Baker’s yeast biomass production with rice as carbon and soy meal as nitrogen sources

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Malaysian Journal of Microbiology, Vol 10(3) 2014, pp. 205-214
Malaysian Journal of Microbiology
Published by Malaysian Society for Microbiology
(In since 2011)
205 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Baker’s yeast biomass production with rice as carbon and soy meal as nitrogen
sources
Inparuban Keturah, Balakumar Sandrasegarampillai and Arasaratnam Vasanthy *
Department of Biochemistry, Faculty of Medicine, University of Jaffna, Sri Lanka.
Email: arva26arva@yahoo.com
Received 11 December 2013; Received in revised form 28 February 2014; Accepted 24 April 2014
Aims: This research was an attempt to produce baker’s yeast biomass utilizing locally available carbon and nitrogen
sources.
Methodology and results: The yeast was grown in different media (30 °C, pH 5.0), which were aerated (100
bubbles/min). In the YPS medium containing 50 g/L; 5.53 g/L biomass was obtained, when sucrose was replaced with
rice flour hydrolysate (50 g/L), the biomass obtained was 5.72 g/L. With 3.45 g/L of bacteriological peptone 6.02 g/Land
7.5 g/L of yeast extract, 7.12 g/L of biomass were obtained. Highest biomass (7.98 g/L) was obtained when rice protein
was hydrolyzed with 10.0 mL/L Neutrase, a protease. Replacing bacteriological peptone (3.45 g/L) and yeast extract (7.5
g/L) with refluxed soy meal or soybean suspension, recorded 6.50 and 6.38 g/L of biomass respectively. Increase in
reducing sugars to 200 g/L increased the biomass to 12.38 g/L. Double the amount of soy meal protein hydrolysate
increased the biomass to 15.90 g/L. Replacing (NH4)2HPO4 with refluxed soy meal suspension gave similar biomass
production (15.58 g/L). Thus replacing commercial bacteriological peptone, yeast extract and (NH4)2HPO4 with refluxed
soy meal suspension is possible for baker’s yeast biomass production. Further by optimizing the concentrations of
sugar and nitrogen sources led to 2.57 folds increase in baker’s yeast biomass production.
Conclusion, significance and impact study: Locally available rice flour hydrolysate and soy meal protein provides
better alternatives to commercial carbon and nitrogen sources to produce baker’s yeast.
Keywords: baker’s yeast, biomass, rice flour hydrolysate, soybean, soy meal
INTRODUCTION
Saccharomyces cerevisiae strains have been selected for
many years for their dough leavening characteristics
(Randez-Gil et al., 1999). In Sri Lanka, supply of baker's
yeast, depends on import. Hence, it is important to go for
local production of Baker’s yeast biomass. This study has
focused on utilizing the locally available carbon and
nitrogen sources for baker’s yeast biomass production.
Most common source for baker’s yeast production is the
molasses, a by-product from sugar production (Imrie,
1969). Alternative sources have been introduced
(Champagne et al., 1990; Ejofor et al., 1996; Ferrari et al.,
2001; Alemzadeh and Vosoughi, 2002; Bekatorou et al.,
2006; Betroti and Hosseini, 2007; Gelinas, 2012). When
starchy materials are to be used for yeast growth, the
starch has to be first converted to sugars (Kim and
Hamdy, 1985). Hydrolysates of starch based carbon
sources such as wheat; corn rice, manioc and soybean
were tested for baker’s yeast biomass production and rice
flour hydrolysate was found to be the best (Thurairatnam
et al., 2006). In this studies rice flour hydrolysate obtained
by two-steps procedure (Arasaratnam et al., 2012) was
used as the carbon source for Baker’s yeast production
with nutrients supplementation.
MATERIALS AND METHODS
Materials
Yeast extract and peptone were from Oxoid, U.K. α-
Amylase (TermamylR, 120 L, activity 120 KNU/g),
glucoamylase (San 240 L, activity 159.9 AGU/mL) and
Neutrase 0.5 L (0.5 AU/g mettallo-protease of Bacillus
subtilis) were from Novo Industries, Denmark. Table
sugar, polished rice, soy meal [Delmage Distributors (Pvt)
Ltd, Colombo, Sri Lanka] and soybean were purchased in
the local market. Polished rice, soy meal and soybean
were pulverized in a domestic grinder.
Microorganism
The baker’s yeast, Fermipan was from Gist-Brocades,
Waterings-1, Delft-Holland, The Netherlands.
Unit definition for enzyme activities
One Kilo Novo Unit (1 KNU) of α-amylase is the amount of
enzyme, which hydrolyzes 5.26 g of starch (Merk Amylum
Soluble Erg.B.6 Batch No.994 7255) per hour at 37 °C in
buffer solution (pH 5.6) containing 0.0043 M calcium.
*Corresponding author

Mal. J. Microbiol. Vol 10(3) 2014, pp. 205-214
206 ISSN (print): 1823-8262, ISSN (online): 2231-7538
One Novo Amylo Glucosidase Unit (1 AGU) is the
amount of enzyme which hydrolyzes one µmole maltose
per minute at 25 °C and at pH 4.3.
One Anson unit (AU) is the amount of enzyme which
at 25 °C digests hemoglobin at an initial rate such that
there is liberated per minute an amount of trichloro acetic
acid soluble product which gives the same colour with
phenol reagent as one mille equivalent of tyrosine.
Analytical methods
Reducing sugar (Miller, 1959), total sugar (Pearson,
1980), amino acid (Rick, 1974), soluble proteins (Lowry et
al., 1951) nitrogen content (Pearson, 1980), ethanol
(Varley et al., 1980), and dry weight of the yeast cells
(gravimetric method) were measured using standard
methods.
Activation of yeast cells
The yeast cells were activated as before (Inparuban et al.,
2010).
Yeast Peptone Salt (YPS) medium
The medium contained (g/L); yeast extract, 2.5;
bacteriological peptone, 1.15; (NH4)2HPO4, 0.25 and
MgSO4·7H2O, 0.025 at pH 5.0 with 50 g/L sucrose as
carbon source (Inparuban et al., 2010).
Preparation of inoculums
As described before (Inparuban et al., 2010).
Cultivation of baker’s yeast in YPS medium
The YPS medium (1 L) in a 2 L flask was inoculated with
10 mL of activated yeast cells and incubated at 30 °C with
aeration (200 bubbles/min) (Inparuban et al., 2010).
Samples were analyzed for dry weight of yeast cell,
ethanol production and residual sugar.
Cultivation of baker's yeast in Rice flour hydrolysate
(RFH)-YPS medium
RFH was prepared by enzymatic hydrolysis (Arasaratnam
et al., 2012) and strained through a muslin cloth. RFH was
analysed for reducing sugar, total sugar, amino acid,
soluble proteins and nitrogen contents.
RFH diluted to have 50 g/L reducing sugars was
supplemented with YPS medium (Medium I, Table 1). The
medium was inoculated with activated yeast cells and
aerated (200 bubbles/min) at 30 °C. The dry weight of
cells and ethanol production were monitored. YPS
medium with 50 g/L sucrose was used as control
(Inparuban et al., 2010) (Table 1).
Effect of bacteriological peptone in RFH-YPS medium
In the rice flour hydrolysate (RFH) containing YPS
medium (RFH-YPS medium) the concentration of
bacteriological peptone was changed (Media I-IV, Table
1).
Effect of yeast extract in RFH-YPS medium
In RFH-YPS medium with optimized concentration of
peptone, the concentration of yeast extract was changed
(Media VI-IX, Table 1).
Effect of hydrolyzing the rice protein in RFH with
Neutrase
To sterile rice flour hydrolysate with minerals (as of YPS
medium) different amounts of Neutrase (Table 1, Media X-
XIII) was added and inoculated with Baker’s yeast.
Neutrase untreated medium containing optimized
amounts of yeast extract and bacteriological peptone was
used as the control.
Supplementing with soy proteins
Refluxing soy meal and soybean suspension with
hydrochloric acid
Soy meal (10 g) and soybean (10 g) flour were mixed with
100 mL of 6 N HCl separately and was refluxed for 40
mins. The mixture was cooled and neutralized using
NaOH. The sample was centrifuged and analyzed for
amino acid content.
Effect of refluxed soy meal and soybean as protein source
in RFH medium
The refluxed soy meal and soybean suspension
equivalent to the total amino acid contents of optimum
yeast extract and bacteriological peptone were taken
instead of the commercial nitrogen sources (Media XIV &
XV, Table 1). Medium with the optimized amounts of yeast
extract and bacteriological peptone was kept as the
control.
Effect of sugar concentration of the RFH containing
refluxed soy meal suspension
With refluxed soy meal suspension (Medium XV) different
amounts of rice flour hydrolysate was taken having
reducing sugar concentration equivalent to 100 (Medium
XVI), 150 (Medium XVII), 200 (Medium XVIII) and 250
g/L(Medium XIX) (Table 1).
Effect of refluxed soy meal suspension in RFH medium
To RFH containing different concentrations of reducing
sugars, double the amount of refluxed soy meal powder
suspension was added (Media XX – XXIII, Table 1).

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207 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Substitution of (NH4)2HPO4 with refluxed soy meal
suspension based on the nitrogen content of (NH4)2HPO4
The constituents of Medium XXII (Table 1) were kept
constant except (NH4)2HPO4. Refluxed soy meal
suspension with the nitrogen content equivalent to 0.25
g/L (NH4)2HPO4 was replaced for (NH4)2HPO4 (Medium
XXIV, Table 1). Medium XXII was used as the control.
Statistical Analysis
Results obtained for specific volume, moisture content
and proximate composition of breads were analysed
statistically by ANOVA and sensorial data were
statistically analysed by Friedman test using SAS
analytical package.
RESULTS AND DISCUSSIONS
Cultivation of baker's yeast in rice flour hydrolysate
(RFH)
Utilization of RFH as a carbon source
When rice flour hydrolysate was used as the carbon
source, maximum biomass (dry weight 5.72 g/L) was
obtained at 28 h. The control medium gave a maximum of
5.5 (±0.03) g/L at 28 h. The maximum amount of alcohol
(20.2 g/L) was produced at 26 h, and there after it
declined with time. The initial sugar concentration of the
medium was 50 g/L, and reached zero at 26 h. According
to the results obtained, rice flour hydrolysate proved itself
a suitable carbon source for yeast growth. In this present
study, the yeast cultivation medium was supplemented
with yeast extract and bacteriological peptone as the
nitrogen sources. Therefore the influence of these
nitrogen sources on yeast cell biomass production was
examined.
Effect of bacteriological peptone in RFH-YPS medium
Highest biomass (dry weight) was obtained with 3.45 g/L
bacteriological peptone at 28 h (6.02 g/L) (Medium IV,
Figure 1). The increase in ethanol production did not
follow the biomass production with different bacteriological
peptone concentrations. The initial media contained 50
(±0.644) g/L of reducing sugars, which reached zero at 26
h except with 0.575 g/L bacterial peptone. The results
indicated that bacteriological peptone increases the
growth of yeast up to a point. The optimum bacteriological
peptone concentration of 3.45 g/L under the experimental
conditions was selected for further studies.
Effect of yeast extract concentration in RFH-YPS
Highest cell mass was obtained with 7.5 g/L yeast extract
at 24 h (Medium VIII) (Figure 2) while the highest alcohol
production was also obtained with 10 g/L of yeast extract
at 24 h. The initial sugar concentration in the medium was
50 (±0.555) g/L. All the media showed similar variation in
the residual sugar concentration, and the residual sugar in
the media reached zero between 26 and 28 h. From the
results it is evident that the amount of yeast extract has a
positive effect on yeast growth and after a limit it has a
negative influence on the cell mass production. Hence 7.5
g/L of yeast extract was selected as the optimum amount
for further studies.
Rice flour contained 7.58% of total protein, of which
only 0.8% was in the soluble form (soluble protein) and
0.8% were free amino acids. Therefore, a major part of
the protein was insoluble, which cannot be utilized by the
yeast. It has been well documented that yeast are also
unable to metabolize peptides (except a limited number of
dipeptides) (Hammond, 1993). Thus in order to transform
the insoluble protein of rice flour hydrolysate into soluble
form, protein hydrolysis was carried out with Neutrase.
Effect of hydrolyzing the rice protein in RFH with
Neutrase
When Neutrase of varying concentrations (Table 1) was
introduced into the media, highest yeast cell mass
obtained was 7.21 (±0.15) g/L at 26 h with 10.0 mL/L
Neutrase (Figure 3). RFH treated with 10 mL/L of
Neutrase (Medium XIII) recorded slightly higher biomass
production (12.08%) than the control (Medium VIII). A
slight variation of alcohol production has been observed
with the increase in Neutrase concentration. In all media,
the sugar content decreased to zero at 26 h. Bacterial
protease, Neutrase 0.5 L catalyzed the hydrolysis of
peptide bonds in protein forming soluble peptides and
amino acids (Thurairatnam et al., 2006). Even though the
enzyme hydrolysed rice protein can be used as a good
nitrogen source due to the high price of Neutrase,
alternative measures have to be taken to use the locally
available nitrogen sources.
Refluxing soy protein
Soybean (Glycine max) is relatively an inexpensive and
abundant source of proteins and lipids, which can be
easily assimilated into cellular materials (Bajpai et al.,
1988). Soy meal, which is rich in protein, and available
locally, was selected (Hammond, 1993).
As majority of the proteins in soy meal powder and
soybean flour were insoluble (Table 2) they have to be
hydrolyzed. When refluxed in presence of 6 N HCl under
pressure, the free amino acid contents of soya meal flour
and soya bean flour have increased. By considering the
free amino acid contents of the medium and with the
optimum amount of yeast extract and bacteriological
peptone, equivalent amount of free amino acid from
refluxed soy meal and soy bean suspensions were
supplemented to the RFH medium.

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208 ISSN (print): 1823-8262, ISSN (online): 2231-7538
0.00
5.00
10.00
15.00
20.00
25.00
0.58
1.15
2.30
3.45
Dry Biomass / Ethanol (g/L)
Peptone (g/L)
Dry
Biomass
Ethanol
Figure 1: Effect of bacteriological peptone concentration on the growth of S.
cerevisiae (biomass production) and ethanol production in fermentation
medium containing (g/L); yeast extract, 2.5; (NH4)2HPO4, 0.25 and
MgSO4·7H2O, 0.025 at 30 °C and pH 5.0, aerated (100 bubbles/min). The
rice flour hydrolysate (with 50 g/L reducing sugar) was used as the carbon
source. Here in the media containing 0.58 and 1.15 g/L bacteriological
peptone highest biomass production was obtained at 28 h while with 2.3 and
3.45 g/L bacteriological peptone highest biomass production was obtained at
26 h. At the respective period no residual sugar was in the media.
0.00
5.00
10.00
15.00
20.00
25.00
0.58
1.15
2.30
3.45
Dry Biomass / Ethanol (g/L)
Peptone (g/L)
Dry
Biomass
Ethanol
Figure 2: Effect of yeast extract concentration on the growth of S. cerevisiae
(biomass production) and ethanol production in fermentation medium
containing (g/L); bacteriological peptone, 3.45; (NH4)2HPO4, 0.25 and
MgSO4·7H2O, 0.025 at 30 °C and pH 5.0, aerated (100 bubbles/min). The
rice flour hydrolysate (with 50 g/L reducing sugar) was used as the carbon
source. Here in the media containing 1.25 and 2. 5 g/L yeast extract highest
biomass production was obtained at 28 h while with 5.0 and 7.5 g/L yeast
highest biomass production was obtained at 24 h and with 10.0 g/L yeast
highest biomass production was obtained at 26 h. At the respective period
no residual sugar was in the media.

Mal. J. Microbiol. Vol 10(3) 2014, pp. 205-214
209 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Table 1: Yeast cultivation in RFH medium with different constituents of varying amounts and, the biomass and ethanol produced and sugar utilized. The
time at which maximum dry biomass, and ethanol produced, and highest sugar utilized are given in parenthesis.
A 120 mL/L of rice flour hydrolysate contained 50 g/L of reducing sugars, whereas 17.8 mL/L of refluxed soy meal suspension contained 1.4 g/L nitrogen. In Medium XXIV 0.25
g/L (NH4)2HPO4 was replaced with 0.688 mL/L refluxed soy meal suspension, which was equivalent to the nitrogen content of 0.053 g/L.
Constituents
changed
Medium
Constituents
Sucrose
(g/L)
RFH
(Reducing sugar)
(g/L)
Yeast
Extract
(g/L)
Peptone
(g/L)
(NH4)2HPO4
(g/L)
MgSO4·7H2O
(g/L)
Neutrase
(mL/L)
Soy bean
hydrolysate
(mL/L)
Soy meal
hydrolysate
(mL/L)
Control
50
-
2.5
1.15
0.25
0.025
-
-
-
Peptone
I
-
50
2.5
1.15
0.25
0.025
-
-
-
II
-
50
2.5
0.575
0.25
0.025
-
-
-
III
-
50
2.5
2.3
0.25
0.025
-
-
-
IV
-
50
2.5
3.45
0.25
0.025
-
-
-
V
-
50
2.5
4.6
0.25
0.025
-
-
-
Yeast
Extract
VI
-
50
1.25
3.45
0.25
0.025
-
-
-
IV
-
50
2.5
3.45
0.25
0.025
-
-
-
VII
-
50
5.0
3.45
0.25
0.025
-
-
-
VIII
-
50
7.5
3.45
0.25
0.025
-
-
-
IX
-
50
10.0
3.45
0.25
0.025
-
-
-
Neutrase
X
-
50
-
-
0.25
0.025
2.5
-
-
XI
-
50
-
-
0.25
0.025
5.0
-
-
XII
-
50
-
-
0.25
0.025
7.5
-
-
XIII
-
50
-
-
0.25
0.025
10.0
-
-
Soy
hydrolysate
XIV
-
50
-
-
0.25
0.025
-
22.86
-
XV
-
50
-
-
0.25
0.025
-
-
17.8
Reducing
sugar
XVI
-
100
-
-
0.25
0.025
-
-
17.8
XVII
-
150
-
-
0.25
0.025
-
-
17.8
XVIII
-
200
-
-
0.25
0.025
-
-
17.8
XIX
-
250
-
-
0.25
0.025
-
-
17.8
Reducing
sugar
Double Soy
meal
hydrolysate
XX
-
100
-
-
0.25
0.025
-
-
35.6
XXI
-
150
-
-
0.25
0.025
-
-
35.6
XXII
-
200
-
-
0.25
0.025
-
-
35.6
XXIII
-
250
-
-
0.25
0.025
-
-
35.6
(NH4)2HPO4
XXIV
-
200
-
-
-
0.025
-
-
36.276

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210 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Table 2: Total and soluble protein and amino acid contents of soybean powder and soy meal powder.
ND- Not determined
Effect of refluxed soy meal and soybean suspensions
as nitrogen source in RFH-medium
Refluxed soy protein, with amino acid content equivalent
to that of the Medium VIII was incorporated.
Comparatively, the medium containing refluxed soya meal
(Medium XV) gave higher biomass than the medium with
refluxed soybean (Medium XIV). On the other hand, the
biomass production of the above mentioned media was
reduced than in the control medium (Medium VIII) with
optimum yeast extract and bacteriological peptone. The
maximum cell mass yields obtained in the media with
refluxed soya meal and soya bean were 6.5 (±0.12) and
6.4 (±0.04) g/L respectively at 26 hours, whereas, ethanol
production in the media was higher in refluxed soy bean
meal containing medium (Medium XIV, Figure 4). The
initial reducing sugar content in all the media was 50
(±0.278) g/L, and it reached zero in 26 h.
Protein hydrolysates have sometimes been used as
sources of nitrogen. For instance, the use of wheat starch
(after hydrolysis) as sugar source and hydrolyzed wheat
gluten as nitrogen source has been practiced (Reed and
Peppler, 1973). At this instance, when the soybean is
refluxed with 6 N HCl, along with the proteins, starch also
could have been hydrolysed to sugars or oxidzed, and
there is also possibility for the Mailard reaction. These by-
products could have had a negative effect on the biomass
production or converted to useless substances. This could
be the reason for the reduction in the biomass production
in refluxed soybean suspension substituted medium than
in refluxed soy meal suspension substituted medium. As
the medium with refluxed soy meal suspension gave
higher biomass when compared with refluxed soybean
suspension, it was selected for further studies.
Effect of sugar concentration of RFH with refluxed
soy meal suspension as nitrogen source
Biomass yields were maximum when the sugars were
completely utilized. Further increase in biomass
production could have been possible if additional sugar is
available. Hence it was decided to increase the sugar
concentration to analyze the performance of yeast cells.
When the sugar content was increased highest, cell
mass [12.38 (± 0.13) g/L] production was recorded in the
medium with 200 g/L sugar (Medium XVIII), whereas
highest ethanol productions was [40.2 (±0.8) g/L] recorded
in the medium with 250 g/L (Medium XIX, Figure 5). The
results indicated that, the biomass production was
increased with increase in sugar concentration and this
present experiment has established that the decreased
biomass production in the previous media (Medium I to
Medium XIX) was due to inadequate sugar content.
Comparing biomass productions among the treatments,
medium with 250 g/L sugar (Medium XIX) gave less
biomass production. This may be due to the osmotic effect
of high sugar concentration (Balakumar and Arasaratnam,
2011). Ethanol productions were also increased with
increased sugar concentration and showed an inverse
relationship with the cell mass production. The substrate
uptake in medium with 100, 150, 200 and 250 g/L of sugar
concentrations were 93.8, 95.8, 81.8 and 75.6%
respectively. Ethanol production in all treatments differed
significantly (p<0.05). The residual sugar content showed
significant difference in all treatments (p<0.05). The next
attempt was to increase the nitrogen source to the media
with high sugar concentrations. This was an approach to
find out whether the nitrogen sources were inadequate to
cope up with high sugar concentrations in order to satisfy
the increasing yeast population.
Effect of doubling refluxed soy meal suspension in
RFH medium
In this present study, along with increasing sugar
concentrations, the amount of amino acid content in
refluxed soy meal suspension equivalent to that of the
optimum concentration of yeast extract and bacteriological
peptone was doubled. Here the cell mass productions
were comparatively higher (Figure 6). Again medium with
200 g/L sugar (Medium XXII) gave the highest biomass of
15.6 (±0.06) g/L. This illustrates that, the nitrogen content
in the media XVI, XVII, XVIII, and XIX were inadequate.
Ethanol production has not significantly increased with
increasing sugar concentration with double the amount of
refluxed soy meal suspension. The substrate uptake was
100% with 100 (Medium XX) and 150 g/L sugar (Medium
XXI) while in medium with 200(Medium XXII) and 250 g/L
sugar (Medium XXIII) it was 92.2 and 91.6% respectively.
The dry weights, alcohol productions and residual sugar
contents were significantly different among themselves
(p<0.05). This shows that the yeast cells have utilized
more sugars and produced more biomass with the
increased nitrogen source content. As Medium XXII with
200 g/L of sugars gave the highest yield, it was selected
for further studies.
Content
Non-refluxed
Refluxed
Soybean
Soy meal
Soybean
Soy meal
Total Protein (%, w/w)
42.3
58.58
ND
ND
Free Amino Acid (g/L)
1.28
1.31
382.6
525.7
Soluble Protein (g/L)
1.36
1.52
ND
ND

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211 ISSN (print): 1823-8262, ISSN (online): 2231-7538
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
2.50
5.00
7.50
10.00
Dry Biomass/ Ethanol (g/L)
Neutrase (mL/L)
Dry Biomass
Ethanol
Figure 3: Effect of Neutrase concentration on the hydrolysis of rice protein
hydrolysis and their effect on the growth of S. cerevisiae (biomass
production) and ethanol production in fermentation medium containing (g/L);
MgSO4·7H2O, 0.025 at 30 °C and pH 5.0, aerated (100 bubbles/min). Here
the rice flour hydrolysate (with 50 g/L reducing sugar) was used as the
carbon source after the protein hydrolysis. Here in all the media containing
different concentrations of Neutrase, highest biomass production was
obtained at 26 h. At the respective period no residual sugar was in the
media.
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
2.50
5.00
7.50
10.00
Dry Biomass/ Ethanol (g/L)
Neutrase (mL/L)
Dry Biomass
Ethanol
Figure 4: Effect of replacing yeast extract and bacteriological peptone with
either soybean hydrolysate (22.86 mL/L) and soy meal hydrolysate (17.8
mL/L) in fermentation medium containing (g/L) (NH4)2HPO4, 0.25 and
MgSO4·7H2O, 0.025 at 30 °C and pH 5.0, aerated (100 bubbles/min). The
rice flour hydrolysate (with 50 g/L reducing sugar) was used as the carbon
source. Here in the media containing soybean hydrolysate and soy meal
hydrolysate, highest biomass production was obtained 26 h. At the
respective period no residual sugar was in the media.

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212 ISSN (print): 1823-8262, ISSN (online): 2231-7538
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
100
150
200
250
Dry Bioimass / Ethanol / Residual
Sugar (g/L)
Reducing sugar (g/L)
Dry Biomass
Ethanol
Residual sugar
Figure 5: Effect of different concentration of sugar in rice flour hydrolysate
on the dry biomass and ethanol production in fermentation medium
containing (g/L) (NH4)2HPO4, 0.25 and MgSO4·7H2O, 0.025 at 30 °C and pH
5.0, aerated (100 bubbles/min). Soy meal hydrolysate (17.8 mL/L) was used
instead of optimized amounts of bacteriological peptone and yeast extract.
In all the media highest biomass was obtained at 30 h, and at 32 h highest
ethanol produced and residual sugar present in the media are presented.
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
100
150
200
250
Dry Biomass / Ethanol / Residual Sugar
(g/L)
Reducing sugar (g/L)
Dry Biomass
Ethanol
Residual sugar
Figure 6: Effect of different concentration of sugar in rice flour hydrolysate
on the dry biomass and ethanol production in fermentation medium
containing (g/L) (NH4)2HPO4, 0.25 and MgSO4·7H2O, 0.025 at 30 °C and pH
5.0, aerated (100 bubbles/min). Double the concentration of soy meal
hydrolysate (35.6 mL/L) was used. In all the media highest biomass and
ethanol were obtained at 30 h, and residual sugar present in the media 30 h
are presented.

Mal. J. Microbiol. Vol 10(3) 2014, pp. 205-214
213 ISSN (print): 1823-8262, ISSN (online): 2231-7538
Effect of substitution of (NH4)2HPO4 in RFH medium
with refluxed soy meal suspension based on the total
nitrogen content
The next attempt was to substitute (NH4)2HPO4 with soy
meal hydrolysate. The nitrogen content of soy meal
hydrolysate was 5.46 g/Land the nitrogen content from the
added 0.25 g/L of (NH4)2HPO4 in 1 L medium was 0.053
g/L. Hence, the amount of soy meal hydrolysate
equivalent to this fraction was 9.7 mL for 1 L medium. The
total nitrogen content of medium with 3.45 g/L of
bacteriological peptone and 7.5 g/L of yeast extract
(optimum concentrations) was 1.4 g/L (Medium VIII). So it
was decided to substitute the nitrogen content from
(NH4)2HPO4 with equivalent amount of nitrogen from soy
meal hydrolysate. Biomass of 15.69 g/L was obtained in
the Medium XXIV whereas the control (Medium XXII)
gave 15.9 g/L. This replacement of (NH4)2HPO4 with soy
meal hydrolysate was therefore satisfactory. Nitrogen is
generally added in the form of ammonia or ammonium
salts, such as ammonium sulfate or ammonium
phosphate, or sometimes in the form of urea. Nitrogen
from such sources is readily assimilated and with the
exception of urea they are interchangeable (Reed and
Peppler, 1973). According to the statistical analysis of this
study, the dry weights recorded in both, test (Medium
XXIV) and control media (Medium XXII) were not
significantly different (p>0.05). The residual sugar content
showed non-significant difference in both the treatments
(p>0.05). Hence the replacement of (NH4)2HPO4 with
refluxed soy meal suspension did not lead to negative
effect. So this replacement was selected as a suitable
condition for yeast cultivation in the following steps.
CONCLUSION
Baker’s yeast is not produced in Jaffna peninsula and this
was an attempt to produce baker’s yeast with local raw
materials. This studies, proved that the rice flour
hydrolysate commercial can replace sucrose, and refluxed
soy meal suspension can replace bacteriological peptone,
yeast extract and (NH4)2HPO4. With the local raw
materials 2.57 fold increase in baker’s yeast biomass
production could be obtained and this can reduce the
importation of baker’s yeast.
ACKNOWLEDGEMENT
The financial assistance and support of the University of
Jaffna, Sri Lanka is greatly acknowledged.
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