Characteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation

Abstract

Among the native yeasts found in alcoholic fermentation, rough colonies associated with pseudohyphal morphology belonging to the species Saccharomyces cerevisiae are very common and undesirable during the process. The aim of this work was to perform morphological and physiological characterisations of S. cerevisiae strains that exhibited rough and smooth colonies in an attempt to identify alternatives that could contribute to the management of rough colony yeasts in alcoholic fermentation. Characterisation tests for invasiveness in Agar medium, killer activity, flocculation and fermentative capacity were performed on 22 strains (11 rough and 11 smooth colonies). The effects of acid treatment at different pH values on the growth of two strains ("52"--rough and "PE-02"--smooth) as well as batch fermentation tests with cell recycling and acid treatment of the cells were also evaluated. Invasiveness in YPD Agar medium occurred at low frequency; ten of eleven rough yeasts exhibited flocculation; none of the strains showed killer activity; and the rough strains presented lower and slower fermentative capacities compared to the smooth strains in a 48-h cycle in a batch system with sugar cane juice. The growth of the rough strain was severely affected by the acid treatment at pH values of 1.0 and 1.5; however, the growth of the smooth strain was not affected. The fermentative efficiency in mixed fermentation (smooth and rough strains in the same cell mass proportion) did not differ from the efficiency obtained with the smooth strain alone, most likely because the acid treatment was conducted at pH 1.5 in a batch cell-recycle test. A fermentative efficiency as low as 60% was observed with the rough colony alone.

Keywords: S. cerevisiae; ethanol; fermentation; indigenous yeasts.

Figure 1

A. Colony (grown on YPD medium) and cells (400× magnification by optical microscopy) of a smooth colony (left) and a rough colony (right) of S. cerevisiae. B. Strains of S. cerevisiae grown on YPD medium after 3 days at 30 °C and 2 days at room temperature. Aspects of the colonies before washing; after washing, showing the “spots” on agar medium (invasive growth); and a transverse cut of the agar medium after washing, showing cells growing into the agar (100× magnification).

Figure 2

Flocculation (%) of the smooth and rough colony strains of S. cerevisiae in the presence (grey bar) or absence (black bar) of calcium ions.

Figure 3

A. Average productivity (g alcohol/L.h); B, C. Average alcohol production (g/100 mL) by rough and smooth colonies (total of 22 strains) of S. cerevisiae, over the entire period of fermentation (48 h) and throughout the course of fermentation, respectively. The experiments were carried out in sugar cane juice, 16ºBrix, pH 4.3, during 48 h of fermentation at 30 °C. Different letters above the bars denote significant differences at 5% by Tukeys test.

Figure 4

Growth (cfu/mL) of two yeast strains of S. cerevisiae (52, in black, rough colony; PE-02, in grey, smooth colony), submitted to acid treatment with sulphuric acid at different pH values (1.0, 1.5 and 2.0) at 30 °C for 2 h at 160 rpm. The determination of cfu numbers (in YPD medium) was performed before the acid treatment, immediately after the acid treatment (0 h) and after 18 and 36 h of incubation of the treated cells in multiplication medium (sugar cane juice), at 30 °C and 160 rpm. Different capital and lowercase letters indicate significant differences (p < 0.05) among the treatments for the smooth colony and rough colony, respectively.

Figure 5

Effect of the yeast strain (52, rough; PE-02, smooth; and mixed) on alcohol content (A), total reducing sugars (B), final pH (C) and fermentative efficiency (D) in fermentations carried out in sugar cane juice, 16% (w/v) total reducing sugars, pH 4.3, at 30 °C, over six 12-hfermentative cycles, with acid treatment of the yeast cells (at pH 1.5). Different letters above the bars denote significant differences at 5% by Tukeys test. The values are the means of six fermentative cycles.

Figure 6

Alcohol content (A), total reducing sugars (B) and final pH (C) in fermentations with strains of S. cerevisiae (52, rough strain n; PE-02, smooth strain ▲; and mixed ◆) in sugar cane juice, 16% (w/v) of total reducing sugars, pH 4.3, at 30 °C, over six 12-h fermentative cycles, with acid treatment of the ferment (at pH 1.5). “C0” signifies the start of the fermentation cycles. Other samples were taken at the end of the fermentation cycles.

Figure 7

Concentration of yeasts (cfu/mL) during fermentations conducted in sugar cane juice inoculated with the S. cerevisiae strains 52 (rough colony, in black) and PE-02 (smooth colony, in grey), in pure (A) and mixed fermentations (B), over six 12-h fermentative cycles, 16% (w/v) of total reducing sugars, pH 4.3, at 30 °C, with acid treatment of the ferment (at pH 1.5). The yeast counts were performed on YPD medium. “C0” signifies the start of the fermentation cycles. Other samples were taken at the end of the fermentation cycles.