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Energy Procedia 00 (2017) 000–000
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Energy
EnergyProcedia
Procedia138 (2017) 000–000
00 (2017) 883–888
www.elsevier.com/locate/procedia

2017 International Conference on Alternative Energy in Developing Countries and

Emerging Economies 2017 AEDCEE, 25 – 26 May 2017, Bangkok, Thailand

Bioethanol production
The 15th International from cassava
Symposium starch
on District byand
Heating enzymatic
Cooling
hydrolysis, fermentation and ex-situ nanofiltration.
Assessing the feasibility of using the heat demand-outdoor
temperature function
Wangporafor a long-term
Prayoonyongdistrict
, Chularatheat demand, forecast
a a
Jinnaphat , Paritta Sakdaronnarong
b a
Anawat Sungpet , Woranart Jonglertjunya*
a,b,c a
I. Andrić Department
*, A. ofPina
a
Chemical FerrãoaFaculty
, P.Engineering, , J. Fournier b
., Mahidol
of Engineering, B. Lacarrière c
, O. Le Correc
University, Thailand
b
a Department of Chemical Engineering, Faculty of Engineering, King Mongkut's University of Technology
IN+ Center for Innovation, Technology and Policy Research - Instituto Superior Técnico, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal
b Thonburi, Thailand
Veolia Recherche & Innovation, 291 Avenue Dreyfous Daniel, 78520 Limay, France
c
Département Systèmes Énergétiques et Environnement - IMT Atlantique, 4 rue Alfred Kastler, 44300 Nantes, France

Abstract

Abstract Cassava starch were liquefied and saccharified by alpha-amylase and gluco-amylase,
respectively, before fermentation for bioethanol production. Response surface methodology
(RSM)
District heating was used
networks to optimize
are commonly the condition
addressed of liquefaction
in the literature as one of theandmost
saccharification on sugar
effective solutions for decreasing the
greenhouse gas emissions from
concentrations. theeffects
The buildingofsector.
amount These
of systems
enzyme,require high investments
liquefaction which
temperature areliquefaction
and returned through the heat
sales. Due to the on
time changed climate
dextrin conditions and
concentrations andbuilding renovation
the effects policies, of
of amount heat demand saccharification
enzyme, in the future could decrease,
prolonging the investment return period.
temperature and saccharification time on glucose concentrations were measured and studied.
The main scope of this paper
Maximum is tocontent
glucose assess the
wasfeasibility
273.1g/lofwhen
using cassava
the heat demand – outdoor
starch (30 %w/v)temperature function
was liquefied for heat demand
by 0.9
forecast. Themg/g
district of Alvalade, located inatLisbon
of alpha-amylase/starch 85 °C(Portugal),
for 180 min wasand
usedsaccharified
as a case study. Themg/g
by 1.5 district is consisted of 665
of gluco-
buildings that vary in both construction
amylase/starch at 60 °Cperiod for and
90 typology. Three weather scenarios
min. Saccharomyces cerevisiae(low,(S.medium, high) and
cerevisiae) and three district
renovation scenarios
Zymomonas mobilis (Z. mobilis) were studied in batch mode to prove ethanol efficiency. The values were
were developed (shallow, intermediate, deep). To estimate the error, obtained heat demand
compared with results from a dynamic heat demand model, previously developed and validated by the authors.
batch culture was inoculated at 30±1 °C and agitated at 70 rpm with a Rushton turbine in 2-
The results showed that when only weather change is considered, the margin of error could be acceptable for some applications
liter and 10-liter working volume baffled bioreactors. Microbial cells and ethanol solutions
(the error in annual demand was lower than 20% for all weather scenarios considered). However, after introducing renovation
were then separated from fermentation broths using microfiltration (membrane Model M-
scenarios, the error value increased up to 59.5% (depending on the weather and renovation scenarios combination considered).
M1812PS20) and nanofiltration (membrane Model M-N1812A5 and M-N1812A9),
The value of slope coefficient increased on average within the range of 3.8% up to 8% per decade, that corresponds to the
respectively.
decrease in the The batch
number of heating hoursmode resultsduring
of 22-139h showed that theseason
the heating log phase was on
(depending approximately
the combination 16h.
of weather and
Maximum ethanol produced after 72h period of fermentation by S. cerevisiae in 10-liter
renovation scenarios considered). On the other hand, function intercept increased for 7.8-12.7% per decade (depending on the
bioreactor
coupled scenarios). Thewas 43.5
values g/l, resulted
suggested couldinbeanused
ethanol yield the
to modify of 0.44 withparameters
function a fermentation
for theefficiency of
scenarios considered, and
85.4 %.
improve the accuracy of heat demand estimations.
© 2017 The Authors. Published by Elsevier Ltd.
© 2017 The © 2017 The
Authors. Authors.
Published
Peer-review byPublished by Elsevier Ltd.
Elsevier Ltd.
under responsibility of the scientific committee of the 2017 International Conference on
Peer-review Peer-review
under
Alternative under responsibility
responsibility
Energy inof­Dthe ofCountries
the
Scientific
eveloping Organizing
Committee
and of Committee of 2017 AEDCEE.
The 15thEconomies.
Emerging International Symposium on District Heating and
Cooling.
Keywords: Ethanol, S.cerevisiae, Z.mobilis, cassava starch, nanofiltration
Keywords: Heat demand; Forecast; Climate change

* Corresponding author. Tel.: +2-889-2138-6113; fax: +2-889-2138-6129.

E-mail address: woranart.jon@mahidol.ac.th

1876-6102 © 2017 The Authors. Published by Elsevier Ltd.

Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the scientific committee of the 2017 International Conference on Alternative Energy in
­Developing Countries and Emerging Economies.
10.1016/j.egypro.2017.10.116
884 Jinnaphat Wangpor et al. / Energy Procedia 138 (2017) 883–888
2 Author name / Energy Procedia 00 (2017) 000–000

1. Introduction

Bioethanol fuel is mainly produced by fermenting the sugar components of biomass

which included a rapid and significant answer to these problems [1] such as sugarcane juice,
cassava starch and others carbon sources[2, 3]. Cassava is grown in Thailand is divided into
two types includes bitter and sweet. Sweet cassavas have low quantity of hydrocyanic acid
but the bitter cassavas have a lots of hydrocyanic acid is highly toxic and bitter unfit for
human consumption or use to feed for animals directly [4]. Cassava is more than enough for
the needs of people in the country. Cassava can be processed and converted into value-added
components such as methane (biogas) and ethanol [5, 6], by far the most widely used in the
worldwide for transportation sector. Cassava starch was traditionally hydrolyzed to produce
ethanol by acids but the specificity of the enzymes was a mild reaction condition [3, 7, 8].
The alpha-amylase and gluco-amylase were biocatalysts that can be used for liquefaction and
saccharification of cassava starch. S. cerevisiae are the most common yeasts in fermented
ethanol. Moreover, a few researchers have studied ethanol fermentation by using Z. mobilis
that provides many advantages over the yeast, such as; high cell growth rate and higher
ethanol tolerance [3, 9, 10].
The fermentation broth is composed of mainly sugar, yeast, ethanol, water and by-
product of fermentation. Ethanol can be separated from fermentation broth by membrane
filtration. Separation technologies have been explored for ethanol removal from fermentation
broth during fermentation to meet the requirement of reducing the cost of ethanol
purification [11, 12]. This research is concerned with ethanol production from batch
fermentation of liquefied and saccharified cassava starch using bioreactor follow by a
membrane filtration unit.

2. Methodology

2.1 Liquefaction and Saccharification

Cassava starch (30 %w/v) was added into the solution of 0.8g/L DAP, 0.1g/L Urea and 0.5g/L MgSO4.7H2O. It
was controlled pH to 5 by acetate buffer. The independent variables for optimization of liquefaction and
saccharification process were shown in Table 1. The optimization of temperature, time and amount of enzyme were
performed via response surface methodology by using Central Composite Design (CCD) [10, 13]. The samples were
collected and analyzed to measure the concentration of dextrin and glucose.

Table 1. Levels of variables were tested by the Central Composite Design (CCD)

Levels
Independent variables
-1.6818 -1 0 1 1.6818
o
X 1, Liquefaction temperature ( C) 40 50 60 70 80
X 2, Liquefaction time (min) 30 60 90 120 150
X 3, Alpha-amylase (mg/g dry starch) 0.3 0.6 0.9 1.2 1.5
X 4, Saccharification temperature (oC) 65 75 85 95 105
X 5, Saccharification time (min) 60 90 120 150 180
X 6, gluco-amylase (mg/g dry starch) 0.3 0.6 0.9 1.2 1.5

2.2 Response surface methodology

Three variables were studied at five levels (-1.6818, -1, 0, +1, 1.6818). 20 treatments were established based on
a computer simulation. The mathematical relationship of the response on these variables can be evaluated by the
following quadratic polynomial equation, as shown in Eq.1-2 [10, 13]:

Yn=Ca+C1X1+C2X2+C3X3+C12X1X2+C13X1X3+C23X2X3 +C11X12+C22X22+C33X32 (1)

Ym=Cb+C4X4+C5X5+C6X6+C45X4X5+C46X4X6+C56X5X6 +C44X42+C55X52+C66X62 (2)
Where Ca and Cb = constant, C1, C2, C3, C4, C5 and C6 = linear coefficients, C12, C13, C23, C45, C46 and C56 = cross
product coefficients and C11, C22, C33, C44, C55 and C66 = quadratic coefficients.
 3 Author name / Energy Procedia 00 (2017) 000–000
Jinnaphat Wangpor et al. / Energy Procedia 138 (2017) 883–888 885

2.3 Ethanol production from cassava starch in batch fermentation

S. cerevisiae and Z. mobilis were obtained from Thailand Institute of Scientific and Technological Research.
The culture media for S. cerevisiae and Z. mobilis were YMA (yeast extract 0.3%, malt extract 0.3%, peptone 0.5%,
glucose 1%, and agar 2%) and Zymomonas medium (yeast extract 0.5%, peptone1%, glucose 2%, and agar 1.5%),
respectively. Sterilized cassava starch solution (including 0.8 g/L DAP, 0.1 g/L Urea, 0.5 g/L MgSO4.7H2O) was
controlled pH to 5 by acetate buffer, liquefied by 0.9 mg/g (dry starch) alpha-amylase at 85oC for 180min, and then
was saccharified by 1.5 mg/g (dry starch) gluco-amylase at 60 oC for 90 min (optimal conditions obtained from sec.
2.1). The hydrolysis of cassava starch was used for ethanol production in batch fermentation. The experimental
conditions were temperature of 30±1°C and agitated at 70 rpm with a Rushton turbine in 2 and 10 liter-working
volume baffled tanks.

2.4 Membrane system for ethanol separation

Microfiltration membrane (M-M1812PS20) with molecular cut off of 20,000Da and Nano-filtration membrane
(M-N1812A5 and M-N1812A9) with molecular cutoff of 200-400Da were purchased from Applied Membranes
INC., USA. The membrane module was spiral wound membrane with 0.45m2 in effective surface area. The filtration
system was disinfected by circulating 2% Potassium meta-bisulphite solution for 20min followed by cleaning with
DI water till neutral pH was reached. 10-liters of fermentation broth solution were used for each experimental
condition. Fermentation broth was fed through the microfiltration membrane with trans-membrane pressure of 1-3
bar, and then passed through nanofiltration membrane with trans-membrane pressure of 3-6 bar.

2.5 Analysis
The dextrin, glucose and ethanol concentrations were determined with a HPLC system equipped with a
AMINEX HPX-87H, BioRad. The temperatures of column oven and refractive index detector were set at 60 and
40oC, respectively. The mobile phase was 5 mM sulfuric acid. The flow-rate was 0.6ml/min and the sample injection
was 20µl. The cell concentration was measured by cell number methods. The total reducing sugar was determined
with 3,5-dinitrosalicylic acid method (DNS) by UV/Visible Spectrophotometer Lambda25. It measures the
absorbance at 540 nm against a reagent blank.

2.6 Statistics analysis

The analysis of variance (ANOVA) of the regression parameters of the predicted response surface quadratic
model for dextrin, maltose and glucose concentrations were generated using Microsoft Excel 2010. The optimized
values of three independent variables for maximum response were calculated using the numerical optimization
package of the same software. Origin pro 8.5.1 was used for weighting coefficient experimental 3D plots generate.

3 Results and discussion

3.1 Optimization by response surface methodology

The liquefaction and saccharification of starch were shown in three-dimensional response

of surface plots in Figs. 1-2. For liquefaction step, the highest dextrin concentration of 275.7
g/l and 91.9 % g dextrin/g starch was observed from cassava starch (30 %w/v) that was
liquefied by 0.9 mg/g starch of Alpha-amylase at 85 oC and 180 min. For saccharification
step, the highest glucose concentration of 273.1 g/l and 91 %g glucose/g starch was observed
from cassava starch (30 %w/v) that was saccharified by 1.5 mg/g starch of gluco-amylase at
60 oC and 90 min. Moshi et al [3]stated that the amount of the α-amylase, time, the initial
concentration of starch and temperature has effect to efficient digestion of cassava starch.
These authors also reported reducing sugars of 65 % was achieved by using α-amylase
8U/ml for digesting cassava starch (30 g/l) for 60 min at 70-80 oC. Comparison of this
experiment with that of Ajibola et al [9] shows that cassava was liquefied by using α-
amylase 4 ml at 80 oC for 60 min, and cassava was then saccharified by using amylo-
glucosidase at 40 oC for 48 h. The amount of total enzymes was 1.0 %v/v, equivalent to the
amount of dextrin highest concentration of glucose.
The set of coefficients for the liquefaction and saccharification of starch was calculated
with 10 coefficients, using the multiple linear regression analysis. All the terms regardless of
their significance was derived from the following equations:
886 Jinnaphat Wangpor et al. / Energy Procedia 138 (2017) 883–888
4 Author name / Energy Procedia 00 (2017) 000–000

Y1 = -2378.59+50.20X1+6.83X2+878.74X3-0.08X1X2-10.59X1X3-7.88X2X3-0.23X12+
0.003X22+6.56X32 (3)
2
Y2 = -1248.65+41.48X4+2.18X5+559.00X6-0.03X4X5-3.91X4X6-2.30X5X6-0.33X4 -
0.0002X52-110.44X62 (4)
Where Y1 and Y2 were the predicted dextrin from liquefaction step and glucose
concentration from saccharification step, respectively. X1 – X6 were the independent
variables affect these objectives. The fitness of the model was examined by the coefficient of
determination R2. The R2 value of Y1 and Y2 were 95.97 and 92.46%, respectively. The
adjusted R2 value of Y1 and Y2 were 91.50 and 90.65%, respectively. Both R2 and adjusted
R2 values of regression model were higher than 90% which were considered to be a high
correlative.

Fig.1. Response surface curve for dextrin Fig.2. Response surface curve for glucose
concentration in liquefaction process showing concentration in saccharification process showing
interactions between temperature and time at interactions between temperature and amount of
X3(amount of enzyme) =0 enzyme at X5(time) = 0

3.2 Ethanol production from cassava starch in 2L-batch fermentation

Sterilized cassava starch (20 %w/v) that was liquefied by using α-amylase (0.3 mg/g dry
starch) at 90 oC for 90 min, and then was saccharified by gluco-amylase (0.5 mg/g dry starch)
at 60 oC for 60 min was found to be glucose concentration of 75 g/L. Ethanol fermentation
from hydrolysis of cassava starch using S. cerevisiae in a 2 liter-working volume baffled tank
(Fig. 3) has yielded greater concentration, yield and number of microorganism rather than
that of using Z. mobilis in 72 hours fermentation (data not shown). This may due to S.
cerevisiae can produce ethanol from maltose and glucose [14] which comes from the
hydrolysis of cassava starch. In contrast, Z. mobilis only uses glucose to produce ethanol.
Their ethanol production of 45.2 g/l and yield of 0.23 with a fermentation efficiency of 52.1
% in 72h fermentation were much greater in comparison to that Z. mobilis was 30.9 g/l, yield
of 0.15 and fermentation efficiency of 30.3 %.

3.2 Ethanol production from cassava starch in 10L-batch fermentation

Sterilized cassava starch (10%w/v) that was liquefied by using α-amylase (0.9 mg/g dry
starch) at 85 oC for 180 min, and then was saccharified by gluco-amylase (1.5 mg/g dry
starch) at 60 oC for 90 min was found to be glucose concentration of 85 g/L. Ethanol
fermentation from hydrolysis of cassava starch using S. cerevisiae in a 10 liter-working
volume baffled tank was shown in Fig. 4. The results were found to be ethanol production of
43.5 g/l, yield of 0.44 and fermentation efficiency of 85.4 % in 72h fermentation.
 5 Author name / Energy Procedia 00 (2017) 000–000
Jinnaphat Wangpor et al. / Energy Procedia 138 (2017) 883–888 887

Fig. 3. Ethanol concentration, yield and number of Fig. 4. Ethanol concentration, yield and number of
microorganism in the broth as a function of microorganism in the broth as a function of
fermentation time using S. cerevisiae in 2 liter- fermentation time using S. cerevisiae in 10 liter-
working volume of bioreactor. working volume of bioreactor.

3.3 Membrane Separation

Permeate flux of fermentation broth by using M-M1812PS20 microfiltration based on

trans-membrane pressure was shown in Fig. 5. The results shown that the permeate flux of
M-M1812PS20 microfiltration with trans-membrane pressure of 2 bar was greater than
among these conditions. The permeate solution from microfiltration with trans-membrane
pressure of 2 bar was then separated ethanol using the nanofiltration with trans-membrane
pressure of 4-6 bar for M-N1812A9 and 3 bar for M-N1812A5. The samples of permeate and
retentate were collected every five minute for 90 minutes. The ethanol and total reducing
sugar were shown in Fig.6a and 6b. The results shown that ethanol was separated from
fermentation broth using M-N1812A9 at trans-membrane pressure of 6 bar gave the highest
efficiency. In addition, total reducing sugar could be passed through M-N1812A9 membrane
at high trans-membrane pressure (6 bar). In order to purify ethanol, the lowest total reducing
sugar was favorable. The rejection of total reducing sugar observed by using M-N1812A9
nanofiltration with trans-membrane pressure of 5 bar was 91.78%. Weng Y.H. et al. showed
that high retention of sugar (>80%) was observed by nanofiltration membrane [15].

a b

Fig. 5. The permeate flux of Fig. 6. The weight of substance in the permeate per the weight of
fermentation broth as a function substance in the retentate (Gi/Go); (a) ethanol and (b) total reducing
of time using M-M1812PS20 sugar as a function of time using M-N1812A5(A5) and M-
N1812A9(A9)

4. Conclusion

Cassava starch were liquefied by alpha-amylase and saccharified by gluco-amylase for

sugar production. The effects of temperature, time and amount of enzyme was optimized for
sugar concentration (dextrin and glucose concentration). The optimum liquefaction
conditions for dextrin concentration were 0.9 mg/g of alpha-amylase, 85 °C and 180 minutes.
The saccharification conditions for glucose concentration were 1.5 mg/g of gluco-amylase,
60 °C and 90 minutes. Maximum ethanol produced from hydrolysis of cassava starch in 10
liters-bioreactor after 72h period of fermentation by S. cerevisiae was 43.5 g/l with an
888 Jinnaphat Wangpor et al. / Energy Procedia 138 (2017) 883–888
6 Author name / Energy Procedia 00 (2017) 000–000

ethanol yield of 0.44 and a fermentation efficiency of 85.4 %. The ethanol filtration was
carried out by microfiltration with trans-membrane pressure of 2 bar and nanofiltration (M-
N1812A9) with trans-membrane pressure of 5 bar. The rejection of total reducing sugar was
91.78 %.

5. Acknowledgement

This research project is supported by Mahidol University, Thailand. J. Wangpor thanks a

scholarship from the 60th Year Supreme Reign of His Majesty King Bhumibol Adulyadej.

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