarbon compounds and many studies show that the production of surface-active compounds makes an important contribution. In a recent study, G. amicalis HS-11 was able to remove 92.85% of the diesel oil provided as the sole carbon source after 16 days of incubation, with a corresponding reduction in surface tension due to the production of extracellular surfactants. Microscopy suggested that these surfactants play a role in the emulsification and uptake of the hydrocarbons. Plant-based bioassays also showed that toxicity of the diesel oil decreased. This illustrates the potential of this strain and perhaps other gordoniae for use in the bioremediation of contaminated environments, or industrial wastewaters [82].

The properties and actions of biosurfactants make them particularly relevant to the petroleum industry. MEOR is perhaps the most well-known application in this area. Biosurfactants, or biosurfactant-producing microorganisms, are used to extract some of the oil remaining in reservoirs after primary and secondary processing has been carried out. Mechanisms include reduction of capillary forces holding the oil in porous rock, stabilisation of desorbed oil in water and increased viscosity of oil for easier removal [83]. Dietzia sp. ZQ-4, a hydrocarbon-degrading, surfactant-producing MACA isolated from an oil reservoir, demonstrated potential for use in ex situ oil recovery. Fermentation broth significantly increased oil displacement efficiency by 18.82% in rock cores and performed well within the range reported for other strains. However, injection of the strain itself was not so successful, and field trials testing nutrient injection did not always result in an increase in the population of Dietzia sp. ZQ-4, indicating that an in-situ approach may not be viable although it may be possible to optimise this strategy further [72]. Biosurfactants produced by various rhodococci strains recovered from oil-polluted soils have been shown to be effective at recovering trapped oil from oil-saturated sand packs. Glycolipids produced by strain ST-5 recovered up to 86% [84] and a mix of glycolipids and extracellular lipids produced by strain TA6 up to 86% [24] using the sand pack column method. Studies on biosurfactant produced by R. ruber IEGM 231 showed that 2.5 times greater washing activity could be achieved than with synthetic surfactant Tween-60 in soil columns spiked with polyaromatic carbons (PAHs) and alkanes. The biosurfactant maintained activity at a high (5% w/w) contamination level and consistently removed 0.3–0.5 g PAHs per kg dry soil in a single run of washing [71].

Biosurfactants may also be used to de-emulsify water–oil emulsions that form during oil production in the oilfields, as well as during transportation, and processing and offer a more ecologically friendly solution than chemically synthesized de-emulsifiers. A lipopeptide bio-demulsifier produced by Dietzia sp. strain S-JS-1 grown on waste frying oil achieved 88.3% of oil separation ratio in water/oil emulsion and 76.4% of water separation ratio in oil/water emulsion [75].

Biosurfactants have been shown to reduce phytotoxicity of heavy metals, and pre-treatment of seeds could allow plants to be grown successfully in contaminated soil, facilitating phytoremediation of the environment. Crude biosurfactant from R. ruber IEGM 231 mitigated the toxic effects of high concentrations of molybdenum on oat, white mustard, and vetch seeds. Germination increased up to 4.5 times and shoot and/or root length up to 2.5 times when seeds were pre-treated with a biosurfactant emulsion and grown under conditions of molybdenum contamination [85]. Similar results have been recorded for other heavy metals such as copper [86].

The use of biosurfactants in environmental and industrial applications is limited by the current high costs of production, and the large amounts of biosurfactant required. However, using waste and/or renewable substrates would be cheaper, and a highly purified product is not essential so costs of downstream processing can also be reduced. In addition, different approaches such as selective stimulation of biosurfactant producers in situ, and inoculation of biosurfactant-producing cultures, are being explored [87]. This could potentially overcome some of the challenges associated with accessing the cell-bound biosurfactants produced by MACA such as Rhodococcus spp.

Application	Examples of MACAs	Reference/s
Bioremediation: enhanced hydrocarbon solubility and degradation	D. maris As-13-3; D. maris WR3; G. amicalis HS-11; Gordonia cholesterolivorans AMP 10; N. otitidiscaviarum; R. erythropolis 3C-9; R. pyridinivorans NT2	[15]; [33]; [34]; [32]; [17]; [29]; [23]
Bioremediation: soil washing	Gordonia sp. strain BS29; R. ruber (IEGM 231)	[30]; [71]
MEOR	Dietzia sp. ZQ-4; Gordonia sp. JE-1058; R. ruber Z25; Rhodococcus sp. strain TA6	[72]; [73]; [74]; [24]
Bio-demulsification: treatment of water-oil emulsions generated during processing of petroleum	Dietzia sp. S-JS-1	[75]
Paraffin control in oil transport pipelines	G. amicalis LH3	[76]
Bioflocculation (e.g., for oil recovery from wastewater)	R. erythropolis S-1	[77]

Table 6.

Various potential environmental applications of biosurfactants produced by MACA.

5.3 Challenges to commercialisation

Currently, commercial production of biosurfactants is not economically competitive with chemical surfactant production as there are various challenges to overcome. Bioprocesses presently achieve low biosurfactant productivity and yield and substrates are expensive [6]. Foam formation can cause serious operational issues and downstream biosurfactant recovery can be technically involved and costly. Development work to optimise bioprocesses should focus on enhancing biosurfactant yield and potency. Approaches include the search and discovery of novel biosurfactant-producing organisms and strain improvement by various genetic engineering methods and/or stress-fermentation including co-cultivation [84]. Yield can also be enhanced through the optimisation of culture conditions and costs reduced through the introduction of renewable or waste products [6, 28, 77] as cheaper feed stocks. The effects of biosurfactants on human health and the environment also require further assessment to ensure safe production and use.

6. Conclusions

Biosurfactants offer an attractive proposition for biotechnological application across various sectors and are considered superior to synthetic surfactants. Diverse MACA produce biosurfactants with interesting properties that have been explored in the context of biomedicine and environmental remediation. However, many MACA have not yet been investigated for biosurfactant production and various potential applications are yet to receive significant research. Rapid, reliable methods for high throughput screening for biosurfactant production are essential as are robust standard methods for biosurfactant purification and characterisation. Efforts to evaluate and expand the knowledge of structural characteristics and gene regulation of biosurfactants are warranted to improve their effectiveness and productivity. Commercial-scale production will need to employ various existing and new strategies to become economic and sustainable. Cutting-edge technologies such high-throughput omics-based tools should accelerate the development of commercial production of biosurfactants. Furthering our understanding of biosurfactants produced by MACA will facilitate their commercial exploitation thereby contributing to a sustainable bio-based economy.

Conflict of interest

The authors declare that there is no conflict of interest.