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Isolation and Molecular Characterization of

Xylitol Producing Wild Yeast Strains from
Different Fermented Fruit Juices
Article October 2016
DOI: 10.22205/sijbs/2016/v2/i4/103447

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South Indian Journal Of Biological Sciences 2016; 2(4); 415-423

ONLINE ISSN: 2454-4787

Research Article
Isolation and molecular characterization of xylitol
producing wild yeast strains from different
fermented fruit juices
Kaliyan Barathikannan 1, Ameer Khusro 1, Paul Agastian 1, *

Research Department of Plant Biology and Biotechnology, Loyola College, Nungambakkam, Chennai-34, Tamil
Nadu, India.

* Corresponding author: Paul Agastian E-mail: agastianloyolacollege@gmail.com

Received 21 February 2016; Revised 12 June 2016; Accepted 14 June 2016

Abstract
This study was aimed to isolate potential yeast species from commercial fruits available in
different places of Chennai, India. Fruit juice samples were cultured in yeast extract dextrose
(YED) media. A total of 33 yeast isolates originating from fruit juice were identified by molecular
approaches and screened for the potential yeast for fermenting condition for xylitol production.
For detailed and final identification, the ITS-PCR products were subjected to sequencing followed
by Blast analysis. These genera including Candida (13 isolates), Pichia (7 isolates), Meyerozyma (5
isolates), Saccharomycetes (2 isolates), Hanseniaspora (3 isolates), Saccharomycetales (2 isolates) and
Debaryomyces (1 isolates) as well as three related anamorphic species assigned to phylogenetic
analyzed frominternal transcribed spacer (ITS) region. Based on biochemical test and xylanase
assay, out of 33 isolates, six isolates showed maximum enzyme activity. Among 6 isolates,
Candida tropicalis strain LY15 showed above >300 colony xylose utilization test. HPLC analysis
confirmed the xylitol production from Candida tropicalis strain LY15, indicating the potential role
of the isolate at industrial scale.
Keywords: Fruits juice, Yeast strains, Xylose assimilation, Phylogenetic analysis

1. Introduction
Yeasts have played an important role in industrial development for thousands of years. Yeast is a
group of fungi containing unicellular form as predominant. Yeast is abundant in the environs is
most habitually isolated from sugar rich samples. Several good examples contain fruits berries
and exudates from plants. Certain yeast strains are found in association with soil and insects. In
evaluating a yeast strain for industrial use, exact physiological properties are essential. Most of
the yeasts belong to sub division Ascomycotina of the kingdom Mycotina. The population of
microflora on the substrate always depends upon the pH of the substrate. Yeasts are known to
inhabit fruits because of their acidic nature (Saigal 1994). Fruit surfaces associated yeast isolates
convert wide range of sugars into alcohol with high tolerance of alcohol. Though yeast of
415

different genera Kloeckera, Hansensiaspora, Candida, Pichia are involved but in most cases,
Saccharomyces species dominate the final stage of the fermentation than any other yeast species.
Many types of yeasts are used in various industries such as food, baking and fermentation for
xylitol production. Fungi are thought to respond to oligosaccharides that are initially released
from complex polymers. Xylitol has been considered to trigger the expression of xylanolytic
enzymes (Aro et al., 2005; Margolles-Clark et al., 1997). Traditional methods like morphological,
physiological and biochemical studies used for taxonomic identification of yeast isolates
(Kurtzman et al., 2006; Barnett et al., 1990). A wide range of yeast species belonging to the genus
Candida such as Candida boidinii (Vandeska et al. 1995), Candida guilliermindii (Zagustinaet al.,
2001; Rodrigues et al. 2003), Candida parapsilosis (Oh et al., 1998), Candida peltata (Saha and
Bothast1999) and Candida tropicalis (Kim et al., 2002; Lopez et al. 2004) are well-known for
potential xylitol producer. The present investigation was aimed at the identification and
characterization of 33 indigenous yeast isolates from different fruits collected from different
locations of Tamil Nadu, India. Yeast species were identified and characterized through
molecular tools such as ITS1-5.8S. In this work, more than 33 isolates were investigated for their
xylanase producing ability and growth characteristics on xylose.
2. Materials and methods
2.1. Collection of samples
The different fruits were collected randomly from different fruits of Chennai, India. (Table 1) The
samples were kept in sterile bottles at 4C.
2.2. Isolation of yeasts
The collected fruits were washed and rinsed in distilled water. They were then cut, squeezed and
the juice was extracted in separate sterile flasks and allowed for seven days of fermentation
(Perez & Saguir, 2012). After test they were diluted serially and 0.1 ml of the diluted samples (103, 10-4and 10-5) were plated each on Yeast Extract Dextrose Agar (Yeast Extract 1%, Dextrose 2%,
Agar 2%, pH 6.5; Himedia, Mumbai, India; pH: 7.00.2 and Chloramphenicol: 100 mg/L) medium
and incubated at 30C for 24 to 48 h. The yeast isolates were re-streaked on YED agar to obtain
pure cultures and they were stored as slants supplemented with chloramphenicol for further
studies.
2.3. Morphological characterization
Isolates were sub-cultured on YED agar to check for purity and incubated at 28 2C for 48 h.
Purified cultures were routinely maintained on YED agar slants kept at 4C. The isolates were
stained by lactophenol-cotton blue and methylene blue (0.4%) in order to observe under phase
contrast microscope. The colonies were checked for their colour, outline and other features.
2.4. ITS1 gene amplification and sequence analysis
ITS1 gene amplification was carried out according to the method of (Jeyaram et al., 2008).Yeast
cells from 48 h old fresh colonies growing on YED agar were collected with the sterile tip of
toothpick and suspended in the PCR mixture and used for PCR analysis. Using 1 l each of
forward and reverse primers (ITS1 5-TCCGTAGGTGAACCTGCGG-3, ITS4 5TCCTCCGCTTATTGATATGC-3) (200 pmoll1) (GeNie, BGC02), 0.5 l (3U) of Taq DNA
polymerase (GeNei, MME5J) and 18.5 l of autoclaved deionised water (MilliQ, Millipore India).
The amplified product was cooled at 4 C.

416

Table .1 Isolation and Morphological Colony Characteristics of wild yeast isolates

Isolates Name

Source of Isolation

SAP
SAP9

Manilkara zapota
(Naseberry)

Colony
(10-3)

Morphology

53

Creamy white &

Smooth
Light orange &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
White & Rough
Creamy white &
Smooth
Light yellow &
Smooth
White & Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Light Pink & Smooth

56

SAP

58

FNE43

88

FNE42
FNE41

Phyllanthus emblica
(Indian
gooseberry)

67
59

FNE48

48

NPOM1
FPOM3

54
86

GUA6
RP1

Punica granatum
(Pomegranate)

81
79

POM5

43

POM7

57

FWM17
ORA5

57
Citrus sinensis
(Watermelon)

86

ORA7

68

WM18

68

TAMR
FPA26
FKI35
KIVI2

Tamarindus indica
(Tamarind fruit)
Anana scomosus
(Pineapple)

BG28
FBUT31
PAPY
APP
MANG
B.B

GUA9

64
59

Actinidia deliciosa
(Kiwi)

KIVI6
BG22
BG26

38

57
46

Vitis vinifera (Black

Grapes)

75
73
73

Persea americana
(Butter fruit)
Carica papaya
(Papaya)
Malus
domestica(Apple)
Mangifera indica
(Mango)
Vaccinium
corymbosum
(Blueberry)
Psidium guajava
(Guava)

56
75
78
67
82

80

SETH1

Annona squamosal L.
(Custard Apple)

80

SUR

Saccharum officinarum
L. (Sugarcane)

71

Creamy white &

Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
White & smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth
Creamy white &
Smooth

417

Species identification

ITS

Accession
number

Saccharomycetes sp LY4

438

KJ535098

Saccharomycetes sp LY5

421

KJ535099

Candida inconspicua LY6

390

KJ535100

Candida tropicalis LY1

480

KJ535095

Candida tropicalis LY2

Candida tropicalis LY7

480
508

KJ535096
KJ535101

Saccharomycetales sp LY12

506

KJ734196

Pichia membranifaciens LY3

Candida intermedia LY9

399
487

KJ535097
KJ535103

Candida intermedia LY10

335

KJ535104

Meyerozyma caribbica LY11

540

KJ535105

Meyerozyma caribbica LY24

699

KP053245

Meyerozyma caribbica LY25

684

KP053246

Candida parapsilosis LY14

499

KJ734198

Candida tropicalis LY29

754

KP058928

Pichia membranifaciens LY30

637

KP058929

Pichia manshurica LY33

561

KP058932

Hanseniaspora guilliermondii
LY17
Candida tropicalis LY8

748

KP053238

504

KJ535102

Candida tropicalis LY13

468

KJ734197

Saccharomycetales sp LY18

423

KP053239

Pichia sp LY19

572

KP053240

Candida parapsilosis LY16

Debaryomyces hansenii LY20

493
807

KJ734200
KP053241

Candida saitoana LY21

672

KP053242

Candida tropicalis LY15

505

KJ734199

Hanseniaspora uvarum LY22

803

KP053243

Meyerozyma caribbica LY23

642

KP053244

Meyerozyma guilliermondii LY26

650

KP053247

Pichia kudriavzevii LY27

903

KP053248

Hanseniaspora opuntiae LY28

960

KP053249

Pichia manshurica LY31

678

KP058930

Pichi amembranifaciens LY32

610

KP058931

Products were analyzed on 1% agarose gel containing 0.7 g/mL ethidium bromide and visualized
under UV light. The approximate sizes of amplicons were calculated using standard molecular
weight markers (StepUp 100 bp, GeNei, MDB13). PCR products were purified using PCR
purification kit (GeNei, KT153L). Molecular identification of the new isolate was based on
sequence analysis of the ITS 1 rDNA region. The nucleotide sequences of the ITS1 regions in the
rRNA gene were analyzed directly by sequencing the PCR-amplified fragments. The sequencing
of PCR product was carried out using the ABI 3730xl 96-capillary DNA Analyzers (Applied
Biosytems, Foster city, USA). Chromas 2.1 were used for reading and editing the DNA sequences.
The ITS regions were successfully amplified and sequenced from genomic DNA of the isolated
yeast. The BLAST searches of the sequences were performed at the National Centre for
Biotechnology Information (NCBI) GenBank data library (http://www.ncbi.nlm.nih.gov/BLAST)
using BLASTN with default settings. Sequences were trimmed using Bio EditVersion 7.1.7
software. Phylogenic tree was generated by the neighbor-joining (NJ) method using MEGA
software.
2.5. Biochemical Characterization
Isolates were grown in YEPD for 48 h and then used for further morphological and biochemical
properties of the isolates according tobiochemical and physiological peculiarities following the
manuals for yeast identification by (Kreger-van Rij, 2013) for identification.
2.6. Screening for Xylanase activity on solid media
All the isolates were individually inoculated on 2% (w/v) agar (DifcoBacto) plates containing
xylan from beechwood (X4252 Sigma, USA) (0.1% w/v). All plates also contained minimal salts:
KH 2 PO 4 (1.5% w/v), (NH4)2 SO 4 (0.5% w/v), MgSO 4 .7H2O (0.06% w/v) and CaCl 2 .2H 2 O (0.06%
w/v) (Peterson et al., 2012). This process was replicated for each isolate at each temperature at
28C for 48 hrs. Xylanase activity was visualized by staining the plates with Congo red 1% (w/v)
for 5 mins followed by destaining with 1M NaCl for 15 min. Isolates showing maximum
xylanase productions were selected for xylose assimilation test and xylose reductase assay.
2.7. Xylose assimilation tests
In order to screen xylitol-producing isolates, xylose assimilation tests were performed in complex
medium (yeast extract 20 g/l, xylose 20 g/l, agar 25 g/l.) with xylose as the sole carbon resource.
About six hyper-xylanase producing isolates were selected for xylose assimilation test. These
assays were carried out on both solid and liquid media. Yeast cells were pre-cultured overnight
in liquid YED (dextrose, 20 g/1) medium at 28C and 140 rpm). The pre-cultures were diluted 105 times and then a 200 l drop of each culture was laid on the YPX (xylose, 20 g l) agar plate.
After three days of incubation at 30C, the number of colonies on plate was counted and the
maximal number was number found to be>300.
2.8. Analytical method
Xylose, xylitol and ethanol concentration were determined dual-detection HPLC analytical
approach was employed using the Agilent 1100 series HPLC (Agilent 1100; Hewlett-Packard,
Waldbronn, Germany) on an anion exchange column (HPX-87H) packed with sulfonated
polystyrene-divinyl benzene. Acetonitrile and sulfuric acid were used as the components of the
mobile phase in the HPLC analysis, and other materials including, xylose and xylitol the
standards for the hydrolysates. Xylose and xylitol were used as standards for the dual-detection

418

analysis using HPLC in experiments related to the bioconversion process. Under acidic
conditions using dilute sulfuric acid solution (as catalyst) in a concentration of up to 0.55 N and
at a temperature of up to 130C within a period of 150 minutes and a 10% (w/w) solid
concentration. To simulate the hydrolysate to a xylitol bioconversion medium, xylitol and ethanol
were added to the hydrolysate with concentrations of 50 mg. These samples centrifuged to
separate suspended materials and filtered through 0.2 m pore-sized syringe filters. For the
mobile phase, 8 mmol l-1 H2SO4was used. A mobile phase flow rate of 0.5 ml/min at 30C was
used to determine the concentrations of the major and minor components. This system was
equipped with an RI detector.
2.9. Statistical analysis
All the experiments were carried out in triplicate and data presented are MeanSD.
3. Results and Discussion
In this present work a total of 33 isolates of yeast isolated from different fruit samples. They were
belongs toAscomycota and further identified using biochemical and molecular tests
simultaneously. The fruits are extremely productive in natural ecosystem. From the cultures of
appropriate dilutions of fruit juice samples, each colony having different morphology was
selected for further identification. The amplified PCR products turned out to be approximately
600 bp in length. The partial ITS sequences determined in this study have been deposited in the
GenBank database. The sequence was submitted to NCBI database based on sequence similarity
of the 33 isolates under Ascomycetes. These isolates belong to seven genera including Candida (13
isolates), Pichia (7 isolates), Meyerozyma (5 isolates), Saccharomycetes (2 isolates), Hanseniaspora (3
isolates), Saccharomycetales (2 isolates) and Debaryomyces (1 isolates). Table 1 also indicates the
number of colonies obtained on the agar plates after serial dilution (10-3). Among these 33
isolates, six isolates (listed in Table 1) were chosen for further study based on its xylitol
production. Figure 1 showed phylogenetic relationship among 33 yeast isolates. The biochemical
characteristics of 33 yeast isolates were performed (Table 1). FBUT31, APP, POM5, BG22, TAMR
and PAPY were found to be positive for most of the biochemical and sugar tests carried out.
These isolated were selected for the further experimental analysis. Other isolates showed
negative response for the specific biochemical and sugar assimilation properties (Table 2 and 3).
Xylanase productions for 33 yeasts isolates were performed on agar plate. All the isolates were
showing significant xylanase production. The isolate Candida tropicalis strain LY15 (FBUT31),
Meyerozyma caribbicastrain LY23 (APP), Meyerozyma caribbicastrain LY24 (POM5), Candida
parapsilosis strain LY16 (BG22), Hanseniaspora guilliermondii strain LY17 (TAMR) and Hanseniaspora
uvarum strain LY22 (PAPY) were identified as hyper-xylanase producer based on zone of
clearance (Figure 2). All the 6 isolates selected were tested for their abilities to assimilate xylose.
Isolates showed more colony growth on solid medium with xylose as the sole carbon source.
Yeasts is considered to be the best xylitol producers and have been extensively studied among
xylose-utilizing microorganisms. (Winkelhausen&Kuzmanova, 1998) summarized 22 yeast
isolates selected for xylitol production from previous studies. Most of them belong to the genus
Candida. (Rangaswamy&Agblevor, 2002) secreting xylitol, but xylitol production was found to be
very high in Candida tropicalis strain LY15.
Xylose uptake was reduced with the unavailability of co-substrate. Xylose transport is
also affected by the higher concentration of substrate. In our study 33 isolates belonging to six
different genera not only produce xylitol with high conversion efficiency but also were easily
studied for large-scale xylitol production.

419

Fig.1. Phylogenetic (NJ) tree showing the positions of the new isolates and related species based on ITS
sequences.

According to Guo et al. (2006), C. trophicali and C. maltose APP were selected as promising
xylitol producers with potential for industrial application. Both isolates showed different
characteristics of xylose utilization. C. maltose Xu316 exhibited a higher xylose consumption rate
while C. guiiliermondii Xu280 had a higher xylitol yield and produced lower concentration of byproduct. Our present investigation favours the finding of Altamirano et al. (2000) who
demonstrated that C. tropicalis isolates were found to be a potential producer of xylitol (de Mello
Loureno et al., 2014). Azuma et al. (2000) reported xylitol production of 256 g L-1using Candida
sp. over a period of 276 h in a flask culture, which is the highest previous reported xylitol yield
from xylose using fed-batch fermentation. Kang et al., 2005 reported a 154 g L-1 xylitol
production from 200 g L-1 xylose in 60 h using C. tropicalis HY200. The rate of xylitol production
of our isolates varies from the previous researches. It may be due to the reason that the capacities
of xylitol production of selected isolates depend on the existence of a compound regulation of
xylose catabolism even in the same species. Out of 33 isolates of 6 different genera, one species
was further selected for xylose reductase assay based on maximum xylitol production.

420

Table 2. Biochemical characteristics of wild yeast strains

Isolate
Name
FNE43
FNE42
NPOM1
SAP7
SAP
SAP
FNE41
FPA26
FPOM3
GUA6
RP1
FNE48
FKI35
FWM17
FBUT31
BG22
TAMR
KIVI2
KIVI6
BG26
BG28
PAPY
APP
POM5
POM7
MANG
B.B
GUA9
ORA5
ORA7
SETH1
SUR3
WM18

+++
++
+++
+
++
+++
-

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

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

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

+
+
+
+
+
+

+
+++

++
+++
+
+++

+
+
+++

+
+++
+++

+
+
-

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

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

+++
+++
+
+
+

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

+++
+++
+++
++
++
-

10

11

12

++
+++

+
+
+
+
++
++
+
+

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

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

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

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

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

+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+

+
+
+
-

+
+
+
+
+
-

++
+
+
++
++
+++

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

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

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

+
++
++
+
+
++

+
+
+

+
+
+
++
++

+
+

+++
+
+
+

++
++
+
+
+
+

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

+
++
++
+
+
++

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

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

++
+
+
+
+
-

1; ONPG, 2; lysine utilization, 3; orinthine utilization, 4; urease, 5; phenylalanine, 6; nitrate reduction, 7; H 2 S production,
8; citrate utilization, 9; voges proskauer's, 10; methyl red, 11; indole, 12; malonate utilization

Table 3. Assimilation of different carbon sources by yeast strains isolated from commercial fruits.
Isolate
Name
FNE43
FNE42
NPOM1
SAP7
SAP
SAP
FNE41
FPA26
FPOM3
GUA6
RP1
FNE48
FKI35
FWM17
FBUT31
BG22
TAMR
KIVI2
KIVI6

1
+
+
++
++
+
++
++
+
+
+
+++
+++
+++
+++
-

10

11

+
++
++

++
++
++

+
+
+

++
+
+

++
+
-

+
+
-

++
+++
++

+++
+
++

++
+++
+++

++

++

++

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

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

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

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

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

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

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

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

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

421

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

12
+++
+++
+
++
++
+
++
+
++
+++
+
+
+++
+++
+

BG26
BG28
PAPY
APP
POM5
POM7
MANG
B.B
GUA9
ORA5
ORA7
SETH1
SUR3
WM18

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

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

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

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

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

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

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

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

+
++
+++
+
+++
++
++
++
+
+++
++
-

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

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

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

1; Esculin hydrolysis, 2; Arabinose, 3; Xylose, 4; Adonitol, 5; Rhamnose, 6; Cellobiose, 7; Melibiose, 8; Saccharose, 9;

Raffinose, 10; Trehalose, 11; Glucose, 12; Lactose

Fig.2. Different steps of yeast characterization. A. serial dilution of plates (104), B. Pure cultures, C.
Methylene blue stain under Light Microscope (100X), D. Xylanase activity in Plate.

4. Conclusion
Xylitol production involves complicated metabolic regulation including xylose transport and key
enzymes. Novel isolates with high levels of xylitol production were identified from different
fruits by screening of naturally occurring xylose-utilizing micro-organisms. All isolates exhibited
different characteristics of xylose fermentation. Based on the above results, out of 33 different
isolates, Candida tropicalis strain LY15 were identified as potential isolate for xylitol production.
Further study is in progress to optimize the production of xylose reductase from Candida tropicalis
strain LY15 using various agro-wastes.

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Acknowledgements
We would like to thank Department of Biotechnology, India (Indo-Spain Collaborative project,
Ref. No. - BT/IN/Spain/19/PA/2013) for financial assistance.
Conflict of interest statement
We declare that we have no conflict of interest.
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