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Journal of Bioscience and Bioengineering

VOL. xx No. xx, 1e7, 2015


www.elsevier.com/locate/jbiosc

Production of raw starch-degrading enzyme by Aspergillus sp. and its use in


conversion of inedible wild cassava our to bioethanol
Anselm P. Moshi,1, 2, 3 Ken M.M. Hosea,2 Emrode Elisante,2 Gashaw Mamo,1 Linda nnby,1 and
Ivo Achu Nges1, *
Division of Biotechnology, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden,1 Department of Molecular Biology and Biotechnology, College of Natural and Applied Sciences,
University of Dar es Salaam, P. O. Box 35179, Tanzania,2 and Tanzania Industrial Research and Development Organization (TIRDO), Kimweri Avenue, P. O. Box 23235,
Dar es Salaam, Tanzania3
Received 11 March 2015; accepted 3 September 2015
Available online xxx

The major bottlenecks in achieving competitive bioethanol fuel are the high cost of feedstock, energy and enzymes
employed in pretreatment prior to fermentation. Lignocellulosic biomass has been proposed as an alternative feedstock,
but because of its complexity, economic viability is yet to be realized. Therefore, research around non-conventional
feedstocks and deployment of bioconversion approaches that downsize the cost of energy and enzymes is justied. In
this study, a non-conventional feedstock, inedible wild cassava was used for bioethanol production. Bioconversion of
raw starch from the wild cassava to bioethanol at low temperature was investigated using both a co-culture of Aspergillus sp. and Saccharomyces cerevisiae, and a monoculture of the later with enzyme preparation from the former. A
newly isolated strain of Aspergillus sp. MZA-3 produced raw starch-degrading enzyme which displayed highest activity
of 3.3 U/mL towards raw starch from wild cassava at 50 C, pH 5.5. A co-culture of MZA-3 and S. cerevisiae; and a
monoculture of S. cerevisiae and MZA-3 enzyme (both supplemented with glucoamylase) resulted into bioethanol yield
(percentage of the theoretical yield) of 91 and 95 at efciency (percentage) of 84 and 96, respectively. Direct bioconversion of raw starch to bioethanol was achieved at 30 C through the co-culture approach. This could be attractive since
it may signicantly downsize energy expenses.
2015, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Aspergillus sp.; Raw starch degrading enzyme; Wild inedible cassava; Bioethanol; Co-culture; Monoculture]

The production of bioethanol as a bioenergy carrier is highly


promoted as an alternative solution to energy security and environmental pollution among nations (1). These efforts are however
hampered by the high costs of feedstock and bioconversion of
biomass to bioethanol (2e5). The major costs in the bioconversion
steps are ascribed to energy in the form of heat expended in
feedstock pretreatment and commercial enzymes, which are used
in hydrolysis (6,7). As an example the cost of energy incurred in
processes such as gelatinization, liquefaction and saccharication
of corn starch performed prior to yeast fermentation, is estimated
to correspond to 30e40% of the total cost of production (8). One
practical way of reducing the cost of bioethanol produced from
starchy biomass may be the use of in-house produced enzymes
capable of degrading raw starch at low temperatures. This approach
has two advantages; rstly, it reduces the amount of energy
expended in aforementioned processes (e.g., gelatinization, liquefaction, saccharication) of starch biomass prior to yeast fermentation. Secondly, it reduces the costs of (often expensive)
commercial enzymes, as the use of these tend to increase the cost of
bioethanol production (9). Besides, conventional enzymaticliquefaction of starch is performed at high temperature (e.g.,

* Corresponding author. Tel.: 46 46224849; fax: 46 462227413.


E-mail address: achuing28@yahoo.com (I.A. Nges).

90e110 C) (10) followed by saccharication performed at lower


temperatures, e.g., at 60e70 C (11) thereafter, the hydrolysate is
cooled down to approximately 30 C for yeast fermentation. The
heat energy and enzyme cost in such processes constitutes the
second major cost for the bioethanol production. Therefore, enzymes with specic advantages, e.g., hydrolysis of raw starch at low
temperatures are worth exploring. Such benets, however, may be
specic to given situations and substrate used and may need to be
veried through techno-economical analysis prior to large-scale
application.
Another option is direct ethanol-production by a co-culture of
amylolytic microbe, e.g., amylolytic mould and an effective ethanolproducing microorganism (e.g., Saccharomyces cerevisiae). Simultaneous hydrolysis of starch with an amylolytic organism and
fermentation by an effective fermenting organism is an attractive
technique for bioconversion of starch to bioethanol (12,13). The
advantage of simultaneous hydrolysis and fermentation is that a
multi-stage process for conversion of starch into ethanol is performed in one bioreactor. The glucose produced during saccharication is simultaneously assimilated by the yeast to avoid build-up
of an osmotic pressure, which otherwise tend to inhibit the
fermentation (14).
Once a suitable amylolytic microorganism has been identied,
whether to (i) co-culture it directly with yeast for bioethanol production or (ii) to cultivate it and produce enzymes for hydrolysis of

1389-1723/$ e see front matter 2015, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2015.09.001

Please cite this article in press as: Moshi, A. P., et al., Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of
inedible wild cassava our to bioethanol, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.001

MOSHI ET AL.

J. BIOSCI. BIOENG.,

starch or biomass prior to yeast fermentation needs to be evaluated.


This is because the two strains used in co-cultures may not be
compatible and or may not possess similar culture requirements
such as pH, temperature, nutrient or oxygen demand (8,15,16).
Therefore, in order to have a stable co-culture certain requirements
must be met. Firstly, the two strains must be compatible and able to
grow together (15). The compatibility aspects of combinations of
various strains have been studied (16). In one study, six strains of
S. cerevisiae were co-cultured with different strains of other yeasts.
In the afore mentioned study, none of the six strains of S. cerevisiae
tested were found to inhibit growth of Pichia stipitis or Candida
shehatae, and none of the ve tested C. shehatae strains were found
to have an inhibitory effect on the growth of Saccharomyces species
(16). However, among the six tested P. stipitis strains, ve demonstrated killer activity against Saccharomyces species, and three of
these ve strains showed killer activity against S. cerevisiae (15).
Secondly, the fermentation conditions such as pH, temperature and
oxygen supply for the two strains should be compatible (15). For
example, Zymomonas mobilis ferments glucose at pH 7 and temperature of 37 C, but these conditions are not compatible with
those of xylose-fermenting yeasts (P. stipitis and C. shehatae), which
need pH 5 and temperature 30 C. Conversely, the pH and temperature at which S. cerevisiae ferments glucose to ethanol are
compatible with those of xylose-fermenting yeasts. Furthermore, in
comparison with pure culture, interactions between the different
microorganisms play a decisive role in co-culture systems. The interactions can occur either through direct cell-to-cell communications or by signal substances in the fermentation broth (17).
Particularly, stable co-culture could be controlled by metabolic interactions (i.e., syntrophic relationships, or competition for substrates) and other interactions (i.e., growth promoters or inhibitors
such as antibiotics) (18).
Wild cassava is cheap and unconventional feedstocks, which
can be use for bioenergy production. Characteristics and usability
of wild cassava (Manihot glaziovii) for bioethanol production has
been reported in our previous study (19). The wild cassava
M. glaziovii has many advantages as bioenergy crop. It is very
hardy, a fast grower, free from insect and fungi attacks, requires
little or no attention once established and thrives in poor, dry and
rocky soils unsuited to almost other crops (20). Moreover, the
types recently found in Tanzania possess big tubers with high
content of readily degradable carbohydrates that are up to 89% of
dry matter. Besides, it is inedible because it is presumed to contain
brous roots and is very bitter compared to the domesticated
cassava, Manihot esculentum (19).
In this study, we investigated the bioconversion of the wild
inedible cassava to bioethanol by S. cerevisiae in (i) a co-culture
mode with an amylolytic strain of Aspergillus sp., and compare it
with (ii) a monoculture mode employing an enzyme preparation
from the Aspergillus sp. The study tests the potency of the amylolytic enzyme when secreted in situ during the co-culture fermentation and when prepared and used together with the fermenting
microorganism.

MATERIALS AND METHODS


Collection and preparation of wild cassava tubers
The wild cassava our
was processed from tubers obtained from Kisarawe, Tanzania. The tubers were
processed into our as detailed elsewhere (10). Subsequently, the our samples
were sieved through 850 mm mesh prior to use.
Microorganism isolation and identication
Soil samples were collected
from the rhizosphere of wild cassava plants, M. glaziovii, at Kisarawe district in East
coastal region (Pwani) Tanzania. Amylolytic isolates were screened by plating the
soil samples on nutrient medium supplemented with agar 15 g/L and commercial
starch 10 g/L (21). Antibiotics (tetracycline and chloramphenicol), 0.025% (w/v) were
added into the medium after sterilization to inhibit bacterial contamination.
Afterwards, the plates were incubated at 28 C, for 36 h. Amylolytic isolates were

selected by ooding the agar plates with KI/I2 (2%) solution. Isolates having a
higher ratio of clearing zone to colony size were grown in liquid culture and the
level of amylase production was determined from cell free culture supernatant
uid. Strains MZA-3 and GDA5011 were further identied by molecular
techniques (22). The internal transcribed spacer (ITS) region of these isolates was
amplied by polymerase chain reaction (PCR) and sequenced. The primers used in
the PCR reaction were ITS1 (50 -CCGTAGGTGAA CCTGCGG-30 ) and ITS4 (50 TCCTCCGCTTATTGATA TG-30 ). The PCR reaction-conditions were as follows: 95 C
for 4.5 min, then 30 cycles at 95 C for 30 s, 40 C for 30 s, and 72 C for 1 min,
with a nal extension of 10 min at 72 C.
Enzyme production
The medium for enzyme production comprised of (g/L)
(NH4)2SO4 (2.5), KH2PO4 (3.0), CaCl2 (0.13), MgSO4 (0.2), tryptone (2), and potato
starch (10). The culture was cultivated in 250 mL E-ask with 100 mL reaction
volume which was incubated at 28  2 C, 115 rpm, 36 h. Afterwards, 10% (v/v) of the
inoculum was inoculated into 250 mL of the same medium in 1 L E-ask and
incubated in a shaker incubator at 28  2 C, 110 rpm, 36 h. Subsequently, the culture
was centrifuged at 5500 g, 4 C, 15 min (RC5C, SORVAL Centrifuge, USA) to remove
mycelia and spores, and the supernatant was used for enzyme assays.
Enzyme production was optimized for temperature and pH. The pH was varied
from 3.0 to 7.0 at 30 C and afterwards, the established optimal pH was used to
cultivate the strain at temperature range from 25 C to 40 C. Samples were taken
after 1 h and analysed for reducing sugars by absorbance measurement at 540 nm
according to Millers (23). Since the strains were isolated from rhizosphere of the
wild cassava MGK in Kisarawe Tanzania, where the temperature is about 30 C and
was found to grow well at pH 5.5, the temperature and pH for cultivation and
enzyme production was chosen to fall within this range. Amyloglucosidase (AMG)
was purchased from Novozymes (Copenhagen, Denmark).
Partial purication
The cell-free supernatant uid was precipitated using
solid ammonium sulphate to 60% saturation. The precipitate was recovered and
dissolved in a minimum volume of deionised water and dialysed overnight
against water. The dialysate was maintained in Tris-HCL buffer pH 7.0 at 4 C and
used for enzyme assay and hydrolysis studies.
Enzyme assay
Culture supernatant 94 mL was added to 656 mL of 0.5% (w/v)
soluble starch in 0.05 M sodium acetate buffer (pH 5.6) and incubated at 50 C for
30 min. Afterwards, 750 mL of 3, 5-dinitrosalicylic acid (DNS) reagent was added and
incubated in boiling water for 5 min. Subsequently, the amount of reducing sugars
released was determined by absorbance measurement at 540 nm according to
Millers (23). One unit of the enzyme activity was dened as the amount of enzyme
that released 1 mmol of reducing sugar equivalent to glucose per minute under the
assay conditions.
Simultaneous hydrolysis and fermentation of wild cassava our
Table 1
presents ve operation-conditions which were employed in the hydrolysis and
fermentation of wild cassava our. Therefore, in the rst operation condition,
270 g/L of cassava our in basal nutrient media was inoculated with 10% (v/v)
active culture of MZA-3 and incubated at 30 C, 110 rpm for 24 h. Afterwards, 10%
(v/v) of yeast culture was added and fermentation started at 32 C. The second
operation-condition was identical to the rst except that glucoamylase (0.33
AMG/g of our) was added prior to yeast fermentation. In the third operationcondition, the MZA-3 enzyme preparation 0.29 U/g of our was used to hydrolyze
the wild cassava our (270 g/L suspended in de-ionized water) at 50 C, 110 rpm,
24 h followed by yeast fermentation at 32  2 C. The fourth operation condition
was exactly as the third except that glucoamylase (0.33 AMG/g of our) was
added prior to yeast fermentation at 32  2 C. Operation condition ve (control
experiment), involved direct fermentation of wild cassava our using only
glucoamylase and S. cerevisiae.
The cassava our used for each operation was separately sterilized by Co60
irradiation according Lin et al. (21) and afterwards cooled to room temperature in
sterile cabinet prior to hydrolysis and fermentation.
In all cases, fermentation was performed using automatic gas potential test
system (AGPTS) as described in a previous study (10). Briey, in AGPTS, the volume
of CO2 produced during fermentation is detected by a sensor, measured and registered on-line in AGPTS software. Subsequently, the volume of CO2 was normalized to

TABLE 1. Experimental plan for bioconversion of wild cassava to bioethanol under


different operation conditions 1e5.
Operation
condition
1
2
3

4
5

Pretreatment

Fermentation by
(S. cerevisiae)

Slurry (270 g/L) inoculated with a culture


of MZA-3 and incubated (30 C, 24 h)

Without glucoamylase

Slurry (270 g/L) incubated with MZA-3


enzyme preparation (0.29 U/g of our,
50 C, 24 h)
Slurry (270 g/L) incubated without
enzyme at 50 C, 24 h

Plus glucoamylase
Without glucoamylase

Plus glucoamylase
Plus glucoamylase

Please cite this article in press as: Moshi, A. P., et al., Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of
inedible wild cassava our to bioethanol, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.001

VOL. xx, 2015

RAW STARCH-DEGRADING ENZYME FOR BIOETHANOL PRODUCTION

standard temperature and pressure and thereafter converted to moles using the
common gas law;
PV nRT

W  TS  SC
100  100  0:9

(2)

released sugars g=L


 100
Initial sugars g=L

(3)

ISg

(1)

where P is pressure, V is volume, n is number of moles, T is absolute temperature and


R is gas constant (8.314 J K1 mol1).
Since ethanol and CO2 are produced in equal moles during fermentation the
moles of CO2 are converted to moles of ethanol by applying a factor of (46/44), and
nally to ethanol concentration (g/L) as elaborated elsewhere (10).
Analytical methods
All water-soluble products and remaining glucose
during fermentation were analysed by HPLC as detailed elsewhere (24). CO2 was
monitored and measured online by Automatic gas potential test system (AGPTS).
The monomeric sugars released from enzyme hydrolysis were determined by
HPLC (JASCO Corporation, Tokyo, Japan) equipped with a Bio-Rad Aminex HPX87P column and a refractive index detector (RID). The column temperature was
maintained at 85 C. The mobile phase was HPLC-grade water at a ow rate of
0.6 ml/min. Total solids (TS), volatiles solids (VS) and pH were determined using
standard methods (25).

%TY

Production and preparation of MZA-3 enzyme The effect of


temperature and pH on production of raw starch degrading enzyme
by MZA-3 was studied. During the optimization of enzyme
production, the pH and temperature at which highest enzyme
yield (U/mL) was reached, was found to be 5.5 and 30 C,
respectively (Fig. 2A and B). The resulting enzyme preparation

RESULTS AND DISCUSSION


Isolation and identication of microorganisms
Two isolates designated as MZA-3 and GDA 50011 were found to display
high amylase activity and were selected for further studies. The
isolates were identied to genus level based on morphological
properties and internal transcribed spacer (ITS) sequence identity.
When isolates MZA-3 and GDA5011 were grown on potato
dextrose agar (PDA), colonies displayed white mycelia and green
spores and when observed under microscope, features distinct to
Aspergillus sp. such as conidiophores with swollen heads, external
bore spores and septate hyphae were observed (26). Database
similarity search for the ITS sequence of isolate MZA-3 and
GDA5011 revealed that both isolates belong to Aspergillus species
which supports the morphological characterization. Isolate MZA3 is closely related (99%) to Aspergillus awamori (accession no.
LM653116.1), whereas GDA5011 is closely related (99%) to
Aspergillus nomius (accession no. JN709035.1), Aspergillus avus
(accession no. KC907367.1) and Aspergillus oryzae (accession no.
KF619561.1). ITS sequences of the organisms with high sequence
identity were retrieved from Genebank and a phylogenetic tree
that reveals the taxonomic position of these isolates was
generated (Fig. 1). Sequence alignment and phylogenic tree were
inferred using the Molecular Evolution Genetic Analysis (MEGA4)
software (27).
During hydrolysis of inedible wild cassava using isolates MZA-3
and GDA5011 yield of reducing sugars, glucose equivalent as
percent of theoretical yield (%TY) was 66.4 and 13.5, respectively.
Therefore, isolate MZA-3 was chosen for further studies. The %TY
was estimated from initial reducing sugars glucose equivalent predetermined using the weight of sample (W) (g) hydrolysed, predetermined TS and starch content (SC) of the wild cassava, and a
hydro factor for starch (hexose, i.e., 0.9) using the equations; Initial
sugar glucose (IS) equivalent (g);

64 GDA5011
57 Aspergillus nomius
100

Aspergillus flavus
Aspergillus oryzae
Gliocladium cibotii

100

Aspergillus sp. r308


Aspergillus tubingensis
MZA-3

100 Aspergillus awamori

0.6

0.5

0.4

0.3

0.2

0.1

0.0

FIG. 1. Phylogenic tree showing the taxonomic positions of isolate GDA5011 and MZA-3.

FIG. 2. The effect of temperature (A) and pH (B) on MZA-3 enzyme production and
MZA-3 enzyme activity at temperature range 25e100 C with pH xed at 5.5 (C).

Please cite this article in press as: Moshi, A. P., et al., Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of
inedible wild cassava our to bioethanol, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.001

MOSHI ET AL.

J. BIOSCI. BIOENG.,

displayed the highest activity at 50 C as shown in Fig. 2C, and pH


5.5. The use of a liquefying enzyme at pH 5.5 could reduce the
amount of acid otherwise used to lower the pH from the
liquefying to the saccharifying pH-range (usually, liquefaction
with a-amylase is performed at near neutral pH, whereas
saccharication with glucoamylases is performed around pH 5.0).
This again points towards potential cost reduction gained from
downstream processing (28). Moreover, hydrolysis of raw starch
at 50 C may confer benets through energy saving by avoiding
energy intensive steps such as liquefaction of starch performed at
90e110 C, after gelatinization, which is carry out at even higher
temperatures depending on amylose and moisture content
(10,11). Thus, the enzyme preparation from isolate MZA-3 may
offer some technical advantages. It should be noted that pH 5.5 is
favourable for most species of lactic acid bacteria (29). This may
cause contamination during saccharication and this need to be
considered especially in large scale operations.
Hydrolysis of wild cassava our with MZA-3 enzyme:
enzyme dosage, substrate concentration and retention
time
The maximal enzyme dosage, substrate concentration and
retention time for the MZA-3 enzyme preparation with regards to
cassava our were 3.3 U/mL, 150 g/L and 120 min, respectively
(Fig. 3). By combining the mentioned maximal conditions, the
highest yield of reducing sugar (glucose equivalent) was 66% of
the theoretical value. HPLC analysis revealed that the products of
hydrolysis of the wild cassava using MZA-3 were maltose 42.3%,
maltotriose 11.0%, glucose 6.1% and unidentied sugars 3.2%. This
yield is comparable to that observed with 0.18 KNU (T) (per gram
of cassava our) of commercially available thermostable aamylase at 90 C (2 h) (10). KNU (T) refers to Kilo Novo a-amylase
units (Termamyl), in which one KNU (T) is the amount of aamylase which under standard conditions (pH 7.1, 37 C)
dextrinises 5.36 g of starch dry substance per hour (30). Lin et al.
(21) reported a yield of 80% (reducing sugar) from hydrolysis of
cassava our using similar concentration (of cassava our) with a
different enzyme-preparation from Penicillium sp. However, the
retention time was 18 fold longer (36 h) as compared to 2 h
achieved in the present study. Therefore, MZA-3 enzyme
preparation is relatively more efciency compared to that
reported by Lin et al. (21). In conventional conversion of starchybiomass to bioethanol, the costs for (i) enzymes and (ii) heating
(energy) constitute the second major cost after that of feedstock.
The liquefaction and partial saccharication of starch by bacterial
thermostable a-amylase is one of the most cost intensive unit
operations. It is estimated that the cost of ethanol production
from both sugar cane (Brazil) and corn (USA) is between $0.3 and
0.4 per liter in which enzymes contributes $0.027 (31) equalling
approximately 11e15% of total production cost. In addition the
heat energy expended in gelatinization and liquefaction of starch
80e125 C (32) signicantly raises the cost of starch hydrolysis.
Therefore, hydrolysis of raw starch at low temperature (50 C) can
minimize the energy cost and is expected to favorably contribute
to the economy of starch processing industries.
The yield of reducing sugars at low substrate loading (50 g/L)
increased with increasing enzyme dosage (Fig. 3A) wherein the
highest yield was obtained at 5 U/mL.
The effect of substrate concentration on the yield of reducing
sugars was assessed by considering different substrate concentrations as presented in Fig. 3B. The highest amount of reducing sugar
was measured at the our concentration of 150 g/L. When concentration was increased from 150 to 200, 250, 300 and 350 g/L,
reducing sugars (%TY) was; 43%, 37%, 24% and 16%, respectively
(Fig. 3B). When retention time was extended from 2 to 6 h with
substrate concentration xed at 250 g/L, reducing sugar (%TY)
reached 70%. This implies that the enzyme preparation from isolate

FIG. 3. The effect of MZA-3 enzyme dose (A), (B) substrate concentration, and in (C)
retention (incubation) time on hydrolysis of cassava our starch, to release reducing
sugars, percentage of theoretical yield (%TY).

MZA-3 could have advantage for industrial liquefaction of raw starch


at low temperature, wherein starch concentrations in the range of
150e250 g/L is employed (28). However, a techno-economic analysis will be required to consider all factors such as retention time,
enzyme dosage, substrate, energy, media and chemicals versus
sugar yield to ascertain the economics of the process.
Fermentation of raw starch from wild cassava under various
operational conditions: effect on bioethanol yield Different
operational conditions were investigated with regards to ethanol
yield from wild cassava (Table 1). Fig. 4 shows the ethanol yields for
the studied operation conditions 1 to 5. Results revealed that
ethanol yield (%TY) signicantly improved (from 14% to 91%)
when co-culture of Aspergillus sp. MZA-3 and S. cerevisiae was
supplemented with an external glucoamylase (operation

Please cite this article in press as: Moshi, A. P., et al., Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of
inedible wild cassava our to bioethanol, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.001

VOL. xx, 2015

RAW STARCH-DEGRADING ENZYME FOR BIOETHANOL PRODUCTION

FIG. 4. Effect of different combinations of co-culture of strain MZA-3 and S. cerevisiae as


well as glucoamylase supplementation on theoretical ethanol yield (%TY). Bars 1 to 5
refer to operation conditions: 1, co-culture of S. cerevisiae and Aspergillus sp. (MZA-3),
no glucoamylase; 2, co-culture of cerevisiae and Aspergillus sp. (MZA-3), with glucoamylase; 3, monoculture of S. cerevisiae enzyme preparation from Aspergillus sp.,
MZA-3, no glucoamylase; 4, monoculture of S. cerevisiae enzyme preparation from
Aspergillus sp., MZA-3, with glucoamylase; 5, control experiment consisting of only
glucoamylase and yeast but no MZA-3 enzyme.

conditions 1 and 2). Theoretical yield of ethanol (%TY) was


calculated based on initial estimated glucose wherein each gram
of glucose yields 0.511 g of ethanol. This corresponded to an
ethanol concentration of 11.4 and 65.7 g/L (Fig. 5), respectively. A
similar evaluation employing a monoculture of S. cerevisiae and
the enzyme preparation from isolate MZA-3 (conditions 3 and 4),
ethanol yield (%TY) improved from 18% to 95% (Fig. 4) which
corresponded to ethanol concentrations of 10.8 and 75.5 g/L,
respectively (Fig. 5). In order to conrm the needed synergy
between the MZA-3 enzyme and the glucoamylase observed in
both co- and monoculture, a control experiment containing only
wild cassava our and glucoamylase (operation condition 5) was
included. This resulted in an ethanol yield (%TY) of less than 0.5%
(Fig. 4). This suggested that the starch a-(1,4) glycosidic linkages
were broken by the MZA-3 enzyme primarily to oligomers but
not to glucose. It is plausible therefore to state that the a-amylase
released by strain MZA-3 breaks the a-(1,4) glycosidic bonds in
starch randomly to produce maltodextrins, maltose, maltotriose
and small amounts of fermentable sugars (glucose). However, the
yeast cannot ferment these oligomers. Thus, addition of

FIG. 5. Kinetics and pattern of simultaneous saccharication and fermentation of wild


cassava our using coculture of an Aspergillus sp. MZA-3 and S. cerevisiae and monoculture of S. cerevisiae with enzyme preparation from the Aspergillus sp. MZA-3 with
and without the aid of glucoamylase. Operation conditions: 1, co-culture of S. cerevisiae
and Aspergillus sp. (MZA-3), no glucoamylase; 2, co-culture of cerevisiae and Aspergillus
sp (MZA-3), with glucoamylase; 3, monoculture of S. cerevisiae enzyme preparation
from Aspergillus sp., MZA-3, no glucoamylase; 4, monoculture of S. cerevisiae enzyme
preparation from Aspergillus sp., MZA-3, with glucoamylase.

glucoamylase which converts the oligomers released by aamylase to glucose resulted in the higher ethanol yield.
The combination of a-amylase and glucoamylase has previously
been reported to act synergistically in enhancing hydrolysis of
maize and potato starches (33). In that study, it was shown that
only 20% of glucose was obtained from cooked starch after 60 h
incubation when amyloglucosidase was used, as compared to 100%
glucose when combination of amylase and amyloglucosidase was
used.
The ethanol yield obtained for the co-culture fermentation in
the present study is comparable to that reported for two strains of
yeast Saccharomyces diastaticus and S. cerevisiae 21 (12). However,
Abouzied et al. (13) reported a higher ethanol yield using a coculture of Aspergillus niger and S. cerevisiae, compared to a monoculture containing only the former.
Slightly more ethanol was obtained with the monoculture in
which MZA-3 enzyme was supplemented with glucoamylase (95%)
than the co-culture (91%) (Fig. 4). The discrepancy can be explained
by how the carbon source was allocated for cell growth in the coculture and the monoculture systems. The stoichiometric massbalance analysis indicated that the moles of biomass
(C5H7NO2P0.074) (34) produced per mole of glucose was higher for
the co-culture (0.07) than for the monoculture (0.03) (Table 2). The
carbon recovery for the co-culture was comparable to the monoculture for reactors supplemented with glucoamylase (operation
conditions 2 and 4), 99% and 101%, respectively (Table 2).
Kinetics and pattern of fermentation with co-culture and
monoculture The kinetics and pattern of fermentation displayed by the four operation conditions described in Table 1 is
presented in Fig. 5. The kinetics of fermentation for both monoand co-culture was monitored through CO2 production, which
was measured and recorded online using the AGPT. The volume
of CO2 was afterwards, converted to ethanol as described in
materials and methods. This system has been veried using HPLC
and proved be accurate and reliable for fermentation at both
mesophilic and thermophilic conditions (10,35). It saves the
drudgery, time and cost of sampling and preparing samples for
HPLC or GC analysis. Furthermore, it allows measurement and
recording of both CO2 and ethanol every minute, hence providing
data vital for study of kinetics of fermentation and dynamics of
biomass degradation.
The kinetics and pattern between the mono and co-culture
fermentations were very different. The monoculture supplemented with glucoamylase (operation condition 4) experienced a
lag phase of approximately 8 h, perhaps, due to adaptation of the
yeast in high disaccharide concentration, then after, the exponential phase was rapid and completed within 16 h. Consequently, high
nal ethanol concentration and productivity were achieved, 75 g/L
and 4.5 g/L/h, respectively. Conversely, the co-culture supplemented with glucoamylase (operation condition 2) experienced no
lag phase. However, it was relatively slow, completing the exponential phase in 60 h, and resulted in nal ethanol concentration of
65 g/L with productivity of 1.2 g/L/h. Operation conditions 1 and 3,
i.e., co- and monoculture without glucoamylase, displayed low nal
ethanol concentration and productivity, of 11.4, 11.5 g/L and 0.22
and 0.41 g/L/h, respectively (Fig. 5). Therefore, the use of the MZA-3
enzyme preparation for liquefaction at 50 C followed by SSF with
S. cerevisiae at 30 C seems to be more efcient than the co-culture
approach. However, the cost of enzyme extraction and preparation
and then after, the energy expended in hydrolysis prior to SSF need
to be gauged against the cost of longer fermentation time at 30 C in
a techno-economical analysis to decide on the best approach. The
difference in the fermentation pattern observed could be explained
by the interaction of the two microorganisms present in medium.
Cells present in a medium communicate with each other either by

Please cite this article in press as: Moshi, A. P., et al., Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of
inedible wild cassava our to bioethanol, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.001

MOSHI ET AL.

J. BIOSCI. BIOENG.,

TABLE 2. Product prole and stoichiometric mass balance of fermentation of starch from cassava our under various operation conditions.
Operation
conditions

Type of culture

Co-culture

Co-culture

Monoculture

Monoculture

Stoichiometric balance
C6H12O6 / 0.3CH3CH2OH 0.0CH3COOH
0.0C3H8O3 0.3CO2 0.01C5H7NO2P0.074
C6H12O6 / 1.8CH3CH2OH 0.0CH3COOH
0.1C3H8O3 1.7CO2 0.07C5H7NO2P0.074
C6H12O6 / 0.5CH3CH2OH 0.0CH3COOH
0.0C3H8O3 0.3CO2 0.40C5H7NO2P0.074
C6H12O6 / 1.9CH3CH2OH 0.0CH3COOH
0.1C3H8O3 1.9CO2 0.03C5H7NO2P0.074

direct cell-to-cell-interactions (36) or through the signal substances in the fermentation broth (17). These interactions may be
synergistic, e.g., through their different enzyme systems and
biochemical pathways (37) or antagonistic through inhibitory
substances or competition for substrate (38). Therefore, in addition
to techno economic analysis to compare the economics of the two
approaches, there is a need of specic studies to unravel how the
two microorganisms interact and how the operation conditions can
be optimized for optimum performance.
Raw starch from wild inedible cassava was successfully converted to bioethanol through co-culture of S. cerevisiae and a newly
isolated strain of Aspergillus sp. MZA-3. This was compared with a
monoculture of S. cerevisiae, using enzyme preparation from the
Aspergillus sp. MZA-3. Both approaches when supplemented with
commercial glucoamylase resulted into high ethanol yields (91%
and 95% of TY), and carbon recovery (99% and 101%), respectively.
Although the co-culture displayed longer fermentation time and
relatively low volumetric productivity, 1.2 g/L/h versus 4.5 g/L/h for
the monoculture, it could be of interest since direct bioconversion
of raw starch to ethanol is performed at 30  2 C in a single reactor,
signicantly downsize energy and operational costs. Our future
studies focus on the interactions of the two microorganisms in the
co-culture fermentation to ascertain optimum performance.
Furthermore techno-economical analysis is required to ascertain
the economics of the two approaches.

ACKNOWLEDGMENTS
This research was supported by the Swedish International
Development Cooperation Agency (Sida) and the Swedish Agency
for Research and Cooperation with Developing Countries (SAREC)
as part of the collaborative project Renewable Energy between
Sweden and Tanzania.

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