Comparison of Cassava Starch with Corn As a Feedstock for Bioethanol Production

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Article Comparison of Cassava Starch with Corn as a Feedstock for Bioethanol Production

Sarocha Pradyawong 1, Ankita Juneja 2, Muhammad Bilal Sadiq 1 , Athapol Noomhorm 1 and Vijay Singh 2,*

1 Department of Food Engineering and Bioprocess Technology, Asian Institute of Technology, Pathum Thani 12120, Thailand; [email protected] (S.P.); [email protected] (M.B.S.); [email protected] (A.N.) 2 Department of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-217-333-9510

Received: 9 October 2018; Accepted: 10 December 2018; Published: 13 December 2018

Abstract: Cassava is a high potential feedstock for bioethanol production in Asian countries, primarily due to high yield of carbohydrate per unit land, and its ability to grow on marginal lands with minimal agrochemical requirements. The objective of this study was to compare the bioethanol production from cassava starch with corn starch using a conventional and a raw/granular starch hydrolyzing process (GSH). The fermentation performance of cassava starch was compared with three corn starch types with different amylose: Amylopectin ratios. The ﬁnal ethanol concentration with cassava starch was similar to that of two corn starch types, dent corn and waxy corn for both processes. For the cassava starch, the ethanol concentration achieved with GSH process was 2.8% higher than that in the conventional process. Cassava starch yielded the highest fermentation rates of the four starches investigated, during the conventional process. Ethanol production and fermentation proﬁles comparable with corn, a widely used feedstock, makes cassava starch an attractive substrate for bioethanol production.

Keywords: bioethanol; corn; cassava starch; granular starch hydrolysis; fermentation

1. Introduction Increased energy consumption and raising environmental concerns have led the world to look for alternative energy sources, especially in the transportation sector. Bioethanol is considered as one of the most promising renewable transportation fuels, and is already blended into gasoline in several countries. The global production of bioethanol in 2017 was estimated as 27.05 billion gallons, with the US contributing about 58% (15.8 billion gallons) [1]. Since the economics of bioethanol are highly dependent on the feedstock price, there has been an ongoing effort to search for low cost and easily available raw materials [2]. Most of the bioethanol in the United States is produced from corn. However, some countries, particularly in continental Asia, the use of another starch crop, cassava, for energy purposes is encouraged. Cassava or tapioca (Manihot esculenta), is a plant native to South America, with high nutritional value due to its starchy tuberous roots. Global production of cassava is about 281 million tons a year, with Asia contributing about one third of this production [3]. More than half of this global supply is contributed by Africa, where cassava is a primarily used as a food source. Brazil is third largest producer of cassava in the world [3]. One of the major advantages of cassava is its drought-tolerance and capability of growing on marginal soils and degraded lands, and still containing third highest yield of carbohydrate per hectare after sugarcane and sugar beet [4]. The low agro-chemical requirements reduce the energy input for growth of cassava biomass making the use of this crop energy-efﬁcient. Another species of the same genus (Manihot glaziovii) is also considered

Energies 2018, 11, 3476; doi:10.3390/en11123476 www.mdpi.com/journal/energies Energies 2018, 11, 3476 2 of 11 Energies 2018, 11, x FOR PEER REVIEW 2 of 11 consideredas a bioethanol as a bioethanol source for itssource ability forto its grow ability on to non-arable grow on non-arable lands [5]. lands Due to [5]. high Due drought to high anddrought heat andtolerance, heat tolerance, high yield high of starch, yield andof starch, its capability and its of capability growing onof growing poor soils on without poor soils high without maintenance, high maintenance,cassava is thus cassava gaining is attentionthus gaining as a attention bioethanol as source a bioethanol all over source the world. all over the world. The conventional method employed employed for for ethanol ethanol pr productionoduction from from cassava cassava roots roots usually usually requires requires gelatinization ofof starchstarch followedfollowed by by liquefaction liquefaction and and sacchariﬁcation saccharification and and consequently consequently fermenting fermenting the thesugars sugars formed formed with with yeast yeast or bacteria or bacteria [6,7]. [6,7]. Apart Apart from from the conventional the conventional conversion conversion method, method, raw starch raw starchhydrolysis hydrolysis has also has been also used been for used production for production of ethanol of fromethanol cassava from starch cassava using starch mixed using culture mixed of culturemicroorganisms of microorganisms [8,9]. Both [8,9]. processes Both processes (conventional (conventional and raw and starch raw hydrolysis) starch hydrolysis) are used are to convertused to convertstarch to starch ethanol to forethanol both cornfor both and corn cassava. and Sincecassav corna. Since is a well-establishedcorn is a well-established feedstock forfeedstock ethanol for in ethanolthe United in the States United [10 ],States it is imperative[10], it is imperative to know theto know practical the practical yields of yields cassava of cassava in comparison in comparison to corn tostarch corn to starch understand to understand the industrial the industrial scale feasibility scale feasibility of cassava of as cassava a feedstock as a forfeedstock bioethanol for production.bioethanol production.The cost of cassavaThe cost ethanol of cassava is theoretically ethanol is estimatedtheoretically as $0.237/Lestimated [ 11as]. $0.237/L The theoretical [11]. The ethanol theoretical yield ethanol(kg/ha/year) yield (kg/ha/y from cassavaear) from has beencassava estimated has been as threeestimated times as that three of corntimes [12 that]. However, of corn [12]. there However, is a lack thereof studies is a lack comparing of studies the ethanolcomparing production the ethanol experimentally production fromexperimentally these two ethanol from these producing two ethanol starch producingcrops. Bioethanol starch crops. production Bioethanol from production cassava starch from has cassava been attempted starch has withbeenvarious attempted mixed with strains various of mixedbacteria strains and yeast of bacteria [13,14], and however, yeast there[13,14], are however, limited studies there are employing limited studies process employing similar to cornprocess dry similargrind, which to corn is dry widely grind, used which process is widely for bioethanol used process production for bioethanol in US. Although production both cassavain US. Although and corn bothshare cassava the traits and of corn starchy share crops, the theretraits areof starchy some structural crops, there differences are some in structural both the starches,differences which in both are theshown starches, in Table which1, major are oneshown being in Table the ratio 1, major of amylose one being to amylopectin. the ratio of Amylose amylose isto a amylopectin. linear chain Amylosepolysaccharide is a linear of glucose chain polysaccharide molecules connected of glucose by α molecules-1,4 glycosidic connected bonds, by whereas α-1,4 glycosidic amylopectin bonds, is a whereaswater soluble, amylopectin highly branched is a water polymer, soluble, where highly glucose branched moelecules polymer, are connectedwhere glucose with αmoelecules-1,4 glycosidic are connectedbonds and with the branching α-1,4 glycosidic takes place bonds with andα -1,6the branching glycosidic bonds,takes place occurring with α every-1,6 glycosidic 24 to 630 glucosebonds, occurringunits (Figure every1). 24 The to extraction630 glucose of units corn (Figure starch is 1). complex The extraction due to theof corn need starch for steeping is complex to loosendue to thethe needprotein for matrices steeping surrounding to loosen the the protein starch, whereasmatrices cassavasurrounding starch the is easier starch, to extractwhereas due cassava to low starch quantity is easierof proteins to extract and fatsdue into thelow tuber quantity [15]. of proteins and fats in the tuber [15].

FigureFigure 1. StructuresStructures of of amyl amyloseose and amylopectin. The objective of this study was to compare the fermentation proﬁle and ethanol production of The objective of this study was to compare the fermentation profile and ethanol production of cassava starch with corn starch, using two approaches: the conventional and the granular starch cassava starch with corn starch, using two approaches: the conventional and the granular starch hydrolysis (GSH) process (Figure2). For a broader comparison, three commercially used corn types hydrolysis (GSH) process (Figure 2). For a broader comparison, three commercially used corn types with different amylose content (dent corn, waxy corn and high amylose corn) were used. Regular

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dent corn starch contains about 20 to 30% amylose and 70 to 80% amylopectin, whereas waxy corn has <5% and high amylose corn has >35% amylose content [16].

Table 1. Comparison of normal corn starch and cassava starch [17].

Corn Starch Cassava Starch Energies 2018, 11, 3476 3 of 11 Content of starch (% db) 64 to 78 30 to 35 Average granular diameter (μm) 10 15 with different amyloseAmylose content content (dent (% corn, of total waxy starch) corn and high20 to amylose30 corn) 17 were used. Regular dent corn starch contains aboutDegree 20 of to polymerization 30% amylose (DPn) and 70 to 80% 3000 amylopectin, whereas 800 waxy corn has <5% Gelatinization temperature range (°C) 75 to 80 65 to 70 and high amylose corn has >35% amylose content [16].

Figure 2. Schematics of the laboratory-scale conventional dry grind and GSH (granular starch hydrolysis)Figure 2. Schematics process for of ethanol the laboratory-scale production. conventional dry grind and GSH (granular starch hydrolysis) process for ethanol production. Table 1. Comparison of normal corn starch and cassava starch [17]. 2. Materials and Methods Corn Starch Cassava Starch 2.1. Materials Content of starch (% db) 64 to 78 30 to 35 Average granular diameter (µm) 10 15 Dent cornAmylose starch content(Melojel, (% of~30% total starch)amylose), waxy 20 corn to 30 starch (Amioca, 17 ~1% amylose), high amylose corn Degreestarch of(Hylon polymerization V, ~65% (DPn) amylose) and cassava 3000 starch (Novation 800 3600, Ingredion, Gelatinization temperature range (◦C) 75 to 80 65 to 70 Indianapolis, IN, USA) were obtained from Ingredion Incorporated (Westchester, IL, USA). Enzymes, including conventional α-amylase (Spezyme® CL, with reported activity of 15,225 AAU/g; AAU: 2.alpha Materials amylase and units), Methods conventional glucoamylase (Distillase® SSF, with reported activity of 380 GAU/g; GAU: glucoamylase unit), and GSH enzyme (StargenTM 002, with an activity of 570 GAU/g; 2.1. Materials GAU: glycoamylase unit), were provided by DuPont Industrial Biosciences (Palo Alto, CA, USA). ConventionalDent corn active starch dry (Melojel, yeast was ~30% obtained amylose), from waxy the cornFermentis-Lesaffre starch (Amioca, Yeast ~1% Corporation amylose), high(Milwaukee, amylose WI, corn USA). starch All (Hylon starch V, samples ~65% amylose) and enzymes and cassava were starchstored (Novationat 4 °C until 3600, use. Ingredion, All the Indianapolis,chemicals used IN, were USA) of were analytical obtained grade from and Ingredion were purchased Incorporated from (Westchester, Sigma Aldrich IL, USA).(St. Louis, Enzymes, MO, ® includingUSA). conventional α-amylase (Spezyme CL, with reported activity of 15,225 AAU/g; AAU: alpha amylase units), conventional glucoamylase (Distillase® SSF, with reported activity of 380 GAU/g; GAU:2.2. Conventional glucoamylase Process unit), and GSH enzyme (StargenTM 002, with an activity of 570 GAU/g; GAU: glycoamylase unit), were provided by DuPont Industrial Biosciences (Palo Alto, CA, USA). Conventional active dry yeast was obtained from the Fermentis-Lesaffre Yeast Corporation (Milwaukee, WI, USA). All starch samples and enzymes were stored at 4 ◦C until use. All the chemicals used were of analytical grade and were purchased from Sigma Aldrich (St. Louis, MO, USA). Energies 2018, 11, 3476 4 of 11

2.2. Conventional Process For liquefaction and simultaneous saccharification and fermentation (SSF), a conventional dry grind process as described previously in literature [18,19] was used, with slight modifications (Figure2). In summary, all four starch (30 g) samples were mixed with distilled water to make a slurry with 25% solids on dry basis. All the experiments were performed in triplicate. The pH of the slurry was adjusted to 5.7 using 2 N sulfuric acid, and 10 µL of Spezyme® CL was added for liquefaction. The liquefaction was performed in shaker water bath at 85 ◦C for 2 h with continuous agitation at 100 rpm. The liquefied slurry was then transferred in 1-L flask and cooled down to 32 ◦C and pH was adjusted to 4.2 using 2 N sulfuric acid. Distillase® SSF (22.56 µL), urea (0.4 g/100 g of starch), yeast malt broth (4 g/100 g of starch), and 5 mL yeast culture (1.8 × 108 cells/mL) were then added for SSF. Yeast culture was prepared by dispersing 11 g of active dry yeast and 1 g yeast malt broth in 89 mL of distilled water and agitated at 50 rpm and 30 ◦C for 20 min. The broth was fermented at 32 ◦C for 72 h with continuous agitation at 100 rpm in shaking water bath. Fermentation was monitored by drawing 1-mL sample from the fermentation slurry at 0, 2, 4, 6, 8, 20, 24, 48, and 72 h of SSF.

2.3. Granular Starch Hydrolysis (GSH) GSH of starch slurry (25% dry solids content) was carried out by the method previously described in thecliterature [18,19], with slight modiﬁcations (Figure2). To summarize, the pH of slurry was adjusted to 4.2 by using 2 N sulfuric acid and 37.7 µL StargenTM 002 was added to slurry and temperature was maintained at 48 ◦C for 2 h with continuous agitation at 100 rpm. The slurry was then cooled down to 32 ◦C followed by pH adjustment to 4.2 using 2 N sulfuric acid. StargenTM 002 (37.7 µL), urea (0.4 g/100 g of starch), yeast malt broth (4 g/100 g of starch), and 5 mL yeast (1.8 × 108 cells/mL) were then added to carry out fermentation. Yeast culture was prepared similarly as described above. The broth was fermented at 32 ◦C for 72 h with continuous agitation at 100 rpm in shaking water bath. Fermentation was monitored by drawing 1-mL sample from the fermentation slurry at 0, 2, 4, 6, 8, 20, 24, 48, and 72 h of SSF.

2.4. HPLC analysis Samples collected during both conventional and GSH process, were centrifuged (model 5415 D, Eppendorf, Westbury, NY, USA) at 11,000 rpm for 3 min. The supernatant was then filtered through 0.2-µm syringe filter into 1-mL HPLC vials. The vials were immediately stored at −20 ◦C until analysis. The filtrate was analyzed using HPLC with an ion-exclusion column (Aminex HPX-87H, Bio-Rad, Hercules, CA) and refractive index detector (model 2414, Waters Corporation, Milford, MA, USA). The mobile phase was 0.005 M sulfuric acid at 50 ◦C at a flow rate of 0.6 mL/min. Each sample was analyzed to determine the concentrations of ethanol, glucose, maltose, maltotriose, and glycerol. The HPLC was calibrated with standards and each sample was injected twice for analysis.

2.5. Fermentation Rates and Ethanol Yields Ethanol concentration was measured and was plotted against time. Fermentation rates were calculated during the initial linear phase of fermentation (up to 8 h) by calculating the slope of the plot. Starch-to-ethanol conversion efﬁciencies were calculated as the ratio of actual ethanol yields with the theoretical ethanol yield (Equation (1)):

EEtOH ηEtOH = ·100 (1) ETh_EtOH where ETh_EtOH is theoretical ethanol yield, L/kg dry starch; EEtOH is the actual ethanol yield, L/kg dry starch. Energies 2018, 11, 3476 5 of 11

Energies 2018, 11, x FOR PEER REVIEW 5 of 11 Theoretical yields were estimated based on the starch content, assuming complete starch conversionFinal and ethanol 100% fermentationconcentration and efﬁciency. fermentation Actual rate ethanol for all yieldsfour starches were calculatedwas analyzed based by analysis on methods describedof variance elsewhere (1-way [20 ANOVA)]. and Fisher’s least significant difference (LSD) tests (SAS version 9.4, Cary, NC, USA). The level selected to show the statistical significance in all cases was 5% (p < 0.05). 2.6. StatisticalAll the experiments Analysis were performed in triplicates.

Final3. Results ethanol concentration and fermentation rate for all four starches was analyzed by analysis of variance (1-way ANOVA) and Fisher’s least significant difference (LSD) tests (SAS version 9.4, Cary, NC, USA).3.1. Conventional The level Dry selected Grind toProcess show the statistical significance in all cases was 5% (p < 0.05). All the experimentsThe werefermentation performed profiles in triplicates. for all starches during the conventional dry grind process are shown in Figure 3. The final ethanol concentration for dent corn starch (133.2 g/L) was the highest among 3. Resultsthe four starches analyzed. However, statistically there was no significant difference in the final ethanol concentrations of the three starches—dent corn, waxy corn and cassava starch—which 3.1. Conventionalindicated theDry suitability Grind Processof cassava starch to be used as a comparable starch crop for bioethanol Theproduction fermentation in Asian profiles countries. for The all starchesethanol yield during with the high conventional amylose corn dry starch grind was process lower arethan shown the in Figureother3. The three final starches. ethanol This concentration was expected for due dent to cornthe high starch amylose:amylopectin (133.2 g/L) was the ratio highest in the among high the amylose corn starch, which has previously been reported to decrease the ethanol yield [21]. Since four starches analyzed. However, statistically there was no significant difference in the final ethanol amylose is a linear chain polymer, it aligns itself with strong hydrogen bonds, making it difficult for concentrationsthe enzymes of to the act, three resulting starches—dent in reduced corn,hydrolys waxyis of corn amylose and cassava[21]. High starch—which temperatures provided indicated the suitabilityduring ofliquefaction cassava starch of starches to be in used conventional as a comparable dry grind starchprocess crop assist for in bioethanolbreaking down production these in Asianhydrogen countries. bonds The and ethanol make the yield amylose with available high amylose to enzymes. corn The starch final was ethanol lower produced than theby cassava other three starches.(124.4 This g/L) starch was expected was higher due than to that the repo highrted amylose:amylopectin previously in literature ratio (~71 g/L) in the [22]. high amylose corn starch, whichAbout has 5 previouslyg/L and 1 beeng/L, glucose reported was to decreaseleft unco thenverted ethanol in case yield of [ 21dent]. Since corn amyloseand cassava, is a linear chainrespectively; polymer, it whereas aligns itself complete with conversion strong hydrogen was observed bonds, for makingwaxy corn it and difficult high amylose for the enzymescorn starch to act, resulting(Figure in reduced3). However, hydrolysis glucose of released amylose in [21the]. first High 8 temperaturesh is highest for provided cassava duringstarch (119.5 liquefaction g/L), of indicating better breakdown of starch in to polysaccharides during liquefaction. In contrast, high starches in conventional dry grind process assist in breaking down these hydrogen bonds and make amylose corn starch released least glucose during fermentation, which can be attributed to the the amylose available to enzymes. The final ethanol produced by cassava (124.4 g/L) starch was higher inefficient hydrolysis due to high amylose content. Similar trend of low glucose formation with high thanamylose that reported content previously was observed in literaturein an earlier (~71 study g/L) [21]. [22 ].

FigureFigure 3. Comparison 3. Comparison of ethanol of ethanol and and glucose glucose proﬁles profiles of dent of dent corn corn (DC), (DC), waxy waxy corn corn (WC), (WC), high high amylose cornamylose (HAC), corn and (HAC), cassava and starch cassava (CS) starch during (CS) conventional during conventional fermentation fermentation (SSF (SSF at 30 at◦ 30C for°C for 72 72 h with glucoamylaseh with glucoamylase and yeast). and yeast).

AboutThe 5 g/Linitial and plot 1 g/L,of ethanol glucose vs time was (up left to unconverted 8 h) provides in a case linear of trend dent corn(Figure and 4a), cassava, slope of respectively; which was used to calculate the fermentation rate. All starches for conventional process showed good linear whereas complete conversion was observed for waxy corn and high amylose corn starch (Figure3). However, glucose released in the first 8 h is highest for cassava starch (119.5 g/L), indicating better breakdown of starch in to polysaccharides during liquefaction. In contrast, high amylose corn starch Energies 2018, 11, 3476 6 of 11 released least glucose during fermentation, which can be attributed to the inefficient hydrolysis due to high amylose content. Similar trend of low glucose formation with high amylose content was observed in an earlier study [21]. The initial plot of ethanol vs time (up to 8 h) provides a linear trend (Figure4a), slope of which wasEnergies used 2018 to calculate, 11, x FOR PEER the fermentationREVIEW rate. All starches for conventional process showed good6 of 11 linear fit for first 8 h of fermentation. For GSH process, high amylose corn was the only starch that did not fit thefit for linear first 8 plot h of(R fermentation.2 = 0.47), where For GSH other process, fits could high amylose be investigated. corn was the The only fermentation starch that did rates not of all fit the linear plot (R2 = 0.47), where other fits could be investigated. The fermentation rates of all four four starches are shown in Table2. Highest fermentation rate was observed for cassava starch (0.965), starches are shown in Table 2. Highest fermentation rate was observed for cassava starch (0.965), followed by high amylose corn starch (0.931). During the initial hours of fermentation, the fermentation followed by high amylose corn starch (0.931). During the initial hours of fermentation, the ratefermentation of cassava israte observed of cassava to is be obse statisticallyrved to be higher statistically than higher that of than dent that corn. of dent Despite corn. low Despite final low ethanol concentration,final ethanol high concentration, fermentation high rate fermentation for high amylose rate for cornhigh starchamylose can corn be duestarch to can the betterbe due functioning to the of thebetter yeast functioning in presence of the of yeast low glucosein presence concentrations. of low glucose concentrations.

FigureFigure 4. Fermentation4. Fermentation rates rates calculated calculated as as slopes slopesof of ethanolethanol concentrationconcentration plo plottedtted with with time time during during the ﬁrstthe 8 hfirst of 8 fermentation h of fermentation during during (a) conventional(a) conventional and and (b ()b GSH) GSH (granular (granular starch hydrolysis) hydrolysis) process. process.

Table 2. Fermentation rates for ethanol production during the conventional and GSH process.

Starch Type Conventional GSH

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Table 2. Fermentation rates for ethanol production during the conventional and GSH process.

Starch Type Conventional GSH Energies 2018, 11, x FOR PEER REVIEW 7 of 11 Dent corn 0.7415 b 0.9355 a Waxy cornDent corn 0.6490.7415c b 0.9355 a0.876 b High amyloseWaxy corn corn 0.9310.649a c 0.876 b 0.102 c CassavaHigh amylose corn 0.9650.931a a 0.102 c 0.928 a Cassava 0.965 a 0.928 a Similar letters represent statistically similar values at p < 0.05. Similar letters represent statistically similar values at p < 0.05.

The fermentationThe fermentation efficiencies efficiencies of starchof starch to ethanolto ethanol conversion conversion in in conventional conventional process process rangedranged from 62.8%from to 81.9%, 62.8% highestto 81.9%, being highest for being dent for corn dent starch corn and starch lowest and forlowest high for amylose high amylose corn starch corn starch (Figure 5). The efficiency(Figure 5).of The dent efficiency corn starch of dent was corn higher starch than was previously higher than determined previously determined efficiency ofefficiency dent corn of grits (62.4%)dent [23 corn]. The grits fermentation (62.4%) [23]. efficiencyThe fermentation of cassava efficiency starch of by cassav conventionala starch by method conventional was calculatedmethod as 74.5%,was which calculated was higher as 74.5%, that which earlier was reported higher results that earlier of 68% reported efficiency results with ofSaccharomyces 68% efficiency cerevisiae with [8], but lowerSaccharomyces than another cerevisiae reported [8], but valuelower ofthan 82.88% another [11 reported]. In case value of GSH of 82.88% process, [11]. the In efficiency case of GSH of dent cornprocess, starch was the 74.1%,efficiency comparable of dent corn to starch previously was 74.1%, reported comparable in literature to previously (76.7%) reported [23]. The in literature fermentation (76.7%) [23]. The fermentation efficiency of cassava starch was calculated as 77%, which was 3.7% efficiency of cassava starch was calculated as 77%, which was 3.7% higher than that with conventional higher than that with conventional process. Previously reported literature has shown fermentation process.efficiency Previously of cassava reported ranging literature from 82–99%, has shownbased on fermentation the variety of cassava efficiency and of microorganism cassava ranging used from 82–99%,[9,11]. based on the variety of cassava and microorganism used [9,11].

DC WC HAC CS 100

90 81.86 76.97 80 75.56 74.49 74.16 76.81

70 62.84 60 50 40 30 Fermentation Efficiency (%) Efficiency Fermentation 20 11.76 10 0 Conventional GSH

FigureFigure 5. Fermentation 5. Fermentation efﬁciencies efficiencies of of four four starchesstarches (dent corn corn (DC), (DC), waxy waxy corn corn (WC), (WC), high high amylase amylase corncorn (HAC) (HAC) and and cassava cassava starch starch (CS) (CS) with with conventional conventional and and GSH GSH method. method.

ConcentrationsConcentrations of of other other sugars sugars and and sugarsugar alcoholalcohol produced produced in inthe the conventional conventional process process are are presentedpresented in Table in Table3. Final 3. Final glycerol glycerol production production was was highesthighest among among all all sugars sugars and and sugar sugar alcohols alcohols for for all four starch types. Glycerol formation by Saccharomyces cerevisiae under anaerobic conditions is all four starch types. Glycerol formation by Saccharomyces cerevisiae under anaerobic conditions is caused by the need for reoxidation of NADH [24]. Highest glycerol concentration was observed in causeddent by corn the (8.02 need g/L), for reoxidationwhich was statistically of NADH simila [24].r Highest to that produced glycerol with concentration cassava, whereas was observed waxy in dentcorn corn starch (8.02 produced g/L), which the lowest was statisticallyglycerol (5.30 similar g/L). Maltotriose to that produced concentration with at cassava,the beginning whereas of SSF waxy cornwas starch in range produced of 22.08–33.94 the lowest g/L, similar glycerol to maltose (5.30 g/L). concentration, Maltotriose except concentration for high amylose at the corn beginning starch, of SSF waswhere in no range maltose of 22.08–33.94 was detected g/L, throughout similar fermenta to maltosetion. concentration, At the end of the except fermentation, for high no amylose maltose corn starch,was where detected no for maltose waxy corn was starch, detected and both throughout dent corn fermentation. and cassava starch At had the less end than of the1 g/L fermentation, maltose no maltoseconcentration. was detected Similarly, for maltotriose waxy corn concentration starch, and bothin all dent four corn starch and varieties cassava atstarch the end had of lessthe than 1 g/Lfermentation maltose concentration. was below 5 Similarly,g/L. Concentration maltotriose of DP concentration4+ at the beginning in all fourof SSF starch was varietiesobserved atto thebe end of theabout fermentation 250 g/L for was all starches below 5 except g/L. Concentrationhigh amylose corn of DP4+starch, at where the beginning the starting of concentration SSF was observed was to 184 g/L. However, the concentration of DP4+ at the end of fermentation was less than 1 g/L for all be about 250 g/L for all starches except high amylose corn starch, where the starting concentration starches. was 184 g/L. However, the concentration of DP4+ at the end of fermentation was less than 1 g/L for all starches. Table 3. Concentrations of other sugars in conventional process.

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Table 3. Concentrations of other sugars in conventional process.

Time (hours) Maltotriose (g/L) Maltose (g/L) Glycerol (g/L) Energies 2018, 11DCS, x FOR WCSPEER REVIEW HACS CS DCS WCS HASC CS DCS WCS HACS8 of 11 CS

0Time (hours) 30.2 22.08 Maltotriose 33.94 (g/L) 29.11 32.36 28.67 Maltose (g/L) 0.00 31.89 0.00 Glycerol 0.00 (g/L) 0.00 0.00 2 36.81DCS 16.10 WCS 1.66HACS 10.65 CS 72.96 DCS 82.55WCS HASC 0.00 CS 94.03 DCS 0.90 WCS 0.00HACS 0.00 CS 0.81 40 21.8 30.2 2.96 22.08 0.5733.94 2.9829.11 90.9332.36 85.2228.67 0.00 0.00 31.89 93.50 0.00 1.960.00 0.00 0.00 1.980.00 1.75 62 11.12 36.81 0.64 16.10 0.361.66 1.1310.65 80.5972.96 82.7882.55 0.00 0.00 94.03 92.94 0.90 2.960.00 0.00 0.00 3.100.81 2.86 84 9.305 21.8 0.36 2.96 0.270.57 0.562.98 96.2790.93 76.3285.22 0.00 0.00 93.50 86.50 1.96 3.930.00 0.001.98 3.51.75 4.00 206 0.45 11.12 0.95 0.64 0.810.36 0.801.13 47.6580.59 30.9582.78 0.00 0.00 92.94 43.20 2.96 5.750.00 4.553.10 4.362.86 5.85 248 0.68 9.305 1.42 0.36 0.000.27 1.090.56 35.6496.27 19.5976.32 0.00 0.00 86.50 30.23 3.93 6.080.00 4.62 3.5 4.564.00 5.99 4820 1.63 0.45 1.58 0.95 0.470.81 2.150.80 1.7747.65 30.95 0.95 0.00 0.00 43.20 1.20 5.75 7.704.55 5.204.36 5.615.85 6.92 7224 0.51 0.68 0.35 1.42 0.310.00 0.401.09 0.8535.64 19.59 0.00 0.00 0.00 30.23 0.42 6.08 8.024.62 5.304.56 5.845.99 7.12 DCS: Dent48 Corn 1.63 Starch; 1.58 WCS: Waxy0.47 Corn2.15 Starch; 1.77 HACS: 0.95 High Amylose0.00 1.20 Corn Starch;7.70 CS:5.20 Cassava5.61 Starch.6.92 72 0.51 0.35 0.31 0.40 0.85 0.00 0.00 0.42 8.02 5.30 5.84 7.12 DCS: Dent Corn Starch; WCS: Waxy Corn Starch; HACS: High Amylose Corn Starch; CS: Cassava 3.2. GSH ProcessStarch.

The3.2. GSH ethanol Process and glucose profiles for all four starches during GSH process are shown in Figure6. Similar toThe the ethanol conventional and glucose process, profiles the for final all four ethanol starches concentrations during GSH process for cassava are shown starch in (127.9Figure g/L) with GSH6. Similar process to the was conventional similar to process, dent corn the (122.6 final ethanol g/L) and concentrations waxy corn for (126.9 cassava g/L). starch High (127.9 amylose g/L) corn starchwith was GSH found process to yield was lowestsimilar ethanolto dent corn concentrations (122.6 g/L) and (22.6 waxy g/L), corn due (126.9 to theg/L). inefficiency High amylose of corn enzymes to cleavestarch the was bonds found of to long yield polymericlowest ethanol chain concentratio of amylose.ns (22.6 Similar g/L), finaldue to ethanol the inefficiency concentration of enzymes for high amyloseto cleave corn starchthe bonds (17.3 of g/L)long polymeric were observed chain byof am Sharmaylose. Similar et.al. [21 final]. The ethanol ethanol concentration concentration for high for high amyloseamylose corn corn during starch GSH (17.3 process g/L) were was foundobserved to beby aboutSharma 79% et.al. lower [21]. than The thatethanol for conventionalconcentration for process. This canhigh be amylose attributed corn during to the factGSH that process the was hydrolysis found to in be this about case 79% was lower inefficient. than thatUnlike for conventional conventional process,process. GSH This process can isbe carriedattributed out to at the low fact temperatures, that the hydrolysis which isin notthis sufficient case was toinefficient. break the Unlike hydrogen conventional process, GSH process is carried out at low temperatures, which is not sufficient to break bonds. Since the enzymes need a certain length of amylose/amylopectin polymer to attach and cleave the hydrogen bonds. Since the enzymes need a certain length of amylose/amylopectin polymer to the bonds,attach theand amylosecleave the in bonds, this case the amylose was inaccessible in this case to was the inaccessible enzymes. to the enzymes.

FigureFigure 6. Comparison 6. Comparison of ethanol of ethanol and glucoseand glucose profiles profiles of dent of dent corn corn (DC), (DC), waxy waxy corn corn (WC), (WC), high high amylose cornamylose (HAC), corn and cassava(HAC), and starch cassava (CS) starch during (CS) granular during granular starch hydrolysisstarch hydrol (GSH)ysis (GSH) fermentation fermentation (SSF at (SSF at 30 °C for 72 h with GSH enzyme and yeast). 30 ◦C for 72 h with GSH enzyme and yeast). Statistically, the final ethanol concentrations for dent corn, waxy corn and cassava starch were Statistically, the final ethanol concentrations for dent corn, waxy corn and cassava starch were similar. For cassava starch, the final ethanol concentrations with GSH process was 2.8% higher than similar. For cassava starch, the final ethanol concentrations with GSH process was 2.8% higher than that from conventional process, which suggests that with this starch source, the GSH process can be that fromused conventionalto obtain higher process, ethanol whichyields. suggestsGSH process that occurs with thisat lower starch temperatures source, the than GSH conventional process can be usedprocess, to obtain and higher eliminates ethanol the yields.liquefaction GSH step, process thereby occurs reducing at lower the energy temperatures and cost thanfor bioethanol conventional

Energies 2018, 11, 3476 9 of 11

Energies 2018, 11, x FOR PEER REVIEW 9 of 11 process, and eliminates the liquefaction step, thereby reducing the energy and cost for bioethanol production,production, which can be anan addedadded beneﬁtbenefit forfor usingusing GSHGSH processprocess forfor bioethanolbioethanol productionproduction from cassava. In In the current study, thethe ﬁnalfinal ethanolethanol concentrationconcentration withwith cassavacassava starchstarch (127.9(127.9 g/L)g/L) waswas higher than previouslypreviously reportedreported inin literatureliterature [[8,19].8,19]. InIn thethe casecase ofof thethe GSHGSH process,process, thethe initialinitial 88 hh ofof fermentationfermentation had a linearlinear trendtrend ofof ethanolethanol productionproduction with time for for all all starches, starches, except except high high amylose amylose corn corn starch starch (Fig (Figureure 4b).4b). This This could could be beattributed attributed to the to thefact factthat thatno glucose no glucose was detected was detected after 4 after h in 4this h incase, this an case,d subsequently and subsequently,, no ethanol no ethanolchange was change observed. was observed. High amylose High amylose corn starch, corn therefore starch, therefore had the had lowest the lowestfermentation fermentation rate (Table rate (Table2). Similar2). Similar to conventional to conventional process, process, cassava cassava had the had highest the highest initial initial fermentation fermentation rates, rates, which which were werestatistically statistically similar similar to that to of that dent of dentcorn. corn. InIn contrast contrast to to conventional conventional process, process, higher higher sugars sugars such such as maltose, as maltose, maltotriose maltotriose and DP4+ and DP4+were werenegligible negligible in case in of case GSH of process, GSH process, due to duesimultaneous to simultaneous consumption consumption of these ofsugars. these Glycerol sugars. Glycerolproduction, production, however, however, was observed was observed in GSH in process GSH process (Figure (Figure 7). Concentration7). Concentration of glycerol of glycerol at the at end the endof the of thefermentation fermentation was was highest highest and and statistically statistically similar similar for for waxy waxy corn corn and and cassava. cassava. High High amylose corn starchstarch produced produced lowest lowest glycerol, glycerol, primarily primarily because because of incomplete of incomplete hydrolysis hydrolysis and reduced and amountsreduced ofamounts sugars of released sugars forreleased yeast consumption.for yeast consumption. With inadequate With inadequate yeast growth yeast due growth to lack due of to substrate, lack of glycerolsubstrate, production glycerol prod is reduceduction is [24 reduced]. [24].

9 Glycerol 8 7 6 5 4 3

Concentration (g/L) Concentration 2 1 0 DC WC HAC CS Starch variety

Figure 7.7. Final concentration of glycerol with four starchstarch varieties (dent corn (DC), waxy corn (WC), high amylose corncorn (HAC),(HAC), andand cassavacassava starchstarch (CS))(CS)) forfor GSHGSH (granular(granular starchstarch hydrolysis)hydrolysis) process.process.

4. Conclusions This studystudy investigatedinvestigated the bioethanol production potential of cassava in comparisoncomparison with thethe most widelywidely usedused bioethanolbioethanol substrate,substrate, corn.corn. Both Both conventional and and GSH process were used to evaluate the fermentation profileprofile of cassavacassava starchstarch alongalong withwith threethree corncorn varietiesvarieties withwith varyingvarying amylose: amylopectin amylopectin ratio. ratio. The The final final ethanol ethanol concentration concentration with with cassava cassava starch starch was wassimilar similar to two to twocorn cornstarch starch varieties, varieties, dent dent corn corn and and waxy waxy corn corn starch, starch, for for both both conventional conventional and and GSH GSH process. Ethanol concentrationconcentration with with cassava cassava starch starch for GSH for processGSH process was 2.8% was higher 2.8% than higher that for than conventional that for process.conventional For both process. conventional For both and conventional GSH process, and the GSH fermentation process, the rate fermentation for the initial rate 8 h offor conventional the initial 8 processh of conventional fermentation process was higherfermentation for cassava was higher starch thanfor cassava any of starch the corn than starch any varieties.of the corn Overall, starch thevarieties. fermentation Overall, profile the fermentati of cassavaon starch, profile in of terms cassava of ethanol starch, production in terms of and ethanol formation production of glycerol, and wasformation similar of to glycerol, that of dent was corn similar and to waxy that corn of de starch,nt corn which and suggestswaxy corn the starch, potential which of cassava suggests starch the usepotential for commercial of cassava production starch use for of bioethanol.commercial production of bioethanol.

Energies 2018, 11, 3476 10 of 11

Author Contributions: S.P. carried out all the experiments, M.B.S. helped design the experiments, A.J. analyzed the results and wrote the manuscript, A.N. and V.S. supervised the study, VS. edited the manuscript. All authors read and approved the final manuscript. Funding: This research was funded by a grant from National Institute of Food and Agriculture (NIFA) as a part of the NIFA (Hatch) Project No. ILLU-741-325—“Processing Corn and Cellulosic Biomass for Food, Fuel and Industrial Products”. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Renewable Fuel Association. Ethanol Industry Outlook. 2017. Available online: http://www.ethanolrfa. org/resources/industry/statistics/#1454098996479-8715d404-e546 (accessed on 16 June 2018). 2. Olguin-Maciel, E.; Larqué-Saavedra, A.; Pérez-Brito, D.; Barahona-Pérez, L.F.; Alzate-Gaviria, L.; Toledano-Thompson, T.; Lappe-Oliveras, P.E.; Huchin-Poot, E.G.; Tapia-Tussell, R. Brosimum Alicastrum as a Novel Starch Source for Bioethanol Production. Energies 2017, 10, 1574. [CrossRef] 3. Woiciechowski, A.L.; Medeiros, A.B.P.; Rodrigues, C.; de Souza Vandenberghe, L.P.; de Andrade Tanobe, V.O.; Dall’Agnol, A.; Gazzoni, D.L.; Soccol, C.R. Feedstocks for Biofuels. In Green Fuels Technology; Soccol, C.R., Brar, S.K., Faulds, C., Ramos, L.P., Eds.; Springer International Publishing: AG, Switzerland, 2016; pp. 15–39. 4. Okudoh, V.; Trois, C.; Workneh, T.; Schmidt, S. The potential of cassava biomass and applicable technologies for sustainable biogas production in South Africa: A review. Renew. Sustain. Energy Rev. 2014, 39, 1035–1052. [CrossRef] 5. Sebayang, A.H.; Hassan, M.H.; Ong, H.C.; Dharma, S.; Silitonga, A.S.; Kusumo, F.; Mahlia, T.M.I.; Bahar, A.H. Optimization of Reducing Sugar Production from Manihot glaziovii Starch Using Response Surface Methodology. Energies 2017, 10, 35. [CrossRef] 6. Moshi, A.P.; Hosea, K.M.; Elisante, E.; Mamo, G.; Mattiasson, B. High temperature simultaneous saccharification and fermentation of starch from inedible wild cassava (Manihot glaziovii) to bioethanol using Caloramator boliviensis. Bioresource Technol. 2015, 180, 128–136. [CrossRef][PubMed] 7. Pervez, S.; Aman, A.; Iqbal, S.; Siddiqui, N.N.; Qader, S.A.U. Saccharification and liquefaction of cassava starch: An alternative source for the production of bioethanol using amylolytic enzymes by double fermentation process. BMC Biotechnol. 2014, 14, 49. [CrossRef][PubMed] 8. Reddy, O.V.S.; Basappa, S. Direct fermentation of cassava starch to ethanol by mixed cultures of Endomycopsis fibuligera and Zymomonas mobilis: Synergism and limitations. Biotechnol. Lett. 1996, 18, 1315–1318. [CrossRef] 9. Ueda, S.; Zenin, C.T.; Monteiro, D.A.; Park, Y.K. Production of ethanol from raw cassava starch by a nonconventional fermentation method. Biotechnol. Bioeng. 1981, 23, 291–299. [CrossRef] 10. Kurambhatti, C.V.; Kumar, D.; Rausch, K.D.; Tumbleson, M.E.; Singh, V. Ethanol production from corn fiber separated after liquefaction in the dry grind process. Energies 2018, 11, 2921. [CrossRef] 11. Sriroth, K.; Wanlapatit, S.; Piyachomkwan, K. Cassava bioethanol. In Bioethanol; Lima, M.A.P., Ed.; InTechOpen Publishing: London, UK, 2012; pp. 3–32. 12. Wang, W. Cassava production for industrial utilization in China–present and future perspective. In Proceedings of the Cassava research and development in Asia: Exploring New Opportunities for an Ancient Crop: Seventh Regional Cassava Workshop, Bangkok, Thailand, 28 October–1 November 2002; pp. 33–38. 13. Khamkeaw, A.; Phisalaphong, M. Hydrolysis of cassava starch by co-immobilized multi-microorganisms of Loog-Pang (Thai rice cake starter) for ethanol fermentation. Food Sci. Biotechnol. 2016, 25, 502–516. [CrossRef] [PubMed] 14. Yuangsaard, N.; Yongmanitchai, W.; Yamada, M.; Limtong, S. Selection and characterization of a newly isolated thermotolerant Pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate. Antonie Van Leeuwenhoek 2013, 103, 577–588. [CrossRef][PubMed] 15. Eliasson, A.C. Starch in Food: Structure, Function and Applications; Woodhead Publishing Limited: Oxford, UK, 2004. 16. Wu, X.; Zhao, R.; Wang, D.; Bean, S.; Seib, P.; Tuinstra, M.; Campbell, M.; O’brien, A. Effects of amylose, corn protein, and corn fiber contents on production of ethanol from starch-rich media. Cereal Chem. 2006, 83, 569–575. [CrossRef] Energies 2018, 11, 3476 11 of 11

17. Breuninger, W.F.; Piyachomkwan, K.; Sriroth, K. Tapioca/cassava starch: Production and use. In Starch, 3rd ed.; Elsevier: Amsterdam, The Netherlands, 2009; pp. 541–568. 18. Sharma, V.; Rausch, K.D.; Graeber, J.V.; Schmidt, S.J.; Buriak, P.; Tumbleson, M.; Singh, V. Effect of resistant starch on hydrolysis and fermentation of corn starch for ethanol. Appl. Biochem. Biotechnol. 2010, 160, 800–811. [CrossRef][PubMed] 19. Wang, P.; Singh, V.; Xue, H.; Johnston, D.B.; Rausch, K.D.; Tumbleson, M.E. Comparison of raw starch hydrolyzing enzyme with conventional liquefaction and sacchariﬁcation enzymes in dry-grind corn processing. Cereal Chem. 2007, 84, 10–14. [CrossRef] 20. Kumar, D.; Moy, K.; Singh, V. Ethanol yield calculation method–“an unaccounted factor” responsible for yield variations. In Proceedings of the 2017 ASABE Annual International Meeting, Spokane, WA, USA, 16–19 July 2017; American Society of Agricultural and Biological Engineers: St. Joseph, MI, USA; p. 1701287. 21. Sharma, V.; Rausch, K.D.; Tumbleson, M.E.; Singh, V. Comparison between granular starch hydrolyzing enzyme and conventional enzymes for ethanol production from maize starch with different amylose: Amylopectin ratios. Starch-Stärke 2007, 59, 549–556. [CrossRef] 22. Sriroth, K.; Piyachomkwan, K.; Wanlapatit, S.; Nivitchanyong, S. The promise of a technology revolution in cassava bioethanol: From Thai practice to the world practice. Fuel 2010, 89, 1333–1338. [CrossRef] 23. Juneja, A.; Kumar, D.; Singh, V. Germ soak water as nutrient source to improve fermentation of corn grits from modiﬁed corn dry grind process. Bioresour. Bioprocess. 2017, 4, 38. [CrossRef][PubMed] 24. Nordström, K. Yeast growth and glycerol formation. Acta Chem. Scand. 1966, 20, 1016–1025. [CrossRef] [PubMed]

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