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HAYATI Journal of Biosciences September 2012 Available online at:
Vol. 19 No. 3, p 145-149 http://journal.ipb.ac.id/index.php/hayati
EISSN: 2086-4094 DOI: 10.4308/hjb.19.3.145

SHORT COMMUNICATION

Yeast Isolation for Bioethanol Production

EKA RURIANI1‡, TITI CANDRA SUNARTI2, ANJA MERYANDINI3∗∗

1
Department of Agroindustrial Technology, Faculty of Agricultural Engineering and Technology,
Bogor Agricultural University, Bogor 16680, Indonesia
2
Biology Department, Faculty of Mathematic and Natural Science; Research Centre for Natural Resources and
Biotechnology, Bogor Agricultural University, Bogor 16680, Indonesia

Received March 30, 2012/Accepted September 10, 2012

We have isolated 12 yeast isolates from five different rotten fruits by using a yeast glucose chloramphenicol
agar (YGCA) medium supplemented with tetracycline. From pre-screening assay, four isolates exhibited higher
substrate (glucose-xylose) consumption efficiency in the reaction tube fermentation compared to Saccharomyces
cerevisiae dan Saccharomyces ellipsoids as the reference strains. Based on the fermentation process in gooseneck
flasks, we observed that two isolates (K and SB) showed high fermentation efficiency both in sole glucose and
mixed glucose-xylose substrate. Moreover, isolates K and SB produced relatively identical level of ethanol
concentration compared to the reference strains. Isolates H and MP could only produce high levels of ethanol in
glucose fermentation, while only half of that amount of ethanol was detected in glucose-xylose fermentation.
Isolate K and SB were identified as Pichia kudriavzeevii (100%) based on large sub unit (LSU) ribosomal DNA D1/
D2 region.

Key words: yeast, glucose-xylose substrate, ethanol

___________________________________________________________________________

INTRODUCTION Zhao et al. (2008) had also stated that enzymatic hydrolysis
only uses low energy input, has low polution effects and
The utilization of ethanol as an alternative fuel has no side products such as furfural or HMF are detected.
escalated recently because of some conceivable reasons. The application of yeast for ethanol conversion from
Ethanol is a clean and renewable type of fuel which can starch-containing materials or other sugar sources,
be produced economically and environmentally friendly including cassava, sweet potato, sago palm, and
(Tian et al. 2009). Many agricultural by-products can be unfermented palm juice, has been conducted previously.
used as potential raw material for bioethanol production. Yet, the application of lignocellulose materials, such as
The production of bioethanol from agricultural by- corn cob, for ethanol production should be carried out in
products is very prospective because the raw materials detail because the hemicellulose and lignin content may
do not compete with other food-source materials which influence the final products. The hydrolysis reaction of
contain sugar and starch. Ethanol can be made from oil or hemicellulose generates xylose (pentose sugar/C5) which
biomass conversion by microbes through a fermentation can not be converted into ethanol by commercial yeast
process (Ohgren et al. 2006). (Saccharomyces cerevisiae). Therefore, in this study we
Saccharification of agricultural by-products can be screen for yeast which will be able to convert glucose and
done either through acid hydrolysis or enzymatic xylose mixed-substrates.
hydrolysis. Taherzadeh and Karimi (2007) reported that
enzymatic hydrolysis is more beneficial than acid MATERIALS AND METHODS
hydrolysis. This is due to the absence of sugar
degradation into Hydroxy Methyl Furfuraldehyde (HMF) Yeast Isolation. Yeast isolates were recovered from
or furfural, milder reactions (low temperature, neutral pH), five different rotten fruits, including (i) apple, (ii)
potential for high results in a reaction, and low watermelon, (iii) melon, (iv) papaya, and (v) pineapple.
maintenance expense (no corrosive instruments are used). About 1 gram of each fruit was used as yeasts source and
_________________ further serially diluted in reaction tube using NaCl 0.85%
‡
Current address: Department of Agriculture Product solution until 10-4 of dillution. About 100 μl of each of the
Processing, Faculty of Agriculture Technology, Jember University,
Jalan Kalimantan 37, Jember 68121, Indonesia
last two serial dillutions was then spread on top of yeast
∗ malt extract agar (YMEA) with a composition consisting
Corresponding author. Phone/Fax: +62-251-8622833,
E-mail: [email protected] of 5 g/l malt extract agar and 23 g/l yeast extract agar.
146 RURIANI ET AL. HAYATI J Biosci

Then, the composition was further incubated for 48 hours measuring tube. The reduction in the volume of water
at 30 oC. Expected yeast isolates were then purified and was proportional with the volume of CO2 formed during
screened by using a selective medium. fermentation and simply used to calculate the content of
Yeast Screening. Pre-screening of yeast isolates was CO 2. Besides the content of CO 2, several different
conducted by using the selective yeast glucose observations were also conducted during fermentation
chloramfenicol agar (YGCA with the following composition: such as the substrate consumption [using the
glucose 20 g/l, yeast extract 5 g/l, chloramphenicol 0.1 g/ dinitrosalicilic acid (DNS) method ] (Breuil & Saddler 1985),
l, agar 15 g/l) supplemented with tetracycline (0.05 g/l). and the ethanol content (using gas chromatography).
Each isolate was simply streaked on the YGCA medium Estimation of the Ethanol Content using Gas
and subsequently incubated for 96 hours at 30 oC. All Chromatography (GC). Injection volume for the GC
grown isolates were screened as yeast culture and further (Agilent Technologies 6890N) assay was done in a rate of
assayed for their capability in converting glucose and 0.8 ml/min within the capiler column (HP-Innowax, length
xylose mixed substrate (1:1) without aeration at 30 oC. For 60 m, diameter 0.25 mm and film thickness 0.25 μm) with
this assay, yeast isolates were previously prepared in a helium (He) as the carrier gas. The GC system was attached
potato dextrose agar (PDA) medium and further with a flame ionization detector (FID, 250 οC), while the
subcultured in a potato dextrose broth (PDB) medium for temperature of the injection port was controlled at 200 oC.
24 hours. About 10% (v/v) of the yeast culture was then The ethanol content was calculated by comparing the
inoculated in a reaction tube containing pure glucose- retention time of the sample to the ethanol standard.The
xylose (10%:10%) enriched with a solution of sodium standard curve was made by using pure ethanol with
phosphate potassium (0.04%) and ammonium sulphate methanol as the solvent.
(0.15%). The culture was fermented in a closed system,
whereas the utilization of the substrate was periodically RESULTS
monitored during fermentation, by using the DNS method
(Miller 1959). Selected isolates were chosen particularly Yeast Isolation. By using the YMEA medium, 23
based on substrate consumption compared to the isolates were recovered. The presence of bacterial
reference yeast isolates (S. ellipsoides and S. cerevisiae) morphology was observed around the targeted yeast.
and assayed for bioethanol production afterwards. Therefore, further screening assay using selective medium
Yeast Identification. Yeast isolates were partially was necessary to obtain pure yeast colonies.
identified by using large sub unit (LSU) ribosomal DNA Yeast Screening. Further screening in the YGCA
D1/D2 region. DNA isolation was conducted by employing medium supplemented with tetracycline resulted in 12
a DNA extraction kit of Nucleon PHYTOpure (Amersham isolates being able to show the yeast morphological
Life Science). Primer NL1 (5’-CATATCAATAAGCGG character. Therefore, those 12 isolates were continuosly
AGGAAAG-3’) and primer NL4 (5’-GTCCGTGTTTCAA assayed for their capability to use the glucose-xylose
GACGG-3’) (O’Donnell 1993) were used for PCR substrates in simple fermentation within the reaction tube.
amplification. PCR products were subsequently purified After 72 hours of fermentation, the presence of yeast
based on the polyethyleneglicol (PEG) precipitation growth (indicated by the development of gas bubbles,
method (Hiraishi et al. 1995) and followed with a the turbidity, and the aroma of yeast) was observed for all
sequencing process using ABI PRISM 3130 Genetic experiments. All isolates were capable of using the
Analyzer (Applied Biosystems). Sequences were further substrate with varying substrate consumption efficiency.
used for taxa identification using the BLAST program However, only four isolates showed a higher level of
(http://www.ncbi.nlm.nih.gov/BLAST/) and compared to substrate consumption compared to the reference strains
the GenBank database. (Table 1).
Bioethanol Production. The production of bioethanol Bioethanol Production. Four selected isolates from
was carried out in a modified erlenmeyer flask (250 ml). previous assays were observed for their bioethanol
The flask was connected with a water-containing production. The observation was done by quantifying
measuring tube by a plastic hose and reversely submersed the alteration of the CO2 volume every 3 hours for the first
in a plastic tank containing water. Two different substrates 12 hours and every six hours for a further 48 hours (Table
were used for this fermentation assay: sole glucose (10%) 2). The production of CO 2 differed among isolates.
and a mixture of glucose-xylose (10%:10%). The
fermentation medium was also enriched with a solution of
sodium phosphate potassium (0.04%) and ammonium Table 1. Yeast isolates with higher level of substrate consumption
than reference strains during glucose:xylose (10%:10%)
sulphate (0.15%), as was also used in previous assays.
fermentation
About 10% (v/v) of the yeast culture in the PDB medium
was transferred to the fermentation medium within the Isolate Origin Morphological characters ΔS/S0
modified erlenmeyer flask and subsequently incubated in Reference S. cerevisiae Yellowish, watery 0.34
Reference S. elipsoides Yellowish, watery 0.33
a waterbath shaker (120 rpm) at 30 oC. By using this K Apple White, wide, without core 0.39
modified flask, the CO 2 content yielded from the H Papaya White, small spot 0.40
fermentation reaction would be released through the MP Melon Yellowish-white, watery 0.33
plastic hose which further lowers the water volume in the SB Watermelon White, wide 0.64
Vol. 19, 2012 SHORT COMMUNICATION 147

Interestingly, all isolates produced their highest content augmented CO2 from isolate SB using either glucose or
of CO2 at 18 hours of fermentation, and the number the mixed substrate was found to be relatively similar,
descended afterwards. while isolate K showed a larger volume of CO2 using the
Estimation of Ethanol Content using Gas mixed substrate compared to the sole glucose substrate.
Chromatography (GC). The ethanol content during In contrast, isolates H and MP yielded higher ethanol
fermentation was different among isolates, yet the content content using the sole glucose substrate compared to the
of ethanol from glucose fermentations were higher than glucose-xylose mixed substrate which only resulted in half
that of glucose-xylose fermentations, with the exception of the yield. Therefore, we assumed that the two isolates
of isolate K (Table 3). could only convert glucose in mixed substrate
Based on Table 3, we noticed that isolate K and SB fermentation.
exhibited a relatively identical ethanol content from either Yeast Identification. Based on homology analysis,
glucose or glucose-xylose fermentations. Therefore, we isolate K and SB both, similarly, possessed a high
assumed that those two isolates were capable in using percentage of Pichia kudriavzeevii (100%).
both glucose or xylose, in particular, during ethanol
fermentation. Moreover, the accumulation of CO2 of those DISCUSSION
two isolates seemed to be equivalent with the production
of ethanol (Figure 1). Interestingly, the volume of the Generally, yeast cells use monosaccharide for their
growth, yet only a few of the monosaccharide compounds
can be converted into ethanol. D-glucose is the best
Table 2. The content of CO2 (ml) during fermentation using glucose
and glucose-xylose (1:1) mixed substrate susbtrate for either the growth of yeast cells or
fermentation for ethanol production. Mosier et al. (2005)
CO2 Volume (ml)
Isolate
and Hisamatsu et al. (2006) reported that hexose sugar,
Hour
K SB H MP including glucose, galactose and mannose, can be
G GX G GX G GX G GX fermented by many wild microorganisms, yet pentose
0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 sugar, such as xylose and arabinose, can only be fermented
3 5.0 26.3 7.5 12.5 3.5 12.5 6.0 16.3 by a small number of wild microorganisms, frequently
6 9.0 45.0 15.0 55.0 8.0 17.5 19.0 18.0
9 15.0 48.5 37.5 25.5 27.3 22.5 40.0 34.5 resulting in low ethanol yield. The yeast Issatchenkia
12 18.5 18.5 32.5 30.8 32.8 52.5 37.5 43.8 orientalis MF 121 is one of the potential isolate for
18 47.5 92.5 106.0 109.8 106.3 102.5 133.8 95.0 fermenting those types of substrates. This isolate is acid
24 35.0 52.5 50.0 21.5 53.8 63.8 62.5 61.3 tolerant (pH 2) and halotolerant (5%), however MF 121
30 35.0 35.0 34.0 20.0 33.8 53.8 46.3 42.5
36 38.0 30.0 33.0 17.5 23.8 32.5 35.0 30.0 cannot use susbtrate D-xylose, D-galactose, or cellobiose
42 26.0 12.5 17.0 10.0 13.8 15.0 16.3 17.5 for ethanol production. This research was conducted to
48 16.0 10.0 9.5 10.0 26.3 17.5 23.3 15.0 explore the sources of yeast isolates and further determine
G: Pure glucose, GX: glucose-xylose mixed substrate. their activity in assimilating glucose-xylose mixed
susbtrates because the content of these two sugar
compound are relatively high within agricultural by-
Table 3. The content of ethanol during glucose and glucose-xylose
fermentation by selected yeast isolates using GC analysis
products.
Fruits are food materials that contain a high level of
ΔS/S0 Ethanol content (%)
Isolate glucose. It is suitable for yeast cells to grow on because
Glucose Glucose-xylose Glucose Glucose-xylose
yeast grows optimally in simple sugars, including glucose,
K 0.52 0.50 1.09 1.32
H 0.58 0.53 3.13 1.51 and can even grow in complex sugars, such as sucrose
SB 0.48 0.44 1.68 1.14 (Chavan et al. 2009; Ocon et al. 2010). Yeast cells are both
MP 0.45 0.50 2.49 1.58 saprophyte and parasite. The damage to fruits due to

a b
CO2 volume (ml)

CO2 volume (ml)

Figure 1. The accumulation of augmented CO2 derived from 48 hours of glucose fermentation ( ) and glucose-xylose fermentation
( ) by isolate K (a) and isolate SB (b).
148 RURIANI ET AL. HAYATI J Biosci

physicall collisions may cause structural defects in the multiplying the volume of CO2 with the coefficient 1.045
fruit’s tissue. This condition will lead to the colonization as in the Gay-Lussac equation.
of yeast arround those defective areas. Yeast-causing The observation of the CO2 volume was conducted
damage is often indicated by the development of both an for both pure glucose fermentation and glucose-xylose
acid and alcohol aroma and the development of a unique fermentation. These observations were mainly used as
layer in the surface area of the fruit, such as a defect in the additional data for the two potential isolates. Based on
fruit extract. The colonization of yeast will lead to the the results, it was observed that these two isolates could
rotting of the fruit. produce CO2 during fermentation. In addition, it was also
Stringini et al. (2008) had isolated yeast from both the suggested that the volume of CO2 was equivalent with the
fruits and leaves of papaya, cacao, banana, soil, sugar ethanol content. As the duration of fermentation
cane extract, and other sources from agricultural wastes. increased, the level of CO2 also escalated, yet after a certain
151 isolates were isolated by using two isolation methods, period of incubation the increasing level of CO2 was not
YPD medium and enrichment mediums, one of which used significant. It can be assumed that these two isolates were
chloramphenicol for inhibiting the bacterial growth. able to convert sugar into ethanol through enzymatic
In this study, for the pre-screening assay, YGCA reactions. Moreover, the ethanol production was found
medium added with tetracycline to was used to inhibit the todecrease as the level of substrate decreased.
growth of bacteria and molds. The growth of the yeast At the begining of the fermentation step, yeast cells
isolates varied with some isolates growing better than need oxygen for growth, yet after the accumulation of
others. In fact, several isolates were unable to grow in the CO 2, the reaction turns anaerobic. During anaerobic
YGCA medium. We assumed those extinguish isolates growth, yeast cells metabolize glucose into ethanol mostly
were bacteria, since bacteria are commonly unable to grow through Embden Meyerhoff Parnas. Each mol of glucose
in chloramphenicol and tetracycline-containing medium. will generate two moles of ethanol, CO2 and ATP. Therefore,
Three isolates from apple, papaya and melon exhibited theoritically, each gram of glucose yields 0.51 g of ethanol.
relatively similar substrate consumption efficiency In fact, the production of ethanol is less than 90-95%
compared to the reference strains. In fact, one isolate from because most of the nutrition is used to synthesize
watermelon had two times the substrate consumption biomass and maintain the reaction. Moreover, side
efficiency compared to the reference strains. Therefore, it reactions can also occur resulting in glycerol and succinate
was assumed that the capability of the isolate to convert with 4-5% of substrate consumption. Ethanol can also
susbtrate during the fermentation process was high inhibit the sustainability of the fermentation reaction as
because the level of substrate left was low. However, the its level reaches 13-15%, yet it depends on temperature
high efficiency of substrate consumption may not be an and type of yeast.
indication of substrate convertion to bioethanol, since it
could be a result of the substrate utilization for cell growth ACKNOWLEDGEMENT
and the development of side products such as acids and
flavour compounds. This research was funded by Directorate General of
Based on the results, isolates K and SB produced a Higher Education, Ministry of Education with the research
relatively identical level of ethanol content compared to project of Penelitian Strategis Sesuai Prioritas Nasional
the reference strains. It was suggested that those two for Anja Meryandini. We thank Atit Kanti for identifying
isolates could convert two type of substrate into ethanol, yeast isolates.
indicating their capability in utilizing xylose. Meanwhile,
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