Fuel 202 (2017) 260–270

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Full Length Article

Simultaneous saccharification and fermentation of ethanol from potato

waste by co-cultures of Aspergillus niger and Saccharomyces cerevisiae in
biofilm reactors
Gulten Izmirlioglu a, Ali Demirci a,b,⇑
a
Department of Agricultural and Biological Engineering, The Pennsylvania State University, University Park, PA 16802, United States
b
The Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, PA 16802, United States

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Ethanol was produced in biofilm SSF Ethanol Production by A. niger and S. cerevisiae

reactors by A. niger and S. cerevisiae. 45

40
70

60
Ethanol and Glucose (g/L)

Plastic composite supports were 35

Enzyme Activity (U/ml)

50
30

employed for promoting biofilm 25

20
40

30

formation of co-culture. 15
10
20

Optimum conditions for maximum 5

10

0 0

ethanol production in biofilm reactor 0 10 20 30 40

Time (h)
50 60 70 80

were pH 5.8, 35 °C, and no aeration.

Glucose (g/L) Ethanol (g/L) Enzyme Activity (U/ml)

Max ethanol production of 37.93 g/L

was achieved with 0.41 g etoh /g
starch yield.

Biofilm reactor with PCS

Biofilm formation on PCS

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

Article history: Novel approaches for bioethanol production from industrial wastes have gained attention due to not only
Received 17 January 2017 maximize the ethanol production productivity, but also reduce the production cost. Part of the produc-
Received in revised form 10 April 2017 tion cost includes cost of the enzymes needed for saccharification step during the starch hydrolyzation,
Accepted 11 April 2017
which can be significant cost depending on the enzymes’ performances. Therefore, in this study, simul-
taneous saccharification and fermentation (SSF) of ethanol by co-cultures of Aspergillus niger and
Saccharomyces cerevisiae was assessed in a potato waste based medium by using biofilm reactors. The
Keywords:
plastic composite supports (PCS) were studied for biofilm formation. Effects of temperature, pH, and aer-
Biofilm
SSF
ation rates in biofilm reactors were evaluated by response surface methodology and the optimal condi-
Co-culture tions were found to be 35 °C, pH 5.8, and no aeration. The maximum ethanol concentration of 37.93 g/L
Bioethanol was achieved at the end of 72 h fermentation, with a 0.41 g ethanol/g starch yield. Finally, biofilm forma-
Potato waste tion of co-culture on PCS was also evaluated by scanning electron microscope. In conclusion, the results
Biofuel demonstrated that PCS can be utilized for SSF processes for ethanol production in biofilm reactors with
co-cultures by using starchy industrial wastes.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction

⇑ Corresponding author at: Department of Agricultural and Biological Engineer- Bioethanol is one of the alternative renewable fuels to
ing, The Pennsylvania State University, University Park, PA 16802, United States. substitute petroleum fuels and has already been used in the
E-mail address: demirci@psu.edu (A. Demirci).

http://dx.doi.org/10.1016/j.fuel.2017.04.047
0016-2361/Ó 2017 Elsevier Ltd. All rights reserved.
 G. Izmirlioglu, A. Demirci / Fuel 202 (2017) 260–270
transportation industry. Bioethanol production reached 54 billion
liters in 2014 in the USA [1]. Currently, bioethanol is mainly pro-
duced from sugar and starchy food crops, such as sugar cane (Bra-
zil), corn (USA), wheat (European countries), and sorghum since
processing these crops are well established with higher ethanol
yields. However, using food crops for bioethanol production is of
concern due to agricultural land and water use [2], as well as the
high costs of food crops [3]. Lignocellulosic biomass (agricultural
and forestry residues) and algae are promissing substrates in terms
of cost and availability, however, bioethanol production from these
substrates, currently, faces some processing difficulties, which
yield lower ethanol productions at higher production costs in com-
parison to simple sugars and starchy food crops. Utilization of
wastes and by-products of food industry, therefore, has great
potential for bioethanol production due to the fact that these sub-
strates contain sufficient amounts of fermentable sugars and/or
hydrolysable starch, and are inexpensive. Potato industry waste,
especially, has great potential for bioethanol production due to
high starch content. The most notable examples can be potato
pulp, potato processing water, peels, and waste potato mash [4,5].
The conventional ethanol production from starch requires a
two-step pretreatment process, liquefaction and saccharification,
before fermentation. Alpha-amylase is employed for liquefaction,
while glucoamylase is used for saccharification step to produce
glucose monomers. Because liquefation and saccharification are
usually performed at elevated temperatures (around 95 °C and
60 °C, respectively), energy demand is high [6]. Another drawback
is enzyme inhibition caused by accumulation of glucose during
saccharification process [7]. Therefore, simultaneous saccharifica-
tion and fermentation (SSF) can be an alternative to prevent
enzyme inhibition, provide shorter processing times, decrease
energy demand, and eventually reduce production costs [8,9]. For
SSF, either commercial enzymes or amylase producing microor-
ganims can be employed along with ethanol producing microor-
ganisms. However, the biggest challenge of co-culturing is
different nutrient and culture requirements of the microorganisms
[6], and must be studied for an efficient ethanol production. Oberoi
et al. [10] studied SSF ethanol production from banana peels using
enymes and S. cerevisiea and reported that under optimum condi-
tions (9 FPU/g-cellulose cellulase, 72 IU/g-pectin pectinase, 37 °C
and 15 h), 28.2 g/L of ethanol was produced. In another study,
SSF ethanol production from damaged sorghum and rice grains
by co-cultures of A. niger and S. cerevisiae were evaluated, and
the maximum ethanol production was reported as 29 and 20.9 g/
L from damaged sorghum and damged rice grains, respectively
[11].
Biofilm, the natural attachment of microorganisms to a surface,
could resolve the problems of co-culturing process. It is well docu-
mented that microorganisms in a biofilm are resistant to extreme
environmental conditions such as pH and temperature, contami-
nants, hydraulic shocks, antibiotics, and toxic substances [12]
and co-immobilized microorganisms might adapt to their new
environmental conditions better than single culture systems [8].
Because biofilm formation occur naturally, providing the right sup-
port material play a crucial role for a successful biofilm formation
[13]. The characteristics of a desirable solid support can be listed as
inexpensive, favorable to microorganisms, easily available, resis-
tant to mechanical force, sufficient surface area, and porosity
[13]. Plastic composite supports (PCS) are one of the supports
made from polypropylene and agricultural products which were
developed at Iowa State University [14]. In this support,
polypropylene provides the matrix for the mixture of agricultural
products. PCS is suitable for long run fermentations because of
its robustness, strength, longevity, as well as the slow nutrient
leaching [12]. Application of PCS in biofilm reactors for various
value-added products have already been studied in bench-scale,
261
and showed improvements in nisin, ethanol, bacterial cellulose,
pullulan, lactic acid and lysozyme productions [15–20].
In this study, growth conditions of co-cultures of Aspergillus
niger and Saccharomyces cerevisiae were optimized for maximum
ethanol production from industrial potato waste in PCS biofilm
reactors. Even though, PCS has been investigated for various
value-added products, it has been only used for single culture sys-
tems. To the best of the authors’ knowledge, this is the first study
that PCS biofilm reactors were used for co-culturing as well as SSF
process for ethanol production.
2. Materials and methods
2.1. Microorganisms and media
Saccharomyces cerevisiae (ATCC 24859) was obtained from the
American Type Culture Collection (Manassas, VA, USA). The culture
was grown in a medium containing 20 g/L of glucose, 6 g/L of yeast
extract, 0.3 g/L of CaCl22H2O, 4 g/L of (NH4)2SO2, 1 g/L of MgSO4-
7H2O, and 1.5 g/L of KH2PO4 at 30 °C for 24 h [21]. The activity
of the culture was maintained by storing at 4 °C and sub-
cultured biweekly, while stock cultures were kept in 20% glycerol
at 80 °C.
Aspergillus niger van Tieghem (NRRL 330) was obtained from the
USDA’s Agricultural Research Service (Peoria, IL). It was grown on
Petri plates containing malt extract agar (20 g/L of malt extract,
20 g/L of glucose, 1 g/L of peptone, and 20 g/L of agar) at 30 °C
for 120 h [22]. A. niger spore suspension was prepared as follows:
Fungi were grown on malt extract agar plates for 5 days at 30 °C.
At the end of the incubation, A. niger spores were suspended by
using 2 ml of 0.1% sterile peptone water per plate. Then, spore sus-
pension (1%), which had 106 CFU/ml was used to inoculate. In order
to maintain viability, the cultures were stored at 4 °C and sub-
cultured every three weeks. Stock spore cultures were grown as
described above, however washed off with 20% glycerol solution
and kept in 20% glycerol at 80 °C.
For PCS selection, the growth medium for each microorganism
was used during the culture tube fermentation. The glucose con-
centration of S. cerevisiae medium was increased to 50 g/L. The fer-
mentation medium that was used for biofilm reactor runs
consisted of 92.37 g/L (dry basis) of industrial waste potato mash,
59.42 g/L of malt extract, and 0.159 g/L of FeSO47H2O [23]. The
industrial waste potato mash slurry was blended for 3 min and
strained with a regular kitchen strain to remove large peels, then
the slurry was supplemented with malt extract and FeSO47H2O.
Then, the medium was autoclaved for 30 min at 121 °C.
2.2. Plastic composite support (PCS)
The PCS tubes evaluated in this study were manufactured at
Iowa State University (Ames, IA) using a twin-screw corotating
Brabender PL2000 extruder (model CTSE-V; C.W. Brabender Instru-
ments, Inc., South Hackensack, NJ) [14]. The polypropylene, organic
and inorganic ingredients were mixed and extruded at 13 rpm
through a medium pipe die at high barrel temperatures, 200,
220, and 200 °C. The PCS tubes had 2.5 mm wall thickness and
10.5 mm outer diameter. According to the prior studies conducted
in our lab and other published work, four types of PCSs were
selected. Table 1 shows the compositions of PCS tubes studied in
this study. The common ingredients for all of the PCS tubes were
polypropylene (PP) 50% (w/w), soybean hulls (SH), soybean flour
(SF), and salts (S) while yeast extract (YE), dried bovine albumin
(BA) and dried bovine red blood cell (RBC) were the ingredients
only some of the PCS tubes contained.
262 G. Izmirlioglu, A. Demirci / Fuel 202 (2017) 260–270

Table 1
The composition of plastic composite supports (PCS).

Support Types Ingredients of extrusion mixture (% [w/w])

PPa SHb SFc YEd BAe RBf Saltsg
SH-SF-S 50 40 10 +
SH-SF-YE-S 50 40 5 5 +
SH-SF-YE-BA-S 50 35 5 5 5 +
SH-SF-YE-RBC-S 50 35 5 5 5 +
a
PP: Polypropylene resins (Quantum USI Divisions, Cincinnati, OH).
b
SH: Ground (20-mesh), vacuum dried (48 h at 110°C; 30 in of mercury) soybean hulls (Cargill Soy Processing Plant, Iowa Falls, Iowa).
c
SF: Defeated soybean flour (Archer Daniels midland, Decature, III.).
d
YE: Yeast extract (Ardamine Z; Champlain Industries Inc.).
e
BA: Dried bovine albumin (American Protein Crop., Ames, Iowa).
f
RB: Dried bovine red blood cells (American Protein Crop., Ames, Iowa).
g
Salts: Mineral salts; 2 g/kg of sodium acetate, 1.2 g/kg MgSO47H2O, and 0.06g/kg MnSO47H2O.

2.3. Culture tube fermentation for PCS selection
Four types of PCSs (Table 1) were evaluated for both of the
microorganisms separately in response to ethanol production,
enzyme activity, released glucose, and biomass formation in cul-
ture tube fermentation in triplicate. The PCS tubes were cut into
approximately 3 mm rings, and 3 g of those PCS rings were used
for each replicate. PCS rings were dry-sterilized in culture tubes
for 1 h at 121 °C. Under aseptic conditions, the sterilized medium
(15 ml) was added to the sterile PCS rings and incubated at 30 °C
for 24 h to hydrate them. The soaking medium was discarded
and the fresh sterile medium was decanted aseptically. Fermenta-
tions were started with either 1% (v/v) 24-h grown S. cerevisiae or
120-h grown A. niger spore suspension. Fermentation was carried
out at 30 °C at 150 rpm in a shaker incubator 24 h for S. cerevisiae,
120 h for A. niger. Five repeated-batch fermentation runs were con-
ducted by replacing the broth with fresh sterile medium every 24 h
or 120 h to promote the biofilm formation. Ethanol production,
enzyme activity, and released glucose concentrations were evalu-
ated under three consecutive repeated-batch fermentations fol-
lowing biofilm formation, while the biomass was determined at
the end of three consecutive repeated-batch fermentations. The
PCS type that was selected to conduct the biofilm reactor was cho-
sen according to the biomass production on the PCS (log CFU/g
PCS), ethanol production (g/L), enzyme activity (U/ml) and released
glucose concentrations in the fermentation broth.
2.4. SSF in biofilm reactor
The selected PCS type was used to build the biofilm reactor as
demonstrated in Fig. 1. The PCSs were cut into 6.5 cm long tubes
and 12 PCS tubes were attached to the agitator shaft in a grid-
like fashion creating six rows of two parallel tubes. Sartorious Bio-
stat B Plus bioreactor (Allentown, PA) with a 2 L vessel was used.
The reactor vessel was sterilized with deionized (DI) water at
121 °C for 60 min after the PCS installation. After the sterilization,
water was pumped out and 1.5 L of pre-sterilized fermentation
medium (121 °C for 30 min) was pumped in aseptically to the reac-
tor vessel. After inoculation with 3% (v/v) 24-h grown culture of S.
cerevisiae (106 CFU/ml) [21] and 1% 120-h grown spore suspen-
sion of A. niger, five repeated batch fermentations were completed
to allow the biofilm formation on the PCS. The fermentation runs
for biofilm formation were carried out at pH 5, 30 °C, 150 rpm, with
no aeration for 72 h. pH was controlled by addition of either 2 N
NaOH or 2 N HCl. Once biofilm formed, batch fermentations were
subsequently performed under the conditions designed by Box-
Behnken design of surface response methodology to evaluate tem-
perature (25–35 °C), pH (4–6), and aeration rate (0–1.5 vvm)
(Table 2). Samples were taken every 6 or 12 h throughout of the
72-h fermentation. Each condition was replicated, and ethanol pro-
duction was used as the response for optimization. The identified
optimum conditions were validated twice experimentally. Even
though glucose levels were measured for co-culture fermentation,
glucose concentration showed constant levels throughout the fer-
mentation since released glucose was consumed by S. cerevisiae
simultaneously. In addition, other sugars including maltose, fruc-
tose, arabinose, xylose, galactose were analyzed for these valida-
tion runs to have a better picture in terms of sugar profile during
the SSF process.
After all the fermentation runs were completed, the biofilm
reactor was disassembled and the PCS tubes were removed asepti-
cally. The biofilm formation on PCS tubes were visually evaluated
by scanning electron microscope (SEM).
2.5. Analysis
2.5.1. Biomass on PCS
The biofilm population on the PCS was enumerated by stripping
sand method [16]. The PCS rings in the culture test tubes were
washed in 100 ml of sterile 0.1% (w/v) peptone water by turning
the tubes upside-down 10 times. Then, PCS rings were aseptically
transferred into a 50-ml test tube containing 5 g of sterile sand and
9 ml of sterile peptone water. Then, the tubes were vortexed three
times in 30-s intervals. A serial of dilutions of sand stripped sam-
ples were carried out in 0.1% sterile peptone water and, then, S.
cerevisiae and A. niger cells were spirally plated on potato dextrose
agar (Difco, MD) and malt agar, respectively. Spiral plating was
done to enumerate the cell population using a spiral auto-plater
(Model 4000, Spiral Biotech, Norwood, MA) and Q-count software
(Version 2.1; Spiral Biotech, Norwood, MA). Plates were incubated
at 30 °C for 24 h and 72 h for S. cerevisiae and A. niger, respectively.
Q-count software (Version 2.1; Spiral Biotech) was used for enu-
meration. Results were indicated as log10 CFU/g PCS.
2.5.2. Ethanol and glucose
Samples of the culture tube fermentation for PCS selection were
evaluated for glucose and ethanol using the YSI Biochemistry Ana-
lyzer (Model 2700, Yellow Springs, OH). Samples were diluted up
to 20-fold by using DI water to bring the concentration levels of
glucose and ethanol to the equipment’s measurement range. Then,
the samples were analyzed using the YSI Biochemistry Analyzer.
However, samples of the fermentations in biofilm reactors were
analyzed for ethanol and glucose with a Waters’ high pressure liq-
uid chromatography (HPLC) equipped with a refractive index
detector (Waters, Milford, MA). Separation of ethanol and glucose
was carried out using Bio-Rad Aminex HPX-87H column
(300 mm  7.8 mm; Bio-Rad, Richmond, CA) with 0.8 ml/min of
0.012 N sulfuric acid as mobile phase. The detector and column
 G. Izmirlioglu, A. Demirci / Fuel 202 (2017) 260–270 263

Fig. 1. Diagram of the PCS biofilm reactor and actual image of PCS tubes on the agitator shaft before (left) and after (right) biofilm formation.

Table 2
Effect of growth parameters on SSF ethanol production in biofilm reactors with PCS by A. niger and S. cerevisiae.

Experimental Predicted
Temperature (°C) pH Aeration (vvm) Ethanol (g/L) Glucoamylase Activity (U/ml) Ethanol (g/L) Glucoamylase Activity (U/ml)
30 4 0 13.08 26.5 10.82 19.61
30 5 0.75 15.07 7.36 18.03 5.29
30 6 0 37.58 40.11 35.52 38.86
35 5 1.5 15.94 25.67 12.35 16.92
25 4 0.75 10.12 2.92 8.80 1.07
35 4 0.75 10.09 0.10 11.62 7.50
25 5 1.5 12.86 0.88 12.12 1.48
25 6 0.75 24.00 7.67 22.48 0.18
35 5 0 22.84 22.11 23.58 21.51
30 5 0.75 18.35 3.73 18.03 5.29
30 6 1.5 22.86 7.58 25.12 14.47
25 5 0 5.32 5.99 8.91 14.74
30 4 1.5 11.14 24.89 13.20 26.15
35 6 0.75 33.24 14.11 34.56 15.96
30 5 0.75 20.68 4.79 18.03 5.29

temperatures were constant at 35 and 65 °C, respectively. The
samples were centrifuged 4 °C for 20 min at 5200g in order to sep-
arate the particles and the cells from the samples. Then, super-
natant was filtered with 0.2 lm nylon syringe filters (VWR,
Radnor, PA), and injected to the HPLC.
2.5.3. Enzyme activity
Extracellular glucoamylase activity in the culture broth was
determined by measuring the glucose released from the starch as
described by Lemmel et al. [24] with minor modification. In order
to remove the cells, samples were centrifuged at 4 °C for 20 min at
5200g, and the supernatant was used for glucoamylase activity
analysis. Culture supernatant (0.5 ml) was mixed with 0.5 ml of
1.0% potato starch dissolved in a 0.01 M acetate buffer at pH 4.8.
Tubes were incubated in a 30 °C water bath for 30 min and then
placed in boiling water for 15 min to stop the enzyme activity.
Finally, the released glucose was measured using the YSI Glucose
Analyzer. One unit of glucoamylase activity is defined as the
amount of enzyme that releases 1 mmol of glucose (0.18 mg) from
starch in 30 min at 30 °C.
2.5.4. Sugar analysis
Sugar profile for the validation run at the optimum conditions
was determined by Dionex IC 3000 Ion Exclusion Chromatography
264 G. Izmirlioglu, A. Demirci / Fuel 202 (2017) 260–270
(Dionex Corporation, Sunnyvale, CA). Samples were filtered
through a 0.2 lm nylon filters after serially diluted 103 fold. Sepa-
ration was carried out by high pH anion exchange at 30 °C using
CarboPac PA20 guard (3 x 30 mm) and analytical (3  150 mm)
columns with 200 mM sodium hydroxide (NaOH) for 40 min at a
flow rate of 0.5 ml/min, and dropped to 50 mM during the sample
injection. Sugars were detected by pulsed amperometric [electro-
chemical] detection at gold working electrodes, using quadruple
waveform.
2.5.5. Scanning electron microscope (SEM)
Exterior and interior surfaces of the PCS tubes were visually eval-
uated for biofilm formation by using SEM. The biofilm structure was
maintained by chemical fixation of the cells. PCS tubes were soaked
in 2.5% gluteraldehyde in 0.1 M phosphate buffer (pH 7.2) with
0.02% Triton X-100 and stored at 4 °C for overnight. After primary
fixative discarded, samples were washed 3–5 times with phosphate
buffer. Samples, then, were serially washed with 25%, 50%, 70%, 85%,
95% and 100% ethanol for 5 min. Later, critical point drying were
carried out [25,26]. Finally, SEM micrographs of critical-point dried
PCS tubes were taken with Zeiss Sigma Variable Pressured Field
Emission Electron Scanning Microscope (VP-FESEM).
2.5.6. Statistical analysis
Fermentations were analyzed using ethanol production,
enzyme activity, released glucose concentrations, and biomass as
responses. Minitab Software (Version 16.1, State College, PA) was
used for ANOVA and the Tukey’s comparison test to determine
the PCS type for biofilm formation. During the statistical analysis
of Box-Behnken design, the ANOVA and regression analysis were
obtained to determine the coefficients of the predictive model
and significant terms (Minitab Statistical Software). The optimum
conditions were determined by using the Response Optimizer tool
in Minitab.
3. Results and discussion
3.1. PCS selection
The selection of PCS type was carried out in culture tube fer-
mentation to investigate the four different PCS types for A. niger
and S. cerevisiae separately. The released glucose concentrations,
glucoamylase activity, and biomass for A. niger on each PCS types
were presented in Fig. 2(A). The released glucose concentrations
were ranged from 4.4 g/L to 7.60 g/L depending on the PCS type,
and the PCS composed of SH-SF-YE-BA-S resulted the highest glu-
cose release, 7.60 g/L. It was followed by SH-SF-S (7.30 g/L) and SH-
SF-YE-S (6.13 g/L). The lowest glucose release was obtained from
the PCS with SH-SF-YE-RBC-S (4.40 g/L). The differences among
the means of released glucose of SH-SF-S, SH-SF-YE-BA-S and SH-
SF-YE-S were not statistically significant. However, the PCS with
SH-SF-YE-RBC-S produced significantly lower glucose than SH-SF-
S and SH-SF-YE-BA-S (p-value < 0.05), though the difference
between SH-SF-YE-RBC-S and SH-SF-YE-S did not show a
significance (p-value > 0.05). Enzyme activity, on the other hand,
did not show any statistical difference among the PCS types
(p-value > 0.05). The highest enzyme activity was found to be
6.87 U/ml and obtained from the PCS composed of SH-SF-YE-BA-
S. The lowest enzyme activity was 6.34 U/ml and observed with
the PCS with SH-SF-S. On the other hand, the biomass on PCS types
were varied from 2.73 log10 CFU/g PCS to 4.75 log10 CFU/g PCS,
and revealed significant difference among the PCS types
(p-value = 0.00). The highest biomass formation was observed on
the PCS with SH-SF-YE-S, 4.75 log10 CFU/g PCS and followed by
SH-SF-S (4.38 log10 CFU/g PCS) and SH-SF-YE-RBC-S (2.97 log10
CFU/g PCS). The lowest biofilm formation was resulted from the
PCS with SH-SF-YE-BA-S (2.73 log10 CFU/g PCS).
For S. cerevisiae, the ethanol productions and biomass for each
PCS types were presented in Fig. 2(B). The ANOVA test revealed
that PCS types had statistically significant effect on ethanol pro-
ductions (p-value < 0.05). The highest ethanol production
(21.68 g/L) was observed with the PCS composed of SH-SF-YE-
RBC-S and followed by SH-SF-YE-S (21.58 g/L ethanol). The lowest
ethanol production, on the other hand, obtained from the PCS with
SH-SF-YE-BA-S (20.7 g/L). No statistically significant difference was
found among the PCSs that were composed of SH-SF-S, SH-SF-YE-S,
and SH-SF-YE-RBC-S. However, the ethanol production of the PCS
composed of SH-SF-YE-BA-S was significantly lower than the PCSs,
SH-SF-YE-S and SH-SF-YE-RBC-S (p-value < 0.05). Yet, no signifi-
cant difference was determined between SH-SF-YE-BA-S and SH-
SF-S in terms of ethanol production. For S. cerevisiae, the biomass
formations on PCS were varied from 6.70 log10 CFU/g PCS to 6.93
log10 CFU/g PCS, however, showed no significant difference among
the PCS types (p-value > 0.05).
To form the biofilm in the reactor, a PCS that is favorable to not
only A. niger, but also S. cerevisiae was targeted and, the PCS with
SH-SF-YE-S was found to be capable of promoting the biomass for-
mation as well as the product production for both of the microor-
ganisms. Even though glucose conversion by A. niger on the
selected PCS was lower than other two PCSs (SH-SF-S and SH-SF-
YE-BA-S), the difference was not statistically significant. In terms
of enzyme activity, no difference was observed among the PCSs.
Additionally, the highest biomass was obtained on selected PCS,
which was also statistically significant. In regards to S. cerevisiae,
none of the PCSs showed significant differences on biomass pro-
duction; however, the highest ethanol production was observed
with selected PCS. Therefore, SH-SF-YE-S was selected to form
the biofilm in the reactor for next phase of the study.
Although no significant differences were detected among the
PCS types in terms of the enzyme activity for A. niger, glucose con-
centration and biofilm formation were varied. A possible explana-
tion for various glucose concentrations might be a variety of
amylase enzymes could be produced that resulted significant dif-
ferences in released glucose concentrations. It was reported by
Amirul et al. [27] that two different forms of glucoamylase as well
as other amylases (a-glucosidase and a-amylase) were secreted by
Aspergillus niger van Tieghem in a liquid medium containing raw
tapioca starch as the carbon source. On the other hand, ethanol
production showed variance while biofilm formation did not reveal
any significant difference among the PCS types. It can be because of
the slow leaching of nutrients from PCS into the fermentation
broth [28] that caused variation in ethanol production. Another
interesting finding was that glucose concentration and enzyme
activity on the PCS of SH-SF-YE-BA-SALTS showed no significant
differences with other PCS even though the biomass of Aspergillus
was significantly lower on the PCS of SH-SF-YE-BA-SALTS com-
pared to the other PCS types. Several studies have shown that
the PCSs provide nutrients during the fermentation while support-
ing the biofilm formation [12,16]. Nutrients leached from bovine
albumin (BA) might promote enzyme secretion and resulted simi-
lar glucose conversions with other PCSs even with less biomass.
These results are in agreement with findings of Demirci et al.
[16] who also suggested the PCS composed of SH-SF-YE-S for etha-
nol production by S. cerevisiae in biofilm reactors after assessing
eleven compositionally different PCS types.
3.2. Optimization of growth parameters by response surface
methodology in biofilm reactor
After determining the type of PCS to be used, biofilm reactors
were constructed as described earlier with the selected PCS type
 G. Izmirlioglu, A. Demirci / Fuel 202 (2017) 260–270 265

8
a
a
7 a a
a a
ab
6

5 b
b a SH-SF-Salts
4 SH-SF-YE-Salts

3 d SH-SF-YE-BA-SALTS
c
SH-SF-YE-RBC-SALTS
2

0
Glucose (g/L) Glucoamylase Acvity Biomass (log CFU /g PCS)
(U/ml)
(A)
25
ab a a
b
20

15

SH-SF-Salts
10
a a a a SH-SF-YE-Salts

5 SH-SF-YE-BA-SALTS

SH-SF-YE-RBC-SALTS
0
Ethanol (g/L) Biomass (log CFU/g PCS)
(B)
Fig. 2. Effects of different PCS compositions on the glucose, glucoamylase activity, and biomass for A. niger (A) and ethanol production and biomass for S. cerevisiae (B) in test
tubes (n = 3). (Different letters represents the significant difference between treatments (p-value < 0.05)).

(SH-SF-YE-S). Then, effects of temperature (25–35 °C), pH (4–6),
and aeration rate (0–1.5 vvm) were evaluated to optimize the
SSF of ethanol in biofilm reactors by co-cultures of A. niger and S.
cerevisiae in industrial waste potato mash medium. Even though
ethanol fermentation requires no oxygen, aeration was studied to
determine the effect of aeration on glucoamylase production.
Table 2 provides the experimental and predicted results of ethanol
production and enzyme activity obtained from the Box-Behnken
design matrix. It is apparent from the table that ethanol production
and enzyme activities were highly affected by fermentation condi-
tions, and ranged from 5.32 g/L to 37.58 g/L for ethanol and from
0.1 U/ml to 40.11 U/ml for enzyme activity. A positive effect of
temperature was observed in which higher temperature profiles
revealed higher results for both ethanol productions and enzyme
activity. This can be seen under the conditions of pH 6, 0.75 vvm
aeration, and 25 °C in which 24 g/L ethanol and 7.67 U/ml enzyme
activity observed, while at pH 6, 0.75 vvm and 35 °C, ethanol pro-
duction and enzyme activity raised to 33.24 g/L and 14.11 U/ml,
respectively. In case of pH, it was also observed that higher pH pro-
files promoted the production of ethanol as well as enzyme activ-
ity. As a notable example of this, 37.58 g/L ethanol and 40.11 U/ml
enzyme activity were obtained at pH 6, 30 °C and 0 vvm aeration.
At pH 4, however, both the values of ethanol production and
enzyme activity dropped down to 13.08 g/L and 26.5 U/ml, respec-
tively (when temperature and aeration kept constant). In contrast
to temperature and pH, increased rates of aeration caused a
decrease in ethanol production while increased the enzyme activ-
ity. For instance, 22.84 g/L ethanol attained at pH 5, 35 °C, and 0
vvm aeration, while ethanol production decreased to 15.94 g/L,
when aeration increased to 1.5 vvm at the constant pH and tem-
perature. Enzyme activity, however, increased from 22.11 U/ml
to 25.67 U/ml in presence of aeration (1.5 vvm) compared to no
aeration (0 vvm) under same pH and temperature profiles, 5 and
35 °C, respectively.
Second order polynomial equation (Eq. (1)) for ethanol produc-
tion was obtained using Minitab software and shown below:
g 
Ethanol ¼ 15 þ 5:87X 1  42:8X 2 þ 50:2X 3  0:1119X 21
L
þ 4:13X 22  1:77X 23 þ 0:464X 1 X 2  0:963X 1 X 3
 4:26X 2 X 3 ð1Þ
where X1, X2 and X3 are temperature (°C), pH and aeration rate
(vvm), respectively. The results of ANOVA indicated that the model
is reliable with R2 value of 93.68% and significant linear effects. The
insignificant lack of fit proved that experimental data is well fitted
to the model (p-value = 0.322). Fig. 3 is the representation of
response surface plots, the effects of each factor as well as interac-
tions among the factors. As it can be seen in Fig. 3, a linear correla-
tion observed between pH and ethanol production, resulting higher
ethanol productions at pH 6. Ethanol production also showed a lin-
ear increase with increased temperature up to about 34 °C, and then
266 G. Izmirlioglu, A. Demirci / Fuel 202 (2017) 260–270

Fig. 3. Response surface plot showing the interactions of pH, temperature and aeration and their effect on ethanol production.

reaches its plateau around 35 °C. The relationship between aeration
and ethanol production, on the other hand, showed linear decrease
in which ethanol productions decreases at increased aeration rates.
The response optimizer tool in Minitab software was used to
determine the optimum level for the each factor to maximize the
ethanol production. Fig. 4 illustrates the optimization graph for
maximum ethanol production. Based on this, the optimum condi-
tions were determined as pH 5.8, 35 °C, and 0 vmm, which esti-
mated 37.95 g/L ethanol production. The suggested optimum
condition (pH 5.8, 35 °C, and 0 vmm) was validated experimen-
tally. The averages of the results under the optimum conditions
were presented in Fig. 5. The maximum ethanol production was
measured as 37.93 g/L with a 0.410 g ethanol/g starch yield and
1.044 g/L/h ethanol productivity. Even though the optimum tem-
perature suggested by the model was the highest point in the stud-
ied range, as it is seen in Fig. 4, ethanol production does not show a
linear trend with temperature and reaches its plateau around
35 °C. Because of that, 35 °C was accepted as the optimum temper-
ature without further investigation. However the pH showed a lin-
ear effect on ethanol production (Fig. 4), and the optimum value for
pH was very close to the highest limit of evaluated range,
additional fermentation was carried out at pH 6.5 at 35 °C, and 0
vmm, as well as at uncontrolled pH to confirm this is indeed
the optimum condition. The ethanol productions for pH 6.5 and
 G. Izmirlioglu, A. Demirci / Fuel 202 (2017) 260–270 267

Fig. 4. Optimization graph for maximum ethanol production.

45 70
40 60
Ethanol and Glucose (g/L)

Enzyme Activity (U/ml)

35
50
30
25 40
20 30
15
20
10
5 10

0 0
0 10 20 30 40 50 60 70 80
Time (h)
Glucose (g/L) Ethanol (g/L) Glucoamylase Activity (U/ml)

Fig. 5. Product formation curve for ethanol fermentation from industrial potato waste under the statistically optimized conditions in biofilm reactor by A. niger and S.
cerevisiae.

uncontrolled pH were significantly lower than the suggested opti-
mum pH, 32.98 g/L and 11.09 g/L, respectively. Thus, the optimal
pH was proven to be 5.8.
During the course of study, the focus was only on the glucose
analysis as the main sugar. However, other sugars were also ana-
lyzed for the validation runs. Sugar analysis revealed the presence
of maltose in the fermentation broth, from beginning to the end of
the fermentation (9.7 g/L). Since the concentrations of maltose
were not constant (5.3 g/L to 14.55 g/L), it can be concluded that
maltose was a by-product of the fermentation as well as a sub-
strate. Insignificant amounts of arabinose (30.60 mg/L), xylose
(77.16 mg/L), galactose (84.20 mg/L), and fructose (320 mg/L) were
also determined in the fermentation broth samples. The concentra-
tions of those sugars were constant throughout the SSF process.
These results suggest that those sugars were not by-products of
the fermentation, however, released from potato and/or peels dur-
ing the sterilization of the medium. The fermentation time to reach
the maximum enzyme activity was reduced from 96 h to 24 h in
biofilm reactors, which also decreased the total fermentation time
to 72 h instead of 120 h [23], which makes the overall process 40%
shorter.
The optimal pH was determined as 5.8. These results are in
agreement with findings of Liu and Shen [29] and Germec et al.
[30] who reported pH 5 and 5.18 were the optimum pH for ethanol
production by immobilized S. cerevisiae. In contrast to earlier find-
ings, a relatively high temperature was found to be optimal for
ethanol production. A possible explanation of this can be the ele-
vated temperature requirements of glucoamylases (50–70 °C) for
optimal activity [31]. Furthermore, biofilm formation increases
the resistance of microorganisms to extreme environmental condi-
tions such as pH, temperature, toxic substances [13], and explain
the higher productivity of S. cerevisiae at 35 °C. Another important
finding was no aeration suggested for maximum ethanol produc-
tion by the model. It is well known that ethanol productivity of
S. cerevisiae decreases in the presence of oxygen; however, A. niger
requires some amounts of aeration. The results of this study fur-
ther supports the idea of co-immobilization can enhance the adap-
tation of co-cultures to the harsh and inhibiting environments [8].
In our recent study, growth conditions for ethanol production in
biofilm reactors by S. cerevisiae were optimized in an industrial
potato waste based medium. Commercial enzymes were employed
during hydrolyzation of the starch for saccharification before fer-
mentation. In that study, under optimum conditions (pH 4.2,
34 °C, and 100 rpm), 37.05 g/L of ethanol was obtained with
92.08% theoretical yield and 2.31 g/L/h ethanol productivity. The
results of our current study indicated that comparable ethanol
yields can be achieved with SSF of ethanol by co-cultures of A. niger
and S. cerevisiae in biofilm reactors without enzyme addition. Fuji
268 G. Izmirlioglu, A. Demirci / Fuel 202 (2017) 260–270

Fig. 6. Scanning electron micrographs of SSF of ethanol by co-cultures of A. niger and S. cerevisiae on the exterior and interior surfaces SH-SF-YE-S PCS tubes in fermentation
medium. A: Exterior surface of PCS tubes before cell growth (control). B: Interior surface of PCS tubes before cell growth (control). C: Co-culture biofilm on the exterior surface of
PCS tubes at 60X magnification. D: Enlargement of cell clusters shown in square in image C at 1000X magnification. E: Enlargement of the square in image D at 2500 X
magnification showing the spores and hyphae. F: Enlargement of A. niger spores (shown in blue square in E) at 20 K X magnification. G: Enlargement of fungal hyphae (red square
in E) at 15 K X magnification. H: Co-culture biofilm on the interior surface of PCS tubes at 60X magnification. I: Enlargement of cell clusters shown in image H showing the cells.
 G. Izmirlioglu, A. Demirci / Fuel 202 (2017) 260–270
Fig. 6 (continued)
et al. [32] reported 25.5 g/L maximum ethanol production by
immobilized co-cultures of A. awamori and S. pastorianus using cel-
lulose carriers. The results of current study, thus, indicates that PCS
biofilm reactors can successfully employed for co-culture systems,
which provided 37.93 g/L ethanol production with a 0.410g etha-
nol/g starch yield and 1.044 g/L/h ethanol. Furthermore, compara-
ble amounts of ethanol can be produced using SSF process with
application of PCS biofilm reactors while utilizing the industrial
wastes.
3.3. SEM evaluation
The co-culture biofilm formations on selected PCS were visually
evaluated with scanning electron microscopy (Fig. 6). For the com-
parison, external and internal surfaces of control PCS tubes before
microbial growth were shown in Fig. 6A and B, respectively. A por-
ous surface promote the biofilm formation [12]. As it can be seen in
Fig. 6A and B, PCS is highly porous and promoted biofilm formation
resulting very dense formation of biofilm on the outer surface of
the PCS, especially within the pores (Fig. 6C). A closer look at the
PCS at 1000X and 2500X magnification clearly revealed the spores
and mycelia of A. niger (Fig. 6D and E). Fig. 6F is the enlargement of
A. niger spores at 20 K X magnification, while Fig. 6G is the enlarge-
ment of mycelia at 15 K X magnification SEM micrographs also
showed that S. cerevisiae cells bounded mostly on the mycelia.
On the other hand, biofilm formation at 60X magnification in the
interior surface of the PCS shown in Fig. 6H. As it can be seen in
Fig. 6I, microbial cells were captured at 6700X magnification on
the interior surface of PCS. Furthermore, interior surface was more
favorable for S. cerevisiae cells than A. niger since no mycelia or
spores were observed. This might be due to the lack of oxygen in
the interior surface, which is necessary for A. niger however not
required for yeast.
4. Conclusions
The present study was designed to determine the PCS composi-
tion for biofilm formation of co-cultures of A. niger and S. cerevisiae
and the effects temperature, pH, and aeration rates on ethanol pro-
duction in SSF of ethanol in PCS biofilm reactors from industrial
potato waste. The response surface methodology was used to
determine the model to predict the ethanol production under
different combinations of temperature, pH and aeration rates.
The study has identified that PCS with SH-SF-YE-S were capable
of promoting biofilm formation for both cultures of A. niger and
269
S. cerevisiae. In a PCS biofilm reactor, optimum conditions for
maximum ethanol production of SSF of potato waste by co-
cultures of A. niger and S. cerevisiae was 35 °C, pH 5.8, and no aer-
ation (0 vvm). A maximum of 37.93 g/L ethanol was produced
under optimum conditions, with 0.410 g ethanol/g starch yield
and 1.044 g/L/h productivity. The results suggest that PCS biofilm
reactors can be used for co-immobilization of co-culture systems.
SEM images revealed that when mold and yeast are allowed to
form a biofilm, hyphae of the mold provide surface area for the
yeasts’ attachment. Also, under biofilm conditions, culture require-
ments of the microorganisms show differences compared to sus-
pended cells, as it was seen in our study that A. niger did not
require aeration in biofilm. Overall, bioethanol production from
starchy industrial wastes can be improved with application of bio-
film reactors while the production cost is reduced with integra-
tions of SSF process and co-culturing.
Acknowledgements
This work was supported in part by the Turkish Ministry of Edu-
cation and the Pennsylvania Agricultural Experiment Station. The
authors gratefully acknowledge Keystone Potato Products (Hegins,
PA, USA) and Pennsylvania Grain Processing, LLCÒ (Clearfield, PA,
USA) for supplying waste potato mash and enzymes, respectively.
Authors want to thank Kay Dimarco, Agricultural and Biological
Engineering, Pennsylvania State University for her help in sugar
analysis. We also would like to thank to John J. Cantolina of Micro-
scopy and Cytometry Facility at Hucks Institutes of the Life
Sciences, Pennsylvania State University for his help in SEM
protocols.
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