in the Mycobacterium avium, M. intracellulare group, nocardia (Nocardia asteroids), and corynebacteria (Corynebacterium diphtheria, C. matruchotii, and C. xerosis) and the observed or potential pathogenicity of these producer strains, as well as the high toxicity of the biosynthesized glycolipids, significantly restricted their use, specifically in the biomedical field, in which is mandatory they are GRAS (Generally Regarded As Safe). For this purpose, non-pathogenic producing bacteria have been studied, in particular, representatives of Rhodococcus, Gordonia, Dietzia, Tsukamurella, Skermania, Williamsia, among others (Kuyukina and Ivshina 2019; Kuyukina et al. 2020). Although all of these microorganisms can be used for the production of trehalose lipids, Nocardia and Rhodococcus are the most widely explored (Patil and Pratap 2018).
In the case of Rhodococcus and related genera, the trehalose lipids biosynthesized are usually bound to the cell envelope and are produced mainly when the microorganisms are grown on hydrocarbon sources. This ability to access hydrocarbons is related to the microorganism’s cell surface hydrophobicity. Cells with high hydrophobicity directly contact oil drops and solid hydrocarbons, while low hydrophobicity allows the adhesion of microbial cells to the micelles or emulsified oils, formed due to the presence of extracellular biosurfactants (Franzetti et al. 2010). White et al. (2013) evaluated the production of trehalose lipids by a marine bacterium Rhodococcus sp. PML026 using sunflower oil as a hydrophobic substrate.
The capacity to produce a metabolite is bestowed by the genes of the microrganism, affecting the yield of biotechnological products. A production process is commercially viable and profitable after the yield of the final product by the producer organisms is high, in fact the bioindustrial production process is often dependent on the use of hyperproducing microbial strains, even with cheap raw materials, optimized medium and culture conditions, and efficient recovery processes (Bouassida et al. 2018). The genetic manipulation to improve production of biosurfactants was highlighted in 2016 by Paulino et al. (2016).
Moreover, when good yields from natural producer strains are lacking, the industrial production process will depend on the availability of recombinant and mutant organisms. To economize the production process further and to obtain products with better commercially important properties, recombinant hyperproducers have been developed. However, only a few mutant and recombinant varieties with enhanced biosurfactant production characteristics are reported in the literature (Mukherjee et al. 2006). Transposons (Koch et al. 1991), chemical mutagens (e.g., N-methyl-N0 nitro-Nnitrosoguanidine (Lin et al. 1998), radiation (Iqbal et al. 1995) or by selection are strategies used to produce mutant varieties.
The first report about the use of genetic engineering was the insertion and expression of the Vitreoscilla hemoglobin gene (vgb) in Gordonia amarae. This resulted in enhancement of trehalose lipid production in a medium supplemented with 1% hexadecane (Dogan et al. 2006). The elucidation of biosynthetic pathways through the identification of putative acyl coenzyme A transferase, fructose-biphosphate aldolase, and alkane monooxygenase genes and genetic manipulation allowed an increase in succinoyl trehalolipid production by Rhodococcus sp. strain SD-74 (Inaba et al. 2013).
The screening of biosurfactant producers can be carried out using several methods (qualitative and/or quantitative) with variable precisions and aims. Some examples of these methods are the following: (i) hemolytic assay (HA) (Carrillo et al. 1996), (ii) the N-Cetyl-N,N,N-trimethylammoniumn bromide (CTAB) agar plate method (Pinzon and Ju 2009), (iii) cell surface hydrophobicity methods, that is to say bacterial adherence to hydrocarbons (BATH) assay (Desai and Banat 1997) and microbial adherence to hydrocarbons (MATH) assay (Siegmund and Wagner 1991), (iv) the oil spreading method (Morikawa et al. 2000), (v) emulsification activity (Varjani et al. 2014), (vi) tilted glass slide (Satpute et al. 2010), (vii) hydrocarbon overlay agar (HOA) (Satpute et al. 2008), (viii) aximetric drop shape analysis (ADSA) (Satpute et al. 2010), (ix) direct colony thin layer chromatographic, turbidity assay (Satpute et al. 2008), (x) tensiometric measurements, like surface and interfacial tension (Varjani et al. 2014; Varjani and Upasani 2016). The primary screening method for biosurfactant producers were hemolytic assay and CTAB agar test methods (Mulligan et al. 1984; Satpute et al. 2010).
1.5 Factors Affecting Trehalose Lipid Production
The factors affecting trehalose lipids production can be divided in two major groups: (i) nutritional and (ii) environmental.
i) Nutritional factors
The carbon source can influence the biosurfactant synthesis either by induction or repression, it has considerable bearing on the produced type of biosurfactants. Furthermore nitrogen can play an important role in the regulation of biosurfactant synthesis (Cameotra and Makkar 1998; Ribeiro et al. 2013; Santos et al. 2016).
1.5.1 Carbon Source
The growth of the microorganisms for trehalose lipid production requires a carbon source. This carbon source influences not only the quantity but also the quality of the formed trehalose lipids. When carbon sources and insoluble substrates are combined the intracellular diffusion and production of different substances are simplified (Santos et al. 2016). For instance, in 1996 a study (Espuny et al. 1996) proceeded to test the effect of different carbon sources where two different mixtures were used, P-120 and P-147. P-120 was composed of n-decane, n-undecane, dodecane, n-tridecane, n-tetradecane. The second mixture, P-147, had in its composition n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane. In this study, the higher yield was accomplished using n-tridecane alone (Espuny et al. 1996).
In several studies, n-hexane was used as the carbon source (Christova et al. 2015; Janek et al. 2018; Kuyukina et al. 2016; Tokumoto et al. 2009). Another study showed that Rhodococcus erithropolis, using n-hexane as the carbon source was able to produce mono- and di-succinyl trehalose lipids (Kitamoto et al. 2002; Uchida et al. 1989).
Kuyukina et al. (2015) used as the carbon source, n-dodecane and n-hexadecane and obtained 6.5 and 9.4 g L-1 of trehalose lipids, showing that the best method uses hydrocarbons in the bioprocess. Rhodococcus wratislaviensis BN38 grown in n-hexadecane produced 3.1 g L-1 of 2,3,4,2′-trehalose tetraesters containing succinic acid. These trehalose lipids showed high surface activity reducing the surface tension to 28.6 mN m-1 in mixture and 24.4 mN m-1 when purified at a CMC value of 5 mg L-1.
In a different study, Marqués et al. (2009) carried out a study with Rhodococcus erythropolis 51T7 in a medium containing 2% of tetradecane, enabled the production of trehalose tetraester in a concentration of 0.48–1.12 g L-1 and a reduction in the surface tension to 27.9 mN m-1 at a CMC value of 37 mg L-1.
Nevertheless, other sources have been used such as n-alkanes (Inaba et al. 2013), which have been used for the production of TL-1 and TL-2, and n-decane to produce trehalose-tetraesters (Kitamoto et al. 2002), glucose or vegetable oil (Santos et al. 2016), glyceryltrioleate (Kügler et al. 2014).
Furthermore, using sugars as carbon sources directly may affect the carbohydrate moiety of trehalose lipids, which might have a good effect on the production and be less expensive.
Different carbon sources, like glycerol, diesel oil, and vegetable oil, showed feasibility as trehalose lipids producers (Bajaj et al. 2014). Additionally the authors (Bajaj et al. 2014) correlated the possibility of using trehalose lipid bioproducers as potential agents in bioremediation process.
A further target for production is the use of low-cost substrates. Experiments on R. erythropolis 16 LM.USTHB showed the conversion of used sunflower frying oil, a cheap renewable source, into extracellular glycolipids with the ability to lower the surface tension of crude broth to 31 mN m-1 (23 Franzetti et al. 2010).
Kügler et al. (2014) performed a study using non-pathogenic actinomycetes Tsukamurella spumae and Tsukamurella pseudospumae using sunflower oil as the carbon source in a fermentation medium. They found trehalose lipids with C4–C6 and C16–C18