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Characteristics of Saccharomyces cerevisiae Yeasts Exhibiting Rough Colonies and

Pseudohyphal Morphology in Respect to the Alcoholic Fermentation

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Characteristics of rough colonies of S. cerevisiae yeasts

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Vanda Renata Reis, Ana Paula Guarnieri Bassi, Jessica Carolina Gomes da Silva,

Sandra Regina Ceccato-Antonini*

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Dept Tecnologia Agroindustrial e Socio-economia Rural, Centro de Cincias Agrrias,

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Universidade Federal de So Carlos, Via Anhanguera km 174 13600-970 - Araras So

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Paulo Brazil

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*Corresponding author:

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Phone: +55 19 35432676

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E-mail: antonini@cca.ufscar.br

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ABSTRACT

Among the native yeasts more commonly found in the alcoholic fermentation, rough colonies

associated with pseudohyphal morphology belonging to Saccharomyces cerevisiae species are

highlighted. This work aimed to perform the morphological/physiological characterization of

S. cerevisiae strains which exhibited rough and smooth colonies, searching for alternatives

that could contribute to the management of these yeasts (rough colonies) in the alcoholic

fermentation. Characterization tests for invasiveness in Agar medium, killer activity,

flocculation and fermentative capacity were carried out with 22 strains (11 rough and 11

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smooth colonies). The effect of the acid treatment using different pH values over the growth

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of two strains (52 rough and PE-02 smooth) was also evaluated as well as a batch

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fermentation with cell recycling. The invasiveness in YEPD Agar medium occurred in low

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frequency; ten out of eleven rough yeasts exhibited expressive flocculation; none of the

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strains showed killer activity; rough strains presented lower and slower fermentative capacity

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comparing to smooth strains, in a 48-hour cycle in batch system using sugar cane juice. The

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rough strain was severely affected by the acid treatment in pH values of 1.0 and 1.5, but the

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same did not occur with the smooth colony. The fermentative efficiency in mixed

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fermentation (smooth and rough strain in the same cell mass proportion) did not differ from

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the efficiency obtained with the smooth strain alone, probably due to the acid treatment

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conducted in pH 1.5, in a batch cell-recycle test. A fermentative efficiency as low as 60% was

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observed with the rough colony alone.

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Keywords: S. cerevisiae, ethanol, fermentation, wild yeasts

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INTRODUCTION

Although many efforts to search for new microorganisms, the yeast Saccharomyces cerevisiae

remains the most utilized in the ethanol production in Brazil. It is a robust yeast, capable to

afford stressful conditions, with high fermentation efficiency, rapid growth, effective sugar

metabolization, ability to produce and consume ethanol, tolerance to high ethanol

concentrations and low levels of oxygen, osmotolerance, thermotolerance, and cell activity in

acidic environments, which are fundamental for its industrial utilization [3].

In Brazil, a continuous sugar cane harvest season takes place over a period of about 200 days

to produce fuel ethanol. Very high yeast cell concentrations producing alcohol concentrations

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of 6-11% (v/v) in short fermentation times lasting 6 to 10 hours is one of the main conditions

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of Brazilian distilleries. Another peculiarity is the separation of yeast cells from the fermented

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broth by centrifugation and continuous recycling. The concentrated yeast receives a treatment

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with diluted sulfuric acid for 1-2 hours at pH 2.0-2.5 to kill contaminant bacteria and is re-

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pitched into fermentation tanks. The fermented broth is distilled to obtain anhydrous ethanol

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for fuel [2].

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One of the main concerns in the sugar and ethanol industry is how to avoid and deal with the

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contaminant microorganisms, mainly bacteria and wild yeasts (Saccharomyces and non-

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Saccharomyces), both competing with the selected yeast strains to survive in the fermentors

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[9]. The peculiarities of the Brazilian fermentation process for ethanol production, as the

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successive recycling of yeast cells along the sugar cane harvest and the difficulties to sterilize

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large volumes of juice and water, allow the contaminant microorganisms to enter into the

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process [1].

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Contamination by non-Saccharomyces yeasts might be controlled more easily than by

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indigenous (wild) strains of Saccharomyces, because in the latter the strategies to circumvent

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the contamination can affect the industrial yeast strain, once their metabolism is very similar.

Besides, non-Saccharomyces strains are normally eliminated from the process. However, the

indigenous Saccharomyces sometimes dominate the process, replacing the main strain, which

is not always harmful, since their fermentative performance is somehow similar [4].

Additionally, Saccharomyces yeasts possess a range of responses to enable survival in

deleterious circumstances including filamentation, invasive growth, flocculation, and biofilm

development [16], which are undesirable characteristics for the industry. However, there are

many reports concerning the application of flocculating yeast in the fermentation industry,

especially for beer, wine and distilled beverages [8] but for the ethanol production the use is

still limited [29]. Anyway, the definition of yeast flocculation must be revisited in order to

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distinguish between the flocculent yeast types.

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Yeast flocculation is defined as a nonsexual, homotypic, reversible and multivariate process

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of cell aggregation into multicellular masses called flocs, with the subsequent rapid

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sedimentation from the medium in which they are suspended [24, 25]. This process has

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gained special interest for its biotechnological relevance and was recently reviewed in many

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aspects [8, 24, 29]. In Saccharomyces cerevisiae cells, aggregation as sexual aggregation, co-

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flocculation and cell chain formation should not be confused with flocculation. Of particular

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interest in the present work, cell chain formation is derived from a failure of the younger bud

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to separate from the mother cell, resulting in an aggregate formation of approximately 30-50

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cells, covalently linked, and presenting no re-aggregation after mechanical dispersion [24,

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25]. This is the reason by which the rough variant of S. cerevisiae cells with chain of cells -

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here studied will not be referred to flocculent cells, although the known ability of the cells to a

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rapid sedimentation is called flocculation.

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The colony and cellular morphologies of natural and industrial populations of S. cerevisiae

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strains vary in response to different environmental stimuli. Variants of smooth colonies to

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rough colonies are quite common in this species. Among one thousand strains grown on

YEPD medium, 2.5% exhibited a rough colony phenotype [10]. In Candida albicans, the

colony morphology is known to be related to the cell types: smooth colonies contain only

blastopores while rough colonies consist of different proportions of true hyphae and

pseudohyphae [20]. In fact, the formation of pseudohyphae influences the colony

morphology, which appears as a central body from which numerous branches depart [10].

Saccharomyces cerevisiae strains exhibiting rough colonies and pseudohyphal morphology

have been frequently associated with disturbances in the fermentation process depending on

the fermentation system and other operational conditions. However, a little is known about

their competitive status comparing to typical strains of S. cerevisiae (smooth and bright

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colonies and dispersed cells).

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In this context, this work aimed to evaluate strains of S. cerevisiae exhibiting rough colonies

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and pseudohyphal morphology in comparison to typical strains of this species concerning

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their morphological and physiological characteristics, especially their fermentative

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performance in batch system using sugar cane juice submitted to cell recycle and acid

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treatment, as developed in industrial conditions. Although these yeast strains are quite

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common in the alcoholic fermentation environment, and the problems coming from their

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installation in the system are well known, the present work is the first scientific contribution

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to the understanding of their role in the process. The results may hopefully highlight possible

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ways of management of these S. cerevisiae strains during the fermentation when necessary.

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MATERIAL AND METHODS

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Microorganisms

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Yeast cultures exhibiting smooth and rough colonies were collected from different ethanol-

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producing units and submitted to PCR and sequencing of the ITS region, using the primers

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ITS-1 and ITS-4 in the rDNA [28] to confirm the species S. cerevisiae. After confirmation,

eleven strains exhibiting smooth colonies and dispersed cells (2, 3, 12, 15, 17, 18, 33, 37, 38,

39 and 47) and eleven strains exhibiting rough colonies and pseudohyphal morphology (4, 6,

7, 8, 9, 10, 16, 19, 35, 36 and 52) were chosen for the experiments, as illustrated in Fig. 1.

Intraspecific variation was verified by amplification of microsatellite loci confirming that the

strains were different (data not shown).

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Morphological and physiological characterization

Invasive growth

The yeast strains were cultivated in YEPD medium at 30C for 3 days, following incubation

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at room temperature for 2 days. The Petri dishes were photographed and after the agar surface

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was washed with distilled water to take the colonies off. The dishes were photographed again

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to verify the presence of colony spots in the agar (a signal of invasiveness into the culture

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medium). Transversal cuts were made in parallel with the spots to visualize the filamentous

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structures at the optical microscope.

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Flocculation

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The flocculation assay was performed according to [25] with modifications. After the growth

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in the multiplication medium, the cells were collected by centrifugation, washed twice with

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sodium citrate buffer (50 mM; pH 3.0) containing 5 mM EDTA and with water at 4C. The

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cells were resuspended in cold distilled water and diluted or concentrated until an OD (600

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nm) of 2.0 was reached. The flocculation (sedimentation) of the cells was determined in the

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absence or presence of calcium chloride solution (10 mM). After a vigorous shaking, samples

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from the upper portion of the tube were taken at 0 and after 10 minutes from the resting and

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the OD was determined. The flocculation percentage (%) was calculated as following:

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Flocculation (%) = [(OD0min OD10min) X 100] / OD0min.

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Killer character

The yeast strains were tested for the production of killer toxins in buffered methylene blue-

YEPD (citrate-phosphate buffer, pH 4.5-4.7, 3% methylene blue) at 30C. The strains to be

tested were inoculated with sterile toothpicks on the medium surface previously inoculated

with the sensitive strains S. cerevisiae NCYC1006 and Torulopsis glabrata ATCC15126. The

killer activity was detected when an inhibition zone and or a blue zone around the test yeast

colony was observed.

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Fermentative tests in batch system without cell recycle

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The inoculum was prepared in 50-mL Falcon tubes containing 20 mL of multiplication

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medium (sugar cane juice with around 4% of reducing sugars), inoculating two loops of the

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yeast strain. The tubes were incubated at 30C, at 160 rpm, for 12 hours. Following, the

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medium was centrifuged for 5 minutes at 3,400 rpm, discarding the supernatant and

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resuspending the cells in 5 mL of the multiplication medium, which were inoculated in 125-

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mL Erlenmeyers containing 45 mL of the same medium, at 30C and 160 rpm, for 12 hours.

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Following, the entire volume was transferred to 50-mL Falcon tubes previously sterilized,

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weighed and centrifuged under the same conditions. The cells were washed twice with

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distilled water. After the last wash, the tubes were weighed to estimate the wet mass in grams.

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A concentration of 10 g of wet mass/liter of fermentation medium was utilized in a proportion

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of 20% (v/v) as the inoculum. Flask fermentations (500-mL Erlenmeyers containing 200 mL

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of final volume of fermentation medium constituted of sugar cane juice with around 16% w/v

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of total reducing sugars) were maintained at 30C for 48 hours. Samples were taken out at

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each 12 hours and centrifuged. Soluble solids (Brix), by refratometer; pH, in a digital pH-

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meter; and alcohol content (g/100 mL), after the sample distillation and determination of

alcohol content in the hydroalcoholic solutions by densimeter, were determined in the

supernatant.

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Effect of the acid treatment over the yeast growth

For this test, the yeast strains PE-02 (here named 17) and 52 were selected, representing the

smooth colony and rough colony of S. cerevisiae, respectively. Initially, the cells of each

phenotype were cultivated in multiplication medium overnight, following the centrifugation

and transference of the cell mass to tubes containing acid solution (diluted sulfuric acid) in

pH values of 1.0; 1.5; and 2.0, in triplicates. The tubes were incubated at 30C, 160 rpm, for 2

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hours. After the acid treatment, the cells were washed twice with sterile distilled water,

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inoculated in Erlenmeyers flasks containing the multiplication medium and incubated at

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30C, 160 rpm, for 36 hours. Samples were taken before the acid treatment, after the acid

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treatment, and after 18 and 36 hours of cultivation in the multiplication medium, and

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inoculated in YEPD medium. After incubation at 30C for 72 hours, the colonies were

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counted and the results expressed as colony forming units/mL.

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Fermentative tests in batch system with cell recycle

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For this test, the yeast strains named PE-02 (here named 17) and 52 were selected,

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representing the smooth colony and rough colony of S. cerevisiae, respectively. The strains

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were utilized in pure fermentations of each and in a mixed fermentation, with both in the

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proportion of 1:1 regarding the cell mass utilized as inoculum. The fermentation tests were

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conducted as described in the item Fermentative tests in batch system without cell recycle,

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however, after the completion of each fermentative cycle (12 hours), the cells were recovered

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by centrifugation (3,400 rpm, 5 minutes) and treated with sulfuric acid solution, pH 1.5, at

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30C, 160 rpm, for 2 hours. Following the cell mass was washed twice with sterile distilled

water and resuspended in fermentation medium to initiate a new fermentative cycle. Six 12-

hour fermentative cycles were carried out, analyzing in the supernatant after each

fermentative cycle: soluble solids (Brix), by refratometr; pH, in a digital pH-meter; alcohol

content (g/100 mL), after the sample distillation and determination of alcohol content in the

hydroalcoholic solutions by densimeter; and total reducing sugar, by the 3,5-dinitrosalicylic

acid method, after hydrolysis of the samples. The fermentative efficiency was calculated

based on the alcoholic content of the fermented medium and the consumption of total

reducing sugar in relation to the theoretical efficiency of Gay-Lussac (51,11 g ethanol/100 g

glucose). Before centrifugation, the fermented broth was plated in YEPD medium to evaluate

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the number of smooth and rough colonies.

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Statistical analysis

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The results were analyzed by analysis of variance and Tukeys test to compare the averages

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(p<0.05).

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RESULTS AND DISCUSSION

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Invasive growth

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All the selected yeast strains were tested regarding their ability to grow invasively in YEPD

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medium. Only six strains showed invasive growth, and it was not a particular feature of

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smooth or rough colony (Fig. 1).

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Carbon limitation causes a developmental switch allowing the cells to penetrate the surface of

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an agar medium. This is called invasiveness, which means that in response to long incubation

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periods in rich medium where the concentration of glucose or other fermentable carbon source

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decreases, haploid cells of S. cerevisiae elongate and invade the agar in order to forage for

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nutrients [5, 14, 19, 21]. Industrial strains of S. cerevisiae exhibited invasive growth, both

haploid and diploid strains [22]. In the present work, the rough colonies were expected to

present wide expression of invasiveness due to the pseudohyphal morphology, but this result

was not found. Others have also observed invasive growth regardless the colony phenotypes

[10].

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Flocculation

All the yeast strains were grown in multiplication medium (diluted sugar cane juice) and the

analysis of cell flocculation was determined in the presence or absence of Ca2+ ions. Eight

strains (4, 8, 9, 10, 16, 19, 36 and 52) showed a high flocculation rate (>50%) both with and

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without calcium, and 4 of them had a flocculation rate above 90% in these conditions (strains

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8, 9, 16 and 52). Another group (strains 7 and 35) showed a flocculation rate from 20 to 35%,

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which increased significantly after the addition of Ca2+ ions, indicating a calcium-dependent

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flocculation. Finally, the remaining strains (total of 12) had a little or no flocculation (0-3%)

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regardless the presence of calcium ions (Fig. 2).

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Ten out eleven rough yeast colonies expressed high flocculation; concerning the smooth

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strains, no one showed flocculation. This characteristic flocculation - is not desirable for the

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alcoholic fermentation process because it makes difficult the conversion of sugar to ethanol.

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For a maximum conversion, the permanence of the yeasts suspended in the fermentation

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broth and not flocculated is essential [18]. Yeast flocculation is one of the worst problems

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presented in a fermentation process with cell recycle by centrifugation. This phenomenon

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causes loss of cells in the centrifuge and consequently the substrate is deviated for the cell

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reposition, causing decrease in the alcohol yield [1].

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This kind of yeast strain (rough colony, pseudohyphal morphology) is sometimes called

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flocculent yeast, however, it does not follow the definition of flocculation, in despite of the

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cell settlement at the bottom of the flasks. The cell aggregates result from the inability of the

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buds to separate from the mother cell after the cell division. It is quite different from the

flocculation caused by environmental conditions and their interactions with the yeast cell

wall. The presence of adhesins decorating the cell wall is a major requirement for flocculation

at the end of fermentation in brewing yeasts [26]. An extreme variation in the pH of the

medium [24] or the addition of special enzymes like papain, cause the release of the cells

clustered in aggregates. These treatments do not affect the condition of cell aggregates

displayed by the strains here studied. There is indeed a multiplicity of factors that affects

flocculation, like cations, pH, temperature, oxygen, sugars, ethanol, genealogical and cultural

age, cell density and mechanical agitation [24], which seems not affect the rough colonies

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exhibiting pseudohyphal morphology. Interestingly, the yeasts with this phenotype can also

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float on the top of the fermentors constituting a scum, resulting in the arrest of CO2 inside the

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tank and the possibility of medium release with loss of sugar and ethanol. Indeed,

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Saccharomyces flor yeasts are among the most tolerant organisms to ethanol, and are able to

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produce a biofilm that has acquired the ability to float, probable an adaptation to the extreme,

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selective pressures imposed by the conditions inside sherry wine barrels. The yeast cells are

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strongly aggregated, showing a flocculation phenotype [16].

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Killer phenotype

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The yeasts were also evaluated concerning the production of killer toxins, however, no strain

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(smooth or rough colonies) inhibited both sensitive yeast strains. Two strains of S. cerevisiae

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exhibiting rough colonies and pseudohyphal morphology, isolated from the ethanol

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fermentation, also displayed neutral reaction to a group of standard killer strains, in different

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pH values [12]. Others studied 165 flocculent yeasts and found only 3% of them as killer

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toxin producers (C. Steckelberg, M.G.S. Andrietta. 2011. Abstr. Simposio Nacional de

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Bioprocessos, Caxias do Sul, RS, Brazil). These authors credited this result to the hard

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conditions found in the fermentation tanks (high acidity and alcohol content), which could

impair the toxin activity. However, others showed a strain isolated from the fermentative

process with high killer activity against other yeasts, besides exhibiting killer activity along

the fermentative cycle regardless the unfavorable conditions [13].

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Fermentative tests in batch system without cell recycle

The fermentative capability of the rough and smooth colonies of S. cerevisiae was analyzed in

batch system without cell recycle. The results of alcohol productivity for each of the yeast

strain are seen in Fig. 3, showing a better performance of the smooth colonies. Considering all

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the yeasts divided in two groups (smooth and rough), it was observed that the alcoholic

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content was higher for the smooth colonies in the periods of 12 and 24 hours of fermentation,

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however in 36 and 48 hours, there was no significant difference between the two colony

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phenotypes. Generally, the smooth colonies presented significantly higher alcoholic content in

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the fermented must than that displayed by the rough colonies (Fig. 3).

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The data of productivity showed in Figure 3 considered the maximum productivity displayed

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by each strain during the 48-hour fermentation test. The range for the smooth colonies was

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1.3-2.2 g/l.h while for rough colonies the range was 1.1-1.7 g/l.h, showing higher variability

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among the smooth colonies than with rough colonies. Values of 2.22 to 2.84 g/l.h of ethanol

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productivity were found for 19 yeast strains isolated from Brazilian distilleries belonging to

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the Saccharomyces sensu stricto group, with great variability of kinetics values in terms of

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strain [3]. In the present work, in spite of the similar variability, the average ethanol

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production of rough colonies was significantly lower than smooth colonies of S. cerevisiae

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when 11 strains of each colony phenotype were analyzed.

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Effect of the acid treatment over the yeast growth

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To reduce bacterial contamination, when fermentation ceases, yeast cells are centrifuged to

result a yeast cream with 60-70% (wet weight basis/v) of cells. This yeast suspension is

diluted with water (1:1) and treated with sulfuric acid to pH 1.8-2.5 for 2 hours with agitation.

The yeast cream is ready to be re-used as starter for a next fermentation cycle so the recycling

is a peculiar trait of Brazilian process over a production season lasting 200-250 days [6].

However in spite of the relative tolerance to low pH displayed by yeasts, the acid treatment

can cause physiological disturbances in yeast cells, with mineral leakage and decreasing level

of intracellular trehalose resulting in cell viability decrease [15]. Of particular interest is to

evaluate whether a differential sensitivity to low pH can be found between the rough and

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smooth colony of S. cerevisiae in order to manage the contaminant strains with rough

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colonies during the step of acid treatment.

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For this test, the strains 17 (smooth) and 52 (rough) were chosen to verify the effect of acid

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treatment on their growth. The first strain, also named PE-02, is a selected yeast strain with

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dominance and permanence in the process during all the harvest season [6] , and the second

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was chosen because it presented high flocculation rate, as described above, besides high

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resistance to sugar concentration and sensitivity to low pH (data not shown). The results

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showed that the rough strain was severely affected by low pH values as 1.0 and 1.5, with

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significant decrease in the number of colony forming units. There was a recovery in the

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growth when the yeast was transferred to multiplication medium (sugar cane juice) after the

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acid treatment, however, the initial number of colonies before the treatment was not reached

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(Fig. 4).

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Different results were obtained for the smooth strain (PE-02). This yeast strain did not show

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significant decrease in the growth after the acid treatment, but in pH 1.0; on the contrary, a

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significant increase in the number of colonies was observed after the acid treatment in pH 2.0.

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After 36 hours of incubation in multiplication medium, the cells treated with sulfuric acid had

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their number of colonies increased in relation to the initial value (before the treatment), as

seen in Fig. 4.

These results point out to a satisfactory strategy to control the contamination by a rough strain

of S. cerevisiae. Lowering the pH value from 2.0 (which is commonly utilized in industrial

conditions [1]) to 1.5, for example, would result in low numbers of this contaminant yeast

inside the fermentation tanks, without impairing the main yeast (here represented by PE-02).

The growth recovery of the rough strain after the treatment occurred in a longer period of

time than that elapsed in a fermentative cycle, normally between 8-10 hours.

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Fermentative tests in batch system with cell recycle

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The same yeast strains as above were utilized in this experiment, conducted in batch system

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with cell recycle and acid treatment of the ferment. The results are depicted in Figs. 5, 6 and

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7.

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The fermentation with the rough strain showed lower ethanol production, higher

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concentration of residual sugar and fermentative efficiency around 60%. With the mixed

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fermentation with rough and smooth strains (equal amounts of cell mass of each strain in the

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initial fermentation cycle), a significant higher production of ethanol was observed in relation

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to the rough strain alone, however, it was lower than the value found with the smooth strain

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alone. The fermentative efficiency of the mixed fermentation was similar to the fermentation

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with PE-02 alone, but different from the rough alone. These results corroborate the ones

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obtained for the fermentation in a single 48-hour cycle, in which the rough strain had similar

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fermentative behavior in relation to the smooth strain but only after 36 hours of fermentation.

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In a batch system with cell recycle at each 12 hours of fermentation, the rough yeast was not

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able to complete the fermentation and consume all the available sugars (Fig. 5).

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The profile displayed by the rough strain, as slow fermentation, high residual sugar and lower

fermentative efficiency has been frequently correlated with contamination by this kind of S.

cerevisiae colony phenotype (data not shown). The cell morphology, with clusters or flocs,

becomes the contact cell nutrients more difficult, which in turns affects the fermentation. A

floc, when suspended in a liquid medium, presents a negative gradient of nutrients from the

periphery to the centre. Nutrient limitation and waste product accumulation are problems

suffered by the cells in the centre of the floc [24].

Another important point to be considered is the protection guaranteed to the cells within flocs,

increasing survival rates [23]. This is a particularly important characteristic conferring more

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adaptability of the rough colonies to the fermentation system.

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Indigenous Saccharomyces species are more difficult to control than non-Saccharomyces in

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the fermentation because the metabolism of the first is very similar to the one displayed by the

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selected yeast strain, besides the fact that they carry undesirable features such as flocculation,

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low fermentation yield and incomplete sugar consumption [6], as observed in this work for

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rough S. cerevisiae strains. Nowadays, a product or process that can be utilized to avoid or

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reduce contaminations by indigenous Saccharomyces without affecting the selected yeast

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strain is not known [1]. In this regard, a particular sensitivity towards a specific condition

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could be employed to manage the growth of these contaminants, and in this work a

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differential sensitivity to a lower pH may be of good application in industry.

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The final pH of the fermentation broth was statistically similar or lower, respectively, when

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the mixed culture of both yeasts or the pure culture of the smooth strain was utilized, and

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significantly different from the pure culture of the rough strain. The lower pH reflects in a

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more efficient fermentation (Fig. 6).

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Concerning the yeast growth during the fermentation process, the smooth strain showed the

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same growth performance in pure culture or in mixed culture with the rough strain, however

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the last strain displayed a different profile. The initial number of colonies was approximately

similar in pure or mixed fermentations, for each strain, however for the rough colony, there

was an increase in growth till the second fermentative cycle, showing stabilization further. In

the mixed culture, an increase in the colony number was also observed till the second

fermentation cycle, but the number of rough colonies increased further, which it did not occur

in the pure fermentation with this strain (Fig. 7). An important point should be emphasized

concerning the growth monitoring of rough colonies. Equal proportion of cell mass was added

to the fermentation medium at the start of the fermentation for each colony phenotype,

however, the number of colonies is different among them especially due to the fact that with

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the rough colony, a group of cells (pseudohyphae) will generate only one colony, in such a

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way that the number of colonies will always be lower than the number of yeast cells.

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In the mixed fermentation, the sugar consumption was lower and slower comparing to the

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pure culture of the smooth strain, indicating the effect of the contamination by the rough

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strain. The latter may have utilized the sugar for biomass production, because the growth was

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increased along the fermentative cycles, resulting in lower alcohol production. This growth

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behavior was not observed in the pure culture of the rough strain, which could indicate that

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the smooth strain was more efficient in the sucrose hydrolysis, releasing reducing sugars as

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glucose and fructose which are readily utilized by the rough strain in a slower rate. In pure or

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mixed culture with the rough strain, the growth of the smooth strain had approximately the

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same profile (Fig. 7).

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If this consideration is true, the question arisen is in what extension (and if) the acid treatment

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at pH 1.5 was effective to control the rough strain growth. The results described in the last

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item showed that the rough strain was effectively affected by the cell treatment at pH 1.5,

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which did not occur with the smooth strain. In view of this result, a greater rough strain

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growth would be expected if the acid treatment was not carried out or a less stressful

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condition was adopted. In fact, the overall fermentative efficiency was not affected by the

contamination of the rough strain (in equal amounts of mass cell of rough smooth strains at

the initial step), despite of the significant greater residual sugar concentration in the mixed

fermentation.

Metabolic functions associated with this kind of colony variant were seldom studied.

Comparisons among the strain M28 (a natural isolate from Tuscany, Italy) and the colony

variants (filigreed or rough and smooth) segregated from a single tetrad showed that all of the

segregants were vigorous, but there were differences in the gene expression patterns between

them. The ammonia permease, MEP2, had the greatest affinity for ammonia and was most

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prominently overexpressed in the rough segregants while the amino acid transporters were

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abundantly expressed in smooth strains [11]. However, there was no detectable difference in

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growth rate in any progeny. Given the different metabolic profiles observed, their fitness may

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differ in other, non-nutrient-rich conditions, which require further investigation [17].

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Shorter fermentation time and more cell recycles are important points to be considered,

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because although the alcoholic fermentation is a well-known process, a little is known about

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the response of the yeasts to the stressing conditions adopted by the Brazilian distilleries,

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which simply not occur in other fermentation processes [1]. The understanding of how the

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stressing factors affect the contaminant yeasts without effect upon the main strain is still a

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challenge, but it is undoubtedly a way to manage the contamination by yeasts during the

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process. In this context, the contribution of the present work is to show that this kind of wild

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yeast strain of S. cerevisiae, displaying rough colony, pseudohyphal morphology and high

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flocculation rate, is a slow-fermenting strain, resulting in low yields of alcohol in the

23

fermentation system adopted in Brazilian industries, with cell recycle after a short

24

fermentation period of time not longer than 8-12 hours [7]. The contamination with this yeast

25

strain results in a lower ethanol production and higher residual sugar concentration, but a

17

more drastic acid treatment between the cell recycles (pH 1.5) could avoid more harmful

effects of the contamination.

Other levels of contamination should be tested in order to verify the acceptable number of

rough strain to have a little or no effect upon the fermentation yield.

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6

ACKNOWLEDGMENTS

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8

This study was supported by FAPESP (fellowship grants to A.P.G. Bassi and J.C.G.
Silva and research support 2009/14617-4) and CAPES (fellowship grant to V.R. Reis).

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FIGURE LEGENDS

Figure 1. A. Colony (growing in YEPD medium) and cells (magnification of 400X at optical

microscopy) of a smooth colony (left) and a rough colony (right) of S. cerevisiae strains. B.

Strains of S. cerevisiae grown on YEPD medium after 3 days at 30C and 2 days at room

temperature. Aspect of the colonies before washing; after washing, showing the spots on

agar medium (invasive growth); and transversal cut of the agar medium after washing

showing cells growing into the agar (100X magnification).

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Figure 2. Flocculation (%) of the S. cerevisiae strains in the presence (black bar) or absence
(gray bar) of calcium ions.

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Figure 3. A. Productivity (g alcohol/L.h); B, C. Average alcohol production (g/100 mL)

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presented by rough and smooth colonies (total of 22 strains) of S. cerevisiae. The experiments

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were carried out in sugar cane juice, 16Brix, pH 4.3, during 48 hours of fermentation at

15

30C. Different letters over the bars mean significant difference at 5% by Tukeys test.

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Figure 4. Growth (CFU/mL) of two yeast strains of S. cerevisiae (52, in black, rough colony;

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PE-02, in gray, smooth colony), submitted to the acid treatment with sulfuric acid in different

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pH values (1.0; 1.5; 2.0), at 30C, for 2 hours, at 160 rpm. The determination of CFU (in

20

YEPD medium) was carried out before the acid treatment, right after the acid treatment (0

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hours) and after 18 and 36 hours of incubation of the treated cells in multiplication medium

22

(sugar cane juice), at 30C, 160 rpm.

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Figure 5. Effect of the yeast strain (52, rough; PE-02, smooth; and mixed) over the alcoholic

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content (A), total reducing sugars (B), final pH (C) and fermentative efficiency (D) in

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fermentations carried out in sugar cane juice, 16% (w/v) of total reducing sugars, pH 4.3, at

30C, along six 12-hour fermentative cycles, with acid treatment of the ferment (at pH 1.5).

Different letters over the bar mean significant difference at 5% by Tukeys test.

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5

Figure 6. Alcohol content (A), total reducing sugars (B) and final pH (C) in the fermentations

with strains of S. cerevisiae (rough strain , smooth strain

juice, 16% (w/v) of total reducing sugars, pH 4.3, at 30C, along six 12-hour fermentative

cycles, with acid treatment of the ferment (at pH 1.5).

and mixed

) in sugar cane

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Figure 7. Number of yeasts (CFU/mL) during the fermentations carried out in sugar cane

11

juice inoculated with the strains 52 (rough colony, in black) and PE-02 (smooth colony, in

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gray), in pure (A) and mixed culture (B), along six 12-hour fermentative cycles, 16% (w/v) of

13

total reducing sugars, pH 4.3, at 30C, with acid treatment of the ferment (at pH 1.5). The

14

yeast countings were taken in YEPD medium.

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