final yeast paper nidhi
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Isolation and Comparative Kinetic Study of Various Yeast Strains Isolated
From Different natural Sources
Sunil Kr.Verma,Ajeet Kr.Tiwari,Nidhi,Prashant Shukla,
Nitya Gupta,Prarthna,Dr.Sujeet Pratap Singh
Amity institute of Biotechnology, Amity University Lucknow, Uttar Pradesh
ABSTRACT
Recently, the interest in bioethanol as automobile fuel has risen from the perspectives of
measures for global warming and energy security. Bioethanol is produced by fermenting thesugar obtained from the raw materials, using yeasts. Bioethanol production from renewable
sources, such as sugar cane makes it a biofuel that is both renewable and environmentally
friendly .one of the strategies to reduce production cost and to make ethanol fuel economicallycompetitive with fossil fuels .the aim of this work was to investigate the kinetics study of various
yeast strain so that it can help in various fields. Such as presently whole world is facing energy
crisis due to fat dwindling fossil fuels. The picture in our country is more serious since we areimporting more than 70% of our crude oil requirement as a result there is a need to search for
alternative renewable source of energy. The most important alternate is alcohol(Bioethanol)
which is viewed as a future fuel and is produced mainly by fermentation of yeast.
KEYWORDS:Ethanol; costs; environment;growth kinetics;yeast strains
Introduction
Yeasts are a heterogonous group of fungi that superficially appear to be homogeneous. Yeasts grow in a
conspicuous unicellular form that reproduces by fission, budding, or a combination of both. True yeastsreproduce sexually, developing ascospores or basidiospores under favorable conditions. The majority ofascomycetous and basidiomycetous yeasts isolated by the lab go unrecognized because most of them areheterothallic. In most instances, only one of the mating types is isolated and therefore no asci or basidiaare produced Yeast-like fungi (imperfect yeasts) reproduce only by asexual means. The identification of
these fungi is based upon a combination of morphological and biochemical criteria. Morphology is
Primarily used to establish the genera, whereas biochemical assimilations are used to differentiate the
various species. Many opportunities may be explored using different costless renewable waste
materials with a lot of usable substrate for microorganisms to grow upon, and then produceuseful products for society (for example, agricultural food waste, wood chips,molasses, whey
permeate, rice straw, and newspaper waste).
Most renewable energy source (carbon source) can be used in a fermentation process with
microorganisms to produce bio-ethanol. Ethanol production benefits the society and theenvironment. Currently, ethanol blended in fuels represents more than 12% of the U.S. motor
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Gasoline sales. Ethanol blended fuel is also widely marketed across the world as a high-octane
Enhancer and oxygenate that reduces air pollution and improves automobile performance. The
necessity to isolate and select yeasts in nature without genetic modifications was researched bymany investigators to ascertain if those yeast strains were suitable for high bio-ethanol
production. Eighteen yeast strains from six genera were tested by Saigal D. (1994) to verify their
ability to grow on 20% glucose media at 40
C.(11)
Material and methods:
Isolation of yeast from various sources:-.
Requirements
YPD media, 500 ml beaker/conical flask, petriplates, paraffin tapes, distilled water,
cotton, electric balance and autoclave.
Petri Dish: A round, shallow dish used to grow bacteria. Culture: To grow living organisms in a prepared mixture of nutrients (media). Media: Substance containing nutrients needed for cell growth. Agar: Jelly to which food has been added for the growth of microbes. Inoculate: To introduce a microbe to an environment where it can grow. Sterile/Aseptic Technique: Laboratory procedures used in handling cultures, media and
equipment that prevent contamination.
For any fermentation industry it is essential that microorganism be used is a single culture so that
by-product formation is strictly avoided. Presence of undesired microorganism in any
fermentation process adversely affects its efficiency at the same time product quality also
deteriorates. Hence prior to using any microorganism it should be in purified state.
The isolation of yeast for use in the production of alcohol can be done from any saccharine
material which is partially deteriorates. In these present study the isolation if yeast has been
carried out from five different sources:
Grape Orange
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Tadi Yeast (Saccharomyces cerevisiae used as control) Sugarcane
MAKING OF YPD MEDIA:-
Yeast- 10 gm
Peptone- 20 gm
Dextrose- 20 gm
pH was adjusted to 6.5 for 1000ml media. Agar was added up to 1.5% i.e. 15 gm in 1000ml.
250 ml + 250 ml YPD media was weighted for the substances- YPD extract- 5gm, peptone- 10
gm, Dextrose- 10 gm, pH should be 6.5 and agar- 7.5 gm in 500 ml.
Procedure-
Media prepared was poured in the petriplates after it was autoclaved. This was done in laminar
air flow. All the plate was sealed after the culture media was solidified using paraffin tape this
was kept for 24 hours. Before the YPD media was prepared the beaker, petriplates and tips was
kept in the autoclave. Next day contamination was checked, if any plates were contaminated or
not.
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Fig 1: YPD Media
Five different sources of yeast was taken-
Grape Orange Tadi Yeast(Saccharomyces cerevisiae used as control) Sugarcane
Using serial dilution, the sample for each sources was taken up to 12 dilution .i.e.12 test tube was
taken with each having 9 ml distilled water and 1 ml sample was added to the first test tube and
from the first test tube 1 ml sample was added to the second test tube and so on.
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Fig.2: Serial dilution
The yeast was streaked using the inoculums loop in petriplates in laminar airflow from the
selected dilution say 6th
, 7th
and 8th
dilution. These petriplates was kept in incubator for overnight
and the result was checked.
Fig 3: Petriplates containing yeast
250 ml liquid YPD media was prepared and 3 ml liquid YPD was poured in 10 test tubes. The
test tube and conical flask containing media was autoclaved.
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All the petridish in the incubator are checked for growth. Most of the plates got contaminated. So
two of the petriplates was taken containing YPD media in which streaking was not done. Again
serially dilute the sugarcane juice from 1 to 8 dilution. The dilution test tube 3 to 8 was taken.
The petriplates containing YPD media was divided in 3 parts. Then the plate was streaked in the
laminar air flow and then incubated at 25C. 12 more petriplates was made using YPD media in
which antibiotics ampicillin was added in 6 plates so that no other bacteria can grow. It was kept
overnight. Then the plate kept in the incubator was checked,all show growth. Again 12
petriplates was checked for contamination,and it was found that all were contamination free. In
these 12 petriplates,6 plates contain antibiotics and 6 does not contain antibiotics. Serial dilution
of sample of grape,orange,tadi and yeast strain was done upto 9 dilution and the test tube of
dilution 6th , 7th
,8th
and 9th
for each sample was taken for streaking in petriplate containing
antibiotics. After streaking the petriplate was incubated for 24 hours. After 24 hours yeast is
isolated and kept in liquid YPD media.
Growth Curve:-Principle-
Yeast growth pattern can be studied in vitro. Depending upon nutritional status, yeast exibit
different growth patterns. For eg. In batch culture yeast show a growth like sigmoid curve.
However the same yeast culture display growth in straight line in fed batch culture. Secondly in a
freshly inoculated medium broth, yeast take time to adjust in the new enviroment. This gap of
time is called lag phase,thereafter it uses the nutrients of the medium and multiply very fast
showing showing exponential growth. This period is called exponential growth orlog phase of
the growth. Then the growth become stagnant.This stage is called stationary phase. After a few
days nutrient of the medium starts diminishing, therefore fresh medium containing nutrients
should be added.If these are not added growth rate retards. There comes a stage when there is no
increase or decrease in the number of yeast cells. This is called stationary phase. Finally,due to
continuous accumulation of toxic metabolites there occurs death of yeast cells,this phase is called
death phase..
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The later two phases are accomplished due to accumulation of toxic inhibitory secondary
metabolites of yeast. In laboratory, yeast growth can be demonstrated by plotting a graph
between yeast number (measured as optical density spectrophotometrically) and time duration.
Procedure-
For growth curve, 1.5L of YPD media was prepared and it was autoclaved (for less
contamination pour it in 3 flask of 1000ml each with 500ml media). Then it was transfered in 6
flask and 6 test tube of 225ml and 25ml respectively. All the flask and the tube was sealed by
cotton plug, and it was autoclaved. Then the strain of yeast was added in each test tube
containing 25ml YPD and it was kept in the shaker for overnight.
Liquid YPD media was taken which contain samples of orange, grape, tadi, sugercane etc and
250 ml YPD liquid media was taken. 250 ml YPD media was divided into two part 25ml in one
beaker and 225ml in the other for each sample. Then the liquid YPD media was added in 25 ml
beaker and this was poured to 225ml YPD media then 4 ml of this liquid was taken in the
apendop and the reading in UV spectrophotometer was taken at 600nm wavelength. OD with
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reference water at 600nm wavelength was taken. Then 2ml of culture from apendop was taken
and it was centrifuge at 14495rpm/5 min/RT. Then 0.5ml of supernatent was taken and added to
0.5ml of DNS, this mixture is shaked well and it is boiled for sometimes till the color get
changed. It was then diluted with 9 ml of distilled water and was vortexed. Then the OD at
540nm was taken with DNS as reference. And the OD was noted for each at 600nm and at
540nm wavelength.
Result and Discussion:
After running of DNA-
The isolated DNA samples of yeast obtained from different sources were loaded in the agrose gel
and the band was observed. The finest DNA observed was of a strain of yeast V.awaste which
was loaded in the first well from the left side. The second loaded sample was of tadi loaded in
the second well and the DNA obtained from the tadi sample contain some impurities. The third
well loaded was the sample of orange and the DNA observed was with much more impurities
then the second one.The fourth was ladder which is for calculating unknown base pair. RNA is
seen at the second end.
Fig 5: DNA band of yeast different samples
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The result of Growth Curve-
Yeast growth results in turbidity which is in index of yeast growth. The cells suspended in the
culture interprets the passage of light allowing less light to reach the photoelectrical cell.The
amount of light energy transmitted through the suspension is measured as percentage of
transmission or spectrophotometer at 0.1% to 100%. The density of cell suspension is expressed
as absorbance or optical density which is directly proportional to cell concentration. Absorbance
is a logarthic value and is used to plot a graph of yeast growth.
Growth curve table :-
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
GRAPES 0.307 0.319 0.338 0.377 0.513 0.7 1.027 1.125 1.131
ORANGE 0.041 0.169 0.201 0.219 0.261 0.305 1.313 0.85 1.009
TADI 0.195 0.203 0.215 0.26 0.433 0.462 0.538 1.07 1.09
YEAST 0.202 0.289 0.292 0.298 0.584 0.68 1.068 1.101 1.121
SUGARCANE 0.433 0.494 0.644 0.739 0.765 0.841 1.106 1.928 1.896
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Growth curves:
Grapes
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
GRAPE 0.307 0.319 0.338 0.377 0.513 0.7 1.027 1.125 1.131
Orange
Tadi
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
ORANGE 0.041 0.169 0.201 0.219 0.261 0.305 1.313 0.85 1.009
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
GRAPES
GRAPES
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
GRAPES
GRAPES
0
0.2
0.4
0.6
0.8
1
1.2
1.4
To T1 T2 T3 T4 T5 T6 T7 T8
ORANGE
ORANGE
0
0.2
0.4
0.6
0.8
1
1.2
1.4
To T1 T2 T3 T4 T5 T6 T7 T8
Series1
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TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
TADI 0.195 0.203 0.215 0.26 0.433 0.462 0.538 1.07 1.09
Yeast (Saccharomyces cerevisiae used as control)
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T
YEAST 0.202 0.289 0.292 0.298 0.584 0.68 1.068 1.101 1.12
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
TADI
TADI
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
TADI
TADI
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
yeast
yeast
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Sugarcane
Comparisons
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
SUGARC. 0.433 0.494 0.644 0.739 0.765 0.841 1.106 1.928 1.896
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
To T1 T2 T3 T4 T5 T6 T7 T8
SUGARCANE
sugarcane
0
0.5
1
1.5
2
2.5
To T1 T2 T3 T4 T5 T6 T7 T8
SUGARCANE
sugarcane
0
0.5
1
1.5
2
2.5
To T1 T2 T3 T4 T5 T6 T7 T8
GRAPES
ORANGE
TADI
yeast
sugarcane
0
1
2
3
4
5
6
7
To T1 T2 T3 T4 T5 T6 T7 T8
sugarcane
yeast
TADI
ORANGE
GRAPES
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DNS TEST OBSERVATION TABLE
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
GRAPES 0.513 0.502 0.498 0.338 0.329 0.319 0.256 0.246 0.203
ORANGE 0.417 0.376 0.357 0.219 0.201 0.196 0.069 0.057 0.046
TADI 1.05 0.538 0.467 0.391 0.347 0.33 0.205 0.195 0.189
Yeast 0.841 0.802 0.798 0.584 0.567 0.494 0.297 0.278 0.267
Sugarcane 1.106 1.067 0.849 0.765 0.656 0.645 0.478 0.467 0.443
0
1
2
3
4
5
6
7
ToT1
T2T3
T4T5
T6T7
T8
sugarcane
yeast
TADI
ORANGE
GRAPES
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Graphs:
Grapes
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
GRAPES 0.513 0.502 0.498 0.338 0.329 0.319 0.256 0.246 0.203
Orange-
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
ORANGE 0.417 0.376 0.357 0.219 0.201 0.196 0.069 0.057 0.046
0
0.1
0.2
0.3
0.4
0.5
0.6
To T1 T2 T3 T4 T5 T6 T7 T8
GRAPES
GRAPES
0
0.1
0.2
0.3
0.4
0.5
0.6
To T1 T2 T3 T4 T5 T6 T7 T8
GRAPES
GRAPES
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
To T1 T2 T3 T4 T5 T6 T7 T8
ORANGE
ORANGE
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
To T1 T2 T3 T4 T5 T6 T7 T8
ORANGE
ORANGE
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Tadi-
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
TADI 1.05 0.538 0.467 0.391 0.347 0.33 0.205 0.195 0.18
Yeast-
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
Yeast 0.841 0.802 0.798 0.584 0.567 0.494 0.297 0.278 0.267
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
TADI
TADI
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
TADI
TADI
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
To T1 T2 T3 T4 T5 T6 T7 T8
YEAST
yeast
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
To T1 T2 T3 T4 T5 T6 T7 T8
YEAST
yeast
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Sugarcane-
TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8
SUGARCANE 1.106 1.067 0.849 0.765 0.656 0.645 0.478 0.467 0.443
Comparison:-
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
SUGARCANE
sugarcane
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
SUGARCANE
sugarcane
0
0.2
0.4
0.6
0.8
1
1.2
To T1 T2 T3 T4 T5 T6 T7 T8
GRAPES
ORANGE
TADI
yeast
sugarcane
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Conclusion:
The isolation of yeast from various sources such as grape, sugarcane, yeast sample, tadi and
orange was done using serial dilution and then isolation of DNA from the different yeast strain
using YPD media. Then the isolated DNA samples were loaded on agrose gel and the DNA band
obtain after running is studied. Then kinetics study of different strain of yeast was also done. A
sigmoidal curve of yeast is seen.
Future Recommendations:
From the isolated DNA in future we will do RAPD for further studying the nature of the yeast
extract and also alcohol tolerance.
0
0.5
1
1.5
2
2.5
3
3.5
4
ToT1
T2T3
T4T5
T6T7
T8
sugarcane
yeast
TADI
ORANGE
GRAPES
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References:
1. Technology Evaluation and Norms Study in industrial Alcohol Industry Report 1993.2. P.J.Manohar Rao (2004), All India Seminar on Commercial Derivative from sugarcane
and its Co-products p.8
3. Study report of committee set up for assessing the viability of ethanol project 2003.4. Lehning , A.L. (1984), Principles of Biochemistry, CBS Publisher, New Delhi,399.5. P.J.Manohar Rao(1997), Industrial Utilization of Sugarcane and its coproducts ISPCK
Publisher and Distributors, New Delhi, p.239.
6. Mathur, R.B.L.(1975),Handbook of cane sugar Technology p.65.7. D.R. Berry and C. Brown, "Physiology of yeast growth" in Yeast Biotechnology (Allen
∓ Umwin, Boston, Massachusetts, 1987).8. B. Kirsop, "Maintenance of yeast cultures" in Yeast Biotechnology (Allen ∓
Umwin, Boston, Massachusetts, 1987).
9. B. E. Kirsop, "Maintenance of Yeasts" in Maintenance of Microorganisms (AcademicPress London, 1984).
10.E. O. Morris, "Yeast Growth" from some unknown yeast textbook.11.Saigal D. 1994. Isolation and selection of thermotolerant yeasts for ethanol production.
Indian Journal of Microbiology 34: 193-203.
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