Characterization of Ethanol Producing Yeasts for Their Efficiency in Ethanol production, Salt Tolerance, and Utilization of Glucose and Xylose

Yeasts are the mainstay in ethanol production industry. Search for efficient yeast strains that are salt tolerant and utilize both hexoses (glucose) and pentoses (xylose and arabinose) is important in fermentation industry. In this regard,12 yeast strains, viz., CDBT1-12, were isolated from various sources and characterized. Molecular characterization of the yeast strains was done by sequencing 26S rRNA gene, D1D2 region. Out of 12 isolates, 10 were found to be Saccharomyces cerevisiae , CDBT7 was Wikerhamomyces anomalous , and CDBT8 was Cyberlindnera fabianii . The yeast isolates were characterized in terms of their ethanol production efficiency, salt tolerance and ability to utilize of glucose and xylose. All the strains were found to be good ethanol producers. Yeast strain CDBT2 was found to have tolerance for high salt (up to 15%) and ethanol (up to 16%) concentrations. Yeast strain CDBT7 was found to utilize both glucose and xylose without compromising on ethanol production efficiency. The CDBT7 strain was also salt tolerant (up to15%).Yeast strain CDBT2 when grown in an electrochemical cell with low levels of applied external voltage, alcohol dehydrogenase (ADH1) and pyruvate decarboxylase (PDC1) mRNA levels were increased by 2.78 ± 0.80 and 1.12 ± 0.37 fold, respectively. We believe, the latter observation is novel and it has not been reported previously. It also further supports our previous observation of increase in level of alcohol production by CDBT2 strain in the presence of applied electrical current. Blastn


Introduction
Yeast strains are the common dwellers of most of nutrient rich media/sources such as fruits, tree bark, soils, etc., [Alfenore et al, 2002]. They form one of the important class of microorganisms that are more complex than bacteria. Unlike other fungi, yeasts are ovoid single cells that are about 8 µm long and 5 µm in diameter. Their doubling times are 1-3hunder optimal growing conditions [Morris et al, 1992]. According to published news reports the global market for yeast products has reached nearly $7.6 billion in 2017and it is increasing rapidly and expected to grow to nearly $10.7 billion by 2022 [Globe news wire, 2018]. Yeasts are used for baking, brewing and various other industrial applications. Such applications include manufacturing of shoyu, miso and production of various fermentation products such as enzymes, vitamins, capsular polysaccharides, carotenoids, polyhydric alcohols, lipids, glycolipids, citric acid, etc [Turker, 2014] and as eukaryotic system to produce novel compounds. Given the importance of the yeasts and yeast by products described above, extensive research has been undertaken to identify, catalog and preserve yeast strains worldwide [James et al, 1995].
The process of identification of yeasts involves sequence analysis of ribosomal RNA genes that are conserved. The ribosomal RNA genes coding for both 18 s and 26 s RNA have been extensively analyzed and the analysis has proven that it is not only important in establishing them as useful molecular markers for studying evolutionary relationships between organisms but also useful tools for molecular characterization of yeasts [Ciardo et al, 2006]. Early studies related to characterization of yeasts and their classifications have shown a widespread pattern of disparity between phenotypes and genotypes. For the purpose of clarity and to systematically classify yeasts, analysis of genes coding for 18S rRNA [James et al, 1995], internal transcribed spacer (ITS) of18s rRNA [Ciardo et al, 2006] and the DNA sequences for domains 1 and 2 (D1/D2) of 26 s rRNA [Kurtzman et al, 2015] have proven to be optimal.
Many efforts have been made to isolate and characterize yeasts from various climates of Nepal with applications in baking and brewing [Karki et al, 2017], however their molecular characterization and systematic evaluation of their application, especially, in brewing is lacking. An important parameter in selecting brewing yeast is its tolerance to salt and ethanol [Kodama et al, 2013], because these are known to damage, the lipid layers and thus destabilize the cultures [Stanley et al, 2013]. Our laboratory has been working towards characterizing various yeast isolates from Nepal, and assesses their ability to tolerate salt and ethanol, and the effect of yeast to electric voltage supply especially on ethanol production [Joshi et al, 2019]. Further, we are also interested in isolating yeasts that utilize both glucose and xylose for alcohol fermentation from xylose containing substrate like lignocellulosic biomass. In the present study, 12 yeast isolates collected from various sources have been characterized by (i) nucleotide sequencing of domains 1 and 2 (D1/D2) of 26 s rRNA genes, (ii) their ability to tolerate salt and ethanol as well as utilization of glucose and xylose as substrates for ethanol production, and (iii) effect of voltage supply on alcohol dehydrogenase and pyruvate decarboxylase expression.

Materials And Methods Sample collection
Samples were collected from different yeast sources available in Kathmandu valley, Nepal as shown in Table 1. All the samples were collected during the months of September and October, and the samples were placed in sterile zip lock bags and stored at 4 o C until further analysis. Table 1 Substrates from which the yeast strains described herein were isolated.

Studies on Glucose and Xylose Utilization and Ethanol Production
All of the isolated yeasts were cultured separately in PYN media supplemented with glucose or xylose as a carbohydrate source. The growth of yeast was observed by measuring absorbance at 600 nm (turbidity changes) as described by Sherman (2002). Successively, ethanol production was also measured using the protocol of Seo and associates [Seo et al, 2009]. The culture broth was centrifuged at 10000 x g for 15 min. One mL of the supernatant was added to1 mL tri-n-butyl phosphate (TBP). The mixture was vortexed for 15 min. Finally, the vortexed mixture was centrifuged at 10000 x g for 15 min to separate layers. About 750 µL of upper layer was transferred to another tube and mixed with equal volume of acidified 5% potassium dichromate reagent. The process of vortexing and centrifuging was repeated. Then the lower layer was pooled and absorbance was measured at 595 nm using spectrophotometer.

Study of Salt and Ethanol Tolerance by Yeast Isolates
All the isolated yeasts were cultured separately in PYN media supplemented with 0-22% sodium chloride or ethanol respectively and allowed to grow at pH 4.5 and temperature 28 o C for 96 h [Balakumar and Arasaratnam, 2012]. Microbial growth patterns were observed spectrophotometrically for changes in turbidity (Thermo-Scientific, USA) at 600 nm against medium blank [Sherman, 2002].   Table 2.

Isolation of yeasts and molecular characterization Morphological Study
From the eight different substrates (Table 1) tested, 12 different yeast colonies (CDBT1 to CDBT12) were isolated ( Table 3). The isolated yeast were white or creamy colonies with variability in consistency and texture as described by Cletus and associates [Cletus et al., 2011]. All isolates have cottony or rubbery like appearance (Fig. 1, Table 3). All the yeast were multiplied by budding and were good ethanol producer (Table 4). CDBT7 and CDBT8, in addition to glucose, were also found to utilize xylose. CDBT2, CDBT3, CDBT7 and CDBT11 were found to tolerate high salt concentrations (15%). All the yeast strains showed normal growth in the presence of ethanol up to 4%, except CDBT8 that can only tolerate 2% ethanol (Fig. 2). CDBT2 was found to grow normally in the presence of 6% ethanol. Almost all yeast strains were found to grow in media with 14% ethanol, with the exception of CDBT2 in which can resist up to 16% ethanol in the medium. Overall, from biochemical characterization, CDBT2 and CDBT7 were found to be potent strain for ethanol production as they are high salt and ethanol tolerant, can produce ethanol from glucose and xylose as well. The 680 bp amplified 26S rDNA products were confirmed by electrophoresis in 1.0% agarose gel ( Figure.3). The sequences were edited by BioEdit software [Hall, 1999] and analyzed by NCBI blast.
Out of twelve yeasts, ten of them were Saccharomyces cerevisiae and CDBT7 and CDBT8 were Wikerhamomyces anomalous and Cyberlindnera fabianii respectively (Table 3). A phylogenetic tree was developed to see the relatedness between the yeasts (Fig. 4)

Alcohol Dehydrogenase and Pyruvate Decarboxylase Expression Analysis in Normal and Electrochemically Enhanced Fermentation by RT-PCR
Previously, we have demonstrated enhancement of ethanol production in CDBT2 cultures supplied with low levels of applied electrical current. The rationale for the increased levels of ethanol could be over expression of key alcohol fermentation genes, viz., ADH1 and PDC1. Accordingly, total RNA was isolated from CDBT2 strain cultured under normal conditions and in an electrochemical cell under applied current. The cDNA was prepared from isolated RNA. Both the RNA and cDNA preparations were confirmed by running in 1% agarose gel (Fig. 5). Real time qPCR was used to quantify the relative expression of genes Pyruvate Decarboxylase (PDC1) and Alcohol Dehydrogenase (ADH1) in normal and electrochemically enhanced yeasts.
Gene expression was analyzed taking same amount of template for both reference/ housekeeping gene TFC1 and test genes PDC1 and ADH1. Relative expression of PDC1 and ADH1 was then calculated comparing the expression of TFC1 gene as reference gene. Gene expression in CDBT2 cultured under normal growth conditions was used as control and CDBT2 cultured in an electrochemical cell under applied electricity was used as test sample. The C t data obtained in Table 5 clearly revealed high expression of both the genes than in normal condition. When the obtained C t data were used to calculate the relative expression of ADH1 and PDC1 genes using the protocol given by Yuan et al. (2006), ADH1 and PDC1 genes were found to express 2.78 ± 0.80 and 1.12 ± 0.37 fold more than normal fermentation indicating that external voltage supply during growth of yeast enhanced enzyme expression.  Additionally, like CDBT7, W. anomalous strain isolated from sugar beet thick juice was found to have a comparable ethanol yield, but needed longer fermentation time and can utilize xylose [Ruyters et al, 2015]. CDBT2 is found to be potent yeast strain for ethanol production using glucose as substrate. It is also tolerant to high salt and ethanol concentrations. Yeast strain CDBT 7 is also tolerant to high salt and ethanol concentrations; however, it can utilize both glucose and xylose to produce ethanol.
Selection of ethanol tolerance strain is must when the yeast is used for industrial production of ethanol [Ekunsanmi and Odunfa, 1990 Figure 1 Yeast isolates grown onYMA media -colony morphologies of representative yeast isolates.   Phylogenetic tree based on sequences of the D1/D2 region of the rDNA 26S gene. The tree shows the position of CDBT isolates to be closely related yeast species. The tree was constructed based on the genetic distances obtained according to MEGA6 using the neighbor-joining method.