RESEARCH ARTICLE
Biomass production and alcoholic fermentation performance of
Saccharomyces cerevisiae as a function of nitrogen source
Ruben Martı́nez-Moreno1,2, Pilar Morales1, Ramon Gonzalez1, Albert Mas2 & Gemma Beltran2
1
Instituto de las Ciencias de la Vid y del Vino, Logroño, Spain; and 2Department of Bioquimica i Biotecnologia, Faculty of Enologia, Universitat
Rovira i Virgili, Tarragona, Spain
Correspondence: Albert Mas, Department
Bioquimica i Biotecnologia, Faculty of
Enologia, Universitat Rovira i Virgili,
Tarragona 43007, Spain. Tel.: 34977558688;
fax: 34977558232; e-mail: [email protected]
Received 10 October 2011; revised 7 March
2012; accepted 7 March 2012.
Final version published online 10 April 2012.
DOI: 10.1111/j.1567-1364.2012.00802.x
Editor: Isak Pretorius
Keywords
wine yeast; nitrogen requirements; organic
nitrogen; stuck fermentation.
Abstract
Nitrogen limitation is one of the most common causes for stuck or sluggish
fermentation. A broad range of values have been reported as the minimum
nitrogen concentration necessary for the completion of alcoholic fermentation.
We have analyzed the minimum nitrogen concentration required to yield the
maximum biomass (nitrogen reference value) using a microwell plate reader to
monitor fermentation with different nitrogen sources and sugar concentrations.
The biomass yield was dependent on the amount of available nitrogen, the nature of nitrogen source, and the sugar concentration in the medium. Nevertheless, achieving the maximum biomass was not sufficient to ensure the
completion of the alcoholic fermentation, because the fermentation of
280 g sugar L 1 stuck, regardless of the nature and concentration of nitrogen
source. However, a mixture of five amino acids (Leu, Ile, Val, Phe and Thr) as
the nitrogen source allowed for maximum sugar consumption. Analysis of cell
vitality by impedance showed a significant improvement in the vitality for cells
fermenting using this amino acid combination.
YEAST RESEARCH
Introduction
Assimilable nitrogen is essential for yeast metabolism and
growth. Nitrogen availability is directly related to biomass
production during the yeast exponential growth phase at
early stages of alcoholic fermentation (Cantarelli,
1957a, b; Varela et al., 2004; Hernandez-Orte et al.,
2005). Nitrogen is often the limiting factor in grape juice
fermentation (Varela et al., 2004; Bell & Henschke, 2005;
Carrau et al., 2008; Hazelwood et al., 2008). Low nitrogen
content in the must may result in sluggish or stuck fermentations (Bisson & Butzke, 2000). To prevent these
problems, a common practice is to supplement the must
with nitrogen, preferably at early stages of alcoholic fermentation (Jiranek et al., 1995). Several studies have been
performed to find the optimal nitrogen concentration in
must to guarantee complete fermentation (Cantarelli,
1957a, b; Bely et al., 1990; Mendes-Ferreira et al., 2004).
The absolute minimum amount of nitrogen required for
alcoholic fermentation is very difficult to determine.
In fact, the available references to date report ranges
from 120 to 140 mg yeast available nitrogen (YAN) L 1
(Bely et al., 1990) and from 200 to 267 mg YAN L 1
FEMS Yeast Res 12 (2012) 477–485
(Mendes-Ferreira et al., 2004), that is twice as much.
These differences could be attributed to several factors,
such as the yeast strain used, or the quality of the nitrogen source (amino acids and ammonium are present in
different concentrations according to grape variety and
vintage conditions). The nitrogen requirements of different yeast strains are well known among wine yeast suppliers, and commercial strains are categorized according to
the amount of nitrogen they require. Other aspects such
as enological practices (pressing, macerations, etc.) alter
nitrogen availability and the nitrogen concentration in
the must and thus are also considered when determining
the quality and concentration of the nitrogen source. Furthermore, although a nitrogen deficit can result in stuck
or sluggish fermentation, excess nitrogen can lead to
other problems, including the production of unwanted
metabolites such as ethyl carbamate or biogenic amines
(Ribéreau-Gayon et al., 2004). Thus, the amount of nitrogen supplied to the must needs to be sufficient for fermentation, but not in excess. Consequently, it is highly
recommended that the wine producers understand the
nitrogen requirements of each yeast strain and routinely
evaluate the YAN in the must.
ª 2012 Federation of European Microbiological Societies
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478
R. Martı́nez-Moreno et al.
Another aspect to be considered is that the nitrogen
requirements are also related to must sugar concentration. Until recently, the differences in sugar concentration
for a given grape variety in a certain location were limited, with a rather constant concentration for every vintage. However, during the last decades and because of
global warming, larger differences in sugar concentration
have been observed with a trend toward increased sugar
content. Although grapes can be harvested earlier, if
appropriate phenolic ripeness is sought, sugar concentrations will be higher and thus change the nitrogen requirements of the yeast and the nitrogen availability in the
must (Jones & Davis, 2000; Jones et al., 2005; Mira de
Orduña, 2010). In light of this evidence, there is a need
for a better understanding of how yeast nitrogen metabolism is altered by increased levels of sugar.
In this study, we sought to determine the minimum
amount of nitrogen necessary to obtain the maximum
yeast biomass under different conditions of nitrogen
availability and sugar concentrations. We used a microwell spectrophotometer to quantify the growth parameters in synthetic musts with different sugar and nitrogen
concentrations to determine yeast nitrogen requirements.
Synthetic must was used to avoid interference of uncontrolled factors that are present in complex natural
musts. On the other hand, the effects of different nitrogen sources at different sugar concentrations on fermentation kinetics, population size, and cell vitality during
fermentation were also analyzed in laboratory fermentations.
and 60 mg L 1 potassium disulfite (Merck). The media
were filtered with 0.22 lm pore size nitrocellulose filters
and adjusted to pH 3.5 with 10 N KOH. All media preparations were derived from MS300 as described by Bely
et al. (1990).
The carbon source used was a 50 : 50 mixture of glucose and fructose. The concentrations tested were 200,
240, and 280 g sugar L 1.
Four different nitrogen sources were used: (1) ammonium chloride as an inorganic nitrogen source (I), (2)
arginine as a simple organic nitrogen source (O), (3) a
cocktail of leucine – 20.4% (w/v), isoleucine – 13.3% (w/
v), valine – 20.2% (w/v), phenylalanine – 12.1% (w/v),
and threonine – 34% (w/v) as a mixed organic nitrogen
source (M), and (4) a mixture of 19 amino acids and
ammonium chloride as a control synthetic grape must
[Control Nitrogen Content (C)]. The proportions of each
amino acid and ammonium described by Beltran et al.
(2004) were used as a reference to formulate the control
must, and the concentration of each amino acid in the M
mixture was proportional to the concentration of the
same amino acid in the C cocktail.
For cell vitality experiments, a specific medium was
designed to mimic the middle-late fermentation stage
(MF medium) containing 2% glucose, 4.5% fructose,
8.8% (v/v) ethanol, 0.6% citric acid, 0.6% malic acid,
0.17% YNB without amino acids and ammonium sulfate,
and anaerobic factors (15 mg L 1 ergosterol, 5 mg L 1
sodium oleate, and 0.5 mL L 1 Tween 80), adjusted to
pH 3.5 with 10 N KOH.
Materials and methods
Nitrogen reference value (NRV) determination
Yeast strain
The active dry wine yeast (ADWY) strain Saccharomyces
cerevisiae EC1118 (Lallemand Inc., Canada) was used
throughout this study. Before each experiment, 1 g of
ADWY was rehydrated in 10 mL of sterile water at 37 °C
for 30 min, in accordance with the manufacturer’s specifications. For all assays performed using a microwell plate
–reading spectrophotometer, 0.1 g of ADWY was rehydrated in 10 mL of sterile water at 37 °C for 30 min.
Culture media
Sixteen different types of culture media were used in this
study. The basic medium composition included a carbon
source, a nitrogen source, 0.6% citric acid, 0.6% malic
acid (all of them from Sigma-Aldrich), 0.17% YNB without amino acids and ammonium sulfate (Difco), anaerobic factors [15 mg L 1 ergosterol (Sigma), 5 mg L 1
sodium oleate (Sigma), 0.5 mL L 1 Tween 80 (Scharlau)],
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Yeast was grown in synthetic musts containing different
nitrogen sources to determine the minimum amount of
nitrogen necessary to obtain the maximum biomass.
Microfermentations were carried out in a 96-well plate
(Brand/plates microplates, sterile – ref. 781662) and monitored in a microwell spectrophotometer (Omega PolarStar, BMG Labtech) for 4 days at 28 °C. The initial
optical density (OD) was adjusted to 0.2 at 600 nm, corresponding approximately to 2 9 106 cell mL 1 of rehydrated dry yeast. Yeast growth was determined by the
measurement of the OD at 600 nm every 30 min after
shaking (100 r.p.m.) for 40 s in a final volume of 250 lL
per well (sterile medium was used as blank). The growth
was analyzed in three different sugar concentration (200,
240, and 280 g sugar L 1) and with increasing nitrogen
concentrations (0, 5, 10, 20, 40, 60, 80, 100, 120, 140,
160, 180, 200, and 220 mg N L 1) of the four different
nitrogen sources (I, O, M, and C). Each combination
(nitrogen source 9 nitrogen concentration 9 sugar concentration) was performed in quadruplicate.
FEMS Yeast Res 12 (2012) 477–485
479
Nitrogen availability and alcoholic fermentation
Data from the microwell spectrophotometer software
were transformed with the polynomial curve
0.205*x2 + 1.1086*x
0.0107 for cory = 0.1736*x3
recting the nonlinearity of the optical recording at higher
cell densities. This polynomial curve was obtained from
previous studies using serial dilutions of yeast suspensions
as described by Warringer & Blomberg (2003).
Fermentation conditions
Fermentations were performed in 250-mL borosilicate
glass bottles (Schott AG, Mainz, Germany) containing
200 mL of medium and capped with closures that
enabled carbon dioxide to escape and samples to be
removed. Depending on the sugar concentration, different
nitrogen concentrations of each nitrogen source were
used (200 g sugar L 1, 140 mg N L 1; 240 g sugar L 1,
160 mg N L 1; 280 g sugar L 1, 180 mg N L 1). Fermentations were performed in triplicate at 28 °C with
continuous orbital shaking (100 r.p.m.). The initial OD
was adjusted to 0.2 at 600 nm, corresponding to approximately 2 9 106 cell mL 1 of rehydrated dry yeast. A
2-mL sample was taken daily and filtered through membrane filters of 0.22 lm pore size (Sarstedt, Germany) for
analysis of the main metabolites, and a 3-mL sample to
monitor the medium density using a Densito 30 PX densitometer (Mettler Toledo, Switzerland). Fermentations
were considered complete when the density was below
995 g L 1 and/or the sugar content was below 4 g L 1.
At that point, a 1-mL sample was removed and used to
determine the population of viable and total cells. Cells
were diluted and spread onto YPD agar medium (2%
glucose, 2% peptone, 1% yeast extract, and 2% agar)
using a Whitley Automatic Spiral Plating apparatus (AES
Laboratoire, France) to assess viability. Plates were incubated at 28 °C for 48 h, and the CFU were quantified
using the ProtoCOL SR/HR 1.27 counting system software, supplied by Symbiosis (Cambridge, UK). Total cells
counts were determined by counting on a BlauBrand
Thoma counting chamber (Brand GMBH + CO KG,
Wertheim, Germany).
and inoculated at an OD at 600 nm of 0.500 ± 0.014 into
a glass vial containing 5 mL of MF Medium. The vial
containing the sample was placed into the measuring cell
with 2 mL of a solution of 0.2% KOH solution, and the
measuring cell was immediately tightly sealed and incubated. The BacTrac measured the impedance level every
10 min and drew a curve to show the percentage decrease
in impedance over time. The time taken to reach
decreases of 10% and 40% in impedance at 20 °C was
selected as threshold value for 140-h samples (10% and
20% for the 270-h sample with 280 g sugar L 1 condition). Each condition (nitrogen source 9 sugar concentration) was tested in quadruplicate.
Vitality data were analyzed by one-way ANOVA and the
Tukey test (P < 0.05) for means comparison using SPSS
Inc. (Chicago, IL).
Analytical methods
The concentrations of the main extracellular compounds,
that is, glucose, fructose, glycerol, and ethanol, were
determined throughout the fermentation by HPLC using
a Surveyor Plus chromatograph (Thermo Fisher Scientific,
Waltham, MA) equipped with a refraction index and a
photodiode array detector (Surveyor RI Plus and
Surveyor PDA Plus, respectively) and a HyperREZTM XP
Carbohydrate H + 8 lm column (Thermo Fisher Scientific) assembled to its corresponding guard column. The
column was maintained at 50 °C, and 1.5 mM H2SO4 at
a flow rate of 0.6 mL min 1 was used as the mobile
phase. Samples were diluted 5- or 10-fold and analyzed in
duplicate.
The nitrogen concentration in all of the media used
was confirmed by HPLC analysis. The nitrogen concentrations were analyzed in duplicate by HPLC according to
the method of Gomez-Alonso et al. (2007). Amino acid
and ammonium concentrations were transformed into
yeast assimilable nitrogen (YAN, expressed as mg N L 1)
according to assimilable nitrogen atoms of each amino
acid. One nitrogen atom per amino acid was considered
except Arg (three atoms), Lys (two atoms), Gln (two
atoms), and Pro (no assimilable nitrogen).
Cell vitality determination
Cell vitality (fermentation activity) was analyzed by modification of the protocol described by Rodriguez-Porrata
et al. (2008) using the BacTrac 4300 microbiological analyzer (SY-LAB Instruments, Austria). This device measures variations in electrical impedance of a solution
owing to the CO2 produced by yeast during the fermentation process (Ribeiro et al., 2003).
Cells were removed from fermentation bottles 140 or
270 h after inoculation. Cells were washed in sterile water
FEMS Yeast Res 12 (2012) 477–485
Statistical analysis
Statistical analysis was performed using the SPSS win 19.0
program (SPSS Inc.).
The corrected OD data were used in a multivariate
analysis as the dependent variable, and the amount of
nitrogen, nature of the nitrogen source, and sugar concentration as fixed factors (Tukey test; P < 0.05). Principal components analysis (PCA) was used to determine
the influence of each studied factor on the OD.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
R. Martı́nez-Moreno et al.
480
6.0
(a)
5.0
4.0
OD 600 nm
The OD obtained at NRV values was analyzed by multivariate analysis using OD as the dependent variable and
the nitrogen source and sugar concentration as factors
(Tukey test; P < 0.05).
One-way ANOVA was carried out on fermentation
parameters and vitality test data obtained with the various nitrogen sources. The means were compared by the
Tukey test (P < 0.05).
3.0
2.0
1.0
Results
0.0
NRV and maximal biomass production
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
(b)
5.0
OD 600 nm
4.0
3.0
2.0
1.0
0.0
(c)
6.0
5.0
4.0
OD 600 nm
Our aim was to determine the minimum amount of
nitrogen required to obtain a maximal biomass yield for
a given must composition. We have named this parameter the NRV. It was calculated by studying cell growth in
a range of nitrogen concentrations (5–220 mg L 1) and
then plotting the maximal OD reached against the nitrogen concentration available (Fig. 1). The nitrogen concentration for which the slope was below 0.02 was taken as
the NRV for that particular condition.
The NRV was calculated for the combination of three
different sugar concentrations and four different nitrogen
sources (Fig. 1). The results are summarized in Table 1.
As expected, the NRV depended on the composition of
the medium. It increased with the initial sugar concentration of the must, but it was also dependent on the nature
of the nitrogen source. A clear trend for the dependence
of the NRV on the nature of the nitrogen source could
not be established, probably because of interactions
between the two factors under study (initial sugar concentration and nature of the nitrogen source). For example, both the lowest and the highest NRV values were
found for ammonium (they depended on the sugar concentration) and no differences were found in NRV for
must containing 240 g L 1 sugar.
ANOVA analysis confirmed the interaction between initial
sugar content and nitrogen source for maximum biomass
production. PCA analysis showed that as well as nitrogen
concentration, which explained 86% of the variance, the
nature of the nitrogen source and sugar concentration
explained 65% and 35% of the residual variance, respectively. When multivariate analysis was performed using
OD at the NRV concentration as dependent variable, significant differences were detected in sugar content, with
the intermediate sugar concentration giving rise to the
highest biomass production levels for each nitrogen
source (Table 1). The highest sugar concentration tested
did not produce more biomass, which indicated that high
osmotic pressure had some inhibitory effect on growth.
Clear and significant differences were observed among
nitrogen sources, and the production of biomass was
6.0
3.0
2.0
1.0
0.0
0
50
100
150
200
250
[N] mg L–1
Fig. 1. Maximum biomass production of Saccharomyces cerevisiae
strain EC1118 in the presence of different nitrogen sources and sugar
concentrations. (a) 200 g sugar L 1; (b) 240 g sugar L 1; (c)
280 g sugar L 1. Each value represents the mean ± standard deviation
from four growth curves. Nitrogen sources: M; O; C; I.
highest for the complete synthetic must and lowest for
single nitrogen sources, especially ammonium.
Fermentation kinetics and nitrogen sources
To better understand the interactions between the different nitrogen sources and sugar content, we decided to go
beyond biomass production and study fermentation
kinetics using a synthetic must with varying sugar content
and nitrogen levels that would ensure a high biomass
production for any of the nitrogen sources.
FEMS Yeast Res 12 (2012) 477–485
481
Nitrogen availability and alcoholic fermentation
Table 1. Nitrogen reference values (NRV, calculated as slope < 0.02),
and maximum biomass shown as OD600 nm obtained for each
combination of sugar concentration and nitrogen source
200 g L
NRV
OD600
1
240 g L
NRV
OD600
1
280 g L
NRV
OD600
1
Signif.
nm
nm
nm
M
O
C
I
Signif.
140
3.91 ±
0.10
140
3.70 ±
0.09
160
4.79 ±
0.11
120
3.32 ±
0.09
A
160
4.72 ±
0.12
160
4.25 ±
0.11
160
5.2 ±
0.13
160
3.90 ±
0.10
B
180
4.29 ±
0.11
b
200
3.41 ±
0.09
c
180
4.68 ±
0.12
a
200
3.12 ±
0.08
d
A
Different letters indicate significant differences (P < 0.05) for OD values. Upper case letters are used for sugar concentration differences,
and lower case letters for nitrogen sources.
The total fermentation duration was similar for all fermentations that used a sugar concentration of 200 g
sugar L 1, and the values of the main metabolites did not
show any significant differences (Table 2). The largest difference observed was in the significantly higher maximum
fermentation rate (MSFR) for the complete must. However, no significant differences were found in ethanol
yields.
The results were more variable at higher sugar concentrations, and not all of the nitrogen sources equally supported the fermentation activity. At a sugar concentration
of 240 g L 1, fermentations performed with single nitrogen sources (O and I) took much longer (4 days) than
the fermentations with more complex nitrogen sources
(M and C). The sample with the highest maximum fermentation rate was again the control must, although at
this concentration of sugar, the ammonium sample also
sustained a high fermentation rate. At this concentration
(240 g sugar L 1), significant differences were detected in
ethanol yields, and the control must presented the worst
one (Table 2a). At 280 g sugar L 1, all of the fermentations were stuck. Thus, we repeated the same fermentations with a higher nitrogen concentration (300 mg N L 1).
Nevertheless, these fermentations still did not go to completion. Significant differences between nitrogen sources
were observed in the amounts of residual sugars: the
residual sugar concentration was always lower with the
mixture of complex amino acids and higher with ammonium as the only nitrogen source. As a consequence, the
ethanol concentration was also higher when the mixture
of complex amino acids was present, whereas with
ammonium, the ethanol concentration was from one to
three percentage points lower. There was no difference
FEMS Yeast Res 12 (2012) 477–485
between the two nitrogen concentrations tested when
ammonium was used as the nitrogen source, whereas in
the other nitrogen conditions, the ethanol concentration
was generally one percentage point higher when
300 mg L 1 of nitrogen was used, because of a higher
consumption of residual sugars on those fermentations.
The analysis of ethanol yields did not show significant
differences among the nitrogen conditions. The different
levels of ethanol and residual sugars could be easily
related to the amount of viable cells. In fact, no viable
cells were recovered at the end of fermentation except
from the fermentation with mixed organic nitrogen
source. The presence of viable cells would explain why
the residual sugar was always lower in fermentations with
the mixed organic nitrogen source. Surprisingly, the maximum fermentation rate was always the lowest in fermentations with mixed organic nitrogen source, almost half
of the rate of the values obtained from the other fermentations. The amount of nitrogen available in the must
with 280 g sugar L 1 had a clear effect on the MSFR values. Unexpectedly, at this high sugar concentration, the
maximum fermentation rates were lower when the nitrogen availability was higher.
The effect of the mixed organic nitrogen source was
interesting for several reasons. These fermentations started
later than fermentations with other sources, which correlated with a longer lag phase in nitrogen uptake (results
not shown). Moreover, the fermentation rate with the
mixed organic nitrogen source was slightly lower but was
maintained for longer periods of time, allowing for a similar fermentation length at low sugar concentrations and
a better performance at higher sugar concentrations.
Although the differences in fermentation performances
between mixed nitrogen sources (C and M) and single
sources (I and O) could be partly attributed to the production of higher biomass, not all of the differences could
be explained by the biomass.
Vitality test
The difference between the high biomass and fermentation performance could also be explained by the vitality
of the cells once they have reached the maximum population density. The cell vitality was measured as the changes
in impedance observed when cells had reached the maximum population during the fermentation process
(140 h). The vitality was analyzed by measuring the
changes in impedance at 10% or 40% of the total impedance change possible. For comparative purposes, the values (in hours) were normalized and referred to the
control values (synthetic must). The positive values
indicate that longer times are required to achieve the
same impedance drop, whereas negative values mean that
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
2.53 ± 0.64a
85.56 ± 3.56a
6.46 ± 0.64a
0.43 ± 0.02a
1.30E+08
3.44E+07
26.46
55.73 ± 3.68a
274
280 g sugar L
33.7 ± 0.96a
105.81 ± 1.77c
9.5 ± 0.08a
0.43 ± 0.02a
1.56E+08
5.33E+00
3.42E 06
46.54 ± 1.25a
Residual sugar (g L 1)
Ethanol (g L 1)
Glycerol (g L 1)
Ethanol yield (g sugar g etanol 1)
Total cells L 1
Viable cells L 1
% survival
MSFR (g L 1 day 1)
T100 (h)
b
Residual sugar (g L 1)
Ethanol (g L 1)
Glycerol (g L 1)
Ethanol yield (g sugar g etanol 1)
Total cells L 1
Viable cells L 1
% survival
MSFR (g L 1 day 1)
1
1
O
54.50 ± 0.79b
96.24 ± 1.99b
9.7 ± 0.07a
0.43 ± 0.02a
8.08E+07
0.00E+00
0
88.59 ± 5.82b
(180 mg N L 1)
3.83 ± 0.58a
84.86 ± 2.67a
6.16 ± 0.71a
0.43 ± 0.02a
6.40E+07
1.89E+07
29.53
58.58 ± 4.54a
274
(140 mg N L 1)
52.3 ± 1.2b
98.13 ± 1.41b
9.8 ± 0.11a
0.43 ± 0.02a
8.38E+07
0.00E+00
0
86.38 ± 6.03b
2.6 ± 0.17a
82.35 ± 2.39a
6.17 ± 0.19a
0.42 ± 0.03a
8.48E+07
2.49E+07
29.36
97.43 ± 0.58b
274
C
60.40 ± 1.54c
90.20 ± 0.93a
8.7 ± 0.10a
0.41 ± 0.01a
3.48E+07
0.00E+00
0
85.38 ± 2.38b
3.73 ± 0.55a
78.97 ± 3.02a
6.3 ± 0.35a
0.40 ± 0.04a
5.68E+07
1.78E+07
31.34
56.78 ± 3.46a
274
I
MSFR, major sugar fermentation rate; T100, time taken by cells to complete the fermentation process.
a,b mean statistically significant differences set at P < 0.05.
200 g sugar L
a
M
1
12.7 ± 0.25
112.57 ± 1.37
10.5 ± 0.05
0.42 ± 0.01
1.08E+08
5.20E+02
4.81E 04
34.05 ± 1.92
280 g sugar L
1
2.02 ± 1.05a
102.44 ± 2.26a
6.96 ± 0.35a
0.43 ± 0.02a,b
3.84E+07
3.58E+07
93.22
57.3.47 ± 0.06a
257
240 g sugar L
M
37 ± 0.6
105.74 ± 1.39
10.9 ± 0.02
0.44 ± 0.01
9.10E+07
0.00E+00
0
60.40 ± 0.16
(300 mg N L 1)
3.76 ± 0.45a
100.09 ± 2.47a
7.3 ± 0.19a
0.42 ± 0.02a,b
1.87E+07
6.63E+06
35.4
59.81 ± 0.17a
350
(160 mg N L 1)
O
25 ± 0.5
102.84 ± 1.41
11.0 ± 0.06
0.40 ± 0.01
1.37E+08
0.00E+00
0
63.50 ± 0.10
3.23 ± 0.75a
97.65 ± 1.02a
6.83 ± 0.11a
0.41 ± 0.01b
4.57E+07
8.13E+06
17.79
83.09 ± 3.54b
257
C
60 ± 1.2
90.90 ± 1.44
11.4 ± 0.03
0.41 ± 0.02
3.55E+07
0.00E+00
0
62.15 ± 0.08
3.8 ± 0.15a
105.03 ± 1.97a
7.40 ± 0.23a
0.44 ± 0.02a
3.13E+07
6.97E+05
2.22
75.79 ± 2.38b
350
I
Table 2. Main parameters of the laboratory fermentations performed with different nitrogen sources at three different sugar concentrations. (a) Fermentations with 200 and 240 g sugar L 1;
(b) Fermentations using 280 g sugar L 1 with 180 and 300 mg N L 1
482
R. Martı́nez-Moreno et al.
FEMS Yeast Res 12 (2012) 477–485
483
Nitrogen availability and alcoholic fermentation
200 g L–1
*
(a)
*
240 g L–1
*
280 g L–1
*
280 g L–1α
200 g L–1
*
(b)
*
*
240 g L–1
*
–1
280 g L
*
280 g L–1β
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
Fig. 2. Change in impedance as a measurement of cell vitality.
Values are expressed relative to control fermentations. Positive values
indicate that the samples took longer to reduce impedance by 10%
(a) or 40% (b) after 140 h of fermentation. Exceptionally, impedance
was determined after 270 h of fermentation (a) or after 270 h of
fermentation but with a decrease in impedance of 20% (b). Nitrogen
sources were M (■), O (□) and I (■).
the cells require less time and, thus, have higher vitality
(Fig. 2). In the case of cells from the fermentations using
280 g sugar L 1, the analysis of impedance was performed using different thresholds (10% and 20%) because
the cells stopped producing CO2 before they reached the
40% threshold, and a second measurement was also taken
at 270 h because the fermentations were much longer.
It became evident that at low sugar concentrations, the
mixed amino acid source (M) and the inorganic source
(I) displayed significantly lower vitality than the control
source, which had similar vitality to that of the single
organic source (O). The differences disappeared in the
middle sugar concentration (except for ammonium,
which still had the lowest vitality). However, at the highest sugar concentration, the mixed amino acid source
(M) allowed for the highest levels of vitality and reached
values at 270 h that are statistically significant.
Discussion
A variety of approaches have been used to determine the
nitrogen concentration that would allow for optimal wine
fermentation. These approaches include measuring the fermentation rate in a nitrogen-depleted must (Cantarelli,
1957a), analyzing the CO2 production (Bely et al., 1990),
measuring the residual sugar in a nitrogen-supplemented
model media (Mendes-Ferreira et al., 2004) or adding different forms of nitrogen to a natural must (Arias-Gil
FEMS Yeast Res 12 (2012) 477–485
et al., 2007). We propose that analysis of biomass production is a mechanism to determine the minimum nitrogen
concentration needed to obtain the maximum biomass
possible for a given S. cerevisiae strain (EC1118), based on
the concept that biomass determines the fermentation rate
(Varela et al., 2004). We have used the real-time monitoring of biomass production (changes in OD600 nm) in a
microwell plate–reader spectrophotometer during the
exponential growth and early stationary phases. The
principal advantage of this method is the possibility to
perform a large screening with high reproducibility at low
cost (only 0.250 mL of medium per well is needed).
Our results agree with other studies that found the
nitrogen threshold concentration to be approximately
140 mg YAN L 1 (Bely et al., 1990; Mendes-Ferreira
et al., 2004; Beltran et al., 2005). This value was estimated
using the strains S. cerevisiae PYCC 4072 and S. cerevisiae
QA23. The relationship between nitrogen concentrations
and initial sugar concentration has been studied before
with NOPA assays (Bisson & Butzke, 2000), although the
nitrogen requirements were not specific to any yeast
strain. In our study, the nitrogen requirements of S. cerevisiae EC1118 were determined on the basis of the biomass production, and we found that it was affected by
three factors: the amount of nitrogen available, the nature
of the nitrogen source, and the sugar concentration in the
medium. From our results, there was no obvious correlation between the NRV values and biomass production
levels. The NRV values for each nitrogen source were
highest for the must with the highest initial sugar content, while the biomass production levels were highest for
the intermediate level (240 g L 1). It is well known that
S. cerevisiae has limited tolerance to high osmotic pressure (Ribéreau-Gayon et al., 2004), and this could be a
reason for the limited growth at the highest sugar concentration. In general, nitrogen requirements are considered to be strain dependent (Manginot et al., 1998;
Beltran et al., 2005). This method could easily be adapted
to determine the nitrogen requirements of other yeast
strains. It should be emphasized that the maximum biomass obtained was dependent on the nature of the nitrogen source, with the mixed nitrogen source producing
the highest biomass, in agreement with previous reports
that observed that mixed organic improved the fermentation performance (Arias-Gil et al., 2007).
Although our experimental design allowed for the analysis of biomass, it did not indicate the amount of nitrogen required to finish fermentation. We analyzed
laboratory fermentations using synthetic must with
increasing concentrations of sugar and nitrogen. Fermentations with 200 g sugar L 1 and 140 mg N L 1 and fermentations with 240 g sugar L 1 and 160 mg N L 1
finished with similar parameters (e.g. fermentation length,
ª 2012 Federation of European Microbiological Societies
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R. Martı́nez-Moreno et al.
484
MSFR or sugar consumption). In contrast, fermentations
with 280 g sugar L 1 were stuck. However, the stuck fermentations were not caused by inadequate nitrogen
content because fermentations with the same sugar concentration and moderate but not limiting nitrogen availability (Bell & Henschke, 2005) did not finish either,
regardless of the nitrogen source used. The reason for the
stuck fermentations is most likely a combination of different factors, such as high osmotic pressure, ethanol concentration, medium chain fatty acids production, and the
presence of potassium sulfite in the medium at the beginning of fermentation and/or other undetermined factors.
Although it has been reported that both ammoniacal
and amide nitrogen produce high fermentation rates
(Cantarelli, 1957b), we found that the combination of
both organic and inorganic forms of nitrogen in the mixture produced the highest fermentation rate. It has been
reported that growth in media containing amino acids is
better because yeasts can adapt their metabolism and
avoid amino acid synthesis (Albers et al., 1996). In general, yeast can modify their metabolism based on the
available nitrogen in the medium (Jimenez-Marti et al.,
2007). The metabolic adaptation can be observed at the
level of biomass production. In fact, our study found that
the highest yeast population size was always observed in
fermentations with amino acids as the main nitrogen
source. However, when the nitrogen source is a single
compound (in our case arginine or ammonium), a considerable reduction in growth is observed. The presence
of a combination of amino acids allowed for a partial
recovery of the maximum biomass, despite the fact that
growth was particularly slow.
On the other hand, it is well known that nitrogen
catabolite represion (NCR) depends on the availability of
‘good’ nitrogen sources, which are absent from this medium with mixed organic nitrogen sources (Godard et al.,
2007), and thus NCR will not be active. However,
branched-chain and aromatic amino acids (main components of mixed organic nitrogen source) cannot support
high growth rates because their permeases (Bap1p, Bap2p
and Tat1p, Tat2p) are tightly regulated (Bell & Henschke,
2005; Beltran et al., 2005). Therefore, these amino acids
are taken up and assimilated slowly throughout the fermentation process (Hazelwood et al., 2008). A combination of NCR, change in nitrogen metabolism and amino
acid synthesis could explain the long lag phase and the
low MSFR observed in presence of these amino acids.
However, it is not possible to completely explain these
dynamics or the high cell survival.
Attributing the different fermentation kinetics observed
to changes in maximum biomass would be an oversimplification and would not explain these differences. However, differences in fermentation can also be attributed to
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
differences in yeast vitality, that is, the difference in the
ability of the yeast cells to modify their environment
(Ribeiro et al., 2003). One way to analyze yeast vitality is
to determine the changes in the impedance of the medium, which is directly dependent on the metabolic
activity of the yeast cells. Different studies have been performed to determine yeast vitality using methodologies
such as the BacTrac system (Redon et al., 2008; Rodriguez-Porrata et al., 2008) and/or flow cytometry (Attfield
et al., 2000). These works have mainly focused on the
study of vitality in the ADWY after rehydration. In this
study, we have incorporated several changes to enable us
to determine the cell vitality at any specific time during
alcoholic fermentation. Our results can help to explain
why, in the presence of a combination of different amino
acids and despite the slow growth and fermentation rate,
the fermentation can proceed further and can even produce larger amounts of ethanol in low nitrogen availability. Certainly, the metabolism of these amino acids
during fermentation is different than those considered as
good nitrogen (ammonium) or intermediate nitrogen
(arginine) sources, yet this slow metabolism helps to
maintain the cell survival in adverse conditions. The right
combination of both good and poor nitrogen sources
(control must) yields the best results in terms of quick
fermentation and biomass production in most conditions.
However, in extreme conditions, such as those present in
high sugar concentration musts, formulations based on
mixtures of complex amino acids (precursors for
aromatic compounds) could be a better solution for the
nitrogen additions to natural musts.
We have observed that S. cerevisiae EC1118 was not able
to completely ferment a synthetic must with
280 g sugar L 1, regardless of the nature and the amount of
nitrogen source. These conditions, S. cerevisiae EC1118 and
high initial sugar concentration must (280 g sugar L 1),
could be used as a model to study different parameters and
characteristics of stuck fermentations.
The methodology proposed in this work to determine
nitrogen requirements is relatively easy and inexpensive
and could be used to determine the best nitrogen sources
and concentrations for different wine yeast strains. However, it has to be emphasized that obtaining the maximum biomass does not guarantee the success of the
fermentation, and each variable or additional stress
should be analyzed in experimental fermentations before
considering the maximum biomass as sufficient for industrial fermentations.
Acknowledgements
This work was supported by a predoctoral fellowship from
the JAE Program (CSIC) to R. M-M, by the DEMETER
FEMS Yeast Res 12 (2012) 477–485
Nitrogen availability and alcoholic fermentation
project (Ingenio2010-CENIT) and by grant AGL200907331 from the Ministerio de Educación y Ciencia, Spain.
The authors acknowledge Cristina Juez, Laura López, and
Braulio Esteve for excellent technical assistance and to
Zoel Salvadó, Marta Sancho and Manuel Quirós for their
valuable advice.
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