in hydrophobic moiety, in a low concentration of product (approximately 1.3 g L-1). Kundu et al. (2013) performed a study using the Rhodococcus pyridinivorans NT2 strain isolated from effluent sediment contaminated with pesticides and evaluated it for biodegradation of 4-nitrotoluene. The biosurfactant produced, a trehalose lipid, exhibited surface activity, which allowed the reduction of surface tension of the media from 71 to 29 mN m-1, with a CMC value of the 30 mg L-1. The most abundant trehalose lipid derivatives produced by the mentioned strain were trehalose-succinic acids and 2,3,4,2′-trehalose tetraester analogues. Moreover, an emulsification index of 90–95% was obtained with long-chain hydrocarbons (diesel, liquid paraffin, motor oil, groundnut oil, and soybean oil) while shorter-chain alkanes resulted in a lower emulsification index (50–80%). The microbial characteristics of Rhodococcus pyridinivorans NT2 contributed to its potential use in bioremediation field, despite the low concentration in the production of trehalose lipids (45 mg L-1) (Kundu et al. 2016a, 2016b).
1.5.2 Nitrogen Source
In fermentative processes, the carbon/nitrogen (C/N) ratio usually affects the accumulation of metabolites. High carbon/nitrogen (C/N) ratios limit bacterial growth. On the other hand, low carbon/nitrogen ratios lead to the synthesis of cellular material and limit the accumulation of products (Santos et al. 2016).
In the specific case of trehalose lipids, different nitrogen sources can be used.
In 1988, Ramsay et al. (1988) used sodium nitrate instead of ammonium sulfate, as a nitrogen source, with good results.
In another study (Uchida et al. 1989) the effect of the different nitrogen sources was evaluated. The cell growth was not affected considerately by ammonium sulfate, ammonium dihydrogen phosphate, ammonium nitrate, or urea. Nevertheless, potassium nitrate allowed a higher yield (Uchida et al. 1989). In another work, making use of limited nitrogen conditions allowed trehalose mycolates to be formed by Rhodococcus erythropolis, to produce anionic trehalose tetraesters (Lang and Philp 1998).
In fact, nitrogen sources played a key role in biosurfactant production. Wang et al. (2019) noticed that when organic nitrogen urea was used as the nitrogen source, in the Rhodococcus qingshengii strain FF growth, the yield of trehalose lipids was always higher compared to the use of inorganic nitrogen, ammonium nitrate. When n-hexadecane was used as the sole carbon source, the yield of trehalose lipids was 1.62 g L-1 with urea as the nitrogen source, which was 2.36 times the yield obtained when inorganic nitrogen ammonium nitrate was used (Wang et al. 2019). Through screening different types of carbon and nitrogen sources, Wang et al. (2019) identified, hexadecane:oleic acid (m : m = 1 : 1) and urea, as the best carbon and nitrogen sources respectively.
ii) Environmental factors
The environmental parameters for the fermentative process are of great importance. Environmental factors like pH, temperature, agitation and oxygen availability, play vital roles in microbial growth and glycolipid production as they show their effects on cellular growth and activity (Varjani and Upasani 2017). For trehalose lipid production in a bioreactor with control systems (e.g. digital), these parameters were stable during bacterial growth (Pacheco et al. 2010).
Although trehalose lipids produced by Rhodococcus sp. formed emulsions that were stable at pH 2–10 and temperatures of 20–100 ºC (Mnif and Ghribi 2015; White et al. 2013), the temperatures were stabilized between 28 ºC and 30 ºC during growth and the pH was neutral and stable as well, leading to higher yields (Janek et al. 2018; Kügler et al. 2014; Kuyukina et al. 2016).
In order to avoid the problem of low concentration of trehalose lipids in biotechnological processes, the use of statistical methods is an alternative approach to optimize the factors that affect growth and production, increasing yields and reducing process costs. Through response surface methodology (RSM), Mutalik et al. (2008) achieved an increase from 3.2 to 10.9 g L-1 in the concentration of trehalose lipids, using Rhodococcus spp. MTCC2574 as a biocatalyst and n-hexadecane as a substrate (Mutalik et al. 2008).
1.6 Downstream Process
The downstream process is probably the major problem to overcome in bioprocesses, as normally the purification of the target biological compound can account for over half of the manufacturing cost in many biotechnology processes (Desai and Banat 1997). In fact, even if production is optimized related to media and culture conditions, the manufacturing process is incomplete without an efficient and economical strategy for the recovery of the bioproducts. For many biotechnological products, the downstream processing costs account for 60% of the total production costs.
In the recovery of biosurfactants several conventional methods have been used over time, like acid precipitation, solvent extraction, crystallization, ammonium sulfate precipitation and centrifugation (Desai and Banat 1997). One of the main problems in these recovery processes is the toxicity of the solvents (e.g. acetone, methanol, chloroform) in nature and which are harmful to the environment. In recent years an alternative to those solvents was the use of methyl tertiary-butyl ether (MTBE), a cheap and less toxic solvent, mainly used in the recovery of biosurfactants produced by Rhodococcus (Kuyukina et al. 2001; Philp et al. 2002).
In the case-study of the trehalose lipid downstream process, although a variety of methods are available, the most commonly used is solvent extraction. In fact, extraction is one of the most used to obtain a crude extract free from the aqueous culture medium. One of the main problems is the number of impurities, often co-extracted during extraction along with several structural types of the target biosurfactant. Afterwards, they need to be evaluated by separating and removing the impurities. There are different mixtures of solvent systems that can be used, the three most frequently used are chloroform-methanol, methyl tert-butyl ether (MTBE), or a mixture of ethyl acetate-methanol (Franzetti et al. 2010). When the goal is the production of trehalose lipids to be applied in the biomedical field, some of the solvent/extraction systems may represent a problem, due to their toxicity.
Other interesting methods have been reported, such as foam fractionation (Davis et al. 2001), ultrafiltration (Sen and Swaminathan 2005), adsorption–desorption on polystyrene resins and ion exchange chromatography and adsorption–desorption on woodbased activated carbon (Dubey et al. 2005). These methods are based on the surface activity of trehalose lipids or their ability to form micelles and/or vesicles and are particularly useful for large-scale continuous recovery of extracellular biosurfactants, with high purity from culture broth. In particular in the pharmaceutical industry, but also in the food and cosmetic industries, individually purified trehalose lipids are required. In these applications the trehalose lipids can be obtained performing multistep recovery by further purification steps. Silica gel column chromatography is a relatively inexpensive method that can be combined with other methods to purify trehalose lipids. Using this technique milligram to kilogram quantities can be obtained free from impurities and can also be used to separate structural types of trehalose lipids for further analysis.
1.7 Identification and Characterization
Glycolipids can be identified and characterized using a broad range of techniques from simple colorimetric assays to sophisticated techniques, like mass spectrometry (MS) and nuclear magnetic resonance (NMR).
The choice of the chemical and structural analysis of glycolipids depend on the purpose. The experimental procedure mainly rely on the following key steps: (i) extraction of glycolipids from the culture medium, (ii) detection; (iii) separation and purification of the crude product (iv) structural analysis.
In the first step colorimetric methods can be used to evaluate the presence of the biosurfactants in either the culture medium or the extract. Detection can be carried out using assays based on the detection of sugar moieties, such as anthrone (Hodge and Hofreiter 1962) or orcinol assay without the need for extraction. The anthrone method is a colorimetric technique based on the reaction of anthrone with the sugar, forming a coloured complex that can be quantified in the visible region, by spectrophotometry. However, interferences from chemicals and carbon sources can