Oleaginous yeasts from Ethiopia

Yeasts are a polyphyletic group of basidiomycetous and ascomycetous fungi with the ability to grow unicellularly. This diverse group of microorganisms represents a part of the microbiota in all natural ecosystems, such as soils, freshwaters and marine waters from the ocean surface to the deep sea (Kutty and Philip 2008). They are widely distributed in the different natural ecosystems and environments. Some yeasts also colonize more extreme environments, such as low temperatures, low oxygen availabilities, and oceanic waters (Butinar et al. 2007). Currently there are more than 2000 recognized yeast species (http://www.mycobank.org). Yeasts grow typically in moist environments where there is an abundant supply of sugar-rich sources. They are common inhabitants of leaf, fruit surfaces and roots as well as various types of food. Few types of yeast have the capacity to degrade polymers like starch and cellulose. Yeasts are found in the atmosphere as well as certain restricted habitats. Some yeasts inhabit both agricultural and forest soil (Sláviková and Vadkertiová 2003). They also occur in the intestinal flora of mammals and some insects (Martini 1992).

In a similar fashion, oleaginous yeasts have been isolated from diverse natural habitats such as various soils, water bodies and flower surfaces (Breuer and Harms 2006; Dai et al. 2007; Pan et al. 2009; Amaretti et al. 2010).

In the current study, most of the screened and identified oleaginous yeasts were isolated from soil (9), flower (4), fruit (3) and leaf (2) (Table 1). So that soil can be a good source for isolation of oleaginous yeasts.

Conventionally the presence of lipids from microorganisms is estimated using Sudan Black B or Sudan III (Thakur et al. 1988) or Nile red (Kimura et al. 2004) staining. These techniques only provide preliminary information and do not allow precise insight into intracellular lipid content of the lipid accumulation ability of the tested microorganism. So that there may be a high proportion of false-positive is results with respect to lipid content (Pan et al. 2009).

After screening the potential oleaginous yeasts, identification and characterization was undertaken using traditional (morphological and physiological) as well as molecular methods. Morphological and physiological characterization test results were then compared with the criteria set by Yarrow (1998), Barnett et al. (2000), Kurtzman et al. (2003, 2011) and lead to the identification of isolated and screened oleaginous yeasts at the genus level. Their identification was confirmed by sequencing both ITS domain and D1/D2 domains of the 26S rRNA gene of the large subunits of each strain.

In the current study, the 18 yeast strains were identified by sequence analysis of both ITS and D1/D2 domain of the large subunit. The tree obtained by analyzing ITS domain is not shown here since the same relationship among species was obtained. However, it has helped in identifying SY52. On the other hand, sequence analysis of D1/D2 the 26S rRNA gene of the large subunit of PY39, SY89 and SY94 indicates that these strains are species of C. curvatus, R. kratochvilovae and R. dairenensis, respectively. On the other hand the remaining oleaginous yeasts strains were identified as R. mucilaginosa (Fig. 2).

Sequence analysis of ITS domain and/or D1/D2 domain of the 26S rRNA gene techniques were applied in identifying yeasts for various purposes. For example, Pan et al. (2009) identified 20 oleaginous yeasts by sequencing the D1/D2 domains of 26S r DNA (rRNA gene). The nucleotide sequences of ITS 1 and 2 regions in the rRNA gene were determined by directly sequencing PCR-amplified fragments for species (17 species and five varieties) in the genus Trichosporon (Sugita et al. 1999). Comparative sequence analysis suggests that six medically relevant species, T. asahii, T. asteroides, T. cutaneum, T. inkin, T. mucoides and T. ovoides. Some other investigators were able differentiate the different species within Saccharomyces genera (Oda et al. 1997; Fernandez-Espinar et al. 2000). Fernandez-Espinar et al. (2000) identified flour yeasts using such method. Yeasts involved in the spoilage of yoghurt were also identified using this method (Caggia et al. 2001). Leaw et al. (2006) also identified medically important yeasts using ITS1 and ITS2 sequencing. They were able to identify 373 strains (86 species). Mohamed et al. (2014) identified three isolates of R. mucilaginosa and three of R. glutinis using ITS.

Biomass production and lipid accumulation of oleaginous microorganisms are affected by the carbon source available during fermentation (Mamatha 2009). For the production of maximum cell biomass and lipid yield in oleaginous yeasts, medium with an excess of carbon source and limited amount of nitrogen sources has a significant influence (Ratledge 2004). In this study, the 18 screened and identified oleaginous yeasts were further tested for their lipid production capacity by growing them in a nitrogen-limited media (Table 3). It was observed that yeasts varied in their lipid accumulating capacities with the maximum exhibited by C. curvatus PY39 (46.51 ± 0.70 %) and minimum exhibited by R. mucilaginosa PY61 (16.99 ± 0.85 % L). Other strains gave intermediate values. According to earlier works, oleaginous microorganisms differ in their ability in accumulating oil. This is the difference in their biochemistry and genetic constitution of their cells (Wynn and Ratledge 2006; Nigam and Singh 2014). The difference can be exhibited not only among different oleaginous species but among strains of the same species.

On other research work, a stain of R. mucilaginosa namely R. mucilaginosa TJY15a which was isolated from surface of marine fish was grown on hydrolysate of cassava starch. The cells of this oleaginous yeast contained 47.9 % w/w oil during batch cultivation, whereas 52.9 % (w/w) of lipid was obtained during the fed-batch cultivation (Li et al. 2010). Enshaeieh et al. (2015) tested lipid producing capacity of another strain of R. mucilaginosa on xylose, grass and leaves hydrolysate as substrates. Under similar conditions, R. mucilaginosa TJY15a accumulated 55.4 % (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/L within 48 h (Zhao et al. 2011). On the other hand, a strain of R. kratochvilovae HIMPA1 was isolated from Himalayan permafrost soil showed that boosted TAG accumulation in the lipid droplets (Patel et al. 2014). Patel et al. (2014) observed enhanced total lipid content and lipid yield of 55.56 %, 8.39 ± 0.57 g/L when the yeast was grown in Hemp seeds aqueous extract. This result was better than 41.92 %, 6.2 ± 0.8 g/L which was obtained when the yeast was grown in industrially used glucose synthetic medium. A biomass of 11 g/L and lipid content of 46 % (w/w) were exhibited by C. curvatus when grown on diluted (25 %) prickly-pear juice for 35 h in batch culture (Hassan et al. 1994). Another strain of this species exhibited a biomass of 69 g/L and lipid content of 48 % (w/w) of intercellular lipid when grown on a crude glycerol in a fed-batch(ThiruM and Rangaswamy 2011).

The results in this research work suggest that much more oleaginous yeasts can be isolated from Ethiopian environment. The 18 yeast strains that we isolated have the potential to be used for the production of single cell oil and hence biodiesel at the industrial level. Traditional identification methods (morphological and physiological) must be complemented with molecular method of identification especially the use of ITS and D1/D2 domains of the 26S rRNA gene sequencing. To improve the production of lipid from oleaginous yeasts, cultivation conditions for lipid production by oleaginous yeasts should be optimized, oleaginous yeasts should be subjected to mutagenesis or genetically engineered to select the best strains with the highest lipid production capacity, and cheap substrates like lignocellulosic wastes and sugar factory by-products have to be used for cultivation purpose to minimize cost of production.