Stability of the process of simultaneous saccharification and fermentation of corn flour.

Stability of the process of simultaneous saccharificationand fermentation of corn flour.The effect of structural changes of starch by stillage recycling and scaling up of theprocessDaria Szymanowska-Powałowska a ,⇑,Gra _zynaLewandowicz a ,Piotr Kubiak a ,Wioletta Błaszczak b a Department of Biotechnology and Food Microbiology,Poznan University of Life Sciences,Wojska Polskiego 48,60-627Poznan,PolandbInstitute of Animal Reproduction and Food Research of the Polish Academy of Sciences,Division of Food Science,Tuwima 10,10-747Olsztyn,Polandh i g h l i g h t sStillage recycling does not affect effectiveness of repeated SSF process. Amount of unhydrolyzed residual starch was similar in every SSF cycles. The repeated SSF process is stable in scaling up and in industrial scale.a r t i c l e i n f o Article history:Received 15December 2011Received in revised form 22December 2012Accepted 18November 2013Available online 2December 2013Keywords:EthanolSimultaneous saccharification and fermentationNative corn starcha b s t r a c tIntensive development of the transport sector and a rise in the prices of fossil fuels boost the demand for fuels from alternative sources of energy,including biofuels.New energy-efficient technologies of fuel pro-duction from renewable resources are developed.The aim of the present study was to examine the fac-tors influencing the effectiveness of the process of simultaneous saccharification and fermentation of corn flour with full stillage recycling.The effect of structural changes of starch granules during the long-term repeated SSF process as well as the scale of the process were mercially avail-able STARGEN 001enzymatic preparation and Saccharomyces cerevisiae strain Red Star Ethanol Red were used in the experiment.The results proved that raw material quality is of the utmost importance for the effectiveness of this processes.Bacterial contamination of the raw material caused decreased ethanol productivity despite similar substrate utilization.Process scale turned out to be a second significant fac-tor influencing the SSF outcome.Increase of bioreactor volume resulted in decreased productivity.Repeated stillage recycling and the resulting concentration of broth ingredients has a lesser impact on the process.Ethanol content and the amount of residual starch was independent of the number of oper-ation cycles.Formation of porous granules is predominant as starch undergoes hydrolysis.The affinity of the amylolytic enzyme used towards crystalline and amorphous regions is equal.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionThe economical competitiveness of different technologies for fuel ethanol production depends on the cost of the first stage of this process –obtainment of a solution of fermentable sugars.Three types of raw material requiring different processing can be used for ethanol manufacturing.Sugar crops (sugarcane or sugar beet)require only the extraction process before fermentation.From starch-containing plants (cereal grains,potato tubers,cas-sava roots)the polysaccharide first has to be extracted and then hydrolyzed.Lignocellulosic biomass (wood,straw)has to undergo the most complicated pretreatment prior to fermentation that includes removal of lignin (non-saccharide fraction)followed by hydrolysis of cellulose.The latter process is far more difficult than the saccharification of starch.Therefore,despite the fact that most of agricultural by-products are lignocellulosic materials,produc-tion of second generation bioethanol is currently economically not competitive [1].For this reason,almost all bio-ethanol is pro-duced from grain and sugarcane.Moreover,much effort is made to decrease the cost of the production of this biofuel using starchy raw materials,i.e.to improve the hydrolysis stage.The conventional process of enzyme hydrolysis of starch to pro-duce fermentable sugars involves following steps:gelatinisation,liquefaction with thermostable a -amylase,and saccharification [2].The energy consumption of theses processes usually amounts to about 30–40%of all energy required for ethanol production [3].However,recently a new enzyme mix –STARGEN 001–has0016-2361/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.fuel.2013.11.034Corresponding author.Tel.:+48618466010;fax:+48618466003.E-mail address:darszy@up.poznan.pl (D.Szymanowska-Powałowska).been developed by Genencor International that hydrolyzes granu-lar starch.Employing this enzyme makes it possible to effectively perform the process of simultaneous hydrolysis of native starch and ethanol fermentation[4].During this process sugars liberated by hydrolysis are instantly consumed by yeast.The process of simultaneous saccharification and fermentation(SSF)is energy-and water-saving and results in higher ethanol productivity by avoiding the loss of fermenting sugars,which may occur during heating of fermentation broth(i.e.in the Maillard reaction).There are a few important factors determining economic effec-tiveness of bioethanol production,one of the most important being full utilization of sugars and the related efficient conversion of granular starch into ethanol.The key role in the hydrolysis of gran-ular starch is played by their supramolecular structure,crystallin-ity and the presence of complexing agents[5,6].These factors are determined by starch origin.The comparison of the four cheapest commercial starches in their native form in terms of their suscep-tibility to amylolysis sets them in the following diminishing order: corn P wheat>cassava>potato[7,8].The second important factor is water management.Firstly,the growing global water shortage leads to an increase in its costs.Sec-ondly,distillery stillage is ranked among especially burdensome industrial effluents,particularly in large biorefineries.One of the methods of water cost reduction applied by distilleries is the reuti-lization of stillage in the production process which enables not only to reasonably utilize this by-product,but also to significantly reduce the demand for production water.In order to improve the economic outcome of the SSF process,the zero-discharge fermen-tation system is introduced.This means that the solid-containing whole stillage isfirst separated using a decanter centrifuge.After-wards,the solid phase is dried in a drum dryer to produce DDGS (distillers dry grain solids),a valuable co-product used for animal feed,whereas the liquid phase is evaporated in a double-effect evaporator[9].Another way of improving the zero-discharge fer-mentation system is the application of the repeated SSF process with complete recycling of stillage liquid fraction.This approach assumes liquid phase recirculation into the simultaneous sacchar-ification and fermentation process[9].It should be emphasized that coupling hydrolysis to fermentation in time and space through SSF reduces the costs and duration of the process and eliminates the necessity of using two separate vessels[10].The number of reports on the subject reflects the awareness of the key role played by the efficient hydrolysis of granular starch [11–16].In spite of much effort of different research groups within thefield,a lot of questions remain answered.Still it is not clear which regions of starch granules,crystalline or amorphous,are more susceptible to amylolysis[12,11,17,18].Moreover,most of the reports utilize experimental systems significantly different from those occurring in industrial practice.The aim of the present study was to examine the factors influencing the effectiveness of simultaneous saccharification and fermentation of cornflour with full stillage recycling.The effects of structural changes of starch granules during the long-term repeated SSF process as well as scal-ing up of the process were investigated.2.Materials and methods2.1.MaterialsCommercially available cornflour(BIO CORN,Zie˛bice,Poland) was used as raw material for lab scale and pilot plant fermentation experiments.In the industrial scale corn grain ground using1mm mesh size was used.The corn grain contained69%starch in dry mater and was significantly contaminated with soil and sand. Freeze-dried distiller’s yeast–Red Star Ethanol Red(Saccharomyces cerevisiae)obtained from Lesaffre Company(France)was used for the production of ethanol from corn mashes.A mixture of granu-lar-starch hydrolyzing enzymes,containing a-amylase and gluco-amylase was used(STARGEN001,Genencore International,USA) and fungal acid protease GC106(Genencore International,USA) were used.2.2.Simultaneous saccharification and fermentation processThe SSF experiments were performed in a5L bioreactor BioFlo III(New Brunswick,USA),containing4L of the fermentation med-ium.Cornflour was suspended in unsterilized water to obtain the mash of the concentration of250g LÀ1.The fermentation broth pH was adjusted to5.0by the addition of10%H2SO4.In all cases,the medium was supplemented with acid protease GC106(40l L kgÀ1 cornflour dry matter)and STARGEN001(2.05mL kgÀ1cornflour dry matter).The fermentation was started with the addition yeast (0.5g LÀ1of the fermentation medium).The batch SSF fermenta-tions were performed in anaerobic,non-sterile conditions at 35°C with medium agitation rate of200rpm for72h.After the fer-mentation period was completed,mash containing ethanol was pumped to a continuous distillation column(UOP3CC,Armfield, UK).At the top of this column,operating at78.5°C and reflux ratio of4:1,carbon dioxide is stripped from the ethanol solution and leads to an ethanol concentration of93–95%(volume based)in a side stream.The liquid fraction collected at the bottom of the dis-tillation column,wasfirst cooled down to30°C and then centri-fuged at4000g for20min.Supernatant was used instead of water to prepare the mash using native corn starch in the subse-quent SSF batch.The amount of recirculated stillage was kept con-stant,amounting for75%replacement of fresh water.The procedure was repeated twenty times over a period of60days, using a fresh yeast culture for inoculation during each run.Samples from the4th,the7th and the20th cycles of fermentation were ta-ken for analyses.Studies on the scaling up of fermentation processes comprised fur stages:using laboratory bioreactors of5L,then30L,next 150L,andfinally1500L.The procedure of the simultaneous hydrolysis and fermentation followed the scheme presented above.Industrial scale experiment was made at a agricultural distillery located near Poznan,Poland.The distillery was equipped with a mill coupled to a weighing tank,a mash tub with a stirrer,a jet–cooker,fermentation tanks,a system of pumps,a decantation cen-trifuge,slop tanks and a distillation column.First,the mash tub of 8000L wasfilled with a weighed amount of maize grain taking into consideration moisture content of the raw material.Next,water was added to make up to afinal volume of8000L.In the produced sweet mash of a concentration of250g LÀ1,pH was adjusted to5.0 using10%H2SO4solution.In the next stage4L of STARGEN001, 0.2L acid protease GC106and4kg lyophilised yeasts were added. The entire volume was pumped to a fermentation tank.Fermenta-tion was run for72h,with no adjustment of pH or temperature. Mash was stirred every15min using screw pumps coupled to fer-mentation tanks.Fermented mash was distilled on a column by Chemat,Poland.After distillation the eluate was centrifuged using a WD350decanter(Spomasz Wronki,Poland).The solid fraction (precipitate)was dried and used as animal feed,while the liquid fraction was used as a substitute of process water for the prepara-tion of another batch of mash.All the experiments were performed in triplicate.The block diagram of the experiment was given at Fig.1.2.3.Analytical methodsAnalysis of sugars,ethanol and glycerol concentration were per-formed using on HPLC(Merck Hitachi,Germany)equipped withD.Szymanowska-Powałowska et al./Fuel119(2014)328–334329refractive index detector and an Aminex HPX-87P column(Bio-Rad,USA)thermostated at30°C.5mM sulfuric acid solution was used as the mobile phase at aflow rate of0.6mL minÀ1.The starch content was analyzed according to the enzymatic method developed by Holm et al.[19].The yeast cell populations were determined by a direct microscopic count in a counting chamber after staining with methylene blue.Pure starch was isolated from grain meal using the procedure described by Varatharajan et al.[20].The purified granules were dried and then deposited on a copper disc,coated with gold using a Jeol JEE-400vacuum evaporator,and analyzed with a scanning electron microscope(Jeol JSM5200)under10kV accelerating voltage.The X-ray diffractometry was performed with a TUR62Carl Zeiss X-ray diffractometer under the following conditions:X-ray tube Cu Ka(Nifilter);voltage30kV;current15mA;scanning from Q=2°to18°.To avoid the influence of relative humidity on relative crystallinity,the starch samples were placed in a desiccator and conditioned in the atmosphere of relative humidity of92%for48h.The FTIR measurements were performed in the solid state with an FTIR Bruker IFS113v spectrometer under the following condi-tions:KBr pellet(200mg1.5mgÀ1),resolution2cmÀ1.3.Results and discussionOur previously reported study on the process of simultaneous saccharification and fermentation of raw corn starch using GSH (granular starch hydrolyzing)enzymes proved that although glyc-erol,lactic and acetic acid as well as inorganic ions content slightly increases with the increase in the number of recycling cycles,the yield of ethanol production does not decrease[9].Moreover,the resulting increase in osmotic pressure did not influence yeast con-dition and enzyme activity.A slight increase in the amount of residual starch,which negatively affects the outcome of the fer-mentation,was observed.The application of the zero-discharge system,however,allows the solid-containing whole stillage to serve as animal feed after being separated and dried,thus mitigat-ing the aforementioned influence.When the cost-effectiveness of the SSF is considered,the com-plete hydrolysis of starch to fermentable sugars is a priority.In or-der to determine the effect of stillage liquid fraction recycling on the effectiveness of starch hydrolysis,structural changes of starch granules were assessed after three selected batches.The4th,the 7th and20th cycle of the fermentation process were chosen ran-domly to this purpose.As shown in Table1,no significant changes of the fermentation characteristics were observed during the course of the fermentation between the4th,the7th and20th cycle. Moreover,the results show that the SSF process ran correctly,i.e. starch hydrolysis ran parallel to the fermentation process.Together with the formation of starch hydrolysis products,ethanol concen-tration in the mash was increasing.After72h of the process,eth-anol concentration amounted to about11.5%v/v(90g LÀ1).Thefinal alcohol concentration is somewhat lower than than stated by the producer.According to Genencor International Inc. STARGEN001used on corn mash of the concentration of33%dry mass gives thefinal ethanol concentration of16–18%.This has also not been confirmed by other authors.Moreover,the results of Wang et al.obtained with STARGEN001are similar to ours[21]. These authors compared the process performed according to STAR-GEN001technology with the traditional process of ethanol pro-duction with liquefaction at a temperature of90–120°C, followed by saccharification with the use of two different combina-tions of liquefying and saccharifying enzymes(DG1and DG2; a-amylase and glucoamylase),and completed by fermentation. Using both methods,the authors obtained similarfinal ethanol concentrations of14.1–14.2%(v/v)with significantly different su-gar profiles in the analyzed systems.It should be emphasized that the data presented in Table1proved no accumulation of disaccha-rides,so lack of glucoamylase activity was not the reason for the lowerfinal concentration of ethanol in this experiment in compar-ison with the data of Genencor International Inc.However,there was a part of starch which was resistant to enzymatic hydrolysis.The most important factor determining starch resistance to enzymatic attack is its supermolecular structure[22].Not only the type of crystal structure(A or B)but also the distribution of semi-crystalline and crystalline layers influences the rate of hydro-lysis[17].On the basis of the‘‘blocklet’’concept of the supramolec-ular organization of starch it is postulated that residual,not hydrolyzed material,exhibits a higher degree of crystallinity than native one[17].However,data reported by Zhang et al.[18]points to even digestion of both crystalline and amorphous regions in na-tive cereal starches during hydrolysis by the mixture of a-amylase and amyloglucosidase.Our studies using X-ray diffractometry,IR spectroscopy and scanning electron microscopy did not confirm the thesis that hydrolysis of starch preferentially takes place in amorphous regions.X-ray diffraction patterns of native corn starch as well as starch isolated after4th,the7th and the20th cycle of the fermentation process(data not shown)did not reveal any changes neither in the type of crystal structure nor in the degree of crystal-linity.However,different mechanism of enzymatic hydrolysis cat-alyzed by enzymes of different biological origin could results in different affinity to crystal and amorphous regions of starch[23]. Likewise,IR spectroscopy did not show any changes caused by the simultaneous saccharification and fermentation process,both in the functional groups as well as in thefinger-print range(data not shown).Even the bands1047cmÀ1and1022cmÀ1,considered as sensitive to the amount of crystalline and amorphous starch fractions did not change in the result of the hydrolysis process [24].However,the FTIR technique does not enable differentiation between the A-and B-type of crystallinity and is less a powerful tool in crystal structure research than X-ray diffraction[25,26].As observed in the SEM images of starch samples isolated after the7th cycle of the fermentation(Fig.2)the predominant part con-sisted of granules with the pores.This is a typical observation, reported by different research groups[7,27–29].However,pyra-mid-shaped residuals,also described by Zhang et al.,are present at the very last stage of the hydrolysis[18].The latter as well as undigested starch granules were less prevalent(Fig.2).The above observation suggests that the limits of the SSF were not reached and the degree of hydrolysis can be enhanced.Before commercialization,developed technologies usually un-dergo a scale-up procedure.Our research was was performed in330 D.Szymanowska-Powałowska et al./Fuel119(2014)328–334four stages,in bioreactors of the following volumes,5L,through 30and 150up to 1500L.The most important parameters determining the effectiveness of the technology investigated into were ethanol and starch concentration in the fermentation broth.The data pre-sented in the Table 2proved that the process performed very stable independently of the volume of the bioreactors.Only during the last 24h a slight decrease in process kinetics took place.However,the decrease of final concentration of ethanol with the increase of bioreactor volume,by about 7%,14%and 22%in case of bioreactors of the volume of 30,150and 1500L,respectively,was observed (Table 2).Nevertheless,obtained results are satisfying.They are similar to those obtained by Siquiera et al.[30]in the study of scal-ing-up the process of ethanol fermentation using soya molasses.Higher values of final concentration of ethanol (19%v/v,which cor-responds to 155g L À1),obtained in the SSF process,was reported by Lamsal et al.[4].However,that experiment was performed in significantly different conditions:lower scale of the process (100ml of suspension),higher concentration of corn (30%of d.m),and longer fermentation time (96h).Our previous experience shows that prolonging the fermentation over 72h also results in the increase of ethanol concentration in the fermentation broth,however,it is accompanied by decreasing fermentation rate which makes the process ineffective,therefore,it was not included in this study.In contrast to ethanol concentration,utilization of starch was not dependent on the scale of the process and varied in a very nar-row range from 89%to 90%.It corresponded to the residual starch concentration in the fermentation broth of 21–23g L À1(Table 2).The concentration of low molecular weight sugars was also low and did not exceed 3.5%.The permanent presence of glucose and maltose proved that there were no lack of the carbon source and that the fermentation could be continued.The most important parameter –the ethanol volumetric productivity decreased with the increase of the scale of the process from 1.62g L À1h À1to 1.29g L À1h À1.The plateau of this parameter usually was observed after 48th hour of the fermentation process,in case of the biggest bioreactor even after 24th.Similar values of productivity were re-ported by Devantier et al.[31]for barley mashes,and Mojovic´et al.[32]for 20%corn mashes.Higher values of productivity were ob-tained by Nicolic et al.[33],but using gelatinised corn starch as raw material.Concluding,the simultaneous saccharification and fermentation undergoes the scale-up stably.Noteworthy,despite the lack of high temperature processing,bacterial contamination was very low as suggested by the insignificant acetic and lacticT a b l e 1T i m e c o u r s e o f S S F p r o c e s s e s w i t h s t i l l a g e r e c y c l i n g a f t e r h y d r o l y z e d i n t h e 4t h ,t h e 7t h a n d t h e 20t h c y c l e o f t h e S S F p r o c e s s .P r o c e s s p a r a m e t e rF e r m e n t a t i o n t i m e (h )0244872O p e r a t i o n a l c y c l eO p e r a t i o n a l c y c l eO p e r a t i o n a l c y c l eO p e r a t i o n a l c y c l e4t h7t h 20t h 4t h 7t h 20t h 4t h 7t h20t h4t h7t h20t hE t h a n o l (g L À1)0.00.00.056.2±0.353.1±0.455.2±0.577.3±0.876.2±0.878.1±0.987.4±0.991.1±1.090.2±1.1S t a r c h (g L À1)210.1±2.1210.2±2.2210.2±2.1130.4±1.6157.6±1.7146.3±1.677.1±0.991.4±0.887.5±0.943.2±0.240.1±0.241.3±0.2D i s a c c h a r i d e s [(g /L *)10À1]2.0±0.15.0±0.23.0±0.21.0±0.11.0±0.11.0±0.15.0±0.21.0±0.11.0±0.14.0±0.31.0±0.13.0±0.3G l u c o s e (g L À1)15.1±0.112.9±0.111.3±0.117.8±0.123.6±0.221.4±0.29.7±0.111.4±0.110.8±0.12.1±0.19.2±0.11.9±0.1G l y c e r o l (g L À1)15.4±0.122.0±0.233.0±0.218.0±0.223.6±0.235.2±0.320.1±0.326.5±0.336.7±0.323.0±0.327.4±0.337.3±0.3L a c t i c a c i d [(g L À1)Â10À1]7.0±0.69.0±0.78.0±0.89.0±0.911.0±0.910.0±0.69.0±0.311.0±0.410.0±0.510.0±0.512.0±0.911.0±0.7A c e t i c a c i d [(g L À1)Â10À1]1.0±0.11.0±0.11.0±0.12.0±0.13.0±0.14.0±0.32.0±0.23.0±0.23.0±0.23.0±0.23.0±0.24.0±0.3p H5.05.05.03.73.63.73.73.53.64.03.73.8Y e a s t [(c f u Â108m L À1)Â10À1]9.0±0.89.0±0.79.0±0.813.0±0.914.0±1.114.0±1.219.0±1.717.0±1.417.0±1.318.0±1.518.0±1.218.0±1.4V o l u m e t r i c p r o d u c t i v i t y (g E t O H L À1h À1)–––2.32.22.31.61.61.61.21.21.22.Scanning electron micrographs (SEM)of corn starch in the 7th cycle of process.D.acids concentrations(Table2).However,it was probably due to high quality raw material(cornflour)used in the pilot plant experiment.Significantly different conditions were employed during the last experiment performed in a distillery.Firstly,corn grains were of rather low quality.They were partially underdeveloped and con-taminated with soil.The concentration of starch in grains was of near69%,whereas in the case of lab-scale experiments it was near 86%.Moreover,the moisture content was similar to critical value for microbial growth,thus indicating partial decomposition.How-ever,such quality of the raw material is typical for distillery prac-tise.It results in a relatively low pH value of the fermentation mash (5.1–5.2)at the beginning of the process with no need of its adjust-ment.The pH lowered to3.3–3.6after three days of the fermenta-tion(Table3).While it is typical of ethanol fermentation to observe a pH drop,the increased microbial contamination and the resulting acid formation added caused increased acidification[34].The reports of other authors concerning industrial scale exper-iments pointed out the very low efficiency of the process.For example,Suresh et al.[35],using25%mashes containing damaged wheat or sorgo grains,reached thefinal ethanol concentration of 4.4%and3.5%(v/v)respectively.Higher concentration of ethanol (6.7%w/v)was reported by Montesinos and Navarro[36].However that process was preceded by liquefaction at the temperature of 60°C.Summarizing,the distillery experiment shows the potential of the SSF process,which could be competitive to the traditional tech-nology.Stillage recycling appears to be economically justified as it minimizes water consumption and waste emission,thus contribut-ing greatly to the overall production costs.Keeping appropriate technological standards could make it possible to obtain afinal ethanol concentration of12%(w/v).As the high temperature sub-strate pretreatment is omitted,the process becomes less energy demanding but bacterial contamination monitoring and stricter hygiene regimes become critical[37,38].Contamination problems can be avoided through the use of antibiotics[39,40],it is,how-ever,banned in Europe(in contrast to America)due to the utiliza-tion of stillage for fodder.4.ConclusionsThe described repeated batch SSF process with stillage recycling conducted with cornflour and the use of STARGEN001enzyme preparation runs efficiently in spite of the accumulation of glyc-erol,organic acids and inorganic ions in the fermentation broth. The most important factor influencing effectiveness of this pro-cesses is the quality of the raw material.Bacterial contamination of the raw material caused lower ethanol productivity despite sim-ilar utilization of starch.Instead of ethanol,bacterial metabolites ctic and acetic acids were produced.The scale of the process proved to be another important factor influencing the effectiveness of the simultaneous saccharification and fermentation.The in-crease of bioreactor volume resulted in decrease of productivity accompanied by similar level of starch utilization.The effect of the number of recirculation cycles is a less important factor.Etha-nol content as well as the amount of residual starch is independent of the number of operation cycles.Hydrolysis of starch undergoes with predominant formation of porous granules and an additional small amount of undigested granules and pyramid-shaped residu-als.Crystalline and amorphous regions are evenly digested. References[1]Mussato SI,Dragone G,Guimarães PMR,Silva JPA,Carneiro LM,Roberto IC,et al.Technological trends,global market,and challenges of bio-ethanol production.Biotechnol Adv2010;2010(28):817–30.[2]Wang P,Singh V,Xu L,Rausch KD,Tumbleson ME,Johnston parison ofenzymatic(E-Mill)and conventional dry-grind corn processes using a granular starch hydrolyzing enzyme.Cereal Chem2005;82:734–8.[3]Lim LH,Macdonald DG,Hill GA.Hydrolysis of starch particles usingimmobilized barley[alpha]-amylase.Biochem Eng J2003;13:53–62.[4]Lamsal BP,Wang H,Jahnson LA.Effect of corn preparation methods on dry-gring ethanol production by granular starch hydrolysis and portioning of spent beer solids.Bioresource Technol2011;102:6680–6.[5]Oates CG.Towards and understanding of starch granule structure andhydrolysis.Trends Food Sci Technol1997;8:375–82.[6]Dona AC,Pages G,Gilbert RG,Kuchel PW.Digestion of starch:In vivo andin vitro kinetic models used to characterize oligosaccharides or glucose release.Carbohydr Polymer2010;80:599–617.[7]Sarikaya E,Higasa T,Motoyasu A,Mikami parison of degradationabilities of a-and b-amylases on raw starch granules.Process Biochem 2000;35:711–5.[8]Piyachomkwan K,Wunsuksri R,Wanlapatit S,Chatakanonda P,Sriroth K.Application of granular starch hydrolyzing enzymes for ethanol production.In: Yuryev V,Bertoft E,Tomasik P,editors.Starch.progress in basic and applied sciences.Kraków:Polish Society of Food Technologist,Małopolska Branch;2007.p.183–90.[9]Białas W,Szymanowska D,Grajek W.Fuel ethanol production from granularcorn starch using Saccharomyces cerevisiae in a long term repeated SSF process with full stillage recycling.Biores Technol2010;101:3126–31.[10]Kobayashi F,Sawada T,Nakamura Y,Ohnaga M,Godliving M,Ushiyama T.Saccharification and alcohol fermentation in starch solution of steam-exploded potato.Biotechnol Appl Biochem1998;69:177–89.[11]Uthumporn U,Zaidul ISmM,Karim AA.Hydrolysis of granular starch at sub-gelatinisation temperature using a micture of amylolytic enzymes.Food Bioproducts Proces2010;88:47–54.[12]Chen G,Zhang B.Hydrolysis of granular corn starch with controlled pore size.JCereal Sci2012;56:316–20.[13]Adams JMM,Teunissen PJM,Robson G,Dunn-Coleman N,Donnison IS.Scanning wlectron microscopy and fermentation studies on selected known maize starch mutants using STARGEN TM enzyme blends.Bioenergy Res 2012;5:330–40.[14]Naguleswaran S,Li J,Vasanthan T,Bressler D,Hoover R.Amylolysis of largeand small granules of native triticale,wheat and corn starches using a mixture of a-amylase and glucoamylase.Carbohydr Polymers2012;88:864–74. [15]Sarian FD,van der Kaaij RM,Krajl S,Wijbenga DJ,Binnema DJ,van der MaarelMJEC,et al.Enzymatic degradation of granular potato starch by Microbacterium aurum strain B8.A.Appl Microbiol Biotechnol 2012;93:645–54.[16]Adejumo AL,Aderibige AF,Layokun SK.Cassava starch:production,physicochemical properties and hydrolysation–a review.Adv Food Energy Security2011;2:8–17.[17]Gallant DJ,Bouchet B,Baldwin PM.Microscopy of starch:evidence of a newlevel of granule organization.Carbohydr Polymer1997;32:177–91.[18]Zhang G,Ao Z,Hamaker BR.Slow digestion property of native cereal starches.Biomacromolecules2006;7:3252–8.[19]Holm J,Björck I,Drews A,Asp NG.A rapid method for the analysis of starch.Starch/Stärke1986;38:224–6.[20]Varatharajan V,Hoover R,Li J,Vasanthan T,Nantanga KKM,Seetharaman K.Impact of structural changes due to heat-moisture treatment at different temperatures on the susceptibility of normal and waxy potato starches towards hydrolysis by porcine pancreatic alpha amylase.Food Res Inter 2011;44(9):2594–606.[21]Wang P,Singh V,Xue H,Johnston DB,Rausch KD,Tumbleson parisonof raw starch hydrolyzing enzyme with conventional liquefaction and saccharification enzymes in dry-grind corn processing.Cereal Chem 2006;84:10–4.[22]Planchot V,Colonna P.Buleon.A Enzymatic hydrolysis of a-glucan crystallites.Carbohydr Res1997;298:319–26.[23]Robertson GH,Wong DWS,Lee CC,Wagschal K,Smith MR,Orts WJ.Native orraw starch digestion:a key step in energy efficient biorefining of grain.J Agr Food Chem2006;54:353–65.[24]van Soest JJG,Tournois H,de Wit D,Vliegenthart JFG.Short-range structure in(partially)crystalline potato starch determined with attenuated total reflectance Fourier-transform IR spectroscopy.Carbohydr Res1995;279: 201–14.[25]Sevenou O,Hill SE,Farhat JR,Mitchell anisation of the external region ofthe starch granule as determined by infrared spectroscopy.Int J Biol Macromol 2002;31:79–85.[26]Lewandowicz G,Soral-S´mietana M.Starch modification by iterated syneresis.Carbohydr Polymer2004;56(4):403–13.[27]Planchot V,Colonna P,Gallant DJ,Bouchet B.Extensive degradation of nativestarch granules by alpha-amylase from Aspergillus fumigates.J Cereal Sci 1995;21:163–71.[28]Huber KC,Miller JN.Visualisation of channels and cavities of corn andsorghum starch granules.Cereal Chem1997;74(5):537–41.[29]Huber KC,Miller JN.Channels of maize and sorghum starch granules.Carbohydr Polymer2000;41:269–76.[30]Siqueira PF,Karp SG,Carvalho JC,Sturm W,Rodríguez-León JA,Tholozan JL,et al.Production of bio-ethanol from soybean molasses by Saccharomyces cerevisiae at laboratory,pilot and industrial scales.Bioresource Technol 2008;99:8156–63.D.Szymanowska-Powałowska et al./Fuel119(2014)328–334333。