Bioresource Technology 289 (2019) 121614

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

On the evaluation of different saccharification schemes for enhanced T

bioethanol production from potato peels waste via a newly isolated yeast
strain of Wickerhamomyces anomalus
⁎
Imen Ben Atitallaha,b, Georgia Antonopoulouc, , Ioanna Ntaikouc, Maria Alexandropoulouc,
Moncef Nasria, Tahar Mechichib, Gerasimos Lyberatosc,d
a
Laboratory of Enzyme Engineering and Microbiology, National School of Engineers of Sfax, University of Sfax, BP 1173, 3038 Sfax, Tunisia
b
Laboratory of Biochemistry and Enzymatic Engineering of Lipases, National School of Engineers of Sfax, University of Sfax, 3038 Sfax, Tunisia
c
Institute of Chemical Engineering Sciences, Stadiou, Platani, Patras GR 26504, Greece
d
School of Chemical Engineering, National Technical University of Athens, GR 15780 Athens, Greece

A R T I C LE I N FO A B S T R A C T

Keywords: The present study focuses on the exploration of the potential use of potato peels waste (PPW) as feedstock for
Potato peels waste bioethanol production, using a newly isolated yeast strain, Wickerhamomyces anomalus, via different sacchar-
Enzymatic saccharification ification and fermentation schemes. The saccharification of PPW was performed via thermal and chemical (acid,
Pretreatment alkali) pretreatment, as well as via enzymatic hydrolysis through the use of commercial enzymes (cellulase and
Bioethanol
amylase) or enzymes produced at lab scale (alpha-amylase from Bacillus sp. Gb67), either separately or in
W. anomalus
mixtures. The results indicated that the enzymatic treatment by commercial enzymes led to a higher sacchar-
ification efficiency (72.38%) and ethanol yield (0.49 g/gconsumed sugars) corresponding to 96% of the maximum
theoretical. In addition, acid pretreatment was found to be beneficial for the process, leading also to high hy-
drolysis and ethanol yields, indicating that PPW is a very promising feedstock for bio-ethanol production by W.
anomalus under different process schemes.

1. Introduction discharged as by-product (Lappalainen et al., 2015). These residues,

Biomass - based fuels, the so-called biofuels are among the key re-
newable energy sources which could be produced from zero-cost ma-
terials (Kurambhatti et al., 2018). Among different biofuels, bioethanol
and its blends has been most widely used as alternative liquid fuel for
transportation (Bazoti et al., 2017). The key factor and at the same time
one of the most important challenges for sustainable bioethanol pro-
duction is the selection of suitable, zero-cost raw materials to be used as
renewable feedstocks (Xu et al., 2018; Ntaikou et al., 2018). In this
context, lignocellulosic biomass, agricultural residues and industrial by-
products represent viable substrates for ethanol production due to their
great availability and low cost (Yu et al., 2019).
In recent years, a significant increase in residue production of the
potato processing industry has been noticed, primarily due to the in-
creased demands for fast and convenience food (Dos Santos et al.,
2012). During processing of the potato crops for production of potato
crisps and chips, French fries or starch, important fractions (20–50%) of
the raw product, mainly peels i.e. potato peels waste (PPW), are
generated by the abrasion peeling process, contain a large quantity of
starch, cellulose, hemicellulose and lignin which could be potential
sources for the secondary production of high added value bio-products
or biofuels, i.e. biobutanol or bioethanol (Arapoglou et al., 2010). In
order to overcome the structural and compositional barriers for con-
verting non-soluble carbohydrates of PPW to ethanol, two extra steps
towards the saccharification of biomass, which could take place either
independently or in combination, are necessary i.e. a hydrolysis step,
where enzymatic conversion of polymeric carbohydrates into soluble
sugars takes place and a pretreatment step for enhancing the enzymatic
hydrolysis (Carrere et al., 2016). Up to now, limited research has been
conducted on the effect of the chemical or thermal pretreatment on
PPW. Ben Taher et al. (2017) applied an acid, an alkali, and a hydro-
thermal pretreatment for 30 min at 121 °C on PPW and found that the
acid pretreatment led to higher starch and lignocellulose reduction,
which however led to a lower concentration of reducing sugars and
saccharification yield. This was probably due to the fact that under
these severe acid pretreatment conditions tested, a degradation of the

⁎
Corresponding author.
E-mail address: geogant@chemeng.upatras.gr (G. Antonopoulou).

https://doi.org/10.1016/j.biortech.2019.121614
Received 1 April 2019; Received in revised form 3 June 2019; Accepted 6 June 2019
Available online 07 June 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
I. Ben Atitallah, et al.
reducing sugars released from hemicellulose solubilization was prob-
ably taken place, towards other more oxidized compounds, contributing
thus to the lower saccharification yield.
For starchy-based substrates, such as PPW, with a low lignin and a
high cellulose and starch content, saccharification could be accom-
plished only by an enzymatic hydrolysis step. Enzymatic processes are
generally carried out at mild conditions, leading to high sugars yields
(Arapoglou et al., 2010), due to the absence of secondary reactions and
the subsequent formation of inhibitory compounds, commonly reported
as by-products under severe chemical or thermal pretreatments
(Antonopoulou et al., 2016). Regarding enzymatic hydrolysis of PPW,
in the study of Khawla et al. (2014) an enzymatic mixture of amylases
that perform liquefaction (starch hydrolysis to maltodextrins and mal-
tose) and saccharification (conversion to pure glucose by glucoamy-
lases) was used at different conditions and it was found that the higher
the enzyme concentration used, the higher was the fermentable redu-
cing sugar content obtained. In addition, for the saccharification of
PPW, Arapoglou et al. (2010) used different commercial enzymatic
mixtures of cellulolytic and amylolytic enzymes, in order to achieve
cellulose hydrolysis along with starch hydrolysis.
It is well known that enzymatic hydrolysis and fermentation to-
wards bioethanol production could be accomplished either separately
(separate hydrolysis and fermentation (SHF)) or simultaneously (si-
multaneous saccharification and fermentation (SSF)) (Schell et al.,
2016), with certain advantages for both processes (Gubicza et al., 2016;
Sewsynker-Sukai and Gueguim Kana, 2018). Regarding fermentation of
PPW to bioethanol production, wild type strains of Saccharomyces cer-
evisiae have been used (Arapoglou et al., 2010; Khawla et al., 2014),
while Chintagunta et al. (2016) and Izmirlioglu and Demirci (2016)
studied the use of co-cultures of Aspergillus niger and S. cerevisiae, for
enhancing amylolytic activity and total ethanol yield from PPW at SSF.
According to the authors, co-cultures of both microorganisms increased
the ethanol production efficiency, by preventing the accumulation of
inhibitory concentrations of reducing sugars. On the other hand, the
exploitation of proper microbial strains exhibiting advanced perfor-
mance and high ethanol yield is of crucial importance for process sus-
tainability (Raja Sathendraa et al., 2019; Ntaikou et al., 2018). Wick-
erhamomyces anomalus also known as Hansenula anomala or Pichia
anomala belongs to the group of the so-called non-Saccharomyces or
‘wild’ yeasts (Daniel et al., 2011) and was found to be a robust mi-
crobial strain for the production of bioethanol (da Conceicao et al.,
2015; Zha et al., 2013; Ben Atitallah et al., 2018).
Thus, in the present study the newly isolated yeast W. anomalus X19
was used for the first time, to produce ethanol from PPW. For the
saccharification of PPW, thermal and chemical pretreatment and en-
zymatic hydrolysis or combinations of these pretreatments were per-
formed, aiming to maximum saccharification of PPW. The effect of each
method on saccharification efficiency and carbohydrates fractionation
of the substrate was evaluated, in a comparative way, for the first time.
Pretreated and enzymatic hydrolyzed PPW were used for exploring the
valorization of PPW towards bioethanol production, at the maximum
degree, using W. anomalus X19 at different conditions, either through
SSF or SHF. The possibility of using lab-scale produced enzymes, in-
stead of using commercial ones, was explored at all possible process
schemes. Moreover, fermentation experiments were performed either
using the whole slurry or the separated fractions obtained after pre-
treatment, investigating thus all the possible process schemes, in order
to achieve, if possible, the maximum energy recovery in the form of
ethanol.
2. Materials and methods
2.1. PPW preparation
The PPW used in the present study was obtained from local fast food
restaurants located in Sfax, Tunisia, using potatoes of Spunta variety.
2
Bioresource Technology 289 (2019) 121614
Prior to use, PPW was initially washed with water to remove undesir-
able particles and dried at 40 °C for 48 h. Then, it was chopped with a
lab grinder (IKA, M 20 Universal Mill), sieved to obtain particles with a
size < 1 mm and the obtained powder was air-dried.
2.2. Chemical and thermal pretreatment conditions
PPW was subjected to three different pretreatment methods: acid,
alkaline and thermal. For all pretreatment methods used, the mass/
volume ratio of solid to aqueous solution was 5%. Acid and alkaline
pretreatments were performed through the addition of H2SO4 and
NaOH (0.1% w/v) at 121 °C for 1 h and 80 °C for 24 h respectively,
while thermal pretreatment was conducted for 1 h at 121 °C and 80 °C
for 24 h without addition of any chemicals, based on previous studies
(Antonopoulou et al., 2015). All pretreatment experiments were per-
formed in duplicate. Depending on the process scheme followed, either
a) the whole pretreatment slurry or b) the two fractions obtained after
separation through filtering with 0.7 μm (liquid and solid fractions
(solid PPW or SPPW) obtained after pretreatment), were used for
ethanol production.
2.3. Enzymatic hydrolysis
2.3.1. Production of alpha-amylase from Bacillus sp. Gb67
The bacterium Bacillus sp. Gb67 (Accession number: MH447984.1)
was recently isolated from desert soils in southern Tunisia and main-
tained at 4 °C on Luria-Bertani (LB) agar slants containing (g/L): pep-
tone 10.0, yeast extract 5.0, NaCl 5.0 and agar 18.0. The inoculation
was performed in 250 mL Erlenmeyer flasks containing 50 mL LB broth
medium (pH 7.0) and incubation was carried out over night at 37 °C in
a rotatory shaker set at 200 g.
Regarding amylase production, the experimental conditions had
been previously optimized in terms of the culture medium composition
(salts, carbon and nitrogen source and their concentration) and the
physico-chemical characteristics (pH, temperature, and agitation rate).
Thus, cultures were conducted at 37 °C for 24 h in 250 mL flasks
containing 50 mL of optimized culture medium having the following
composition (g/L): wheat, 30.0; peptone, 2.0; K2HPO4, 0.5; KH2PO4,
0.5; CaCl2, 2.0; NaCl, 0.5. The pH was adjusted to 8.0 prior to ster-
ilization. The media were autoclaved at 120 °C for 20 min. The cultures
were centrifuged at 8000 g for 15 min at 4 °C and the supernatants were
used for alpha-amylase activity determination. The alpha -amylase ac-
tivity was detected by measuring the reducing sugars released during
the enzymatic hydrolysis of starch (Bhange et al., 2016). The reaction
mixture contained 0.5 mL of appropriate diluted crude enzyme and
0.5 mL of 1% (w/w) soluble starch (sigma-Aldrich) in 0.1 M phosphate
buffer (pH 7.0). After 10 min of incubation at 50 °C, the concentration
of reducing sugars was measured. One unit (FAU) of amylolytic activity
was defined as the amount of enzyme releasing 1 µmol of reducing end
groups per minute.
2.3.2. Enzymatic hydrolysis of PPW
Hydrolysis was performed in 100 mL Erlenmeyer flasks, using a
working volume of 20 mL. Dried PPW with a mass/volume ratio of solid
to aqueous solution of 5%, was used and treated with a) commercial
cellulase blend (CEL) (Cellic CTec2-CEL, 223 FPU/mL, Sigma-Aldrich),
b) commercial alpha -amylase (C.A.) from Aspergillus oryzae (fungal
alpha - amylase, ≥ 800 FAU/g, 120 FAU/mL, Sigma-Aldrich) and c)
alpha - amylase from Bacillus sp. Gb67 (B.A.), 11 FAU/mL, or combi-
nation of the cellulotytic and amylolytic enzymes at different conditions
such a) CEL and C.A. at pH 4.8, either at 30 °C or 50 °C and b) CEL and
B.A. at the same conditions. Additionally, a two-step enzymatic hy-
drolysis was performed at the optimum conditions for each enzyme, i.e.
PPW was initially treated with B.A. for 24 h (pH 7.0, 30 °C) and then
with CEL (pH 4.8, 50 °C) for another 24 h. 0.1 M sodium acetate buffer
was used for pH maintaining at 4.8, while a phosphate buffer at the
I. Ben Atitallah, et al. Bioresource Technology 289 (2019) 121614

Table 1
Enzymatic hydrolysis and fermentation schemes which were used in this study (CEL: commercial cellulase blend - Cellic CTec2-CEL, C.A.: commercial alpha-amylase
from Aspergillus oryzae, B.A: alpha- amylase from Bacillus sp. Gb67).
Abbreviations Enzymes Enzymatic hydrolysis conditions used

CEL Cellulase 50 °C, pH: 4.8, 100 FPU/g cellulose

C.A. α-amylase 50 °C, pH: 4.8, 100 FAU/g starch
B.A. α-amylase 30 °C, pH: 7, 100 FAU/g starch
CEL + C.A. Mixture 50 °C, pH: 4.8,100 FPU/g cellulose and 100 FAU/g starch
30 °C, pH: 4.8,100 FPU/g cellulose and 100 FAU/g starch
CEL + B.A. Mixture 50 °C, pH: 4.8,100 FPU/g cellulose and 100 FAU/g starch
30 °C, pH: 4.8,100 FPU/g cellulose and 100 FAU/g starch
B.A. + CEL 2 step process B.A. 100 FAU/g starch at pH: 7.0, 30 °C for 24 h and then addition of CEL 100 FPU/g cellulose at pH: 4.8, 50 °C for another 24 h.

Abbreviations Scheme Fermentation conditions used

C.A. + CEL, (SSF) 2 step process C.A. 100 FAU/g starch at pH: 4.8, 50 °C for 24 h and then addition of CEL 100 FPU/g cellulose and simultaneous ethanol production at pH:
5, 30 °C through SSF
C.A. + CEL, (SHF) 2 step process C.A. 100 FAU/g starch and CEL 100 FPU/g cellulose at pH: 4.8, 50 °C for 24 h and then ethanol production at pH: 5, 30 °C through SHF
B.A + CEL, (SSF) 2 step process B.A. 100 FAU/g starch at pH: 7.0, 30 °C for 24 h and then addition of CEL 100 FPU/g cellulose and simultaneous ethanol production at pH: 5,
30 °C through SSF
B.A + CEL, (SHF) 3 step process B.A. 100 FAU/g starch at pH: 7.0, 30 °C for 24 h, addition of CEL 100 FPU/g cellulose at pH: 4.8, 50 °C for another 24 h. and then ethanol
production at pH: 5, 30 °C through SHF

Table 2
Changes of PPW composition (g/100 g TSinitial) after pretreatment/hydrolysis methods applied in this study (CEL: commercial cellulase blend - Cellic CTec2-CEL, C.A.:
commercial alpha-amylase from Aspergillus oryzae, B.A: alpha- amylase from Bacillus sp. Gb67).
Pretreatment, hydrolysis Conditions Cellulose Hemicellulose Lignin Starch Material Recovery

Thermal 80 °C, 24 h 30.5 ± 0.4 2.0 ± 0.0 3.9 ± 0.1 29. 9 ± 0.1 60.5 ± 0.4
Thermal 120 °C, 1 h 27.7 ± 0.8 2.0 ± 0.1 4.0 ± 0.2 28.1 ± 0.2 56.3 ± 0.6
H2SO4 0.1% w/v 10.6 ± 0.4 0.8 ± 0.0 4.3 ± 0.1 4.0 ± 0.1 29.9 ± 2.1
NaOH 0.1% w/v 14.4 ± 0.3 1.1 ± 0.1 2.2 ± 0.0 11.5 ± 0.2 34.9 ± 0.6
CEL + C.A. 50 °C pH 4.8, 24 h 8.2 ± 0.8 1.2 ± 0.0 3.6 ± 0.0 3.0 ± 0.0 32.6 ± 0.0
B.A. + CEL B.A at 30 °C, pH 7.0, 24 h and CEL at 50 °C, pH 4.8 for 24 h. 14.5 ± 0.1 0.8 ± 0.0 3.6 ± 0.5 8.2 ± 0.0 46.2 ± 0.2

Table 3 2.4. Fermentation experiments

Analysis of liquid fraction obtained after pretreatment/hydrolysis of PPW (in g/
100 g TSinitial) applied in this study (CEL: commercial cellulase blend - Cellic 2.4.1. Preculture preparation
CTec2-CEL, C.A.: commercial alpha-amylase from Aspergillus oryzae, B.A: alpha- One loopful of a single colony of the yeast strain W. anomalus X19
amylase from Bacillus sp. Gb67, HMF: hydroxyl-methyl-furfural). (MH237950.1) (Ben Atitallah et al., 2018) was transferred from the
Pretreatment, Conditions Sugars (g/ Reducing HMF (mg/100 agar plate into 50 mL of sterile yeast peptone dextrose medium (YPD)
hydrolysis 100 sugars (g/ gTSinitial) containing (g/L): glucose, 20.0; yeast extract, 10.0 and peptone, 10.0.
gTSinitial) 100 gTSinitial) The inoculum was cultivated overnight in a 250 mL Erlenmeyer flask in
Thermal 80 °C, 24 h 33.6 ± 0.1 1.8 ± 0.0 n.d a rotator shaker (150 g) at 30 °C until reaching OD550 ∼ 2.000, corre-
Thermal 120 °C, 1 h 41.3 ± 0.3 4.2 ± 0.0 0.1 ± 0.0 sponding to a biomass concentration of 4 g/L.
H2SO4 0.1% w/v 53.3 ± 0.9 6.1 ± 0.1 201.9 ± 22.8
NaOH 0.1% w/v 43.2 ± 0.1 4.8 ± 0.0 n.d
2.4.2. Bioethanol experiments
CEL + C.A. 50 °C pH 4.8, 68.4 ± 0.0 58.8 ± 0.0 n.d
24 h For the fermentation experiments, 160 mL serum vials with a
B.A. + CEL B.A at 30 °C, 57.4 ± 0.1 42.6 ± 0.1 n.d working volume of 25 mL were used. The experiments were performed
pH 7.0, 24 h in duplicate in batch mode and incubated at 150 g and 30 °C. The vials
and CEL at were sealed with rubber stoppers and equipped with 0.22 µm filters for
50 °C, pH 4.8
for 24 h.
CO2 venting and sterilization. In all experiments, cells were harvested
from pre-culture of the W. anomalus strain X19, at 5% v/v of the final
fermentation volume of each experiment, leading to an initial biomass
same concentration was used for pH 7. Details for the enzymatic hy- concentration of 0.2–0.25 g/L. For the inoculation, the estimated vo-
drolysis schemes are presented in Table 1. To avoid microbial con- lume of preculture was centrifuged at 4500 g for 15 min and the yeast
tamination, all the experiments were supplemented with 2% sodium pellet was re-suspended in a solution containing KH2PO4, MgCl2. 6 H2O
azide. The samples were centrifuged and analyzed for the reducing and (NH4)2SO4 each at concentrations of 1 g/L.
sugars content and the enzymatic hydrolysis yield was determined as The experiments were performed at raw, pretreated and en-
described in Ben Taher et al. (2017) according to the equation: zymatically hydrolyzed PPW samples. Specifically, raw, chemically or
thermally pretreated PPW and SPPW at a solid loading of 5% w/v were
Hydrolysis yield (%) used for ethanol production through SSF or SHF, using the enzymatic
Reducing sugars released × 0.9 mixtures of CEL and C.A. at pH 4.8, either at 30 °C (SSF) or at 50 °C for
= × 100 24 h (SHF). The liquid fractions obtained after chemical and thermal
Cellulose , hemicellulose and starch content in PPW (1)
pretreatment as well as the chemically or thermally pretreated PPW
(the whole slurry obtained after pretreatment) were also used for
ethanol production without addition of enzymes. Furthermore, dif-
ferent enzymatic schemes were used for ethanol production, presented

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Table 4
Enzymatic hydrolysis yield during the different enzymatic schemes tested,
calculated at the end of each experiment and at 24 h of hydrolysis (CEL: com-
mercial cellulase, C.A.: commercial alpha-amylase, B.A: alpha- amylase from
Bacillus sp.).
Enzymes Enzymatic hydrolysis yield (%)

At the end of experiment At 24 h

CEL 29.64 ± 0.22 (30 h) 27.60 ± 0.35

C.A. 45.30 ± 0.34 (30 h) 42.96 ± 0.02
B.A. 17.12 ± 0.05 (30 h) 14.81 ± 0.13
CEL + C.A. (50 °C) 72.38 ± 0.46 (48 h) 53.16 ± 1.08
CEL + C.A. (30 °C) 64.90 ± 0.18 (48 h) 40.99 ± 0.13
CEL + B.A. (50 °C) 40.13 ± 0.96 (48 h) 33.03 ± 0.32
CEL + B.A. (30 °C) 32.97 ± 0.06 (48 h) 26.29 ± 1.33
B.A. + CEL (2 step) 57.72 ± 0.26 (72 h in 52.66 ± 0.06 (48 h in
total) total)

30 °C through SHF, c) B.A. at pH 7.0, 30 °C for 24 h and then addition of

CEL and simultaneous ethanol production at 30 °C through SSF and d)
B.A. at pH 7.0, 30 °C for 24 h, addition of CEL at pH 4.8, 50 °C for 24 h
more and then ethanol production at 30 °C, through SHF (two stage
enzymatic process). In all experiments the amylolytic and cellulolytic
enzyme loadings were 100 FAU/g starch and 100 FPU/g cellulose, re-
spectively.
During the fermentation experiments, the initial pH was adjusted to
5.0, by using NaOH or HCl solution (6 N). For substrate and products
analysis, samples were withdrawn at regular time intervals from the
fermentation cultures and were centrifuged at 6000 g for 10 min. The
resulting supernatant was used for measurement of ethanol and residual
sugars concentrations.

2.5. Analytical methods

Total solids (TS) and volatile solids (VS) were determined according
to Standard Methods (APHA, 1995). Soluble carbohydrates content
determination was performed through the phenol–sulfuric acid method
described by DuBois et al. (1956), while, reducing sugars concentration
was estimated by the DNS (3,5-dinitrosalicylic acid) method and was
expressed as glucose equivalents (Miller, 1959). Quantification of
starch was made via a total starch assay kit (Megazyme). Extractives,
cellulose, hemicellulose and lignin content were analyzed at the raw
and pretreated PPW samples, following the National Renewable Energy
Laboratory (NREL) (1998) standard laboratory analytical procedures
(LAP) for determination of extractives and structural carbohydrates in
biomass (Sluiter et al., 2008a,b) at conditions presented by
Antonopoulou et al. (2015). Detection and quantification of ethanol,
furfural, hydroxyl-methyl-furfural (HMF) and aliphatic acids such as
formic and acetic acids as well as sugar monomers (glucose, xylose and
arabinose) were performed using an HPLC-RI with an Aminex HPΧ-87H
column (Biorad) at 60 °C using H2SO4 0.004 mM, as an eluent, at a flow
rate of 0.7 mL/min.

3. Results and discussion

3.1. Chemical composition of raw PPW

Fig. 1. Reducing sugars content during enzymatic hydrolysis of PPW using a)
cellulose (CEL); alpha -amylase from Bacillus sp. Gb67 (B.A) and commercial
amylase (C.A) at the optimum conditions for each enzyme, b) CEL and C.A. at
50 °C; CEL and C.A. at 30 °C; CEL and B.A. at 50 °C; CEL and B.A. at 30 °C and c)
the two-step enzymatic hydrolysis at the optimum conditions for each enzyme
(B.A, pH 7.0 at 30 °C for 24 h and CEL, pH 4.8 at 50 °C for extra 24 h).
in Table 1, including a) C.A. at pH 4.8, 50 °C for 24 h and then addition
of CEL and simultaneous ethanol production at 30 °C through SSF, b)
C.A. and CEL at pH 4.8, 50 °C, for 24 h and then ethanol production at
The composition of raw PPW used in the present study was: TS (%):
84.40 ± 0.10, VS (g/100 g TS): 90.40 ± 0.20, cellulose (g/100 g TS):
34.30 ± 0.60, hemicellulose (g/100 g TS): 5.90 ± 1.90, lignin (g/
100 g TS): 4.30 ± 0.13, starch (g/100 g TS): 44.80 ± 0.70, extractives
(g/100 g TS): 40.30 ± 1.90 (g/100 g TS), ash (g/100 g TS):
11.00 ± 0.10. Comparable compositional analysis has been reported in
other studies using PPW as feedstock (Khawla et al., 2014; Ben Taher
et al., 2017). However, different analysis has been reported from
Chintagunta et al. (2016) in which PPW collected from India contained

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Fig. 2. Ethanol production from raw and pretreated PPW a) without enzymes Fig. 3. Ethanol production from a) the liquid fraction obtained after pretreat-
addition, b) at SSF (CEL and C.A. at 30 °C simultaneously with fermentation) ment, b) the SPPW at SSF (CEL and C.A. at 30 °C, simultaneously with fer-
and c) at SHF (CEL and C.A. at 50 °C for 24 h, prior to fermentation). mentation) and c) the SPPW at SHF (CEL and C.A. at 50 °C for 24 h, prior to
fermentation).

only 5.69 ± 1.6% cellulose and 28.52 ± 0.17% starch, indicating that
3.2. Compositional analysis of pretreated and hydrolyzed PPW
the main chemical composition of PPW depends on the potato crops
growth location, the influence of climatic conditions and on the
The composition changes and weight loss of PPW reveal the pre-
methods adopted for potato processing. PPW used in this study has an
treatment effectiveness and is correlated to the pretreatment severity.
holocellulose content of 40.2 g/100 g TS, which, together with the
Table 2 summarizes the effect of chemical, thermal and enzymatic
starch, accounts for 85% g carbohydrates/g TS, which upon proper
pretreatment on the fractionation of PPW in terms of lignin, cellulose,
hydrolysis/pretreatment, could be fully recovered in the form of
hemicellulose and starch, as well as on the material recovery, expressed
ethanol.

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carbohydrate content. From Table 2 it is obvious that the use of com-

Fig. 4. Ethanol production from enzymatic treated PPW using C.A. (pH 4.8, at
50 °C) for 24 h and then addition of CEL and simultaneous ethanol production
at 30 °C through SSF (C.A and CEL, SSF); CA. and CEL (pH 4.8, at 50 °C) for 24 h
(C.A. and CEL, SHF); B.A. (pH 7.0, at 30 °C) for 24 h and then addition of CEL
and simultaneous ethanol production at 30 °C through SSF (B.A. and CEL, SSF)
and B.A. (pH 7.0, at 30 °C) for 24 h and CEL (pH 4.8, at 50 °C) for 24 h, in a two-
stage process and then ethanol production at SHF, (B.A. and CEL, SHF).
as g/100 gTS of the untreated biomass. As shown in Table 2, the ad-
dition of chemicals at the same conditions with the thermal treatment
caused a significant decrease in the percentage of material recovery, i.e.
from 56.30% ± 0.60 and 60.50% ± 0.40 after thermal treatment at
120 °C, 1 h and 80 °C, 24 h, respectively, the recovery decreased to
29.90% ± 2.10 and 34.90% ± 0.60, when using H2SO4 and NaOH,
respectively. The composition of thermally pretreated PPW was slightly
modified as compared to that of untreated PPW. The cellulose, hemi-
cellulose and lignin contents of PPW after 24 h of treatment at 80 °C
were 30.50 ± 0.40, 2.00 ± 0.00 and 3.90 ± 0.10 g/100 g TSinitial,
respectively while the same fractions were 27.70 ± 0.80, 2.00 ± 0.10
and 4.00 ± 0.20 g/100 g TSinitial, respectively after 1 h of treatment at
120 °C. However, both thermal pretreatment methods influenced the
starch content, causing a reduction of starch to 29.90 ± 0.10 and
28.10 ± 0.20 g/100 g TS initial, respectively. These results are in ac-
cordance with those obtained by Ben Taher et al. (2017) who reported
that thermal pretreatment is effective for starch solubilization. Re-
garding chemical pretreatments, acid pretreatment caused a reduction
of cellulose and starch to 10.60 ± 0.40 and 4.00 ± 0.10 g/100 g
TSinitial, respectively, while alkali pretreatment led to a cellulose content
of 14.40 ± 0.30 g/100 g TSinitial and a starch content of
11.50 ± 0.20 g/100 g TSinitial.
Beyond chemical pretreatments, also enzymatic hydrolysis through
the use of cellulolytic and amylolytic enzyme mixtures, resulted in high
biomass loss, due to the high solubilisation efficiency of the
mercial alpha -amylase was more effective for the reduction of starch
content, compared to the alpha -amylase from Bacillus sp. Gb67. Thus,
the starch content was 3.00 ± 0.00 g/100 g TSinitial after 24 h incuba-
tion with the mixture of commercial enzymes (CEL + C.A.) at 50 °C pH
4.8, corresponding to 93.3% solubilization efficiency of the initial
starch content, while incubation for 24 h with the alpha -amylase
produced from Bacillus sp. Gb67 at 30 °C, pH 7.0 and then with CEL for
another 24 h at 50 °C, pH 4.8 (B.A. + CEL) in a two-step process, led to
a starch content of 14.50 ± 0.10, corresponding to 67.6% starch re-
moval. It is apparent that the hydrolytic efficiency of commercial
amylase is much higher than the respective of the amylase produced at
lab-scale, at the same enzymatic loading (100 FAU/g starch). The lower
saccharification efficiency of B.A. could be attributed to a) to the dif-
ferent origin of both amylases used. i.e. the Bacillus amylase was bac-
terial amylase while the C.A. was fungal amylase, b) the different de-
gree of purification of both enzymes, since the commercial amylase is a
totally purified enzymatic mixture and c) the different optimum con-
ditions applied, since at higher temperatures the rate of hydrolysis is in
general, more favorable. In addition, the mixture of the commercial
enzymes enhanced the cellulose removal efficiency (76.1%), rather
than the respective of the enzymatic mixture involving B.A., in the two-
step process, (57.7%).
Apart from the characterization of the solid fractions, a complete
analysis of the liquid fractions obtained after pretreatment/hydrolysis
was also carried out, in order to determine concentrations of soluble
and reducing sugars and possible inhibitors, such as furfural and HMF,
which are common by-products of acid pretreatment (Antonopoulou
et al., 2015). As shown in Table 3, the enzymatic hydrolysis led to the
highest fermentable sugars concentration (68.40 ± 0.00 and
58.80 ± 0.00 g/100 g TSinitial were the soluble and reducing sugar
content for the mixture of two commercial enzymes) and 57.4 ± 0.10
and 42.6 ± 0.10 g/100 g TSinitial were the respective concentrations for
the two-step enzymatic process of B.A. and CEL. Previous studies on
enzymatic hydrolysis of potato waste showed similar high reducing
sugars yields, when mixtures of enzymes were applied (Arapoglou
et al., 2010). This high sugars content, is also confirmed by the values of
the material recovery, presented in Table 2. After acid hydrolysis, the
concentration of soluble and reducing sugars was 53.3 ± 0.9 and
6.1 ± 0.1 g/100 g TSinitial, while after alkali pretreatment it was
43.2 ± 0.1 and 4.8 ± 0.0 g/100 g TSinitial, respectively. Ben Taher
et al. (2017) who applied a hydrothermal, an acid and an alkaline
pretreatment on PPW found that the hydrothermal method, while
leading to lower starch and cellulose reduction compared to the other
methods, resulted to higher reducing sugars concentration, and thus to
higher enzymatic hydrolysis yield. The same authors also found, that
during acid pretreatment, HMF at a concentration of 1.7 g/L was re-
leased. It is worth mentioning that HMF comes from glucose degrada-
tion at high temperatures and is a common by-product during acid or
hydrothermal pretreatment, at high temperature and low pH value
(Monlau et al., 2014). In the present study, 201.9 ± 22.8 mg/100 g

Table 5
Ethanol yields (g/100 g TS initial) of all pretreatment/hydrolysis schemes applied in this study (CEL: commercial cellulase, C.A.: commercial alpha-amylase, B.A:
alpha- amylase from Bacillus sp.).
Whole slurry Separate fractions

No enzymes Enzymes Liquid Solid

SSF SHF SSF SHF

Thermal (80 °C) 10.9 ± 0.1 27.9 ± 0.5 37.4 ± 0.1 8.6 ± 0.0 10.0 ± 0.1 14.1 ± 0.3
Thermal (120 °C) 12.8 ± 0.0 28.9 ± 1.1 31.9 ± 0.1 11.6 ± 0.0 10.1 ± 0.1 14.5 ± 0.2
H2SO4 19.4 ± 0.1 34.9 ± 0.5 44.9 ± 0.1 10.2 ± 0.0 8.2 ± 0.0 10.9 ± 0.0
NaOH 15.4 ± 0.1 33.4 ± 0.8 41.9 ± 0.2 12.6 ± 0.1 8.9 ± 0.0 11.5 ± 0.1
C.A. + CEL – 30.2 ± 0.5 40.1 ± 0.2 – – –
B.A + CEL – 19.6 ± 0.0 25.5 ± 0.3 – – –

6
I. Ben Atitallah, et al.
Table 6
Ethanol yields (g/g consumed sugars) of all pretreatment/hydrolysis schemes applied in this study (CEL: commercial cellulase, C.A.: commercial alpha-amylase, B.A:
alpha- amylase from Bacillus sp.).
Whole slurry
No enzymes Enzymes
SSF SHF
Thermal (80 °C) 0.19 ± 0.0 0.36 ± 0.0 0.35
Thermal (120 °C) 0.20 ± 0.0 0.35 ± 0.0 0.36
H2SO4 0.24 ± 0.0 0.41 ± 0.0 0.44
NaOH 0.22 ± 0.0 0.37 ± 0.0 0.37
C.A. + CEL – 0.49 ± 0.0 0.47
B.A + CEL – 0.30 ± 0.0 0.40
TSinitial or 0.101 g/L of HMF was produced during acid pretreatment,
which was much lower than the respective amounts obtained from Ben
Taher et al. (2017). The discrepancy in the values could be attributed to
the different concentrations of acid which were used in the two studies,
since the release of these compounds depends strongly on the pre-
treatment severity (Monlau et al. 2014). Furfural or aliphatic acids such
as formic and acetic acids were not detected in any samples, which is
expectable since their production is in general associated with xylose
degradation or hemicellulose solubilization due to bond cleavage
(Antonopoulou et al., 2015), respectively, and in the case of PPW, the
hemicellulose concentration is low.
3.3. Enzymatic hydrolysis of raw PPW
In Fig. 1, the production of reducing sugars versus time during the
enzymatic hydrolysis experiments, using the amylolytic and cellulolytic
enzymes separately (Fig. 1a), in mixtures (Fig. 1b), or in the two-step
enzymatic hydrolysis process (Fig. 1c) is depicted. It should be men-
tioned that for the experiments of Fig. 1a, the optimum conditions of
each enzyme were used, while for the mixtures of enzymes (Fig. 1b),
two different temperatures were used, i.e. 30 °C which is the optimum
for B.A. and W. anomalus (in the case of SSF experiments) and 50 °C,
which is the optimum for commercial enzymes. In order to optimize the
individual conditions for B.A and CEL, a two-step process scheme was
studied, at the optimum conditions. In Table 4, the hydrolysis yield
calculated for two different experimental times (24 h of enzymatic hy-
drolysis and at the end of each experiment), is presented. For the two-
stage enzymatic hydrolysis process, the 24 h is referred to the time after
addition of the CEL (48 h in total, taking into account the first step with
B.A.).
It is obvious that the use of commercial amylase led to higher re-
ducing sugars release (42.78 ± 0.32 g/100 g TSinitial), compared to the
B.A. (16.17 ± 0.05 g/100 g TSinitial) (Fig. 1a). In the case of the enzy-
matic mixtures (Fig. 1b), the use of commercial enzymes led to more
promising results, indicating also that the operational conditions play a
crucial role on the enzymatic hydrolysis efficiency. Thus, at the op-
timum conditions for the commercial enzymes (pH 4.8; 50 °C), the re-
ducing sugars release after 48 h of hydrolysis was 68.36 ± 0.43 g/
100 g TSinitial, corresponding to 72.38 ± 0.46% enzymatic hydrolysis
yield, while at 30 °C it was 61.30 ± 0.17 g/100 g TSinitial
(64.90 ± 0.18%). Similarly, a higher temperature enhanced the effi-
ciency of the mixture of CEL with B.A. (37.89 ± 0.90 g/100 g TSinitial at
50 °C and 31.13 ± 0.05 g/100 g TSinitial at 30 °C, after 48 h of hydro-
lysis). The same profile was observed for sugars release and hydrolysis
yield, after 24 h of hydrolysis, where mixture of CEL + C.A. exhibited
the optimum yield compared with all enzymatic mixtures. From Fig. 1c
it is obvious that the separation of both hydrolytic steps, applying also
the optimum conditions of each enzyme, enhanced the saccharification
and the hydrolysis yield (52.66 ± 0.06% after 24 h hydrolysis with
CEL). The experimental results obtained are in accordance with the
results of Arapoglou et al. (2010) who reported that the addition of
7
Bioresource Technology 289 (2019) 121614
Separate fractions
Liquid Solid
SSF SHF
± 0.0 0.20 ± 0.0 0.29 ± 0.0 0.30 ± 0.0
± 0.0 0.24 ± 0.0 0.33 ± 0.0 0.31 ± 0.0
± 0.0 0.20 ± 0.0 0.40 ± 0.0 0.40 ± 0.0
± 0.0 0.23 ± 0.0 0.37 ± 0.0 0.39 ± 0.0
± 0.0 – – –
± 0.0 – – –
Celluclast (pool of cellulases) in a mixture of amylases, increased the
release of reducing sugars significantly, due to the additional cellulose
degradation. Regarding the amylolytic enzymes used, Izmirlioglu and
Demirci (2016) and Khawla et al. (2014) reported that enzymatic
mixtures of alpha -amylases perform liquefaction, decreasing the visc-
osity of the enzymatic slurry, and together with the enzymes of amy-
loglucosidase, are necessary for promising hydrolysis yields and en-
hanced bioethanol production from starchy-based substrates, such as
PPW.
3.4. Bioethanol production
3.4.1. Ethanol production from chemically or thermally pretreated PPW
In the present study, either the whole slurry obtained following
different pretreatment schemes or the separate fractions after pre-
treatment, were studied for ethanol production using W. anomalus X19.
Referred to the whole slurry fermentation, in Fig. 2a, the ethanol profile
of raw PPW as well as the whole slurry obtained after different che-
mically and thermally pretreated PPW without addition of enzymes, is
presented. In all cases, cumulative bioethanol production after chemical
or thermal pretreatment methods was much higher than the respective
of the raw biomass. For instance, ethanol production of raw PPW was
0.55 ± 0.07 g/L and increased to 9.16 ± 0.20 g/L or 7.27 ± 0.19 g/L
due to acid and alkali pretreatment, after approximately 40 h of fer-
mentation, respectively. Fig. 2b presents the ethanol concentration
from the whole slurry of chemically or thermally pretreated PPW
during fermentation with W. anomalus X19 using SSF (simultaneously
addition of CEL and C.A.) at initial pH 4.8, at 30 °C and Fig. 2c presents
the ethanol concentration using SHF (enzymatic hydrolysis with CEL
and C.A. at pH 4.8, at 50 °C for 24 h and subsequently fermentation
with W. anomalus X19 at 30 °C). As shown in the figures, the separation
of hydrolysis from the fermentation step (SHF) led to higher ethanol
production from the whole pretreatment slurry of PPW, due to the
optimum conditions which were applied in both separate processes. It
can also be seen that the ethanol concentrations were enhanced after
acid pretreatment either using SSF or SHF (16.59 ± 0.21 and
21.17 ± 0.19 g/L, respectively). This could be attributed to the higher
solubilization which occurred during acid pretreatment, leading to
higher sugars concentration (Table 3). In addition, the concentration of
the HMF produced during acid pretreatment was low, compared with
those reported in the literature to cause yeast inhibition (Delgenes
et al.,1996; Antonopoulou et al., 2016; Ben Taher et al., 2017).
In order to exploit different processes which could lead to promising
ethanol production, fermentation experiments with both fractions ob-
tained after separation of the chemically and thermally pretreated
slurry, were carried out. The liquid fractions were used for ethanol
production without addition of enzymes, while the solid ones (SPPW)
were used for ethanol production using SSF (CEL and C.A. at initial pH
4.8, at 30 °C) and using SHF (enzymatic hydrolysis with CEL and C.A. at
pH 4.8, at 50 °C for 24 h and subsequently fermentation with W.
anomalus X19 at 30 °C) (Fig. 3). Ethanol production from the liquid
I. Ben Atitallah, et al.
fractions obtained after pretreatment, despite the fact that they contain
high sugars concentration, led to low ethanol concentrations, compared
to the solid ones. In the case of SPPW obtained after thermal and
chemical pretreatments, ethanol concentration-versus -time followed
the same trend with the whole fraction of PPW (Fig. 2), with higher
ethanol production at SHF than at the SSF concept, and with the acid
pretreatment leading to higher ethanol concentration (17.27 ± 0.01 g/
L using SHF and 12.94 ± 0.04 g/L the respective concentration using
SSF).
3.4.2. Ethanol production from enzymatically hydrolyzed PPW
Enzymatically hydrolyzed PPW were used for ethanol production
(Fig. 4). The use of commercial amylases (Fig. 4a and b, where C.A. was
used together with CEL, either via SSF or SHF (the term is referred to
CEL, which in the case of SSF, was added simultaneously with the W.
anomalus and in the case of SHF, CEL with C.A. performed hydrolysis
for 24 h at 50 °C and then ethanol production was carried out at 30 °C
through W. anomalus)), similarly to the saccharification efficiency, led
to higher ethanol concentration compared to alpha - amylase from
Bacillus sp., when contained in the enzymatic mixtures with CEL
(12.00 ± 0.21 g/L). In addition, in the case of enzymatic hydrolysis
with B.A and CEL, in a two-stage process, the ethanol concentration was
higher (10.19 ± 0.32 g/L) when compared with the B.A. and CEL via
SSF, which was 7.83 ± 0.19 g/L.
3.4.3. Comparison of ethanol production yields from all pretreatment and
hydrolysis schemes
In Tables 5 and 6, the influence of all pretreatment/hydrolysis
schemes was evaluated in terms of ethanol production yield, expressed
either in g/g TSinitial of raw PPW or in g/gconsumed sugars. It should be
pointed out that, despite the high ethanol concentrations, the ethanol
yields from the pretreated SPPW (expressed as g/g TSinitial) were low,
due to the high solubilization and thus low percentage material re-
covery, which was observed during pretreatment, especially with the
use of chemicals, as this recovery was taken into account in the cal-
culations. In order to calculate the final yields, the ethanol produced
from liquid and the solid fractions (Table 5) should be added. From
Table 5, it can be seen that the higher ethanol yields were obtained
from chemically pretreated samples, under SHF (44.9 ± 0.1 g/100 g
TSinitial), and this was comparable with the yield of alkali-treated PPW
at SHF (41.9 ± 0.2 g/100 g TSinitial). Enzymatic hydrolysis using com-
mercial enzymes also led to high ethanol yields at SHF (40.1 ± 0.2 g/
100 g TSinitial), while the use of B.A. led to the lower ethanol production
efficiency, especially under SSF (19.6 ± 0.0 g/100 g TSinitial). High
ethanol yields could also be confirmed by the values presented in
Table 6, where it can be seen that W. anomalus X19 led to very high
ethanol yields, given though that the maximum theoretical ethanol
yield is 0.511 g/gconsumed sugars. In the case of enzymatic treatment with
commercial enzymes, the ethanol production yield was 0.49 g/gconsumed
sugars, corresponding to 96% of the maximum theoretical, while acid
pretreatment led to a yield of 0.44 (86% of the theoretical) and enzy-
matic hydrolysis using B.A led to a yield of 0.40 ± 0.0 g/gconsumed sugars
(78% of the theoretical). The latter could enhance the process economy,
since the possibility of substituting commercial enzymes by onsite
produced enzymes would reduce significantly the process cost. How-
ever, for the selection of the process scheme for a full-scale plant, it is
necessary to take into account both economic (based on enzymes costs)
and technical (based on ethanol yield) aspects.
The yields obtained in the current study are comparable with those
reported by Arapoglou et al. (2010) who used S. cerevisiae to produce
ethanol from enzymatically hydrolyzed PPW (0.46 g/g of sugar con-
sumed). Much lower yields have been reported by Ben Taher et al.
(2017) who obtained 0.17 and 0.26 g/gconsumed sugars, when using acid
and enzymatic hydrolysis of PPW, respectively, for ethanol production
from S. cerevisiae. These authors attributed the low ethanol yields to the
high concentrations of HMF in the medium (1.7 g/L), which inhibited
8
Bioresource Technology 289 (2019) 121614
the yeast growth, leading to low ethanol production efficiency. On the
other hand, Khawla et al. (2014) obtained a yield of 65% of the max-
imum theoretical when using mixtures of commercial enzymes and 70%
of the respective one with a mixture of enzymes produced in the lab.
The higher yields obtained in the present study could be attributed to
the process optimization which has been carried out, testing different
pretreatment and hydrolysis methods at different schemes (separation
of the fractions, SSF or SHF), which have been evaluated in a com-
parative way, maximizing thus the ethanol production efficiency of the
new isolated strain of W. anomalus X19.
4. Conclusions
The present study demonstrated that PPW is an attractive by-pro-
duct to produce bioethanol using the newly isolated yeast W. anomalus
X19. Results showed that acid treatment at SHF or enzymatic hydrolysis
with commercial enzymes, led to high ethanol yields, corresponding to
86 and 96% of the maximum theoretical. The possibility of using en-
zymes produced at lab- scale was also evaluated at different process
schemes, giving also promising results in terms of the enzymatic hy-
drolysis efficiency and ethanol yield. Further evaluation of the process
economy is needed in order to assess the optimum scenario.
Acknowledgements
This work was funded by the project “Research infrastructure for
Waste Valorization and Sustainable Management of Resources,
INVALOR” (MIS 5002495) which is implemented under the “Action for
the Strategic Development on the Research and Technological Sector”,
funded by the Operational Programme “Competitiveness,
Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed
by Greece and the European Union (European Regional Development
Fund). Dr. Georgia Antonopoulou acknowledges the financial support
of the Stavros Niarchos Foundation within the framework of the project
ARCHERS (“Advancing Young Researchers’ Human Capital in Cutting
Edge Technologies in the Preservation of Cultural Heritage and the
Tackling of Societal Challenges”) while Imen Ben Atitallah acknowl-
edges the Ministry of Higher Education and Scientific Research,
Tunisia.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biortech.2019.121614.
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