**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 antiadhesion and antibiofilm, anti-inflammatory, antimicrobial (anti-bacterial, antifungal 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.

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

**Figure 5.** *Promising medical and environmental applications for biosurfactants produced by MACA.*

#### *Biosurfactant Production by Mycolic Acid-Containing Actinobacteria DOI: http://dx.doi.org/10.5772/intechopen.104576*

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



#### *Biosurfactant Production by Mycolic Acid-Containing Actinobacteria DOI: http://dx.doi.org/10.5772/intechopen.104576*

#### **Table 5.**

*Biomedical research on biosurfactants produced by MACA.*

commercialisation [80]. It is not unreasonable to expect that rhamnolipids produced by MACA may also exhibit such properties. Indeed, the search for nonpathogenic 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


**Table 6.**

*Various potential environmental applications of biosurfactants produced by MACA.*

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.

#### **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.
