ISOLATION AND IDENTIFICATION OF MICROORGANISMS FROM...

Chapter 2

ISOLATION AND

IDENTIFICATION OF

MICROORGANISMS FROM

HANDIA

Chapter II: Isolation And Identification Of Microorganisms From Handia

Page//35

CHAPTER 2

ISOLATION AND IDENTIFICATION OF MICROORGANISMS FROM HANDIA

2.1 Introduction

The key organism used as starter culture for wine production is Saccharomyces

cerevisiae. Wines produced in different countries differ greatly in their quality depending on the

type of wine yeast strains used. The quality is determined by flavour, taste, viscosity and

appearance. Wine is generally produced by S. cerevisiae which was previously isolated from

grapes. Considering the gradual demand of consumers for wines of various flavours, the isolation

of microorganisms from sources other than grapes may be of great benefit in the production of

new types of wine. Keeping this in mind microorganisms were isolated and identified from

Handia, a traditional Indian fermented alcoholic beverage. The microbial composition of Handia

is not yet explored. It is anticipated that wine produced with indigenous cultures will be of low

cost. Although there are many similar fermented alcoholic beverages produced indigenously all

over the world, no attempt has been made to evaluate the potential of the microorganisms from

these beverages for application in wine production.

In this study the microorganisms present in Handia have been identified to species level.

Traditionally, identification of microorganism has been classified on the basis of phenotypic

properties which include morphology, fermentation of various carbohydrates and the ability to

grow at different temperatures and pH (Barnett et al., 1990; Kurtzman et al., 2006). However,

the conventional phenotypic methods are not reliable for correct identification of yeasts.

Alternatively, many molecular techniques have been developed and used for rapid and reliable

identification of yeasts (Quesada and Cenis.1995; Loureiro Malfeito-Ferreira., 2003). Molecular

identification is based on 26S rRNA gene sequence analysis (Kurtzman and Robnett, 1997,

1998; Fell et al., 2000; Kurtzman et al., 2003). One of the most powerful methods for yeast

identification is PCR-RFLP analysis of the ribosomal rRNA genes (5S, 5.8S, 18S and 26S)

(White et al., 1990; Hopfer et al., 1993; Molina et al., 1993; James et al., 1996; Redecker et al.,

1997; Wyder and Puhan, 1997; Belloch et al., 1998; Guillamon et al., 1998; Kurtzman and

Robnett, 1998; Gonzalez et al., 2006; Couto et al., 2005) and the non-coding ITS (Internal


Page//36

Transcribed Spacer) regions (Sabate et al., 2002; Cadez et al., 2002; Hopfer et al., 1993; Molina

et al., 1993; Redecker et al., 1997; Wyder and Puhan, 1997).

The aim of this study in this chapter is to isolate and identify the microorganisms from

Handia samples collected from different localites of West Bengal, India. Yeast species were

identified by morphological, biochemical and molecular techniques. Molecular identification

was based on 26S rRNA gene sequence analysis, PCR-RFLP analysis of 5.8S ITS region and

M13 genomic fingerprinting techniques. The bacterial isolates were identified by morphological,

biochemical and sequence analysis of 16S rRNA gene.

2.2 Materials and methods

2.2.1 Collection of Handia samples

Indigenously producing Handiasamples were aseptically collected in previously sterilized

250 mL flasks at different stage of fermentation from different districts (Bankura, Hooghly,

Burdwan and Birbhum) of West Bengal, India. The samples were brought to the laboratory in ice

and kept at 40C.

2.2.2 Isolation of microorganisms from Handia and culture media

One mL of each sample of Handia was transferred to 10 mL of YPD (0.5% Yeast extract,

1% Peptone and 2% Dextrose, w/v, pH 6.5) and TGE medium (1% Tryptone, 1% Glucose and

1% Yeast extract, w/v, pH 6.5). After incubation at 25 and 370C for 24 h each sample was then

serially diluted in saline water up to 10-8. A 50 µL from appropriate dilutions was spread on YPD

and TGE-agar plates in duplicates. The plates were incubated overnight at 25 and 370C for the

appearance of colonies. A few morphologically distinct colonies were picked up from each plate

and repeatedly streaked on the same agar medium to obtain pure cultures. Each pure culture was

maintained on YPD and TGE agar at 40C.


Page//37

2.2.3 Standard yeast cultures

Saccharomyces cerevisiae MTCC 178, S. cerevisiae MTCC 180, S. cerevisiae MTCC

211 and Issatchenkia orientalis MTCC 642 (Pichia kudriavzevii) were obtained from Microbial

Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh,

India. The strains were maintained on YPD agar at 40C. All these strains were grown on YPD

medium.

2.3 Identification of microorganisms from Handia

2.3.1 Phenotypic analysis

2.3.1.1 Morphology

The shape and size of the microorganisms isolated from Handia were examined by

scanning electron microscopy (SEM). Cells were harvested after 12 h of incubation at 300C in 5

mL of TGE and YPD medium. The cell pellet obtained after centrifugation was washed twice

with physiological saline solution (0.9%). Cells were fixed in 3.0% glutaraldehyde and 5.0%

DMSO buffered with 0.05 M acetate (pH 5.0), dehydrated in graded series of absolute ethyl

alcohol (10, 25, 35, 45, 55, 65, 75, 85 and 95%), and then dried by the critical point method.

Dried cells were coated with gold and examined in a HITACHI S-530 scanning electron

microscope. The instrumentation Centre at the University of Burdwan, West Bengal, India

provided this facility.

2.3.2 Biochemical and Molecular characterization of yeast isolates

2.3.2.1 Biochemical characteristics of yeast isolates

Biochemical characteristics of yeast isolates were determined based on their ability to

utilize a variety of carbon compounds. Each experiment was repeated three times.

2.3.2.1.1 Carbohydrate utilization profile of the yeast isolates

Fermentation of glucose, fructose, trehalose, xylose, arabinose, D-mannitol, raffinose,

sucrose, maltose, lactose, D-mannose, galactose, innuline and inositol were tested separately in

different tubes. Each sugar was used at 2%, except for raffinose which was 4%. The fermentation


Page//38

basal medium used in this test consisted of 4.5 g yeast extract and 7.5 g peptone (g/L) and 20 g

sugar (40g for raffinose) in 1 L of distilled water. The final working medium was made by

addition of 4 mL of bromothymol blue from 50 mg/75 mL to 100 mL of fermentation medium.

The pH of the medium was adjusted to 7 to 8. At this pH, the solution had dark greenish colour.

The medium was dispensed into tubes (4 mL in each tube) and sterilized at 1210C for 15 min.

The tubes were inoculated at 2% from each culture grown overnight in YPD medium and

incubated at 250C for 7 days. The tubes were inspected at frequent intervals. Positive results

were indicated by the change of colour in the indicator from dark green to yellow and continuous

evolution of gas in the forms of bubble. The change in colour green to yellow was scored as a

positive result. In the control sugar was not added.

2.3.2.2 Molecular characterization of yeast isolates

2.3.2.2.1 Isolation of genomic DNA from yeast

Genomic DNA was isolated from pure cultures of each isolate. Each pure culture in 5 mL

of YPD medium was grown at 370C for 6 h and the cells were harvested by centrifugation at

5000 × g for 7 min. The cell pellet was washed in 5 mL of TE buffer (Tris, 10mM; EDTA,

0.5mM; pH 8.0). The cell pellet obtained after centrifugation at 5000 × g for 7 min was

resuspended gently in 400 µL of lysis buffer (SDS 1% and sodium acetate 88 mM) mixture.

After vigorous mixing the suspension was incubated for 15 minutes at room-temperature

followed by addition of 400 µL of TE-saturated phenol (pH 8). The sample was mixed by gently

tapping the sample and incubated for 10 min at 650C. After incubation the sample was

centrifuged at 5000 × g for 7 minutes at 40C to separate phases. The aqueous supernatant was

transferred to a fresh microcentrifuge tube and 20 µL RNase (10mg/ mL) (Genei, Bangalore,

India) was added with an additional incubation for 30 minute at 370C. After incubation with 10

µL Proteinase K (Genei, India) at 500C for 1 h an equal volume of phenol/chloroform (1:1)

mixture (500 µL) was added with gentle mixing .The mixture was centrifuged at 5000 × g for 7

min at 40C. The upper phase was transferred into a fresh tube and one tenth volume of sodium

acetate (3 M) and an equal volume of isopropanol were added. The sample was kept in room-

temperature for 15 min for precipitation of DNA. The sample was centrifuged at 10000 × g for

15 min and the DNA pellet was washed in 1 mL of 70% ethanol. After centrifugation at 10000 ×


Page//39

g for 15 min, DNA was resuspended in TE or sterile double distilled water. The concentration of

DNA was measured following O.D. at 260 nm (UV/Vis spectrophotometer, Beckman Coulter,

DU 730, California, USA).

2.3.2.2.2 PCR amplification of 26S rRNA gene

In order to identify the phylogenetic position of the yeast isolates, the variable D1 and D2

regions of the 26S rRNA gene (at the 5′ end of the nuclear large subunit of the rRNA gene) were

amplified by PCR with the conserved fungal primer pair NL1 (5′-

GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′).

The polymerase chain reactions of 26S rDNA was performed in a reaction volume of 50 µL

containing 0.5 µM primer NL-1 and 0.5 µM primer NL-4 at a concentration of 0.2 mM each, 10

mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate

(dNTPs), 1.25 U of Pfu DNA polymerase (Fermentas, Hanover, MD, USA), and approximately

50 ng of genomic DNA. The amplification was carried out in 36 cycles with a Thermal Cycler

(Applied Biosystem 2720, Foster city, CA) under the following conditions: initial denaturation

at 950C for 5 min, followed by 36 cycles at 950C for 1 min, 520C for 1 min, 720C for 2 min and a

final extension at 720C for 10 min. The primers were synthesized commercially from Bangalore

Genei Pvt. Ltd., India. These primers (NL1 and NL4) was previously used to amplify the

variable D1/D2 domain of the large subunit (26S) ribosomal DNA by Kurtzman and Robnett

(1998).

PCR products were checked by electrophoresis on 1% (w/v) agarose gel. A 100-bp DNA

ladder was used as the molecular marker (New England BioLabs inc.,). Gel was stained with

ethidium bromide, visualized and photographed.

2.3.2.2.3 Partial sequencing of the 26S rDNA and sequence analysis

PCR products purified by QIAquick PCR purification kit (Qiagen, Hilden, Germany) was

sequenced commercially from Chromous Biotech Pvt. Ltd, Bangalore, India using the NL1

primer. Sequence comparisons were performed using the BLASTprogram

(http://www.ncbi.nlm.nih.gov/BLAST) (Altschul et al., 1990).


Page//40

2.3.2.2.4 PCR amplification of 5.8S-ITS region

The 5.8S ITS region was amplified by PCR from the genomic DNA of S. cerevisiae

using the conserved primers, used in previous study for taxonomic identification of yeasts (White

et al. 1990). The sequence of ITS1 primer is 5′-TCCGTAGGTGAACCTGCGG-3′ and the

sequence of ITS4 is 5′-TCCTCCGCTTATTGATATGC-3′. The primers were synthesized

commercially from Clonitec, India. PCR amplification of 5.8S-ITS region was carried out in a

total volume of 50 µL containing 50 ng template DNA, 1.5 U of Pfu DNA polymerase

(Fermentas, Hanover, MD, USA), 0.5 µM of each primer, 0.2 mM of each dNTPs, 1.5 mM

MgCl2, 10 mM Tris-HCl and 50 mM KCl. The PCR amplification was performed with a total of

35 cycles in a Thermal Cycler (Applied Biosystem 2720, Foster city, CA). The cycling program

consisted of an initial denaturing at 950C for 5 min followed by 35 cycles of denaturation at 950C

for 30 s, annealing at 550C for 30 s and elongation at 720C for 1 min. The PCR was ended with a

final extension at 720C for 10 min. The amplified DNA was electrophoresed on 1% agarose gel.

Gel was stained with ethidium bromide, visualized and photographed.

2.3.2.2.5 Partial sequence analysis of the 5.8S ITS region

PCR amplified product of 5.8S ITS region from Pichia kudriavzevii H21L was purified

by QIAquick PCR purification kit (Qiagen, Hilden, Germany). The DNA was sequenced

commercially from Chromous Biotech Pvt. Ltd, Bangalore, India using the ITS1 and ITS4

primers. Sequence comparisons were performed using the BLASTprogram

(http://www.ncbi.nlm.nih.gov/BLAST) (Altschul et al., 1990). Multiple sequence alignment of

5.8S ITS region of P. kudriavzevii H21L with the homologous DNA sequences retrieved from

GenBank was performed using the online software package

(http://www.ebi.ac.uk/Tools/clustalw2/index.htmL) (Larkin et al., 2007). Restriction sites within

DNA sequence was analyzed with the NEBcutter program

(http://tools.neb.com/NEBcutter/index.php3).

http://www.ebi.ac.uk/Tools/clustalw2/index.html

http://tools.neb.com/NEBcutter/index.php3


Page//41

2.3.2.2.6 PCR/RFLP analysis of Internal Transcribed Spacer (ITS) region of the yeast

isolates

A second molecular approach was taken to identify the yeast isolates to species level. It

involved PCR amplification of the internal transcribed (ITS1–5.8S rDNA–ITS2) region and

restriction analysis of the PCR products. The restriction enzymes HaeIII, HinfI and PstI

(Fermentas) were used to digest the DNA fragments. The restriction patterns generated after

digestion were compared. Aliquots of PCR products were digested separately with the restriction

endonucleases according to the manufacturer‘s instructions. These restriction enzymes were

chosen because previous works have shown that the DNA banding profiles with these restriction

enzymes (HaeIII and HinfI) can resolve the yeast isolate at species level (Guillamon et al., 1998;

Esteve-Zarzoso et al., 1999; Granchi et al., 1999). The reaction mixture contained 20µL of the

PCR products, 2.5 µL of 10X buffer, 1.5 µL of restriction enzyme and 1 µL of sterile deionized

water. The restriction enzymes HaeIII, HinfI and PstI were used separately to digest the

amplification products of PCR. The digestion reaction was incubated at 370C water bath for 2

hour. Restriction fragments (RFLP products) were analyzed by electrophoresis in 3% (w/v)

agarose gels. The DNA fragments were separated by applying 15 µL of each digested PCR

products with 1.5 µL of loading buffer to 3% agarose gel containing 0.5 µL/ mL ethidium

bromide. DNA markers 100 bp (100-bp ladder; New England BioLabs inc.,) for the calculation

of the DNA fragments size. The gel was run 1X TAE (Tris-acetic acid-EDTA) buffer for 2 h at

80 V, viewed on an UV transilluminator and photographed on UV transilluminator.

2.3.2.2.7 RAPD-PCR fingerprinting using M13 primer

In the third approach the isolates of S. cerevisiae and P. kudriavzevii were differentiated

by RAPD-PCR fingerprinting using M13 primer. The PCR amplification was carried out in 50

µL of reaction mixture containing approximately 50 ng of genomic DNA, 5 µL of PCR buffer

(10X), 5 µL of dNTPs (2.5 mM each), using 4 µL M13 primer (5′-GAGGGTGGCGGTTCT-3′)

(Sigma- Aldrich, India), 1µL (2.5 U/μl) of Pfu DNA polymerase (Fermentas, USA), and 18.5 µL

of deionised water. The amplification was performed with a total of 35 Cycles in a Thermal

cycler. The cycling program comprised an initial denaturation at 940C for 5 min, followed by 35

cycles of denaturation (940C for 1 min), annealing (20 s at 400C), extension (1 min at 720C) and


Page//42

a final extension of 720C for 5 min. The amplified DNA product was separated on 1% agarose

(Genei, Bangalore, India) gel. A 1 Kb DNA ladder (Genei, Bangalore, India) was used as

standard. The M13 primer has previously been used in DNA fingerprinting workby several

researcher in order to determine geneological relationship of yeasts at strain level (Messner et al.,

1994; Mayer et al., 1991; Prillinger et al., 1999; Andrighetto et al., 2000).

2.3.3 Physiological, Biochemical and Molecular characterization of bacterial isolates

Three bacterial isolates B16, B4, and BA were identified by phenotypically and

genotypially. For physiological and biochemical characterization, Gram-staining, Voges

Preoskauer test, Methyl Red test, citrate and sugar utilization test, growth in 2, 4 and 6.5% NaCl

were conducted. The morphology of cells was studied by scanning electron microscopy. The

genotypical method involved analysis of the 16S rRNA gene sequences (Weisburg, 1991) of the

isolates.

2.3.3.1 Physiological and Biochemical characterization of bacterial isolates

Gram staining

Gram-positive and Gram-negative bacteria were differentiated by Gram staining. A drop

of distilled water was added to a microscope slide and then overnight-grown bacterial suspension

were applied into the water and spread thinly along the entire area of the slide. Once the water

dried, the slide was heat fixed by quickly moving the slide through a sprit lamps three times.

Crystal violet (crystal violet, 5 g/L) was applied to the slide for one min and then rinsed with

distilled water. Grams Iodine (iodine, 10 g/L; potassium iodide, 20 g/L) was applied to the slide

for one min and rinsed with distilled water. The slide was further rinsed with 95% ethanol for

five seconds and immediately rinsed with distilled water. Safranin (5 g/L) stain was added to the

slide for two min followed by a rinsing with distilled water. A light microscope was used at 400x

magnification to observe the stained slides.


Page//43

Voges-Proskauer Reaction

Cells were inoculated in MRVP broth (0.7% polypeptone, 0.5% K2HPO4, 0.5% glucose)

and incubated at 37oC. After 48 h 1mL of culture was taken and mixed thoroughly with 0.6 mL

of 5% (wt/vol) α-napthol. 40% aqueous KOH was added to it and incubated in a slanted position

for 15 to 60 min. A strong red color that develops at the surface of the medium was scored as a

positive result.

Citrate utilization test

Cells were inoculated in the Simon‘s citrate medium (NaCl, 5 g/L; MgSO4,7H2O, 0.2

g/L; NH4H2PO4, 1 g/L; K4HPO4, 1g/L; Na-citrate, 5g/L; Bromothymolblue, 0.02 g/L; yeast

extract, 50 mg/L; agar, 20 g/L; pH 6.8 ) and incubated for 7 days. Growth and bright blue

coloration of the medium are indicative of positive test while no growth indicated negative.

Carbohydrate utilization profile of the bacterial isolates

The carbohydrate utilization ability of the bacterial isolates was determined based on

their growth on specific sugar and acid production. To performed this experiment a basal

medium (bovine extract, 10g/L; neopepton, 10g/L; yeast extract, 5g/L; K2HPO4, 2g/L;

CH3COONa+3H2O, 5g/L; diamonium citrate, 2g/L; MgSO4, 0.2g/L; MnSO4, 0.05g/L; tween 80,

1 mL) supplemented with different carbon sources (glucose, sucrose, maltose, mannitol and

lactose, each at 1%) was prepared with the addition of bromocresol-purple (0.004%) (Tserovska

et al., 2002). A five mL of this sterile medium was inoculated at 2% with an overnight culture of

each isolates. The inoculated medium was incubated at 370 C. After incubation for at least 24 h,

the changes in growth and colour change was recorded. Yellow coloration of the medium

indicated positive test. The test was negative when colour was not changed.

2.3.3.2 Molecular characterization of bacterial isolates

2.3.3.2.1 Isolation of Genomic DNA from bacteria

Genomic DNA was isolated by lysozyme-proteinase K procedure (Smith et al., 1981).

Briefly, each pure culture was grown overnight at 370C in TGE medium. Cells were centrifuged


Page//44

at 8000 rpm at 40C for 10 min and resuspended in TE (10 mM Tris and 1 mM EDTA, pH 8.0)

buffer containing lysozyme (1 mg/mL, Genei, Bangalore, India). After incubation at 370 C for 30

min, the lysate was treated with RNase (Genei, Bangalore, India) at 370C for 30 min and then

with proteinase K (Genei, Bangalore, India) and sarkosyl (Sigma) at 500C for 1 h. The digested

sample was successively extracted with phenol, phenol-chloroform and chloroform-isoamyl

alcohol. The crude DNA in the aqueous layer was recovered by isopropanol precipitation. DNA

pellet obtained after centrifugation was washed with 70% ethanol to remove salts and finally

resuspended in TE or sterile double distilled water. The concentration of DNA was measured

following OD at 260 nm.

2.3.3.2.2 PCR amplification of 16S rRNA gene

Bacteria-specific universal primers were used for the amplification of 16S rDNA gene of

all bacterial isolates. The forward primer was 27F (5′-AGAGTTTGATCATGGCTC-3′) and the

reverse primer was 1327R (5′- CTAGCGATTCCGACTTCA-3′) (Villani et al., 2001). The PCR

reaction was carried out in a total volume of 50 µL containing 50 ng of the genomic DNA, 1.25

U of Pfu DNA polymerase (Fermentas, Hanover, MD, USA), 0.2 mM of each primer, 0.2 mM

each of dNTPs, 1.5 mM MgCl2, 10 mM Tris-HCl and 50 mM KCl. The 16S rDNA gene was

amplified in 35 cycles with a Thermal Cycler; initial denaturing at 950C for 5 min, followed by

35 cycles at 950C for 1 min, a primer annealing step at 600C for 1 min and an extension step at

720C for 2 min and a final extension at 720C for 5 min. The primers were synthesized

commercially from Clonitec, Genuine chemical corp., India.

PCR products were checked by electrophoresis on 1% (w/v) agarose gel. A 100-bp DNA

ladder was used as the molecular marker (New England BioLabs inc.,). PCR products were

purified by QIAquick PCR purification kit (Qiagen, Hilden, Germany).

2.3.3.2.3 Partial sequencing of the 16S rRNA gene and sequence analysis

The purified PCR product was sequenced commercially (Chromous Biotech Pvt. Ltd.,

India) using 27F (5΄-AGAGTTTGATCATGGCTC-3΄) primer. Sequence comparisons were

performed using the BLASTprogram (http://www.ncbi.nlm.nih.gov/BLAST) (Altschul et al.,

1990).


Page//45

2.4 Results and discussion

2.4.1 Isolation of microorganisms from Handia

A total 15 microorganisms were isolated from ‗Handia‘. The isolates were designated as

G1, G4, H3, H8, H11, H12, H15, H17, KpY, 18VSL, 18VLL, B16, B4, BA and H21L. Table 2.1

describes the sources of the collected sample.

Table 2.1 Designation of the isolated microorganisms and their sources.

Sl. No. Designation Place of collection District Province

1 G1 Putidanga, Chatra Bankura West Bengal

2 G4 Putidanga, Chatra Bankura West Bengal

3 H3 Paschimpara Hooghly West Bengal

4 H8 Paschimpara Hooghly West Bengal

5 H11 Rajamela Bankura West Bengal

6 H12 Ranigang Burdwan West Bengal

7 H15 Ukharidihi Bankura West Bengal

8 H17 Bolpur (Pearsonpally) Birbhum West Bengal

9 KpY Mathura, Kalipur Hooghly West Bengal

10 18 VSL Vurkhunda Hooghly West Bengal

11 18 VLL Vurkhunda Hooghly West Bengal

12 H21L Rangamati Bankura West Bengal

13 B4 Paschimpara Hooghly West Bengal

14 BA Paschimpara Hooghly West Bengal

15 B16 Vurkhunda Hooghly West Bengal

2.4.2 Phenotypic identification

2.4.2.1 Morphology

The morphology of the microorganisms isolated from Handia was examined by

Scanning Electron Microscopy. Based on the shape, size, bud and bud scar the isolates G1, G4,


Page//46

H3, H8, H11, H12, H15, H17, KpY, 18VSL, 18VLL and H21L were grouped under yeast. The

isolates B16, B4 and BA were grouped under bacteria based on size and shape. The average

diameter of all bacterial isolates was 0.7 µm. The bacterial isolate B16 exhibited coccoid

morphology, occurred in chain. B4 cells were coccoid, arranged in tetrads and BA were rod-

shaped. The size of the yeasts varied from 2-3.6 µm in diameter. All the yeast isolates formed

buds. Bud scar was seen in only isolate 18VSL. The isolate G1, G4 and H21L were ovoidal to

elongate. The isolates H3, H8, H11, H12, H17, KpY, 18VSL, and were found to be oval in

shape. Scanning electron microscopic photograph of the isolates form Handia are shown in Fig.

2.1.A- 2.1.E and the morphological characteristics of these microorganisms are shown in Table

2.2.

Figure 2.1.A. SEM (Scanning electron microscopic photograph) of the isolates from Handia: The

photograph A represents the isolate G1 and B represents G4.


Page//47

Figure 2.1.B. SEM (Scanning electron microscopic photograph) of the isolates from

Handia: The photograph C represents the isolate H3; D represents H8; E represents

H11; and F represents H12.


Page//48

Fig.2.1.C. SEM (Scanning electron microscopic photograph) of the isolates from Handia:

The photograph G represents the isolate H15; H represents H17; I represents KpY; and J

represents 18VSL.


Page//49

Figure 2.1.D. SEM (Scanning electron microscopic photograph) of the isolates from Handia:

The photograph K represents the isolate 18VLL; L represents B16; M represents B4 and N

represents BA.


Page//50

Figure 2.1.E. SEM (Scanning electron microscopic photograph) of the isolates form

Handia: The photograph O represents the isolate H21L.


Page//51

Table 2.2 Scanning electron microscopic characteristics of microorganisms isolated from

Handia.

Isolate Shape Size Buds

G1 Ovoidal to elongate 3.15 – 3.26 × 7.36 – 8.42 µm Polar

G4 Ovoid / ovoidal to elongate 2.29 – 3.79 × 5.0 – 5.41 µm Polar

H3 Ovoal / round to oval 1.8 – 2.1 × 3 – 3.9 µm Lateral

H8 ovoal / round to oval 1.54 – 1.8 × 2.12 – 2.31 µm Lateral

H11 Ovoal / round to oval 2.26 – 2.58 × 2.12 – 2.62 µm Lateral

H12 Ovoal / round to oval 2.0 – 2.64 × 2.98 –4.67 µm Lateral

H15 Globose / oval 2.38 – 2.72 × 2.5 – 3.0 µm Polar

H17 Oval / globose 2.66 – 3.0 × 4.18 – 4.56 µm Polar

KpY Oval 3.61 × 5.28 µm Polar

18VSL Oval / globose 2.63 – 3.68 × 3.42 – 3.95 µm Polar

18VLL Oval to elongate / rounded 3.15 × 4.21 – 6.31 µm Lateral

H21L Ovoidal to elongate 1.62 – 1.78 × 4.1 – 4.2 µm Polar

B16 Coccoid 0.5×1.46-2.31 µm

B4 Spherical (coccus) 0.76×1.11-1.31 µm

BA Rod shaped 0.5 × 1.46 – 2.31 µm

2.4.3 Biochemical and molecular characterization of the yeast isolates

2.4.3.1 Biochemical characterization of the yeast isolates by carbohydrate utilization test

The yeast strains isolated from Handia were screened for their ability to ferment carbon

sources. In this study, 14 different types of sugars were used viz, glucose, fructose, trehalose,

xylose, arabinose, D-Mannitol, raffinose, sucrose, maltose, lactose, D-mannose, galactose,

innuline and inositol. The sugar utilization profiles of the isolates are shown in Table 2.3.


Page//52

Table 2.3 Carbon source utilization of the yeast isolates

Carbon

source

G1 G4 18VSL Kp

Y

H15 H17 H21L H3 H8 H11 H12 18VLL

Glucose + + + + + + + + + + + +

Fructose + + + + + + + + + + + -

Glactose + + + + + + + + + + + +

D-

Mannose

+ + + + + + + + + + + +

Sucrose + + + + + + + - - - - +

Lactose - - - - - - - - - - - -

Xylose - - - - + - - + + + + +

Trehalose - - - - - - - + + + + +

Maltose + + + + + + + - - - + +

Raffinose - - - - + + - - - - - -

Inuline + + + + + + + + + + + +

Arabinose - - - - - - - - - - - -

Mannitol + + - - - - - - - - - +

Inositol - - - - - - - - - - - -

‗+‘ indicates growth and ‗– ʼ indicates absence of growth

All yeast isolates could utilize glucose, galactose, D-mannose and innuline but none of

them utilized lactose, arabinose and inositol. Fructose was utilized by all the isolates except for

the strain 18VLL. Isolates H3, H8, H11 and H12 did not utilize sucrose; however, all other

isolates utilized sucrose. H15, H3, H8, H11, H12 and 18VLL could utilize xylose. Xylose was

not utilized by the rest of the isolates G1, G4, 18VSL, KpY, H17and H21L. The isolates H3, H8,

H11, H12 and 18VLL were able to utilize trehalose which was not utilized by the isolates G1,

G4, 18VSL, KpY, H15, H17 and H21L. Isolates G1, G4, 18VSL, KpY, H15, H21L, H12 and

18VLL utilized maltose whereas H3, H8 and H11 could not. H15 and H17 were the only

isolates which utilized raffinose. Mannitol was utilized only by G1, G4 and 18VLL. The sugar

utilization profiles were similar to those reported previously by Barnett et al. (2000); Mpofu et


Page//53

al. (2008). Based on these results 18VSL, KpY, H15, H17 were tentatively identified as the

members of the genus Saccharomyces, G1 and G4 as Hanseniaspora sp. and H21L as Pichia sp.

H3, H8, H11, H12 and 18VLL were tentatively identified as the species of Candida. However,

the identification of these strains to the species level was not possible using conventional

phenotypic methods.

2.4.3.2 Molecular characterization of the yeast isolates

2.4.3.2.1 Analysis of 26S rRNA gene sequence

In order to identify the yeast isolates to the species level the 26S rRNA gene was

amplified by PCR using NL1 and NL4 primers, as mentioned in the experimental sections. The

PCR amplification yielded 0.6 kb DNA fragment (Fig. 2.2), similar to results reported previously

for yeasts (Kurtzman and Robnett, 1998). The PCR products for each isolate were sequenced.

The BLASTanalysis showed 100% identity of 26S rRNA gene sequence of all the isolates with

the corresponding sequences in the GenBank Fig. 2.3.A- 2.3.K with the exception of H21L

which shared 99% homology with Pichia kudriavzevii (Fig. 2.3L). The level of percent identity

of all yeast strains with the database is shown in Table 2.7.

Figure 2.2 PCR amplified product of 26S rRNA gene, separated on 1% agarose gel. Lane 1,

isolate KpY; Lane 2, 18VSL; Lane 3, H15; Lane 4, H17; Lane 5, H21L; Lane 6, H3; Lane 7,

H8; Lane 8, H11; Lane 9, H12; Lane 10, 18VLL; Lane 11, G4; Lane 12, G1; Lane 13, S.

cerevisiae MTCC180 and Lane M, 1kb DNA ladder.


Page//54

>dbj|AB618029.1| Hanseniaspora guilliermondii gene for 26S rRNA, partial

sequence

Length=578

Score = 998 bits (540), Expect = 0.0

Identities = 540/540 (100%), Gaps = 0/540 (0%)

Strand=Plus/Plus

Query 7 CCTTAGTAACGGCGAGTGAAGCGGTAAAAGCTCAAATTTGAAATCTGGTACTTTCAGTGC 66

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 17 CCTTAGTAACGGCGAGTGAAGCGGTAAAAGCTCAAATTTGAAATCTGGTACTTTCAGTGC 76

Query 67 CCGAGTTGTAATTTGTAGAATTTGTCTTTGATTAGGTCCTTGTCTATGTTCCTTGGAACA 126

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 77 CCGAGTTGTAATTTGTAGAATTTGTCTTTGATTAGGTCCTTGTCTATGTTCCTTGGAACA 136

Query 127 GGACGTCATAGAGGGTGAGAATCCCGTTTGGCGAGGATACCTTTTCTCTGTAAGACTTTT 186

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 137 GGACGTCATAGAGGGTGAGAATCCCGTTTGGCGAGGATACCTTTTCTCTGTAAGACTTTT 196

Query 187 TCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCAAAGTGGGTGGTAAATTCCATCTAAAGC 246

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 197 TCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCAAAGTGGGTGGTAAATTCCATCTAAAGC 256

Query 247 TAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTT 306

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 257 TAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTT 316

Query 307 TGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACAT 366

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 317 TGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACAT 376

Query 367 GGTGTTTTTTGCATGCACTCGCCTCTCGTGGGCTTGGGCCTCTCAAAAATTTCACTGGGC 426

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 377 GGTGTTTTTTGCATGCACTCGCCTCTCGTGGGCTTGGGCCTCTCAAAAATTTCACTGGGC 436

Query 427 CAACATCAGTTCTGGCAGCAGGATAAATCATTAAGAATGTAGCTACCTCGGTAGTGTTAT 486

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 437 CAACATCAGTTCTGGCAGCAGGATAAATCATTAAGAATGTAGCTACCTCGGTAGTGTTAT 496

Query 487 AGCTTATTGGAATACTGCTAGCTGGGATTGAGGACTGCGCTTCGGCAAGGATGTTGGCAT 546

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 497 AGCTTATTGGAATACTGCTAGCTGGGATTGAGGACTGCGCTTCGGCAAGGATGTTGGCAT 556

Figure 2.3.A. Sequence alignment of 26S rRNA gene of Hanseniaspora guilliermondii G1from

BLAST data, compairing with the sequences in GenBank.

http://www.ncbi.nlm.nih.gov/nucleotide/325302537?report=genbank&log$=nuclalign&blast_rank=1&RID=8CXJGDH101N


Page//55

>dbj|AB618029.1| Hanseniaspora guilliermondii gene for 26S rRNA, partial sequence Length=578


Identities = 557/557 (100%), Gaps = 0/557 (0%)

Strand=Plus/Plus

Query 5 CTTAGTAACGGCGAGTGAAGCGGTAAAAGCTCAAATTTGAAATCTGGTACTTTCAGTGCC 64

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 18 CTTAGTAACGGCGAGTGAAGCGGTAAAAGCTCAAATTTGAAATCTGGTACTTTCAGTGCC 77

Query 65 CGAGTTGTAATTTGTAGAATTTGTCTTTGATTAGGTCCTTGTCTATGTTCCTTGGAACAG 124

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 78 CGAGTTGTAATTTGTAGAATTTGTCTTTGATTAGGTCCTTGTCTATGTTCCTTGGAACAG 137

Query 125 GACGTCATAGAGGGTGAGAATCCCGTTTGGCGAGGATACCTTTTCTCTGTAAGACTTTTT 184

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 138 GACGTCATAGAGGGTGAGAATCCCGTTTGGCGAGGATACCTTTTCTCTGTAAGACTTTTT 197

Query 185 CGAAGAGTCGAGTTGTTTGGGAATGCAGCTCAAAGTGGGTGGTAAATTCCATCTAAAGCT 244

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 198 CGAAGAGTCGAGTTGTTTGGGAATGCAGCTCAAAGTGGGTGGTAAATTCCATCTAAAGCT 257

Query 245 AAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTT 304

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 258 AAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTT 317

Query 305 GAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATG 364

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 318 GAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATG 377

Query 365 GTGTTTTTTGCATGCACTCGCCTCTCGTGGGCTTGGGCCTCTCAAAAATTTCACTGGGCC 424

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 378 GTGTTTTTTGCATGCACTCGCCTCTCGTGGGCTTGGGCCTCTCAAAAATTTCACTGGGCC 437

Query 425 AACATCAGTTCTGGCAGCAGGATAAATCATTAAGAATGTAGCTACCTCGGTAGTGTTATA 484

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 438 AACATCAGTTCTGGCAGCAGGATAAATCATTAAGAATGTAGCTACCTCGGTAGTGTTATA 497

Query 485 GCTTATTGGAATACTGCTAGCTGGGATTGAGGACTGCGCTTCGGCAAGGATGTTGGCATA 544

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 498 GCTTATTGGAATACTGCTAGCTGGGATTGAGGACTGCGCTTCGGCAAGGATGTTGGCATA 557

Query 545 ATGGTTAAATGCCGCCC 561

|||||||||||||||||

Sbjct 558 ATGGTTAAATGCCGCCC 574

Figure 2.3.B. Sequence alignment of 26S rRNA gene of Hanseniaspora guilliermondii G4 from


http://www.ncbi.nlm.nih.gov/nucleotide/325302537?report=genbank&log$=nuclalign&blast_rank=1&RID=8CYKJ7G201S


Page//56

>gb|HQ641276.1| Candida glabrata strain UL314 26S ribosomal RNA gene,

partial sequence

Length=561


Identities = 446/446 (100%), Gaps = 0/446 (0%)

Strand=Plus/Plus

Query 1 ATGCTTAGTACGGCGAGTGAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTTGGTGC 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 9 ATGCTTAGTACGGCGAGTGAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTTGGTGC 68

Query 61 CCGAGTTGTAATTTGGAGAGTACCACTTTGGGACTGTACTTTGCCTATGTTCCTTGGAAC 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 69 CCGAGTTGTAATTTGGAGAGTACCACTTTGGGACTGTACTTTGCCTATGTTCCTTGGAAC 128

Query 121 AGGACGTCATGGAGGGTGAGAATCCCGTGTGGCGAGGGTGTCAGTTCTTTGTAAAGGGTG 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 129 AGGACGTCATGGAGGGTGAGAATCCCGTGTGGCGAGGGTGTCAGTTCTTTGTAAAGGGTG 188

Query 181 CTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAG 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 189 CTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAG 248

Query 241 CTAAATACAGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACT 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 249 CTAAATACAGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACT 308

Query 301 TTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACA 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 309 TTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACA 368

Query 361 TGGTGTTTTGCGCCCCTTGCCTCTCGTGGGCTTGGGACTCTCGCAGCTCACTGGGCCAGC 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 369 TGGTGTTTTGCGCCCCTTGCCTCTCGTGGGCTTGGGACTCTCGCAGCTCACTGGGCCAGC 428

Query 421 ATCGGTTTTGGCGGCCGGAAAAAACC 446

||||||||||||||||||||||||||

Sbjct 429 ATCGGTTTTGGCGGCCGGAAAAAACC 454

Figure 2.3.C. Sequence alignment of 26S rRNA gene of Candida glabrata H3 from BLAST

data, compairing with the sequences in GenBank.

http://www.ncbi.nlm.nih.gov/nucleotide/335882946?report=genbank&log$=nuclalign&blast_rank=1&RID=89K9A5XP01S


Page//57

>gb|HM591730.1| Candida glabrata isolate 001713 26S ribosomal RNA gene,

partial sequence

Length=564


Identities = 561/561 (100%), Gaps = 0/561 (0%)

Strand=Plus/Plus

Query 1 GGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTTGGTGCCCGAGTTGTA 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 2 GGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTTGGTGCCCGAGTTGTA 61

Query 61 ATTTGGAGAGTACCACTTTGGGACTGTACTTTGCCTATGTTCCTTGGAACAGGACGTCAT 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 62 ATTTGGAGAGTACCACTTTGGGACTGTACTTTGCCTATGTTCCTTGGAACAGGACGTCAT 121

Query 121 GGAGGGTGAGAATCCCGTGTGGCGAGGGTGTCAGTTCTTTGTAAAGGGTGCTCGAAGAGT 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 122 GGAGGGTGAGAATCCCGTGTGGCGAGGGTGTCAGTTCTTTGTAAAGGGTGCTCGAAGAGT 181

Query 181 CGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATACAG 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 182 CGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATACAG 241

Query 241 GCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAG 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 242 GCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAG 301

Query 301 AGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGTGTTTTG 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 302 AGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGTGTTTTG 361

Query 361 CGCCCCTTGCCTCTCGTGGGCTTGGGACTCTCGCAGCTCACTGGGCCAGCATCGGTTTTG 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 362 CGCCCCTTGCCTCTCGTGGGCTTGGGACTCTCGCAGCTCACTGGGCCAGCATCGGTTTTG 421

Query 421 GCGGCCGGAAAAAACCTAGGGAATGTGGCTCTGCGCCTCGGTGTAGAGTGTTATAGCCCT 480

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 422 GCGGCCGGAAAAAACCTAGGGAATGTGGCTCTGCGCCTCGGTGTAGAGTGTTATAGCCCT 481

Query 481 GGGGAATACGGCCAGCCGGGACCGAGGACTGCGATACTTGTTATCTAGGATGCTGGCATA 540

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 482 GGGGAATACGGCCAGCCGGGACCGAGGACTGCGATACTTGTTATCTAGGATGCTGGCATA 541

Query 541 ATGGTTATATGCCGCCCGTCT 561

|||||||||||||||||||||

Sbjct 542 ATGGTTATATGCCGCCCGTCT 562

Figure 2.3.D. Sequence alignment of 26S rRNA gene of Candida glabrata H8 from BLAST data, compairing with the sequences in GenBank.

http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?maps=blast_set&db=nr&na=1&gnl=gb%7CHM591730.1%7C&gi=333495232&term=333495232%5Bgi%5D&taxid=5478&RID=3279U9CH016&QUERY_NUMBER=1&log$=nuclalign


Page//58

>gb|HM591715.1| Candida glabrata isolate 001207 26S ribosomal RNA gene,

partial sequence

Length=576


Identities = 358/358 (100%), Gaps = 0/358 (0%)

Strand=Plus/Plus

Query 10 ATGCCTTAGTACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTTGGT 69

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1 ATGCCTTAGTACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTTGGT 60

Query 70 GCCCGAGTTGTAATTTGGAGAGTACCACTTTGGGACTGTACTTTGCCTATGTTCCTTGGA 129

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 61 GCCCGAGTTGTAATTTGGAGAGTACCACTTTGGGACTGTACTTTGCCTATGTTCCTTGGA 120

Query 130 ACAGGACGTCATGGAGGGTGAGAATCCCGTGTGGCGAGGGTGTCAGTTCTTTGTAAAGGG 189

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 121 ACAGGACGTCATGGAGGGTGAGAATCCCGTGTGGCGAGGGTGTCAGTTCTTTGTAAAGGG 180

Query 190 TGCTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAA 249

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 181 TGCTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAA 240

Query 250 AGCTAAATACAGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAA 309

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 241 AGCTAAATACAGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAA 300

Query 310 CTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCA 367

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 301 CTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCA 358

Figure 2.3.E. Sequence alignment of 26S rRNA gene of Candida glabrata H11 from BLAST


http://www.ncbi.nlm.nih.gov/nucleotide/333495218?report=genbank&log$=nuclalign&blast_rank=1&RID=89M1AFZ101N


Page//59

>gb|HQ641276.1| Candida glabrata strain UL314 26S ribosomal RNA gene,

partial sequence

Length=561

Score = 623 bits (337), Expect = 2e-175

Identities = 337/337 (100%), Gaps = 0/337 (0%)

Strand=Plus/Plus

Query 9 ATGCTTAGTACGGCGAGTGAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTTGGTGC 68

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 9 ATGCTTAGTACGGCGAGTGAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTTGGTGC 68

Query 69 CCGAGTTGTAATTTGGAGAGTACCACTTTGGGACTGTACTTTGCCTATGTTCCTTGGAAC 128

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 69 CCGAGTTGTAATTTGGAGAGTACCACTTTGGGACTGTACTTTGCCTATGTTCCTTGGAAC 128

Query 129 AGGACGTCATGGAGGGTGAGAATCCCGTGTGGCGAGGGTGTCAGTTCTTTGTAAAGGGTG 188

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 129 AGGACGTCATGGAGGGTGAGAATCCCGTGTGGCGAGGGTGTCAGTTCTTTGTAAAGGGTG 188

Query 189 CTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAG 248

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 189 CTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAG 248

Query 249 CTAAATACAGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACT 308

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 249 CTAAATACAGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGAACT 308

Query 309 TTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAA 345

|||||||||||||||||||||||||||||||||||||

Sbjct 309 TTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAA 345

Figure 2.3.F. Sequence alignment of 26S rRNA gene of Candida glabrata H12 from BLAST


http://www.ncbi.nlm.nih.gov/nucleotide/335882946?report=genbank&log$=nuclalign&blast_rank=1&RID=89MPZ1RD01S


Page//60

>gb|HQ711330.1| Saccharomyces cerevisiae strain Y5-3 26S ribosomal RNA gene,

partial sequence

Length=612


Identities = 573/573 (100%), Gaps = 0/573 (0%)

Strand=Plus/Plus

Query 1 ACCGGGATTGCCTTAGTAACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTA 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 31 ACCGGGATTGCCTTAGTAACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTA 90

Query 61 CCTTCGGTGCCCGAGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGT 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 91 CCTTCGGTGCCCGAGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGT 150

Query 121 TCCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTT 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 151 TCCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTT 210

Query 181 GTAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATT 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 211 GTAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATT 270

Query 241 CCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGAT 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 271 CCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGAT 330

Query 301 GAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATT 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 331 GAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATT 390

Query 361 TGATCAGACATGGTGTTTTGTGCCCTCTGCTCCTTGTGGGTAGGGGAATCTCGCATTTCA 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 391 TGATCAGACATGGTGTTTTGTGCCCTCTGCTCCTTGTGGGTAGGGGAATCTCGCATTTCA 450

Query 421 CTGGGCCAGCATCAGTTTTGGTGGCAGGATAAATCCATAGGAATGTAGCTTGCCTCGGTA 480

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 451 CTGGGCCAGCATCAGTTTTGGTGGCAGGATAAATCCATAGGAATGTAGCTTGCCTCGGTA 510

Query 481 AGTATTATAGCCTGTGGGAATACTGCCAGCTGGGACTGAGGACTGCGACGTAAGTCAAGG 540

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 511 AGTATTATAGCCTGTGGGAATACTGCCAGCTGGGACTGAGGACTGCGACGTAAGTCAAGG 570

Query 541 ATGCTGGCATAATGGTTATATGCCGCCCGTCTT 573

|||||||||||||||||||||||||||||||||

Sbjct 571 ATGCTGGCATAATGGTTATATGCCGCCCGTCTT 603

Figure 2.3.G. Sequence alignment of 26S rRNA gene of Saccharomyces cerevisiae H15 from


http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?maps=blast_set&db=nr&na=1&gnl=gb%7CHQ711330.1%7C&gi=331031305&term=331031305%5Bgi%5D&taxid=4932&RID=3270T4UZ012&QUERY_NUMBER=1&log$=nuclalign


Page//61

>gb|EU386759.1| Saccharomyces cerevisiae strain C545 26S ribosomal RNA gene,

partial sequence

Length=575


Identities = 245/245 (100%), Gaps = 0/245 (0%)

Strand=Plus/Plus

Query 13 CTTAGTACGGCGAGTGAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTCGGTGCCCG 72

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1 CTTAGTACGGCGAGTGAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTCGGTGCCCG 60

Query 73 AGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGG 132

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 61 AGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGG 120

Query 133 ACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTGTAAAGTGCCTTC 192

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 121 ACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTGTAAAGTGCCTTC 180

Query 193 GAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTA 252

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 181 GAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTA 240

Query 253 AATAT 257

|||||

Sbjct 241 AATAT 245

Figure 2.3.H. Sequence alignment of 26S rRNA gene of Saccharomyces cerevisiae H17 from


http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=166236994&dopt=GenBank&RID=WZ54S15S014&log$=nuclalign&blast_rank=1


Page//62

>gb|HQ641267.1| Saccharomyces cerevisiae strain UL139 26S ribosomal RNA

gene, partial sequence

Length=556


Identities = 266/266 (100%), Gaps = 0/266 (0%)

Strand=Plus/Plus

Query 8 GGCATGCTTAGTACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTCG 67

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 6 GGCATGCTTAGTACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTCG 65

Query 68 GTGCCCGAGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGTTCCTTG 127

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 66 GTGCCCGAGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGTTCCTTG 125

Query 128 GAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTGTAAAG 187

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 126 GAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTGTAAAG 185

Query 188 TGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCT 247

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 186 TGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCT 245

Query 248 AAAGCTAAATATTGGCGAGAGACCGA 273

||||||||||||||||||||||||||

Sbjct 246 AAAGCTAAATATTGGCGAGAGACCGA 271

Figure 2.3.I. Sequence alignment of 26S rRNA gene of Saccharomyces cerevisiae KpY from


http://www.ncbi.nlm.nih.gov/nucleotide/335882937?report=genbank&log$=nuclalign&blast_rank=1&RID=8ACE5K4201N


Page//63

>gb|JF757233.1| Saccharomyces cerevisiae strain 18-9 26S ribosomal RNA gene,

partial sequence

Length=592


Identities = 366/366 (100%), Gaps = 0/366 (0%)

Strand=Plus/Plus

Query 1 CTTCGGTGCCCGAGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGTT 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 81 CTTCGGTGCCCGAGTTGTAATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGTT 140

Query 61 CCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTG 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 141 CCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTG 200

Query 121 TAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTC 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 201 TAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTC 260

Query 181 CATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATG 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 261 CATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATG 320

Query 241 AAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTT 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 321 AAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTT 380

Query 301 GATCAGACATGGTGTTTTGTGCCCTCTGCTCCTTGTGGGTAGGGGAATCTCGCATTTCAC 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 381 GATCAGACATGGTGTTTTGTGCCCTCTGCTCCTTGTGGGTAGGGGAATCTCGCATTTCAC 440

Query 361 TGGGCC 366

||||||

Sbjct 441 TGGGCC 446

Figure 2.3.J. Sequence alignment of 26S rRNA gene of Saccharomyces cerevisiae 18VSL from


http://www.ncbi.nlm.nih.gov/nucleotide/346655388?report=genbank&log$=nuclalign&blast_rank=1&RID=89FSP8BR016


Page//64

>gb|EF644470.1| Candida tropicalis isolate T3 26S ribosomal RNA gene,

partial sequence

Length=568


Identities = 297/297 (100%), Gaps = 0/297 (0%)

Strand=Plus/Plus

Query 1 GCTTAGTAGCGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGCTCTTTCAGAGT 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 30 GCTTAGTAGCGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGCTCTTTCAGAGT 89

Query 61 CCGAGTTGTAATTTGAAGAAGGTATCTTTGGGTCTGGCTCTTGTCTATGTTTCTTGGAAC 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 90 CCGAGTTGTAATTTGAAGAAGGTATCTTTGGGTCTGGCTCTTGTCTATGTTTCTTGGAAC 149

Query 121 AGAACGTCACAGAGGGTGAGAATCCCGTGCGATGAGATGATCCAGGCCTATGTAAAGTTC 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 150 AGAACGTCACAGAGGGTGAGAATCCCGTGCGATGAGATGATCCAGGCCTATGTAAAGTTC 209

Query 181 CTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAA 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 210 CTTCGAAGAGTCGAGTTGTTTGGGAATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAA 269

Query 241 GCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAG 297

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 270 GCTAAATATTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAG 326

Figure 2.3.K. Sequence alignment of 26S rRNA gene of Candida tropicalis 18VLL from


http://www.ncbi.nlm.nih.gov/nucleotide/150026639?report=genbank&log$=nuclalign&blast_rank=1&RID=89P8DB49013


Page//65

>gb|FJ919397.1| Pichia kudriavzevii isolate D-6 26S ribosomal RNA gene,

partial sequence

Length=600


Identities = 564/566 (99%), Gaps = 1/566 (0%)

Strand=Plus/Plus

Query 1 TGCATATCAAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCTCAGTAGCGGCGAGTG 60

|||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 2 TGCATATCCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCTCAGTAGCGGCGAGTG 61

Query 61 AAGCGGCAAGAGCTCAGATTTGAAATCGTGCTTTGCGGCACGAGTTGTAGATTGCAGGTT 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 62 AAGCGGCAAGAGCTCAGATTTGAAATCGTGCTTTGCGGCACGAGTTGTAGATTGCAGGTT 121

Query 121 GGAGTCTGTGTGGAAGGCGGTGTCCAAGTCCCTTGGAACAGGGCGCCCAGGAGGGTGAGA 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 122 GGAGTCTGTGTGGAAGGCGGTGTCCAAGTCCCTTGGAACAGGGCGCCCAGGAGGGTGAGA 181

Query 181 GCCCCGTGGGATGCCGGCGGAAGCAGTGAGGCCCTTCTGACGAGTCGAGTTGTTTGGGAA 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 182 GCCCCGTGGGATGCCGGCGGAAGCAGTGAGGCCCTTCTGACGAGTCGAGTTGTTTGGGAA 241

Query 241 TGCAGCTCCAAGCGGGTGGTAAATTCCATCTAAGGCTAAATACTGGCGAGAGACCGATAG 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 242 TGCAGCTCCAAGCGGGTGGTAAATTCCATCTAAGGCTAAATACTGGCGAGAGACCGATAG 301

Query 301 CGAACAAGTACTGTGAAGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGCACGT 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 302 CGAACAAGTACTGTGAAGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGCACGT 361

Query 361 GAAATTGTTGAAAGGGAAGGGTATTGCGCCCGACATGGGGATTGCGCACCGCTGCCTCTC 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 362 GAAATTGTTGAAAGGGAAGGGTATTGCGCCCGACATGGGGATTGCGCACCGCTGCCTCTC 421

Query 421 GTGGGCGGCGCTCTGGGCTTTCCCTGGGCCAGCATCGGTTCTTGCTGCAGGAGAAGGGGT 480

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 422 GTGGGCGGCGCTCTGGGCTTTCCCTGGGCCAGCATCGGTTCTTGCTGCAGGAGAAGGGGT 481

Query 481 TCTGGAACGTGGCTCTTCGGAGTGTTATAGCCAGGGCCAGATGCTGCGTGCGGGGACCGA 540

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 482 TCTGGAACGTGGCTCTTCGGAGTGTTATAGCCAGGGCCAGATGCTGCGTGCGGGGACCGA 541

Query 541 GGACTGCGGCCGTGTAG-TCACGGAT 565

||||||||||||||||| ||||||||

Sbjct 542 GGACTGCGGCCGTGTAGGTCACGGAT 567

Figure 2.3.L. Sequence alignment of 26S rRNA gene of Pichia kudriavzevii H21L from BLAST

data, compairing with the sequences in GenBank. The changes in the nucleotides are shown by

green colour.

http://www.ncbi.nlm.nih.gov/nucleotide/229609542?report=genbank&log$=nuclalign&blast_rank=2&RID=CM6PC9W5016


Page//66

The partial sequence of 26S rRNA gene of the isolate H21L (accession number

JN108878) was deposited in NCBI GenBank. Based on the results the isolate G1 was identified

as Hanseniaspora guilliermondii, G4 as Hanseniaspora guilliermondii, H21L as Pichia

kudriavzevii (Issatchenkia orientalis), H3 as Candida glabrata, H8 as C. glabrata, H11 as C.

glabrata, H12 as C. glabrata, 18VLL as C. tropicalis, H15 as Saccharomyces cerevisiae, H17 as

S. cerevisiae, KpY as S. cerevisiae and 18VSL as S. cerevisiae.

Since 26S rRNA gene sequence analysis could not differentiate the isolates of

Saccharomyces cerevisiae, Pichia kudriavzevii and Hanseniaspora guilliermondii at strain level,

another molecular approach was taken.

2.4.3.2.2 PCR/ RFLP analysis of Internal transcribed Spacer (5.8 S ITS) region

In order to further confirm species identification intraspecific variation of S. cervisiae

KpY, S. cervisiae 18VSL, S. cervisiae H15, S. cervisiae 17, P. kudriavzevii H21L and H.

guilliermondii G4 was studied by restriction analysis of the PCR amplified product of their 5.8S

ITS region located in between 18S rRNA and 26S rRNA genes (White et al., 1990). PCR-RFLP

analysis of 5.8S-ITS rDNA is a reliable technique for the differentiation of yeasts at species level

(Guillamon et al., 1998; Esteve-Zarzoso et al., 1999; Granchi et al., 1999; Fernandez-Espinar et

al., 2000; Clemente-Jimenez et al., 2004; de Llanos et al., 2004; Combina et al., 2005; Villa-

Carvajal et al., 2006; Nisiotou and Nychas, 2007; Zott et al., 2008). The DNA banding patterns

obtained after restriction digestion for each isolate were compared with the reference strains

(Saccharomyces cervisiae MTCC 178, S. cerevisiae MTCC 180, S. cerevisiae MTCC 211 and

Pichia kudriavzevii MTCC 642) and literature.

The results showed that PCR amplification of ITS region of the genomic DNA of S.

cerevisiae MTCC 211, S. cerevisiae MTCC 180, S. cerevisiae MTCC 178, S. cerevisiae H15, S.

cerevisiae H17, S. cerevisiae 18VSL and S. cerevisiae KpY yielded a fragment of 880 bp (Fig. 2.

4, lane 2 to 6 and lane 9 and 10) similar to results reported previously (Clemente-Jimenez et al.,

2004; Pulvirenti et al., 2001; Esteve-Zarzoso et al., 1999). Upon digestion with restriction

enzyme Hae III, four bands were generated (Fig. 2.5; Lane 2 to Lane 6 and Lane 9 and Lane 10).

The fragment sizes were approximately 320 bp, 240 bp, 180 bp and 140 bp (Table 2.4). The


Page//67

banding pattern is in accordance with the result reported previously by Esteve-Zarzoso et al.

1999; Jeyarama et al., 2008). The digestion with Hinf I produced three bands of 370, 370 and

130 bp (Fig. 2.6, Lane 4 to Lane 10; and Table 2.4), which is in agreement with the findings

reported in literature (Pulvirenti et al., 2001; Esteve-Zarzoso et al., 1999); Jeyarama et al., 2008).

The PCR product of 5.8S-ITS region of S. cerevisiae remained uncut after Pst I digestion (Fig.

2.7, Lane 4 to Lane 10; and Table 2.4). Although this technique could not differentiate the four

isolates of S. cerevisiae at strain level, their identity to the species level was confirmed.

The PCR amplification of 5.8S ITS region of the genomic DNA of both P. kudriavzevii

MTCC 642 and P. kudriavzevii H21L and yielded 500 bp fragment (Fig. 2.4, Lane 1 and Lane 8;

Table 2.4), similar to the results reported previously (Latorre-Garcia et al., 2007). Digestion of

this product from P. kudriavzevii H21L with Hae III yielded two fragments of approximately

380 and 100 bp (Fig. 2.5, Lane 8; Table 2.4) whereas Hinf I produced fragments of 250 and 150

bp (Fig. 2.6, Lane 3; Table 2.4). The strain P. kudriavzevii MTCC 642 displayed a restriction

pattern consisting of approximately 320 and 100 bp with Hae III (Fig. 2.5, Lane 1; Table 2.4)

and 280 and 130 bp fragments with Hinf I (Fig. 2.6, Lane 2; Table 2.4). The PCR products of

both P. kudriavzevii H21L and P. kudriavzevii MTCC 642 remained uncut after digestion with

PstI (Fig. 2.7, Lane 3 and Lane 2; Table 2.4). Although the 5.8S-ITS pattern of P. kudriavzevii

H21L generated with Hae III was found to be similar to published strains (EL-Sharoud et al.,

2009), it differed from that of P. kudriavzevii MTCC 642.

The PCR amplification of 5.8S ITS region of the genomic DNA of H. guilliermondii G4

yielded 775 bp (Fig. 2.4; lane 7) fragment (Table 2.4). The result is in agreement with that H.

guilliermondii 11029T reported earlier (Esteve-Zarzoso et al., 2001). The PCR product remained

uncut after digestion with HaeIII (Fig. 2.5; Lane 7; and Table 2.4). The result is in accordance

with the works reported previously by Esteve-Zarzoso et al., 2001. The digestion with Hinf I

produced four bands of approximately 385 bp, 200 bp, 160 bp and 100 bp (Fig. 2.6, Lane 1; and

Table 2.4) similar to previous result (Esteve – Zarzoso et al., 2001). The PCR product remained

uncut after digestion with Pst I (Fig. 2.7, Lane 1; and Table 2.4). The results of PCR-RFLP

analysis of 5.8S ITS region is summarized in Table 2.4.


Page//68

Figure 2.4 PCR of 5.8S ITS region using ITS1 and ITS4 primers, PCR products were separated on 1% Agarose gel. From left to right Lane 1, Pichia kudriavzevii MTCC 642; Lane 2, Saccharomyces cerevisiae MTCC 211; Lane 3, S. cerevisiae MTCC 180; Lane 4, S.

cerevisiae MTCC 178; Lane 5, S. cerevisiae H 15; Lane 6, S. cerevisiae H 17; Lane 7, Hanseniaspora guilliermondii G4; Lane 8, P. kudriavzevii H21L; Lane 9, S. cerevisiae 18VSL; Lane 10, S. cerevisiae KpYand Lane M, 100 bp DNA ladder.

Figure 2.5 PCR/RFLP of ITS region with Hae III restriction enzyme, products was

separated on 3% agarose gel. From left to right: Lane 1, Pichia kudriavzevii MTCC

642; Lane 2, Saccharomyces cerevisiae KpY; Lane 3, S. cerevisiae 18 VSL; Lane 4,

S. cerevisiae H 15; Lane 5, S. cerevisiae H 17; Lane 6, S. cerevisiae MTCC 178;

Lane 7, Hanseniaspora guilliermondii G4; Lane 8, P. kudriavzevii H21L; Lane 9, S.

cerevisiae MTCC 180; Lane 10, S. cerevisiae MTCC 211 and Lane M, 100 bp DNA.

ladder .


Page//69

Figure 2.7 PCR/RFLP of ITS region with pstI restriction enzyme, product were separated on

3% agarose gel. From left to right: Lane 1, Hanseniaspora guilliermondii G4; Lane 2, P.

kudriavzevii MTCC 642; Lane 3, Pichia kudriavzevii H21L; Lane 4, Saccharomyces

cerevisiae KpY; Lane 5, S. cerevisiae 18VSL; Lane 6, S. cerevisiae H15; Lane 7, S.

cerevisiae H17; Lane 8, S. cerevisiae MTCC 178; Lane 9, S. cerevisiae MTCC 180; Lane 10,

S. cerevisiae MTCC 211 and Lane M, 100 bp DNA ladder.

Figure 2.6 PCR/RFLP of ITS region with HinfI restriction enzyme, products was

separated on 3% agarose gel. From left to right: Lane 1, Hanseniaspora guilliermondii

G4; Lane 2, Pichia kudriavzevii MTCC 642; Lane 3, P. kudriavzevii H21L; Lane 4,

Saccharomyces cerevisiae KpY; Lane 5, S. cerevisiae 18VSL; Lane 6, S. cerevisiae H15;

Lane 7, S. cerevisiae H17; Lane 8, S. cerevisiae MTCC 178; Lane 9, S. cerevisiae MTCC

180; Lane 10, S. cerevisiae MTCC 211 and Lane M, 100 bp DNA ladder.


Page//70

Table 2.4 Molecular typing of yeast strains isolated from Handia

Strains PCR of PCR/RFLP PCR/RFLP PCR/RFLP

ITS (bp) of ITS with of ITS with of ITS with

Hae III (bp) Hinf I (bp) pst I (bp)

S. cerevisiae KpY 880 320 – 240 – 180 – 140 370 – 370- 130 880

S. cerevisiae 18VSL 880 320 – 240 – 180 – 140 370 – 370- 130 880

S. cerevisiae H15 880 320 – 240 – 180 – 140 370 – 370- 130 880

S. cerevisiae H17 880 320 – 240 – 180 – 140 370 – 370- 130 880

S. cerevisiae MTCC 178 880 320 – 240 – 180 – 140 370 – 370- 130 880



P. kudriavzevii H21L 500 380 – 100 250 – 150 500

P. kudriavzevii MTCC 642 500 320 – 100 280 – 130 500

H. guilliermondii G4 775 775 385 – 200 – 160 - 100 775

The RFLP fingerprinting profile of 5.8S ITS region of the isolate P. kudriavzevii H21L

was found to be different from that of P. kudriavzevii MTCC 642 but simiar to the previous

works. When 26S rRNA gene sequence of P. kudriavzevii H21L was analyzed, it was found that

the strain differed from the sequences of P. kudriavzevii strains in GenBank. In order to confirm

whether H21L is a new strain, 5.8S-ITS region of P. kudriavzevii H21L was thus sequenced.


Page//71

2.4.3.2.3 Analysis of 5.8S ITS rRNA gene sequence

In order to confirm the identity of P. kudriavzevii H21L, The PCR amplified product of

5.8S ITS region from P. kudriavzevii H21L was sequenced and analyzed by BLASTprogram

(Fig. 2.9). It was found that H21L had 98% identity with the P. kudriavzevii ZA020 in the

GenBank. The 5.8 S-ITS sequence of Pichia kudriavzevii H21L was deposited to GenBank

(accession number JN164664).

2.4.3.2.4 RAPD-PCR using M13 primer

S. cerevisiae KpY, S. cerevisiae 18VSL, S. cerevisiae H15 and S. cerevisiae H17, S.

cerevisiae MTCC 178, S. cerevisiae MTCC 180, S. cerevisiae MTCC 211, P. kudriavzevii H21L,

P. kudriavzevii MTCC642 and H. guilliermondii G4 were genetically characterized by RAPD-

PCR using M13-PCR in order to determine their geneological relationship at strain level by

RAPD-PCR using M13 primer (Lieckfeldt et al., 1993). Genomic amplification with primer M13

generated a variable number of PCR products, typically consisting of 7 to 9 distinct bands

distributed within the approximate 500 to 4000 bp region (Fig. 2.8). In RAPD-PCR analysis the

banding pattern was found to be unique for each isolate of S. cerevisiae (Fig. 2.8, Lane 7-10).

The result suggests that all four isolates of S. cerevisiae are different at strain level. RAPD-PCR

of each strain of S. cerevisiae also revealed different genotypic patterns from the standard strains,

S. cerevisiae MTCC 211, S. cerevisiae MTCC 180 and S. cerevisiae MTCC 178 (Fig. 2.8, Lane

4- 6). The result suggests that each strain of S. cerevisiae isolated from Handia is not only

different among themselves but also show differences from the standard strains.

The banding pattern of P. kudriavzevii H21L was different from P. kudriavzevii MTCC

642. The P. kudriavzevii MTCC 642 (Fig. 2.8; Lane 2) showed eight distinct bands whereas it

was nine for P. kudriavzevii H21L (Fig. 2.8; Lane 3) However, both the strains shared one band

in common. The result suggests that these two strains of P. kudriavzevii were different at strain

level.


Page//72

Since the two strains of P. kudriavzevii (P. kudriavzevii H21L and P. kudriavzevii

MTCC 642) showed different banding patterns with identical restriction enzymes, the strain P.

kudriavzevii H21L was further characterized genetically by sequencing of its 5.8S-ITS region. In

BLASTanalysis, partial nucleotide sequence (456 bp) P. kudriavzevii H21L (accession no.

JN164664) showed 98% identity with the sequence of P. kudriavzevii in GenBank (accession no.

FJ697171)(Fig. 2.9). The result suggests that strain H21L is closely related to P. kudriavzevii.

The nucleotide change at position 111 (T in place of C) created two new restriction sites (ApoI

and Tsp5091) in ITS1 region of P. kudriavzevii H21L (JN164664) whereas three restriction sites

(SalI, TaqI and AccI) were lost due to the nucleotide change (T in place of C ) at position 98.

Although Issatchenkia sp. YF04A (DQ667976) possessed the ApoI and Tsp5091 sites at 111

positions, it differed from H21L in having the SalI, TaqI and AccI sites at position 98 (Fig. 2.10).

The alteration of restriction sites at these two positions of P. kudriavzevii H21L will serve as a

marker to distinguish this organism from other strains of P. kudriavzevii in the database. Hence,

P. kudriavzevii H21L isolated from Handia was considered to be a new strain.

Figure 2.8 RAPD-PCR patterns generated by M13 primer. Lane M, 1 Kb DNA

ladder; Lane 1, H. guilliermondii G4; Lane 2, P. kudriavzevii MTCC 642; Lane 3, P.

kudriavzevii H21L; Lane 4, S. cerevisiae MTCC 211; Lane 5, S. cerevisiae MTCC

180; Lane 6, S. cerevisiae MTCC 178; Lane 7, S. cerevisiae H17; Lane 8, S.

cerevisiae H15; Lane 9, S. cerevisiae 18 VSL and Lane 10, S. cerevisiae KpY.


Page//73

>gb|FJ697171.1| Pichia kudriavzevii isolate ZA020 18S ribosomal RNA gene,

partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and

internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene,

partial sequence

Length=517


Identities = 448/456 (98%), Gaps = 0/456 (0%)

Strand=Plus/Plus

Query 1 GGGGGACCTGCGGAAGGATCATTACTGTGATTTAGTACTACCCTGCGTGAGCGGAACGAA 60

||| ||||||||||||||||||||||||||||||||||||| ||||||||||||||||||

Sbjct 13 GGGTGACCTGCGGAAGGATCATTACTGTGATTTAGTACTACACTGCGTGAGCGGAACGAA 72

Query 61 AACAAAAACACCTAAAATGTGGAATATAGCATATAGTTGACAAGAGAAATTTACGAAAAA 120

||||||||||||||||||||||||||||||||||||| |||||||||||| |||||||||

Sbjct 73 AACAAAAACACCTAAAATGTGGAATATAGCATATAGTCGACAAGAGAAATCTACGAAAAA 132

Query 121 CAAACAAAACTTTCAACAACGGATTTTTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAA 180

|||||||||||||||||||||||| | |||||||||||||||||||||||||||||||||

Sbjct 133 CAAACAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAA 192

Query 181 TGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCCCCTTGC 240

||||||||||||||||||||||||||||||||||||||||||||||||||||| | ||||

Sbjct 193 TGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCACATTGC 252

Query 241 GCCCCTCGGCATTCCGGGGGGCATGCCTGTTTGAGCGTCGTTTCCATCTTGCGCGTGCGC 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 253 GCCCCTCGGCATTCCGGGGGGCATGCCTGTTTGAGCGTCGTTTCCATCTTGCGCGTGCGC 312

Query 301 AGAGTTGGGGGAGCGGAGCGGACGACGTGTAAAGAGCGTCGGAGCTGCGACTCGCCTGAA 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 313 AGAGTTGGGGGAGCGGAGCGGACGACGTGTAAAGAGCGTCGGAGCTGCGACTCGCCTGAA 372

Query 361 AGGGAGCGAAGCTGGCCGAGCGAACTAGACTTTTTTTCAGGGACGCTTGGCGGCCGAGAG 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 373 AGGGAGCGAAGCTGGCCGAGCGAACTAGACTTTTTTTCAGGGACGCTTGGCGGCCGAGAG 432

Query 421 CGAGTGTTGCGAGACAACAAAAAGCTCGACCTCAAA 456

||||||||||||||||||||||||||||||||||||

Sbjct 433 CGAGTGTTGCGAGACAACAAAAAGCTCGACCTCAAA 468

Figure 2.9 Sequence alignment of 18S rRNA gene of Pichia kudriavzevii H21L from BLAST

data, compairing with the sequences in GenBank. The changes in the nucleotides are shown by

green colour.

http://www.ncbi.nlm.nih.gov/nucleotide/224611728?report=genbank&log$=nuclalign&blast_rank=1&RID=YTJ91E0501S


Page//74

FJ515204 GGTGAACCTGCGGAAGGATCATTACTGTGATTTAGTACTACACTGCGTGAGCGGAACGAA 60

DQ667972 GGTGAACCTGCGGAAGGATCATTACTGTGATTTAGTACTACACTGCGTGAGCGGAACGAA 60

FM178339 GGTGAACCTGCGGAAGGATCATTACTGTGATTTAGTACTACACTGCGTGAGCGGAACGAA 60

HM053475 GGTGAACCTGCGGAAGGATCATTACTGTGATTTAGTACTACACTGCGTGAGCGGAACGAA 60

FJ697171 GGTGA-CCTGCGGAAGGATCATTACTGTGATTTAGTACTACACTGCGTGAGCGGAACGAA 59

DQ667976 GGTGAACCTGCGGAAGGATCATTACTGTGATTTAGTACTACACTGCGTGAGCGGAACGAA 60

JN164664 GGGGGACCTGCGGAAGGATCATTACTGTGATTTAGTACTACCCTGCGTGAGCGGAACGAA 60

** * *********************************** ******************

FJ515204 AACAAAAACACCTAAAATGTGGAATATAGCATATAGTCGACAAGAGAAATCTACGAAAAA 120

DQ667972 AACAAAAACACCTAAAATGTGGAATATAGCATATAGTCGACAAGAGAAATCTACGAAAAA 120

FM178339 AACAAAAACACCTAAAATGTGGAATATAGCATATAGTCGACAAGAGAAATCTACGAAAAA 120

HM053475 AACAAAAACACCTAAAATGTGGAATATAGCATATAGTCGACAAGAGAAATCTACGAAAAA 120

FJ697171 AACAAAAACACCTAAAATGTGGAATATAGCATATAGTCGACAAGAGAAATCTACGAAAAA 119

DQ667976 AACAACAACACCTAAAATGTGGAATATAGCATATAGTCGACAAGAGAAATTTACGAAAAA 120

JN164664 AACAAAAACACCTAAAATGTGGAATATAGCATATAGTTGACAAGAGAAATTTACGAAAAA 120

***** ******************************* ************ *********

FJ515204 CAAACAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAA 180

DQ667972 CAAACAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAA 180

FM178339 CAAACAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAA 180

HM053475 CAAACAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAA 180

FJ697171 CAAACAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAA 179

DQ667976 CAAACAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAA 180

JN164664 CAAACAAAACTTTCAACAACGGATTTTTTGGTTCTCGCATCGATGAAGAGCGCAGCGAAA 180

************************ * *********************************

FJ515204 TGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCACATT 238

DQ667972 TGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCACATT 238

FM178339 TGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCACATT 238

HM053475 TGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCACATT 238

FJ697171 TGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCACATT 237

DQ667976 TGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCACATT 238

JN164664 TGCGATACCTAGTGTGAATTGCAGCCATCGTGAATCATCGAGTTCTTGAACGCCCCTT 238

***************************************************** * **

Figure 2.10 Multiple sequence alignment of 5.8S ITS region of Pichia kudriavzevii H21L with

the homologous sequences in the GenBank. The green nucleotides represent changes in

restriction sites. The numbering is according to the 5′ end of the submitted sequence (JN164664)

of 5.8S-ITS of P. kudriavzevii H21L.

2.4.4 Physiological, Biochemical and molecular characterization of the bacterial isolates

2.4.4.1 Physiological and Biochemical characterization of the bacterial isolates

All the bacterial strains were found to be Gram-positive (Table 2.5). The isolates BA and

B16 are MR positive while B4 are negative. B4 was the only isolate which was VP (Voges-

Proskauer reaction) positive. Among all the isolates, B4 had the ability to utilize citrate. All

bacterial isolates were capable of growth in the presence of 6.5% NaCl except for B16 which

grew at 4% NaCl. The growth of all the strains was positive on TGE and MRS medium.


Page//75

Table 2.5 Physiological and biochemical characteristics of the bacterial isolates.

Tests BA B4 B16

Gram staining + + +

MR test + - +

VP test - + -

Citrate utilization - + -

Growth in TGE + + +

Growth in MRS + + +

Growth in 2% NaCl + + +

Growth in 4% NaCl + + +

Growth in 6.5% NaCl + + _

‗+‘ indicates growth; ‗- indicates no growth

Biochemical characterization by carbohydrate utilization test

All the bacterial strains fermented glucose, fructose, galactose, D-mannose, sucrose and

maltose as carbon source (Table 2.6). Lactose was utilized by the isolate B16 only. None of the

strains utilized xylose, trehalose, raffinose and arabinose. The isolates B4 and B16 utilized

innuline and it was not utilized by the isolate BA. The isolate B4 and BA utilized D-mannitol

and inositol whereas B4 and B16 could not utilize D-mannitol and inositol. The sugar utilization

profiles of the isolates B4, BA and B16 were similar to previous results (Garvie, 1986;

Schillinger et al., 1989; Zhou et al., 2008). However, the isolates could not be identified based

on these data.


Page//76

Table 2.6 Carbohydrate utilization profile of the bacterial isolates.

Carbon source B4 B16 BA

Glucose + + +

Fructose + + +

Galactose + + +

D-mannose + + +

Sucrose +/- + +

Lactose - + -

Xylose - - -

Trehalose - - -

Maltose + + +

Raffinose - - -

Inuline + + -

Arabinose - - -

D-mannitol + - +

Inositol + - +

‗+‘ indicates growth; ‗-‘ indicates no growth; ‗+/-‘ indicates negligible growth.

2.4.4.2 Molecular characterization of the bacterial isolates

2.4.4.2.1 Analysis of 16S rRNA gene sequence

In order to identify the yeast isolates to the species level the 16S rRNA gene was

amplified by PCR using 27F and 1327R primers, as mentioned in the experimental sections. The

size of the 16S rRNA from all the bacterial strains was 1300 bp. The PCR products for each

isolate were sequenced. The BLASTanalysis showed 100% identity of 16S rRNA gene sequence

of all the isolates with the corresponding sequences in the GenBank. Fig 2.11.A-2.11.C. Based

on the results the isolate B4 was identified as Kocuria sp., BA as Brevibacillus agri, and B16 as


Page//77

Leuconostoc mesenteroides. The level of percent identity of all bacterial strains with the

database is shown in Table 2.7.

>gb|JN390957.1| Kocuria sp. A 71(2011) 16S ribosomal RNA gene, partial

sequence

Length=963


Identities = 527/527 (100%), Gaps = 0/527 (0%)

Strand=Plus/Plus

Query 1 GCGAACGGGTGAGTAATACGTGAGTAACCTGCCCTTGACTCTGGGATAAGCCCGGGAAAC 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 75 GCGAACGGGTGAGTAATACGTGAGTAACCTGCCCTTGACTCTGGGATAAGCCCGGGAAAC 134

Query 61 TGGGTCTAATACTGGATGCTACATGTCACCGCATGGTGGTGTGTGGAAAGGGTTTACTGG 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 135 TGGGTCTAATACTGGATGCTACATGTCACCGCATGGTGGTGTGTGGAAAGGGTTTACTGG 194

Query 121 TCTTGGATGGGCTCACGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACG 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 195 TCTTGGATGGGCTCACGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACG 254

Query 181 ACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTC 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 255 ACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTC 314

Query 241 CTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCC 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 315 CTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCC 374

Query 301 GCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCCACAAGT 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 375 GCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCCACAAGT 434

Query 361 GACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 435 GACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG 494

Query 421 GGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCT 480

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 495 GGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCT 554

Query 481 GCTGTGAAAGCCCGGGGCTTAACCCCGGGTGTGCAGTGGGTACGGGC 527

|||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 555 GCTGTGAAAGCCCGGGGCTTAACCCCGGGTGTGCAGTGGGTACGGGC 601

Figure 2.11.A. Sequence alignment of 16S rRNA gene of Kocuria sp. B4 from BLAST data,

compairing with the sequences in GenBank.

http://www.ncbi.nlm.nih.gov/nucleotide/343469115?report=genbank&log$=nuclalign&blast_rank=1&RID=88YHAVUB01S


Page//78

>gb|HM629394.1| Brevibacillus agri strain B-J-NA4 16S ribosomal RNA gene,

partial sequence Length=1330


Identities = 601/601 (100%), Gaps = 0/601 (0%)

Strand=Plus/Plus

Query 1 ACGGGTGAGTAACACGTAGGCAACCTGCCTCTCAGACTGGGATAACATAGGGAAACTTAT 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 64 ACGGGTGAGTAACACGTAGGCAACCTGCCTCTCAGACTGGGATAACATAGGGAAACTTAT 123

Query 61 GCTAATACCGGATAGGTTTTTGGATCGCATGATCTGAAAAGAAAAGATGGCTTTTCGCTA 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 124 GCTAATACCGGATAGGTTTTTGGATCGCATGATCTGAAAAGAAAAGATGGCTTTTCGCTA 183

Query 121 TCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGC 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 184 TCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGC 243

Query 181 GACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAG 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 244 GACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAG 303

Query 241 ACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAA 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 304 ACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAA 363

Query 301 CGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTCAGGGACGAACACGT 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 364 CGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTCAGGGACGAACACGT 423

Query 361 ACCGTTCGAACAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGT 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 424 ACCGTTCGAACAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGT 483

Query 421 GCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGC 480

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 484 GCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGC 543

Query 481 GCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGGGCTCAACCCCGGTTCGCATC 540

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 544 GCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGGGCTCAACCCCGGTTCGCATC 603

Query 541 GGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAAT 600

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 604 GGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAAT 663

Query 601 G 601

|

Sbjct 664 G 664

Figure 2.11.B. Sequence alignment of 16S rRNA gene of Brevibacillus agri BA from BLAST


http://www.ncbi.nlm.nih.gov/nucleotide/313116304?report=genbank&log$=nuclalign&blast_rank=1&RID=89EDW3R301N


Page//79

> JN990379.1 Leuconostoc mesenteroides strain CR1_1417 16S ribosomal RNA gene, partial sequence

Length=1417

Score = 852 bits (461),Expect = 0.0

Identities = 461/461 (100%), Gaps = 0/461 (0%)

Strand=Plus/Plus

Query 1 TGAGTGGCGAACGGGTGAGTAACACGTGGACAACCTGCCTCAAGGCTGGGGATAACATTT 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 42 TGAGTGGCGAACGGGTGAGTAACACGTGGACAACCTGCCTCAAGGCTGGGGATAACATTT 101

Query 61 GGAAACAGATGCTAATACCGAATAAAACTTAGTGTCGCATGACACAAAGTTAAAAGGCGC 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 102 GGAAACAGATGCTAATACCGAATAAAACTTAGTGTCGCATGACACAAAGTTAAAAGGCGC 161

Query 121 TTCGGCGTCACCTAGAGATGGATCCGCGGTGCATTAGTTAGTTGGTGGGGTAAAGGCCTA 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 162 TTCGGCGTCACCTAGAGATGGATCCGCGGTGCATTAGTTAGTTGGTGGGGTAAAGGCCTA 221

Query 181 CCAAGACAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACAC 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 222 CCAAGACAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACAC 281

Query 241 GGCCCAAACTCCTACGGGAGGCTGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGAT 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 282 GGCCCAAACTCCTACGGGAGGCTGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGAT 341

Query 301 GGAGCAACGCCGCGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTTGTATGGGAAG 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 342 GGAGCAACGCCGCGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTTGTATGGGAAG 401

Query 361 AACAGCTAGAATAGGAAATGATTTTAGTTTGACGGTACCATACCAGAAAGGGACGGCTAA 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 402 AACAGCTAGAATAGGAAATGATTTTAGTTTGACGGTACCATACCAGAAAGGGACGGCTAA 461

Query 421 ATACGTGCCAGCAGCCGCGGTAATACGTATGTCCCGAGCGT 461

|||||||||||||||||||||||||||||||||||||||||

Sbjct 462 ATACGTGCCAGCAGCCGCGGTAATACGTATGTCCCGAGCGT 502

Figure 2.11.C. Sequence alignment of 16S rRNA gene of Leuconostoc mesenteroides B16 from



Page//80

Table 2.7 Comparison of 26S and 16S rRNA-gene sequence of yeast and bacterial isolates to the

closely related species.

Isolates Identity

(%)

Number of bases of

26S rRNA gene

sequenced

Number of

bases of 16S

rRNA gene

sequenced

Closest relative Closest relative

accession number

KpY 100 265 Saccharomyces cerevisiae HQ641267

18VSL 100 366 S. cerevisiae JF757233

H15 100 573 S. cerevisiae HQ711330

H17 100 244 S. cerevisiae EU386759

G1 100 539 Hanseniaspora guilliermondii AB618029

G4 100 556 H. guilliermondii AB618029

H21L 99 565 Pichia kudriavzevii FJ919397

H3 100 446 Candida glabrata HQ641276

H8 100 561 C. glabrata HM591730

H11 100 357 C. glabrata HM591715

H12 100 336 C. glabrata HQ641276

18 VLL 100 297 Candida tropicalis EF644470

BA 100 601 Brevibacillus agri HM629394

B4 100 527 Kocuria sp. JN390957

B16 100 461 Leuconostoc mesenteroides JN990379


Page//81

2.5 Conclusion

In microbiological analysis it was found that Handia fermentation is a result of a

microbial consortium composed of a variety of yeast strains and bacteria. Among yeasts, both

Saccharomyces and non-Saccharomyces yeasts (H. guilliermondii, P. kudriavzevii, C. glabrata

and C. tropicalis) were found in Handia. Among bacterial species B. agri, L. mesenteroides and

Kocuria sp. were found to be associated with Handia fermentation. S. cerevisiae, H.

guilliermondii and P. kudriavzevii have been previously reported as starter cultures for wine

production (Zironi et al., Kim et al., 2008 1993). It has been previously demonstrated that the

specific characteristic and quality of foods and beverages is strain-specific (Beh et al., 2006).

The identification of four different strains of S. cerevisiae from Handia is thus important in this

regard. It is interesting to note that Handia contained a new strain P. kudriavzevii H21L, which

differed from any other reported strain in the database. This was demonstrated by partial

sequence analysis of 26S and 18S rRNA genes.

S. cerevisiae is considered to be generally regarded as safe (GRAS) organism because of

a long history of safe use in food industry. However, the presence of C. glabrata and C.

tropicalis in Handia is a matter of concern to the safety of this drink because of their implications

in human health and disease (Silva et al., 2011). The presence of C. glabrata and C. tropicalis

may thus pose a risk to human health upon consumption of Handia. The poor hygienic condition

and uncontrolled technical process may be the reason for such microbial contamination of

Handia. Pathogenic strains were excluded from this study,

This is the first report to explore the microbial composition of Handia. The study offers

the opportunity to exploit the biotechnological potential of these microorganisms in the

production of beer, wine and other alcoholic beverages.


Page//82

2.6 References

1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search

tool. J Mol Biol 1990; 215: 403–410.

2. Andrighetto C, Psomas E, Tzanetakis N, Suzzi G, Lombardi A. Randomly amplified

polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy

products. Lett Appl Microbiol 2000; 30: 5–9.

3. Barnett JA, Payne RW, Yarrow D. Yeasts: Characteristics and identification. Cambridge

University Press, Cambridge, 2nd. Ed, 1990, p.1002.

4. Barnett JA, Payne RW, Yarrow D. Yeasts: characteristics and identification, Cambridge

University Press, Cambridge, 2000.

5. Beh A L, Fleet GH, Prakitchaiwattana C, Heard GM. Evaluation of molecular methods

for the analyses of yeasts in foods and beverages. Adv Exp Me Biol 2006, 571, 69-106.

6. Belloch C, Barrio E, Garcia MD, Querol A. Phylogenetic reconstruction of the genus

Klyuveromyces: restriction map analysis of the 5.8S rRNA gene and the two ribosomal

internal transcribed spacers. Syst Appl Microbiol 1998; 21:266–273.

7. Clemente-Jimenez JM, Mingorance-Cazorla L, Martinez-Rodriguez S, Las Heras-

Vazquez FJ, Rodriguez-Vico F. Molecular characterization and oenological properties of

wine yeasts isolated during spontaneous fermentation of six varieties of grape must. Food

Microbiol 2004; 21: 149–155.

8. Cadez N, Raspor P, deCock AWAM, Boekhout T, Smith MT. Molecular identification

and genetic diversity within species of the genera Hanseniaspora and Kloekera. FEMS

Yeast Res 2002; 1: 279–289.

9. Combina M, Mercado L, Borgo P, Elia A, Jofré V, Ganga A, Martinez C, Catanis C..

Yeasts associated to Malbec grape berries from Mendoza, Argentina. Int J Food

Microbiol 2005; 98: 1055–1061.

10. Couto MMB, Rezinho RG, Duarte. Partial 26S rDNA restraiction analysis as a tool to

characterize non-Saccharomyces yeasts present during red wine fermentation. Int J Food

Microbiol 2005; 102: 49-56.


Page//83

11. de Llanos FR, Fernandez-Espinar MT, Querol A. Identification of species of the genus

Candida by analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed

spacers. Antonie Leeuwenhoek 2004; 85: 175–185.

12. EL-Sharoud WM, Belloch C, Paris D, Querol A. Molecular Identification of Yeasts

Associated with Traditional Egyptian Dairy Products. J Food Sci 2009, 0, M1-M6.

13. Esteve-Zarzoso B, Belloch C, Uruburu F, Querol A. Identification of yeasts by RFLP

analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Int J

Syst Bactriol 1999; 49: 329–337.

14. Esteve-Zarzoso B, Peris-Toran MJ, Ramon D, Querol A. Molecular characterisation of

Hanseniaspora species. Antonie Leeuwenhoek 2001; 80: 85–92.

15. Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A. Biodiversity and

systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2

domain sequence analysis. Int J Syst Evol Microbiol 2000; 50: 1351–1371.

16. Fernandez-Espinar MT, Esteve-Zaroso B, Querol A, Barrio E. RFLP analysis of the

internal transcribed spacers and the 5.8S rRNA gene region of the Saccharomyces: a fast

method for species identification and the differentiation of flour yeasts. Antonie

Leeuwenhoek 2000; 78: 87-97.

17. Garvie EI. Genus Leuconostoc. In: Sneath et al, (Eds) Bergy‘s Manual of systematic

Bacteriology, 1986; p. 1071-1075.

18. Gonzalez SS, Barrio E, Querol A. Molecular identification and characterization of wine

yeasts isolated from Tenerife (Canary Island, Spain). J Appl Microbiol, 2006.

doi:10.1111/j.1365-2672.2006.03150.x.

19. Granchi L, Bosco M, Messini A, Vincenzini M. Rapid detection and quantification of

yeast species during spontaneous wine fermentation by PCR-RFLP analysis of the rDNA

ITS region. J Appl Microbiol 1999; 87: 949– 956.

20. Guillamon JM, Sabate J, Barrio E, Cano J, Querol A. Rapid identification of wine yeast

species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region.

Archiv Microbiol 1998; 169: 387-392.


Page//84

21. Hopfer RL, Walden P, Setterquist S, Highsmith WE. Detection and differentiation of

fungi in clinical specimens using polymerase chain reaction (PCR) amplification and

restriction enzyme analysis. J Med Vet Mycol 1993; 31: 65–75.

22. James S A, Collins M D, Roberts IN. Use of an rRNA internal transcribed spacer region

to distinguish phylogenetically closely related species of the genera Zygosaccharomyces

and Torulaspora. Int J Syst Bacteriol 1996; 46:189–194.

23. Jeyarama K, Singh WM, Capece A, Romano P. Molecular identification of yeast species

associated with ‗Hamei‘— A traditional starter used for rice wine production in Manipur,

India. Int J Food Microbiol 2008; 124:115–125.

24. Kim DH, Hong YA, Park HD. Co-fermentation of grape must by Issatchenkia orientalis

and Saccharomyces cerevisiae reduces the malic acid content in wine. Biotechnol Lett

2008; 30:1633 – 1638.

25. Kurtzman CP, Boekhout T, Robert V, Fell JW, Deak T. Methods to identify yeasts. In

Yeasts in Food: Beneficial and Detrimental Aspects (T. Boekhout and V. Robert, eds.)

pp. 69–121, Woodhead Publishing, Cambridge, U.K, 2003.

26. Kurtzman CP, Fell JW. Yeast systematics and phylogeny: implications of molecular

identification methods for studies in ecology. In C. Rosa, & G. Péter (Eds.),

Biodiversity and Ecophysiology of Yeasts: The Yeast Handbook. New York: Springer,

2006, p. 11-30.

27. Kurtzman CP, Robnett CJ. Identification and phylogeny of ascomycetous yeasts from

analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie

Leeuwenhoek 1998; 73: 331–371.

28. Kurtzman CP, Robnett CJ. Identification of clinically important ascomycetous yeasts

based on nucleotide divergence in the 5‘end of the large-subunit (26S) ribosomal DNA

gene. J Clin Microbiol 1997; 35: 1216–1223.

29. Kurtzman CP. rRNA sequence comparisons for assessing phylogenetic relationships

among yeasts. Int J Syst Bacteriol 1992; 42: 1–6.

30. Larkin M A, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H,

Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG.

Clustal W and Clustal X version 2.0., Bioinformatics, 2007; 23:2947-2948.


Page//85

31. Latorre-Garcia L, Castillo-Agudo L, Polaina J. Taxonomical classification of yeasts

isolated from kefir based on the sequence of their ribosomal RNA genes. World J

Microbiol Biotechnol 2007; 23: 785–791.

32. Lieckfeldt E, Meyer W, Börner T. Rapid identification and differentiation of yeasts by

DNA and PCR fingerprinting. J Basic Microbiol 1993; 33: 413- 425.

33. Loureiro V, Malfeito-Ferreira M. ―Spoilage yeasts in the wine industry‖. Int J Food

Microbiol 2003; 86: 23–50.

34. Messner R, Prillinger H, Altmann F, Lopandic K, Wimmer K, Molnár O, Weigang F.

Molecular characterization and application of random amplified polymorphic DNA

analysis on Mrakia and Sterigmatomyces species. Int J Syst Bacteriol 1994; 44: 694–703

35. Meyer W, Koch A, Niemann C, Beyermann B, Epplen JT, Börner T. Differentiation of

species and strains among filamentous fungi by DNA fingerprinting. Curr Genet 1991;

19: 239–242.

36. Molina FI, Jong S-C, Huffman JL. PCR amplification of the 3‘ external transcribed and

intergenic spacer of the ribosomal DNA repeat unit in three species of Saccharomyces.

FEMS Microbiol Lett 1993; 108: 259–264

37. Mpofu A, Kock JLF, Pretorious EE, Pohl CH, Zvauya R. Identification of yeasts isolated

from Mukumbi, A Zimbabwean traditional wine. J Sustain Dev Africa 2008; 10: 88-102.

38. Nisiotou AA, Nychas GJE. Yeast populations residing on healthy or Botrytis-infected

grapes from a vineyard in Attica, Greece. Appl Environ Microbiol 2007; 73: 2765–2768.

39. Prillinger H, Moln´ar O, Eliskases-Lechner F, Lopandic K. Phenotypic and genotypic

identification of yeasts from cheese. Antonie Leeuwenhoek 1999; 75: 267–283.

40. Pulvirenti A, Caggia C, Restuccia C, Gullo M, Giudici P. DNA fingerprinting methods

used for identification of yeasts isolated from Sicilian sourdoughs. Ann Microbiol 2001;

51: 107-120.

41. Quesada MP, Cenis JL. . ―Use of random amplified polymorphic DNA (RAPD)–PCR in

the characterization of wine yeasts‖. Am J Enol Vitic 1995; 46: 204-208.

42. Redecker D, Thierfelder H, Walker C, Werner D. Restriction analysis of PCR-amplified

internal transcribed spacers of ribosomal DNA as a tool for species identification in

different genera of the order Glomales. Appl Environ Microbiol 1997; 63: 1756–1761.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Lieckfeldt%20E%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Meyer%20W%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22B%C3%B6rner%20T%22%5BAuthor%5D


Page//86

43. Sabate J, Cano J, Esteve-Zarzoso B, Guillamon JM. Isolation and identification of yeast

associated with Vineyard and winery by RFLP analysis of ribosomal genes and

mitochondrial DNA. Microbiol Res 2002; 157: 267–274.

44. Schillinger U, Holzapfel W, Kandler O. Nucleic acid hybridization studies on

Leuconostoc and heterofermentative lactobacilli and description of Leuconostoc

amelibiosum sp. nov. Syst Appl Microbiol 1989; 12:48-55.

45. Silva S, Negri M, Henriques M, Oliveira R, Willams DW, Azeredo J. Candida glabrata,

Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and

antifungal resistance. FEMS Microbiol Rev, 2011, (doi: 10.1111/j. 1574-

6976.2011.00278.x.

46. Smith CA, Cooper PK, Hanawalt PC. DNA repair. In A Laboratory Manual of Research

Procedures ed. Friedberg, E.C. and Hanawalt, P.C., New York: Marcel Dekker, 1981; 1:

289–305.

47. Tserovska L, Stefanova S, Yordanova T. Identification of lactic acid bacteria isolated

from Katyk, goats milk and Cheese. J Cult Collect 2002; 3: 48-52.

48. Villa-Carvajal M, Querol A, Belloch C. Identification of species in the genus Pichia by

restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal

DNA gene. Antonie Leeuwenhoek 2006; 90: 171–181.

49. Villani F, Aponte M, Blaiotta G, Mauriello G, Pepe O, Moschetti G. Detection and

characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae

isolated from raw cow‘s milk. J Appl Microbiol 2001; 90: 430–439.

50. Weisberg WG, Barns SM, Pelletier BA, Lane DJ. 16S ribosomal DNA amplification for

phylogenetic study. J Bacteriol 1991; 173: 697–703.

51. White TJ, Bruns T, Lee S, Taylor JW. Amplification and direct sequencing of fungal

ribosomal RNA genes for phylogenetics. In: Innis, M. A., Gelfand, D. H., Sninsky, J. J.

and White, T. J. (Eds), PCR Protocols: A guide to methods and applications. Academic

Press Inc San Diego, CA, USA, 1990, pp. 315–322.

52. Wyder MT, Puhan Z. A rapid method for identification of yeasts from Kefyr at species

level. Milchwissenschaft. 1997; 52: 327–330.


Page//87

53. Zhou G, Luo X, Tang Y, Zhang L, Yang Q, Qiu Y, Fang C . Kocuria flava sp. nov. and

Kocuria turfanensis sp. nov., airborne actinobacteria isolated from Xinjiang, China. Int J

Syst Evol Microbiol 2008; 58: 1304–1307.

54. Zironi R, Romano P, Suzzi G, Battistutta F, Comi G. Volatile metabolites produced in

wine by mixed and sequential cultures of Hanseniaspora guilliermondii or Kloeckera

apiculata and Saccharomyces cerevisiae. Biotechnol Lett 1993; 15: 235 – 238.

55. Zott K, Miot-Sertier C, Claisse O, Lonvaud-Funel A, Masneuf-Pomarede I. Dynamics

and diversity of non-Saccharomyces yeasts during the early stages in winemaking. Int J

Food Microbiol 2008; 125: 197–203.

ISOLATION AND IDENTIFICATION OF MICROORGANISMS FROM...

Documents

Transcript of ISOLATION AND IDENTIFICATION OF MICROORGANISMS FROM...