[email protected]

Bioethanol production froand fermentation with ma

Jelena D. Pejin a,, Ljiljana V. MojoSvetlana B. Nikolic b, AleksandraaUniversity of Novi Sad, Faculty of Technology, BulevarbUniversity of Belgrade, Faculty of Technology and MetacUniversity of Ni, Faculty of Technology, Bulevar Oslob

ns in trre addedded beffect oe is no

to determine the effect of magnesium or calcium ions content in triticale

duction from triticale increase triticales amylase activity as well as yeast enzyme activity. All this showsction with the addi-lysis, which

All rights re

1. Introduction

Bioethanol is regarded as a promising alternative energy source,which is both renewable and environmentally friendly. Duringbioethanol production, the composition of media affects the

Corresponding author. Tel.: +381 21 485 3721.E-mail address: [email protected] (J.D. Pejin).

Fuel 142 (2015) 5864

Contents lists availab

Fuethat when triticale with high amylolytic enzymes activity is used in bioethanol prodution of magnesium ions there is no need to use commercial enzymes in starch hydrothe use of triticale as a raw material for bioethanol production more economical.

2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.fuel.2014.10.0770016-2361/ 2014 Elsevier Ltd. All rights reserved.makes

served.Received 10 August 2013Received in revised form 23 October 2014Accepted 28 October 2014Available online 8 November 2014

Keywords:TriticaleBioethanol yieldMagnesiumCalcium

mashes on glucose and maltose content after liquefaction as well as on bioethanol yield after fermenta-tion. Triticale variety Odyssey was used in this study. Liquefaction and saccharication in this study wereperformed without using any additional saccharifying enzymes, i.e. the triticale starch was hydrolyzedonly by the enzymes present in triticale grain. Glucose and maltose content increased with the increaseof magnesium and calcium ion content in mash. Glucose and maltose content increased by 30.16% and9.58%, 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 calciumions were added, compared to the control sample. According to the obtained results for glucose and malt-ose content increase during liquefaction, the supplementation of mashes with calcium ions had greaterinuence on the activity of triticales amylases than the supplementation of mashes with magnesiumions. The present investigation shows that magnesium and calcium ions addition to triticale mashesimproved bioethanol production during SSF processing. When 160 mg/L of magnesium ions were addedbioethanol content increased by 31.22% compared to the control sample while when 160 mg/L of calciumions were added bioethanol content increased by 21.04%. High percentage of the theoretical bioethanolyield (92.19%) was achieved after fermentation when 160 mg/L of magnesium ions were added to triti-cale mash. The obtained results show that the addition of magnesium and calcium ions in bioethanol pro-Article history: The aim of this study wash i g h l i g h t s

The effect of magnesium or calcium io When 160 mg/L of magnesium ions we When 160 mg/L of calcium ions were a Magnesium ions had more signicant When magnesium ions are added ther

a r t i c l e i n f om triticale by simultaneous saccharicationgnesium or calcium ions addition

vic b, Duanka J. Pejin a, Suncica D. Kocic-Tanackov a, Dragia S. Savic c,P. Djukic-Vukovic b

Cara Lazara 1, 21 000 Novi Sad, Serbiallurgy, Karnegijeva 4, 11 000 Belgrade, Serbiao -denja 124, 16 000 Leskovac, Serbia

iticale mashes on bioethanol yield.d bioethanol content increased by 31.22%.ioethanol content increased by 21.04%.n bioethanol yield than calcium ions.need to use commercial enzymes.

a b s t r a c tjournal homepage: www.elsevier .com/locate / fuelle at ScienceDirect

l

release of metal ions during ethanolic fermentation is a dynamic

content after liquefaction as well as on bioethanol yield after fer-

el 1physiological state and, consequently, the fermentation perfor-mance of the microorganism employed [1]. Bioethanol is producedby fermentation of sugar, starch or cellulosic biomass and its utili-zation can signicantly reduce fossil fuels use. It is expected to beone of the dominating renewable biofuels in the transportationsector within the twenty years to come [2]. The production of bio-ethanol is increasing over the years, and has reached the level of85.2 billion litres in the year 2012 [3]. The governmental supportsfor the substitution of fossil fuels with bioethanol produced frombiomass is predicted to result in global production of 125 109 Lof bioethanol by 2020 [4]. The primary benecial aspects of fer-menting biomass-derived sugars to bioethanol as a fuel source isthat it can be produced from renewable plant material that is ableto photosynthetically re-x CO2 produced during bioethanol pro-duction and combustion [5]. Bioethanol production has remarkablyincreased because many countries look for reducing oil imports,boosting rural economies and improving the air quality [2]. Onemajor problem with bioethanol production is the availability ofraw materials for the production. There are several criteria forchoosing raw materials for bioethanol production: price and yieldof raw material, bioethanol yield, starch content, pest and diseasesresistance, suitability for soil and weather conditions, harvestingtransportation and storage options as well as the usability of by-products [6]. The availability of feedstocks for bioethanol produc-tion can vary considerably from season to season and depends ongeographic locations [7]. However, feedstocks for bioethanol pro-duction must be sustainable and must not threaten biodiversityor food security [5].

Yeast strains of Saccharomyces cerevisiae have been extensivelystudied in recent years for fuel bioethanol production, in whichyeast cells are exposed to various stresses such as high tempera-ture, bioethanol inhibition, and osmotic pressure from productand substrate sugars and so on [8].

Triticale is a cereal crop adapted to less favorable soil condi-tions. It is suitable for low input farming because of lower demandson pesticides application [9]. Today, it has been reported that trit-icale is cultivated in more than 30 countries worldwide [10] onaround 3.7 million ha in total, yielding more than 12 million ton-nes a year [11]. Modern triticale varieties have been found to bevery competitive as a feedstock for bioethanol production [12].Triticale crops have a high yield potential as well as a high starchcontent, together with a low content of soluble polysaccharidesand proteins, and is therefore considered to be ideal for bioethanolproduction [13]. There is high activity of triticales own amylolyticenzymes, mainly a-amylase, and this is crucial in starch sacchari-cation [14,15]. Considering the currently prevalent cold tech-nique of saccharication, by means of commercial enzymes, theprocessing of triticale is economically benecial as it enables thereduction of the commercial enzymes consumption [15]. In ourprevious research [16,17] it was shown that the addition of com-mercial enzymes was not necessary during liquefaction and sac-charication step in bioethanol production from triticale varietyOdyssey. Cereal a-amylases are known to be metalloenzymes. Ishas been shown that these enzymes contain covalently bound cal-cium ions which act as an allosteric activator. Besides calcium ions,magnesium ions can also act as a-amylases activator. Studies onbarley a-amylase show that these ions, especially calcium ion helpin maintaining the three-dimensional structure of amylases [18].

The mineral metabolism of yeast is of interest to bioethanolproducers looking to improve yields, increase fermentative capac-ity, and maintain consistency of product quality [19]. Metal ionsespecially divalent cations are necessary for the activation of sev-eral glycolytic enzymes and, in practical terms, if industrial media

J.D. Pejin et al. / Fuis decient in them, the conversion of sugar to bioethanol may besuppressed leading to slow or incomplete fermentation process[20]. Magnesium is involved in many essential physiological andmentation. Triticale variety Odyssey, from experimental elds,Rimski ancevi location (Serbia) was used in this study. The effectof magnesium and calcium ions content in triticale mashes on glu-cose and maltose content after liquefaction and on bioethanol yieldafter fermentation was investigated by adding different amounts ofMgSO47H2O or CaCl2 solution in triticale mashes before liquefac-tion and saccharication. In this study the process was conductedwithout the addition of external amylolytic enzymes, and theliquefaction and saccharication of starch were performed onlyby enzymes present in triticale grain. The bioethanol yield and pro-ductivity were also assessed.

2. Materials and methods

2.1. Materials

Triticale variety Odyssey was obtained from Institute of Fieldand Vegetable Crops Novi Sad (Serbia). Triticale was milled in adry conical mill (Miag-Braunschweig, Germany) type: DOXY 71b/4, mill motor power 0.22 kW, at 1375 r/min. The granulation oftriticale meals was determined by sieving 100 g of milled samplefor 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 fromAlltech Fermin, Senta, Serbia was used as a producing microorgan-ism. Prior to each experiment, the yeast was activated according tothe following procedure: the yeast was measured and suspendedin 0.1% sterile peptone water warmed up to 38 C. The yeast cellcount was determined in Neubauers counting chamber. Fromthe prepared yeast solution, the amount of inoculum needed toobtain 3035 106 CFU/mL in the fermentation medium, was taken[27]. Indirect counting method, i.e. pour plate technique, was usedto determine the number of viable cells. Serial dilutions of theprocess and that the intensity depends on the sugar and bioethanolcontent in the fermentation medium [24]. Calcium is not a require-ment but may stimulate cell growth, protects yeast cell membranestructure and helps maintain membrane permeability underadverse conditions [22]. Calcium, being actively excluded fromthe yeast cell, acts mainly extracellularly for example, calcium isessential for amylase activity [25]. Metal ion deciencies oftenoccur in fermentation media, and studies on optimization of metalions combinations are thus of great practical importance toimprove bioethanol production [1,26].

The aim of this study was to determine the effect of magnesiumor calcium ions content in triticale mashes on glucose and maltosebiochemical functions in yeast cells, including growth, cell division,enzyme activation, stimulation of synthesis of essential fatty acids,regulation of cellular ionic levels, and maintaining membraneintegrity and permeability. Yeasts have a very high growth demandfor magnesium ions, and magnesium accumulation by yeastcorrelates closely with the progress of fermentation [21,22].Magnesium also plays roles in protecting yeast cells againstenvironmental stresses during fermentation such as caused by bio-ethanol, high temperature, or high osmotic pressure [23]. The rateof uptake and utilization of metal ions by the yeast biomassdepends both on the ion content in the medium, as well as on itsbioavailability. It has been established that accumulation and

42 (2015) 5864 59samples were performed, and after the incubation time at 30 C,colonies grown in Petri dishes were used to count the number ofviable cells.

2.3. Triticale analysis

Triticale samples were monitored for the following qualityparameters and the following methods were used for analysis: testweight (g/L) [28], thousand grain weight (g) weight of 1000grains, percentage of grain above the sieve 2.5 mm, protein contentby Kjeldahl method, magnesium and calcium content [29], Fallingnumber (s) [30], starch content after Ewers polarimetric method(% dry matter) [31], and the moisture content in the triticale mealwas determined by the standard drying method in an oven at105 C to a constant mass [29]. All above mentioned standard trit-icale analyses were carried in triplicate (results presented in Tables

by Duncans multiple range test was used to test the hypothesisabout differences between mean values of samples in which nomagnesium or calcium ions were added and samples in whichmagnesium or calcium ions were added. Means were consideredstatistically 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

60 J.D. Pejin et al. / Fuel 11 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 autoamylolyticenzyme activity in our previous research [16,17], liquefactionand saccharication in this study were performed without usingany additional saccharifying enzymes, i.e. the triticale starch washydrolyzed only by the enzymes present in triticale grain. Lique-faction was carried out using automated mashing water bath(Glasblserei, Institut fr Grungs Gewerbe, Berlin). Milled triticalesamples were mixed with distilled water warmed to 60 C (sampleto water ratio 1:3). The samples were kept in the mashing bath at60 C for 65 min for liquefaction. After the liquefaction sampleswere cooled to 20 C.

The samples were transferred to 500 mL glass bottles after theliquefaction. The simultaneous saccharication and fermentation(SSF) process was initiated by adding S. cerevisiae inoculum (toobtain 3035 106 CFU/mL in the fermentation medium) to the liq-ueed samples and carried out for up to 96 h at 30 C. The bottleswere closed with foam burgs to allow venting of the CO2 producedduring the fermentation. After fermentation, the fermented mashwas centrifuged for 15 min (10,000 r/min at 4 C) in a refrigeratedcentrifuge (Sorvall RC 24) and the supernatant was used for deter-mination of bioethanol content [32]. The bioethanol content wasdetermined based on the density of bioethanol distillate at 20 Cand expressed as % w/w [29]. During fermentation, samples wereprepared in the same manner and analyzed for bioethanol content.Analyses were carried out at least in triplicate. Results were repre-sented as mean standard deviation.

2.5. Calculation of important process parameters

On the basis of the bioethanol content after fermentation, thetotal fermentable sugars content (g/100 g of triticales dry matter)were calculated as well as bioethanol yield (g bioethanol/g of trit-icale starch), and percentage of the theoretical bioethanol yield.

Table 1Quality parameters of triticale variety Odyssey.a

Mechanical analysisPercentage of grains over the 2.5 mm sieve (%) 95.6 0.12Thousand grain weight (g) 36.04 0.16Test weight (kg/hL) 81.2 0.21

Chemical analysisMoisture content (%) 11.44 0.09Protein content (% dry matter) 11.60 0.15Falling number (s) 64 0.12Starch content (% dry matter) 66.00 0.11Magnesium ions content (mg/kg dry matter of triticale) 1010.0 0.27Calcium ions content (mg/kg dry matter of triticale) 288.6 0.18a Values represent means standard deviation calculated from threedeterminations.During the fermentation the yeast produces bioethanol accordingto the Gay-Lussac equation. From each gram of glucose consumed,0.51 g of bioethanol can be produced which represents the theoret-ical yield of bioethanol. Starch content in investigated triticale vari-ety was 66.00% of dry matter. Under the optimal conditions ofpretreatment, liquefaction and fermentation, all starch contentshould be converted to fermentable sugars and then to bioethanol.According to the obtained bioethanol content (g/100 g of triticalesdry 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 wascalculated as the ratio between actual bioethanol yield (g/g of trit-icale starch) and theoretical bioethanol yield (0.51 g) multiplied by100 (assuming all starch was converted to glucose and then tobioethanol).

2.6. HPLC analysis of glucose and maltose

The supernatant obtained as previously described was used forglucose andmaltose analysis by HPLC. Prior to the analysis, proteinswere removed from the supernatant [33]. The supernatant was col-lected and ltered through a 0.22 lmmembrane. A 20 lL aliquot ofthe ltrate was applied to an Aminex HPX087H Column (9 lm,7.8 mm ID 300 mm, Biorad Laboratories) for HPLC analysis, usingan Agilent 1100 Series HPLC system equipped with vacuum, degas-ser, binary pump, thermostatted column compartment, variablewavelength detector and RI detector. The temperature was main-tained at 50 C. The absorbance at 214 nm was detected at the owrate of 1 mL min1. The mobile phase was 5 mmol L1 H2SO4 withisocratic elution. Analyses were carried out at least in triplicate.Results are represented as mean standard deviation.

2.7. Statistical analysis

All analyses were carried out in triplicate. Results were repre-sented as mean standard deviation. MS Statistica 4.5 was usedto calculate means, standard deviations and differences betweenthe means. The analysis of variance (one-way ANOVA) followed

Table 2Triticale our particle size distributiona.

Sieve aperture size (lm) (%)

>1000 0.9 0.031000/700 6.1 0.07700/450 21.0 0.15450/250 39.9 0.18250/150 6.8 0.11

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 1000grain weight. Conditions of heat and drought during grain-llinghave been found to decrease 1000 grain weight, whereas cooland moist weather during grain lling has been found to increase1000 grain weight [35]. Test weight is in compliance with 1000grain weight as well as the percentage of grains over the 2.5 mmsieve. Obuchowski et al. [36] showed that triticale starch contentis 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 con-tent is inversely related to the starch content [35]. Protein contentin triticale is generally higher than in its parental species, and thisfact apparently is due to the combination of the protein fractionsfrom wheat and rye [37]. According to Aufhammer et al. [38] sub-strates for bioethanol production should not contain more than11% of protein. This is in agreement with the ndings of Rosenber-ger [39].

Determination of the Falling number is a measurement based

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

3.3. Effect of magnesium and calcium ions addition on simultaneoussaccharication and fermentation (SSF) of liqueed triticale mash

Figs. 3 and 4 present the time course of bioethanol productionin the SSF processing of liqueed triticale mashes by S. cerevisiae,without and with the addition of different magnesium or calciumions contents: 40, 80, 120, or 160 mg/L. As shown in Figs. 3 and4, the bioethanol production proles in all samples were similar.During the SSF process the bioethanol contents obtained insamples with the addition of magnesium ions were signicantlyhigher (p < 0.05) than in the control sample, especially in samplesin which 120 and 160 mg/L of magnesium ions were added. Inthese samples, a maximum bioethanol content of 15.16% and15.19% (v/v), respectively, was achieved after 96 h of the SSF pro-cess. During the SSF process the bioethanol contents obtained insamples with the addition of calcium ions were signicantly higher(p < 0.05) than in the control sample, especially in samples inwhich 120 and 160 mg/L of calcium ions were added. In these sam-ples, 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 pro-cess completed after 72 h since there was no signicant difference

J.D. Pejin et al. / Fuel 1Glucose and maltose contents in triticale mashes after liquefac-tion prepared without and with the addition of different magne-sium and calcium ions content are presented in Figs. 1 and 2. Inall investigated samples, determined maltose contents were muchhigher (approximately 40 times higher) than glucose contentswhich indicates that triticale amylolytic enzymes produce moremaltose. Glucose and maltose content increased with the increaseof magnesium and calcium ion content in mash. Glucose and malt-ose content increased by 30.16% and 9.58%, respectively, when

Fig. 1. Glucose content in triticale mashes after liquefaction prepared without andon the breakdown of the starch gel by the a-amylase present inthe sample. This is indicative of the a-amylases activity of triticale[40]. The low value (64 s) in 2012 indicates a very high activity ofamylolytic 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 lowerthan 700 lm.

3.2. Effect of magnesium and calcium ions addition on glucose andmaltose content in triticale mashes after liquefactionwith the addition of different magnesium and calcium ions content. Experimentalconditions 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.sample. Glucose and maltose content increased by 69.31% and61.66%, respectively, when 160 mg/L of calcium ions were added,compared to the control sample. According to the obtained resultsfor glucose and maltose content increase during liquefaction, thesupplementation of mashes with calcium ions had greater inu-ence on the activity of triticales amylases than the supplementa-tion of mashes with magnesium ions. Enhancement of amylaseactivity by calcium ions is based on its ability to interact with neg-atively charged amino acid residues, which results in stabilizationas well as maintenance of enzyme conformation. Calcium also isknown to have a role in substrate binding [18]. Bush et al. [18]showed that binding of calcium ions to barley amylase is preferredover other cations such as magnesium. Muralikrishna and Nirmala[41] also showed that calcium ions have more positive inuencethan magnesium ions on the activity of cereal a-amylases.Fig. 2. Maltose content in triticale mashes after liquefaction prepared without andwith the addition of different magnesium and calcium ions content. Experimentalconditions 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.

42 (2015) 5864 61in bioethanol content (p > 0.05) compared to bioethanol contentdetermined after 96 h which could be explained by many positiveaspects of increasing magnesium availability in fermentation

cantly different (p < 0.05).

el 142 (2015) 5864Fig. 3. Time course of bioethanol production in the SSF processing of triticalemashes by S. cerevisiae without and with the addition of different magnesium ionscontent. Experimental conditions for SSF process: 30 C, 96 h. Vertical barsrepresent the standard deviation (n = 3) for each data point.

62 J.D. Pejin et al. / Fumedium including: decreasing the lag phase and initial increase ofthe cell number, rate of growth and total bioethanol yield [25].

After 72 and 96 h of SSF processes in which 80, 120, and160 mg/L of magnesium ions were added signicantly higher bio-ethanol contents (p = 0.0070.0198) were obtained compared tothe SSF processes in which corresponding calcium ions wereadded.

The addition of magnesium ions in triticale mashes increasedthe ratio of magnesium to calcium, with higher values givingincreased bioethanol content which is in agreement with resultsobtained by Walker et al. [19] and Bromberg et al. [42].

Bioethanol contents (g/100 g of triticales dry matter) obtainedafter the fermentation of samples of triticale mashes without andwith the addition of different magnesium or calcium ions contentsare given in Figs. 5 and 6. Bioethanol content increased by 30.00%and 31.22% when 120 and 160 mg/L of magnesium ions wereadded, respectively, compared to the control sample (p < 0.05). Bio-ethanol content increased by 19.03% and 21.04% when 120 and160 mg/L of calcium ions were added, respectively, compared tothe control sample (p < 0.05).

Walker et al. [19] demonstrated that by increasing magnesiumto calcium ratio in molasses, used as a raw material for bioethanolproduction, more active yeast metabolism was achieved. This wasmanifested by the elevation in nal bioethanol produced. Rees andStewart [25] investigated the inuence of magnesium (500 ppm)

Fig. 4. Time course of bioethanol production in the SSF processing of triticalemashes by S. cerevisiae without and with the addition of different calcium ionscontent. Experimental conditions for SSF process: 30 C, 96 h. Vertical barsrepresent the standard deviation (n = 3) for each data point.Fig. 5. Effect of magnesium ions addition on bioethanol content obtained after thefermentation 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 signi-or calcium (800 ppm) ions addition in wort on bioethanolproduction and concluded that elevated magnesium contentscaused higher bioethanol content. The addition of calcium ions inwort at such high content decreased bioethanol production.Increasing the calcium to magnesium ratio may have exacerbated

Fig. 6. Effect of calcium ions addition on bioethanol content obtained after thefermentation 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 signi-cantly different (p < 0.05).

Table 3Effect 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 ionscontent (mg/L)

Total fermentable sugars content (g/100 g oftriticales dry matter)*

Magnesium Calcium

0 46.35 0.93a,A 46.35 0.93a,A

40 51.05 1.00b,A 52.49 0.82b,A

80 56.71 0.90c,A 53.66 1.08bc,B

120 60.27 0.62d,A 55.18 0.89cd,B

160 60.85 0.72d,A 56.13 1.00d,B

* Values represent means standard deviation calculated from three determi-nations. Means of fermentable sugars contents with different small letter in a col-umn are signicantly different (p < 0.05). Means of fermentable sugars contentswith different capital letter in a row are signicantly different (p < 0.05).

the inadequate level of magnesium due to competition betweenthese two ions.

On the basis of the bioethanol content after fermentation (Figs. 5and 6), the total fermentable sugars content (g/100 g of triticalesdry matter) (Table 3) were calculated as well as bioethanolyield YP/S (g bioethanol/g triticale starch) and percentage of thetheoretical bioethanol yield (Figs. 7 and 8). Bioethanol yields andfermentation efciencies (the total fermentable sugars contentsand percentage of the theoretical bioethanol yield) were deter-mined by distilling triticale mashes after the fermentation. Thehighest bioethanol yields and therefore the contents of the total fer-mentable sugars were achieved in samples in which 120 and160 mg/L of magnesium ions were added (60.27 and 60.85 g/100 gof triticales dry matter). In samples in which 120 and 160 mg/L ofmagnesium ions were added, the maximum percentage of the the-oretical bioethanol yield of 91.32% and 92.19%, respectively, wasachieved. Percentage of the theoretical bioethanol yield should be9095% [43].

Fig. 7. Effect of magnesium ions addition on the percentage of the theoreticalbioethanol yield obtained after the fermentation of triticale mashes. Experimentalconditions for SSF process: 30 C, 72 h. Vertical bars represent the standarddeviation (n = 3) for each data point. Means of the percentage of the theoreticalbioethanol yield with different small letters above bars are signicantly different(p < 0.05). Percentage of the theoretical bioethanol yield was calculated as the ratiobetween actual bioethanol yield and theoretical bioethanol yield, assuming allstarch was converted to glucose and then to bioethanol.

J.D. Pejin et al. / Fuel 1Fig. 8. Effect of calcium ions addition on the percentage of the theoreticalbioethanol yield obtained after the fermentation of triticale mashes. Experimentalconditions for SSF process: 30 C, 72 h. Vertical bars represent the standarddeviation (n = 3) for each data point. Means of the percentage of the theoreticalbioethanol yield with different small letters above bars are signicantly different

(p < 0.05). Percentage of the theoretical bioethanol yield was calculated as the ratiobetween actual bioethanol yield and theoretical bioethanol yield, assuming allstarch was converted to glucose and then to bioethanol.The results show that magnesium ions addition signicantlyaffects bioethanol yield andpercentage of the theoretical bioethanolyield. Percentage of the theoretical bioethanol yield increased by31.22% (p < 0.05) in a sample in which 160 mg/L of magnesium ionswere added compared to the control sample. This increase in bio-ethanol yield is very important in bioethanol technology. Additionof magnesium ions had more signicant effect on bioethanol yieldincrease (for 10%) than the addition of calcium ions. It can be con-cluded that if magnesium and calcium ions content ratio in triticalemashes is higher, better bioethanol yields are obtained which is inagreement with the results reported by Walker et al. [19].

Future work is needed to optimize magnesium and calcium ionsratio to achieve higher bioethanol yields. Also, additional investiga-tions are needed to scale up the system designs to large batch orcontinuous processes, in order to fully realise the potential benetsof magnesium and calcium ions addition in triticale mashes forbioethanol production. Based on the obtained results the time ofthe SSF processing of triticale mashes with the addition of120 mg/L of magnesium ions may be reduced from 96 to 72 hwhich makes the bioethanol production process more efcientand economical.

4. Conclusions

With an increase in magnesium and calcium ions content in trit-icale mashes glucose and maltose content increased during lique-faction. Glucose and maltose content increased by 30.16% and9.58%, respectively, when 160 mg/L of magnesium ionswere added,compared to the control sample. Glucose and maltose contentincreased by 69.31% and 61.66%, respectively, when 160 mg/L ofcalcium ions were added, compared to the control sample. Accord-ing to the obtained results for glucose andmaltose content increaseduring liquefaction, the supplementation of mashes with calciumions had greater inuence on the activity of triticales amylasesthan the supplementation of mashes with magnesium ions whichshows that calcium is essential for triticales amylases activity.

The present investigation shows that magnesium and calciumions addition to triticale mashes improved bioethanol productionduring SSF processing. When 160 mg/L of magnesium ions wereadded bioethanol content increased by 31.22% compared to thecontrol sample while when 160 mg/L of calcium ions were addedbioethanol content increased by 21.04%. High percentage of thetheoretical bioethanol yield (92.19%) was achieved after fermenta-tion when 160 mg/L of magnesium ions were added to triticalemash. The obtained results show that the addition of magnesiumand calcium ions in bioethanol production from triticale increasetriticales amylase activity as well as yeast enzyme activity. All thisshows that when triticale with high amylolytic enzymes activity isused in bioethanol production with the addition of magnesiumions there is no need to use commercial enzymes in starchhydrolysis, which makes the use of triticale as a raw material forbioethanol production more economical.

Acknowledgement

This work was funded by the Ministry of Education, Science andTechnological Development (TR-31017) Republic of Serbia.

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Bioethanol production from triticale by simultaneous saccharification and fermentation with magnesium or calcium ions addition1 Introduction2 Materials and methods2.1 Materials2.2 Yeast strain2.3 Triticale analysis2.4 Liquefaction and simultaneous saccharification and fermentation (SSF) experiments2.5 Calculation of important process parameters2.6 HPLC analysis of glucose and maltose2.7 Statistical analysis

3 Results and discussion3.1 Triticale analysis3.2 Effect of magnesium and calcium ions addition on glucose and maltose content in triticale mashes after liquefaction3.3 Effect of magnesium and calcium ions addition on simultaneous saccharification and fermentation (SSF) of liquefied triticale mash

4 ConclusionsAcknowledgementReferences