In the present study, we examined the effect of the culture conditions, two different media, and supplementation of the media on the level of free L-carnitine in the biomass of Y. lipolytica strains. The results confirm other findings [7, 22, 35–38] revealing that the fermentation process parameters can have a significant impact on improvement of the nutrient content in the biomass of the studied yeast strains. However, we showed that the concentration of free L-carnitine in the yeast biomass depended primarily on the medium used, and, to some extent, on the strains and culture conditions.
The first medium used was biofuel waste (the SK medium) with high contents of fatty acids. It is known that Y. lipolytica growing in fatty substrates is able to accumulate and store lipids [18, 24, 37, 39–40]. Since extracellular carbon sources were depleted, the yeast utilized its own storage lipids (body lipids) as a carbon and energy source, increasing the production of proteins [19, 20, 41, 42]. Interestingly, a knockout of the sextuple POX genes in Y. lipolytica causes inability of this yeast to degrade storage lipids, leading to over-accumulation of fats in yeast cells [15, 18]. Therefore, the biosynthesis of cellular proteins or polysaccharides and the fat-free biomass production are competitive to lipid accumulation [42, 43]. We found previously that both Y. lipolytica strains (ATCC 9793 and A-101) utilized biofuel waste (the SK medium) to produce biomass with a high concentration of protein and amino acids, especially the industrial A-101 strain [21, 35]. In the present study of both Y. lipolytica strains grown in the biofuel waste at a temperature range from 20°C to 30°C and different pH values (from 4.0 to 7.0), we did not notice a significant influence in the L-carnitine concentrations. The level of L-carnitine was below 1 mg/100 g of wet biomass in all fermentation samples (data not shown). We can hypothesize that, irrespective of the culture conditions, both Y. lipolytica strains used the entire pool of endogenous free L-carnitine to utilize fatty acids from biofuel waste to grow and produce protein-enriched biomass. It is worth emphasizing that carnitine can also be used as a sole nitrogen source, most commonly through the glycine betaine pathway, where glycine conversion to serine is followed by deamination to form pyruvate and ammonia . It was proved that Y. lipolytica grown in biofuel waste was able to produce all amino acids [21, 35]. However, it should be added that the reference Y. lipolytica ATCC 9793 strain did not grow at low pH (4.0 or 5.0) in the biofuel waste, in contrast to the growth in the standard YPD medium. In turn, the Y. lipolytica A-101 strain was able to grow at low pH (4.0 or 5.0) in the biofuel waste. Our results coincide with those reported by Swigers et al. , who found that L-carnitine was strictly required for yeast growth on non-fermentable carbon sources such as acetate, ethanol, fatty acids, and glycerol, which do not contain carnitine. Mutants with deletion of all three CAT genes (Δcit2 strain) were unable to grow in media containing these carbon sources. However, supplementation of yeast extract, which contains a sufficient amount of carnitine, or free L-carnitine into these media, caused growth of the Δcit2 strain due to absorption of carnitine from the environment .
Another study  showed that another oleaginous yeast C. albicans strain, with deletion of all four genes determining the L-carnitine synthesis pathway, was unable to grow on fatty acids and to utilize either acetate or ethanol as carbon sources. In turn, a transfer of the gene encoding acetyl-CoA oxidase from Y. lipolytica to S. cerevisiae enabled S. cerevisiae to grow on fatty acid-rich feedstock . The possibility of biofuel waste utilization as a substrate by Y. lipolytica mainly depended on the strains and culture conditions. Our previous studies revealed that the temperature of 30°C and pH 5.0 were more suitable for production of SPC, amino acids, and B-group vitamins by Y. lipolytica strains cultivated in both YPD and SK media (biofuel waste) than at standard industrial conditions (30°C, pH 6.0) [21, 22, 26, 35]. Noteworthy, the culture parameters (i.e. temperature and pH) also strongly affect lipase activities. The maximum activity of lipases produced by Y. lipolytica is noted at a temperature between 30°C and 40°C and pH 5.0 . Moreover, these culture conditions significantly influence Y. lipolytica lipid accumulation during the primary anabolic growth when cultivated on fatty substrates [36–38].
The standard laboratory YPD medium, used in this study as supplementary, contains yeast extract with only 0.10%-0.15% of fatty acids. Yeast extract is mainly added to the medium as a nitrogen source; however, it also contains several biocomponents, including sufficient amounts of carnitine, to complement the carnitine requirements of yeast [4, 7]. It was reported that the effect of yeast extract on L-carnitine biosynthesis was dissimilar among tested fungal strains. Yeast extract had a slight effect on the increasing in the L-carnitine concentration in Aspergillus oryzae and Rhizopus oryzae biomasses but did not influence L-carnitine production in Neurospora intermedia. Moreover, it was found that, as long as glucose is present in the medium, it serves as the primary energy and carbon source, preventing L-carnitine consumption. However, after glucose exhaustion, although the biomass weight kept increasing, the concentration of free L-carnitine in the biomass started to decrease. Therefore, the prolongation of the cultivation time caused the fungus to consume glucose entirely and then L-carnitine was utilized as a carbon source . In our study, endogenous free L-carnitine in both tested Y. lipolytica strains was detected when they were cultivated in YPD medium. The highest levels of L-carnitine in this medium in the industrial Y. lipolytica A-101 biomass were obtained when it was grown at a temperature of 30°C and pH 7.0, although promising results were also obtained at pH 5.0 (Fig. 1A). The reference ATCC 9793 strain cultivated in YPD at a temperature of 20°C and pH 6.0 contained two times more L-carnitine than the industrial A-101 strain (Fig. 1B). However, a similar amount of L-carnitine in the ATCC 9793 biomass was obtained when the strain was cultivated at the temperature of 30°C and pH 5.0 (Fig. 1A). We showed previously that these conditions were optimal to obtain protein-enriched biomass of Y. lipolytica cultured in biofuel waste [21, 35]. Thus, the ability to produce nutritional elements is not a static property, and it can be considerably affected by fermentation process parameters. However, as shown in Fig. 2, reference Y. lipolytica ATCC 9793 grown in YPD medium, irrespective of the culture parameters, proved to be a better producer of free L-carnitine than another investigated strain.
In the presented work, the effect of precursors for L-carnitine biosynthesis was observed as well (Fig. 2B). Strijbis et al. [3, 9] reported that trimethyllysine (TML) is a main precursor to synthesize carnitine. TML is a component of the first enzyme of the carnitine biosynthesis pathway, namely TML dioxygenase (TMLD), which requires the presence of ascorbate (vitamin C) and iron(II) for its enzymatic activity. The results confirmed that the addition of L-carnitine precursors increased the growth and quantity of yeast biomass growing in the fat-acid-rich medium, i.e. biofuel waste. This confirms the previous reports that the growth of such oleaginous yeasts as C. albicans, Y. lipolytica, or engineered S. cerevisiae strains on non-fermentable fat-rich carbon sources is possible only in the presence of L-carnitine biosynthesis intermediates [3, 6, 12]. In turn, in wild S. cerevisiae, peroxisomal membranes are impermeable to acetyl-CoA, when the yeast is cultivated on fatty acids as carbon source. Therefore, wild S. cerevisiae are not able to grow in fat-rich waste substrates  in contrast to Y. lipolytica. In this respect, production of nutritional yeast biomass by oleaginous species on available inexpensive wastes used as carbon and energy sources (e.g. biofuel waste) is desired by industry in the broad sense.
We also observed a stimulatory effect of chromium on the free L-carnitine production. Trivalent chromium, an essential trace element, as diet components improving glucose uptake and fat metabolism was reported . Moreover, the deficiency of trivalent Cr may induce symptoms comparable to those associated with diabetes in mammals . Our experimental results showed that the introduction of water-soluble chromium (Cr (III)) salt as a component of biofuel waste as the culture medium for Y. lipolytica resulted in production of a slight amount of free L-carnitine by the yeast. This implies that Cr supported L-carnitine metabolism in some way in the yeast cells. However, the issue of the chemical dependencies between Cr and L-carnitine in yeast cells needs further investigations. Additionally, we supplemented zinc and selenium into biofuel waste in the pilot plant scale to obtain zinc- or selenium-enriched yeast biomass, but we did not observe a significant increase in the L-carnitine concentrations.