Wine fermentation is carried out by Saccharomyces cerevisiae. Yeast
cells encounter variable concentration of different carbon sources. The changes
in sugar content affect the fermentation process which can be established by
the study of kinetic behavior of fermentation process (Wang
et al., 2004).
The glucose concentration and level of glycolytic intermediates regulate the
expression of several glucose transporters and some glycolytic gene (Carlson,
1999). Therefore, the main glucose repression pathway ensures that the preferred
sugar are metabolized before the consumption of alternative carbohydrates i.e.,
maltose and galactose (Verstrepen et al., 2004).
Glycerol is another carbon source which has been demonstrated to serve as the
major osmolyte of S. cerevisiae. Glycerol is an important cellular compound
in yeast. It can serve as a carbon source as well as it has a metabolic function
because its synthesis contributes to the maintenance of the cellular redox balance
(Ansell et al., 1997). It is a precursor for
the phospholipids which may in turn play a role in signaling (Siderius
et al., 2000). Glycerol is used by the yeast as the sole source of
carbon and energy. The glycerol kinase is a key enzyme for controlling the glycerol
catabolic pathway in yeast. The structural genes glycerol kinase (GUT1) and
FAD dependent glycerol-3-phosphate dehydrogenase (GUT2) have shown similarity
to prokaryotic and eukaryotic homology (Pavlik et al.,
1993; Ronnow, 1992). The gut1 and gut2 mutants are
unable to use glycerol as a sole source of carbon and energy (Sprague
and Cronan, 1977) which suggests that the phosphorylative pathway is the
major route for glycerol assimilation in S. cerevisiae (Grauslund
et al., 1999). Another important aspect is that glycerol maintains
the signaling competent state of the cell (Siderius et
al., 2000). It was proposed, based on the observation of increased glycerol
intracellular levels at high temperature, of HOG pathway, mutants could be related
with increase of glycerol.
Quantitatively, nitrogen is the second most abundant nutrient in wine fermentations.
It is essential for yeast metabolism and growth. Consequently, lack of nitrogen
triggers sluggish fermentations (Alexandre and Charpentier,
1998; Boulton et al., 1996; Fleet
and Heard, 1993). The timing of the addition is key for ensuring a successful
fermentation (Bely et al., 1990; Salmon,
1989). Early addition affects both the fermentation rate and the biomass
yield. Late addition has a minimal effect on biomass formation but however increases
the fermentation rate (Bely et al., 1990).
Depending on the particular yeast race and fermentation conditions, urea may
be used as a nitrogen source by S. cerevisiae. In this case, urea is
degraded to ammonium ion and CO2 by the multipurpose enzyme ATP-urea
amidolyase (Whitney and Cooper, 1972). Ethyl carbamate
is a well-known carcinogen found in fermented foods and drinks (Ough,
1976) which are produced during wine fermentation in the presence of ethanol
and urea. Therefore, high concentration of urea is avoided during fermentation.
The ammonium salt (diammonium hydrogen phosphate) is also used as a good nitrogen
source for the fermentation. It not only prevents the sluggish fermentation
but it is also used for improvement of fermentation kinetics by time optimization
(Bell and Henschke, 2005).
The present research deals with individual as well as the combined effect of carbon source i.e., glucose and glycerol on palm wine fermentation carried out by S. cerevisiae. The effect of nitrogen supplementation on glucose and glycerol during fermentation is also studied.
MATERIALS AND METHODS
Chemicals: Dextrose, Glycerol (GR), KH2PO4, K2HPO4, MgSO4.7H2O, FeSO4.7H2O, Urea, Di-ammonium hydrogen phosphate were purchased from Merck, India. Yeast extract and Peptone were procured from Himedia, India, 3,5-dinitrosalicylic acid used was from Loba Chemie, India.
Microorganism and culture preparation: Stock culture of Saccharomyces cerivisiae (NCIM 3045) was procured from National Chemical Laboratory (NCL) Pune, India. The culture media prepared consisted of malt extract 0.3, glucose 1.0, yeast extract 0.3, peptone 0.5, all in g 100 mL. The organisms were grown at a temperature of 30°C and pH 6.5. The incubation period was 48 h. After incubation, the culture was stored at 4°C in a refrigerator.
Preparation of fermentation media: The palm juice was collected from rural areas of West Bengal, India. It was put in a cooling bag and preserved at -20°C in a freezer (C340, New Brunswick Scientific) within 2-3 h from the time of collection. For fermentation, carbon, nitrogen and other trace elements were added to the palm juice in appropriate amounts. The three different flasks contained the fermentation media as glucose 1.0, glucose: glycerol (1:1) 1.0, glycerol 1.0, (g/100 mL) respectively and others components kept the same for the three flasks were KH2PO4 0.05, K2HPO4 0.05, MgSO4.7H2O 0.05, FeSO4.7H2O 0.001 (g/100 mL). For optimization of nitrogen sources, the fermentation media contained optimized carbon source along with other trace elements as per previous media.
||Influence of different carbon source on sugar utilization
in palm wine fermentation
Fermentation was performed in a 250 mL flask with 100 mL of fermentation media and inoculated with 1 mL of yeast culture solution. The culture solution was prepared by sterile distilled water mixed with yeast slant and concentration of yeast cells in OD was 0.5. The pH and temperature were adjusted to 5.5 and 32°C, respectively in anaerobic condition. The incubation time was 3 days. The samples were withdrawn at appropriate time intervals for analysis. All the experiments were performed thrice Fig. 1.
Estimation of ethanol, sugar and protein concentration: About 5 mL of
fermented sample was centrifuged (Remi C-24, Mumbai, India) at 6500 g for 10
min. The supernatant solution was used to determine the ethanol concentration
by Gas Chromatography (Perichrom SGE D11, column BP1-dimethyl polysiloxane).
The absorbance of the sugar solution was determined spectrophotometrically (Model
no. 2800 Hitachi, Japan) at 540 nm by DNS method (Plummer,
2007). Protein content of juice was estimated by Lowry method at 650 nm
Estimation of biomass concentration: The Biomass concentration was determined
by the dry weight method. The cells were separated by centrifuging 5 mL of fermented
broth (Remi C-24, Mumbai, India) at 1200 g for 20 min consecutively twice with
saline water. The cells were dried at 65°C for 2 days in hot air oven (Concept
International, Kolkata, India). Dilutions of the culture were made and the absorbance
was measured. The calibration curve correlating absorbance and dry weight gave
a straight line (Chowdhury et al., 2003).
RESULTS AND DISCUSSION
In the process of the palm wine fermentation, the effect of different sugars on S. cerevisiae and their performance in the fermentation process has been studied. The organism has shown varying sugar utilization rate, biomass formation rate, ethanol production rate, when the carbon source (i.e., glucose, glycerol, combination of glucose: glycerol) was varied. Table 1 shows the influence of different carbon sources on metabolic activity of the biomass i.e., sugar utilization rate, rate of ethanol production, rate of protein utilization and yield of product coefficient (Yp/s) that relates the amounts of products formed per unit mass of substrate consumed.
Role of different sugars on palm wine fermentatios: In the palm wine
fermentation, glucose, glycerol and the combined effect of glucose: glycerol
very much influenced the yeast activity. Generally, yeasts are glucophilic organism
but under some specific conditions (e.g., in the presence of nitrogen), rapidly
utilize sugars other than glucose (Berthels et al.,
2004). From the beginning and upto 2nd day, the sugar utilization rate was
higher in glucose containing flask it was 0.0047 g/L/h, rather than glycerol
flask (0.0027 g/L/h) or glucose: glycerol flask (0.0014 g/L/h) (Fig.
1). But on the 3rd day sugar utilization rate (0.0363 g/L/h) was highest
in glycerol flask and the rate decreased from glucose: glycerol to glucose these
were 0.0351 and 0.0279 g/L/h, respectively.
Figure 2 shows that initially the production of ethanol was high in glycerol flask from 2nd day onwards it was 105.79 g L-1. Other two flasks had near about the same ethanol concentration upto 2nd day these were 95.1 g L-1 in glucose flask and 97.67 g L-1 in glucose: glycerol. Finally after 3rd day, glucose containing flask produced highest ethanol which was 124.54 g L-1.
Table 2 shows the statistical significance of ethanol production
(one way ANOVA, Microsoft Excel, Windos 2007) with different sugars. The experimental
results were F ratio value 0.000439, p value 0.999561. The F ratio value was
lower than the value of F critical at the 5% level of significance. Therefore
least significance test was not needed (Gacula and Singh, 1984).
Influence of urea in wine fermentation: Nitrogen helps to avoid sluggish
fermentation (Alexandre and Charpentier, 1998; Boulton
et al., 1996). In this study, it has been found that the sugar utilization
was enhanced by using supplemented nitrogen in the fermentation broth. Figure
3 shows that sugar utilization in the control (in absence of urea) was slower
than urea containing media (6 g L-1).
The rates of sugar utilization in urea (6 g L-1) flask were 0.033,
0.087 and 0.032 g/L/h from the 1st-3rd day, respectively. The highest ethanol
production in 6 g L-1 urea flask was 152.20 g L-1 after
72 h. Another nitrogen source, di-ammonium hydrogen phosphate was also used
in the fermentation broth to study its effect on the process of fermentation.
It was found that di-ammonium hydrogen phosphate accelerated the fermentation
process and increased the ethanol production.
||Influence of different carbon source on ethanol production
in palm wine fermentation
||The experimental values of different substrates utilization
rate for the production of ethanol in palm wine fermentation
|| Analysis of Variance (ANOVA) for different substrate utilization
rate for the production of ethanol (Table 1)
|SS = Sum of Squares, df = degree of freedom, MS = Mean of
Squares, p = probability
||Influence of urea on sugar utilization and ethanol production
in palm wine fermentation. Composition of fermentation media-Glucose 1 g/100
mL, other trace element as per media composition. Control has no urea
Optimum concentration of di-ammonium hydrogen phosphate was found to be 4 g
L-1 and yield of ethanol was 96.38 g L-1 after 72 h. The
rates of sugar utilization were 0.020, 0.063 and 0.0154 g/l/h from 1st-3rd day,
respectively. The 7 g L-1 urea and 5 g L-1 di-ammonium
hydrogen phosphate containing flask produced lower quantity of ethanol than
the 6 g L-1 urea and 4 g L-1 di-ammonium hydrogen phosphate
containing flask it may be due to the higher concentration of nitrogen inhibited
the cellular activity of the yeast and ethanol production was affected (Fig.
3 and 4). It can also be concluded from the result that
urea is a better source of nitrogen than di-ammonium hydrogen phosphate with
respect to ethanol production.
Figure 5 shows α-nitrogen utilization and biomass formation with respect to time. The α-nitrogen utilization was overall high in glucose flask, the concentration of α-nitrogen come down from the 1st-2nd day these were 0.9 and 0.436 g L-1 and the utilized α-nitrogen were 0.3 and 0.464 g L-1 as can be calculated from Fig. 5.
After 2nd day utilization slowed down, α-nitrogen concentration was 0.363
g L-1. In the 3rd day concentration of utilized α-nitrogen it
was 0.073 g L-1 and total utilization upto 3rd day was 0.837 g L-1
(data was not shown in Fig. 5). Whereas other two flasks,
there were α-nitrogen concentration came down from the 1-2 days were 1.04
g and 0.586 g L-1 for glycerol and 1.12 and 0.594 g L-1
for glucose: glycerol flask, respectively. The utilized α-nitrogen concentrations
from 1-2 days were for glycerol 0.164 and 0.45 g L-1 and for glucose:
glycerol were 0.076 and 0.53 g L-1, respectively (data were calculated
from Fig. 5).
of di-ammonium hydrogen phosphate on sugar utilization and ethanol production
in palm wine fermentation. Composition of fermentation media- Glucose
1 g/100 mL, other trace element as per media composition. Control has
no diammonium hydrogen phosphate
utilization and biomass formation in palm wine fermentation. Constituent
of fermentation media- Glucose 1 g/100 mL, other trace element as per
media composition and urea 0.6 g/100 mL
In the 3rd day α-nitrogen concentration were 0.433 and 0.484 g L-1
for glycerol and glucose: glycerol flask, respectively. The total α-nitrogen
utilization upto 3rd day were 0.716 and 0.767 g L-1 for glycerol
and glucose: glycerol flask consecutively (data were not shown in graph). The
highest biomass formation, observed in glucose containing flask upto the 2nd
day was 1.273 g L-1 after that the rate of formation decreased and
stationary phase set in. In case of glycerol upto 2nd day biomass formation
was 1.214 g L-1 and it rapidly decreased and the cells entered death
phase after the 2nd day. But in case of glucose: glycerol, biomass formation
increased up to 3rd day with a stationary phase found in the 2nd day. From zero
to 1st day biomass increased to (0.471 g L-1) but from 1st-2nd day
growth was nearly stagnant it was 0.033 g L-1. Again in the 3rd day
growth became exponential and the biomass concentration increased by 0.26 g
L-1. This has happened probably because there is a mixed substrate
utilization which led to a diauxic growth curve.
The predicted utilization of different carbon sources during the palm wine
fermentation was observed. In general, yeast consumed glucose faster than the
other sources of carbon, though some yeast strains prefer fructose (Schutz
and Gafner, 1995). Results show that sugar utilization rate decreased in
the order of glucose, glycerol and glucose: glycerol upto the 2nd day. The reason
behind this was that the glucose was the first choice than other carbon sources.
The ethanol production was high in glycerol flask and then ethanol concentration
decreased from glucose: glycerol to glucose flask in that order upto the 2nd
day. This is because in case of ethanol production, glycerol was converted to
pyruvic acid faster than the glucose and this pyruvic acid was converted to
ethanol by alcoholdehyrogenase. Therefore glycerol containing flask produced
more ethanol than the glucose flask. On the 3rd day, sugar utilization rate
increased from glucose, glucose: glycerol and glycerol containing flasks and
ethanol production decreased from glucose, glucose: glycerol and glycerol flask
respectively. In the glycerol containing flask, after 2nd day glycerol was depleted
from the media, therefore ethanol production also decreased. But glycerol containing
flask needed carbon source for ethanol production as well as to maintain cellular
activity of yeast cells. But that much energy was not available from glycerol
after the 2nd day. That is why glucose was utilized faster in glycerol flask
to recover that energy. In case of glucose flask, on the 3rd day sugar utilization
rate increased for first two days but that change normally occurred for ethanol
production and routine cellular activity.
The glucose and glycerol penetrated into the cell by different pathways. When
glycerol was the main carbon source for S. cerevisiae, it can permeate
the plasma membrane of cells by three mechanisms-passive diffusion (Gancedo
et al., 1968), facilitated diffusion through the Fps1p channel protein
(Luyten et al., 1995) and glycerol/protein symport
system (Lages and Lucas, 1997). In case of glucose,
it is known that the glucose, glucose-carrier was constitutive (Eddy,
1982). Busturia and Lagunas (1986) showed that the
glucose penetration by facilitated diffusion used different carriers, one of
them had high affinity, others had low affinity (fructose crosses over the membrane
using the same carriers as the glucose but their affinity was lower) (Busturia
and Lagunas, 1986). From the kinetic studies of Bisson
and Fraenkel (1984) and Bisson (1988) it has been
established that the low affinity system was constitutive, it was very efficient
during the growth phase and less efficient during the stationary phase (Bisson
and Fraenkel, 1984; Bisson, 1988). The high affinity
system was repressed by high glucose concentration. The rate of each of this
carrier does not seem clear and these carriers are interconvertive depending
on the metabolic conditions of the cell. From the above said evidences we can
say that there were chances for the competitive inhibition to occur between
glucose and glycerol. Thats why sugar utilization rate was always lower
compared to the other two flasks i.e., glycerol and glucose.
The glucose and glycerol degradation pathway was different. Glycerol degradation
occurs via a phosphorylative pathway (Sprague and Cronan,
1977). Glycerol is converted to glycerol-3 phosphate by cytosolic glycerol
kinase. Glycerol-3 phosphate then passes the outer mitochondrial membrane and
oxidized to dihydroxy acetone phosphate (DHAP) by an inner mitochondrial membrane
enzyme, FAD-dependent glycerol-3 phosphate dehydrogenase. DHPA returns to the
cytosol where it was catabolized by glycolysis to produce pyruvic acid. Glucose
catabolism occurs via EMP pathway (Voet et al., 2008).
More steps are needed for degradation of glucose than the glycerol break down.
The glycerol has highly reduced nature of carbon atoms and conversion of glycerol
into the glycolytic intermediate, Phosphoenol Pyruvate (PEP) generate the twice
amount of reducing equivalents than that produced by the metabolism of glucose
(Syed and Ramon, 2007). This is also one of the reasons for higher rate of ethanol
production in glycerol flask since the first two days.
On the 3rd day, ethanol production was high in glucose flask but sugar utilization rate was high in glycerol and glucose: glycerol containing flask rather than only glucose containing flask, the reason being that glucose is also needed for cellular activity which was not fulfilled by glycerol.
Nitrogen is an essential nutrient that is critical for fermentation efficiency
and generally become limiting during wine fermentation (Salmon,
1989). The depletion of the nitrogen source, in combination with the rapid
turnover of sugar transporters in the stationary phase was thought to be responsible
for subsequent reduction in the fermentation rate observed towards the end of
fermentation (Lagunas et al., 1982). It has been
found that nitrogen deficiency has an impact on the transporter turnover rate
and on the expression of at least one transporter, HXT1 (Bisson,
1999). From the study, we find that 6 g L-1 of urea concentration
was optimum for ethanol fermentation, it produced 152.20 g L-1 amount
of ethanol compared to 4 g L-1 of diammonium hydrogen phosphate producing
96.38 g L-1.
The diammonium hydrogen phosphate is a good nitrogen source whereas urea is
a poor nitrogen source for wine fermentation carried out by S. cerevisiae.
But for S. cerevisiae metabolism the better nitrogen source decreased
the enzyme level and permeases required for the utilization and uptake of poor
nitrogen this mechanism called nitrogen catabolite repression this is the reason
urea produced more ethanol rather than diammonium hydrogenphosphate (Schutz
and Gafner, 1995). The nitrogen source induced the yeast metabolism, therefore
sugar utilization was higher in nitrogen containing media than the control (Alexandre
and Charpentier, 1998; Boulton et al., 1996).
The amino nitrogen is a very much essential compound for fermentation. It is
taken up during the first part of the S. cerevisiae growth phase. Biosynthetic
pools of amino acids are filled and the remaining nitrogenous compounds are
utilized as nitrogen source (Copper, 1982). Nitrogen
catabolite repression is attributed to the action of three proteins, GLN3, URE2
and GAP1 (Magasanik, 1992). GAP1, the general amino acid
permease that transports all biological amino acid across the plasma membrane
(Jauniaux and Grenson, 1990) is regulated at the transcriptional
level by GLN3 and URE2 and is inactivated by dephosphorylation in the presence
of glutamate and glutamine (Stanbrough and Magasanik, 1995).
Proline and arginine are the most abundant amino acid in fruit juice but S.
cerevisiae is not able to completely utilize these two amino acids during
alcoholic fermentation. Derepression for the assimilation of amino acid in URE2
mutant strains of S. cerevisiae lead to better amino acid assimilation
during alcoholic fermentation. But the results showed that α amino nitrogen
utilization rate was high in glucose containing flask and the rate decreased
from glycerol to glucose: glycerol containing flask with time. Therefore, it
may be concluded that nitrogen utilization was also controlled by main constituent
of media i.e., carbon source.
In this study, it has been seen that glucose is the main carbon source for producing highest ethanol upto 3rd day of fermentation. In case glycerol, it helps in faster ethanol production than glucose till the 2nd day, therefore glycerol can be used as an inducer of short time fermentation for ethanol production in industry. It is also seen that organic nitrogen is better than inorganic nitrogen in the yeast mediated fermentation.
The reserarch work is financially supported by the Centre for Advanced studies (CAS I) programme under University Grants Commission (UGC), Govt. of India and Dept. of Food Processing Industries and Horticulture, Govt. of West Bengal, India, Some facilities have been provided by the centre for Medicinal Food and Applied Nutrition of Jadavpur University, India.