ORIGINAL PAPER

The fermentation of sugarcane molasses by Dekkerabruxellensis and the mobilization of reserve carbohydrates

Luciana Filgueira Pereira • Elisa Lucatti •

Luiz Carlos Basso • Marcos Antonio de Morais Jr

Received: 7 October 2013 / Accepted: 12 December 2013 / Published online: 27 December 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The yeast Dekkera bruxellensis is consid-

ered to be very well adapted to industrial environ-

ments, in Brazil, USA, Canada and European

Countries, when different substrates are used in

alcoholic fermentations. Our previous study described

its fermentative profile with a sugarcane juice sub-

strate. In this study, we have extended its physiolog-

ical evaluation to fermentation situations by using

sugarcane molasses as a substrate to replicate indus-

trial working conditions. The results have confirmed

the previous reports of the low capacity of D.

bruxellensis cells to assimilate sucrose, which seems

to be the main factor that can cause a bottleneck in its

use as fermentative yeast. Furthermore, the cells of D.

bruxellensis showed a tendency to deviate most of

sugar available for biomass and organic acids (lactic

and acetic) compared with Saccharomyces cerevisiae,

when calculated on the basis of their respective yields.

As well as this, the acetate production from molasses

medium by both yeasts was in marked contrast with

the previous data on sugarcane juice. Glycerol and

ethanol production by D. bruxellensis cells achieved

levels of 33 and 53 % of the S. cerevisiae, respec-

tively. However, the ethanol yield was similar for both

yeasts. It is worth noting that this yeast did not

accumulate trehalose when the intracellular glycogen

content was 30 % lower than in S. cerevisiae. The lack

of trehalose did not affect yeast viability under

fermentation conditions. Thus, the adaptive success

of D. bruxellensis under industrial fermentation con-

ditions seems to be unrelated to the production of these

reserve carbohydrates.

Keywords Ethanol fermentation � Glycogen �Industrial adaptation �Molasses �Organic acids �Trehalose

Introduction

Alcoholic fermentation is endowed with stressors (low

pH, high temperature, high osmotic pressure at the

beginning and high ethanol concentration at the end),

which are either environmental in origin or result from

the yeast metabolism. Whenever these stressors act

synergistically, particularly during the cell recycling,

the cell viability is severely affected with negative

consequences on the fermentation yield (Dorta et al.

L. F. Pereira � M. A. de Morais Jr (&)

Interdepartmental Research Group on Metabolic

Engineering, Federal University of Pernambuco, Recife,

Brazil

e-mail: marcos.morais@pesquisador.cnpq.br

URL: http://www.ufpe.br/nem

E. Lucatti � L. C. Basso

Department of Biological Sciences, ESALQ-University of

Sao Paulo, Piracicaba, Brazil

M. A. de Morais Jr

Department of Genetics, Federal University of

Pernambuco, Av. Moraes Rego, 1235 Cidade

Universitaria 50, Recife, PE 670-901, Brazil

123

Antonie van Leeuwenhoek (2014) 105:481–489

DOI 10.1007/s10482-013-0100-5

2006; Bai et al. 2008; Basso et al. 2008; De Lucena

et al. 2012).

The yeast Dekkera bruxellensis has proved to be well

suited to industrial fermentation processes and obtained

ethanol yields similar to those of Saccharomyces

cerevisiae in both laboratory media and sugarcane juice

(De Souza Liberal et al. 2007; Pereira et al. 2012). The

ability of this yeast to adapt to industrial processes has

been attributed to its resistance to weak organic acids

(Ciani et al. 2003), the nutritional advantage by the use of

nitrate as nitrogen source (De Barros Pita et al. 2011),

tolerance to ethanol (Jensen et al. 2009), robustness

against variations of pH and temperature (Blomqvist

et al. 2010) and its ability to scavenge limited amount of

glucose at oxygen-limited conditions (Blomqvist et al.

2012). The physiological and metabolic causes of this

kind of adaptation still remain unclear. Despite their

robustness in industrial conditions, D. bruxellensis cells

are very sensitive to storage under in laboratory medium

for long periods (unpublished data) and its growth rate is

lower in laboratory medium than in industrial medium,

such as sugarcane juice (De Barros Pita et al. 2011; Leite

et al. 2012; Pereira et al. 2012). In a recent study, we

observed that cells of the industrial strain D. bruxellensis

GDB 248 are less tolerant to thermal and oxidative

stresses than industrial strain S. cerevisiae JP1 (unpub-

lished data). Moreover, it seems that a combined

treatment with sulphuric acid and ethanol can effectively

eliminate D. bruxellensis cells from mixed cultures with

S. cerevisiae (Meneghin et al. 2013). Thus, a more in-

depth metabolic analysis of this yeast is required in order to

discover the mechanisms that go beyond this adaptation.

Yeast cells that are subjected to stressful fermen-

tation conditions have low level of glycogen reserve

carbohydrates and trehalose. This may affect their

survival in these environments as elevated levels of

these carbohydrates are required to enable yeast to

withstand the stress involved in industrial processes

(Basso et al. 2011). It is well established that the

intracellular content of glycogen and trehalose in yeast

cells varies significantly depending on the growth

conditions, the availability of nutrients in the media

and the presence of stress-inducing factors (Francois

and Parrou 2001). Moreover, studies have shown the

relationship between the ethanol stress and the mobi-

lization of these carbohydrates, since situations may

occur during fermentation that can reduce the treha-

lose content by up to 60 % (Pataro et al. 2002).

Likewise, the increase of the intracellular content of

glycogen during fermentation is linked to its adapta-

tion and tolerance to ethanol (Mansure et al. 1997;

Dake et al. 2010). Recent work has shown that during

the sugarcane juice fermentation that involves re-

pitching, there was a lower rate of sucrose consump-

tion by D. bruxellensis cells, a large deviation in the

determination of biomass carbon, and a similar

tolerance level to ethanol and sensitivity to organic

acids as that of S. cerevisiae (Pereira et al. 2012). In

this study, we evaluated the fermentation profile of D.

bruxellensis in assays performed under conditions of

cell re-pitching fermentation using sugarcane molas-

ses as a substrate. This laid emphasis on the analysis of

intracellular content and the mobilization of trehalose

and glycogen, by drawing a parallel with this

biochemical trait and the adaptability of yeast cells

to the industrial environment.

Materials and methods

Yeast strains and media

Industrial strain Dekkera bruxellensis GDB 248 and

industrial strain S. cerevisiae JP1 were used in this

study (Pereira et al. 2012). The cells were maintained

in YPD medium. Propagation molasses medium

(PMM) was composed of sugarcane molasses diluted

to 4 g(sugar) l-1 with distilled water, while fermen-

tation molasses medium (FMM) was composed of

sugarcane molasses diluted to 170 g(sugar) l-1 with

distilled water. Both were sterilized by autoclaving

(121 �C, 15 min) before use. The sugar in the media

consists of the sum of glucose and fructose (either as

the monomers and the equivalent amount from

sucrose) in the molasses as quantified by HPLC,

unless stated otherwise in the text.

Fermentation assays

Yeast cells were pre-grown in 50 ml YPD at 30 �C for

48 h in a rotatory shaker at 140 rpm and recovered by

centrifugation. The cellular sediment was re-sus-

pended in 50 ml PMM and cultivated in shake flasks

at 30 �C. Saccharomyces cerevisiae and D. bruxell-

ensis cells were cultivated for 48 and 96 h, respec-

tively, and during these periods, yeast cultures were

fed with 100 ml PMM. Following this, the yeast cells

were collected by centrifugation for the separation of

482 Antonie van Leeuwenhoek (2014) 105:481–489

123

yeast biomass from the fermentation wine. Each

fermentation cycle (24 h) consisted of two stages:

the fed-batch stage (6 h) and the batch stage (18 h) as

practiced in industry. In the first cycle, the fed-batch

stage started by re-suspending yeast biomass in 2 ml

fermentation wine, 6 ml distilled water and 28 ml

FMM. The cultures were fed with three portions of

9.3 ml of FMM (with intervals of 2 h) and incubated at

30 �C for 20 h without agitation. After fermentation

cell were collected by centrifugation (800 g for

20 min), weighted, acid treated (addition of sulphuric

acid until pH 2.5) and re-used (the whole biomass) as

inoculum for a next fermentation cycle, as is per-

formed in the industrial process (Basso et al. 2008).

Five fermentation cycles were performed, and samples

were withdrawn at the end of each cycle for assessing

yeast viability (after eosine dying followed by micros-

copy inspection) and metabolite titers.

Metabolite measurements

Sucrose, glucose, fructose and glycerol were deter-

mined by ion exchange chromatography with a

Dionex DX-300 device equipped with CarboPac PA-

1 (4 9 250 mm) column and pulsed amperometric

detector. Mobile phase was 100 mM NaOH at a

constant flux of 0.9 ml min-1 (Basso et al. 2008). The

fermentation yields were calculated as the amount of

metabolite produced by the consumed sugar. Produc-

tion of CO2 was measured by the weight loss

procedure (Basılio et al. 2008). The intracellular

content of glycogen and trehalose was determined as

follows. Yeast cells were submitted to 0.5 M trichlo-

roacetic acid treatment at 0 �C for 20 min for extrac-

tion of reserve carbohydrates. The trehalose was

quantified by Dionex DX-300 device (Basso et al.

2008) while the level of glycogen was determined in

accordance with Rocha-Leao et al. (1984). Organic

acids (lactate and acetate) were measured by HPLC,

using a CG 480-C isocratic equipment with HPX-87H

column, 5 mM sulphuric acid as mobile phase at

65 �C and a RI detector. All the experiments were

performed in biological triplicates with two technical

replicates and the averages (±S.D.) were plotted.

Kinetic assay

Kinetic assays with both yeasts (D. bruxellensis and S.

cerevisiae) were performed during a fermentation

cycle simulating the industrial process and using the

same proportion of substrate, yeast inoculum and

incubation temperature as used in the re-cycling assay.

Substrate was fed at a constant rate during 4 h by

means of a peristaltic pump and the collected samples

(from 0 to 10 h) were centrifuged (4,000 rpm for

10 min) and the supernatants were used for metabo-

lites analysis as mentioned above. The sugar values

(sucrose, glucose and fructose) represented the titers

yeasts were exposed to during a simulation of the fed-

batch process. For glycerol, lactate and acetate values

the amounts of each metabolite found in the initial

yeast suspensions (0 h) were discounted as were the

substrate contributions during the feeding phase. Such

data represent the amount produced or consumed by

yeast during 10 h fermentation.

Results

Sugarcane molasses fermentation with cell re-

pitching

Cell viability during the fermentation cycles was

maintained at around 96 and 98 % for D. bruxellensis

and S. cerevisiae, respectively (Fig. 1a) and the cell

population for both yeasts was kept constant until the

end of each fermentation cycle (Fig. 1b). As previ-

ously reported for sugarcane juice, in sugarcane

molasses, the D. bruxellensis population was also

higher than the S. cerevisiae population (Fig. 1b). The

final pH of the fermentation was lower for D.

bruxellensis (4.8 ± 0.02) than for S. cerevisiae

(5.06 ± \0.01). The mass balance during the cycles

was calculated as 99 and 94 % for D. bruxellensis and

S. cerevisiae, respectively, and distributed in the

fermentation products as shown in Fig. 2. Overall, the

biomass yield was higher for D. bruxellensis than for

S. cerevisiae cultures (Fig. 2a), whereas the yield of

organic acids (lactic?acetic) was much higher for D.

bruxellensis (Fig. 2b), which can explain the lower

medium pH cited above. In the production of these

acids, there was no difference in the final concentra-

tion of lactic acid between the two yeasts, while the

cultures of D. bruxellensis produced almost three

times more acetate than S. cerevisiae (Table 1). The

residual concentration of glucose and fructose were

similar, which is in contrast with the fact that, on

average, only 36 % of the initial sucrose was

Antonie van Leeuwenhoek (2014) 105:481–489 483

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consumed by D. bruxellensis cells during the fermen-

tation cycles (Table 1). As a result, the production of

both ethanol and CO2 was lower in cultures of D.

bruxellensis than in cultures of S. cerevisiae, although

the ethanol yield was similar for both yeasts (Table 1

and Fig. 2c).

With regard to the reserve carbohydrates, trehalose

could not be detected in cells of D. bruxellensis at the

end of each fermentation cycle, while S. cerevisiae had

up to 17 % of cell dry weight (CDW) corresponding to

this metabolite (Table 1). The average intracellular

glycogen content was lower in D. bruxellensis cells

than in S. cerevisise during the cycles (Table 1). These

results show the difference between both yeasts in the

intracellular content of their carbohydrate reserves.

During cultivation under aerobic conditions in YPD

medium, no measureable trehalose was observed in

the D. bruxellensis cells, while lower glycogen content

was detected in this yeast compared with S. cerevisiae

(unpublished data). This suggests it was a metabolic

trait of this yeast.

Fermentation kinetics in sugarcane molasses

The dynamic behaviour of the yeast physiology was

analysed during single batch experiments. After the

fed batch stage, total sugar was quantified as ca

87 g l-1 which represented the dilution of molasses

and the initial consumption by the cells. Samples were

then collected during the fermentations and the results

showed a slower consumption of sucrose by D.

bruxellensis compared with S. cerevisiae (Fig. 3a).

The glucose and fructose were completely consumed

within 6 h of the batch fermentation by both yeasts

(Fig. 3b, c). The initial accumulation of glucose and

fructose in the medium in cultures of S. cerevisiae

(Fig. 3b, c) might be explained by the regulation of

extracellular invertase, while this enzyme is almost

exclusively intracellular in D. bruxellensis (Leite et al.

2012). Glycerol production by cells of D. bruxellensis

was very low when compared with that of S. cerevisiae

(Fig. 4a). The production of acetate was higher in D.

bruxellensis cultures (Fig. 4b), while the lactic acid

production was almost the same for both yeasts

(Fig. 4c).

Trehalose was not detected in the cells of D.

bruxellensis during fermentation, whereas this metab-

olite accounted for up to 14 % of S. cerevisiae CDW

(Fig. 5). On the other hand, glycogen accumulation

was observed in D. bruxellensis cells, although in

smaller amounts than in S. cerevisiae (Fig. 5).

Discussion

The yeast D. bruxellensis has been regarded as well

suited to industrial fermentation processes due to its

persistence in the fermentation tanks even when it is

not used as an inoculant (De Souza Liberal et al. 2007;

Passoth et al. 2007; Basılio et al. 2008; Pereira et al.

2012; De Souza Barros et al. 2012). Although there is a

limited amount of data on the metabolic mechanisms

that confer this adaptability, it is generally agreed that

this trait may be related to greater tolerance in various

forms of stress (Rozpedowska et al. 2011). The

processes for producing ethanol from sugarcane

substrates are characterized by a succession of non-

Saccharomyces yeasts (Da Silva-Filho et al. 2005;

Basılio et al. 2008; Basso et al. 2008). In addition, the

yeast D. bruxellensis showed a higher rate of growth

Fig. 1 Cell viability (a) and cell count (b) of Saccharomyces

cerevisiae JP1 strain (grey columns) and Dekkera bruxellensis

GDB248 (black columns) at the end of each fermentation cycles

in molasses medium. Cell viability was measured by the

proportion of de-stained cells from the total cells observed in the

microscopic chamber field

484 Antonie van Leeuwenhoek (2014) 105:481–489

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than S. cerevisiae and thus achieved greater efficiency

in sugar conversion to biomass in sugarcane juice

medium (Pereira et al. 2012). Similarly, this tendency

for biomass to be produced by this yeast was also

observed in this study when sugarcane molasses

medium was used (Fig. 2a). Current data show that

D. bruxellensis is capable of achieving ethanol yields

close to those of S. cerevisiae (De Souza Liberal et al.

2007; Blomqvist et al. 2010; De Barros Pita et al. 2011),

and this may occur in situations with Lactobacillus

vini consortium (Passoth et al. 2007; De Souza Barros

et al. 2012). However, it is currently stated that high

ethanol production was only observed after long

periods of fermentation due to the slow assimilation

of sucrose from laboratory medium (De Souza Liberal

et al. 2007; Basılio et al. 2008; Blomqvist et al. 2010),

sugarcane juice (Pereira et al. 2012), grape must

(Renouf et al. 2006), and now from sugarcane

molasses (Fig. 3a). The intracellular location of its

invertase requires the sucrose to first enter D. brux-

ellensis cells before hydrolysis (Leite et al. 2012).

Thus, we can attribute this low productivity of D.

bruxellensis cells to inefficient sucrose assimilation.

Dekkera bruxellensis was also regarded as inefficient

for assimilating glucose and fructose during the

fermentation of grape juice when pure cultures were

used (Renouf et al. 2006). However, in the present

study, the level of assimilation of both sugars was in

the range of that observed for S. cerevisiae (Fig. 3b, c).

In S. cerevisiae the hexose transport system is carried

out by various transporters, while only one H?-

symport sucrose transporter (codified by the AGT1

gene) was identified and characterized (Stambuk et al.

2000). This situation may be similar in D. bruxellensis

with the gene AWRI1499_4315 (Tiukova et al. 2013)

and the differences in hexose assimilation might be

strain-dependent, although deficiency in sucrose

assimilation is a metabolic trait of this species (Basılio

et al. 2008; Leite et al. 2012).

Under anaerobic conditions, little or no glycerol is

produced by cells of D. bruxellensis (Blomqvist et al.

2010; Pereira et al. 2012) compared with the already

well-described production of glycerol by S. cerevisiae

during fermentation. This effect is regarded as evi-

dence of the low capacity of D. bruxellensis to restore

the redox balance through the production of reduced

metabolites such as glycerol (Wijsman et al. 1984). In

this study some glycerol was produced by D. bruxell-

ensis cells, although the amount was about three times

lower than in S. cerevisiae (Table 1). Furthermore, and

unlike what occurs in S. cerevisiae, the yeast D.

bruxellensis produces a good deal of acetate under

aerobic conditions (Leite et al. 2012) and very little (or

none) under anaerobic or oxygen-limited conditions

(Uscanga Aguilar et al. 2003; Pereira et al. 2012). For a

long time, it has been known that Dekkera yeasts

require a supply of oxygen to stimulate fermentation—

a phenomenon known as the Custer effect—and that

Fig. 2 Yields for biomass (a), organic acids (lactate?acetate)

(b) and ethanol (c) of S. cerevisiae JP1 strain (grey columns) and

D. bruxellensis GDB248 (black columns) at the end of

fermentation cycles in molasses medium

Antonie van Leeuwenhoek (2014) 105:481–489 485

123

this can be overcome by the presence of electron

acceptors in the medium such as acetoin (Van Dijken

and Scheffers 1984). De Barros Pita et al. (2011) first

suggested that the nitrate in the sugarcane juice could

be an ultimate electron acceptor, which was able to

suppress the Custer effect and increase the production

of ethanol by D. bruxellensis. This hypothesis was

further confirmed by another study (Galafassi et al.

2013). Similarly, we suggest that some components of

molasses could induce the production of acetate by the

cells of D. bruxellensis even under anaerobic condi-

tions (Table 1; Fig. 4b) by the presence of some

unknown final electron acceptor, which does not occur

in the sugarcane juice medium (Pereira et al. 2012).

This induction some sort of anaerobic respiration,

overcoming the Custer effect, may be responsible for

the increasing of fermentation yield observed in

molasses (Fig. 2c) when compared to sugarcane juice

(Pereira et al. 2012). However, we have observed for

long date that D. bruxellensis does not settle industrial

processes that use molasses as substrates.

Trehalose and glycogen are important intracellular

carbohydrate reserves that maintain carbon and energy

sources as well as playing an important role in cell

Table 1 Metabolites at the

end of three cycles of

fermentation (C1, C3 and

C5) of pure cultures of

Saccharomyces cerevisiae

JP1 and Dekkera

bruxellensis GDB 248 in

sugarcane molasses

medium

Metabolite S. cerevisiae D. bruxellensis

C1 C3 C5 C1 C3 C5

Sugar consumed (%) 99.2 99.7 99.1 50.6 36.4 37.5

Residual glucose (g l-1) 0.19 0.45 0.43 0.29 0.1 0.29

Residual fructose (g l-1) 0.1 0.15 0.14 0.3 0.49 0.31

Glycerol (g l-1) 3.3 3.5 3.9 1.4 1.2 1.2

Acetate (g l-1) 0.42 0.58 0.5 1.4 1.4 1.5

Lactic acid (g l-1) 0.61 0.64 0.68 0.97 0.83 0.97

Glycogen (%DW) 3.9 6.3 6.6 3.2 4.5 4.2

Trehalose (%DW) 13.2 16.8 13.3 0.0 0.0 0.0

Ethanol (%v/v) 7.45 8.45 8.28 4.71 4.73 4.8

CO2 (g l-1) 1.97 2.23 2.18 1.15 1.10 1.19

Fig. 3 Kinetics of the consumption of sucrose (a), glucose (b) and fructose (c) by cells of S. cerevisiae JP1 strain (open circles) and D.

bruxellensis GDB248 (closed circles) within a fermentation cycle in molasses medium

486 Antonie van Leeuwenhoek (2014) 105:481–489

123

protection under stress conditions (Parrou et al. 1997;

Francois and Parrou 2001). The lack of trehalose in D.

bruxellensis cells, together with their low glycogen

content, point to the fact that an increase in biomass

yield should be related to high-resolution metabolic

flux, in a shift towards amino acids biosynthesis in this

yeast. If it comes to fruition, the production of D.

bruxellensis cell biomass as a source of a single cell

protein could be economically beneficial when com-

pared with S. cerevisiae (Leite et al. 2012). Another

important biological function of these carbohydrates is

their participation in cell viability and their longevity.

During the alcoholic fermentation processes, various

stresses can affect the viability of the yeast cells

(Parrou et al. 1997; Graves et al. 2007). Elevated levels

of carbohydrate allow the cells to bear the effects of the

acid wash caused by industrial processes and make

them more tolerant of industrial conditions (Basso et al.

2011). Trehalose is a metabolite of paramount impor-

tance for the tolerance of yeast since this substance

enables it to face different situations of stress associ-

ated with the plasma membrane, while maintaining its

integrity (Martini et al. 2006; Elsztein et al. 2008). This

metabolite also seems to be involved in ethanol stress

tolerance (Mansure et al. 1997). There was no detect-

able accumulation of trehalose by D. bruxellensis cells

in the course of the fermentation (Fig. 5) or during the

fermentation cycles (Table 1). Additionally, the

absence of this metabolite does not appear to affect

cell viability during fermentation when ethanol accu-

mulates in the medium (Fig. 1a), or to organic acids

(Pereira et al. 2012). Moreover, the accumulation of

trehalose is involved in the acquisition of thermotol-

erance in S. cerevisiae (De Virgilio et al. 1994). It is

generally accepted that D. bruxellensis is less tolerant

to heat stress than S. cerevisiae (Brendam et al. 2008).

It was also found that D. bruxellensis is more sensitive

to a combination of acid pH and ethanol treatment than

S. cerevisiae (Bassi et al. 2013). When taken together,

these findings show that in D. bruxellensis, sensitivity

to heat and ethanolic stresses could be connected to a

lack of intracellular trehalose.

Fig. 4 Kinetics of the

production of glycerol (a),

acetate (b) and lactate (c) by

cells of S. cerevisiae JP1

strain (open circles) and D.

bruxellensis GDB248

(closed circles) within a

fermentation cycle in

molasses medium

Fig. 5 Kinetics of intracellular content of glycogen (straight

lines) and trehalose (dashed lines) in the cells of S. cerevisiae

JP1 strain (open circles) and D. bruxellensis GDB248 (closed

circles) within a fermentation cycle in molasses medium

Antonie van Leeuwenhoek (2014) 105:481–489 487

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The intracellular content of glycogen in the cells of

D. bruxellensis was one third of that observed for S.

cerevisiae at the end of the cycle of molasses

fermentation (Table 1; Fig. 5). This metabolite is

considered to be the major reserve of energy in S.

cerevisiae (Parrou et al. 1997; Dake et al. 2010) and

also seems to be involved in its tolerance to ethanol

(Francois and Parrou 2001). Thus, in a similar way to

trehalose, glycogen might be involved in the sensitiv-

ity of D. bruxellensis to thermal or oxidative stresses

(manuscript in preparation) and to the combined effect

of ethanol and low pH (Bassi et al. 2013), but not to

organic acid (Pereira et al. 2012).

In conclusion, fermentation of sugarcane molasses

by D. bruxellensis might display some particular

features when related with sugarcane juice, the most

significant being its ability to stimulate acetate

production even in anaerobiosis. It can be compared

to the differences in mineral/chemical composition

between these substrates. In both cases, sucrose

assimilation still remains the main problem in seeking

to complete the fermentation by means of industrial

processes in a reasonable period, without leaving a

high level of residual sugar in the fermented broth.

And finally, the lack of trehalose and the low glycogen

content (as reserve carbohydrates) is a trait that reveals

a particular metabolic regulation regarding the carbon

metabolism in this yeast which should be taken into

account before this yeast is used as a fermenting

micro-organism. Furthermore, this deficiency may be

linked to the sensitivity of this yeast to stressing

agents, although it does not explain its success to settle

to industrial production plants.

References

Bai FW, Anderson WA, Moo-Young M (2008) Ethanol fer-

mentation technologies from sugar and starch feedstocks.

Biotech Adv 26:89–105

Basılio ACM, Araujo PRL, Morais JOF, Silva-Filho EA, Morais

MA Jr, Simoes DA (2008) Detection and identification of

wild yeast contaminants of the industrial fuel ethanol fer-

mentation process. Curr Microbiol 56:322–326

Bassi AP, da Silva JC, Reis VR, Ceccato-Antonini SR (2013)

Effects of single and combined cell treatments based on low

pH and high concentrations of ethanol on the growth and

fermentation of Dekkera bruxellensis and Saccharomyces

cerevisiae. World J Microbiol Biotechnol 29:1661–1676

Basso LC, Amorim HV, Oliveira AJ, Lopes ML (2008) Yeast

selection for fuel ethanol in Brazil. FEMS Yeast Res

8:1155–1163

Basso LC, Basso TO, Rocha SN (2011) Ethanol production in

Brazil: the industrial process and its impact on yeast fer-

mentation. In: Dos Santos Bernardes MA (ed) Biofuel

production—recent developments and prospects. Intech,

Zagreb, pp 85–100

Blomqvist J, Eberhard T, Schnurer J, Passoth V (2010) Fer-

mentation characteristics of Dekkera bruxellensis strains.

Appl Microbiol Biotechnol 87:1487–1497

Blomqvist J, Nogue VS, Gorwa-Grauslund M, Passoth V (2012)

Physiological requirements for growth and competitive-

ness of Dekkera bruxellensis under oxygen-limited or

anaerobic conditions. Yeast 29:265–274

Brendam C, Castro-Martınez C, Delia M-L, Ramon-Portugal F,

Strehaianoet P (2008) Effect of temperature on Brettano-

myces bruxellensis: metabolic and kinetic aspects. Can J

Microbiol 54:11–18

Ciani M, Maccarelli F, Fatichenti F (2003) Growth and fer-

mentation behaviour of Brettanomyces/Dekkera yeasts

under different conditions of aerobiosis. World J Microbiol

Biotechnol 19:419–422

Da Silva-Filho EA, Melo HF, Antunes DF, Dos Santos SKB,

Resende MA, Simoes DA, Morais MA Jr (2005) Isolation

by genetics and physiological characteristics of a fuel

ethanol fermentative Saccharomyces cerevisiae strain with

potential for genetic manipulation. J Ind Microbiol Bio-

technol 32:481–486

Dake MS, Jadhv JP, Patil NB (2010) Variations of two pools of

glycogen and carbohydrate in Saccharomyces cerevisiae

grown with various ethanol concentrations. J Ind Microbiol

Biotechnol 37:701–706

De Barros Pita W, Leite FC, de Souza Liberal AT, Simoes DA,

de Morais MA (2011) The ability to use nitrate confers

advantage to Dekkera bruxellensis over S. cerevisiae and

can explain its adaptation to industrial fermentation pro-

cesses. Antonie Van Leeuwenhoek 100:99–107

De Lucena RM, Elsztein C, Simoes DA, De Morais Jr MA

(2012) Participation of CWI, HOG and Calcineurin path-

ways in the tolerance of Saccharomyces cerevisiae to low

pH by inorganic acid. J Appl Microbiol 113:629–640

De Souza Barros R, Dos Santos BM, De Souza RFR, Da Silva

PQN, Lucena BL, Marcos Morais MA Jr (2012) The

consequences of Lactobacillus vini and Dekkera bruxell-

ensis as contaminants of the sugarcane-based ethanol fer-

mentation. J Ind Microbiol Biotechnol 39(11):1645–1650.

doi:10.1007/s10295-012-1167-0

De Souza Liberal AT, Basılio ACM, Resende AM, Brasileiro

BTRV, Silva-Filho EA, Morais JOF, Simoes DA, Morais

MA Jr (2007) Identification of Dekkera bruxellensis as a

major contaminant yeast in continuous fuel ethanol fer-

mentation. J Appl Microbiol 102:538–547

De Virgilio C, Hottiger T, Dominguez J, Boller T, Wiemken A

(1994) The role of trehalose synthesis for the acquisition of

thermotolerance in yeast I. Genetic evidence that trehalose

is a thermoprotectant. Eur J Biochem 219:179–186

Dorta C, Oliva-Neto P, De-Abreu-Neto MS, Nicolau-Junior N,

Nagashima AI (2006) Synergism among lactic acid, sulfite,

pH and ethanol in alcoholic fermentation of Saccharomy-

ces cerevisiae (PE-2 and M-26). World J Microbiol Bio-

technol 22:177–182

Elsztein C, Menezes JAS, Morais Junior MA (2008) Polyhex-

amethylene biguanide can eliminate contaminant yeasts

488 Antonie van Leeuwenhoek (2014) 105:481–489

123

from fuel-ethanol fermentation process. J Ind Microbiol

Biotechnol 35:967–973

Francois J, Parrou JL, Graves T, Narendranath N, Power T

(2001) Reserve carbohydrates metabolism in the yeast.

J Inst Brew 113:263–271

Galafassi S, Toscano M, Vigentini I, Piskur J, Compagno C

(2013) Osmotic stress response in the wine yeast Dekkera

bruxellensis. Food Microbiol 36:316–319

Graves T, Narendranath N, Power R (2007) Development of a

‘‘stress model’’ fermentation system for fuel ethanol yeast

strains. J Inst Brew 113:263–271

Jensen SL, Umiker NL, Arneborg N, Edwards CG (2009)

Identification and characterization of Dekkera bruxellen-

sis, Candida pararugosa and Pichia guilliermondii isolated

from commercial red wines. Food Microbiol 26:915–921

Leite FCB, Basso TO, Pita WB, Gombert A, Simoes D, Morais

Junior MA (2012) Quantitative aerobic physiology of the

yeast Dekkera bruxellensis, a major contaminant in bio-

ethanol production plants. FEMS Yeast Res 13:34–43

Mansure JJ, Souza RC, Panek AD (1997) Trehalose metabolism

in Saccharomyces cerevisiae during alcoholic fermenta-

tion. Biotechnol Lett 19:1201–1203

Martini S, Ricci M, Bartolini F, Bonechi C, Braconi D, Millucci

L, Santucci A, Rossi C (2006) Metabolic response to

exogenous ethanol in yeast: an in vivo NMR and mathe-

matical modelling approach. Biophys Chem 120:135–142

Meneghin MC, Bassi AP, Codato CB, Reis VR, Ceccato-An-

tonini SR (2013) Fermentative and growth performances of

Dekkera bruxellensis in different batch systems and the

effect of initial low cell counts in co-cultures with Sac-

charomyces cerevisiae. Yeast 30:295–305

Parrou JL, Teste MA, Francois J (1997) Effects of various types

of stress on the metabolism of reserve carbohydrates in

Saccharomyces cerevisiae: genetic evidence for a stress-

induced recycling of glycogen and trehalose. Microbiol

143:1891–1900

Passoth V, Blomqvist J, Schnurer J (2007) Dekkera bruxellensis

and Lactobacillus vini form a stable ethanol-producing

consortium in a commercial alcohol production process.

Appl Environ Microbiol 73:4354–4356

Pataro C, Guerra JB, Gomes FCO, Neves MJ, Pimentel PF, Rosa

CA (2002) Trehalose accumulation, invertase activity and

physiological characteristics of yeasts isolated from 24 h

fermentative cycles during the production of artisanal

Brazilian cachaca. Braz J Microbiol 33:202–208

Pereira LF, Bassi APG, Avansini SH, Neto AGB, Brasileiro

BTRV, Ceccato-antonini SR, De Morais MA (2012) The

physiological characteristics of the yeast Dekkera brux-

ellensis in fully fermentative conditions with cell recycling

and in mixed cultures with Saccharomyces cerevisiae.

Antonie Leeuwenhoek 101:529–539

Renouf V, Falcou M, Miot-Sertier C, Perello MC, De Revel G,

Lonvaud-Funel A (2006) Interactions between Brettano-

myces bruxellensis and other yeast species during the initial

stages of winemaking. J Appl Microbiol 100:1208–1219

Rocha-Leao MHM, Panek AD, Costa-Carvalho VLA (1984)

Glycogen accumulation during growth of Saccharomyces

cerevisiae: catabolite repression effects. IRCS Med Sci

12:411–412

Rozpedowska E, Hellborg L, Ishchuk OP, Orhan F, Galafassi S,

Merico A, Woolfit M, Compagno C, Piskur J (2011) Par-

allel evolution of the make–accumulate–consume strategy

in Saccharomyces and Dekkera yeasts. Nat Commun 2:302

Stambuk BU, Batista AS, De Araujo PS (2000) Kinetics of

active sucrose transport in Saccharomyces cerevisiae.

J Biosci Bioeng 89:212–214

Uscanga Aguilar MG, Delia ML, Strehaiano P (2003) Bretta-

nomyces bruxellensis: effect of oxygen on growth and

acetic acid production. Appl Microbiol Biotechnol

61(157–10):162

Van Dijken JP, Scheffers WA (1984) Studies on alcoholic fer-

mentation in yeasts. In: Houwink EH, Van der Meer RR

(eds) Innovations in biotechnology. Elsevier, Amsterdam,

pp 497–506

Wijsman MR, van Dijken JP, van Kleeff BH, Scheffers WA

(1984) Inhibition of fermentation and growth in batch

cultures of the yeast Brettanomyces intermedius upon a

shift from aerobic to anaerobic conditions (Custers effect).

Antonie Van Leeuwenhoek 50:183–192

Antonie van Leeuwenhoek (2014) 105:481–489 489

123