Fuel 142 (2015) 5864

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Fuel
journal homepage: www.elsevier.com/locate/fuel

Bioethanol production from triticale by simultaneous saccharication

and fermentation with magnesium or calcium ions addition
Jelena D. Pejin a,, Ljiljana V. Mojovic b, Duanka J. Pejin a, Suncica D. Kocic-Tanackov a, Dragia S. Savic c,
Svetlana B. Nikolic b, Aleksandra P. Djukic-Vukovic b
a
b
c

University of Novi Sad, Faculty of Technology, Bulevar Cara Lazara 1, 21 000 Novi Sad, Serbia
University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11 000 Belgrade, Serbia
University of Ni, Faculty of Technology, Bulevar Oslobodenja
124, 16 000 Leskovac, Serbia

h i g h l i g h t s
 The effect of magnesium or calcium ions in triticale mashes on bioethanol yield.
 When 160 mg/L of magnesium ions were added bioethanol content increased by 31.22%.
 When 160 mg/L of calcium ions were added bioethanol content increased by 21.04%.
 Magnesium ions had more signicant effect on bioethanol yield than calcium ions.
 When magnesium ions are added there is no need to use commercial enzymes.

a r t i c l e

i n f o

Article history:
Received 10 August 2013
Received in revised form 23 October 2014
Accepted 28 October 2014
Available online 8 November 2014
Keywords:
Triticale
Bioethanol yield
Magnesium
Calcium

a b s t r a c t
The aim of this study was to determine the effect of magnesium or calcium ions content in triticale
mashes on glucose and maltose content after liquefaction as well as on bioethanol yield after fermentation. Triticale variety Odyssey was used in this study. Liquefaction and saccharication in this study were
performed without using any additional saccharifying enzymes, i.e. the triticale starch was hydrolyzed
only by the enzymes present in triticale grain. Glucose and maltose content increased with the increase
of magnesium and calcium ion content in mash. Glucose and maltose content increased by 30.16% and
9.58%, respectively, when 160 mg/L of magnesium ions were added, compared to the control sample.
Glucose and maltose content increased by 69.31% and 61.66%, respectively, when 160 mg/L of calcium
ions were added, compared to the control sample. According to the obtained results for glucose and maltose content increase during liquefaction, the supplementation of mashes with calcium ions had greater
inuence on the activity of triticales amylases than the supplementation of mashes with magnesium
ions. The present investigation shows that magnesium and calcium ions addition to triticale mashes
improved bioethanol production during SSF processing. When 160 mg/L of magnesium ions were added
bioethanol content increased by 31.22% compared to the control sample while when 160 mg/L of calcium
ions were added bioethanol content increased by 21.04%. High percentage of the theoretical bioethanol
yield (92.19%) was achieved after fermentation when 160 mg/L of magnesium ions were added to triticale mash. The obtained results show that the addition of magnesium and calcium ions in bioethanol production from triticale increase triticales amylase activity as well as yeast enzyme activity. All this shows
that when triticale with high amylolytic enzymes activity is used in bioethanol production with the addition of magnesium ions there is no need to use commercial enzymes in starch hydrolysis, which makes
the use of triticale as a raw material for bioethanol production more economical.
2014 Elsevier Ltd. All rights reserved.

1. Introduction

Corresponding author. Tel.: +381 21 485 3721.

E-mail address: jpejin@uns.ac.rs (J.D. Pejin).
http://dx.doi.org/10.1016/j.fuel.2014.10.077
0016-2361/ 2014 Elsevier Ltd. All rights reserved.

Bioethanol is regarded as a promising alternative energy source,

which is both renewable and environmentally friendly. During
bioethanol production, the composition of media affects the

J.D. Pejin et al. / Fuel 142 (2015) 5864

physiological state and, consequently, the fermentation performance of the microorganism employed [1]. Bioethanol is produced
by fermentation of sugar, starch or cellulosic biomass and its utilization can signicantly reduce fossil fuels use. It is expected to be
one of the dominating renewable biofuels in the transportation
sector within the twenty years to come [2]. The production of bioethanol is increasing over the years, and has reached the level of
85.2 billion litres in the year 2012 [3]. The governmental supports
for the substitution of fossil fuels with bioethanol produced from
biomass is predicted to result in global production of 125
109 L
of bioethanol by 2020 [4]. The primary benecial aspects of fermenting biomass-derived sugars to bioethanol as a fuel source is
that it can be produced from renewable plant material that is able
to photosynthetically re-x CO2 produced during bioethanol production and combustion [5]. Bioethanol production has remarkably
increased because many countries look for reducing oil imports,
boosting rural economies and improving the air quality [2]. One
major problem with bioethanol production is the availability of
raw materials for the production. There are several criteria for
choosing raw materials for bioethanol production: price and yield
of raw material, bioethanol yield, starch content, pest and diseases
resistance, suitability for soil and weather conditions, harvesting
transportation and storage options as well as the usability of byproducts [6]. The availability of feedstocks for bioethanol production can vary considerably from season to season and depends on
geographic locations [7]. However, feedstocks for bioethanol production must be sustainable and must not threaten biodiversity
or food security [5].
Yeast strains of Saccharomyces cerevisiae have been extensively
studied in recent years for fuel bioethanol production, in which
yeast cells are exposed to various stresses such as high temperature, bioethanol inhibition, and osmotic pressure from product
and substrate sugars and so on [8].
Triticale is a cereal crop adapted to less favorable soil conditions. It is suitable for low input farming because of lower demands
on pesticides application [9]. Today, it has been reported that triticale is cultivated in more than 30 countries worldwide [10] on
around 3.7 million ha in total, yielding more than 12 million tonnes a year [11]. Modern triticale varieties have been found to be
very competitive as a feedstock for bioethanol production [12].
Triticale crops have a high yield potential as well as a high starch
content, together with a low content of soluble polysaccharides
and proteins, and is therefore considered to be ideal for bioethanol
production [13]. There is high activity of triticales own amylolytic
enzymes, mainly a-amylase, and this is crucial in starch saccharication [14,15]. Considering the currently prevalent cold technique of saccharication, by means of commercial enzymes, the
processing of triticale is economically benecial as it enables the
reduction of the commercial enzymes consumption [15]. In our
previous research [16,17] it was shown that the addition of commercial enzymes was not necessary during liquefaction and saccharication step in bioethanol production from triticale variety
Odyssey. Cereal a-amylases are known to be metalloenzymes. Is
has been shown that these enzymes contain covalently bound calcium ions which act as an allosteric activator. Besides calcium ions,
magnesium ions can also act as a-amylases activator. Studies on
barley a-amylase show that these ions, especially calcium ion help
in maintaining the three-dimensional structure of amylases [18].
The mineral metabolism of yeast is of interest to bioethanol
producers looking to improve yields, increase fermentative capacity, and maintain consistency of product quality [19]. Metal ions
especially divalent cations are necessary for the activation of several glycolytic enzymes and, in practical terms, if industrial media
is decient in them, the conversion of sugar to bioethanol may be
suppressed leading to slow or incomplete fermentation process
[20]. Magnesium is involved in many essential physiological and

59

biochemical functions in yeast cells, including growth, cell division,

enzyme activation, stimulation of synthesis of essential fatty acids,
regulation of cellular ionic levels, and maintaining membrane
integrity and permeability. Yeasts have a very high growth demand
for magnesium ions, and magnesium accumulation by yeast
correlates closely with the progress of fermentation [21,22].
Magnesium also plays roles in protecting yeast cells against
environmental stresses during fermentation such as caused by bioethanol, high temperature, or high osmotic pressure [23]. The rate
of uptake and utilization of metal ions by the yeast biomass
depends both on the ion content in the medium, as well as on its
bioavailability. It has been established that accumulation and
release of metal ions during ethanolic fermentation is a dynamic
process and that the intensity depends on the sugar and bioethanol
content in the fermentation medium [24]. Calcium is not a requirement but may stimulate cell growth, protects yeast cell membrane
structure and helps maintain membrane permeability under
adverse conditions [22]. Calcium, being actively excluded from
the yeast cell, acts mainly extracellularly for example, calcium is
essential for amylase activity [25]. Metal ion deciencies often
occur in fermentation media, and studies on optimization of metal
ions combinations are thus of great practical importance to
improve bioethanol production [1,26].
The aim of this study was to determine the effect of magnesium
or calcium ions content in triticale mashes on glucose and maltose
content after liquefaction as well as on bioethanol yield after fermentation. Triticale variety Odyssey, from experimental elds,
Rimski ancevi location (Serbia) was used in this study. The effect
of magnesium and calcium ions content in triticale mashes on glucose and maltose content after liquefaction and on bioethanol yield
after fermentation was investigated by adding different amounts of
MgSO4
7H2O or CaCl2 solution in triticale mashes before liquefaction and saccharication. In this study the process was conducted
without the addition of external amylolytic enzymes, and the
liquefaction and saccharication of starch were performed only
by enzymes present in triticale grain. The bioethanol yield and productivity were also assessed.
2. Materials and methods
2.1. Materials
Triticale variety Odyssey was obtained from Institute of Field
and Vegetable Crops Novi Sad (Serbia). Triticale was milled in a
dry conical mill (Miag-Braunschweig, Germany) type: DOXY 71
b/4, mill motor power 0.22 kW, at 1375 r/min. The granulation of
triticale meals was determined by sieving 100 g of milled sample
for 3 min on the set of sieves with the following opening widths:
1000, 700, 500, 250 and 132 lm on Bhler MLU 300 sieve.
2.2. Yeast strain
Instant dry active bakers yeast S. cerevisiae provided from
Alltech Fermin, Senta, Serbia was used as a producing microorganism. Prior to each experiment, the yeast was activated according to
the following procedure: the yeast was measured and suspended
in 0.1% sterile peptone water warmed up to 38 C. The yeast cell
count was determined in Neubauers counting chamber. From
the prepared yeast solution, the amount of inoculum needed to
obtain 3035
106 CFU/mL in the fermentation medium, was taken
[27]. Indirect counting method, i.e. pour plate technique, was used
to determine the number of viable cells. Serial dilutions of the
samples were performed, and after the incubation time at 30 C,
colonies grown in Petri dishes were used to count the number of
viable cells.

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J.D. Pejin et al. / Fuel 142 (2015) 5864

2.3. Triticale analysis

Table 2
Triticale our particle size distributiona.

Triticale samples were monitored for the following quality

parameters and the following methods were used for analysis: test
weight (g/L) [28], thousand grain weight (g) weight of 1000
grains, percentage of grain above the sieve 2.5 mm, protein content
by Kjeldahl method, magnesium and calcium content [29], Falling
number (s) [30], starch content after Ewers polarimetric method
(% dry matter) [31], and the moisture content in the triticale meal
was determined by the standard drying method in an oven at
105 C to a constant mass [29]. All above mentioned standard triticale analyses were carried in triplicate (results presented in Tables
1 and 2). Results were represented as mean standard deviation.
2.4. Liquefaction and simultaneous saccharication and fermentation
(SSF) experiments
Since triticale variety Odyssey exhibited high autoamylolytic
enzyme activity in our previous research [16,17], liquefaction
and saccharication in this study were performed without using
any additional saccharifying enzymes, i.e. the triticale starch was
hydrolyzed only by the enzymes present in triticale grain. Liquefaction was carried out using automated mashing water bath
(Glasblserei, Institut fr Grungs Gewerbe, Berlin). Milled triticale
samples were mixed with distilled water warmed to 60 C (sample
to water ratio 1:3). The samples were kept in the mashing bath at
60 C for 65 min for liquefaction. After the liquefaction samples
were cooled to 20 C.
The samples were transferred to 500 mL glass bottles after the
liquefaction. The simultaneous saccharication and fermentation
(SSF) process was initiated by adding S. cerevisiae inoculum (to
obtain 3035
106 CFU/mL in the fermentation medium) to the liqueed samples and carried out for up to 96 h at 30 C. The bottles
were closed with foam burgs to allow venting of the CO2 produced
during the fermentation. After fermentation, the fermented mash
was centrifuged for 15 min (10,000 r/min at 4 C) in a refrigerated
centrifuge (Sorvall RC 24) and the supernatant was used for determination of bioethanol content [32]. The bioethanol content was
determined based on the density of bioethanol distillate at 20 C
and expressed as % w/w [29]. During fermentation, samples were
prepared in the same manner and analyzed for bioethanol content.
Analyses were carried out at least in triplicate. Results were represented as mean standard deviation.
2.5. Calculation of important process parameters

Table 1
Quality parameters of triticale variety Odyssey.a
Mechanical analysis
Percentage of grains over the 2.5 mm sieve (%)
Thousand grain weight (g)
Test weight (kg/hL)

a
Values represent
determinations.

means standard

deviation

(%)

>1000
1000/700
700/450
450/250
250/150
<150

0.9 0.03
6.1 0.07
21.0 0.15
39.9 0.18
6.8 0.11
25.3 0.19

a
Values represent means standard deviation
calculated from three determinations.

During the fermentation the yeast produces bioethanol according

to the Gay-Lussac equation. From each gram of glucose consumed,
0.51 g of bioethanol can be produced which represents the theoretical yield of bioethanol. Starch content in investigated triticale variety was 66.00% of dry matter. Under the optimal conditions of
pretreatment, liquefaction and fermentation, all starch content
should be converted to fermentable sugars and then to bioethanol.
According to the obtained bioethanol content (g/100 g of triticales
dry matter) after fermentation and its relation to starch content
(66.00% of dry matter); the bioethanol yield (g/g of triticale starch)
was calculated. Percentage of the theoretical bioethanol yield was
calculated as the ratio between actual bioethanol yield (g/g of triticale starch) and theoretical bioethanol yield (0.51 g) multiplied by
100 (assuming all starch was converted to glucose and then to
bioethanol).
2.6. HPLC analysis of glucose and maltose
The supernatant obtained as previously described was used for
glucose and maltose analysis by HPLC. Prior to the analysis, proteins
were removed from the supernatant [33]. The supernatant was collected and ltered through a 0.22 lm membrane. A 20 lL aliquot of
the ltrate was applied to an Aminex HPX087H Column (9 lm,
7.8 mm ID  300 mm, Biorad Laboratories) for HPLC analysis, using
an Agilent 1100 Series HPLC system equipped with vacuum, degasser, binary pump, thermostatted column compartment, variable
wavelength detector and RI detector. The temperature was maintained at 50 C. The absorbance at 214 nm was detected at the ow
rate of 1 mL min 1. The mobile phase was 5 mmol L 1 H2SO4 with
isocratic elution. Analyses were carried out at least in triplicate.
Results are represented as mean standard deviation.
2.7. Statistical analysis

On the basis of the bioethanol content after fermentation, the

total fermentable sugars content (g/100 g of triticales dry matter)
were calculated as well as bioethanol yield (g bioethanol/g of triticale starch), and percentage of the theoretical bioethanol yield.

95.6 0.12
36.04 0.16
81.2 0.21

Chemical analysis
Moisture content (%)
Protein content (% dry matter)
Falling number (s)
Starch content (% dry matter)
Magnesium ions content (mg/kg dry matter of triticale)
Calcium ions content (mg/kg dry matter of triticale)

Sieve aperture size (lm)

11.44 0.09
11.60 0.15
64 0.12
66.00 0.11
1010.0 0.27
288.6 0.18

calculated

from

three

All analyses were carried out in triplicate. Results were represented as mean standard deviation. MS Statistica 4.5 was used
to calculate means, standard deviations and differences between
the means. The analysis of variance (one-way ANOVA) followed
by Duncans multiple range test was used to test the hypothesis
about differences between mean values of samples in which no
magnesium or calcium ions were added and samples in which
magnesium or calcium ions were added. Means were considered
statistically different at 95% of condence level.
3. Results and discussion
3.1. Triticale analysis
In Table 1 are given triticale quality parameters.
Results given in Table 1 show that triticale variety Odyssey had
high share of grains over the 2.5 mm sieve (95.6%). According to
the literature values [34] 1000 grain weight for triticale can be
expected to range from 35 to 55 g of dry matter. The result

J.D. Pejin et al. / Fuel 142 (2015) 5864

obtained for 1000 grain weight in this study is within this interval,
with value of 36.04 g of dry matter. According to Erekul and Khn
[34] genetic factors play the greatest role in determining 1000
grain weight. Conditions of heat and drought during grain-lling
have been found to decrease 1000 grain weight, whereas cool
and moist weather during grain lling has been found to increase
1000 grain weight [35]. Test weight is in compliance with 1000
grain weight as well as the percentage of grains over the 2.5 mm
sieve. Obuchowski et al. [36] showed that triticale starch content
is correlated positively with test weight and 1000 grain weight.
The protein and starch content in Odyssey variety were 11.60%
of dry matter and 66.00% of dry matter, respectively. Protein content is inversely related to the starch content [35]. Protein content
in triticale is generally higher than in its parental species, and this
fact apparently is due to the combination of the protein fractions
from wheat and rye [37]. According to Aufhammer et al. [38] substrates for bioethanol production should not contain more than
11% of protein. This is in agreement with the ndings of Rosenberger [39].
Determination of the Falling number is a measurement based
on the breakdown of the starch gel by the a-amylase present in
the sample. This is indicative of the a-amylases activity of triticale
[40]. The low value (64 s) in 2012 indicates a very high activity of
amylolytic enzymes in triticale grain.
In triticale grain ratio of magnesium to calcium was 3.50
(Table 1).
In Table 2 is given the size distribution of triticale particle.
Triticale our consisted of 93% of particles with average size lower
than 700 lm.
3.2. Effect of magnesium and calcium ions addition on glucose and
maltose content in triticale mashes after liquefaction
Glucose and maltose contents in triticale mashes after liquefaction prepared without and with the addition of different magnesium and calcium ions content are presented in Figs. 1 and 2. In
all investigated samples, determined maltose contents were much
higher (approximately 40 times higher) than glucose contents
which indicates that triticale amylolytic enzymes produce more
maltose. Glucose and maltose content increased with the increase
of magnesium and calcium ion content in mash. Glucose and maltose content increased by 30.16% and 9.58%, respectively, when

Fig. 1. Glucose content in triticale mashes after liquefaction prepared without and
with the addition of different magnesium and calcium ions content. Experimental
conditions for liquefaction: triticale sample to water ratio = 1:3, 60 C, 65 min.
Vertical bars represent the standard deviation (n = 3) for each data point.

61

Fig. 2. Maltose content in triticale mashes after liquefaction prepared without and
with the addition of different magnesium and calcium ions content. Experimental
conditions for liquefaction: triticale sample to water ratio = 1:3, 60 C, 65 min.
Vertical bars represent the standard deviation (n = 3) for each data point.

160 mg/L of magnesium ions were added, compared to the control

sample. Glucose and maltose content increased by 69.31% and
61.66%, respectively, when 160 mg/L of calcium ions were added,
compared to the control sample. According to the obtained results
for glucose and maltose content increase during liquefaction, the
supplementation of mashes with calcium ions had greater inuence on the activity of triticales amylases than the supplementation of mashes with magnesium ions. Enhancement of amylase
activity by calcium ions is based on its ability to interact with negatively charged amino acid residues, which results in stabilization
as well as maintenance of enzyme conformation. Calcium also is
known to have a role in substrate binding [18]. Bush et al. [18]
showed that binding of calcium ions to barley amylase is preferred
over other cations such as magnesium. Muralikrishna and Nirmala
[41] also showed that calcium ions have more positive inuence
than magnesium ions on the activity of cereal a-amylases.

3.3. Effect of magnesium and calcium ions addition on simultaneous

saccharication and fermentation (SSF) of liqueed triticale mash
Figs. 3 and 4 present the time course of bioethanol production
in the SSF processing of liqueed triticale mashes by S. cerevisiae,
without and with the addition of different magnesium or calcium
ions contents: 40, 80, 120, or 160 mg/L. As shown in Figs. 3 and
4, the bioethanol production proles in all samples were similar.
During the SSF process the bioethanol contents obtained in
samples with the addition of magnesium ions were signicantly
higher (p < 0.05) than in the control sample, especially in samples
in which 120 and 160 mg/L of magnesium ions were added. In
these samples, a maximum bioethanol content of 15.16% and
15.19% (v/v), respectively, was achieved after 96 h of the SSF process. During the SSF process the bioethanol contents obtained in
samples with the addition of calcium ions were signicantly higher
(p < 0.05) than in the control sample, especially in samples in
which 120 and 160 mg/L of calcium ions were added. In these samples, a maximum bioethanol content of 13.88% and 14.11% (v/v),
respectively, was achieved after 96 h of the SSF process.
With addition of 120 and 160 mg/L of magnesium ions SSF process completed after 72 h since there was no signicant difference
in bioethanol content (p > 0.05) compared to bioethanol content
determined after 96 h which could be explained by many positive
aspects of increasing magnesium availability in fermentation

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J.D. Pejin et al. / Fuel 142 (2015) 5864

Fig. 3. Time course of bioethanol production in the SSF processing of triticale

mashes by S. cerevisiae without and with the addition of different magnesium ions
content. Experimental conditions for SSF process: 30 C, 96 h. Vertical bars
represent the standard deviation (n = 3) for each data point.

Fig. 4. Time course of bioethanol production in the SSF processing of triticale

mashes by S. cerevisiae without and with the addition of different calcium ions
content. Experimental conditions for SSF process: 30 C, 96 h. Vertical bars
represent the standard deviation (n = 3) for each data point.

medium including: decreasing the lag phase and initial increase of

the cell number, rate of growth and total bioethanol yield [25].
After 72 and 96 h of SSF processes in which 80, 120, and
160 mg/L of magnesium ions were added signicantly higher bioethanol contents (p = 0.0070.0198) were obtained compared to
the SSF processes in which corresponding calcium ions were
added.
The addition of magnesium ions in triticale mashes increased
the ratio of magnesium to calcium, with higher values giving
increased bioethanol content which is in agreement with results
obtained by Walker et al. [19] and Bromberg et al. [42].
Bioethanol contents (g/100 g of triticales dry matter) obtained
after the fermentation of samples of triticale mashes without and
with the addition of different magnesium or calcium ions contents
are given in Figs. 5 and 6. Bioethanol content increased by 30.00%
and 31.22% when 120 and 160 mg/L of magnesium ions were
added, respectively, compared to the control sample (p < 0.05). Bioethanol content increased by 19.03% and 21.04% when 120 and
160 mg/L of calcium ions were added, respectively, compared to
the control sample (p < 0.05).
Walker et al. [19] demonstrated that by increasing magnesium
to calcium ratio in molasses, used as a raw material for bioethanol
production, more active yeast metabolism was achieved. This was
manifested by the elevation in nal bioethanol produced. Rees and
Stewart [25] investigated the inuence of magnesium (500 ppm)

Fig. 5. Effect of magnesium ions addition on bioethanol content obtained after the
fermentation of triticale mashes. Experimental conditions for SSF process: 30 C,
96 h. Vertical bars represent the standard deviation (n = 3) for each data point.
Means of bioethanol contents with different small letters above bars are signicantly different (p < 0.05).

Fig. 6. Effect of calcium ions addition on bioethanol content obtained after the
fermentation of triticale mashes. Experimental conditions for SSF process: 30 C,
96 h. Vertical bars represent the standard deviation (n = 3) for each data point.
Means of bioethanol contents with different small letters above bars are signicantly different (p < 0.05).

or calcium (800 ppm) ions addition in wort on bioethanol

production and concluded that elevated magnesium contents
caused higher bioethanol content. The addition of calcium ions in
wort at such high content decreased bioethanol production.
Increasing the calcium to magnesium ratio may have exacerbated

Table 3
Effect of magnesium or calcium ions addition on the total fermentable sugars content
(g/100 g of triticales dry matter) obtained after the fermentation of triticale mashes.
Experimental conditions for SSF process: 30 C, 96 h.
Added ions
content (mg/L)

Total fermentable sugars content (g/100 g of

triticales dry matter)*
Magnesium

0
40
80
120
160

Calcium
a,A

46.35 0.93
51.05 1.00b,A
56.71 0.90c,A
60.27 0.62d,A
60.85 0.72d,A

46.35 0.93a,A
52.49 0.82b,A
53.66 1.08bc,B
55.18 0.89cd,B
56.13 1.00d,B

*
Values represent means standard deviation calculated from three determinations. Means of fermentable sugars contents with different small letter in a column are signicantly different (p < 0.05). Means of fermentable sugars contents
with different capital letter in a row are signicantly different (p < 0.05).

J.D. Pejin et al. / Fuel 142 (2015) 5864

the inadequate level of magnesium due to competition between

these two ions.
On the basis of the bioethanol content after fermentation (Figs. 5
and 6), the total fermentable sugars content (g/100 g of triticales
dry matter) (Table 3) were calculated as well as bioethanol
yield YP/S (g bioethanol/g triticale starch) and percentage of the
theoretical bioethanol yield (Figs. 7 and 8). Bioethanol yields and
fermentation efciencies (the total fermentable sugars contents
and percentage of the theoretical bioethanol yield) were determined by distilling triticale mashes after the fermentation. The
highest bioethanol yields and therefore the contents of the total fermentable sugars were achieved in samples in which 120 and
160 mg/L of magnesium ions were added (60.27 and 60.85 g/100 g
of triticales dry matter). In samples in which 120 and 160 mg/L of
magnesium ions were added, the maximum percentage of the theoretical bioethanol yield of 91.32% and 92.19%, respectively, was
achieved. Percentage of the theoretical bioethanol yield should be
9095% [43].

63

The results show that magnesium ions addition signicantly

affects bioethanol yield and percentage of the theoretical bioethanol
yield. Percentage of the theoretical bioethanol yield increased by
31.22% (p < 0.05) in a sample in which 160 mg/L of magnesium ions
were added compared to the control sample. This increase in bioethanol yield is very important in bioethanol technology. Addition
of magnesium ions had more signicant effect on bioethanol yield
increase (for 10%) than the addition of calcium ions. It can be concluded that if magnesium and calcium ions content ratio in triticale
mashes is higher, better bioethanol yields are obtained which is in
agreement with the results reported by Walker et al. [19].
Future work is needed to optimize magnesium and calcium ions
ratio to achieve higher bioethanol yields. Also, additional investigations are needed to scale up the system designs to large batch or
continuous processes, in order to fully realise the potential benets
of magnesium and calcium ions addition in triticale mashes for
bioethanol production. Based on the obtained results the time of
the SSF processing of triticale mashes with the addition of
120 mg/L of magnesium ions may be reduced from 96 to 72 h
which makes the bioethanol production process more efcient
and economical.
4. Conclusions

Fig. 7. Effect of magnesium ions addition on the percentage of the theoretical

bioethanol yield obtained after the fermentation of triticale mashes. Experimental
conditions for SSF process: 30 C, 72 h. Vertical bars represent the standard
deviation (n = 3) for each data point. Means of the percentage of the theoretical
bioethanol yield with different small letters above bars are signicantly different
(p < 0.05). Percentage of the theoretical bioethanol yield was calculated as the ratio
between actual bioethanol yield and theoretical bioethanol yield, assuming all
starch was converted to glucose and then to bioethanol.

With an increase in magnesium and calcium ions content in triticale mashes glucose and maltose content increased during liquefaction. Glucose and maltose content increased by 30.16% and
9.58%, respectively, when 160 mg/L of magnesium ions were added,
compared to the control sample. Glucose and maltose content
increased by 69.31% and 61.66%, respectively, when 160 mg/L of
calcium ions were added, compared to the control sample. According to the obtained results for glucose and maltose content increase
during liquefaction, the supplementation of mashes with calcium
ions had greater inuence on the activity of triticales amylases
than the supplementation of mashes with magnesium ions which
shows that calcium is essential for triticales amylases activity.
The present investigation shows that magnesium and calcium
ions addition to triticale mashes improved bioethanol production
during SSF processing. When 160 mg/L of magnesium ions were
added bioethanol content increased by 31.22% compared to the
control sample while when 160 mg/L of calcium ions were added
bioethanol content increased by 21.04%. High percentage of the
theoretical bioethanol yield (92.19%) was achieved after fermentation when 160 mg/L of magnesium ions were added to triticale
mash. The obtained results show that the addition of magnesium
and calcium ions in bioethanol production from triticale increase
triticales amylase activity as well as yeast enzyme activity. All this
shows that when triticale with high amylolytic enzymes activity is
used in bioethanol production with the addition of magnesium
ions there is no need to use commercial enzymes in starch
hydrolysis, which makes the use of triticale as a raw material for
bioethanol production more economical.
Acknowledgement
This work was funded by the Ministry of Education, Science and
Technological Development (TR-31017) Republic of Serbia.

Fig. 8. Effect of calcium ions addition on the percentage of the theoretical

bioethanol yield obtained after the fermentation of triticale mashes. Experimental
conditions for SSF process: 30 C, 72 h. Vertical bars represent the standard
deviation (n = 3) for each data point. Means of the percentage of the theoretical
bioethanol yield with different small letters above bars are signicantly different
(p < 0.05). Percentage of the theoretical bioethanol yield was calculated as the ratio
between actual bioethanol yield and theoretical bioethanol yield, assuming all
starch was converted to glucose and then to bioethanol.

References
[1] Pereira F, Guimaraes P, Teixeira J, Domingues L. Optimization of low-cost
medium for very high gravity ethanol fermentations by Saccharomyces
cerevisiae using statistical experimental designs. Bioresour Technol
2010;101:785663.
[2] Mojovic L, Pejin D, Grujic O, Markov S, Pejin J, Rakin M, et al. Progress in the
production of bioethanol on starch-based feedstocks. CI & CEQ
2009;15:21126.

64

J.D. Pejin et al. / Fuel 142 (2015) 5864

[3] Global Renewable Fuel Alliance. <http://www.globalrfa.com/pr_062612.php>

[accessed 18.03.13].
[4] Balat M, Balat H. Recent trends in global production and utilization of bioethanol fuel. Appl Energy 2009;86:227382.
[5] Walker G. 125th Anniversary review: fuel alcohol: current production and
future challenges. J Inst Brew 2011;117:322.
[6] Jansone I, Malcka S, Miglane V. Suitability of winter triticale varieties for
bioethanol production in Latvia. Agron Res 2010;8:57382.
[7] Balat M, Balat H, z C. Progress in bioethanol processing. Prog Energy Combust
2008;34:55173.
[8] Zhao X, Bai F. Mechanisms of yeast stress tolerance and its manipulation for
efcient fuel ethanol production. J Biotechnol 2009;144:2330.
[9] Hanzalov A, Barto P. Resistance of triticale to wheat leaf rust. Czech J Genet
Plant Breed 2011;47:106.
[10] Mergoum M, Pfeffer H, Pea J, Ammar K, Rajaram S. Triticale crop
improvement: the CIMMYT programme. In: Mergoum M, GmezMacpherson H, editors. Triticale improvement and production: FAO plant
production and Protection Paper 179. Rome: Food and Agriculture
Organization of the United Nations; 2004. p. 1126.
[11] Du Pisani F. Evaluation of structural and functional composition of South
African triticale cultivars (X Triticosecale Wittmack). Master of Science in Food
Science, Department of Food Science, Faculty of AgriSciences, Stellenbosch
University; 2009. <http://hdl.handle.net/10019.1/2221> [accessed 01.04.09].
[12] Eudeus F. Canadian triticale biorenery initiative. In: Proceedings of the 6th
international triticale symposium, 37 September 2006, Stellenbosch, South
Africa. p. 858.
[13] Boros D. Triticale of high end-use quality enhances opportunities to increase its
value in world cereal market. In: Proceedings of the 6th international triticale
symposium, 37 September 2006, Stellenbosch, South Africa. p. 11925.
[14] Mojovic L, Nikolic S, Rakin M, Vukasinovic M. Production of bioethanol from
corn meal hydrolyzates. Fuel 2006;85:17505.
[15] Kucerov J. The effect of year, site and variety on the quality characteristics
and bioethanol yield of winter triticale. J Inst Brew 2007;113:1426.
[16] Pejin D, Mojovic L, Vucurovic V, Pejin J, Dencic S, Rakin M. Fermentation of
wheat and triticale hydrolysates: a comparative study. Fuel 2009;88:16258.
[17] Pejin D, Mojovic L, Pejin J, Grujic O, Markov S, Nikolic S, et al. Increase in
bioethanol production yield from triticale by simultaneous saccharication
and fermentation with application of ultrasound. J Chem Technol Biotechnol
2012;87:1706.
[18] Bush D, Sticher L, van Huysteee R, Wagner D, Jones R. The calcium requirement
for stability and enzymatic activity of two isoforms of barley aleurone aamylase. J Biol Chem 1989;264:193928.
[19] Walker G, Birch R, Chandrasena G, Maynard A. Magnesium, calcium, and
fermentative metabolism in industrial yeasts. J Am Soc Brew Chem
1996;54:138.
[20] Walker G, DeNicola R, Anthony S, Learmonth R. Yeast metal interactions:
impact on brewing and distilling fermentations. In: Institute of Brewing and
Distilling Asia Pacic Section Convention, Hobart, Australia; 2006. <https://
eprints.usq.edu.au/> [accessed 09.05.12].
[21] Walker G. Magnesium as a stress-protectant for industrial strains of
Saccharomyces cerevisiae. J Am Soc Brew Chem 1998;56:10913.
[22] Russell I. Understanding yeast fundamentals. In: Jacques KA, Lyons TP, Kelsall
DR, editors. The alcohol textbook. Nottingham: Nottingham University Press;
2003. p. 85119.
[23] Walker G, Maynard A. Accumulation of magnesium ions during fermentative
metabolism in Saccharomyces cerevisiae. J Ind Microbiol Biot 1997;18:13.

[24] Poreda A, Antkiewicz P, Tuszynski T, Magorzata M. Accumulation and release

of metal ions by brewers yeast during successive fermentations. J Inst Brew
2009;115:7883.
[25] Rees E, Stewart G. The effects of increased magnesium and calcium
concentrations on yeast fermentation performance in high gravity worts. J
Inst Brew 1997;103:28791.
[26] Ciesarov Z, mogrovicov D, Domeny Z. Enhancement of yeast ethanol
tolerance by calcium and magnesium. Folia Microbiol 1996;41:4858.
[27] Wang S, Thomas C, Sosulski K, Ingledew M, Sosulski W. Grain pearling and
very high gravity (VHG) fermentation technologies for fuel alcohol production
from rye and triticale. Process Biochem 1999;34:4218.
[28] International Standard: ISO 7971-2. Cereals determination of bulk density,
called mass per hectoliter Part 2: Routine method. International
Organization for Standardization; 1995.
[29] AOAC Ofcial Methods of Analysis of AOAC International, 17th ed.
Gaithersburg, MD, USA: AOAC International; 2000.
[30] AACC Approved Methods of the American Association of Cereal Chemists,
2008. 10th ed. St. Paul, Minnesota, USA: American Association of Cereal
Chemists, Method 5681 B.
[31] International Standard: ISO 10520. Native starch determination of starch
content Ewers polarimetric method. International Organization for
Standardization; 1997.
[32] Miedl M, Cornne S, Leiper K, Shepher M, Stewart G. Low-temperature
processing of wheat for bioethanol production, Part I. Studies on the use of
commercial enzymes. J Am Soc Brew Chem 2007;65:18391.
[33] Methods of enzymatic bioanalysis and food analysis, D-glucose, UV method,
Boehringer Mannheim GmbH Biochemicals, Mannheim; 1997. p. 49.
[34] Kozak M, Samborski S, Rozbicki J, Madry W. Winter triticale grain yield, a
comparative study if 15 genotypes. Soil Plant Sci 2007;57:26370.
[35] Erekul O, Khn W. Effect of weather and soil conditions on yield components
and bread-making quality (winter wheat Triticum aestivum L.) and winter
triticale (Triticosecale Wittm.) varieties in northeast Germany. J Agron Crop Sci
2006;192:45264.
[36] Obuchowski W, Banaszak S, Makowska A, uczak M. Factors affecting
usefulness of triticale grain for bioethanol production. J Sci Food Agric
2010;90:250611.
[37] Burgos-Hernndez A, Rosas-Burgos C, Ramrez-Wong B, Carbonell-Barrachina
A, Cinco-Moroyoqui F. Identication of a-amylase inhibitors in triticale grain. J
Sci Food Agric 1999;79:16715.
[38] Aufhammer W, Pieper H, Kasser J, Schafer V, Senn K, Kubler E. The suitable of
grains from cereal crops with different N supply for bioethanol production. J
Agron Crop Sci 1996;177:18596.
[39] Rosenberger A. Gewinnung von bioethanol aus Getreide mit Hilfe technischer
enzyme. Getreidetechnologie 2004;58:3035.
[40] Tohver M, Kann A, Tht R, Mihhavelski A, Hakman J. Quality of triticale
cultivars suitable for growing and bread-making in northern conditions. Food
Chem 2005;89:12532.
[41] Muralikrishna G, Nirmala M. Cereal a-amylases an overview. Carbohydr
Polym 2005;60:16373.
[42] Bromberg S, Bower P, Duncombe G, Fehring J, Gerber LA, Lau V, et al.
Requirements for zinc, manganese, calcium, and magnesium in wort. J Am Soc
Brew Soc Chem 1997;55:1238.
[43] Kosaric N, Duvnjak Z, Farkas A, Sahm H, Bringer-Meyer S, Goebel O, et al. In:
Ullmanns encyclopedia of industrial chemistry. WILEY-VCH; 2003. p. 408.