Renewable Energy 146 (2020) 1031e1040

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Valorisation of potato peel wastes for bioethanol production using

simultaneous saccharification and fermentation: Process optimization
and kinetic assessment
Naseeha A. Chohan, G.S. Aruwajoye, Y. Sewsynker-Sukai, E.B. Gueguim Kana*
University of KwaZulu-Natal, School of Life Sciences, Pietermaritzburg, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: This study optimized bioethanol production from potato peel wastes on inputs of temperature, pH and
Received 27 February 2019 solid loading using simultaneous saccharification and fermentation. Subsequently, the kinetics of yeast
Received in revised form growth and bioethanol formation under the optimized conditions were assessed using the logistic and
25 June 2019
modified Gompertz models, respectively. Maximum bioethanol concentration (22.54 g/L) and yield
Accepted 6 July 2019
(0.32 g/g) were observed under optimal process conditions of 40
C (temperature), 5.78 (pH) and 12.25%
Available online 8 July 2019
w/v (solid loading). The logistic model gave a maximum specific growth rate and maximum cell biomass
concentration of 0.20 h1 and 2.43 g/L respectively. Furthermore, the modified Gompertz model dis-
Keywords:
Bioethanol
played a maximum bioethanol production rate, maximum potential bioethanol concentration and a lag
Fermentation time of 1.51 g/L/h, 15.47 g/L and 4.66 h respectively. Optimization and kinetic findings obtained from this
Optimization study provide significant knowledge for the development of simultaneous saccharification and bio-
Potato peels ethanol production processes using starch-based agricultural wastes.
Valorisation © 2019 Elsevier Ltd. All rights reserved.

1. Introduction rich in nutrients. Additionally, its use in biofuel production pro-

Increasing industrialisation and population has led to a
continual surge in the global energy demand [1]. In the present
time, more than 80% of the global energy is produced using fossil
fuels [2]. However, fossil fuels are depleting at an alarming rate and
its combustion causes environmental pollution [3]. Therefore, there
is a need for renewable and sustainable energy sources that do not
affect the environment and its ecosystems. Biofuels have recently
emerged as ideal fuel carriers to meet these energy requirements in
a sustainable manner [4]. More specifically, bioethanol can be used
as an alternative to petroleum sources [5] and has become one of
the most dominating biofuels in industry since the majority of CO2
emissions are currently contributed by the transport sector [6]. In
addition, ethanol is renewable, possesses a higher energy and ox-
ygen content and can be stored easily [7,8].
More recently, agro-industrial and lignocellulosic-based bio-
ethanol production has been prioritized since food security is not
compromised [5]. Agro-industrial substrates such as starch-based
wastes have displayed a high availability, biodegradability and are
* Corresponding author.
E-mail address: kanag@ukzn.ac.za (E.B. Gueguim Kana).
cesses eliminates waste disposal problems. A variety of agro-
industrial feedstocks can be used as substrates for the bioconver-
sion of ethanol. Tuber crop wastes such as potato, sweet potato and
cassava are favourable substrates since they contain sufficient
quantities of starch, which can be hydrolysed to sugars and sub-
sequently fermented to ethanol [9]. Potatoes are especially suitable
as they have a high fermentable carbohydrate yield [10]. Moreover,
potatoes are the third most important food crops in the world after
rice and wheat, all of which are considered staple crops. Due to its
extensive use in industry, large volumes of potato peel wastes
(PPW) are generated. Potato processing industries produce be-
tween 20 and 50% wastes from the raw product [11]. These wastes
are usually discarded in landfills, which cause environmental
problems due to microbial spoilage [12]. The valorisation of PPW
will reduce this waste stream, greenhouse gas emissions and the
carbon footprint while generating value-added products such as
biofuels and bioproducts.
Although bioethanol is produced using waste material, pro-
duction costs are still high making the process economically un-
feasible at large scale. Recently, the simultaneous saccharification
and fermentation (SSF) approach has emerged as a more effective
method for bioethanol production compared to the conventional

https://doi.org/10.1016/j.renene.2019.07.042
0960-1481/© 2019 Elsevier Ltd. All rights reserved.
1032 N.A. Chohan et al. / Renewable Energy 146 (2020) 1031e1040
separate hydrolysis and fermentation (SHF). This is due to the
reduction in energy, time and capital costs [13]. While SHF pro-
cesses encompasses a separate saccharification and fermentation
stage, SSF involves the simultaneous hydrolysis of cellulose to
glucose with immediate conversion into ethanol by the microbial
culture. Additionally, the SSF method minimizes enzyme inhibition
by high glucose concentrations and reduces the risk of microbial
€
contamination [14]. Ohgren et al. [15] reported a 13% increase in the
ethanol yield from the SSF experiments compared to the SHF pro-
cess type.
Improvement in ethanol production has been reported by SSF
processes from starch-based feedstocks [13,16e18]. However, the
optimum SSF operating conditions for both the enzymatic hydro-
lysis and ethanol production stages by the microbe require further
investigation.
Key process parameters including solid loading, temperature,
initial pH, enzyme loading and yeast concentration have shown to
affect the ethanol productivity and yield during the SSF process
[19]. Optimization of these parameters will improve the process
efficiency by enabling maximum productivity and yield at reduced
process costs. Some studies have optimized key input parameters
for the improvement of SSF processes [13,17,20e22]. Parameter
ranges that have previously been studied include solid loading
(5e30% w/v), initial pH (4e7) and temperature (25e50
C). Yin-
gling et al. [17] evaluated the impact of pH (4.0e5.5) using high-
gravity cassava mash and observed a 1.05-fold increase in the
ethanol concentration. On the other hand, Sujeeta et al. [21] studied
the influence of temperature (30e35
C) using PPW and recorded a
1.50-fold improvement in the ethanol concentration. The study by
Zhu et al. [23] investigated the effect of corn stover solid loading
(15e25%) on the bioethanol concentration and displayed a 1.4-fold
enhancement in the ethanol concentration. Even though some
literature on SSF process optimization exists, there has been a
scarcity of sufficient knowledge on agro-industrial residues such as
PPW.
Various approaches have been explored for optimization
including the one variable at a time (OVAT), response surface
methodology (RSM), artificial neural network (ANN) and genetic
algorithm (GA) [10,24,25]. Response surface methodology (RSM) is
a statistical model that analyses the output of several experiments
by studying the influence of the identified parameters with indi-
vidual and interactive effects [26]. Thus, the RSM model could be
used for efficient optimization of bioethanol production from SSF
processes. Bioethanol process optimization using RSM models have
previously been reported [19,22,24]. For instance, the study by
Betiku and Taiwo [24] optimized various fermentation parameters
for optimal bioethanol production from bread fruit starch hydro-
lysate using RSM. Likewise, Wang et al. [19] optimized the simul-
taneous saccharification and fermentation (SSF) process using RSM
for maximum bioethanol production from sweet sorghum bagasse.
In the same vein, Sewsynker-Sukai and Gueguim Kana [22] used
RSM models for the optimization of bioethanol concentration and
conversion from corn cobs in SSF processes.
Furthermore, adequate knowledge on the dynamics of cell
growth and product formation will provide significant improve-
ment in process design and yield [27]. This can be achieved through
the kinetic studies of the SSF processes using the logistic model
which provides information on the microbial growth [28] and the
modified Gompertz model to describe the product formation [29].
Several studies have previously used the logistic model to assess
the growth pattern of S. cerevisiae during bioethanol production
[22,28]. The cell kinetics may be expressed as a function of growth
rate, initial and maximum biomass concentration and time.
Sewsynker-Sukai and Gueguim Kana [22] studied the kinetics of
Saccharomyces cerevisiae on corn cobs and reported a maximum
specific growth rate (mmax) and maximum biomass concentration
(Xmax) of 0.216 h1 and 3.650 g/L respectively. Similarly, Phukoet-
phim et al. [28] observed mmax and Xmax values of 0.154 h1 and
5.146 g/L respectively from sweet sorghum juice. On the other
hand, the modified Gompertz model describes product formation
as a function of production lag time, maximum production rate and
maximum product concentration [29]. The studies by Sewsynker-
Sukai and Gueguim Kana [22] and Phukoetphim et al. [28] re-
ported maximum ethanol production rates of 2.39 g/L/h and 1.82 g/
L/h from corn cobs and sweet sorghum juice respectively.
Therefore, the present study optimized bioethanol production
from potato peels wastes (PPW) on inputs of pH, temperature and
solid loading. Additionally, the kinetics of S. cerevisiae cell growth
and ethanol production under the optimized input conditions were
studied using the logistic and modified Gompertz models
respectively.
2. Materials and methods
2.1. Materials
The PPW used in this study was obtained from a local fast food
takeaway in Pietermaritzburg, South Africa. The substrate was oven
dried at 50
C until dry and was thereafter milled to a particle size
of less than 1e2 mm by a centrifugal miller (Retsch ZM-1, South
Africa). All chemicals used in this study were obtained from Merck
(South Africa). Termamyl (a-amylase) 120 L and Amyloglucosidase
(glucoamylase) with enzyme activities of 3000 U/ml (500 units/
mg protein) and 260 U/ml respectively were provided by the sup-
plier (Sigma-Aldrich, South Africa).
2.2. RSM experimental design and optimization of the SSF process
The RSM (Box-Behnken design) was used for the modelling and
optimization of bioethanol production. The three independent
variables pH (A), temperature (B) and solid loading (C) were the
input parameters with the bioethanol concentration and bio-
ethanol yield as the response outputs. The input parameters were
varied in the range of 4.0e7.0, 28e55
C and 5e20% (w/v) for pH,
temperature and solid loading respectively (Table 1). Input pa-
rameters and ranges were chosen based on previous works by Dash
et al. [20], Yingling et al. [17] and Shanavas et al. [30]. A total of 17
SSF experiments were generated and carried out. For each of the 17
experiments, a control experiment was simultaneously carried out
without inoculation in order to determine the glucose utilisation.
The experimental data were fitted in the quadratic polynomial
model equation, relating the input parameters to the bioethanol
concentration and bioethanol yield, according to Equation (1) using
Design Expert software (Stat-Ease Inc., USA).
Y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b11 x21 þ b22 x22 þ b33 x23
þ b12 x1 x2 þ b13 x1 x3 þ b23 x2 x3
(1)
where Y is the ethanol response, b0 is the intercept, b1 x1 to b3 x3
represents the linear blending portion, b11 x21 to b33 x23 are quadratic
coefficients and b12 x1 x2 to b23 x2 x3 are the interaction coefficients.
The significance of the model was assessed by the Analysis of
Variance (ANOVA).
2.3. Pretreatment of potato peel wastes (PPW)
The milled PPW were pretreated using a soaking assisted ther-
mal pretreatment (SATP) as previously described by Aruwajoye
 N.A. Chohan et al. / Renewable Energy 146 (2020) 1031e1040 1033

Table 1
Box-Behnken design of different process parameters affecting bioethanol concentration and bioethanol yield using PPW.

Run Input parameters Output

pH Temperature (
C) Solid loading (% w/v) Bioethanol concentration (g/L) Bioethanol yield (g/g)

1 5.50 41.50 12.50 24.36 0.29

2 5.50 28.00 5.00 4.03 0.14
3 7.00 28.00 12.50 11.61 0.14
4 4.00 41.50 5.00 8.38 0.28
5 4.00 28.00 12.50 11.46 0.15
6 5.50 41.50 12.50 29.86 0.33
7 5.50 41.50 12.50 26.46 0.31
8 7.00 41.50 5.00 6.64 0.23
9 5.50 28.00 20.00 0.21 0
10 5.50 55.00 5.00 0.36 0
11 4.00 41.50 20.00 0.69 0.16
12 5.50 41.50 12.50 23.09 0.27
13 5.50 55.00 20.00 0.45 0.03
14 4.00 55.00 12.50 0.39 0.03
15 5.50 41.50 12.50 21.8 0.26
16 7.00 41.50 20.00 23.17 0.21
17 7.00 55.00 12.50 0.35 0.05

et al. [31]. Pretreatment was carried out in 100 mL Schott bottles
with a working volume of 25 mL. The PPW solid loadings were
varied according to the experimental design (Table 1) and were
immersed in 3.68% (v/v) HCl. The pretreatment reactors were
incubated at 69.62
C for 2.57 h in a waterbath. Following the
soaking process, the pretreatment reactors were autoclaved at
121
C for 5 min.
2.4. Liquefaction process
The pH of the pretreated slurry was adjusted to neutral (pH 7)
and 125.3 Units/g of alpha amylase enzyme (Termamyl) was
thereafter added. This process was performed at 90
C and 100 rpm
for 60 min [5]. This was followed by a denaturation step that was
carried out at 96
C for 10 min and subsequent cooling to room
temperature. Compositional analysis of the pretreated sample was
determined using the National Renewable Energy Laboratory
method [32]. The total starch was determined using the Megazyme
starch kit (Megazyme, Ireland). The native PPW contained 20%
starch, 10% hemicellulose, 4.03% cellulose and 6.07% lignin.
2.5. Inoculum development and SSF processes
2.5.1. Fermentation medium formulation
Fermentation media (g/L) made up of: yeast extract, 5.0;
peptone, 5.0; (NH4)2SO4, 1.0; KH2PO4 and MgSO4, 1.0 was auto-
claved at 121
C for 15 min.
2.5.2. Microorganism and inoculum preparation
Saccharomyces cerevisiae BY4743 was generously donated by the
Department of Genetics, University of KwaZulu-Natal (Pietermar-
itzburg), South Africa. The S. cerevisiae cells were cultivated in yeast
peptone dextrose (YPD) (100 mL) media containing (g/L); yeast
extract, 10; peptone, 20 and glucose, 20 and were incubated at 30
C
and 120 rpm for 16 h. The cell count was evaluated using a Neu-
bauer counting chamber (Neubauer, Germany) and was
2.56  106 cells/mL.
2.5.3. . SSF process conditions
The SSF processes were carried out in sterilised 100 mL Erlen-
meyer flasks with a working volume of 50 mL according to the
experimental design (Table 1). Flasks containing pretreated potato
peel slurry (25 mL), fermentation media (20 mL), 15 Units/g of
glucoamylase (Amyloglucosidase) and 10% (v/v) inoculum were
incubated at their respective temperatures (Table 1) for 24 h at
120 rpm.
2.6. Determination of biomass, ethanol and glucose concentrations
Biomass concentration was determined by relating the
S. cerevisiae cell count to the dry cell weight. The yeast cells were
grown in YPD broth until it reached exponential phase and was
thereafter diluted accordingly. Each dilution was thereafter
centrifuged at 10000 rpm for 5 min. The supernatant was discarded
and the resulting pellet was oven dried at 90
C. The dry weights
with the corresponding cell counts were used to plot a standard
curve.
The ethanol concentration was measured in the gas phase of the
SSF process by using an ethanol vapour sensor (ETH-BTA, Vernier
Software and Technology, Beaverton, OR, USA). The ethanol yield
was calculated using Equation (2):
g½ethanol maximum ethanol concentration ½g=L
Ethanol yield ¼
g½glucose utilised glucose ½g=L
(2)
Sample aliquots (1 mL) were extracted at the beginning and the
end of the SSF process for glucose analysis. The glucose concen-
tration (g/L) was quantified using the Megazyme glucose assay kit
(Megazyme, Ireland) and the glucose utilisation was determined
using Equation (3):

Initial glucose concentration ðg=LÞ  Final glucose concentration ðg=LÞ

Glucose utilisation ð%Þ ¼  100 (3)
Initial glucose concentration ðg=LÞ
1034 N.A. Chohan et al. / Renewable Energy 146 (2020) 1031e1040

2.7. Validation of the SSF model using PPW 3. Results and discussion

Validation experiments were conducted under the optimized
conditions. Sample analysis was conducted every 2 h. Ethanol,
glucose and biomass concentrations were determined as stated
previously. The validation experiments with their corresponding
control experiments (not inoculated) were performed in duplicate.
Control experiments were set up to estimate the initial glucose
concentration.
3.1. Development of the RSM models
The experimental conditions with the corresponding bioethanol
concentration and yields are presented in Table 1. Experimental
data were used to develop two polynomial equations relating the
ethanol concentration and yield with the input variables as shown
in Equations (7) and (8) below:

Ethanol concentration ¼ þ 25:11 þ 2:61A  3:22 B þ 0:64C

2.8. Kinetic models and calculation of kinetic coefficients  0:047AB þ 6:06AC þ 0:98BC  5:35A2  13:81B2
 10:04C2
2.8.1. The logistic model
The logistic model in the differential form (Equation (4)) and (7)
integrated (Equation (5)) represents the exponential and stationary
phases of growth. This logistic model illustrates the relationship of Ethanol yield ¼ þ 0:29 þ 1:250  103 A  0:040 B  0:031C
biomass (X) to initial cell concentration (X0), maximum cell con-
centration (Xmax) and maximum specific growth rate (mmax) at þ 7:500  103 AB þ 0:025AC þ 0:04BC  0:011A2  0:19B2
specific times (t) during the exponential and stationary phases of  0:061C2
yeast growth.
(8)
 
dX X
¼ mmax 1 X (4) where Y represents ethanol concentration and ethanol yield for
dt Xmax
Equations (7) and (8), respectively and A, B and C represent the pH,
temperature and solid loading respectively.
X0 expðmmax tÞ The validity of the fitted models was evaluated by using the
X¼    (5)
analysis of variance (ANOVA) (Tables 2 and 3). The ethanol con-
1 X0
Xmax ð1  expðmmax tÞÞ
centration and yield models displayed high F-values of 8.75 and
34.09 with low p-values of 0.0046 and < 0.0001 respectively. The
high F values and low p-values (<0.05) indicate the model signifi-
cance [33]. The coefficient of determination (R2) values of the
models were 0.918 (ethanol concentration) and 0.978 (ethanol
2.8.2. The modified gompertz model yield), thus both these models can elucidate for 91.8% and 97.8% of
The experimental data of bioethanol production over time were variations in the observed data respectively.
fitted into the modified Gompertz model using the least squares The significance of the individual parameters was assessed us-
method, (CurveExpert V1.5.5). The model relates the bioethanol ing their p-values (Table 2). Temperature (0.0032) and solid loading
concentration (P) to the potential maximum bioethanol concen- (0.0110) significantly affected the ethanol yield, whilst the inter-
tration (Pm), maximum bioethanol production rate (r p,m) and the active effect of pH and solid loading significantly impacted on the
lag time (tL) as depicted in Equation (6). ethanol concentration (0.0380). The temperature mainly influences
the yeast metabolism and enzyme activity in SSF processes [21].
   
rp;m : expð1Þ Low temperatures cause membrane gelling of the yeast cells and as
P ¼ Pm : exp  exp : ðtL  tÞ þ 1 (6)
Pm a result cell growth is stunted. Alternatively, too high temperatures
trigger thermal lysis of the microbial cells [34], which prevents
where P is bioethanol concentration (g/L), Pm is potential maximum further cell development and metabolic processes. On the other
bioethanol concentration (g/L), r p,m is maximum bioethanol pro- hand, the solid loading parameter affects the efficiency of mass and
duction rate (g/L/h) and tL is the time from the beginning of heat transfer in the SSF process. A high viscosity is usually observed
fermentation to exponential bioethanol production (h). at an elevated solid loading, thus reducing enzyme diffusion. This

Table 2
Analysis of variance (ANOVA) of the developed ethanol concentration model.

Source Sum of squares df Mean square F-value p-value Prob > F Remark

Model 1772.46 9 196.94 8.75 0.0046 Significant

A- pH 54.34 1 54.34 2.41 0.1642
B- Temperature 82.95 1 82.95 3.68 0.0964
C- Solid loading 3.26 1 3.26 0.14 0.7147
AB 9.025  103 1 9.025  103 4.009  104 0.9846
AC 146.65 1 146.65 6.51 0.0380
BC 3.82 1 3.82 0.17 0.6926
A2 120.61 1 120.61 5.36 0.0528
B2 802.96 1 802.96 35.67 0.0006
C2 424.60 1 424.60 18.86 0.0034
Residual 157.59 7 22.51
Lack of Fit 117.60 3 39.20 3.92 0.1100 Not significant
Pure Error 39.98 4 10.00
Cor Total 1930.04 16
 N.A. Chohan et al. / Renewable Energy 146 (2020) 1031e1040 1035

Table 3
Analysis of variance (ANOVA) of the developed ethanol yield model.

Source Sum of squares df Mean square F-value p-value Prob > F Remark

Model 0.20 9 0.023 34.09 <0.0001 Significant

A- pH 1.250  105 1 1.250  105 0.019 0.8948
B- Temperature 0.013 1 0.013 19.25 0.0032
C- Solid loading 7.813  103 1 7.813  103 11.75 0.0110
AB 2.250  104 1 2.250  104 0.34 0.5790
AC 2.500  103 1 2.500  103 3.76 0.0937
BC 7.225  103 1 7.225  103 10.86 0.0132
A2 5.095  104 1 5.095  104 0.77 0.4104
B2 0.15 1 0.15 224.98 <0.0001
C2 0.016 1 0.016 23.56 0.0018
Residual 4.655  103 7 6.650  104
Lack of Fit 1.375  103 3 4.583  104 0.56 0.6698 Not significant
Pure Error 3.280  103 4 8.200  104
Cor Total 0.21 16

negatively affects sugar release for yeast cell growth and bioethanol
formation [35].
3.2. Interactive effects of process parameters on the ethanol
concentration and yield
The ethanol concentration and ethanol yield from the various
experimental runs are shown in Table 1. Ethanol concentration
ranged from 0.21 to 29.86 g/L whilst the yields ranged from 0 to
0.33 g/g. The present SSF process resulted in a high ethanol con-
centration (23.09e29.86 g/L) and yield (0.26e0.33 g/g) when all the
process inputs were at their median values (run 1, 6, 7, 12, 15.).
Alternatively, a low pH (5.50), temperature (28
C) and solid
loading (5% w/v) (run 5) gave low bioethanol concentration and
yield values of 11.46 g/L and 0.15 g/g respectively. A high pH, tem-
perature and solid loading of 7.00, 41.5
C, and 20.00% w/v
respectively (run 16) produced a high ethanol concentration (20 g/
L) and yield (0.21 g/g).
The response surface graphs showing the interactive effects of
the process parameters on the bioethanol ethanol concentration
and yield are shown in Fig. 1AeF. A simultaneous increase in the pH
from 4.00 to 5.80 and temperature from 28.0 to 40.8
C increased
the ethanol concentration from 7.2 to 25.5 g/L (Fig. 1A). Further
increase in the pH (>5.80) and temperature (>40.8
C) resulted in a
decrease in ethanol concentration from 25.5 to 14.2 g/L. Similarly,
incremental variations in the pH (4.00e6.30) and temperature
(28.0e41.5
C) led to an increase in the ethanol yield (0.14e0.29 g/
g) (Fig. 1B). Any further increase of pH and temperature above 6.30
and 41.5
C respectively reduced the process yield (0.29e0.14 g/g).
The effect of temperature on the bioethanol concentration and
yield in the present SSF process can be attributed to thermal lysis of
S. cerevisiae cells above 40
C [17]. Moreover, low temperatures
(<40
C) reduce the enzymatic saccharification efficiency and thus
negatively affect the sugar release and utilisation. SSF processes
require optimum temperature and pH conditions for maximal re-
action of both glucoamylase and S. cerevisiae [14]. Glucoamylase
was reported to have an optimum temperature and pH between 50
and 60
C and 4e5 respectively, whilst S. cerevisiae have tempera-
ture and pH optima between 28 and 32
C and 5e6 respectively
[36]. The high ethanol concentration and yield observed at tem-
peratures and pH values of 40
C and 5.80 respectively can be
accounted for by the required conditions that promote both the
enzyme and yeast activity. Yingling et al. [17] studied the effect of
pH on the bioethanol concentration from cassava mash and
demonstrated that low initial pH (4.5) prevents bacterial contam-
ination. These authors showed that the initial pH significantly
affected the final ethanol concentration during the SSF process but
exhibited a negative effect above a certain threshold (pH 5.5).
The interactive effect of the solid loading and temperature pa-
rameters on the ethanol concentration and yield whilst maintain-
ing the pH at its centre point, is shown in Fig. 1C and D respectively.
The ethanol concentration increased (5.2e25.3 g/L) with a simul-
taneous increase in the solid loading and temperature from 5.0 to
12.7% w/v and 28.0e39.3
C respectively. Any further increases in
solid loading (>12.7% w/v) and temperature (>39.3
C) negatively
affected the ethanol concentration from 25.3 to 12.0 g/L. A similar
trend was observed for the ethanol yield where increasing the solid
loading from 5.0 to 10.5% w/v and temperature from 28.0 to 40.0
C
led to an increase in the yield (0e0.30 g/g). Further increments in
the solid loading (>10.5% w/v) and temperature (>40.0
C)
decreased the yield (from 0.30 to 0 g/g). The low ethanol concen-
trations and yields (solid loading > 10.5% w/v) can be attributed to
poor mixing at high solid loadings, which reduces the enzyme ef-
ficiency and thus glucose recovery. Generally, poor mixing in-
creases the viscosity of the SSF system which causes ineffective
mass and heat transfer [37]. In addition, it leads to the development
of nutrient concentration gradients and may result in sugar osmotic
pressure effects on the S. cerevisiae cells. This causes the yeast cells
to channel their metabolic processes towards survival strategies
such as cell maintenance and biomass growth [22,38]. Sebayang
et al. [39] investigated the effect of solid loading 10e25% w/v on the
ethanol production from Manihot glaziovii starch and observed an
optimum of 23.88% w/v which was higher than the present study
(12.70% w/v). Generally, PPW display high viscosity compared to
the aforementioned tuber and therefore requires a lower solid
loading (12.70% w/v). Furthermore, the study by Sebayang et al.
[39] utilised an SHF system which allows separate hydrolysis of the
substrate prior to bioethanol production and this process type
(SHF) has previously demonstrated higher saccharification effi-
ciency and reduced initial viscosity at the beginning of fermenta-
tion [23].
The interactive effect of solid loading and pH on the ethanol
concentration and yield whilst temperature is kept at its median
value are shown in Fig. 1E and F respectively. It was observed that
simultaneous increases in the solid loading from 5.0 to 13.3% w/v
and pH from 4.0 to 6.0 resulted in an increase in the ethanol con-
centration from 12.7 to 24.5 g/L. A further increase in the solid
loading (>13.3% w/v) and pH (>6.0) reduced the bioethanol con-
centration from 24.5 to 21.0 g/L. The ethanol yield displayed a
similar trend with these process parameters. For instance, varia-
tions in the solid loading from 5.0 to 13.5% and pH from 4.0 to 6.0
displayed an increase in the ethanol yield (0.26e0.29 g/g). In
addition to nutrient concentration gradients, poor mixing due to
elevated solid loadings (>13.0%) have shown to affect enzymatic
1036 N.A. Chohan et al. / Renewable Energy 146 (2020) 1031e1040

Fig. 1. Response surface curves showing the interactive effect of: (A) temperature and pH (ethanol concentration), (B) temperature and pH (ethanol yield), (C) solid loading and
temperature (ethanol concentration), (D) solid loading and temperature (ethanol yield), (E) solid loading and pH (ethanol concentration), (F) solid loading and pH (ethanol yield).
 N.A. Chohan et al. / Renewable Energy 146 (2020) 1031e1040
Table 4
Validation of the optimized ethanol concentration and yield models.
Run pH Temperature (
C) Solid loading (% w/v)
- - - -
1 5.78 40 12.25
saccharification and this directly influences the yeast cell devel-
opment and sugar assimilation in SSF bioprocesses [37]. Negative
effects on the S. cerevisiae cell metabolism impacts on the ethanol
production pathway since it is a primary metabolite and has been
shown to exhibit a mixed growth associated pattern [40]. While
low pH conditions (<5.0) reduce the risk of microbial contamina-
tion, it also favours acetic acid production. This compound is lip-
osoluble and in its undissociated form, which causes intracellular
anion accumulation. The acetic acid then dissociates, triggering a
lower cell pH and has shown to inhibit microbial activity during
bioethanol production [41]. Alternatively, high pH values (>5.8)
result in a high concentration of protons in the fermentation me-
dium, which changes the overall charge on the cell plasma mem-
brane [42]. This can affect the permeability of essential nutrients
from entering the cell, causing inefficient growth and therefore low
conversion of bioethanol. Zhu et al. [23] noted a 1.4-fold increase in
bioethanol concentration during SSF from corn stover, when the
solid loading was increased from 15 to 25% w/v. Substrates such as
PPW are light weight due to their low density and this results in
highly viscous solutions and poor mixing under high substrate
loading. An optimal solid loading of 13% w/v was observed in the
current study.
3.3. Validation of the developed RSM models and comparison with
previous reports
Validation experiments under the optimal conditions were
conducted to maximize the ethanol concentration and yield
(Table 4). The predicted optimum conditions for maximum ethanol
concentration and yield were 5.78, 40
C and 12.25% w/v for pH,
temperature and solid loading respectively corresponding to a
bioethanol concentration and yield of 25.53 g/L and 0.29 g/g
respectively. The optimized conditions produced a slightly lower
ethanol concentration of 22.54 g/L compared to the model predic-
tion of 25.53 g/L. Sujeeta et al. [21] reported similar optimum SSF
input conditions (temperature of 35
C and pH of 6.0) for bio-
ethanol production from PPW, corresponding to an ethanol con-
centration of 2.83% v/v. The ethanol yield observed in this study
was compared with previous reports on starchy substrates
(Table 5). The present work recorded a lower ethanol yield (0.32 g/
g) compared to the studies by Arapoglou et al. [43] (0.46 g/g),
Sanusi et al. [44] (0.42 g/g) and Aruwajoye et al. [45] (0.58 g/g).
Variations in the ethanol yields may be attributed to differences in
the agro-residue composition, which has shown to impact on the
enzymatic saccharification efficiency. Effects on the enzymatic
saccharification influence glucose production and consumption
processes and thus bioethanol formation.
Table 5
Comparison of ethanol yields produced from starch-based substrates.
Substrate Process conditions
Potato peel waste 5.78a, 40
Cb, 12.25% w/vc
Potato peel waste 5.0a, 32
Cb, 2% w/vc
Potato peel waste 7a, 37
Cb,10% w/vc
Cassava peel waste 7a, 30
Cb,10.16% w/vc
a
pH.
b
temperature.
c
solid loading.
1037
Ethanol concentration (g/L) Ethanol yield (g/g)
Predicted Observed Predicted Observed
25.53 22.54 0.29 0.32
3.4. Kinetic assessment
3.4.1. Yeast cell growth using the logistic model
Variations in the growth pattern of S. cerevisiae over time are
shown in Fig. 2A. The lag phase in the microbial growth curve was
observed during the first 2 h of the SSF process. The cell number
increased exponentially from 2 to 16 h. A relatively short time is
required for the amyloglucosidase enzyme (in this case, 2 h) to
release the glucose for consumption by the microbial culture [13].
The stationary phase was reached after 17 h which corresponded
with the depletion of fermentable sugars. In addition, depletion of
other nutrients in the fermentation media and accumulation of
toxic by-products hampers the yeast cell growth [46]. The growth
data fit the regression model with a correlation coefficient (R2) of
0.980. The observed kinetic parameters for biomass growth on PPW
were compared to previous studies in Table 6. A maximum specific
growth rate (mmax) and maximum cell biomass concentration
(Xmax) of 0.195 h1 and 2.428 g/L respectively, were obtained.
Previous kinetic studies on bioethanol production gave comparable
mmax and Xmax values. For instance, Yan et al. [47] studied the ki-
netics of S. cerevisiae on food waste under a sugar concentration of
93.71 g/L and reported a mmax and Xmax of 0.135 h1 and 3.754 g/L
respectively. Similarly, Phukoetphim et al. [28] observed a mmax and
Xmax of 0.154 h1 and 5.146 g/L respectively from sweet sorghum
juice containing 160 g/L of reducing sugar. Likewise, Sewsynker-
Sukai and Gueguim Kana [22] recorded a mmax of 0.216 h1 and
Xmax of 3.650 g/L. Variations in the kinetic coefficients compared to
previous experiments can be ascribed to other factors such as the
difference in yeast strain, substrate type and operating fermenta-
tion conditions including initial enzyme loading pH, temperature
and solid loading.
3.4.2. Bioethanol production kinetics using the modified Gompertz
model
Changes in bioethanol production over time during the SSF
process is shown in Fig. 2B.
A short lag phase of 2 h was observed for bioethanol production
and this coincided with the lag phase of yeast cell growth. This
suggested that 2 h was required for the S. cerevisiae cells to adapt to
the medium and for sufficient glucose to be produced by glucoa-
mylase. The ethanol concentration slightly increased (0.73e8.06 g/
L) after 2e8 h. Thereafter, a sharp increase in the ethanol concen-
tration from 8.06 to 22.53 g/L was observed from 8 to 16 h. The
exponential phase for bioethanol production coincided with a high
glucose utilisation (97%) from 8 to 16 h (Fig. 3). The complex nature
of SSF processes requires the simultaneous release and degradation
of glucose monomers. During the exponential phase, sufficient
Ethanol yield (g/g) Reference
0.32 This study
0.46 [43]
0.42 [44]
0.58 [45]
1038 N.A. Chohan et al. / Renewable Energy 146 (2020) 1031e1040

Fig. 2. (A) S. cerevisiae biomass growth and (B) bioethanol production during SSF process.

Table 6
Kinetic coefficients obtained from the logistic model compared to previous studies.

Substrate mmax (h1) Xo (g/L) Xmax (g/L) Reference

Potato peel waste 0.195 0.441 2.428 This study

Sugar beet juice 0.169 2.469 7.909 [29]
Sweet sorghum juice 0.154 0.587 5.146 [28]
Food waste 0.135 0.381 3.754 [47]
Corn cobs 0.216 0.556 3.650 [22]

Мmax ¼ maximum specific growth rate, Xo ¼ initial cell concentration,

Xmax ¼ maximum cell concentration.

glucose was continuously produced to sustain the metabolic

pathway of the yeast culture for ethanol production. The highest
bioethanol concentration of 23.53 g/L was observed during the
exponential phase and was associated with a rapid conversion of
the glucose monosaccharide during the SSF process. Ethanol con-
centration decreased after the exponential phase (>16 h) corre-
sponding to a gradual deceleration in the yeast growth and the
beginning of the stationary phase. Decreases in the ethanol con-
centration can be attributed to glucose depletion, ethanol oxidation
and organic acid (acetic acid) formation [41]. Reversible oxidation
of ethanol to aldehydes occurs by alcohol dehydrogenases (ADHs)
with a reduction of NADþ or NADPþ [48]. Furthermore, the accu-
mulation of ethanol in fermentation broth may have resulted in the
deactivation of essential ethanol producing enzymes such as py-
ruvate decarboxylase and alcohol dehydrogenase [49]. Experi-
mental data fitted the modified Gompertz model with a high R2
value of 0.98. The observed kinetic parameters were compared to
previous kinetic studies on bioethanol formation (Table 7). The
observed potential bioethanol concentration (Pm) (15.48 g/L) from
PPW and was comparable to previous bioethanol kinetic studies on
kefir (16.37 g/L) [50] and sorghum leaves (17.15 g/L) [51]. However,
bioethanol produced from Manihot glaziovii starch [39] produced a
high Pm value of 87.47 g/L. This relatively higher Pm value is
accounted by the fact that whole tuber of Manihot glaziovii with
high starch content corresponding to a glucose concentration of
170.28 g/L was used. Similar observations were recorded for sugar
beet raw juice [29] and sweet sorghum juice [28]. Despite the high
Pm values (>70 g/L) obtained in the aforementioned reports
Fig. 3. Glucose utilisation by S. cerevisiae during SSF process.
compared to the present study (15.48 g/L), substrates such as
Manihot glaziovii starch, sugar beet raw juice and sweet sorghum
juice threaten global food security and are therefore not feasible for
potential industrial scale application. The maximum bioethanol
production rate (rp,m) value indicates that 1.51 g/L of ethanol was
produced every hour. Similar rp,m values of 1.84 g/L/h and 1.82 g/L/h
were observed using Manihot glaziovii starch [39] and sweet sor-
ghum juice [28] respectively. A longer lag time (tL) of 4.66 h was
observed in this study compared to previous kinetic studies on corn
cobs [22], sugar beet raw juice [29] and sweet sorghum juice [28],
indicating that a longer time was required for S. cerevisiae to adapt
to the fermentation medium with PPW. The longer lag time
observed in the current study was required for the hydrolysis of
starch to glucose using glucoamylase enzymes [29].
4. Conclusion
The present study optimized bioethanol production from potato
 N.A. Chohan et al. / Renewable Energy 146 (2020) 1031e1040 1039

Table 7 THLC0409, Biomass Bioenergy 34 (12) (2010) 1922e1929.

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bioethanol production from corn cobs: process optimization and kinetic
studies, Bioresour. Technol. 262 (2018) 32e41.
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Acknowledgements
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(NRF) of South Africa (Grant number: 11157) for financially facili- and artificial neural network, Renew. Energy 74 (2015) 87e94.
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those of the author and are not necessarily attributed to the NRF. Zymomonas mobilis using response surface methodology and genetic algo-
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