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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).