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Wild strains of fermenting yeast isolated of sugar cane juice from an
Silva, R. O. et al.
Vol. 2, N. 3: pp. 22-27, August 2011
ISSN: 2179-4804
22
Journal of Biotechnology
and Biodiversity
Wild strains of fermenting yeast isolated of sugar cane juice
from an alcohol distillery from Mato Grosso, Brazil
Rosimeire Oenning da Silva1,2, Margareth Batistote3 and Marney Pascoli Cereda4*
1,2
Master Program of Biotechnology; Catholic University of Campo Grande - UCDB; 79117-900; Campo Grande MS - Brasil. 2,3State University of Mato Grosso – UNEMAT; 78390-000; Barra dos Bugres - MT - Brasil. 3State
University of Mato Grosso do Sul - UEMS; CEP: 79.730-000; Gloria de Dourados - MS - Brasil. 4Master Program
of Biotechnology and CeTeAgro - Center of Technology and Agribusiness Analysis, Catholic University of Campo
Grande - UCDB; 79117-900; Campo Grande - MS - Brasil.
ABSTRACT
Ethanol production for fuel is important for the Brazilian economy as a renewable solution to oil derivatives. The
industrial process uses commercially available yeast, most of them isolated from sugar mills from the Brazilian state
of São Paulo. The research information on the availability of yeast appropriate to the climatic conditions occurring
in the Midwest region of Brazil is scarce. The article presents the isolation and morphological characterization of
fermentative wild yeast prospected on the sugar cane juice of a sugar and alcohol mill of Mato Grosso State. The
juice samples were collected in both the hot and rainy (HR) season and in the cold and dry (CD) one. The diluted
sugar cane juice samples were plated on agar LWYN and evaluated for fermentation in test tubes containing
inverted Durham tubes. From 26 morphotypes isolated, 50% were able to ferment sugar cane juice. Greater number
and diversity of yeasts was found in both December (HR) and July (CD). In the remaining months of each season
there were a low number of colonies with a poor diversity.
Key words: Isolation, morphology, bioprospecting, biodiversity, fermentation
INTRODUCTION
The ethanol for fuel is renewable, cheaper and
less polluting them the oil. The yeast plays a
very important role in their production by
fermentation. In biotechnological processes, the
bioprospecting is a valuable tool used to find
agents with potential for industrial process.
With the biotechnology progress yeast strains
may be selected by its desirable characteristics.
The selection of suitable yeast for each kind of
fermentation is an important strategy to ensure
good fermentation and process yield. In Brazil
the commercial production of ethanol for fuel
uses the yeast Saccharomyces cerevisiae as
agent, in granular form or in pressed humid
tablets.
The inoculation allows establishing a high
population of a selected strain of yeasts in order
to ensure their dominance in the process,
resulting in a fast fermentation and high alcohol
production (Dorneles et al., 2005).
Although a new front is opening up for sugar
and ethanol industry in Brazilian Midwest, little
research has been done towards search for
specialized agents adjusted to the environmental
conditions of the region. To support the
implementation of ethanol sector in this region,
the selection of more suitable agents is crucial,
since weather conditions can cause stress of
commercial yeasts strains and interfere in the
fermentation performance.
During the fermentation process is common the
occurrence of wild yeast comes from the sugar
cane juice and the environment. Since the
conditions in the industries lack of asepsis it
favors the predominance of wild yeast in the
fermentation, replacing the inoculated yeast
(Ceccato-Antonin et al., 2004).
_______________________________________________
Author for correspondence: [email protected]
J. Biotec. Biodivers. v. 2, N.3: pp. 22-27, Aug. 2011
Silva, R. O. et al.
This wild yeast makes the bioprospecting an
advantageous tool to obtain strains with
potential for use in alcohol fermentation. The
inoculum using native strains with fermentative
capacity is an alternative for solving the
problems of contamination by yeast with low
fermentative capacity and avoids costly
reductions in alcohol yield (Andrietta et al.,
1995).
The fermentative capacity and power of
dominance in the fermentation are both basic
prerequisites for select yeasts to be used in
fermentation processes of ethanol production
(Silva Filho, 2004).
Pataro et. al. (2000), reports that most of the
yeasts isolated from the fermentation processes
are physiologically adapted to the conditions in
that they are founded. However, it is possible
that this characteristic may differ for yeasts
founded in early or late stages of the
fermentation.
Cabrini and Gallo (1999), isolated yeasts from
the Pedra sugar mill with an alcohol distillery
(Serrana, São Paulo State) from samples of
sugar cane juice, centrifuged suspension of yeast
and wine. The authors identified 72 yeasts
belonging to five genera. The species
Saccharomyces cerevisiae was the predominant
yeast in this industry and the genus
Saccharomyces had highest incidence.
This wild yeast is considered contaminants of
the process. In a distillery in São Paulo State a
strain of Saccharomyces cerevisiae was isolated
from a continuous fermentation process. It was
founded as the responsible for 10% reduction in
fermentation yield and 18% in ethanol
production. The authors point out that not
always those wild yeasts are harmful.
Sometimes strains are unnoted because it are
compatible with the inoculated yeast or even
more resistant to adverse conditions of the
medium that the commercial ones (Parazzi and
Oliveira, 1996).
After the isolation, the preservation of the
available microorganisms is of fundamental
importance in the prospective studies. Among
the preservation methods, the most commonly
used is the continuous ringing, water
conservation, preservation in mineral oil,
freezing at -20 °C, cryopreservation in liquid
nitrogen and lyophilization. According to Borba
(2000), the selection of appropriate methods for
23
maintenance of fungi is based on phenotypic
characteristics inherent to each, as well as the
behavior of each species in relation to the
methods of preservation.
Lyophilization as method for yeast preserving
provides high death rate, but the survival viable
cells remain stable during the storage period
(Costa and Ferreira, 1991; Silva et al., 1992).
Freeze drying is one of the most efficient
methods for obtaining the conservation for
extended periods of time. This efficiency is due
to the removal of intracellular water by
sublimation that prevent ice crystals formation,
which damage the enzymes located in the
cytosol of the cell (Costa and Ferreira, 1991).
If diversity of fermentative yeasts in the state of
Sao Paulo is explored and commercialized, on
the contrary the knowledge about wild yeasts in
Mato Grosso is scarce. To reduce the differences
and make the sector more competitive, it is
important to isolate strains appropriated to the
local weather conditions.
With this objective it was performed the
isolation and morphological characterization of
strains of wild fermentative yeast from sugar
cane juice of a sugar mill with attached distillery
from Barra do Bugres state of Mato Grosso,
Brazil.
MATERIAL AND METHODS
The sugar mill with attached distillery is located
at the city of Barra do Bugres, Mato Grosso, on
15°04'21 south latitude and 57º10'52" west
longitude and 171 km of the Capital Cuiaba. The
industry produces alcohol, sugar, electrical
energy and biodiesel, justifying the harvest
period from March to early December.
Available sugar cane was a mixture of varieties
SP79-1011, RB 86-7515 RB 75-5113, SP 835073, SP-8642 and RB 92-8064. Sample were
collected for yeast selection during all the
harvest period, bimonthly from December 2009
to November 2010, covering the hot and humid
season (September, November, December and
March) and the dry and cold season (May, July).
The data of temperature and relative humidity in
all the sampling period was provided by the
agricultural sector of the same industry.
Collection and plating
The sugar cane juice was collected in sterilized
flakes and the samples transported at low
J. Biotec. Biodivers. v. 2, N.3: pp. 22-27, Aug. 2011
Silva, R. O. et al.
temperature to the Microbiology Laboratory of
the State University of Mato Grosso
(UNEMAT) located near 4 km from the
industry. The culture medium used for isolation
Saccharomyces to develop. The formulation is
2.0 g of malt extract, 4.0 g of yeast extract, 2.0 g
peptone, 1.0 g glucose, 1.0 g potassium
phosphate dibasic, 0.5 g of ammonium chloride,
20.0g agar, 6 mg of crystal violet solution, 0.1 g
of fuchsin-sulfite mixture, 5 mg of nalidixic acid
solution and 50mg of ampicillin solution. The
formula of culture medium described by
(Ceccato-Antonini et al., 2004) was adapt by
adding ampicillin (500mg / l) to inhibit bacterial
growth as described by (Silva and Cereda,
2009).
The sugar cane juice collected was serially
diluted in saline (0.85%) up 10-10. Petri dishes
containing modified LWYN were inoculated
with 0.1 ml of each dilution, spread with a
Drigaslky handle. The plates were incubated at
30 °C for 48 to 72 hours (APHA, 2001).
Isolation and purification of colonies
After the incubation period colonies were
selected from those plates showing the better
distribution. A colony from each morphotypes
was isolated, suspended in 2.0 ml saline (0.85%)
and plated on medium LWYN by at least twice
for confirmation of culture purity.
Morphological characterization
The purified colonies were described in relation
to morphological features such as edge, color,
texture and shine.
Fermentation test
Al colonies of wild yeasts isolated as
morphotypes were evaluated for ethanol
fermentation. The same sugar cane juice was
adjusted to 12°Brix with distilled water. A
volume of 10.0ml of diluted sugar cane juice
December and July presented the greater number
and yeasts diversity, with 12 and 10 isolates
respectively, near 50% showed a strong
fermentation. The climate conditions only
cannot explain the concentration of yeasts in
these months. In September there was founded
less diversity with a few yeasts considered as
morphologically different. This month (Table 1)
showed the lowest values of relative humidity
(18%) and higher temperature (42 ºC). From the
24
and morphological characterization was the Lin
Wild Yeast Medium (LWYN) described as a
differential medium, allowing only the wild
yeast
of
the
genus
was transferred for test tubes containing an
inverted Durham tube. The set was autoclaved
120ºC per 20 minutes. Dilutions were made for
each morphotypes using saline solution tubes
and two heave taken as way to provide cell
concentration corresponding near to the scale
5.0 Mac Farland. Tubes in triplicate were
incubated at 30 °C for 72 hours. The criterion
for selection of yeast as fermentative was the
turbidity of the medium and production and gas
retention in Durham tube.
Maintenance of yeast
Only morphotypes with positive evaluation for
fermentation were received letters as
identification and were kept under refrigeration
(4 °C) in inclined tubes with sterile PDA
medium until its reactivation for further
analysis.
Preservation of yeasts
The evaluated yeasts were preserved by
lyophilization (Costa and Ferreira, 1991) by
suspending the yeasts from PDA inclined tubes
in 3 ml of 10% (w/v) skimmed milk Lakbom,
put in Ependorffs tubes placed in a ultra freezer
for thirty minutes and freeze-dried in a Biotop
Biobrás model 101 L for eight hours.
RESULTS AND DISCUSSION
From 28 morphotypes isolated only 16
presented fermentative metabolism. These
were morphologically described and
designated BB. 01 BB.16. Table 1 present
the morphotypes, the months were they are
collected and the local meteorological
conditions.
colonies grew in the plates, only one was
considered as a different morphotypes, but
without fermentation ability. November
provided isolation of several colonies with
different morphotypes, however, only one was
considered as a different morphotypes if
compared to that had been isolated previously.
J. Biotec. Biodivers. v. 2, N.3: pp. 22-27, Aug. 2011
Silva, R. O. et al.
25
Table 1. Morphotypes isolated from each period and the local meteorological conditions.
Precipitation
Temperature
Air Humidity
Months
Number of
Min - Max
Min - Max
morphotypes
Average
Average
Isolates Fermented
(mm)
(ºC)
AH (%)
December / 2009
10C
05B
1,0 – 49,0
16,2 – 40,0
72A
5,37C
27,5A
March/2010
01A
01A
1,0 – 30,0
22,2 – 39,7
68A
5,26C
27,4A
May/2010
03A
02A
2,0 – 48,0
8,7 – 38,0
31B
2,47B
24,8B
9,9 – 39,2
28B
July/2010
12C
07B
0,0 – 8,40
24,2 B
0,27B
16,2 – 42,6
18B
September 2010
01A
00A
0,0 – 0,0
29,5A
0,00A
01A
1,0 - 100
19,4 – 38,7
73A
Novembro/2010
01A
5,47C
27,3A
Numbers followed by the same letters in column do not differ by Tukey test at 5% probability.
Table 2. Morphological characterization of yeasts colonies cultivated in Lin Wild Yeast Medium(LWYN) incubated at 30 °C for 72 hours.
Strains
Diameter
Texture
Color
Surface
Board
BB. 01
1–7 mm
Rough
Cream
Flat
Irregular
BB. 02
1–5 mm
Rough
Cream
Flat
Irregular
BB. 03
1 - 5 mm
Rough
Cream
Flat
Irregular
BB. 04
1 – 5 mm
Smooth
White
Convex
Regular
BB. 05
1 - 3 mm
Smooth
Cream
Convex
Regular
BB. 06
1 - 3 mm
Smooth
Pink
Convex
Regular
BB. 07
1 – 5 mm
smooth
Pink
Convex
Regular
BB. 08
1 – 2 mm
smooth
Cream
Convex
Regular
BB. 09
1 – 3 mm
smooth
Cream
Convex
Regular
BB. 10
2 – 4 mm
smooth
White
Convex
Regular
BB. 11
2 – 3 mm
smooth
Cream
Convex
Regular
BB. 12
2 – 3 mm
smooth
Cream
Convex
Regular
BB. 13
2 - 3 mm
smooth
Cream
Convex
Regular
BB. 14
1 – 3 mm
smooth
Cream
Convex
Regular
BB. 15
2 – 3 mm
smooth
Cream
Convex
Regular
BB. 16
2 – 3 mm
smooth
Cream
Convex
Regular
It is well reported that the medium and
cultivation conditions may influence the
morphological characteristics of colonies as size,
shape of lips, color or surface texture. As all
morphotypes were cultured in the same medium
and conditions, the comparison is possible.
Table 2 presents the description of yeasts
colonies with fermentative activity. Figure 1
illustrates some of the morphological types of
colonies.
From the characterized colonies 18.7% showed
irregular edges, flat and wrinkled texture.
The medium used may have influenced the color
of the colonies, making difficult to differentiate
the colonies. The major of the colonies (75%)
presented cream color, 12.5% a white color, and
12.5% slightly pink. The diameter of colonies
ranged from 1 to 7 mm.
The large majority (81.3%) had regular edges,
convex surface and smooth texture. These
results agree with those obtained in the yeasts
J. Biotec. Biodivers. v. 2, N.3: pp. 22-27, Aug. 2011
Silva, R. O. et al.
isolation from 46 sugar mill in Sao Paulo State
in the harvests 2002, 2003 and 2004. In this
bioprospecting of 300 wild yeast identified by
karyotyping, 57% showed colonies with regular
edges, while 43% had irregular borders
(Fermentec News, 2005).
Morphological characterization is an important
tool for classic identification of yeasts.
Cid et al. (1994), has analyzed different strains
and observed changes for different phenotypes
as a consequence of morphological differences.
Similarly the same authors isolated and
characterized morphologically 10 strains of wild
yeast from four sugar cane liquor (cachaça)
distilleries in Jaboticabal, São Paulo State. All
yeast strains presented convex colonies with
smooth texture. The color ranged from white to
pink colonies and the diameter of 1-3 mm. It
was observed different growth behavior as
consequence of different mediums used in the
identification of the yeasts.
The characterization of yeast is very important
to found similarities and differences between the
microorganisms. In an environment with high
biodiversity as the found in the alcoholic
fermentation in which many organisms may live
together, the knowledge of the physiological and
morphological characteristics may help to
choice methods and techniques for control or
stimulate the yeast in the process (CeccatoAntonini, 2010).
CONCLUSIONS
It was concluded that bioprospecting of sugar
cane juice is potentially a suitable tool for select
fermentative yeasts. Sixteen morphotypes was
selected by its ability to ferment. The mouths of
December and July were the best for
bioprospecting and presented the high number of
different colonies. This result was not explained
only by the weather conditions. In the best
period it was isolated 56.2% of the yeasts with
worm temperature with high humidity but the
second one (43.8%) presented a wet season
(November to March). The yeasts showed
similar morphological features and the most
frequent colonies were smooth, shiny, with a
convex surface and regular edges.
ACKNOWLEDGEMENTS
The authors thank the plant Barrálcool for
cooperation in research and stock provided; To
26
undergraduate students of Food Engineering of
UNEMAT - Marcel, Deborah, Gracie, and Laide
Stéfanie - for his help with laboratory activities.
Figure 1 - Morphotypes of yeasts colonies grew in
LWYN for 72 hours at 30º C for yeast BB 01 (A),
BB 04 (D) and BB 05 (G), details of colonies (B, E,
H) and cells (C, F, I).
RESUMO
A produção de etanol é importante para a economia
nacional constituindo-se em solução renovável para
complementar derivados de petróleo. O processo
industrial
utiliza
leveduras
disponíveis
comercialmente, a maioria das quais isoladas em
usinas do estado de S. Paulo. Não se têm
conhecimento sobre a disponibilidade de leveduras
selecionadas para as condições climáticas da região
Centro Oeste. O artigo apresenta os resultados do
isolamento e caracterização morfológica de leveduras
selvagens fermentativas obtidas do caldo de cana da
usina do município de Barra do Bugres, Mato
Grosso. As amostras de caldo foram coletadas na
época quente e chuvosa e na fria e seca da mesma
safra. Foram semeadas em agar LWYN e avaliadas
para fermentação em tubos de ensaio contendo tubos
de Durhan invertidos. Apenas aquelas com habilidade
para fermentar percebidas pela presença de bolhas de
gás foram caracterizadas. Maior número e
diversidade de leveduras foram obtidas nos meses de
dezembro e julho, mas, apenas as condições
climáticas não explicaram esses resultados. Concluiuse que o caldo de cana é uma boa fonte para
biodiversidade em leveduras.
Palavras-chave:
Isolamento,
morfologia,
bioprospecção, biodiversidade, fermentação
J. Biotec. Biodivers. v. 2, N.3: pp. 22-27, Aug. 2011
Silva, R. O. et al.
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