jo u r n al homep age: www.elsev ier .com/ locate / indcrop
ilot scale simultaneous saccharification and fermentation at veryigh gravity of cassava flour for ethanol production
hinh-Nghia Nguyen, Thanh-Mai Le, Son Chu-Ky ∗
epartment of Food Technology, School of Biotechnology and Food Technology, Hanoi University of Science and Technology, 1 Dai Co Viet, Hai Ba Trung,anoi 10000, Viet Nam
r t i c l e i n f o
rticle history:eceived 30 August 2013eceived in revised form 5 February 2014ccepted 8 February 2014vailable online 28 March 2014
a b s t r a c t
We developed a simultaneous saccharification and fermentation (SSF) process of cassava flour at veryhigh gravity (VHG). Cassava flour (CF) was dissolved in water to reach 315.4 g/l dry matter, and then themixture was liquefied at 80 ◦C for 90 min by using alpha-amylase (3532 AAU/kg CF) and beta-glucanase(2812 U/kg CF). SSF of liquefied mash of cassava was performed at 30 ◦C with the simultaneous addition oftwo glucoamylases (Distillase ASP at 540 GAU/kg CF and Amigase Mega L at 0.035% w/w), active dry yeast
7
eywords:imultaneous saccharification andermentation (SSF)ery high gravity (VHG)thanol
(1.5 × 10 cells/l), urea (12 mM) and KH2PO4 (4 mM). Under these conditions, the SSF process finishedafter 72 h. The ethanol content achieved 17.2% v/v corresponding to 86.1% of the theoretical ethanol yieldat lab scale and decreased to 16.5% v/v corresponding to 83.6% of the theoretical ethanol yield at pilotscale. Therefore, the SSF of cassava flour under VHG condition could have a great potential for the ethanolindustry in Vietnam and South East Asia.
assava flour
. Introduction
According to the increasing price of oil, bio-ethanol is knowns an ideal candidate to replace the role of fossil fuel. Thus, theesearch on this renewable source becomes growingly importantor humans, especially in terms of improving the productivity, thefficiency and decreasing production cost. In Vietnam and in Southast Asia, cassava is considered an attractive raw material for bio-thanol production thanks to the following advantages: (i) the easef plantation in various soil types and climate conditions; (ii) aery low input and investment for planting; (iii) “all year round”vailability of feedstock in the form of fresh roots and dry chips;iv) a high starch-containing raw materials and a lower proportionf fibers (Sriroth et al., 2007). Indeed, the Vietnamese Ministry ofndustry and Trade declared that bio-fuel production will achieve.8 million tons in 2025, which accounts for 5% of country’s demandMinistry of Industry and Trade, 2007b). Moreover, the governmentlso adapted the policy to improve the beverage ethanol industryn Vietnam. By the development strategy of beverage ethanol pro-uction in Vietnam (Ministry of Industry and Trade, 2007a), ethanol
ndustry will produce 188 million liters ethanol for food industryn 2025. Overall, the beverage and bio-ethanol industry has a greatotential in Vietnam in the future.
Besides the conventional process of ethanol production, simul-taneous saccharification and fermentation (SSF) process has beenwidely used in the world, but only recently introduced to Vietnamin order to augment ethanol yield and shorten time production.Indeed, after liquefaction by alpha-amylase, glucoamylase is addedto the slurry, concomitantly with yeasts, and the SSF is conductedin a single reactor. The presence of yeast along with enzymes mini-mizes the sugar accumulation in the bioreactor. Moreover, sincethe sugar produced during starch or cellulosic breakdown slowsdown alpha-amylase action, higher yields and concentrations ofethanol are possible using SSF (Das Neves, 2006; Klasson et al.,2013; Molaverdi et al., 2013; Scordia et al., 2013; Wang et al., 2013;Yingling et al., 2011a,b). The SSF process has been successfully car-ried out on different substrates such as flax shive (Klasson et al.,2013), sweet sorghum stalk (Molaverdi et al., 2013), giant reed(Scordia et al., 2013), sweet sorghum bagasse (Wang et al., 2013),potato tubers (Srichuwong et al., 2009) and cassava (Chu-Ky et al.,2009; Yingling et al., 2011b). Therefore, it is of interest to improvethe efficiency of the SSF process in the ethanol industry in Vietnam.
Very high gravity (VHG) technology has been introduced toincrease the volumetric productivity and the cost effectivenessof the SSF process. In VHG technology, mash preparation con-tains at minimum of 270 g/l dry matter (Bayrock and Ingledew,
2001). This technology has a great deal of advantages in ethanolproduction: (i) increasing plant capacity and reduction in capitalcosts; (ii) increasing plant efficiency; (iii) reducing risk of con-taminating bacteria (Thomas et al., 1996; Yingling et al., 2011a,b).
3 Distillase ASP Glucoamylase 4.0–4.5 58–65 580 GAU/gc
4 Amigase Mega L Glucoamylase 4.0–4.5 55–60 –
a AAU: Alpha Amylase Unit defined by Dupont (One AAU unit of bacterial alpha-amylase activity is the amount of enzyme required to hydrolyze 10 mg of starch per minuteunder specified conditions).
b U: Unit defined by Dupont (one unit of beta-glucanase activity is defined as the quantity of enzyme which produces reducing sugars equivalent to 1 �mol of dextrosep
he amf
Niwcfeacsdayyemc
ttttettt
puliccd
2
2
RDa
2
Vccw
b
er minute from barley beta-glucan under standard assay conditions).c GAU: GlucoAmylase Unit defined by Dupont (One Glucoamylase Unit (GAU) is t
rom soluble starch substrate under the conditions of the assay).
evertheless, VHG technology causes also some inconvenience,ncluding the high viscosity of starch paste after liquefaction,
hich leads to the resistance to solid–liquid separation, diffi-ulties in handling process, incomplete hydrolysis of starch toermentable sugars and lower fermentation efficiency (Ingledewt al., 1999; Srikanta et al., 1992). Therefore, the success of itspplication depends on the preparation of mash with low vis-osity. For instance, in order to reduce starch paste’s viscosity,weet potato was pretreated in a VHG process by using cell-wallegrading enzymes such as cellulases, pectinase, hemi-cellulasesnd viscosity reduction enzyme (xylanase). As a result, the ethanolield was achieved approximately 90% of the theoretical ethanolield (Srichuwong et al., 2009; Zhang et al., 2010, 2011). Thomast al. (1993) reported that in VHG (dissolved solids 300 g/l) of wheatash fermentation at 20 ◦C for 200 h, maximal final ethanol con-
entration of 23.8% v/v was obtained.In another approach to VHG technology with cassava, optimiza-
ion has been applied to study the effects of some key factorshat influence ethanol production such as gravity, particle size, ini-ial pH, liquefaction and fermentation temperature, liquefactionime and enzyme concentration. Under optimized conditions, highthanol concentration (greater than 15%) and high starch utiliza-ion ratio (c.a. 90%) were obtained (Yingling et al., 2011b). However,he investigation on VHG technology with cassava at a larger scalehan that of laboratory has still been limited.
In this work, our approach is to develop cost-effective ethanolrocesses which are based on: (i) decreasing energy consumed bytilizing enzymes which are capable of hydrolyzing raw starch at
ower temperatures; (ii) saving equipment investment and increas-ng ethanol yield by using SSF process of cassava flour under VHGondition. This work aimed to develop SSF processes under VHGondition of cassava flour at lab and pilot scales for ethanol pro-uction.
. Materials and methods
.1. Microorganism
Commercial active dry yeast Saccharomyces cerevisiae (Ethanoled), kindly provided by Fermentis (France), was used in this study.ry yeast was hydrated in tap water at 38 ◦C for 20 min prior toddition to the liquefied mash of cassava flour.
.2. Materials
Cassava flour was obtained in Tuyen Quang province (Northietnam). After thoroughly dried, cassava chips were ground intoassava flour to the size minor than 0.3 mm, and stored at dry and
ool place in the lab. Starch content of the cassava flour used in thisork was 77 ± 1% and its humidity was 11 ± 1%.
Different kinds of commercial enzyme products kindly providedy Dupont (previously known as Genencor—A Danisco Division)
ount of enzyme that liberates 1 g of reducing sugars calculated as glucose per hour
were used in this work including Spezyme Alpha (containingalpha-amylase from Bacillus licheniformis), Optimash TBG (con-taining beta-glucanase from Talaromyces emersonii) and DistillaseASP (containing glucoamylase from Bacillus licheniformis and Tri-choderma reesei). Amigase Mega L (containing glucoamylase fromAspergillus niger) was provided by DSM – Food Specialties – Bever-age Ingredients. Properties of these enzyme products are presentedin Table 1.
2.3. Simultaneous saccharification and fermentation (SSF) at labscale
Three SSF processes at VHG were developed in this work (Fig. 1).Cassava flour (CF) was mixed with tap water in 2-l fermentor toachieve a concentration of 315.4 g/l dry solid in a final volume of1 l. For all three investigated processes, the liquefaction step wasconducted at 80 ◦C and stirred at 200 rpm for 90 min at pH 5.5. Afterliquefaction, the mash was cooled to room temperature (30 ◦C)before subsequent SSF. The SSF of liquefied cassava mash was per-formed at 30 ◦C in a 2-l fermentor, with the simultaneous additionof glucoamylase, active dry yeast (Ethanol Red at 1.5 × 107 cells/ml),urea (12 mM) and KH2PO4 (4 mM). During the first 8 h of SSF, thefermentation broth was agitated every hour for 5 min at 120 rpm toensure homogenization. After this period, the SSF was conductedunder static condition and finished after 72 h. In our work, threeSSF processes were performed and differentiated as follows:
- SSF1 process: alpha-amylase (Spezyme Alpha) at the dosage of3,532 AAU/kg CF was added to the cassava slurry under VHGcondition, and glucoamylase (Distillase ASP) at the dosage of540 GAU/kg CF was added to conduct SSF.
- SSF2 process was similar to SSF1 process with only one modi-fication as follows: additional beta-glucanase (Optimash TBG) atthe dosage of 2,812 U/kg CF was added to the cassava slurry underVHG condition during liquefaction to reduce viscosity of liquefiedmash of cassava.
- For SSF3 process, both alpha-amylase (Spezyme Alpha) at3,532 AAU/kg CF and beta-glucanase (Optimash TBG) at2,812 U/kg CF were added for liquefaction. For SSF, besidesusing glucoamylase (Distillase ASP) at 540 GAU/kg CF, additionalglucoamylase (Amigase Mega L) at 0.035% w/w was added toimprove the efficacy of hydrolyzing residual starch in the slurry.
2.4. Simultaneous saccharification and fermentation (SSF) atpilot scale
The SSF under VHG condition was upgraded to the pilot scalebased on the results obtained with SSF3 process which was con-
ducted at the lab scale as described in Section 2.3. The pilot scaleexperiment was carried out in a total volume of 100 l using a dou-ble jacket reactor (200 l) for liquefaction and a fermentor (200 l) forSSF, respectively. As the same for SSF3 process, both alpha-amylase
162 C.-N. Nguyen et al. / Industrial Crops and Products 56 (2014) 160–165
Cass ava flo ur (CF)
Mixture of suspension (315.4 g/L)
Liquefac tion at 800C for 90 min
Simultane ous Saccharification and Fermentation (SSF)
at 300C for 72 h
Hydrated in water at 380C
for 20 min
Disti llation
Ethanol
By-product
Spezyme Alpha (3,532 AAU/kg
CF)
Optimash TBG (2,812 U/kg Ethanol Red
(1.5x107 cells/ml)
Distillase ASP (540 GAU/kg
CF)
Amigase Mega L (0.035 % w/w)
Urea (12mM)
KH2PO4(4 mM)
Tap water
SSF1
SSF2
SSF3
F n: SSFA + Ami
(TcLetS1c
2
tsb1mbgHsHwt
ig. 1. Three investigated SSF processes at VHG of cassava flour for ethanol productioSP + Optimash TBG; SSF3 process: Spezyme Alpha + Distillase ASP + Optimash TBG
Spezyme Alpha) at 3,532 AAU/kg CF and beta-glucanse (OptimashBG) at 2,812 U/kg CF were added for liquefaction, For SSF, two glu-oamylases in Distillase ASP at 540 GAU/kg CF and in Amigase Mega
at 0.035% w/w were added to the liquefied mash to improve thefficacy of hydrolyzing residual starch. The SSF under VHG condi-ion at pilot scale was performed at 30 ◦C. During the first 8 h ofSF, the fermentation broth was agitated every hour for 5 min at20 rpm to ensure homogenization. After this period, the SSF wasonducted under static condition and finished after 72 h.
.5. Analytical procedures
To measure reducing sugar, fermentation beer was filtrated,hen reducing sugar was determined by using the DNS (3,5-dinitroalicylic acid) method (Miller, 1959). Residual sugar was measuredy the same method after acid hydrolysis (HCl 2% for 120 min at00 ◦C) of the fermentation beer. Ethanol was distilled from fer-entation beer, and then ethanol concentration was determined
y an ethanol ebulliometer (Dujardin-Salleron, France). Maltose,lucose, acetic acid and lactic acid were determined by usingigh Performance Liquid Chromatography (HPLC) (Agilent 1200
eries, Agilent Technologies, Germany) equipped with an AminexPX 87H column (Bio-Rad, Hercules, USA) at a pressure of 52 barith H2SO4 10 mM as eluent according to the provider’s instruc-
ion. All reagents used for HPLC analysis were of analytical grade.
1 process: Spezyme Alpha + Distillase ASP; SSF2 process: Spezyme Alpha + Distillasegase Mega L.
Concentrations were calculated by means of standard curvesrelated to individual concentration to peak area. The viscosity ofstarch slurry after liquefaction was measured by using ElcometerRV1 Rotational Viscometers (Elcometer, UK) with rotation speedsat 100 rpm and 50 rpm according to the provider’s instruction.Dextrose Equivalent (DE) values were estimated as DE = [reducingsugars] × 100/[total dry matter] (Shariffa et al., 2009).
2.6. Statistical analysis
The mean values and standard deviation were calculated fromthree independent experiments. The significance of the differencebetween the mean values was determined using the analysis ofvariance (ANOVA). The confidence interval for a difference in themeans was set at 95% (P ≤ 0.05) for all comparisons.
3. Results and discussion
3.1. Liquefaction
Liquefaction step aimed to convert the starch into maltodextrins
at high temperature and to reduce the viscosity of starch slurryby using thermo-stable alpha-amylases. In VHG process, starchslurry viscosity during liquefaction plays an important role, whichcan decrease enzyme efficacy in starch hydrolysis, thus reducing
C.-N. Nguyen et al. / Industrial Crops and Products 56 (2014) 160–165 163
Table 2Viscosity of liquefied mash of cassava flour after liquefaction in SSF1 and SSF2processes.
Mash viscosity (cp) Rotation speed (rpm)
100 rpm 50 rpm
SSF1 process (with addition of Spezyme Alpha) 340 400
tpmep2(lttahocs(
(graygvwro(sipttei
Fg
Table 3Evolutions of the concentration of maltose, glucose, lactic acid and ethanol duringSSF3 process.
SSF2 process (with addition of both SpezymeAlpha and Optimash TBG)
270 290
he ethanol yield. Therefore, decreasing starch-paste viscosity isrerequisite for conducting ethanol production at VHG. Differentethods have been previously used to resolve this problem. Indeed,
nzymes decreasing viscosity such as cellulase, hemi-cellulase,ectinase (Srichuwong et al., 2009) or xylanase (Zhang et al., 2010,011) were added into the mash. In another work, Yingling et al.2011b) used gelatinization step with different enzyme doses, fol-owed by autoclaving at 121 ◦C for 15 min to completely breakdownhe starch and to avoid contamination. In the conventional method,he liquefaction step is normally carried out at the boiling temper-ture (roughly 100 ◦C), which demands a great deal of energy foreating and maintaining the mash at the boiling temperature. Inur processes when the liquefaction step was performed at signifi-antly lower temperature (only 80 ◦C), the energy was accordinglyaved that demonstrated one of the advantages of the SSF processIngledew, 2009; Thomas et al., 1996).
In SSF1 process, an alpha-amylase, a liquefying enzymeSpezyme Alpha), was used in liquefaction step whereas a beta-lucanase (Optimash TBG) was added in SSF2 process for viscosityeduction. The efficiency of this beta-glucanase was measured by
reduction in starch slurry viscosity and improvement of ethanolield. The combination of two enzymes alpha-amylase and beta-lucanase in SSF2 process significantly decreased starch slurryiscosity compared to SSF1 process where only one alpha-amylaseas added (270 cp compared to 340 cp at rotation speed of 100 rpm,
espectively) (Table 2). However, DE (Dextrose Equivalent) valuesf these processes after liquefaction were not significantly different12.2 and 11.9, respectively). Moreover, in SSF2 process, residualugar was lower and ethanol concentration was higher than thosen SSF1 process after 72 h fermentation (52.9 g/l and 14.8% v/v com-ared to 74.6 g/l and 13.6% v/v, respectively) (Fig. 2). According tohese results, it is likely that the use of beta-glucanase could lead
o a positive effect (viscosity reduction) on this technology. How-ver, in SSF2 process, an ethanol concentration of 14.8% v/v wasnsufficiently high, which would require further improvement.
0
50
100
150
200
250
300
350
6
8
10
12
14
16
18
0 24 48 72
Suga
r co
ncen
trat
ion
(g/l)
Eth
anol
con
cent
rati
on (%
v/v
)
Time (hours)
SSF1 et hano l concentration (% v/v ) SSF2 ethano l conce ntr ation (% v/v )
SSF3 et hano l concentration (% v/v ) SSF1 residu al sugar (g/l )
SSF2 residu al sugar (g/ l) SSF3 residu al sugar (g/l )
ig. 2. Evolutions of residual sugar and ethanol concentration of the three investi-ated SSF processes.
In a previous work, Srichuwong et al. (2009) studied the VHGprocess of sweet potato at 28% dry matter. In order to decrease mashviscosity, cell-wall degrading enzymes (cellulase, hemi-cellulaseand pectinase) have been used to decrease mash viscosity in a pre-treatment step at 50 ◦C for 50 min. As a result, the mash viscositywas reduced from 300 cp to approximately 50 cp. In another work,Zhang et al. (2010) added xylanase to the liquefied sweet potatomash to reduce mash viscosity. After the treatment at 30 ◦C for90 min, the viscosity of mash decreased from 9,863.2 cp to 498.1 cp.
3.2. Simultaneous saccharification and fermentation (SSF)
In order to improve the ethanol yield in this study, anotherglucoamylase (Amigase Mega L) was added to the mash to con-duct SSF. In general, traditional brewing methods permit only 75to 80% hydrolysis of starch present in the grain. According to theproducer, this glucoamylase permits total hydrolysis of dextrin tofermentable glucose, for all types of starch. Indeed, in SSF3 process,after 72 h of fermentation, ethanol concentration achieved 17.2%v/v, which was equivalent to 86.1% of the theoretical ethanol yield,while the residual sugar decreased to 17 g/l (Fig. 2). In compari-son with the results obtained in SSF2 process (14.8% v/v for ethanolconcentration and 52.9 g/l for residual sugar), a significant improve-ment was obtained using an additional glucoamylase (AmigaseMega L). Moreover, with the exception of the first 24 h of fermenta-tion, it is noted that glucose, maltose and lactic acid concentrationsremained at low levels (Table 3), which demonstrated the advan-tages of the SSF process that a low concentration of reducing sugarcould decrease the osmotic pressure on yeast and reduce risk ofcontamination (Thomas et al., 1996).
Srichuwong et al. (2009) have developed the VHG process ofsweet potato at dry matter of 28%. After 61.5 h, ethanol concen-tration of 16.6% v/v was achieved, which was equivalent to 89.7%of theoretical yield. Zhang et al. (2011) reported that ethanol con-centration of 16.3% v/v corresponding to 91.4% of the theoreticalethanol yield achieved with an initial dry matter of 28%. In theirwork, a pretreatment was carried out before fermentation to reducemash viscosity. In another study, Yingling et al. (2011b) identi-fied gravity, particle size, initial pH, and fermentation temperatureas key factors that significantly increased final ethanol concen-tration for VHG processes of cassava. Moreover, Yingling et al.(2011a) used the response surface methodology to study the VHGof cassava mash at 33% dry matter. After model validation, the max-imum ethanol concentration obtained at the optimal conditions ofhydrolysis was 17.96 ± 0.63% while the maximum starch utiliza-tion ratio was 94.52 ± 0.35%. In their research, the cassava mashwas gelatinized at 80 ◦C for 15 min and liquefied at 75 to 77 ◦C for103–108 min with the utilization of a high dosage of alpha-amylase.
In our work, no pretreatment step was performed. Differentenzymes were used in order to decrease viscosity during lique-faction. In our best process (SSF3 process), ethanol concentrationwas 17.2% v/v corresponding to 86.1% of the theoretical ethanol
yield, which was similar to the average yield achieved in the currentethanol factories in Vietnam. Therefore, SSF3 process was chosento scale up to pilot scale to examine its efficiency. The ethanol pro-ductivities (which are equally important from an industrial point of
164 C.-N. Nguyen et al. / Industrial Crops a
0
50
100
150
200
250
300
350
10
11
12
13
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0 24 48 72 88
Suga
r co
ncen
trat
ion
(g/l)
Eth
anol
con
cent
rati
on (%
v/v
)
Time (h)
Ethano l conce ntrat ion (% v/v) Reducing sugar (g/l) Residual sugar (g/l)
Fp
vaSSh(oen
3
wnaFretitHlefbdvtuaaltb(tctprcZft3
ig. 3. Evolutions of residual sugar, reducing sugar and ethanol concentration of SSFrocess at pilot scale.
iew) of the three investigated SSF processes were also calculatednd equal to 1.49, 1.62 and 1.88 g/l/h ethanol for SSF1, SSF2 andSF3 processes, respectively. The values of ethanol productivity ofSF1, SSF2 and SSF3 processes were respectively 30, 41 and 64%igher than that of the conventional ethanol process (1.15 g/l/h)Chu-Ky et al., 2009). These results were in agreement with thosebtained by Ingledew (2009), Srichuwong et al. (2009), Yinglingt al. (2011a,b) and emphasized the great advantage of VHG tech-ology, namely increased ethanol productivity.
.3. Scale up of SSF at VHG of cassava flour at pilot scale
The main objective of scaling-up was to identify problems thatere not significantly noticed at lab scale, and to verify the mainte-ance of ethanol yield after fermentation. According to the resultst lab scale, SSF3 process was chosen to be scaled up to pilot scale.ig. 3 shows the evolutions of ethanol concentration, reducing andesidual sugars during SSF at pilot scale. After 72 h of SSF, thethanol content reached 16.5% v/v, which corresponded to 83.6% ofhe theoretical ethanol yield and to 1.81 g/l/h of ethanol productiv-ty. These values of ethanol yield and productivity were lower thanhose obtained at lab scale (17.2% v/v and 1.88 g/l/h, respectively).owever, the content of residual sugars at pilot scale (6.9 g/l) was
ower than that at the lab scale (17.0 g/l). This result could bexplained by the fact that the yeast could have used in excess ofermentable sugar under aerobic condition for its growth at theeginning of SSF. Hence, the increased biomass would lead to aecrease in the ethanol yield. In our pilot experiment, due to a largerolume (100 l) than that at lab scale, it needed a longer period ofime to cool down the mash and to transfer the mash from the liq-efaction reactor to the fermentor. The speed and the duration ofgitation should have been too high and long for the SSF processt pilot scale even though these values were identical as those atab scale. Therefore, more oxygen could have been taken up intohe fermentation beer and the time for yeast growth could haveeen longer than usual and leading to an increased yeast biomassIngledew, 2009). It is suggested that the yeast pitching rate, cul-uring and aeration condition needed to be optimized and to beontrolled to improve the process efficiency for ethanol produc-ion at pilot scale. The investigation on the VHG process for ethanolroduction from sweet potato showed that high viscosity causedesistance to solid-liquid separation and lower fermentation effi-iency (Srichuwong et al., 2009). In another work conducted by
hang et al. (2011), the VHG process with sweet potato was per-ormed with 20 to 28% dry matter. After liquefaction at 85 ◦C inhe presence of liquefying enzymes, the SSF was carried out at0 ◦C with the addition of saccharifying enzymes and xylanase for
nd Products 56 (2014) 160–165
viscosity reduction. At lab scale, at dry matter of 28%, an ethanolconcentration of 16.3% v/v was achieved, which was equivalent toan ethanol yield of 90%. However, in that work, the dry matter wasdecreased to only 24% at pilot scale to maintain the ethanol yield at90%. In our work, the dry matter has remained as high as that at pilotscale (315.4 g/l or approximately 30% dry matter) when the processwas scaled up at pilot scale. Since the results at pilot scale were notsimilar to those at lab scale, the process needs to be optimized toimprove the process efficiency at pilot scale.
4. Conclusion
In this work, we have successfully developed SSF processesunder VHG condition (315.4 g/l dry matter) of cassava for ethanolproduction at lab scale and pilot scale (100 l). The ethanol con-tent achieved 17.2% v/v corresponding to 86.1% of the theoreticalethanol yield at lab scale and decreased to 16.5% v/v correspond-ing to 83.6% of the theoretical ethanol yield at pilot scale. Weshowed that combination of four enzymes (alpha-amylase, beta-glucanase and two glucoamylases) led to a significant reduction inmash viscosity and to an increased ethanol yield. However, whenthe process was scaled up to pilot scale, a decrease in ethanol yieldwas observed (from 86.1% at lab scale to 83.6% at pilot scale, respec-tively). It is suggested that in order to improve the ethanol yieldat pilot scale, a pretreatment with additional viscosity-reducingenzymes should be carried out. In addition, yeast growth conditionsshould be also optimized and controlled.
Acknowledgments
This work was supported by Ministry of Education and Train-ing and Ministry of Science and Technology of Vietnam. We thankDupont, DSM and Fermentis for kindly providing us with enzymesand yeast samples, respectively. We also thank Dr Nguyen Tien-Thanh for his technical assistance in HPLC analysis and Dr HoPhu-Ha for her revision of the English text.
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