Sandra Regina Ceccato-Antonini

The physiological characteristics of the yeast Dekkera bruxellensis in fully fermentative conditions with cell recycling and in mixed cultures with Saccharomyces cerevisiae

The yeast Dekkera bruxellensis plays an important role in industrial fermentation processes, either as a contaminant or as a fermenting yeast. In this study, an analysis has been conducted of the fermentation characteristics of several industrial D. bruxellensis strains collected from distilleries from the Southeast and Northeast of Brazil, compared with Saccharomyces cerevisiae. It was found that all the strains of D. bruxellensis showed a lower fermentative capacity as a result of inefficient sugar assimilation, especially sucrose, under anaerobiosis, which is called the Custer effect. In addition, most of the sugar consumed by D. bruxellensis seemed to be used for biomass production, as was observed by the increase of its cell population during the fermentation recycles. In mixed populations, the surplus of D. bruxellensis over S. cerevisiae population could not be attributed to organic acid production by the first yeast, as previously suggested. Moreover, both yeast species showed similar sensitivity to lactic and acetic acids and were equally resistant to ethanol, when added exogenously to the fermentation medium. Thus, the effects that lead to the employment of D. bruxellensis in an industrial process and its effects on the production of ethanol are multivariate. The difficulty of using this yeast for ethanol production is that it requires the elimination of the Custer effect to allow an increase in the assimilation of sugar under anaerobic conditions.

Filamentous growth in Saccharomyces cerevisiae

Fungal dimorphism is a complex phenomenon triggered by a large variety of environmental factors and consists of a reversible alternating pattern of growth between different elliptical and filamentous forms of cells. Understanding the mechanisms that regulate these events is of major interest because of their implications in fungal pathogenesis, cell differentiation and industry. Diploid cells of Saccharomyces cerevisiae transform from budding yeast to pseudohyphae when starved for nitrogen, giving the cells an advantage in food foraging, which is sensed by at least two signal transduction pathways: the MAP kinase (MAPK) and the PKA (cAMP-dependent protein kinase A) pathways. The output of these signalling pathways is the expression of pseudohypha-specific genes, whose expression profiles change and is accompanied by a G2 delay in the cell cycle and a prolonged period of polarized growth. Haploid yeast strains show a similar growth type after prolonged incubation on rich medium plates. The cells form chains and invade the agar on the edge of the colony, but they do not become elongated. This growth type is referred to as haploid invasive growth. Alcohols can also induce filamentous growth in S. cerevisiae, promoting aberrant and elongated morphology. The three forms of filamentous growth are revised in this article and also the pathways involved in sensing, signaling and signal transduction during filamentous growth.

Chlorine dioxide against bacteria and yeasts from the alcoholic fermentation

The ethanol production in Brazil is carried out by fed-batch or continuous process with cell recycle, in such way that bacterial contaminants are also recycled and may be troublesome due to the substrate competition. Addition of sulphuric acid when inoculum cells are washed can control the bacterial growth or alternatively biocides are used. This work aimed to verify the effect of chlorine dioxide, a well-known biocide for bacterial decontamination of water and equipments, against contaminant bacteria (Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides) from alcoholic fermentation, through the method of minimum inhibitory concentration (MIC), as well as its effect on the industrial yeast inoculum. Lower MIC was found for B. subtilis (10 ppm) and Leuconostoc mesenteroides (50 ppm) than for Lactobacillus fermentum (75 ppm) and Lactobacillus plantarum (125 ppm). Additionally, these concentrations of chlorine dioxide had similar effects on bacteria as 3 ppm of Kamoran® (recommended dosage for fermentation tanks), exception for B. subtilis, which could not be controlled at this Kamoran® dosage. The growth of industrial yeasts was affected when the concentration of chlorine dioxide was higher than 50 ppm, but the effect was slightly dependent on the type of yeast strain. Smooth yeast colonies (dispersed cells) seemed to be more sensitive than wrinkled yeast colonies (clustered cells/pseudohyphal growth), both isolated from an alcohol-producing unit during the 2006/2007 sugar cane harvest. The main advantage in the usage of chlorine dioxide that it can replace antibiotics, avoiding the selection of resistant populations of microorganisms.

Bioprospection of yeasts as biocontrol agents against phytopathogenic molds

Yeasts isolated from sugar cane and maize rhizosphe re, leaves and stalks were screened against the phytopathogenic molds Colletotrichum sublineolum and Colletotrichum graminicola , both causal agents of the anthracnose disease in sorghum and maize, respectiv ely. Strains identified as Torulaspora globosa and Candida intermedia were able to inhibit the mold growth, with the firs t species also exhibiting killer activity. No previous report on the application and potentiality of these yeasts as biocontrol agents were found neither the killer phenotype in Torulaspora globosa .

Determination of yeast killer activity in fermenting sugarcane juice using selected ethanol-making strains

ABSTRACT Twenty-four yeasts out of 342 isolated from the fermentative process showed killer activity and three of them were selected for the fermentative efficiency evaluation in batch system with cell recycle, flask and fermentor experiments. The selected three killer strains did not present similar results to those of pressed (baking) yeast concerning ethanol (0.07-0.18; 0.12-0.20; 0.10-0.13; 0.22-0.25 g/g, respectively) and biomass (0.19-0.26; 0.33-0.39; 0.13-0.27; 0.47-0.61 g/g, respectively) yields and fermentative efficiency (12.3-36.3; 21.0-40.0; 19.3-26.3; 47.6-54.0 %, respectively) in sugarcane juice, in flasks. In fermentor, similar behaviour was observed. However, the selected strains showed high cellular viability and killer activity (using cell-free filtrate) along the fermentative cycles, in spite of the unfavourable conditions of the medium, like high pH variation of the medium (from 5.5-6.0 to 3.0-4.0), low aeration and higher temperature (30 0 C), which were not the ideal ones for the production/activity of killer toxins. A Pichia strain (CCA 510) showed the best results among the killer yeasts tested, exhibiting a killer activity against 92% of isolated fermentative yeasts of the process and against the pressed (baking) ferment. It also demonstrated killer activity (using crude toxin preparation) at higher temperatures (38

Release of potassium from rock powder by the yeast Torulaspora globosa

The alteration of minerals in rocks and the availability of elements for plant nutrition require long periods of time, and microorganisms are thought to induce the release of potassium and phosphate from rocks. In this context, this work evaluates the role of the yeast Torulaspora globosa, isolated from the sugar cane rhizosphere, in the solubilization of potassium from alkaline ultramafic rock powder. The experiments were performed in liquid medium, with or without agitation, at 30°C with the following treatments: culture medium + alkaline ultramafic; culture medium + yeast suspension; and culture medium + yeast suspension + alkaline ultramafic. The results showed that as much as 38% of the total potassium in the rock was released in the medium with the yeast during a 15-day period of incubation. Acid production may be the mechanism by which the yeast solubilizes potassium because the total acidity increased during the sampling period. Agitation (which increased oxygen availability) resulted in approximately 20% more biosolubilization of the alkaline ultramafic rock than with the static culture. These data indicate the potential for this yeast in biosolubilization processes and biofertilizer production.

Inhibition of Bacteria Contaminating Alcoholic Fermentations by Killer Yeasts

ABSTRACT The aim of this work was to study the in vitro antibacterial activity possessed by killer yeast strains against bacteria contaminating alcoholic fermentation ( Bacillus subtilis , Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides ), in cell X cell and cell X crude toxin preparations. The bacteria were not inhibited by any S. cerevisiae killer strains (5 out of 11). The inhibition caused by two crude toxin preparations ( Trichosporon figueirae and Candida sp) against L. plantarum was surprisingly high but not in the same extent for B. subtilis , especially with three killer strains (Candida glabrata, Pichia anomala and Candida sp). L. mesenteroides and L. fermentum strains were neither inhibited in cell X cell nor crude toxin X cell tests. The results suggested that killer activity of yeasts might operate over bacteria and it could be used for the biocontrol of contaminating bacteria from alcoholic fermentation if additional tests on toxin application in fermentation shown to be successful. A wider panel of S. cerevisiae killer strains should be used to confirm that they were really unable to control the growth of these Gram-positive bacteria. Key words: Bacteria, killer yeasts, alcohol, fermentation

Fermentative and growth performances of Dekkera bruxellensis in different batch systems and the effect of initial low cell counts in co‐cultures with Saccharomyces cerevisiae

Dekkera bruxellensis is a multifaceted yeast present in the fermentative processes used for alcoholic beverage and fuel alcohol production – in the latter, normally regarded as a contaminant. We evaluated the fermentation and growth performance of a strain isolated from water in an alcohol‐producing unit, in batch systems with/without cell recycling in pure and co‐cultures with Saccharomyces cerevisiae. The ethanol resistance and aeration dependence for ethanol/acid production were verified. Ethanol had an effect on the growth of D. bruxellensis in that it lowered or inhibited growth depending on the concentration. Acid production was verified in agitated cultures either with glucose or sucrose, but more ethanol was produced with glucose in agitated cultures. Regardless of the batch system, low sugar consumption and alcohol production and expressive growth were found with D. bruxellensis. Despite a similar ethanol yield compared to S. cerevisiae in the batch system without cell recycling, ethanol productivity was approximately four times lower. However, with cell recycling, ethanol yield was almost half that of S. cerevisiae. At initial low cell counts of D. bruxellensis (10 and 1000 cells/ml) in co‐cultures with S. cerevisiae, a decrease in fermentative efficiency and a substantial growth throughout the fermentative cycles were displayed by D. bruxellensis. Due to the peculiarity of cell repitching in Brazilian fermentation processes, D. bruxellensis is able to establish itself in the process, even when present in low numbers initially, substantially impairing bioethanol production due to the low ethanol productivity, in spite of comparable ethanol yields. Copyright

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

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.

Hyphal-like extension and pseudohyphal formation in industrial strains of yeasts induced by isoamyl alcohol

As leveduras podem produzir pseudohifas e extensoes semelhantes a hifas sob certas condicoes de crescimento, como a adicao de alcool isoamilico. Esse alcool e o constituinte principal do oleo fusel, um subproduto da fermentacao etanolica. A mudanca de morfologia da levedura para a forma filamentosa pode causar problemas ao processo fermentativo. Neste trabalho, foi estudada a influencia dos alcoois fusel (isoamilico e isopropilico), fontes de nitrogenio (sulfato de amonio e leucina) e glifosate (um maturador quimico para cana-de-acucar) adicionados a um meio complexo sobre linhagens industriais de leveduras isoladas do processo fermentativo. Duas linhagens industriais mostraram transicao para pseudohifas (grupos de celulas) ou extensoes semelhantes a hifas apos adicao de alcool isoamilico, de 0,3% a 0,9% v/v. Todas as alteracoes foram reversiveis quando as leveduras foram reinoculadas em YEPD sem adicao de alcool. Apesar das pseudohifas serem comumente resultantes de meios com limitacao de nitrogenio, observou-se que esta morfologia resultou da adicao de alcool isoamilico. Nao foi detectada nenhuma influencia da fonte de nitrogenio ou alcool isopropilico ou glifosate para quaisquer linhagens estudadas.