This document summarizes research on optimizing liquid sugar production for bioethanol processing from arrowroot tuber using Aspergillus niger and amiloglucosidase. The researchers found that: 1) The highest glucose yield was 12% after 11 days of saccharification using 10-15% A. niger starter. Commercial amiloglucosidase achieved 28.5% glucose yield after 24 hours. 2) The highest fermentation yield was 6% alcohol with 65% production efficiency over 7 days at 27°C using Saccharomyces cerevisiae yeast. 3) Optimal conditions for A. niger saccharification were 10% starter concentration at 38°C for

1. 2nd. ISFAS 2012,4-6 September 2012, Puri Pujangga, UKM, Malaysia
OR21
Optimization of Liquid Sugar Production for Bioethanol Processing from Arrowrot
Using Aspergillus niger and Amiloglucosidase
SAWARNI HASIBUAN, ENDRIANUR RAHMAN ZAIN & ITA NOVITA
Department of Agroindustrial Technology, Faculty of Agribusiness and Food Technology,
Djuanda University, Jl. Tol Ciawi No. 01, PO Box Ciawi 35, Bogor 16720, West Java, Indonesia
Email address: sawarni02@yahoo.com
ABSTRACT
The aim of this research is to analyze the optimum condition of liquid sugar production in bioethanol processing from
arrowrot tuber (Maranta arundinaceae Linn) using Aspergillus niger and amiloglucosidase. The bioethanol processing
from arrowrot tuber consist of saccharification of the sugars in arrowrot tuber essence, and continuous ethanol
fermentation of the saccharified liquid. The aim of this study was to compare saccarification process eficiency using
Aspergilus niger and commercial amiloglucosidase (AMG). The starter adding treatments were 10% (A1), 15% (A2),
and 20% (A3). The hydrolysis was conducted for 11 days in the temperature of 38 oC. The highest glucose result was
achieved after saccharification in A1B3 and A2B3 (12%), and 28,5% in commercial AMG. The highest fermentation
result was contained 6% of alcohol with 65% production efficiency was achieved.
Keywords: bioethanol; liquid sugar; arrowrot tuber; liquification; saccharification
INTRODUCTION various sources of raw material for bioethanol. The
advantage of arrowroot compared to other sources of
Indonesia’s high dependence on crude oil for fuel has starch is because its usage as a source of food is not as
quite a significant impact on the Indonesian economy high as cassava, corn, sweet potatoes, sago, etc.; so its
and people, especially after crude oil prices exceeded competition as a source of food is relatively low.
US$ 100/barrel. With a consumption rate exceeding The general objective of the study is to support
production rate, Indonesia has become a net importer of diversification of raw materials and alternative fuels,
150 million barrels/year (ESDM 2012). In respond to particularly bioethanol in Indonesia. Whereas the
the condition, the government of the Republic of specific objectives to be achieved are (1) to determine
Indonesia issued Presidential Istruction No. 10, 2005 the optimum time of conversion of starch sugar into
regarding the saving of energy use; and Presidential syrup from arrowroot tuber starch, (2) to gain an
Instruction No 1, 2006 as well as Presidential optimum condition for the addition of A. niger starter
Instruction No. 5, 2006 on renewable energy. concentration and Amyloglucosidase in the production
Actually, biofeuls are nothing new to our lives; of glucose syrup from arrowroot tuber, and (3) to
only its technology has developed. One example of the determine the efficiency of bioethanol production from
use of biofuel in acient times was the burning of arrowroot tuber with the addition of S. cerevisae.
jatropha seeds for lighting. Nowadays, the application
of biofuels have changed into more modern and more MATERIALS AND METHODS
popular forms named bioethanol dan biodiesel; both
called biofuels. Biofuel business opportuities in the The materials used in this study were arrowroot flour, A.
world are also highly prospective. Based on Clean Edge níger culture, commercial Amyloglucosidase (AMG), S.
report as quoted by Ron Pernick and Clint Wilder cereviseae yeast, Potato Dextrose Agar (PDA), distilled
(2007), the world biofuel market in 2006 reached US$ water, 70% alcohol, and other chemical materials for
20,5 billion (for ethanol dan biodiesel). analysis.
The arrowroot tuber plant is one alternative source The study was conducted in two stages, i.e. the
of carbohydrate. As a raw material for biofuels, the preliminary research and primary research. The purpose
arrowroot can be processed into bioethanol as a of the preliminary research was to determine the
substitute for premium gas. The arrowroot is one source hydrolysis time of arrowroot flour with the addition of
of starch that can be decomposed into sugar and then A. niger starter and commercial AMG. While the
fermented into bioethanol. In its decomposition, starch objective of the primary research was to obtain the best
needs the help of α-amylase and glycoamylase enzymes. treatment of saccharification with the addition of
After it becomes sugar, then it is fermented into ethanol Aspergillus niger starter and commercial AMG for the
using Saccharomyces cerevisiae yeast. production of bioethanol from arrowroot flour.
This study contributes to overcome national
energy issues, particularly in conducting exploration of
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2. THE HYDROLYSIS OF ARROWROOT FLOUR TABLE 1 Results of the chemical composition of
Arrowroot flour was dissolved, then heated and stirred arrowroot flour used in the study
until gelatinization occurred. Then it was put into an
Composition Average (%)
autoclave and sterilized at a temperature of 121oC for 15
minutes. After sterilization, the erlenmeyer was cooled Water content 13.95
at room temperature. The next process was Ash content 1.00
saccharification, i.e. the decomposition of starch into Protein content 0.93
glucose. The starter A. niger was put into a substrate Fat content 1.79
with concentrations of 10%, 15%, and 20%. At the same Fiber content 4.56
time, a similar process was also carried out using Carbohydrate content 73.53
commercial AMG cultures with concentrations of 5%,
7.5%, and 10%. The average carbohydrate content of arrowroot
flour used in the study was 73.53 g/100 g arrowroot
THE FERMENTATION OF ARROWROOT LIQUID SUGAR flour. The amount of carbohydrate in arrowroot starch
Fermentation was done by putting Saccharomyces depends on the production process and the cultivar used.
cerevisiae yeast - which is able to decompose sugar into The total carbohydrate of every 100 g can reach 85,2
ethanol - into the substrate, in which an anaerobic g/100 g arrowroot flour. The carbohydrate content of
fermentation took place. In order to obtain an optimal arrowroot is lower than cassava flour which reaches
fermentation, the temperature was maintained between 88,2 gram, but higher than rice which is only 78,9
25oC-30oC while the pH was between 4,5-5,5. The grams and wheat which is 77,3 grams. Although it is
separation of ethanol and water was done through very widely used, many people are not familiar with the
distillation at a temperature of 78oC, which is the arrowroot plant; unlike cassava, sweet potatoes or taro,
boiling point of ethanol. The ethanol vapor was passed which are very popular.
through a pipe/hose flown with water, so that it was
PRELIMINARY RESEARCH
condensed into liquid ethanol. The analysis conducted
on the bioethanol products included total sugar level The enzyme used in this study comes from the A. niger
analysis, alcohol level analysis, and fermentation fungus, in addition to the commercial AMG. According
efficiency. to Tatang (2007), the Aspergilus sp fungus would
produce the α-amylase and glucoamylase enzymes
THE EXPERIMENTAL DESIGN which would play roles to decompose starch into
The experimental design used in this study was the glucose or simple sugar.
nested design with two treatments (the kind of starter The total sugar content analysis was conducted
and length of fermentation) and two replications. using a hand refractometer. From the results of the
The mathematical model of the nested design used analysis, with amounts of substrates of 10% (A1), 15%
was: (A2), and 20% (A3) and hydrolysis time of 6 days (B1),
9 days (B2), and 11 days (B3), the average sugar
Yijk = μ + Ai + Bj(i) + Єijk content obtained was between 3% to 12%, as presented
Where :
Yijk : Observation variable as a result of treatments of in Figure 1.
the i-th stage of factor A with the j-th stage of
factor B at the k-th repetition
μ : General mean
Ai : Effects of treatment of the i-th stage of factor A
(addition of starter i = 1, 2, 3)
Bj(i) :Effects of treatment of the j-th stage (length of
fermentation = 1, 2, 3) of factor B nested at the i-
th stage of factor A.
Єijk :Experimental error.
RESULTS AND DISCUSSION
PRELIMINARY RESEARCH
The arrowroot tuber as raw material used in the study
was obtained from Pekalongan, Central Java. Results of
analysis on the arrowroot starch (AOAC 1984) used is
presented in Table 1.
FIGURE 1. Average total sugar content of arrowroot
glucose syrup with A. niger starters.
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3. The results of analysis of variance showed that the the production of bioethanol from sweet potato glucose
amount of starter used gave a significant effect on the syrup conducted by Haryani (2008), with a
total sugar content produced (P<0,05). Treatment A2B3 fermentation time of 72 hours and an agitation of 125
(a substrate of 15% for 11 days) produced the highest rpm; the highest level of ethanol was obtained at a
total sugar, i.e. 12%. Haryani’s study (2008), using the concentration of total initial sugar of 24%, i.e.
α-amylase enzyme (0,8 ml/kg of starch) and AMG amounting to 11,80±0,71%, and the efficiency of
enzyme (10,8 ml/ kg of starch) on a substrate of sweet fermentation of sugar into ethanol reached 96,41%.
potato starch resulted in a total initial sugar of 24% at an The level of ethanol produced was presumably
agitation of 150 rpm. The low total sugar produced in affected by the ratio between carbon and nitrogen that
this study was probably due to the low amount of α- occurred in the fermentation. Factors that affect
amylase and glucoamylase enzymes produced by the A. fermentation are temperature, pH, oxygen, nutrients and
niger fungus starter. media of fermentation. Besides glucose, the medium of
The use of commercial AMG in the production of fermentation was added with other substances that
arrowroot tuber starch syrup was much more effective function as sources of macronutrients dan
compared to the use of the A. niger starter. It can be micronutrients as well as growth factors. Ammonium is
seen in Figure 2 that the sugar content resulting from the the form which is most easily used by yeast (Harrison &
use of commercial AMG could reach 28,5% and Graham 1970).
required a shorter fermentation time, i.e. 24 hours. Bell, Higgins & Attfield (2001) stated that the best
Based on Figure 2, the use of AMG at a concentration sugar level for the process of fermentation is 16-25%,
of 1% was able to produce optimum levels of sugar for which will produce ethanol as much as 6-12%. A
fermentation, i.e. at a range of 18,3 to 24,5%. The concentration of sugar above 25% will slow down the
higher the concentration of starch was used, the higher fermentation, whereas that above 70% will stop the
the level of sugar was produced. Maximum sugar level process of fermentation due to osmotic pressure (Osho
was produced from the use of an arrowroot starch 2005).
solution with a concentration of 30%. The fermentation of ethanol occures anaerobically
using certain yeasts which can convert glucose into
ethanol through the Embden Meyerhof-Parnas Pathway.
One molecule of glucose will form two molecules of
ethanol and carbondioxyide. Based on its weight,
theoretically one gram of glucose will produce 0,51
grams of ethanol.
CONCLUSIONS
Based on results of the study, the optimum time to
produce liquid sugar from arrowroot tuber starch using
Aspergillus niger was 11 days, resulting in a total sugar
content of 12%. The use of commercial AMG in the
production process of liquid sugar from arrowroot tuber
starch was much more effective compared to the use of
the Aspergillus niger starter. Sugar contents resulting
from the use of commercial AMG could reach 28.5%
and required a shorter fermentation time, i.e. 24 hours.
Optimum conditions for the use of Aspergillus
FIGURE 2. Average total sugar content of arrowroot niger in the production of liquid sugar from arrowroot
glucose syrup with AMG starter. tuber were at a starter concentration of 10%, a
fermentation temperature of 38oC, and fermentation
FERMENTATION
time of 9 days; while the use of commercial AMG
The sugar syrup produced from the previous stage was required a starter concentration of 10%, and conversion
used as the primary substrate in the fermentation was completed in 24 hours.
process. The simple glucose model structure facilitates The results of analysis of alcohol level with a
the fermentation process by S. cerevisiae. During fermentation time of 7 days, a temperature of 270C and
fermentation, the cells use sugar for growth and produce an addition of Saccharomyces cerevisiae produced an
ethanol. Ratledge (1991), stated that S. cerevisiae could average alcohol level of 6%. The highest efficiency of
produce ethanol from glucose in anaerobic conditions. fermentation of sugar into alcohol reached 65%.
The highest efficiency of fermentation of sugar
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