final yeast paper nidhi

7/28/2019 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


GRAPE 0.307 0.319 0.338 0.377 0.513 0.7 1.027 1.125 1.131

Orange

Tadi


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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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


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


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


GRAPES 0.513 0.502 0.498 0.338 0.329 0.319 0.256 0.246 0.203

Orange-


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-


TADI 1.05 0.538 0.467 0.391 0.347 0.33 0.205 0.195 0.18

Yeast-


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-


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

&mp; Umwin, Boston, Massachusetts, 1987).8. B. Kirsop, "Maintenance of yeast cultures" in Yeast Biotechnology (Allen &mp;

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.

final yeast paper nidhi

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