Pervez et al.

BMC Biotechnology 2014, 14:49

http://www.biomedcentral.com/1472-6750/14/49

RESEARCH ARTICLE Open Access

Saccharification and liquefaction of cassava

starch: an alternative source for the production of
bioethanol using amylolytic enzymes by double
fermentation process
Sidra Pervez, Afsheen Aman, Samina Iqbal, Nadir Naveed Siddiqui and Shah Ali Ul Qader*

Abstract
Background: Cassava starch is considered as a potential source for the commercial production of bioethanol
because of its availability and low market price. It can be used as a basic source to support large-scale biological
production of bioethanol using microbial amylases. With the progression and advancement in enzymology, starch
liquefying and saccharifying enzymes are preferred for the conversion of complex starch polymer into various
valuable metabolites. These hydrolytic enzymes can selectively cleave the internal linkages of starch molecule to
produce free glucose which can be utilized to produce bioethanol by microbial fermentation.
Results: In the present study, several filamentous fungi were screened for production of amylases and among
them Aspergillus fumigatus KIBGE-IB33 was selected based on maximum enzyme yield. Maximum α-amylase,
amyloglucosidase and glucose formation was achieved after 03 days of fermentation using cassava starch. After salt
precipitation, fold purification of α-amylase and amyloglucosidase increased up to 4.1 and 4.2 times with specific
activity of 9.2 kUmg−1 and 393 kUmg−1, respectively. Concentrated amylolytic enzyme mixture was incorporated in
cassava starch slurry to give maximum glucose formation (40.0 gL−1), which was further fermented using
Saccharomyces cerevisiae into bioethanol with 84.0% yield. The distillate originated after recovery of bioethanol gave
53.0% yield.
Conclusion: An improved and effective dual enzymatic starch degradation method is designed for the production
of bioethanol using cassava starch. The technique developed is more profitable due to its fast liquefaction and
saccharification approach that was employed for the formation of glucose and ultimately resulted in higher yields
of alcohol production.
Keywords: Amylases, Aspergillus fumigatus, Biofuel, Saccharification, Saccharomyces cerevisiae, Starch

Background producing nations. Bioprocessing of renewable resources

Emerging environmental issues raised due to combus- available in a particular region can help in resolving these
tion of petroleum-based fossil fuel and emission of toxic issues. Various renewable resources in terms of agricul-
gases have diverted the attention of scientists and re- tural biomass have been investigated for the production of
searchers towards the utilization of various renewable bioethanol and this development proved beneficent for
resources for the production of bioethanol. In addition the biotechnological industries. Amongst various starchy
to these global concerns, other important factors that materials available throughout the world; corn, sugarcane,
have been kept in preference are the mounting prices of wheat, potato [1], corn stover [2,3], molasses [4] and
the fuels and the current political scenario among the oil purified starch [5] have been successfully utilized for
the commercial production of bioethanol. As the de-
* Correspondence: ali_kibge@yahoo.com
mand and the cost of these starchy crop materials is
The Karachi Institute of Biotechnology & Genetic Engineering (KIBGE),
University of Karachi, Karachi -75270, Pakistan

© 2014 Pervez et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
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unless otherwise stated.
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increasing day by day, it has become indispensible to simpler sugars and afterwards another microbial factory
use substitute raw resources. (yeast or bacteria) is incorporated in the same fermenta-
Cassava is a tropical root crop which is an economically tion flask to produce ethanol. In this case the primary
available fermentable source and is produced by numerous organism (specifically fungal specie) along with amylo-
countries [6]. It is incorporated into animal feed (20.0%) lytic enzymes also excretes other toxic substances and
and about similar proportion is converted into starch for proteases which in result inhibit the growth and per-
industrial purposes whereas; some of the portion is also formance of the second ethanol-producing microorgan-
used as food source in several developing countries. About ism. Along with this, the establishment of appropriate
50.0 to 70.0% starch content is recovered from the cassava temperature for starch hydrolysis, enzymatic activity and
root and due to the low ash content and rich organic na- ethanol production also plays an important role. Current
ture it can be used as an ideal substrate for bioethanol research deals with the production of bioethanol from
production [7-9]. In addition, it can also be easily hy- hydrolysis of an inexpensive renewable resource known
drolyzed by various techniques. As cassava starch does as cassava starch, which is commonly available in Pakistan.
not have much industrial application in food industries The methodology used for the production of ethanol was
as compared to corn starch, therefore it also lacks com- based on double fermentation technique using partially
petition in terms of price and is available throughout purified fungal amylolytic enzymes for the liquefaction
the year due to its flexibility in terms of planting and and saccharification of this starchy material. Keeping all
harvesting [7,10,11]. disadvantages in view, this study was designed in two sep-
In recent years, bioprocessing of various value-added arate steps. In first step, amylolytic enzymes (α-amylase
products using microbial factories have been potentially and amyloglucosidase) were produced using indigenously
explored with reference to extracellular enzymes. Agri- isolated filamentous fungi and were partially purified to
cultural biomass used as a substrate for the production hydrolyze cassava starch into simple fermentable sugars.
of bioethanol has several limitations including high fiber In the next step, the sugar cocktail was fermented using
content which requires high temperature for hydrolysis S. cerevisiae to acquire maximum bioethanol yield.
and this energy intensive procedure also does not pro-
vide desired yields of fermentable sugars. Hydrolysis of Results and discussion
lingocellulosic mass by other expensive pre-treatment In the present study, several different fungal isolates
techniques is also time consuming. Industries also have with amylolytic activities were purified from different
concerns regarding the availability of the biomass through- soil samples and preliminary identification was based
out the year and most of the time its storage in bulk on microbiological studies including cultural and micro-
quantities is not possible due to space shortage. The de- scopic characterization followed by 18S rDNA sequence
velopment of an ideal pre-treatment method for hydro- analysis. Colonial and microscopic characteristics indicate
lyzing poly-phenolic lignin in the feedstock is expensive that all isolates belong to genera Aspergillus. Microscopic
with several aforementioned limitations thus, enzymatic morphology of A. fumigatus KIBGE-IB33 showed colum-
treatment is more preferable. Conventional method used nar and uniseriate conidial heads while, conidiophores are
for the production of bioethanol from cassava starch short and smooth. On the other hand, A. niger KIBGE-
usually requires the basic gelatinization step followed by IB36 showed large, globose, dark brown conidial heads
liquefaction and saccharification. The sugar formed with hyaline and smooth conidiophores. Likewise, conidial
during these processes is further fermented using either heads of A. flavus KIBGE-IB34 are radiate and biseriate
yeast or bacteria. Since, starch derived from any plant whereas, conidiophores are hyaline and coarsely rough-
source is a complex molecule, it require various hydro- ened. A. terreus KIBGE-IB35 has biseriate and globose co-
lytic enzymes for its conversion into simple fermentable nidia with hyaline and smooth conidiophores. A. versicolor
sugars. Among many extracellular hydrolases available, KIBGE-IB37 showed centrally rising, velvety floccose and
microbial amylases are frequently used for its conver- slightly blue-green color colony on PDA with conidio-
sion. For commercial production of amylases Aspergillus phores borne from surface or aerial hyphae.
and Rhizopus species are considered most significant Screening of amylolytic property of the strains was based
sources because the enzymes from these sources are on starch hydrolysis method. Initially, 07 fungal strains
generally thermostable and are available in excessive were selected and among them 05 filamentous fungi in-
quantities [12-14]. cluding A. fumigatus KIBGE-IB33, A. flavus KIBGE-IB34, A.
Despite several advantages of simultaneous saccharifi- terreus KIBGE-IB35, A. niger KIBGE-IB36 and A. versicolor
cation and fermentation using multiple organisms, there KIBGE-IB37showed production for amylolytic enzymes.
are also few shortcomings. In the initial steps, the amy- When these isolates were cultivated in the starch con-
lolytic enzymes are produced using fungi and the starch taining production medium, highest titers of α-amylase
present in the medium is allowed to hydrolyze into (11.0 kUmg−1) and amyloglucosidase (142.0 kUmg−1) were
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produced by A. fumigatus KIBGE-IB33 (Figure 1). This

strain was also capable of producing considerable
amount of glucose (81.0 gL−1) which can be used for
the production of ethanol. The fermentable sugar pro-
duced by this isolate can be easily metabolized by S.
cerevisiae. However, other filamentous fungi produced
lower titers of both α-amylase and amyloglucosidase along
with lower concentration of glucose. Although maximum
α-amylase was produced by A. flavus KIBGE-IB34 but it
did not showed higher glucose formation rate as com-
pared to A. fumigatus KIBGE-IB33 therefore, this isolate
was selected for further studies.
Figure 2 Production of amylolytic enzymes from Aspergillus
Optimization of various cultivation parameters plays fumigatus KIBGE-IB33 and glucose formation using various
an important role. For effective bioethanol production, carbon sources.
fermentation time of the microbial culture and the type
of the renewable carbon source used for the production
of starch hydrolyzing enzymes and ethanol are among most favorable inducer and contribute highest amount
the most important factors. The aforementioned factors of enzyme units (α-amylase: 11.0 kUmg−1; amyloglucosi-
will ultimately direct the overall process cost for the dase: 142.0 kUmg−1) and glucose formation (81.0 gL−1) as
development or the scale up of a new methodology in compared to the other carbon sources tested. These re-
any bioethanol producing industry. Hence, fermentation sults suggest that pure starch based carbon sources in-
time for the production of α-amylase and amyloglucosi- cluding sago starch, soluble starch (potato) and cassava
dase and different types of carbon sources were studied. starch are more suitable for the production of enzyme
Varieties of carbon source have been tested and can play and glucose formation as compared to the different
an important role during microbial fermentation because complex biomass (wheat bran and sugarcane bagasse)
they are the integral components for the production of whereas, no enzyme production was detected when
cellular material and most of the time they are also associ- wheat starch and rice bran were used. This is because
ated with microbial growth [15]. Much interest has been the lingocellulosic tough plant matrix was not pre-
diverted towards the utilization of economically available treated. The cell free filtrate (CFF) collected after fer-
carbon sources in order to fulfill the industrial require- mentation showed negligible titers for cellulase, pectinase
ments. In the current study, to improve the production and xylanase (data not shown) therefore; the starch con-
of α-amylase and amyloglucosidase for starch hydrolysis tent was not accessible for fermentation as compared to
and bioethanol production, seven different carbon the purified starch materials. Fatima and Ali [16] tested six-
sources were utilized (Figure 2). The induction pattern teen fungal species for the production of amyloglucosidase
for both amylolytic enzymes was different in various (activity ranged between: 1.906-12.675 U ml−1 min−1) using
carbon sources suggesting that these hydrolases are in- starch in fermentation medium and the best strain they
ducible. Among all, cassava starch proved to be the identified was A. oryzae llB-6 (12.673 ± 0.998 Uml−1 min−1).
They also noticed a 30% increase in the enzyme activity
when some of the process parameters were altered (pH and
incubation time). Very recently, Puri et al. [17] reported the
use of rice bran: wheat bran (1:1), rice bran: paddy husk
(1:1) for the production of amylase and amyloglucosidase
and the maximum amylase (2.72 IU) and amyloglucosidase
(4.11 IU) activity was achieved when rice bran was
incorporated in the fermentation medium inoculated
with A. oryzae. However, A. fumigatus NTCC 1222 exhib-
ited 341.7 U/mL amylase activity under solid state fermen-
tation when incubated at 35°C (pH-6.0) for 06 days in
nutrient salt solution [18]. In another study, detergent
mediated production of glucoamylase in the presence of
Figure 1 Production of amylolytic enzymes and glucose soluble starch is also reported using A. niger FME under
formation by various filamentous fungi. KIBGE-IB33: A. fumigatus; shake flask system [19]. Several other researchers have also
KIBGE-IB34: A. flavus; KIBGE-IB35: A. terreus; KIBGE-IB36: A. niger; KIBGE-
used cassava starch and cassava pulp as alternative carbon
IB37: A. versicolor.
source for bioethanol production using α-amylase and
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amyloglucosidase [20-23]. The compositional analysis of

cassava starch used in this study is presented in Table 1.
The type and source of starch based materials plays a
crucial role for achieving maximum bioethanol yield. The
starch content in variety of biomaterials will govern the
cost of bioethanol production. Therefore, before consider-
ing the production of bioethanol using a specific source,
the nature of the starch molecule (linkage, granule size
and shape) and the method of extraction employed must
be kept in consideration. However, the values of compos-
itional analysis of starch cannot be compared with other
starch sources because of the variation of the plant source
and the methods used to analyze the structure or content Figure 3 Production of amylolytic enzymes and glucose
of starch. After selection of a suitable carbon source, formation by Aspergillus fumigatus KIBGE-IB33 at different
incubation times.
fermentation time for the production of amylolytic
enzymes was also studied by incubating A. fumigatus
KIBGE-IB33 for different time interval ranging from 02 to
07 days. It was observed that production of α-amylase and CFF some other constitutive enzymes may also be
amyloglucosidase started after 02 days of incubation and present including various types of proteases which could
both the hydrolases were continuously produced up to cause interference during liquefaction and saccharifica-
day 06 and 07, respectively with a maximum titter tion process of cassava starch. Therefore, to avoid any
secreted at day 03 (Figure 3). Afterwards, it was also hindrance, starch hydrolyzing enzymes from the CFF
noticed that as incubation time increases, amylolytic activ- were purified using gradient precipitation in the pres-
ity as well as glucose formation decreases. This might be ence of ammonium sulfate ranging from 20.0% to 80.0%
due to the fact, with the passage of time the nutrients and among it 40% saturation level was selected. Table 2
become depleted and other secondary metabolites are summarizes the purification profile of α-amylase and
formed which eventually alters the pH of the medium and amyloglucosidase. Fold purification of both α-amylase
inhibits both the growth of the fungi as well as enzyme and amyloglucosidase increased up to 4.1 and 4.2 times
secretion [24]. Most of the time, secondary metabo- with a specific activity of 9.2 kUmg−1 and 393 kUmg−1,
lites have a catabolic repression effect. Amylase from respectively. Previously, Slivinski et al. [29] and da Silva
fungal sources is normally produced after 03 to 07 days and Peralta [30] have also reported precipitation of amy-
of incubation but in some cases, enzyme secretion loglucosidase using ammonium sulfate produced by A.
can be extended up to 15 days [25-28]. Prolong incubation niger and A. fumigatus, respectively. Ammonium sulfate
time is one of the drawbacks of using filamentous precipitation method was also used for purification of
fungi at industrial scale level which eventually increases α-amylase from Pencillium chrysogenum and A. niger
process cost. JGI 24, respectively [31,32]. The enzyme kinetic analysis
After fermentation, α-amylase and amyloglucosidase showed that the optimum pH, temperature, Vmax and Km
were partially purified from the CFF. Most of the time in values for α-amylase from A. fumigatus KIBGE-IB33
were 6.0 (citrate buffer, 50.0 mM), 65°C, 25.0 kU ml−1
Table 1 Compositional analysis of commercially available and 0.5 mg ml−1, respectively. Whereas, optimum pH,
cassava starch temperature, Vmax and Km values for amyloglucosidase
Composition Cassava content (%, w/w) were 5.0 (citrate buffer, 50.0 mM), 60°C, 105.0 kU ml−1
Total sugar* 85.2 ± 4.26 and 2.56 mg ml−1, respectively. The optimum pH and
Total protein* 1.4 ± 0.07 temperature for amyloglucosidase activity isolated from
A. niger FME was 5.0 and 45°C, respectively whereas,
Reducing sugar* 6.2 ± 0.31
the Km and Vmax values were determined using soluble
Glucose* nil
starch as substrate as 94 μg ml−1 and 39.02 Umg−1,
Moisture content** 0.9 ± 0.04 respectively [19]. In another recent study, the optimum
Amylose*** 10.7 ± 0.53 pH of amyloglucosidase was 6.0 and the optimum
Amylopectin*** 89.3 ± 4.46 temperature was 60°C along with Km and Vmax values of
*2.0% Cassava solution. 0.046 mg ml−1 and 769 Umg−1 [33]. Looking at the kinetic
**1.0 g Cassava starch. properties of the other recently studied amylolytic
***0.4 g Cassava starch.
±Specifies standard deviation (SD) among three equivalent replicates. Values
enzymes, it is therefore suggested that both the hydrolases
in each set differ significantly: p ≤ 0.05. from A. fumigatus KIBGE-IB33 could be used for industrial
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Table 2 Purification profile of starch hydrolyzing enzymes produced from Aspergillus fumigatus KIBGE-IB33
Steps Total volume (ml) Total enzyme units (kU) Total protein (mg) Specific activity (kU mg−1) Fold purification
Alpha amylase
Crude 500 5500 2500 2.2 1.0
(NH4)2SO4 Precipitation 30 1950 210 9.2 4.1
Amyloglucosidase
Crude 500 229500 2500 91.8 1.0
(NH4)2SO4 Precipitation 30 82500 210 393 4.2

starch saccharification purpose. Both partially purified upon optimum enzyme activity as well as the purity of
enzymes were used for bioethanol production. amylolytic enzymes as crude enzyme takes longer time
Conversion of starchy materials into ethanol is an in- period to completely hydrolyze starch molecule into
tricate process and several attempts have been made to glucose as compared to the purified enzyme (Table 3).
produce bioethanol in commercially feasible quantities This percent saccharification (60.0%) could also be fur-
and to easily scale-up the methodology used. Cassava ther augmented by either improving the purity of en-
starch is a complex molecule containing amylose and zyme or by incorporating other hydrolyase (xylanases,
amylopectin and for the production of bioethanol, first pectinases or cellulases) along with these amylolytic en-
the starch molecules must be hydrolyzed into more sim- zymes [42,43]. As reported earlier further increase in
ple sugars. Some pretreatment techniques including hot percent saccharification could also be achieved if the
water and steam explosion treatment, alkaline and solvent starch slurry was autoclaved before addition of amylo-
pretreatment, acid hydrolysis and enzymatic degradation lytic enzyme [44]. Similarly, Aggarwal et al. [45] and
for the breakdown of complex starch molecule into sim- Soni et al. [46] have also discussed about the role of the
pler sugars have also been studied [34-37]. More recently, purity level of amylolytic enzymes during starch hy-
a new pre-treatment technique known as popping pre- drolysis. In the same way, Shanavas et al. [20] have also
treatment have gained attention for the hydrolysis of previously analyzed the effect of reaction time on sac-
starchy feedstock [38]. However, enzymatic degradation charification of cassava starch and have obtained max-
using different hydrolases is mostly preferred because dur- imum percent saccharification after 30.0 minutes of
ing acid hydrolysis the percent conversion of starch into incubation followed by slight increase when using com-
reducing sugars is low as compared to the enzymatic deg- mercially available starch hydrolyzing enzymes. On the
radation [39-41]. With the progression and advancement contrary, Aggarwal et al. [45] reported maximum per-
in enzymology, amylolytic enzymes are now preferable cent saccharification using crude amylolytic enzymes
over conventional methods because enzymatic treatments after 24 hours of incubation time. Very recently, Gohel
lead towards high yield of glucose with reduced energy et al. [47] used simultaneous saccharification and solid
consumption. Therefore, in the current study gelatinized state fermentation for the production of ethanol using
cassava starch was liquefied using α-amylase and was fur- Indian sorghum feedstock and also incorporated acid
ther saccharified by means of amyloglucosidase. However, fungal protease instead of urea for better ethanol yield.
before breaking starch into simple fermentable sugars, the A large number of microbes including bacteria, yeast
time required for both the processes to occur effectively and fungi are capable of producing ethanol from fermented
was also analyzed by incubating the gelatinized starch
slurry with both partially purified amylolytic enzymes for
different time intervals. Glucose was the main end-
product which is required for production of bioethanol,
therefore the concentration of glucose formation as well
as percent saccharification was monitored throughout this
study. Gelatinized cassava starch was mixed with partially
purified α-amylase (9.2 kUmg−1) and amyloglucosidase
(393.0 kUmg−1). It was observed that as the reaction time
increases, the formation of glucose (40.0 gL−1) as well as
percent saccharification (60.0%) also increased up to
90.0 minutes and beyond that both parameters become
constant (Figure 4). This glucose containing mixture was
Figure 4 Percent saccharification of cassava starch and glucose
further used for the production of ethanol. Efficiency of
formation at different reaction time intervals.
enzymatic liquefaction and saccharification also depends
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Table 3 Optimized conditions for starch hydrolysis in the presence of crude and partially purified amylolytic enzymes
Enzyme* Enzyme Units (kU mg−1) Starch** (gL−1) Function Time (minutes) Temperature (°C) pH
Alpha Amylase (Crude) 2.2 20.0 Liquefaction 120 65 5.0
Alpha Amylase (Partially Purified) 9.2 20.0 Liquefaction 15 65 6.0
Amyloglucosidase (Crude) 91.8 20.0 Saccharification 360 60 5.0
Amyloglucosidase (Partially Purified) 393.0 20.0 Saccharification 90 60 5.0
*Both enzymes were produced by Aspergillus fumigatus KIBGE-IB33.
**Cassava starch.

sugars [48]. Among them S. cerevisiae is widely used for amylolytic enzymes had revealed that this two-step based
production of bioethanol because it is not only able to pro- method can be used to achieve higher yields of bioethanol.
duce high amount of ethanol but it can also tolerate and Further, the process cost can also be reduced by using
survive higher concentrations of ethanol formed in the other inexpensive starchy materials or by establishing pilot
medium [49,50]. However, most of the yeast strains are not programs that will scrutinize the actual feasibility and sus-
capable to directly ferment complex starchy materials [48]. tainability of the overall process developed.
Therefore, in the current study S. cerevisiae was used for
the production of ethanol from glucose which was earlier Conclusions
formed by the action of fungal amylolytic enzymes. Table 4 In conclusion, an improved and effective enzymatic sac-
summarizes the results of bioethanol production after 24 charification of inexpensive cassava starch using amylolytic
and 48 hours of yeast fermentation and it was noticed that enzymes from A. fumigatus KIBGE-IB33 was developed.
the maximum percent yield of bioethanol (84.0%) was ob- The glucose obtained after enzymatic degradation was uti-
tained after 48 hours. After 48 hours, the fermented lized for the bioethanol production using S. cerevisiae. Dual
medium was distilled and the percent yield became 53.0%. systematic enzyme conversion has advantages in terms of
The concentration and purity of the distilled ethanol was reduced energy consumption as well as increased produc-
also analyzed using gas chromatography (GC). tion of fermentable sugar to achieve maximum bioethanol
Several techniques including direct fermentation, simul- yield as compared to other processes. In addition, the
taneous saccharification, simultaneous non-thermal sac- process developed is more rapid as compared to the previ-
charification, ultrasound assisted treatment and solid-state ously conducted studies using liquefaction and saccharifi-
fermentation have been studied previously using different cation of cassava starch.
starchy materials and microbial sources for the production
of bioethanol [20,21,51-56]. Along with this ethanol Methods
has also been produced by repeated batch culture Reagents
through immobilization of S. cerevisiae and S. pastorianus All reagents were of analytical grade and were obtained
IFO0751 on calcium alginate and porous cellulose carriers, from commercial sources. Sago and cassava starch were
respectively [57,58]. Nikolic et al. [54] used ultrasound- purchased from local market, Karachi, Pakistan. Peptone
assisted treatment for direct conversion of corn meal into (Oxoid, England), yeast extract (Oxoid, England), ammo-
bioethanol but the cost related to this method in amount nium sulfate (Serva, Germany) and dipotassium hydro-
of energy consumption is very high. Beside this, the pre- gen phosphate (Serva, Germany) were purchased from a
treatment of multiple biomass or starch flour will also add local vendor. Whereas, magnesium sulfate, sodium hy-
extra budget that will eventually affect the feasibility of the droxide, sodium carbonate, copper sulfate, sodium potas-
bioethanol. The attempt made in the current study by sium tartarate, sulfuric acid and potassium dichromate
consuming commercially available cheap cassava starch were acquired from Scharlau (Spain). Other chemical used
along with saccharification by synergistic effect of fungal including 3′, 5′- dinitrosalicylic acid (DNS) was purchased

Table 4 Production of bioethanol using Saccharomyces cerevisiae

Incubation time (Hour) Glucose concentration (gL−1) Ethanol (%) Theoretical yield (g) Actual yield (g) Yield** (%)
Before distillation
24 40.0 36.0 2.04 1.44 70
48 40.0 43.0 2.04 1.72 84
After distillation
48 40.0 27.0 2.04 1.08 53
*Mass of ethanol formed per mass of glucose consumed.
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from BDH Chemicals (USA) and anthrone from MP presence of starch (cassava) under batch conditions
Biomedicals (France) while, soluble starch and folin using submerged fermentation technique. Production of
ciocalteu reagent were purchased from Merck (Germany). α-amylase, amyloglucosidase and glucose was monitored
at different time intervals (02 to 07 days). Basal medium
Isolation and identification of filamentous fungi used for the production of α-amylase and amyloglucosi-
The natural fungal isolates used in the current study were dase consists of (gL−1): Cassava starch, 20.0; yeast extract,
isolated from different soil samples that were collected 10.0; peptone, 10.0; K2HPO4, 1.0; and MgSO4.7H2O, 1.0.
aseptically from diverse vegetative fields located in Karachi, Initial pH of the medium was adjusted at 7.0 before
Pakistan. All the isolates were obtained after serial platting sterilization at 121°C for 15 minutes. Fresh seed culture
on potato dextrose agar (PDA) at 30°C for 05 days (10.0 ml) was inoculated in 90.0 ml production medium
according to the standard protocols. PDA medium and incubated at 30°C for 03 days under static and anaer-
consist of (gL−1): Boiled potato extract, 300.0 ml; dextrose, obic conditions. It was then further transferred into
20.0 g and agar, 16.0 g. Initially, 07 different fungal species 900.0 ml medium and incubated at 30°C up to 07 days.
were isolated from different samples. Among them 05 The fungal spores were harvested by centrifuging the fer-
filamentous fungi were selected and characterized based mented broth at 40248 × g for 15 minutes at 4°C. The
on colonial morphology, 18S rDNA sequence cataloging supernatant was filtered using 0.45 μ filter under vacuum.
and microscopic analysis using lactophenol blue staining The cell free supernatant containing the amylolytic en-
method [59,60]. After 18S rDNA gene analysis and sequen- zymes was stored at −20°C for further analysis. All the ex-
cing, the sequences were analyzed by similarity search using periments were conducted in triplicates.
BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and were
submitted to NCBI GenBank database. The confirmed
Optimization of physicochemical parameters for
sequences received the following GenBank accession
maximum enzyme yield
numbers: KF905648, KF905649, KF905650, KF905651,
For the enhanced production of amylolytic enzymes, dif-
and KF905652 for A. fumigatus KIBGE-IB33, A. flavus
ferent inducing substrates (carbon sources) and fermenta-
KIBGE-IB34, A. terreus KIBGE-IB35, A. niger KIBGE-IB36
tion time were optimized. For this purpose seven different
and A. versicolor KIBGE-IB37, respectively. All of these
carbon sources including sago starch, soluble starch
isolates were tested for the amylolytic enzyme production
(potato), cassava starch, wheat starch, wheat bran, rice
based on starch-iodine plate method. All fungal isolates
bran and sugarcane bagasse were used in the concentra-
were plated on starch medium plates containing (gL−1):
tion of 20.0 gL−1. A. fumigatus KIBGE-IB33 was incubated
cassava starch, 10.0; yeast extract, 10.0; peptone, 10.0;
for different time intervals ranging from 02 to 07 days at
K₂HPO₄, 1.0; and MgSO4.7H2O, 1.0. The cultures were in-
30°C under static and anaerobic condition for the selec-
cubated at 30°C for 05 days. After incubation the plates
tion of optimum fermentation time. Enzyme titer in terms
were flooded with potassium-iodide solution for the de-
of specific activity and glucose formation were monitored
tection of amylolytic activity. Isolates were selected
in triplicate.
based on clear halo-zone around the fungal growth. All
isolates were preserved on PDA slants at 4°C for fur-
ther analysis and were sub-cultured routinely. Purified Partial purification of amylolytic enzymes
Sacchromyces cerevisiae (baker’s yeast) was purchased The cell free supernatant containing α-amylase and amy-
from the local market, Karachi, Pakistan and was grown loglucosidase was precipitated using salt precipitation
and maintained in YPD medium (gL−1:yeast extract,10.0; method. For this purpose, salt gradient precipitation tech-
Bacto-peptone, 20.0 and glucose, 20.0). nique was employed ranging from 20.0% to 80.0% satur-
ation using ammonium sulfate. 20% salt was incorporated
Inoculum preparation for seed culture gradually in CFF with continuous stirring at 4°C and was
Total viable spores were calculated in order to prepare equilibrated for 18 hours. The precipitates formed were
fungal inoculum. For this purpose spores were transferred centrifuged at 40248 × g for 10 minutes at 4°C and were
using sterile needle from a 05 day old fungal culture dissolved in citrate buffer (50.0 mM, pH-5.0). In the next
grown on PDA slant and re-suspended in 10.0 ml sterile run, again the 20% salt saturation was performed using
distilled water containing 0.1% Tween-20. Each suspen- the same supernatant up to 80% and every time the pre-
sion was serially diluted up to 10−5 in order to make cipitates were equilibrated for 18 hours at 4°C. During
homogenous spore suspension of 106 to 108spores ml−1). each saturation range, the precipitates were monitored
and calculated for both enzymes unit in terms of kU mg−1
Medium used for the production of amylolytic enzymes of protein. The saturation level at which both hydro-
All the selected filamentous fungi were tested for the lyase were precipitated out with maximum unit was
production of α-amylase and amyloglucosidase in the selected (40%).
Pervez et al. BMC Biotechnology 2014, 14:49 Page 8 of 10
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Enzyme assay and total protein estimation with a Lie-big condenser and the in-let cold water
Enzyme activity of α-amylase and amyloglucosidase attached to a chiller. All the experiments were run inde-
was estimated using DNS [61] and GOD-PAP method pendently in triplicate and the results presented are the
[62,63], respectively. One unit of α-amylase is defined as mean of three values.
the “amount of enzyme that liberates 1.0 mM of maltose
per minute under standard assay condition”. Whereas, Determination of bioethanol concentration by gas
one unit of amyloglucosidase is defined as the “amount chromatography (GC)
of enzyme that liberates 1.0 mM of glucose per minute Bioethanol concentration was also verified using gas chro-
under standard assay condition”. The specific units of matography system (GC17A, Shimadzu, Japan) equipped
both amylolytic enzymes are expressed in terms of kilo with flame ionization detector (FID). Column used was
units per mg of protein (kU mg−1). Total protein was TRB-5 (30 × 0.25 mm × 0.25 μm) with nitrogen as a carrier
calculated using Lowry’s et al. [64] method with bovine gas (20 cmsec−1). The temperature of the detector and the
serum albumin as standard. injector were kept at 200°C and 130°C, respectively. The
split ratio was 100:1 and the peak area of the compound
Production of bioethanol was integrated against an external standard of absolute
Bioethanol was produced using glucose, which was ob- ethanol.
tained after hydrolysis of starch (cassava) using partially
purified amylolytic enzymes. Partially purified α-amylase Physico-chemical characteristics of cassava starch
and amyloglucosidase (30.0 ml) was amalgamated in 2.5 The cassava starch used in this study was purchased
liters of pre-gelatinized cassava starch slurry (20.0 gL−1) from the local market in Karachi, Pakistan. For the de-
which was prepared in citrate buffer (pH-5.0, 50.0 mM). termination of total sugar anthrone method was used
The reaction mixture was kept at 65°C for liquefac- [66] whereas, reducing sugar was detected using DNS
tion (15.0 minutes) and at 60°C for saccharification method [61]. Total protein was performed using Lowry’s
(90.0 minutes). The reaction was terminated by boiling the et al. [64] method. Glucose content was estimated using
reaction mixture for 10 minutes. The volume was reduced GOD-PAP method [62,63]. Moisture content was calcu-
up to 2.5 folds and the liberated glucose was detected lated using standard drying method at 105°C until the
before and after concentrating the reaction mixture. weight become constant. Amylose and amylopectin frac-
However, for optimized liquefaction and saccharifica- tions were calculated by iodometric method as suggested
tion condition, the reactions were monitored from previously [67].
15 minutes up to 08 hours with an interval of 15 minutes.
Abbreviations
Percent saccharification of cassava starch was calculated CFF: Cell free filtrate; DNS: 3′, 5′-dinitrosalicyclic acid; FID: Flame ionization
as followed: detector; GC: Gas chromatography; gL−1: Grams per liter; GOD-PAP: Glucose
oxidase per oxidase method; kU mg−1: Kilo units per milligrams; mM: Milli
Glucose moles; PDA: Potato dextrose agar.
Saccharification ð%Þ ¼  100
Substrate
Competing interests
In this concentrated hydrolyzate, yeast extract and pep- Its publication is approved by all authors and they do not have any conflict
of interest regarding any financial, personal or other relationships with any
tone were incorporated in the concentration of 3.0 gL−1 other people or organizations.
and 10.0 gL−1, respectively and the pH was adjusted up to
7.0. S. cerevisiae (2.0%) was cultured in this medium an- Authors’ contributions
SP carried out the major experimental work. AA supervised in acquisition of
aerobically at 30°C up to 48 hours. After 48 hours, ethanol laboratory data and interpretation of data along with finalizing the
was collected through distillation and the distillate was manuscript. SI participated in the analytical analysis of bioethanol. NNS
analyzed for the detection of ethanol. Percent yield of performed enzyme purification. SAQ designed the project and gave the final
approval of the manuscript. All authors have read and approved the final
bioethanol was calculated as followed: manuscript.
Actual Ethanol Produced ðg Þ
Ethanol Yield ð%Þ¼ Acknowledgements
Theoritcal Ethanol from Sugar Consumed ðg Þ Authors are obliged to Dr. Asma Ansari for the identification of the natural
100 isolates used in the current study and also gratefully acknowledge the
financial support from KIBGE, University of Karachi, Karachi, Pakistan.

Analytical method for bioethanol analysis Received: 13 January 2014 Accepted: 20 May 2014
Published: 29 May 2014
For the determination of bioethanol concentration, the
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doi:10.1186/1472-6750-14-49
Cite this article as: Pervez et al.: Saccharification and liquefaction of
cassava starch: an alternative source for the production of bioethanol
using amylolytic enzymes by double fermentation process. BMC
Biotechnology 2014 14:49.

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