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 the sugar 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 economically competitive 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 are importing 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 yeasts reproduce sexually, developing ascospores or basidiospores under favorable conditions. The majority of ascomycetous and basidiomycetous yeasts isolated by the lab go unrecognized because most of them are heterothallic. In most instances, only one of the mating types is isolated and therefore no asci or basidia are 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 produce useful 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 the environment. Currently, ethanol blended in fuels represents more than 12% of the U.S. motor

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 by many 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 40C.(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

 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. ProcedureMedia 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.

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.

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.

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:PrincipleYeast 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 or log 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..

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

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 DNAThe 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

The result of Growth CurveYeast 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) GRAPES

To 0.307

T1 0.319

T2 0.338

T3 0.377

T4 0.513 0.7

T5

T6 1.027

T7 1.125

T8 1.131

ORANGE

0.041

0.169

0.201

0.219

0.261

0.305

1.313

0.85

1.009

TADI YEAST

0.195 0.202

0.203 0.289

0.215 0.292

0.26 0.298

0.433 0.584

0.462 0.68

0.538 1.068

1.07 1.101

1.09 1.121

SUGARCANE 0.433

0.494

0.644

0.739

0.765

0.841

1.106

1.928

1.896

Growth curves:
Grapes
TIME(hrs) GRAPE To 0.307 T1 0.319 T2 0.338 T3 0.377 T4 0.513 T5 0.7 T6 1.027 T7 1.125 T8 1.131

GRAPES
1.2 1 0.8 0.6 0.4 0.2 0 To T1 T2 T3 T4 T5 T6 T7 T8 GRAPES 1.2 1 0.8 0.6 0.4 0.2 0

GRAPES

GRAPES

To T1 T2 T3 T4 T5 T6 T7 T8

Orange
TIME(hrs) ORANGE To 0.041 T1 0.169 T2 0.201 T3 0.219
1.4

T4 0.261

T5 0.305

T6 1.313

T7 0.85

T8 1.009

ORANGE
1.4 1.2 1 0.8 0.6 0.4 0.2 0 To T1 T2 T3 T4 T5 T6 T7 T8 ORANGE 1.2 1 0.8 0.6 0.4 0.2 0 To T1 T2 T3 T4 T5 T6 T7 T8 Series1

Tadi

TIME(hrs) TADI

To 0.195

T1 0.203

T2 0.215

T3 0.26

T4 0.433

T5 0.462

T6 0.538 TADI

T7 1.07

T8 1.09

TADI
1.2 1.2 1 0.8 0.6 0.4 0.2 0 To T1 T2 T3 T4 T5 T6 T7 T8 TADI 1 0.8 0.6 0.4 0.2 0

TADI

To T1 T2 T3 T4 T5 T6 T7 T8

Yeast (Saccharomyces cerevisiae used as control)

TIME(hrs) To T1 T2 T3 T4 T5 T6 T7 T8

YEAST

0.202

0.289

0.292

0.298

0.584

0.68

1.068 yeast

1.101

1.121

1.2 1 0.8 0.6 0.4 0.2 0 To T1 T2 T3 T4 T5 T6 T7 T8 1.2 1 0.8 0.6 0.4 0.2 0

yeast

To T1 T2 T3 T4 T5 T6 T7 T8

Sugarcane

TIME(hrs) SUGARC.

To 0.433

T1 0.494

T2 0.644

T3 0.739

T4 0.765

T5 0.841

T6 1.106

T7 1.928

T8 1.896

SUGARCANE
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 To T1 T2 T3 T4 T5 T6 T7 T8 0 0.5 sugarcane 1 1.5 2 2.5

SUGARCANE

sugarcane

To T1 T2 T3 T4 T5 T6 T7 T8

Comparisons
7 6 2 5 4 3 2 GRAPES 1 0 To T1 T2 T3 T4 T5 T6 T7 T8 0 To T1 T2 T3 T4 T5 T6 T7 T8 0.5 sugarcane yeast TADI ORANGE 1.5 ORANGE TADI 1 yeast sugarcane GRAPES

2.5

5 4 3 2 1 0 To T1 T2 T3

sugarcane yeast TADI ORANGE GRAPES

T4

T5

T6

T7

T8

DNS TEST OBSERVATION TABLE

TIME(hrs) To GRAPES ORANGE TADI Yeast Sugarcane 0.513 0.417 1.05 0.841 1.106

T1 0.502 0.376 0.538 0.802 1.067

T2 0.498 0.357 0.467 0.798 0.849

T3 0.338 0.219 0.391 0.584 0.765

T4 0.329 0.201 0.347 0.567 0.656

T5 0.319 0.196 0.33 0.494 0.645

T6 0.256 0.069 0.205 0.297 0.478

T7 0.246 0.057 0.195 0.278 0.467

T8 0.203 0.046 0.189 0.267 0.443

Graphs: Grapes
TIME(hrs) GRAPES To 0.513 T1 0.502 T2 0.498 T3 0.338 T4 0.329 T5 0.319 T6 0.256 T7 0.246 T8 0.203

GRAPES
0.6 0.6

GRAPES

0.5

0.5

0.4

0.4

0.3

GRAPES

0.3

GRAPES

0.2

0.2

0.1

0.1

0 To T1 T2 T3 T4 T5 T6 T7 T8

0 To T1 T2 T3 T4 T5 T6 T7 T8

OrangeTIME(hrs) ORANGE To 0.417 T1 0.376 T2 0.357 T3 0.219 T4 0.201 T5 0.196 T6 0.069 T7 0.057 T8 0.046

ORANGE
0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 To T1 T2 T3 T4 T5 T6 T7 T8 ORANGE 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

ORANGE

ORANGE

To T1 T2 T3 T4 T5 T6 T7 T8

TadiTIME(hrs) To TADI 1.05 T1 0.538 T2 0.467 T3 0.391 T4 0.347 T5 0.33 T6 0.205 T7 0.195 T8 0.189

TADI
1.2 1 0.8 0.6 0.4 0.2 0 TADI 1.2 1 0.8 0.6 0.4 0.2 0

TADI

TADI

YeastTo T1 T2 T3 T4 T5 T6 T7 T8
TIME(hrs) Yeast To 0.841 T1 0.802 T2 0.798 T3 0.584

To T1 T2 T3 T4 T5 T6 T7 T8

T4 0.567

T5 0.494

T6 0.297

T7 0.278

T8 0.267

YEAST
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 To T1 T2 T3 T4 T5 T6 T7 T8 yeast 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

YEAST

yeast

To T1 T2 T3 T4 T5 T6 T7 T8

SugarcaneTIME(hrs) SUGARCANE To 1.106 T1 1.067 T2 0.849 T3 0.765 T4 0.656 T5 0.645 T6 0.478 T7 0.467 T8 0.443

SUGARCANE
1.2 1 0.8 0.6 0.4 0.2 0 To T1 T2 T3 T4 T5 T6 T7 T8 sugarcane 1.2 1 0.8 0.6 0.4 0.2 0

SUGARCANE

sugarcane

To T1 T2 T3 T4 T5 T6 T7 T8

Comparison:1.2

0.8

GRAPES ORANGE

0.6

TADI yeast

0.4

sugarcane

0.2

0 To T1 T2 T3 T4 T5 T6 T7 T8

4 3.5 3 2.5 sugarcane 2 1.5 1 0.5 0 To T1 T2 T3 T4 yeast TADI ORANGE GRAPES

T5

T6

T7

T8

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.

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 (Academic Press 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.