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Keywords:

  • contaminant yeasts;
  • ethanol;
  • batch fermentation;
  • co-cultures;
  • Dekkera, Saccharomyces

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

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 © 2013 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Dekkera bruxellensis is a yeast with multiple aspects that can be explored in the context of alcoholic beverage and fuel production fermentation processes. This yeast contributes to the flavour development of wine and lambic beer by producing higher alcohol content (du Toit and Pretorius, 2000). Aromas such as ‘smoky’, ‘spicy’ and ‘toffee’ are also associated with these yeasts (Arvik and Henick-Kling, 2002). However, the yeast can cause enormous economic losses for the wine industry by the production of phenolic compounds such as 4-ethylguaiacol and 4-ethylphenol (Heresztyn, 1986; Wedral et al., 2010).

In bioethanol production, D. bruxellensis is commonly cited as one of the most important contaminant yeasts, especially in Brazilian industrial use (Souza-Liberal et al., 2005, 2007; Basilio et al., 2008; Pereira et al., 2012). The Brazilian fermentation process is quite unusual, particularly in one aspect: the yeast cells are intensively recycled, approximately 400–600 times during a production season (Amorim et al., 2011), resulting in very high cell densities inside the fermentation tanks, which contributes to a very short fermentation time (a period of 6–11 h) and high ethanol yields (90–92% efficiency) in high-gravity musts, according to Basso et al. (2008). In this context, the role of D. bruxellensis and its potentially detrimental contribution to alcoholic fermentation should be considered in the light of its important physiological aspects. Leite et al. (2012) defined the three main physiological characteristics of the Dekkera/Brettanomyces genus as the ability to produce ethanol in the presence of oxygen and high sugar concentrations (as studied by van Dijken and Scheffers, 1986, and Rozpedowska et al., 2011), the inhibition of alcoholic fermentation under anaerobic conditions (according to Wijsman et al., 1984) and the production of acetic acid and ethanol in the presence of glucose under aerobic conditions (Aguilar-Uscanga et al., 2003).

An important aspect seldom studied is the level of contamination that causes perturbation of the fermentation process. In addition, the information concerning the performance of this yeast in fermentative processes appears to be contradictory in many situations, particularly due to strain variability and the growth/fermentation conditions utilized (Vigentini et al., 2008). Taking this into account, this work aimed to verify the ability of a particular strain of D. bruxellensis, isolated from the water in an alcohol-producing unit (and not from the fermentation vats), to grow and ferment in simulated industrial conditions (batch system with cell recycling) in pure and co-cultures with Saccharomyces cerevisiae. Physiological characteristics such as ethanol resistance and dependence on aeration to produce ethanol and acids were also studied. This work is novel in that it demonstrates how low cell counts of D. bruxellensis can increase substantially during cell-recycled batch fermentation and produce perturbations in the process. Although the strain produces comparable alcohol yields to S. cerevisiae, this study also discusses the importance of the ethanol productivity parameter to express the real role of D. bruxellensis in alcohol fermentation in Brazil.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Microorganisms

A D. bruxellensis strain (CCA059) isolated from water in an alcohol-producing unit and an industrial strain of S. cerevisiae (PE-2; Basso et al., 2008) were used in the experiments. The first strain was identified by DNA sequencing at the D1/D2 26S locus and ITS region of the ribosomal DNA (White et al., 1994). The strains were maintained on YPD (1% yeast extract, 2% glucose, 2% peptone, 2% agar; for broth, agar was not included) slants at 4 °C with constant transfers to new medium.

Growth of D. bruxellensis in YPD with ethanol

The pre-inoculum was prepared by inoculating 125 ml flasks containing 50 ml YPD broth with two loops of fresh yeast cells and incubating these cultures for 12 h at 30 °C under orbital agitation at 160 rpm. The cell suspension was standardized to a concentration of 106 cells/ml. A 10 ml volume was transferred to 250 ml flasks (in triplicate) containing 80 ml YPD broth with added ethanol (volumes varying from 0 to 8 ml ethanol, total volume 10 ml with sterile distilled water added as necessary) for a final concentration of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% (v/v). The flasks were incubated at 30 °C for 48 h at 160 rpm, in triplicate. Samples were taken every 12 h, and the optical density was determined by measuring the absorbance at 600 nm in a Bio-Mate® spectrophotometer.

Fermentative testing of D. bruxellensis in synthetic medium under different agitation and carbon source conditions

For the inoculum preparation, Falcon tubes containing 20 ml YPD broth were inoculated with two loops of D. bruxellensis and maintained at 30 °C and 160 rpm for 12 h. Next, the yeast suspension was centrifuged for 5 min at 3400 rpm. The supernatant was discarded and the cells were resuspended in 5 ml YPD broth. This volume was introduced into 125 ml Erlenmeyer flasks containing 45 ml YPD broth, which were then incubated at 30 °C and 160 rpm for 12 h. The entire volume of the flask was transferred to sterile and pre-weighed Falcon tubes, which were centrifuged, and the cells were washed twice with sterile distilled water. The tubes containing the cell pellets were weighed to estimate the wet cell mass. A proportion of 10 g of the wet cell mass per litre was utilized in the fermentation tests with the following synthetic medium: 0.5% potassium dihydrogen phosphate, 0.1% potassium chloride, 0.15% ammonium chloride, 0.6% yeast extract, 0.1% magnesium sulphate heptahydrate and distilled water. Glucose or sucrose (10%, w/v) was added as the sole carbon source. Fermentation was carried out in 500 ml flasks with 200 ml of final volume of the fermentation medium and maintained at 30 °C and 100 rpm for a 48 h period with or without agitation (static cultures) for the same period of time, in triplicate. Next, 15 ml samples were taken every 12 h to determine the pH using a digital pH meter, soluble solids (°Brix) using a refractometer, and alcohol content (g/100 ml) after distillation of the samples by measuring the alcohol concentration with a digital densimeter (Anton-Paar).

Fermentative testing of D. bruxellensis and S. cerevisiae in sugar cane medium in a single-cycle batch system

The inoculum was prepared by inoculating two loops of each yeast strain, separately, in 125 ml flasks containing 50 ml multiplication medium (clarified sugar cane juice with approximately 4% (w/v) total reducing sugars, pH 5.5, supplied by a local alcohol-producing unit), which were maintained at 160 rpm at 30 °C for 24 h. The yeast cell mass was separated by centrifugation at 3400 rpm for 5 min and again incubated under the same conditions. After centrifugation, the cell mass was evaluated for the number of cells per millilitre in a Neubauer chamber and diluted (if necessary) in multiplication medium to inoculate the fermentation flasks. Two trials were conducted to evaluate the fermentative performance of D. bruxellensis and S. cerevisiae in pure cultures, at an initial concentration of 107 cells/ml. The tests were carried out in 125 ml flasks with 50 ml final volume of fermentation medium (clarified sugar cane juice with approximately 16% (w/v) total reducing sugars, pH 4.5, supplied by a local alcohol-producing unit), in triplicate, at 30 °C, without agitation. The flasks were randomly sampled every 8 h until 72 h of fermentation, and the fermented broth was then initially analysed by serial plating of the samples in YPD medium to determine the number of colony-forming units per millilitre. Next, the remaining fermented broth was centrifuged at 3400 rpm for 5 min and the supernatant was analysed for pH using a digital pH meter; total reducing sugars (g/100 ml) via dinitrosalicylic acid addition after hydrolysis of the sample with hydrochloric acid, and alcohol content (g/100 ml) after distillation of the samples by measuring the alcohol concentration with a digital densimeter (Anton-Paar).

Fermentative tests of pure and co-cultures of D. bruxellensis and S. cerevisiae in sugar cane medium in a multiple-cycle batch system

The inoculum was prepared in two consecutive steps for each yeast strain separately. Initially, the yeast was streaked onto YPD medium and incubated at 30 °C for 48 h. Then, two loops of the yeast cells were transferred to a tube containing 20 ml saline solution (0.85% NaCl). After homogenization, a volume of 1 ml was transferred to a 250 ml flask containing 150 ml multiplication medium (clarified sugar cane juice with approximately 4% (w/v) total reducing sugars, pH 5.5, supplied by a local alcohol-producing unit), which was maintained at 160 rpm at 30 °C for 24 h. The yeast cell mass was separated by centrifugation at 3400 rpm for 5 min and again incubated under the same conditions. After centrifugation, the cell mass was evaluated for the number of cells per millilitre in a Neubauer chamber and diluted (if necessary) in multiplication medium to inoculate the fermentation flasks. Four trials were conducted to evaluate the fermentative performance of D. bruxellensis and S. cerevisiae in pure and co-cultures, as follows: 108 cells/ml S. cerevisiae, 108 cells/ml S. cerevisiae + 10 cells/ml D. bruxellensis, 108 cells/ml S. cerevisiae + 103 cells/ml D. bruxellensis, and 108 cells/ml D. bruxellensis. The tests were carried out in 500 ml flasks with 200 ml of the final volume being fermentation medium (clarified sugar cane juice with approximately 16% (w/v) total reducing sugars, pH 4.5, supplied by a local alcohol-producing unit), in triplicate, at 30 °C, for 14 fermentation cycles of 12 h each, without agitation. At the end of each cycle, a 2 ml sample was collected to determine the number of colony-forming units per millilitre using serial plating with WLN medium (Wallerstein Laboratory Nutrient Medium, Acumedia®) and/or WLD (WLN + 50 ppm cycloheximide) medium. The latter was used in the trials in which D. bruxellensis was inoculated. The fermented broth was centrifuged at 3400 rpm for 5 min, and the cell mass was resuspended in saline solution to wash the cells, centrifuged again and resuspended in a new fermentation medium to start the second fermentation cycle. This procedure was repeated until the 14th cycle. The supernatant was analysed for pH using a digital pH meter, total reducing sugars (g/100 ml) by the addition of dinitrosalicylic acid after hydrolysis of the sample with hydrochloric acid; and alcohol content (g/100 ml) after distillation of the samples by measuring the alcohol concentration with a digital densimeter (Anton-Paar).

Statistical analysis

Analysis of variance and Duncan's test at 5% significance level were applied to the results of yield and productivity in ethanol.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Growth of D. bruxellensis in YPD medium with ethanol

A slower rate of growth was observed in YPD medium with 3% and 4% added ethanol, and the lag phase was long even in YPD medium without ethanol (Figure 1). With 5%, 6%, 7% and 8% added-ethanol YPD medium, there was no growth after 48 h of cultivation.

image

Figure 1. Growth of D. bruxellensis cells in YPD broth with added ethanol at concentrations of 0%, 1%, 2%, 3% and 4% (v/v), at constant agitation of 160 rpm, for 48 h, at 30 °C (n = 3)

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Fermentative testing of D. bruxellensis in synthetic medium under different conditions of agitation and carbon source

The D. bruxellensis strain displayed higher alcohol production under agitation conditions and with glucose as the sole carbon source. The rate of alcohol production was substantially higher under these conditions compared with static cultures regardless of the carbon source. The decrease in soluble solids was more pronounced with agitation and with glucose, as well as when the final pH of the medium reached a value as low as 2.0 at 48 h of fermentation compared with the pH of 3.5 observed in the static cultures (Figure 2).

image

Figure 2. Fermentative test of D. bruxellensis in synthetic medium. Samples were withdrawn every 12 h for determination of alcohol content, soluble solids and final pH of the synthetic medium inoculated with D. bruxellensis, with glucose (diamonds) or sucrose (squares) as the sole carbon source, in static cultures (closed symbols) or under constant agitation (open symbols) at 100 rpm, for 48 h, at 30 °C (n = 3)

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Fermentative testing of D. bruxellensis and S. cerevisiae in sugar cane medium in single-cycle and multiple-cycle batch systems

In a single-cycle batch system, the S. cerevisiae strain completely exhausted the reducing sugars of the sugar cane medium after 32 h of fermentation, with a production of approximately 5 g alcohol/100 ml. The cell number stabilized at 32 h and the medium pH slightly decreased. In contrast, the D. bruxellensis strain consumed less than 50% of the reducing sugars after 72 h of fermentation, with a production of 2 g/100 ml of alcohol at the end of this period. However, the cell number increased substantially within this period and did not reach stability. The pH variation was similar to that observed for S. cerevisiae (Figure 3).

image

Figure 3. Fermentative test of D. bruxellensis and S. cerevisiae in sugar cane medium in the single-cycle batch system, at 30 °C. Samples (n = 3) were withdrawn every 8 h for determination of reducing sugars (▲), cell number (♦), alcohol production (■) and final pH (□)

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In the multiple-cycle batch system, the yeast S. cerevisiae demonstrated alcohol production stabilization from the fourth fermentative cycle, with low residual reducing sugars and stabilization in medium pH from the start of the fermentation cycles. The cell population varied during the cycles, but no more than 4 log cycles of increased cell number were observed. However, for D. bruxellensis in pure cultures, an increase of 6 log cycles was verified after 14 fermentation cycles. The medium pH decreased substantially with the increasing number of cycles, and slower sugar consumption was also observed. Alcohol production reached 2 g/100 ml at the end of the 12th fermentation cycle (Figure 4).

image

Figure 4. Fermentative tests of pure cultures of S. cerevisiae (upper panel) and D. bruxellensis (lower panel) in sugar cane medium in the multiple-cycle batch system, at 30 °C. Samples (n = 3) were withdrawn at the end of each 12 h cycle for determination of reducing sugars (♦),alcohol production (■), final pH (▲) and number of yeasts

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When the co-culture of D. bruxellensis and S. cerevisiae was evaluated after an initial concentration of 10 cells of D. bruxellensis/ml, the results for pH variation, alcohol production and sugar consumption during the 14 cycles of fermentation were comparable to the pure fermentation with S. cerevisiae. However, at an initial cell population of 1000 D. bruxellensis cells/ml, alcohol production was lower and slower, and the final pH of the medium decreased to below 3. The concentration of residual reducing sugars was low from the first fermentative cycle. The number of yeast cells increased in both fermentation conditions (Figure 5).

image

Figure 5. Fermentative tests of co-cultures of S. cerevisiae (upper shaded areas) and D. bruxellensis (lower shaded areas) in sugar cane medium in multiple-cycle batch system, at 30 °C, with different original cell concentrations of D. bruxellensis (10 CFU/ml, upper panel; 103 CFU/ml, lower panel). Samples (n = 3) were withdrawn at the end of every 12 h cycle for determination of reducing sugars (♦),alcohol production (■), final pH (▲) and number of yeasts

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Comparing the batch systems with and without cell recycling, the ethanol productivity of D. bruxellensis in pure cultures or in co-cultures with S. cerevisiae, even at low initial cell counts of 10 and 1000 cells/ml, was always lower than with S. cerevisiae alone. In regard to ethanol yield, when in the single batch system without cell recycling, D. bruxellensis displayed a similar value to S. cerevisiae under the same conditions. Ten- and 1000-cell D. bruxellensis contaminations in a multiple-cycle batch system had an effect on ethanol yield, and this parameter was substantially lower in pure cultures of D. bruxellensis (Figure 6).

image

Figure 6. Comparative ethanol yields and productivities of pure and co-cultures of S. cerevisiae (Sc) and D. bruxellensis (Db) in sugar cane medium in a batch system with and without cell recycling, at 30 °C. Different upper-case letters above the bars mean significant difference by Duncan's test among the fermentations in the batch system with cell recycling, and lower-case letters for comparisons between fermentations in the batch system without cell recycling (p < 0.05)

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Table 1 lists the values for fermentative efficiency (%), assuming a theoretical value of 0.51 g ethanol per gram of sugar. Every fermentation trial in which D. bruxellensis was inoculated, in pure culture or co-culture with S. cerevisiae, presented lower fermentative efficiency regardless of the batch system utilized.

Table 1. Fermentative efficiency (%) of the fermentation trialsa
Fermentative trialsFermentative efficiencyb
  1. a

    Overall average of each fermentative trial.

  2. b

    Fermentative efficiency = (ethanol yield of the trial/0.51) × 100.

Batch system without cell recyclingS. cerevisiae76.0%
 D. bruxellensis70.5%
   
Batch system with cell recyclingS. cerevisiae86.3%
 S. cerevisiae + 10 cells D. bruxellensis/ml80.3%
 S. cerevisiae + 1000 cells D. bruxellensis/ml78.4%
 D. bruxellensis33.3%

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Dekkera bruxellensis is a slow-growing yeast in rich medium, i.e., at non-limiting glucose concentrations (Blomqvist et al., 2012), but it is highly resistant to stressful conditions (Rozpedowska et al., 2011). The yeast's resistance to ethanol remains controversial. Wine isolates of D. bruxellensis have been shown to be more resistant to ethanol than wine S. cerevisiae strains (Renouf et al., 2006). This may explain the prevalence of the former at the end of wine fermentation, when the ethanol concentration reaches high values, and the medium is nutritionally poor (Silva et al., 2004; Renouf et al., 2009). Pereira et al. (2012) verified that both S. cerevisiae and D. bruxellensis were equally sensitive to the ethanol added in the fermentation medium (concentration of 8%) with cell recycling. The suspension of the ethanol addition caused an increase in cell concentration of both yeasts. Only concentrations of 10% or 13% ethanol completely inhibited the growth of D. bruxellensis (Dias et al., 2003; Conterno et al., 2006). However, other authors have emphasized that the ability to survive in a nutritionally poor environment is the main characteristic responsible for the yeast's capacity to proliferate, and D. bruxellensis is indeed less sensitive to ethanol than S. cerevisiae (Jolly et al., 2006; Barata et al., 2008). The conditions under which the ethanol resistance tests were carried out (in rich medium, minimal medium, single or multiple additions of ethanol, etc.) as well as the strain specificity may account for the differences in results.

In our tests, ethanol was added only once and had an effect on the growth of D. bruxellensis in that it lowered or inhibited growth depending on the concentration. This may be a method to control the growth of this yeast in a fermentation contaminated with high counts (e.g. 106 cells/ml) either during the course of fermentation or during the cell treatment performed between the fermentative cycles in industrial settings. However, it should be noted that the ethanol inside the fermentation tank does not have the same concentration as at the start of the fermentation cycle due to the 6–11 h cycle length. The growing concentration of ethanol inside the tank may have a different effect on the yeast cells than the single addition of ethanol at the start of the fermentation. In other words, the exogenous ethanol may indeed have an effect on D. bruxellensis growth, but it is likely that the ethanol produced by S. cerevisiae during fermentation has a minimal effect on the contaminant yeast. At a concentration of 5% ethanol added to YPD medium, cells of D. bruxellensis were inhibited to grow but the same concentration in fermentation trials with cell recycling seemed not to affect the growth of this yeast.

D. bruxellensis is able to accumulate ethanol as well as acetic acid under aerobic conditions (Rozpedowska et al., 2011; Leite et al., 2012) and can also grow anaerobically (Pereira et al., 2012; Piskur et al., 2012). In spite of the results demonstrated by Pereira et al. (2012), which indicated that this species did not present expressive acid production in fully fermentative conditions, in pure culture or co-culture with S. cerevisiae at an initial concentration of 1000 cells/ml, the strain studied here produced a decreasing final pH of the fermentation as the cycles progressed when compared with S. cerevisiae. This subject still remains unclear and demands further investigation (is it a characteristic of this particular strain?). According to Beckner et al. (2011), the interactions between D. bruxellensis and S. cerevisiae are poorly understood as yet and perhaps the production of acids by D. bruxellensis under certain conditions may somehow affect S. cerevisiae cells.

Controversy persists with D. bruxellensis concerning ethanol accumulation. Under limited oxygen, the ethanol yield of D. bruxellensis is almost the same as S. cerevisiae (Galafassi et al., 2011), and our studies produced the same results but only for the batch system without cell recycling. Cell recycling after fermentation appears to favour the growth of this yeast instead of the fermentation. However, is ethanol yield the best unique parameter to express the fermentative performance of D. bruxellensis? The expression of fermentation in terms of time, using the ethanol productivity measurement, reflects the role of this yeast in cell-recycled fermentative systems. The strain is a low-rate ethanol producer, with an expressive growth in oxygen-limited conditions and substantial residual amounts of sugars are present at the end of the fermentation period. The exhaustion of reducing sugars would extend the fermentation time to an impractical level. Galafassi et al. (2011) recognized that D. bruxellensis cannot compete with S. cerevisiae in batch processes when ethanol productivities are compared. Blomqvist et al. (2010) concluded that a more energy-efficient metabolism under oxygen limitation is a characteristic of D. bruxellensis rather than S. cerevisiae; however, the comparisons were based mainly on ethanol yields (grams of ethanol per gram of sugar), in an one-step batch system. Nevertheless, Blomqvist et al. (2012) verified that an increase oxygenation in continuous fermentation enabled D. bruxellensis to consume more sugar, outcompeting S. cerevisiae in co-cultures. But the number of D. bruxellensis cells remained constant and production of acetic acid was observed.

There is a lack of scientific information about the lower limit of contamination by D. bruxellensis capable of producing disturbances in the fermentation process. For that reason, we decided to test the levels of contamination with as low as 10 and 1000 cells/ml in co-cultures with S. cerevisiae. The outstanding capacity of D. bruxellensis to grow its population within the fermentation conditions was demonstrated in this study, which also showed that an initial contamination of 1000 cells/ml could render slower alcohol production and a decreased final medium pH throughout 14 fermentative cycles lasting 12 h each. These results demonstrate that if a few cells enter the fermentation tank they will be recycled together with S. cerevisiae and may achieve high numbers after some time. This peculiarity of Brazilian fermentation systems – cell repitching – favours the development and establishment of D. bruxellensis in the system.

The characteristic of pronounced growth under fully fermentation systems with cell recycling is likely not the only characteristic to account for the permanence and persistence of this yeast during fermentation. Hellborg and Piskur (2009) verified a greater diversity among strains regarding chromosome number and ploidy than in S. cerevisiae. This may be behind the events responsible for the establishment of the yeasts in sugar-rich anaerobic fermentation habitats. For S. cerevisiae, this was achieved by whole gene duplication, whereas for D. bruxellensis it was apparently achieved through increased ploidy (Piskur et al., 2012). Moreover, the ability to assimilate nitrate may confer an advantage to D. bruxellensis over S. cerevisiae and, since high levels of nitrate are present in sugar cane juice, high colonization of this yeast is expected in sugar cane-based ethanol plants (Pita et al., 2011). In this respect, a recent study has demonstrated that under anaerobic conditions nitrate assimilation abolishes the Custers effect, improving the fermentative metabolism of this yeast (Galafassi et al., 2013). This may be one of the reasons why sometimes the presence of D. bruxellensis in ethanol plants is not associated with loss of ethanol yield, suggesting that the monitoring of nitrate concentrations in sugar cane-based media is highly recommended.

Despite the apparently low difference in ethanol yield between fermentation with S. cerevisiae and fermentation with S. cerevisiae + 1000 cells/ml of D. bruxellensis, a particular point should be examined to verify the potentially detrimental effect of this yeast in fermentations with S. cerevisiae. According to Basso et al. (2008), a difference of approximately 3.1 percentage points in fermentative efficiency (assuming a theoretical value of 0.51 g ethanol per gram of sugar) amounts to an increase of 2.1 million litres of ethanol per crop season in a medium-capacity distillery. In the present work, contamination with 10 or 1000 cells of D. bruxellensis caused a decrease of 6.0 or 7.9 percentage points, respectively, in the fermentative efficiency compared with the fermentative trial with no contamination by D. bruxellensis. This would have a substantial effect on the volume of ethanol produced over a season. A detailed monitoring of the microbiological aspects of the fermentation should be used to detect the presence of D. bruxellensis during the fermentation process. Sampling points, such as the water source, should be considered in the monitoring scheme to avoid the entrance of these yeasts into the fermentation tanks.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (fellowships to M. C. Meneghin, A. P. G. Bassi and C. B. Codato; research support under the responsibility of S. R. Ceccato-Antonini).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
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