Screening of oleaginous yeasts and molecular identification
Amongst the heterotrophic microorganisms, oleaginous yeasts are regarded as alternative source for lipid production because of the relatively high biomass yield, fast lipid accumulation, lipid yield and productivity (Vasconcelos, et al., 2018). The most studied oleaginous yeasts species belong to the genera of Rhodotorula, Rhodosporidium, Yarrowia, Candida, Cryptococcus and Lipomyces (Pan et al., 2009). Oleaginous microorganisms store neutral lipids in the form of TAGs and accounting between 20% and 80% of their cell weight (Ratledge, 2004; Lamers, et al., 2016; Ochsenreither, et al., 2016). Candida ethanolica and P. manshurica are normally associated with alcoholic, vinegar fermentation and cocoa bean fermentation and fermentation (or rotting) of plant materials (Maura, et al., 2016; Xing, et al., 2018; Tolieng, et al., 2018). These yeast species have not been studied extensively for their ability to accumulate lipids.
This study demonstrated the lipid producing capabilities of Candida ethanolica and Pichia manshurica isolates. Vincent et al. (2018) isolated and identified oleaginous yeasts from different sources, fruit surfaces, sugarcane juice, sago effluent and agricultural soil. Six species were identified, P. manshurica (1/21), Candida krusei (8/21), C. parapsilosis (1/21), Pichia guilliermondii (2/21), Clavispora lusitaniae (1/21) and K. marxianus (4/21). The limitation of the study was that the growth kinetics, lipid production and lipid profiles of these species were not assessed. The oleaginocity of the yeasts was based on fluorescent microscopy using Sudan IV staining dye under light microscope (Vincent, et al., 2018).
Qualitative detection of lipids
The C. ethanolica and P. manshurica strains were isolated from sugarcane bagasse and selected based on their ability to grow in a high sugar and limited nitrogen medium. This was because the lipid accumulation capacity of oleaginous microorganisms requires limited or depleti nitrogen to redirect excess carbon to lipid synthesis (Pan, et al., 2009). Nutrition is a major factor; the number of lipid bodies inside the cells is reduced at low C/N ratio (Garay et al., 2014). The cells size differences also influence the fluorescence intensity. The larger cells accumulate more lipid bodies, fluoresce becomes intense and lipid bodies appear more golden (Elfeky, et al., 2019). Yeast cells showing high fluorescence intensity are selected for quantitative analysis of neutral lipids by other methods.
Oleaginous yeast regulates their mechanisms to varying degrees depending on the strain in for adaptation under limited nitrogen conditions in order to alter the lipid metabolism and enhance lipid production. The adaptation introduces the modification in the expression of genes or the enzyme machinery involved in the lipid biosynthesis pathway (Patel, et al., 2020). Gravimetric method determines the microbial lipid content by weight of the extracted lipids relative to the weight of the yeast biomass. It is frequently used as reference standard to validate other methods (Chen, et al., 2018; Patel, et al., 2019). Several laboratory methods for extracting lipids are established, however, there is no single method that ensures 100% recovery of intracellular lipids. The recovery efficiency of the lipids from the oleaginous microbial biomass is hindered by the cell wall, which differs between organisms. In order to disrupt the cell wall, mechanical and chemical methods or these methods in combinations are utilized. Such methods including hot-acid hydrolysis, microwave irradiation, sonication, high pressure homogenization, bead beating and swelling by osmotic pressure have been applied to disintegrate cell wall to make the lipids extractable (Geciova, et al., 2002; Patel, et al., 2018). In this study, the hot-acid method was applied to disintegrate the cell wall of the yeasts in order to increase permeability of the extracting solvent, Folch’s method. All the yeast species, viz. C. ethanolica strains and P. manshurica isolated accumulated lipid content of over minimum 20% (w/w), thus making them oleaginous yeasts. The lipid content of P. manshurica strains was between 50%-67% higher than for C. ethanolica strains (28%-46% w/w).
Few studies as indicated below have reported the production of lipids by C. ethanolica and P. manshurica and the reported lipids content are usually less than 20% (w/w) of dry cell weight. The accumulated lipid is found to be dependent on the species type, strain, substrate and cultivation conditions. For instance, cultivation of C. ethanolica AM320 in inulin hydrolysate (fructose and glucose) produced 15% (w/w) of lipid relative to its cell dry weight (Wang, et al., 2018). Arous, et al. (2017) also reported low lipid content by C. ethanolica M1 and P. manshurica with lipid of 12% (w/w) and 15% (w/w), respectively. These lipids levels are lower than the lipid content obtained in this study. A lipid content of 64.8% (w/w) by P. manshurica DMKU-UbC9(2) strain (Polburee, et al., 2015) growing in glycerol medium was comparable to the lipid content of P. manshurica 15-X obtained in this study. The harvesting time at any stage of cultivation also influences the accumulation of lipids. Harvesting at a late cultivation stage correlates with high lipid content (Liu, et al., 2010). Although there is limited literature on the lipid content of C. ethanolica and P. manshurica, other related yeast strains are reported. Pichia etchellsii BM1 accumulated 25% (w/w) of lipid making it oleaginous yeast (Arous, et al., 2017). Pichia manshurica CHC34, Candida parapsilosis CH08 and Pseudozyma parantarctica CHC28 accumulated high lipids in day 1 of cultivation compared to day 5 of cultivation in glucose medium. The lipids content for the yeasts above, following the same order of names in day 1 and 5 were 11.43%, 24.21%, 43.54% and 9.52%, 9.72%, 39.89%, respectively (Areesirisuk, et al., 2015).
Other candida species, C. tropicalis L2 and C. quercitrasa L3 have accumulated very little lipid in glucose medium, 2.29% and 2.17% (w/w), respectively. An exception was for C. tropicalis S1, which has lipid content of 20.95%. However, cultivation of Trichosporon mycotoxinivorans S2 in glucose + xylose medium resulted in lipid content of 44.86% (w/w), (Sagia, et al., 2020). Similar lipid content (44% w/w) is attained by Rhodosporidium TJUWZ4 cultured in glucose medium (Wang, et al., 2017). Trichosporon sp. (RW) growing in glucose medium yielded 35.98% (w/w), (Brar et al., 2017). In cases of low lipid content, the extraction efficiency of the lipid can be increased by optimized microbial cell wall pretreatment. The choice of microbial cell wall pretreatment technique differs with the type of microorganisms studied because of their cell wall composition, which limit extractability of lipids (Patel, et al., 2019). The extraction of lipids from acid-treated cells with hexane showed higher lipid content of 42% (w/w) and 40% (w/w) for Rhodotorula toruloides and Lipomyces starkeyi, respectively. Similar results were obtained using Folch’s method (Bonturi, et al., 2015). Acid-treatment of L. starkeyi led to higher lipid recovery yield (Kruger, et al., 2018).
The SPV requires cell disruption and lipid extraction. This assay has advantages over Folch extraction and gravimetric quantification. The gravimetric estimation of lipids may erroneously include lipophilic proteins and pigments. Therefore, during the acid-thermal reaction (treatment) of the SPV assay the lipophilic proteins and other products are degraded (Patel, et al., 2019). Candida strains grew better in both media than the Pichia strains. Candida ethanolica E2 and C. ethanolica 6-XP attained the highest biomass of 8.48 g/L and 8.9 g/L, respectively. Biomass and lipids are known to be depended on the type of the medium and conditions used. Guo et al. (2019) cultivated wild type Rhodosporidium toruloides RC 2.1389 and R. toruloides R-ZL2 and R-ZY13 UV mutants were cultured in FM medium. The maximum amount of lipid produced by the wild type yeast RC was 1.56 g/L after 96 h of cultivation. The mutants R-ZL2 and R-ZY13 produced 2.24 and 2.15 g/L. The lipids productivity was low for wild type yeast, 16.29 ± 1.6 mg/L/h. The mutants exhibited high productivity of 23.32 ± 1.8 and 22.40 ± 2.5 mg/L/h for R-ZL2 and R-ZY13 mutants, respectively (Guo et al., 2019). In shake flask cultivation, wild type Lipomyces starkeyi (WT) and UV mutants L. starkeyi A1 and A3 were cultivated in a medium containing 30% glucose and 70% xylose for 168 h. The mutants A1 showed slight improvement in biomass (13.74 g/L) and lipid content (39.60% ± 1.3), whilst mutant A3 showed improvement in lipid content (38.11% ± 1.2) when compared to biomass 12.32 g/L and lipid content 35.02 ± 1.59 for wild type yeast (WT), (Tapia et al., 2012). In fed-batch both the biomass and lipid content increased in all the L. starkeyi yeasts. After 144 h of cultivation, mutant A1 showed higher biomass 88.7 g/L and lipid content of 55% compared to L. starkeyi (WT) with biomass of 76 g/L and lipid content of 43.8%. The lipid productivity for mutant A1 was 0.34 g/L/h higher than 0.26 g/L/h for wild-type (Tapia, et al., 2012).
Carbohydrates and proteins
Yeast responds to stress by accumulating either carbohydrates or lipids. Oleaginous yeast strains produce lipids as a protective mechanism under the stressed conditions, e.g. depleted nitrogen (Shi, et al., 2013). The microbial response in nutrient limitations, particularly nitrogen or phosphorus requires robust metabolism in order to attain maximum cell growth (biomass). In nitrogen limitation, cellular carbohydrates (glycogen and trehalose) increases as well as increasing lipid content while protein and RNA content are reduced (Yu, et al., 2020). Yu et al. (2020) showed that a reduction in mRNA prompted the cells to increase gene-specific translation efficiency of genes (~ 74%) and this could be actively regulated or arising naturally as a result of low ribosomes in the cells.
Our results show that P. manshurica strains 11-XP and 15-X accumulated higher protein content of 42–50%, which corresponded with high lipids (w/w) of the strains and less carbohydrates. Both the P. manshurica 11-XP and C. ethanolica C2 strains revealed higher carbohydrates content of 35% and 29%, respectively. Most microorganisms studied under nutrient stress, such as limited nitrogen the accumulation of lipids was directly proportional to the accumulation of carbohydrates. The increase in lipids was accompanied by increase in carbohydrates contents. Kumar et al. (2017) studied oleaginous yeast Pichia guillierrmondii that was cultivate in different carbon sources (20 g/L); molasses, crude glycerol, distillery waste water and corn steep liquor. The accumulated carbohydrates (35%.8–44.2%) were higher than the protein content (12.4% – 24.6%) in P. guilliermondii. It was found that the type of carbon substrate influenced the levels of the accumulated carbohydrates and proteins (Kumar, et al., 2017). In Cyanobacteria, Arthrospira sp. PCC 8005 limited-nitrogen increased the carbohydrates content (from 14%-74%) in the biomass and decreased protein content from 37% – 10%. Transcriptomic and proteomic analysis indicated that de novo protein synthesis was down-regulated in nitrogen limited culture. Instead, the degraded proteins were partially converted into carbohydrates through gluconeogenesis (Depraetere, et al., 2015).
Our results show that the protein contents are higher than the carbohydrates, suggesting the existence of continuous synthesis and accumulation of protein in those yeasts at the late stage of cultivation (168 h) in limited or depleted nitrogen medium. The recycled proteins or amino acids resulting from cell death of other yeasts, also called autophage was necessary for maintaining cell viability. For instance, Lipomyces starkeyi AS2.1560 in response to limited-nitrogen condition after 96 h of cultivation activated protein degradation process and amino acid biosynthesis to salvage and redistribute nitrogen sources for suboptimal cell growth (Liu, et al. 2010). The requirement for autophagy during starvation may be due to the need to recycle biological polymers (proteins, nucleic acids, carbohydrates, lipid bilayers, etc.) into building blocks for reuse under conditions where they may not be available outside the cell (Abeliovich and Klionsky 2001). Tchakouteu et al. (2014) studied interaction between the synthesis of intracellular total carbohydrates and cellular lipids in Cryptococcus curvatus under nitrogen-limited and nitrogen-excess condition with lactose and sucrose as carbon source. The strain accumulated high quantity of intracellular total sugars (up to 68% w/w) at the early stage of fermentation when nitrogen availability was sufficient and at the end of fermentation when nitrogen is depleted the intracellular total sugar decreased to 20%. In excess nitrogen, the intracellular total sugar remains high (Tchakouteu, et al., 2014). Microbial (including algal) proteins are referred to as single cell proteins (SCP) if the microorganism contains 30% or more of proteins (Glencross, et al., 2020; Lapeña, et al., 2020). Yeast is widely accepted for the production of SCP because of the protein nutritional quality and acceptability among the consumers (Lapeña, et al., 2020). Both SCO and SCP have biotechnological applications in food and animal feed diets as sources of amino acids, omega-3- lipids and bioactive molecules (Glencross, et al 2020). Co-production of SCO together with either carbohydrates or proteins will make the process of lipid production cost-effective and sustainable, particularly if waste carbon substrate is used.
The conversation of fatty acids to fatty acid methyl esters
Fatty acids composition is usually influenced by the yeast strain type and the carbon source utilized (Gientka, et al., 2017). The fatty acids produced by all the C. ethanolica strains and P. manshurica strains are dominated by SFA, followed by MUFA and PUFA. Then again, P. manshirica 15-X produced abundant amount of palmitic, stearic, oleic and linoleic acids making up 82% of the total lipids. This composition was similar to fatty acids produced by Cryptococcus curvatus DSM 70022 and the fatty acids are comparable to those of plant or vegetable oils (Annamalai, et al., 2018). Other oleaginous yeasts such as Trichosporon sp. (RW), Thodotorula glutins strains, Rhodotorula babjevae strains, Lipomyces starkeyi strains and Lipomyces lipofer strains had their lipids dominated by plamitic, stearic, oleic and linoleic acids (Shapaval, et al., 2020; Brar, et al., 2017). Trichosporon mycotoxinivorans S2’s major fatty acids were palmitic, oleic and stearic (Sagia et al., 2010). The fatty acids for Rhodosporidium TJUWZ4 were oleic, palmitic and linoleic acids (Wang, et al., 2017). This confirms that specific yeast strains will produce lipids of different fatty acids compared to the others. Polyunsaturated fatty acids for all yeast strains were dominated by linoleic acid (C18:2n6c), constituted up to 20% of the total fatty acids. In order to improve this content, Kolouchova, et al. (2016) found that under limited nitrogen using (NH4)2SO4 as nitrogen source the unsaturated fatty acids increases with cultivation time. The level of linoleic acid for Candida sp., Rhodotorula glutinis, Yarrowia lipolytica and Trichosporum cutancum was between 15.6% and 53.4%.