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]
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.,
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
4
5
Pretreatment
Fermentation by
(S. cerevisiae)
Without glucoamylase
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
standard temperature and pressure and thereafter converted to moles using the
common gas law;
PV nRT
W TS SC
100 100 0:9
(2)
(3)
ISg
(1)
%TY
64 GDA5011
57 Aspergillus nomius
100
Aspergillus flavus
Aspergillus oryzae
Gliocladium cibotii
100
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.,
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).
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
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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inedible wild cassava our to bioethanol, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.001