selection of high ethanol-producing saccharomyces...

Philippine Journal of Science138 (1): 37-48, June 2009ISSN 0031 - 7683

Key Words: Cluster analysis, DNA fingerprinting, ethanol, polymerase chain reaction, Saccharomyces cerevisiae

*Corresponding author: [email protected]

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Selection of High Ethanol-Producing Saccharomyces cerevisiae Strains, their Fermentation Properties,

and Genetic Differentiation Based on rep-PCRand mt rDNA-PCR

Irene G. Pajares1, Francisco B. Elegado*1, Jose Paolo V. Magbanua1, and Asuncion K. Raymundo2

1National Institute of Molecular Biology and Biotechnology2Institute of Biological Sciences, College of Arts and Sciences

University of the Philippines Los Baños, College Laguna

Ten out of 37 Saccharomyces isolates statistically gave higher ethanol yields after 24 hours using 20% (total sugars) unsterile molasses medium. Saccharomyces cerevisiae 2025 and 2023 produced the highest ethanol concentrations of 8.7 % and 7.5% (v/v), respectively. In sterile molasses medium, ethanol production decreased with these two isolates, but increased with S. cerevisiae 2013, 2012, 2008, 2010 and 2014, with ethanol concentrations of 9.17, 8.88, 8.84, 8.44 and 8.31% (v/v), respectively. After 3 days incubation, all ten isolates survived at 40° C but only S. cerevisiae 2031 tolerated up to 15% (v/v) exogenous ethanol. S. cerevisiae 2010 and 2014 were resistant to the toxin produced by killer strain S. cerevisiae K1 (ATCC 60728). Combining the various test parameters, S. cerevisiae 2008, 2010 and 2014 would be ideal for industrial ethanol production and thus fingerprinted. Amplification of the mitochondrial small subunit ribosomal DNA (mtSSU rDNA) by polymerase chain reaction (PCR) detected polymorphisms among the S. cerevisiae strains. Cluster analysis using UPGMA-SAHN was able to group the high from the low ethanol producers but other properties such as ethanol and temperature tolerance were not distinctly separated. Repetitive sequence-based PCR analysis of the ten selected isolates using REP primers (rep-PCR) also clustered the three best S. cerevisiae isolates.

INTRODUCTIONRapid increase in the pump prices of fossil oil products like gasoline and the growing concerns on climate change, partly brought about by excessive carbon dioxide emission of motor vehicles fueled by fossil oils, have led to increased worldwide interests on the utilization of renewable fuels like ethanol. Fuel ethanol is used as additive to gasoline and as replacement for octane enhancers (such as benzene, butadiene and lead)

that are known as noxious to the environment. Studies show that ethanol has a higher octane rating than gasoline and existing engines need not be modified at 10% ethanol blend. Research on ethanol fermentation, including the search for efficient Saccharomyces cerevisiae strains and other strains of closely related species, has been continuing for years in order to lower down the cost of production. Desired yeast strains have the special property of possessing particularly efficient aerobic and anaerobic metabolic capabilities, making them high ethanol producers. They could also

Pajares et al.: Genetic Characterization of Bioethanol Yeasts

Philippine Journal of ScienceVol. 138 No. 1, June 2009

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possess other industrially-important properties such as ethanol tolerance, thermotolerance and resistance to killer yeasts.

Strain differentiation is in many cases more important than species classification, especially in the advent of patents protecting new hybrids (Rainieri et al. 2003; Stewart 1981). Traditional methods for distinguishing strains of yeasts have relied on biochemical, morphological and physiological criteria (Barnett et al. 2000). However, these methods are frequently affected by culture conditions (Azumi and Goto-Yamamoto 2001) and instability of physiological properties (Ryu et al. 1998). Molecular methods involving the use of polymerase chain reaction (PCR) analysis of genomic, karyotypic and mtDNA polymorphisms have been described to resolve genetic variation between S. cerevisiae strains (Nadal et al. 1996). Another technique is repetitive-sequence based polymerase chain reaction (rep-PCR), which is based on the amplification of DNA sequences located between specific interspersed repeated sequences in prokaryotic genomes (George et al. 1998). DNA primers corresponding to repetitive extragenic sequences, such as repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and BOX element (BOX1A sequences), have been used to generate complex fingerprint patterns. These highly conserved and repeated DNA sequences constitute an important fraction of prokaryotic and eukaryotic genomes and have been used to distinguish and classify even closely related bacterial and fungal strains (Raymundo et al. 2005; Raymundo et al. 1999, McManus and Jones 1995). A recent study on the species identification and genetic diversity analysis of Philippine yeast isolates for wine fermentation was also done using PCR with Randomly Amplified Polymorphic DNA (RAPD) primers (Lim et al. 2006).

Researchers from the National Institute of Molecular Biology and Biotechnology (BIOTECH) at University of the Philippines Los Baños (UPLB), College, Laguna, Philippines, have collected yeast isolates from all over the country and developed them for use in the local alcohol industry (BIOTECH-UPLB, Biofuels Program Annual Report 1988). One of these local strains, S. cerevisiae HBY3 (2030) is still being used by some local distilleries for the fermentation of ethanol from molasses. Strains that have shown better performance than HBY3 under laboratory conditions have been identified and evaluated for other fermentation properties. Molecular markers of these yeast isolates would be useful in the breeding and strain improvement experiments and would prove significant in quality monitoring during alcoholic fermentations. This study was conducted: 1) to select for high ethanol-producing yeast isolates with other desirable

fermentation properties such as tolerance in 12.5 and 15% (v/v) exogenous ethanol, thermotolerance, and resistance to the killer factor produced by S. cerevisiae K1 strain; and 2) to identify the molecular markers of these high ethanol-producing yeast isolates based on the generated banding patterns from rep-PCR, and analysis of their mitochondrial DNA.

MATERIALS AND METHODS

Characterization of Fermentation ParametersYeast Isolates and Culture Media. A total of 37 yeasts, including 32 local isolates, 2 type strains from the American Type Culture Collection (ATCC), 2 reference strains and a killer strain from the National Research Institute of Brewing (NRIB), Japan were used in this study (Table 1). They were obtained from the Philippine National Collection of Microorganisms (PNCM), and Culture Collection of the Environmental Biotechnology Program, BIOTECH, UPLB. All strains were grown and maintained on Yeast Extract Peptone Dextrose (YEPD) broth (1 %yeast extract, 2 % bactopeptone, 2 % dextrose) or YEPD agar (added with 2 % agar), respectively.

Ethanol Production in Batch Culture. Pure cultures of the yeast isolates were transferred and grown in Malt Yeast Extract Glucose Peptone (MYGP) agar slant (0.3% malt extract agar, 0.3% yeast extract, 1% glucose, 0.5% bactopeptone, and 1.5% agar) at ambient condition (~30° C) for 24 h. Then, they were inoculated into 50 mL of sterile molasses yeast build-up medium consisting of (per 100 mL): 5% molasses (equivalent total sugar), 0.14 g K2HPO4, 0.025 g MgSO4, and 0.1 g (NH4)2SO4 at pH of 4.5, and incubated with shaking at 30° C for 24 h to prepare the starter culture (Del Rosario 1987). Ten milliliter of the starter culture were transferred to 90 mL of the molasses culture medium, containing similar nutrients and having the same pH as above but with 20% equivalent total sugar (either sterile or non-sterile) and incubated at conditions similar as above. Ethanol production was analyzed using gas chromatography. The top ten producers of ethanol were selected for further evaluation of other fermentation parameters.

Ethanol Tolerance Test. The effect of ethanol on the growth of the different yeast isolates was evaluated at 12.5 and 15 % (v/v) ethanol concentrations following the method of Ogawa et al. (2000) with some modifications. A loopful of the yeast cultures was inoculated in YEPD broth incubated without shaking at 30° C for 24 h and an aliquot of the resulting culture was diluted at 1:10 with sterile distilled water. One mL of the diluted cell suspension was centrifuged at 3000 X g for 5 min. The



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supernatant was discarded and the pellet was washed once with sterile water and resuspended in 5 mL ethanol medium containing per 100 mL: 1.0 g glucose, 12.5% or 15 % (v/v) ethanol in acetate buffer (pH 4.2) (Ogawa et al. 2000). The cultures were incubated at 30° C and samples were taken every 24 h for 3 days. Viability was determined by plating on YEPD agar after each sampling up to 10-8 dilution. Ethanol tolerance was evaluated based on viability at different exposure time and ethanol concentration.

Thermotolerance Test. The yeast cultures were prepared as in above and incubated at different temperatures: 35° C, 40° C, 45° C and 50° C. Aliquots of the resulting cultures were serially diluted and plated on YEPD agar plate every 24 h for 3 days to determine viability at the different temperatures (Ogawa et al. 2000).

Killer Factor Sensitivity Test. Killer factor resistance was evaluated using the killer yeast strain S. cerevisiae K1 (ATCC 60728) based on the reduction of methylene blue dye described by Adams et al. (1997) with some modifications. A suspension of the killer strain was spread-plated onto the low-pH blue plate (YEPD agar + methylene blue + phosphate citrate buffer). Test strains were streaked on the plates containing the lawn of the killer yeast strain and incubated at 20° C for 3-4 days. Cells affected by the killer toxin appeared as blue streak over a lawn of killer strain and resistant strains appeared as white streak. Slightly resistant strains were surrounded by blue colonies.

Statistical Analysis. Alcohol production of 37 yeast isolates in 20% (equivalent total sugars) unsterile molasses-based media were subjected to ANOVA and the means of triplicate values were compared using DMRT (Gomez and Gomez 1984). Similarly, the fermentation efficiencies of the 10 selected strains were also subjected to ANOVA and DMRT.

DNA Fingerprinting Extraction and Preparation. Yeast DNA was isolated following the method of Adams et al. (1997) with some modifications. Yeast cells were grown for 48 h at 30º C in 5 mL YEPD broth. The cells were collected by centrifugation at 6000 X g for 2 min in a 1.5-mL microfuge tube and the supernatant was discarded. The cells were resuspended in 500 µL of 1 M sorbitol, 0.1 M Na2EDTA (pH 7.5), 20 µL of 2.5 mg/mL zymolyase 100,000 solution and incubated at 37º C with shaking for 2 h. The mixture was centrifuged at 6000 X g for 2 min and the supernatant discarded. The cells collected were resuspended in 500 µL of 50 mM Tris-HCL (pH 7.4), 20 mM Na2EDTA, added with 50 µL of 10% SDS solution and incubated at

Table 1. Saccharomyces cerevisiae isolates used in this study, their sources and ethanol production in 20% unsterile molasses medium.

PNCMa Accession No. Origin Ethanol Producedd (%

v/v)

2006 Cebu 6.12EFGHIJK

2007 Bacbac, San Pedro, Laguna 5.57IJKLMN

2008 Binalbagan, Isabela 7.50BC

2010 Binalbagan, Isabela 7.45BCD

2011 Canlubang, Laguna 5.76GHIJKL

2012 Canlubang, Laguna 6.76BCDDEFG

2013 Canlubang, Laguna 6.90BCDEF

2014 Canlubang, Laguna 6.63CDEFGH

2015 Tarlac 6.47DEFGHI

2016 Bulacan 6.36EFGHIJ

2017 Bulacan 4.69MNOP

2018 Bulacan 4.91LMNOP

2019 Tarlac 6.40EFGHIJ

2020 Tarlac 7.09BCDE

2029 Hagonoy, Bulacan 5.40JKLMNO

2030 Hagonoy, Bulacan 5.70HIJKLM

2031 Infanta, Quezon 6.63CDEFGH

2033 Paombong, Bulacan 4.25PQ

2034 Paombong, Bulacan 3.11R

2040 Unknown sourceb 1.19S

2041 Unknown source 0.32T

2042 Unknown source 5.80LMNOP

2043 Unknown source 6.20EFGHIJ

2045 Unknown source 5.08LMNOP

2224 NRIBc 4.63NOPQ

2229 NRIB 6.27EFGHIJ

2234 NRIB 5.09KLMNOP

2185 Type strain 5.61HIJKLMN

2021 Negros Occidental 3.68QR

2022 Nasugbu, Batangas 6.15EFGHIJ

2023 Canlubang, Laguna 7.73B

2024 Lian Batangas 4.53OPQ

2025 Negros Occidental 8.68A

2049 Unknown source 4.24PQ

2059 Pangasinan 5.42JKLMNO

2096 Pangasinan 4.51OPQ

2115 Type strain 5.47IJKLMNO

aPhilippine National Collection of Microorganisms, National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños (UPLB), College, Laguna, Philippines

bDeposited by Dr. E. J. del Rosario, Institute of Chemistry, UPLB.cReference strain, National Institute of Brewing, Japan.dAverage of 3 replicates, values with the same letter are not significantly different at 5%

level using DMRT



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65° C for 30 min. Each tube was added with 200 µL of 5 M potassium acetate (pH 5.5), incubated on ice bath for 60 min and again centrifuged for 5 min at 6000 X g. The supernatant was transferred to a fresh microfuge tube and an equal volume of 100 % isopropanol was added. The tubes were mixed by inversion and allowed to stand at room temperature for 5 min. Centrifugation for 1 min at 6000 X g was performed to pellet out the DNA. The extracted DNA was washed with 500 µL of 70% ethanol then centrifuged again for 1 min at 6000 X g. The excess liquid was decanted while the DNA pellet was air-dried and resuspended in 100 µL TE buffer (pH 7.5). DNA solution was treated with 15 µL of 10 mg/mL RNAse A and incubated at 37° C for 30 min to remove contaminating RNA from the solution. The isolated DNA was stored at 4° C until use.

PCR Amplification of Mitochondrial rRNA. The primers MS1 (5’-CAGCAGTCAAGAATATTAGTCAATG-3’) and MS2 (5’-GCGGATTATCGAATT AAATAAC-3’) (White et al. 1990; Moncalvo et al. 2000) that amplify a portion of the mitochondrial small ribosomal subunit RNA gene (mt SSU rRNA) were used. The reactions were performed in an Eppendorf Mastercycler following the method described by White et al. (1990). Each 25-µL reaction mixture contained 25 pmoles each of the primers, 2.5 µL of 5 mM dNTPs, 1.5 µL of 50 mM MgCl2, 0.2 µL of 5U/ µL of Taq polymerase and 100 ng DNA template. The following conditions were followed: initial denaturation at 94° C for 1 min; followed by 25 cycles of denaturation at 94 °C for 1 min, annealing at 42 °C for 2 min and extension at 74 °C for 3 min, and a final extension at 74 °C for 10 min (Ward & Gray 1992; White et al. 1990). After the reaction, amplification products were loaded in a gel containing 0.5% agarose and 0.75% Synergel in 0.5x Tris–Borate-EDTA buffer (89 mM Tris, pH 7.8; 89 mM Boric acid; and 2 mM EDTA) and run at 100 volts for 5 h. The gels were stained with 0.5 μg/mL ethidium bromide solution and photographed using a Gel-Doc apparatus (Ultraviolet Products). To determine reproducibility, all samples were subjected to three rounds of independent PCR reactions and gel electrophoresis.

Rep-PCR Amplification. The primers used for rep-PCR assay were REP1-R (5’- IIICGICGICATCGIGC-3’), REP2-L (5’-ICGICTTATCIGGCCTAC-3’), ERIC1-R (5’- ATGTAAGCTCCTGGGGATTCAC-3’), ERIC-2 (5’-AAGTAAGTGA CTGGGGTGAG CG-3’), and BOXA1-R (5’-TAGGGCAAGGCGACGCTGACG-3’) (Versalovic et al. 1994). The reaction mixture consisted of 1x Gitschier buffer (335 mM Tris-Cl pH 8.8, 83 mM (NH4)2SO4, 33.5 mM MgCl2, 33.5 mM EDTA and 150 mM 2-mercaptoethanol), 0.15 mg bovine serum albumin (BSA), 10% DMSO, 2.5 mM total dNTP’s, 50 pmoles of

each primer solution, 2 units of Taq polymerase, 50-100 ng DNA template and HPLC-grade distilled water, in a 25 μL final volume. DNA amplification was performed using Eppendorf Personal Mastercycler and carried out essentially as described by Versalovic et al. (1994) with the following reaction cycles: initial denaturation at 95° C for 7 min; 35 cycles (for REP and ERIC primers) or 30 cycles (for BOX primer) of denaturation at 94° C for 1 min, annealing at 44° C (REP), 52° C (ERIC) and 53° C (BOX), for 1 min, extension at 65° C for 8 min; a single final extension at 65° C for 15 min; and holding at 4° C. The amplification products were resolved through electrophoresis, stained, and photographed as mentioned above. All samples were also run in triplicate trials.

Analysis of Amplification Products. The banding patterns generated by rep-PCR and mt SSU rDNA-PCR were converted to a two-dimensional binary matrix (1, presence; 0, absence of a PCR band). The isolates were grouped using SAHN Cluster Analysis of the Numerical Taxonomy System (NTSYS) (Exeter Software, Setauket, New York). Pair wise comparisons of the strains, based on the presence or absence of unique and shared bands, were used to generate Dice similarity coefficients (Rohlf 1994). Phenetic clustering was performed with the unweighted pair group method with averages (UPGMA) to generate dendrogram using biostatistical analysis program NTSYS-pc version 1.70. The confidence limits of the phenogram was determined by bootstrap analysis using the program WINBOOT developed by the International Rice Research Institute (IRRI) (Yap and Nelson 1996).

RESULTS

Fermentation Properties of the Yeast IsolatesEthanol production by 37 yeast strains after 24 h of fermentation at 30° C in unsterile molasses medium with 20% total sugars showed that S. cerevisiae 2008, 2010, 2012, 2013, 2014, 2015, 2020, 2023, 2025, and 2031 were the ten highest ethanol producers (Table 1). They exceeded significantly the ethanol production of industrial strain S. cerevisiae 2030 (HBY3) by about 1 to 3 % (v/v). These strains were selected and further evaluated for ethanol production using sterile medium, ethanol tolerance, heat tolerance and killer factor resistance.

Among the ten high ethanol-producing isolates tested for their ability to survive exogenous ethanol added at concentrations of 12.5% and 15 % (v/v), local isolates S. cerevisiae 2010, 2020, 2023 and 2025 were observed to be ethanol sensitive at both 12.5% and 15% (v/v) exogenous ethanol concentration (Table 2). There was no observed



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growth for these isolates after 24 h of incubation. S. cerevisiae K11, an ethanol tolerant strain, was used as the reference (Ogawa et al. 2000). Four local isolates (S. cerevisiae 2008, 2013, 2015 and 2031) remained viable after 3 days of incubation at 12.5% (v/v) ethanol. On the other hand, S. cerevisiae 2014 can only survive 2 days of incubation in the medium with 12.5% (v/v) ethanol. But at 15% (v/v) exogenous ethanol concentration, marked reduction in viability was observed even after a day of incubation. Isolates 2015 and 2031 remained viable after 2 days. But only isolate 2031 can endure lethal effect of ethanol at 15 % concentration up to 3 days (Figure 1), thus being the most ethanol tolerant.

Viability of the yeast isolates generally increased at 35° C after 24 h of incubation and slightly decreased after 48 h (data not shown). A decreasing population was observed when the yeasts were incubated at 40º C but they remained viable at 3.5 to 5.5 log CFUmL even after 72 h, suggesting that these strains can still survive and may be thermotolerant at this temperature (Figure 2). However, there was no growth at 45º and 50º C (data not shown).

Killer factor resistance was observed in some of the isolates with the use of the killer yeast strain S. cerevisiae K1 (ATCC 60728) which produces a compound called zymosin that can inhibit other yeast cells (Magliani et al. 1997). Sensitive

isolates absorbed the methylene blue dye and appeared as blue streak while resistant isolates appeared as white streak as shown by the control isolates (labeled at the bottom of Figure 3). Yeast cells with killer factor resistance have active enzyme that decolorizes methylene blue when it penetrates the cells, whereas cells with the absence of the killer factor resistance do not (Painting and Kirsop 1989). S. cerevisiae 2020, 2023, and 2025 were sensitive to the killer factor of S. cerevisiae K1 strain. Isolates 2008, 2012, 2013, 2015 and 2031 were slightly resistant to killer factor. Blue colonies surrounding the streak of these isolates were observed suggesting partial inhibition by the killer factor. S. cerevisiae 2010 and 2014 showed resistance to the killer factor (Figure 3, Table 2).

Yeast DNA FingerprintingThe utilization of MS1 and MS2 primers allowed the detection of polymorphisms in closely related strains of S. cerevisiae through differences of their mitochondrial rDNA genetic patterns (Rickwood et al. 1988). Multiple fragments were produced in the amplification of a sequence of mt SSU rRNA for each strain tested. Unique bands were amplified for S. cerevisiae isolates 2014 and 2023 with sizes 2 Kb and 1.5 Kb, respectively. UPGMA-SAHN cluster analysis of MS1 and MS2- amplified PCR

Table 2. Fermentation properties of the ten selected Saccharomyces cerevisiae isolates.

PNCMa Accession No.

Ethanol Productivity in Sterile Mediumb

Alcohol Tolerancec (incubation period)

Thermotolerance at 40 °Cd

Response to Killer FactorEthanol (%

v/v)Fermentation

Efficiency 13% 15% (incubation period)

2025 5.5 51.10B - (24 h) - (24 h)++ (24 h)

Sensitive+ (72 h)

2023 4.6 49.74B - (24 h) - (24 h) + (72 h) Sensitive

2008 8.8 80.94A + (72 h) + (48 h) - (72 h) + (72 h) Slightly resistant

2010 8.4 79.51A - (24 h) - (24 h) + (72 h) Resistant

2020 7.1 68.29AB - (24 h) - (24 h)++ (24 h)

Sensitive+ (72 h)

2013 9.2 87.22A + (72 h) + (24 h) - (48 h) + (72 h) Slightly resistant

2012 8.9 78.21A + (24 h) - (48 h)

+ (24 h) - (48 h) + (72 h) Slightly resistant

2014 8.3 73.40A + (48 h) - (24 h) + (72 h) Resistant

2031 6.6 67.82AB + (72 h) + (72 h) + (72 h) Slightly resistant

2015 8.2 71.30A + (72 h) + (48 h); - (72 h) + (72 h) Slightly resistant

aPhilippine National Collection of Microorganisms, National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Baños, College, Laguna, Philippines

bEthanol production was evaluated using 20% sterile molasses media (equivalent total sugars) after 24 h fermentation, values with the same letter are not significantly at 5% level using DMRT

c +, still viable up to the incubation period (in hours); -, no viable cell observedd ++, growth up to the incubation period (in hours); +, still viable but with population decrease



Figure 1. Cell viability of the high-ethanol yielding Saccharomyces cerevisiae strains at 15% (v/v) exogenous ethanol as a measure of their alcohol tolerance.

Figure 2. Cell viability of the high ethanol-producing Saccharomyces cerevisiae strains incubated at 40 ºC for 3 days.

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Figure 3. Response of Saccharomyces cerevisiae strains (2008, 2010, 2012, 2013, 2014, 2015, 2023, 2025 and 2031) to the killer toxin produced by S. cerevisiae K1 as manifested by reduction of methylene blue dye.

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products resulted to 10 clusters at 85% similarity level (Figure 4). Cluster 1 consisted of 2 sub-clusters, which was comprised mostly of high ethanol producers. A single isolate, S. cerevisiae 2014 was in cluster II. This isolate was a killer factor resistant strain. The ethanol tolerant local isolate S. cerevisiae 2031 was in cluster III. Cluster IV was comprised of other S. cerevisiae isolates including the industrial strain 2030. Low ethanol producers, S. cerevisiae 2040 and 2034 were grouped together in Cluster V. Clusters VI, VII, IX and X are single member groups. Reference strains KI (Killer yeast) and K11 (ethanol tolerant strain) were grouped together with a local isolate 2006.

Almost similar banding patterns for 10 yeast isolates were observed using ERIC and BOXA1-R primers (data not shown). The amplification of total genomic DNA template of ten high ethanol-producing isolates using REP primers (REP1R-1 and REP2-1) has resulted in almost similar banding patterns among S. cerevisiae strains ranging in size from 0.28 kb to 5kb (Figure 5). Fragments with sizes 1.9 kb, 1.4 kb, 1.3 kb, 1.0 kb, 650 bp, and 600 bp were common to all isolates tested suggesting that these fragments maybe generic. However, REP primers were somewhat useful in detecting genetic variations in strains of S. cerevisiae. For instance, approximately 1.65 kb fragment was present in the good performers: S. cerevisiae strains 2008 and 2010 but absent in other isolates tested. The industrial strains 2030 and 2031 have almost similar profiles except that of the 0.3 Kb band present in strain 2030. The high-ethanol producers in non-sterile molasses, S. cerevisiae 2023 and 2025, shared identical banding profiles.

UPGMA-SAHN cluster analysis of these strains revealed two clusters at 85 % similarity level (Figure 5). The first cluster comprised of eight yeast isolates including industrial strain S. cerevisiae 2030 and ethanol tolerant isolates 2031. S. cerevisiae 2012, 2013 and 2020 were grouped together, but they did not have the same fermentation characteristics. Cluster II comprised of S. cerevisiae 2008, 2010 and 2014.

DISCUSSIONSterilization of media caused a dramatic increase in ethanol production by strains of S. cerevisiae. Sterilization inverts the sucrose into glucose and fructose especially at low pH. However, other strains of S. cerevisiae have marked reduction in ethanol production in sterile molasses medium (Table 2). This could probably mean that these yeast strains perform better in co-existence with other possible contaminating microorganisms in the medium, or they possess a more active invertase system (Halos et al. 1987).

The ten isolates selected from 37 S. cerevisiae based on ethanol production using unsterile molasses medium were evaluated for other fermentation parameters such as alcohol tolerance, thermotolerance and killer factor resistance. The efficiency and extent of ethanol production differ among yeast strains due to varying response to different inhibitory factors. Yield can be affected by end product (ethanol) inhibition, by other metabolites such as organic acids, by osmotic pressures resulting from high sugar concentrations, and by elevated temperatures (Panchal and Tavares 1990). Inhibition of fermentation can also be due to aeration/agitation but which can lead to enhancement of cell growth. The presence of contaminating bacteria or other yeasts, high levels of certain cations, and genetic changes leading to formation of mutants and variants can also affect fermentation performance of the yeasts strains.

Ethanol is toxic to yeast cells and high ethanol tolerance is a desirable trait selected for in industrial strains. Ethanol inhibition is directly related to the inhibition and denaturation of important glycolytic enzymes, as well as to modification of the cell membrane (Maiorella 1985). Small-size yeast cells are generally more ethanol tolerant and faster producers of ethanol than large-size yeast cells. While the evidence is not totally convincing, it is widely believed that yeast cells are genetically predisposed to be either ethanol tolerant or intolerant. Ethanol tolerance and thermotolerance are closely linked and related to the structure of the cell membrane, specifically the lipid content (Panchal and Tavares 1990). Unsaturated lipids enhance alcohol tolerance and membrane fluidity, whereas saturated lipids diminish ethanol tolerance and make membrane more rigid. Certain nutrients and lipids enhance ethanol tolerance and ethanol productivity by the yeasts when added to the broth (Panchal and Tavares 1990). The ethanol tolerance of sake yeast K11 and S. cerevisiae 2031 is possibly due to the effect of more than two genes, in addition to other factors such as increased fatty acids and sterols (Casey and Ingledew 1986; D’Amore et al. 1987) induction of stress proteins (Schuller et al. 1994), and accumulation of trehalose (Kim et al. 1996).

Among the isolates tested, S. cerevisiae 2008, 2010, 2013, 2020 and 2014 were found to be thermotolerant, retaining more than 5 log CFUmL even after 3 days of incubation (Figure 2). Yeasts that grow at 40º C and above are considered to be thermotolerant (Panchal and Tavares 1990). In the alcohol industry, ethanol is harvested after 24 h of fermentation in batch culture. Viability after 24 h at elevated temperature may prove to be significant for the alcohol industry. Yeast metabolism liberates 11.7 kcal of heat for each kilogram of substrate consumed (Maiorella 1985). In the absence of cooling, internal temperature in



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Figure 5. Genetic relatedness among top ten ethanol yielding Saccharomyces cerevisiae strains. The dendrogram was generated from Dice similarity coefficient obtained from the presence and absence of total DNA bands of REP-PCR using UPGMA.

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the fermentation vats can reach up to 42º C. Yeast strains that are able to grow at 40º C would mean significant cost savings during fermentation process. Ethanol recovery costs are also lower when operating at this temperature. D’Amore et al. (1987) estimated that 30-35% of the fermentation costs incurred in ethanol production at 32º C is due to cooling process. At 37º C, production cost is expected to decrease by 10 %. Increasing the tolerance by the producing strain to fermentation temperature to 40º C, therefore, results in greater cost reduction.

Normally in fermentations, contaminant yeasts or bacteria are in competition with the producer yeast, and worst, can increase in proportion over successive fermentations. Although various zymocins are known and yeasts of many genera produce them, under the conditions of brewery and distillery fermentations, Saccharomyces spp. are the most likely killer strains (Campbell 1996). Killer yeast produces a proteinaceous toxin (Spencer and Spencer 1983) that is able to kill susceptible cells but is immune to the activity of its own killer toxins (Magliani et al. 1997). Killer toxin resistance is related to the structure and composition of the cell membrane (Ogawa et al. 2000).

With all the above parameters tested, S. cerevisiae 2008, 2010 and 2014 appear to be the ideal strains that can be offered to the alcohol industry. However, fingerprinting of these isolates should be done for purposes of patenting and Intellectual Property Rights (IPR) protection.

Molecular approaches, such as mt rDNA-PCR and REP-PCR, can distinguish groups of isolates based on their banding patterns. Amplification of a sequence of the small subunit of the mitochondrial rRNA gene (mt

SSU rRNA) using primer pair MS1 and MS2 revealed polymorphisms among closely related strains of S. cerevisiae. Polymorphisms using mitochondrial rRNA-PCR most probably arise from point mutations or small deletions rather than larger rearrangements of the mitochondrial genome (Nadal et al. 1996), which is present as 10-40 molecules in haploid yeast (Petes 1980). The multiplicity of the mtDNA molecules results in a cell being in either one of two states, namely either homoplasmic, in which case all the mtDNA molecules are identical in terms of the genetic markers they carry, or heteroplasmic, in which different mtDNA molecules carrying different genetic markers are present simultaneously (Rickwood et al. 1988). In addition, the mitochondrial genome of S. cerevisiae has been reported to recombine with high frequency (Campbell and Thorness 1998).

Cluster analysis of the mt SSU rRNA profiles of the yeast isolates was partly able to separate high ethanol producing strains from other significantly low producers. However, it would be very difficult to conclude that these profiles were related to the fermentation properties of the isolates. Although high ethanol producers were clustered separately from the low ethanol producers, there was no correlation between their fermentation properties and profiles of PCR amplification of the mitochondrial rDNA.

Good amplifications of ERIC, BOX and rep-PCR genes indicated that these repetitive elements, which are highly conserved in the bacterial kingdom, are also present in fungal genomes, such as yeast. However, the distribution of ERIC, BOX and rep-PCR elements was not very variable in S. cerevisiae. The limited genetic



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diversity detected using ERIC, BOX and rep-PCR among strains of S. cerevisiae may be due to very closely related chromosomal genotypes (McManus and Jones 1995). In a previous study, rep-PCR was only slightly effective in differentiating various isolates of the banana strains of Ralstonia solanacearum (Aves-Ilagan et al. 2003). Through rep-PCR, however, the ideal isolates S. cerevisiae 2008, 2010 and 2014 were clustered separately from the other isolates.

ACKNOWLEDGMENTResearch scholarship grant to the senior author leading to the successful completion of her M.S. degree and a research grant to JPVM (later continued by FBE) from the Philippine Council for Advance Science and Technology Research and Development (PCASTRD), Department of Science and Technology (DOST) is gratefully acknowledged. We also thank T. Ilagan and R. Ignacio for their technical assistance and M.T.M. Perez for her advise in DNA fingerprinting studies.

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