Fuel 134 (2014) 250256

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Ethanol fermentation of waste bread using granular starch hydrolyzing

enzyme: Effect of raw material pretreatment
Witold Pietrzak, Joanna Kawa-Rygielska

Department of Food Storage and Technology, Wrocaw University of Environmental and Life Sciences, Chemonskiego 37/41, 51-630
Wrocaw, Poland

highlights
Waste wheat rye-bread ethanol fermentation using granular starch hydrolyzing enzyme. Three raw
material pretreatment methods studied for ethanol yield improvement.
Separate hydrolysis and fermentation process studied for comparison.
Fermentation of unpretreated waste bread yielded 354.36 g ethanol kg
Application of raw material pretreatment further improved ethanol yield.

raw material.

abstract

article info
Article history:
Received 7 March 2014
Received in revised form 20 May
2014 Accepted 27 May 2014
Available online 12 June 2014
Keywords:
Ethan
ol fuel
Wast
e
bread
Ethanol fermentation
Granular starch hydrolyzing enzyme
(GSHE)

The subject of this research project was assessment of direct starch to ethanol conversion
process course of waste wheat-rye bread using granular starch hydrolyzing enzyme (GSHE).
Several pretreatment meth-ods (enzymatic prehydrolysis, microwave irradiation, sonification) were
used to improve the course of fermentation and were compared with separate hydrolysis and
fermentation (SHF). Due to high water binding capacity of raw material fermentations were
1

conducted at a substrate loading of 150 g kg . Only during enzymatic pretreatment and the SHF
process the raw material was preliminary liquefied so its higher concentrations could be applied.
The dynamics of fermentation was similar in all studied variants. The fermentation of unpretreated
waste bread ended with 80.00% ethanol yield (354.36 g kg
material improved ethanol yield by ca. 38%.

of raw material). Pretreatment of raw

2014 Elsevier Ltd. All rights
reserved.

Saccharomyces cerevisiae

Corresponding author. Tel.: +48 71 320 7764.

E-mail address: joanna.kawa-rygielska@up.wroc.pl (J. Kawa-Rygielska).

1. Introduction
Ethanol is considered as one of the most promising
renewable fuel that can replace fossil fuels-based
transportation fuels. It is most commonly produced by
microbial (most often yeast) cata-lyzed fermentations using
plant biomass as a raw material. Starchy raw materials (i.e.
corn, wheat, sorghum) are still the most common feedstocks
for fuel ethanol production in temperate climate regions of
the world (Europe, North America, Central Asia). However its
use as fuel production resources may affect on the prices of
food prod-ucts manufactured from them [1]. The use of nonedible parts of the plant (straws, stalks), known as the
lignocellulose biomass, as the raw material in distillery is
nowadays considered as the most prom-ising opportunity for
ethanol production that does not affect the prices of
foodstuffs [2]. However, the conversion of lignocellusosic
biomass into fermentable sugars and, subsequently into
ethanol

http://dx.doi.org/10.1016/j.fuel.2014.05.08
1 0016-2361/ 2014 Elsevier Ltd. All rights
reserved.

requires high temperature pretreatment which is often

catalyzed using corrosive, non-ecological or costly agents
like acids, alkali, ionic liquids and others [3]. Moreover the
efficiency of saccharifica-tion and fermentation of
lignocellulose is still much less efficient in comparison to
starches [4], but starchy raw materials are very costly and
the cost of the feedstock can exceed 65% of the price of
final product [5]. The solution to the problems of affecting
food prices by using agricultural crops for fuel production
and the tech-nological difficulties with conversion of

lignocellulosic biomass is utilization of food industry wastes

for production of biofuels. One of the most promising food
waste that can be processed into etha-nol is waste bread. It
contains significant amount of starch that is easily
hydrolyzed to monomeric sugars using amylases, the
amount of starch and simple sugars in bread ranges 500
750 and 3 50 g kg

1
1

respectively [6]. Moreover bread

contains 100150 g kg of protein which, after hydrolysis to

peptides and amino acids, is essential for yeast growth and
accelerated fermentation [7]. Waste bread is also highly
accessible raw material for ethanol processing. The
estimated wastage for bakery products ranges 710% of its
total

W. Pietrzak, J. Kawa-Rygielska / Fuel 134 (2014) 250256

251

production [8], taking into consideration estimated world

annual production of bread which is about 100 million tones
[9] the amount of generated waste can reach even 10 million
tones per year worldwide. The major factor for bread waste
formation is that part of the produced product is left unsold
and is returned to the bakery due to significant level of
staling and large amount of avail-able assortment of bakery
products which are produced in excess to fulfill the
consumers demands [10]. There are limited possibilities for
reprocessing bread waste in the bakeries. Some wastes can
be processed into bread crumbs, as a replacement of part of
flour in sourdough preparation or as animal feed. However
due to often microbial spoilage its use for human and animal
nutrition could be risky for health of the consumers. These
problems are the reason why waste bread is most often left
on landfills or used as a fuel for combustion. The most
promising solution for waste bread utiliza-tion is use it as a
raw material for ethanol fuel production. Earlier studies
shown that waste bread is a high-yielding material for ethanol fermentation [11,12]. The amount of ethanol produced
from bread waste that shown no signs of mould
contamination, depend-ing on processing technology,
1

ranged as mentioned by the authors ca. 350370 g kg of

feedstock dry matter. Kawa-Rygielska and Pietrzak [13]
studied the possibility of using waste bread showing high
level of surface mould contamination for ethanol production,
this resulted in a decrease of ethanol yield in comparison to
1

non contaminated material (ca. 230250 g kg depending

on the raw material loading in the fermentation feed). The
other possibilities for waste bread utilization via
biotechnological processes are in example: solid state
fermentation by Aspergillus awamori for pro-duction of
amylases and proteases [14], biohydrogen production using
rhizosphere microflora [15], succinic acid production by Actinobacillus succinogenes [16] or aromatic compounds
production by
Geotrichum candidum [17].
The typical pretreatment method for enzymatic hydrolysis
of starches to fermentable sugars is based on a two-step
method. In the first step starch is liquefied by heat-stable aamylase (EC 3.2.1.1) in order to decrease the viscosity of
gelatinized starch solu-tion and to produce short-chained
dextrins, by breaking down the a-1,4-glycosidic bonds in the
middle of amylose and amylopectin chains. During the second
step of starch hydrolysis (saccharifica-tion) the dextrins are
saccharified by glucoamylase (amyloglucosi-dase, EC 3.2.1.3)
to obtain monomeric sugars (glucose). Often supportive
enzymes, like proteases, cellulases, pullulnases and oth-ers, are
used to increase the amount of fermentable sugars, decrease
the viscosity of the mash and produce free amino nitro-gen that
is used as a nutrient for yeast [18,19]. After the hydrolysis the
mash is inoculated with yeast and subjected to ethanol fermentation. This kind of process is named separate hydrolysis and
fer-mentation (SHF). The enzymatic pretreatment is a costly
process because the liquefaction step is conducted in high
temperature (80100 LC) what demands large amount of
energy, also the sac-charification step is costly because
glucoamylase acts slowly and optimal temperature for its activity
is ca. 5060 LC. The optimiza-tion for energy demand for starch
hydrolysis for ethanol produc-tion led to development of
simultaneous saccharification and fermentation (SSF) process,
in which liquefied starch slurry is cooled to temperature where
yeast are able to ferment and gluco-amylase is added so the
sacchrification of dextrins and utilization of resulting monomeric
sugars occurs at the same time [20]. The most recent
development in the field of processing starchy raw materials to
ethanol is the direct starch to ethanol conversion using granular

starch hydrolyzing enzyme (GSHE). GSHE is obtained from

genetically modified Trichoderma reesei and it shows the activity
of a-amylase and glucoamylase displaying on the sur-face of
starch granules. Earlier studies shown that the efficiency of
direct starch to ethanol conversion process is comparable to
traditional technologies [21] and its advantage is lower energy

demand because of lack of the starch gelatinization and

liquefac-tion steps. Also some improvements for direct
conversion of starch to ethanol technology were done.
Balcerek and Pielech-Przybylska [22] studied, among other,
the effect of thermal prehydrolysis of triticale meal using aamylase and application of protease on the process of raw
starch hydrolysis and fermentation. They discov-ered that
better efficiency of fermentation was obtained without
thermal activation but with added proteolytic enzyme. Montalbo-Lomboy et al. [23] studied the effect of sonification of
corn meal slurry prior to direct conversion to ethanol. The
results of this research proved that sonification of raw
material improved the ethanol yield by ca. 20% in
comparison to the control samples, moreover the ethanol
yield in sonificated samples was similar to jet-cooked corn
meal. The authors also conducted the economical
evaluation and energy usage for jet-cooking and ultrasonics
instal-lations in industrial environments. They stated that the
overall cost for installation and maintenance for ultrasonic
apparatus is lower in comparison to hydrocooking, also the
energy analysis proved that much less energy is needed for
sonification of raw material than for jet cooking. Also
microwave treatment improves enzy-matic hydrolysis of
starch [24]. The usage of cheap, waste raw material like
bread leftovers in the cost-effective process of direct starch
hydrolysis and fermentation could be very attractive for ethanol fuel production. Until now the usage of GSHE for the
waste bread processing into ethanol was not studied. This
could be very effective solution including high ethanol yield
and vast reduction of processing costs in comparison to
traditional processes.
The object of this study was to asses the ethanol
fermentation course and efficiency of waste bread using
granular starch hydro-lyzing enzyme in comparison to
separate hydrolysis and fermenta-tion process. Also different
methods of raw material pretreatment (enzymatic, ultrasonic

and microwave) was conducted to improve raw starch

hydrolysis and fermentation.
2. Materials and methods
2.1. Raw material
Waste wheat-rye bread (after shelf-life, returns from
shops) was obtained from local bakery. Whole loafs were
manually cut into dices of ca. 24 cm size. Obtained dices
were dried in a forced air oven at 40 LC for 12 h and ground
in a knife mill (Rotary Mill, Brabender, Germany) with 1.5
mm internal mesh sieve. Raw mate-rial was stored in an air
tight jar at room temperature until used. Starch content in
waste bread was determined using Evers polari-metric
1

method [25] and it ranged 689.13 4.52 g kg

of raw
material dry matter. The amount of total sugars (as glucose)
was measured using the DNS method [26] after mild acid
1

hydrolysis (70 LC, 10 min) with 80 g L HCl solution (1:7.5

m/v raw material to HCl solution ratio) and it ranged 866.87
1

2.85 g kg
of raw material dry matter. Raw material
moisture content was measured using WPS 50P weighing
1

dryer (Radwag, Poland) and it ranged 41.43 1.38 g kg .

2.2. Enzymes and yeast

GSHE preparation STARGEN 002 was obtained from
Genencor International (USA). STARGEN 002 contains
Aspergillus kawachii a-amylase expressed in T. reesei and
glucoamylase from T. reesei. As declared by the manufacturer,
the preparation activity is 610 glucoamylase units per gram and
has an optimal temperature range of 3545 LC. Neutrase 0.8 L
(Novozymes, Denmark) is a bac-terial protease form Bacillus
amyloliquefaciensis, its declared activ-ity was 0.8 proteolytic
units per gram. Ceremix 6X MG (Novozymes) is a preparation
displaying multidirectional substrate

252

W. Pietrzak, J. Kawa-Rygielska / Fuel 134 (2014) 250256

specificity
(a-amylase,
b-glucanase,
pentosanase, cellulase, prote-ase). In
separate
hydrolysis
and
fermentation
experiment following enzymes were used (all
produced by Novozymes): Termamyl SC DS
(thermostable a-amylase from Bacillus
licheniformis with declared activity of 240 kilo
novo units per gram) and SAN Extra L
(glucoam-ylase from Aspergillus niger with
declared activity of 400 glucoam-ylase units
per gram) and, mentioned above, Neutrase
0.8 L. Commercial active dry yeast
Saccharomyces cerevisiae Ethanol Red
(Fermentis, France) was used as production
9

inoculum, it con-tained over 20 10 of living

cells per gram as declared by the
manufacturer. Prior to inoculation the yeast
were rehydrated in sterile distilled water (1 g
of yeast preparation in 10 mL of water) at 30
LC for 0.5 h with regular agitation.
2.3. Fermentation experiment variants
2.3.1. Direct conversion of raw
material to ethanol without
pretreatment (Variant I)
Waste bread samples (30 g dry matter
basis) were slurred in ca 150 mL of distilled
water preheated to 35 LC in a 300 mL conical
flasks. The pH of the slurry was adjusted to
1

4.5 using 1 mol L H2SO4 solution. GSHE (2

mL per 1 kg of raw material dry matter) and
1

proteolytic (Neutrase 0.8 L, 0.875 mL kg of

raw material dry matter) preparations were
added and the total weight of the slurry was
adjusted to 200 g with distilled water resulting
in a raw material loading of 150 g of dry
matter per kilogram of fer-mentation medium
(the experiments were conducted at relatively
low raw material loading because used raw
material showed the high water binding
capacity and application of higher substrate
loading resulted in obtaining solid like
consistence of the fermen-tation medium
what was observed earlier [11,27]). Then the
waste bread slurry was inoculated with 2 mL
of yeast suspension giving initial yeast
1

concentration of 1 g kg . The flasks were

sealed with rubber stoppers with fermentation
tube filled with glycerol and a sampling port
as described by Kim et al. [28] and were
placed in a WNB7-45 water bath shaker
(Memmert, Germany) at 35 LC with agitation
speed of 150 rpm (2.5 Hz) for 72 h.

2.3.2. Enzymatic pretreatment (Variant II)

Raw material samples (30 g dry matter
basis) were slurred in ca. 150 mL of distilled
water in a 300 mL conical flasks and pH of
the slurry was adjusted to 4.5 using 1 mol L

H2SO4 solution. Then Ceremix 6X MG

preparation (1.25 g of enzyme per kilogram of
raw material dry matter) was added and the
total weight of the flask content was adjusted
to 200 g with distilled water. The flasks were
sealed with rubber stoppers to avoid water
evaporation and were placed in a water bath

heated to 45 LC for 20 min with con-stant

agitation [12]. Afterwards the slurries were
cooled to 35 LC, inoculated with yeast, GSHE
and protease (Neutrase 0.8 L) were added
and further procedure was the same as
described in the Sec-tion 2.3.1.

2.3.3. Microwave pretreatment (Variant III)

Raw material samples (30 g dry matter
basis) were spread in a thin layer on a Petri
dish and irradiated in a microwave oven
KOR-63X5 (Daewoo, China) for 2 min with
400 W microwave power output and 2450
MHz frequency. After the pretreatment
samples were cooled to room temperature in
a desiccator. Further proce-dure was the
same as in Section 2.3.1.
2.3.4. Ultrasonic pretreatment (Variant IV)
Ground waste bread samples (30 g dry
matter basis) were slurred in ca. 150 mL of
distilled water preheated to 35 LC in a 300
mL conical flasks. The flasks were placed in
an ultrasonic bath SONIC-0.5 (Polsonic,
Poland) for 5 min, as described by the
manufacturer the power of the bath was 250
W and ultrasonic fre-

quency was 40 kHz. After this time the

experimental procedure was the same as in
Section 2.3.1.
2.3.5. Separate hydrolysis and fermentation
(SHF) (Variant V)
Waste bread samples (37.5 g dry matter
basis) were weighted into the mashing cups
and slurred with ca. 180 mL of distilled water.
pH of the slurry was adjusted to 6.0 using 1
1
mol L H2SO4 or NaOH solutions. The cups
were placed in a LB 12 labora-tory mashing
device (Lochner Labor + Technik, Germany)
pre-heated to 45 LC. The mashing was
conducted with 150 rpm (2.5 Hz) stirring
1
speed and 2 LC min heating and cooling
rate. When the temperature of the slurry
reached 60 LC thermostable a-amylase
1

(Termamyl SC DS, 2 mL kg of raw material dry

mat-ter) was added and the bath of the mashing
device was further heated to 85 LC. The starch
liquefaction was conducted for 60 min. Then the
samples were cooled to 55 LC, its pH was
adjusted to 5.8 with 7.11 mol L

H2SO4,
1

glucoamylase (SAN Extra L, 1.25 mL kg of raw

material dry matter) and protease (Neutrase 0.8
1

L, 0.875 mL kg
of raw material dry matter)
were added and the hydrolysis was conducted
for 90 min. After this time the sam-ples were
cooled to 20 LC, the pH of the samples was
1

adjusted to 4.5 using 1 mol L H2SO4 solution,

and total weight of the mashes was adjusted to
250 g using distilled water resulting in a final raw
1

material loading at 150 g kg of mash. 200 g

aliquots of the mashes were transferred to 300
ml conical flasks, inoculated with 2 ml of yeast
1

slurry (yeast concentration of 1 g kg of mash)

and sealed with rubber stopper with fermentation
tube and sampling port. Further procedure was
the same as described in Section 2.3.1.

2.4. Analytical methods

Fermentation dynamics was assessed
gravimetrically measuring the weight loss of
fermentation medium due to CO 2 release as
described earlier [12]. The samples for

physico-chemical analysis (ca. 20 mL

aliquots) were taken every 24 h of
fermentation. The non dissolved solids
concentration was determined as follows: ca.
10 g of fermentation media (with an accuracy
of 0.001 g) were fil-tered through pre-dried
(105 LC, 4 h) and pre-weighted filter paper,
the residual dissolved solids were washed
with ca. 250 mL of dis-tilled water. The filters
with remaining non-dissolved solids were
dried in an oven at 60 LC for 12 h next at 105
LC for 4 h. Afterwards the filters were cooled
to room temperature in a desiccator and
weighted with an accuracy of 0.001 g. The
difference in the weight of the filter with and
without of non-dissolved particles gave the
concentration of them in the fermentation
medium which was expressed as grams of
non-dissolved solids per kilogram. For HPLC,
reducing sugar and dissolved solids analysis
the samples of fermen-tation media were
centrifuged at 6000 rpm (5243g) at 4 LC
using MPW-351R centrifuge (MPW Med.
Instruments, Poland) and clear supernatants
were taken for analyses. The reducing sugars
concen-tration (as glucose) was determined
using the DNS method [26]. The dissolved
solids content (apparent extract) was
determined by the density measurement of
the clarified fermentation medium as
described previously [29]. The concentration
of sugars (glucose, maltotriose and dextrins
(DP4+)), fermentation by-products (glyc-erol,
lactic acid) and ethanol was determined using
high perfor-mance liquid chromatography
(HPLC). Prior to analysis clear supernatant
after centrifugation of fermentation media was
diluted with bidistilled water in a 1:3 v/v ratio
and filtered through nylon syringe filter (0.2
lm). The analysis was performed using
Promi-nence chromatograph (Shimadzu,
Japan) equipped with Rezex ROA-Organic
Acid H + column (300 7.8 mm) (Phenomenex,
USA). Following parameters of HPLC
analysis were applied: injec-tion volume-20
lL, elution temperature-60 LC, mobile phase0.005 mol L

H2SO4, mobile phase flow rate1

0.6 mL min . The analytes were detected

using refractive index detector (RID-10A,

W. Pietrzak, J. Kawa-Rygielska / Fuel 134 (2014) 250256

253
1

Shimadzu)
at
50
LC.
Obtained
chromatograms were integrated using
external standard method with CHROMAX 10
software (Pol-Lab, Poland).
On the basis of obtained results following
process parameters were calculated at 24 h
time intervals: ethanol production rate (r p [g L
1

h ]) and practical ethanol yield (Yp [%; g kg

of sugars; g kg of raw material dry matter]

on the basis of stoichiometric reaction where
1 kg of sugars (as glucose) is converted to
511.1 g of ethanol [30].

2.5. Statistical analysis

All experiments and determinations were
conducted in tripli-cates. Presented results
are mean values with standard deviations (n
= 3). The results were also subjected to oneway analysis of var-iance (ANOVA) at
significance level a = 0.05 using Statistica
10.0 package (StatSoft, Inc., USA). Duncans
test was used to determine statistically
homogenous groups (denoted as subsequent
letters of the alphabet) and lowest significant
differences (LSD).
3. Results and discussion
3.1. Effect of pretreatment method on
physico-chemical properties of fermentation
media
The initial concentration of reducing
sugars and dissolved solids in the
fermentation media was the highest in
samples after sepa-rate hydrolysis and
fermentation (SHF) pretreatment (Fig. 1a and
b respectively). Enzymatic pretreatment using
Ceremix 6X MG preparation resulted in
preliminary hydrolysis and liquefaction of
waste bread giving initial content of sugars
and dissolved solids

Reducing sugars [g L -1]

120

(a)

90

60

30

0
0

20

of ca. 25 and 30 g L respectively. Other

pretreatment methods applied (microwave
and ultrasonic) did not affected initial concentration of sugars and dissolved solids at the
beginning of the fer-mentation in comparison
to unpretreated variant. The microwave and
ultrasonic pretreatment did not influenced the
content of non-dissolved solids (ca. 131 g kg
1
of media) in comparison to samples without
pretreatment (Fig. 1c). Preliminary hydrolysis
of waste bread with Ceremix 6X MG caused
reduction in non-dis-solved particles by ca. 80
1
g kg in comparison to control, micro-waved
and sonificated samples, while samples after
separate two-stage hydrolysis with aamylase, glucoamylase and protease (SHF)
contained the lowest amount of non-dissolved
1
solids among others (ca. 30 g kg ).
Earlier studies shown that different
pretreatment methods influ-ence on the initial
properties of fermentation media in the GSHE
aided fermentations. Balcerek and PielechPrzybylska [22] studied the effect of thermal
prehydrolysis (at 5657 LC) of triticale starch
with
a-amylase
and
non-starch
polysaccharide degrading enzyme on the
course of fermentation with GSHE. The initial
content of reducing sugars in pretreated
media was much higher in compari-son to
samples without prehydrolysis (ca. 11 and 4 g
1

100 mL respectively). Shavanas et al. [21]

came to similar conclusion while studying the
effect of preliminary liquefaction of cassava
starch prior to fermentation with GSHE.
Similar results were achieved in present
study in samples subjected to enzymatic
pretreatment where the polysaccharides
present in the raw material were preli-minary
hydrolyzed by the complex of enzymes in
Ceremix 6X MG preparation (amylases,
proteases, cellulases and others). The advantage of using this preparation was previously
confirmed in the pro-cess of separate
hydrolysis and fermentation of waste bread at
high solids loading [12]. Moreover the content
of non-dissolved solids was much lower in
enzymatically pretreated waste bread slurries

150

(b)

Dissolved solids [g L -1]

II
III
IV
V

I
II
III
IV
V

100

50

0
60

80

20

40

60

Non-dissolved solids kg[g -1 ]

150

80

Time [h]

Time [h]

(c)
I
II
III
IV
V

100

50

0
0

20

40

60

80

Time [h]
Fig. 1. Changes in physico-chemical properties of fermentation media from waste wheat-rye bread during direct starch
conversion to ethanol with GSHE (a) reducing sugars,
(b) dissolved solids, (c) non-dissolved solids (I unpretreated variant, II enzymatic pretreatment, III microwave
pretreatment, IV ultrasonic pretreatment, V SHF process). Results expressed as mean values standard deviation
(n = 3).

254

W. Pietrzak, J. Kawa-Rygielska / Fuel 134 (2014) 250256

Table 1
Carbohydrate profiles during fermentation of media prepared from waste wheat-rye bread (I unpretreated variant, II enzymatic pretreatment, III microwave pretreatment, IV
ultrasonic pretreatment, V SHF process). Results expressed as mean values standard deviation (n = 3).
Variant
I
II
III
IV
V

Glucose (g L )

Maltotriose (g L )

Dextrins (DP4+) (g L )

24 h

48 h

72 h

24 h

48 h

72 h

40.78 3.99
42.99 3.54
52.86 3.95
49.33 0.66
40.10 3.47

2.58 2.03
0.16 0.07
2.42 0.15
4.75 0.90
0.06 0.04

0.09 0.05
0.00 0.00
0.00 0.00
0.00 0.00
0.00 0.00

0.24 0.07
0.47 0.06
0.77 0.16
0.37 0.04
0.89 0.18

0.37 0.07
0.35 0.07
0.58 0.01
0.84 0.33
0.30 0.02

0.36 0.04
0.26 0.03
0.39 0.02
0.11 0.09
0.23 0.01

24 h
8.46
11.23
13.90
9.95
12.57

0.72
0.48
0.66
0.17
0.71

48 h

72 h

7.40 0.98
8.28 0.26
9.75 0.39
8.46 0.26
7.93 0.13

7.71 0.56
8.02 0.38
8.43 0.11
6.90 0.61
7.86 0.16

during baking and staling where the formation of gluten-lipid network

and starch retrogradation occurs which are difficult to reverse using
physical treatment [31].

in comparison to microwave, ultrasonic and unpretreated variants of

the experiment, what suggests that application of relatively lowtemperature and short time enzymatic hydrolysis (unlike in the SHF
process) of waste bread for direct conversion to ethanol using GSHE
could be used in fermentation of media at higher solids loading than in

3.2. Effect of pretreatment method on fermentation course of waste

bread with GSHE

present study (150 g kg ) avoiding the problem of water binding

capacity of raw material. Montalbo-Lamboy et al. [23] studied the effect
of batch and continuous ultrasonic pretreat-ment of corn slurry prior to
GSHE aided fermentation. They discov-ered that both batch and
continuous method increased the initial concentration of glucose in raw
material slurries, what was proba-bly caused by starch gelatinization
during pretreatment. Sadeghi and Shawrang [24] studied the effect of
microwave irradiation on rumen degradability of corn meal. They found
that irradiation of raw material for 3 to 5 min at 800 W power input
increased starch degradation rate. In present experimental conditions
no effect of ultrasonic and microwave pretreatment on initial sugar
release and liquefaction of waste bread was observed. This was
probably caused by the physico-chemical alterations of bread
ingredients

60

(Fig. 1a). Its concentration ranged ca. 53, 10 and 0.2 g L after 24, 48
and 72 h of fermentation respec-tively. In enzymatically pretreated
samples sugars concentration after the first day increased by ca. 12 g
1

L
in comparison to ini-tial state. After subsequent days of
fermentation the reducing sugars content in this variant of experiment
was below

(a)

(b)

Glycerol [g L-1]

45

Ethanol [g L-1]

The fermentation dynamics assessment shown that carbon dioxide

liberation from fermentation media was more advanta-geous for
enzymatically pretreated waste bread and for the SHF process in
comparison to other variants of experiment (data not shown). The
changes in reducing sugars concentration in micro-waved, sonificated
and unpretreated samples was similar during the fermentation process

30
I
II
III

15

4
I
II
III

IV

IV

0
0

20

40

60

80

20

40

Time [h]

60

80

Time [h]

1,5

Lactic acid [g L -1

(c)

I
II
III
IV
V

0,5

0
0

20

40

60

80

Time [h]
Fig. 2. Formation of ethanol (a), glycerol (b) and lactic acid (c) during direct starch to ethanol conversion process of waste wheat rye-bread media with GSHE (I unpretreated
variant, II enzymatic pretreatment, III microwave pretreatment, IV ultrasonic pretreatment, V SHF process). Results expressed as mean values standard deviation (n = 3).
W. Pietrzak, J. Kawa-Rygielska / Fuel 134 (2014) 250256
255

0.47 0.11c
0.52 0.06bc
0.73 0.03ab
0.92 0.01a
0.55 0.02bc
0.21

0.13 0.04a
0.10 0.07a
0.06 0.03a
0.01 0.14a
0.05 0.03a
0.26

48 h

1.61 0.11a
1.81 0.01a
1.62 0.01a
1.36 0.02b
1.81 0.03a
0.19

of malto-triose throughout the fermentation was below 1 g L regardless from

the experimental variant. The dextrins were not completely hydrolyzed and
eventually utilized by yeast by the end of fer-mentation, however its
concentration decreased between the 24th and 48th hour and did not
significantly change by the end of the process. The changes in dissolved
solids in the fermenta-tion media were similar to changes in the concentration
of reduc-ing sugars in particular variants of experiment (Fig. 1b). The nondissolved solids content after the first day of fermentation was similar in all
1

studied variants of experiment (ca. 30 g kg ) and it did not significantly

change by the end of the process (Fig. 1c). The changes in ethanol formation
during fermentation tests were shown in Fig. 2a. It was observed that within
the first 24 h of fermentation of enzymatically pretreated waste bread with
GSHE and separate hydrolysis and fermentation variant, yeast produced the

20.11b
4.47a
5.73a
19.70a
2.42a

(g L1 h 1 )

24 h

72 h

ethanol production rate) (I unpretreated variant, II enzymatic pretreatment, III

0.5 g L . In the SHF variant of experiment yeast did consume half of the
available sugars in the fermentation feed after 24 h of the process and almost
all available sugars after subsequent days. HPLC analysis of carbohydrates in
media during the fermentation also shown that almost all available glucose
was utilized by yeast by the second day of the process (Table 1). The content

highest amount of ethanol (ca. 43.5 g L ). Lower concentration of ethanol (ca.

1

15.12b
7.25a
5.78a
2.45a
6.00a
22.25

waste bread sam-ples, while the lowest amount of alcohol (ca. 32.6 g L ) was
determined in media with subsequently microwave irradiated waste bread.
The production of ethanol by yeast in studied fer-mentation tests lasted by the
second day of the process and did not significantly change by the third day.
The lowest concentra-tion of ethyl alcohol was found in unpretreated samples
1

(51.58 g L ), different methods of raw material pretreatment and the SHF

process resulted in higher amount of ethanol by the end of fermentation (ca.
1

difference.

48 h

258.38 17.39a 333.31

290.20 2.39a 373.15
259.24 1.38a 375.51
217.53 2.80b 365.05
290.24 4.63a 378.71
29.99
30.75

24 h

practical ethanolyield, r

(g kg 1 of raw material)

72 h

354.36
389.41
384.60
366.78
385.98

38.8 g L ) was found after the same time in unpretreated and sonificated

5558 g L ). The concentration of fermentation by-products, glycerol and

lactic acid were shown in Fig. 2b and c respectively. Within the first 24 h of
fermentation yeast did produce ca. 90% of total glycerol in all studied variants
of experiment, however less glycerol was formed in media with unpretreated
and microwaved waste bread than in the case of other studied methods. The
final glycerol concentration was sim-ilar in all studied fermentation media (ca.
1

aThemeanvaluesgiveninlineswithdifferentletterindexaresignificantlydifferent( = 0.05). LSD lowestsignificant

72 h
48 h

298.06 20.06a 384.50 17.44b

334.77 2.76a 430.45 18.36a
299.06 1.59a 433.18 6.67a
250.94 3.23b 421.12 2.83a
334.81 5.34a 436.87 6.92a
35.46
27.40
80.00 4.54b
87.92 1.01a
86.83 1.29a
82.81 4.45a
87.14 0.55a
34.59

24 h
24 h

48 h

72 h

(g kg
p

(%)

I
58.33 3.93a 75.25 3.41b
II
65.52 0.54a 84.24 1.64a
III
58.53 0.31a 84.78 1.31a
IV
49.11 0.63b 82.42 0.55a
V
65.53 1.05a 85.50 1.35a
LSD
6.77
6.94
5.36

of sugars)

408.79 23.20b
449.21 5.15a
443.66 6.61a
423.11 22.72a
445.26 2.79a

studied variants of experiment throughout the process (ca. 1 g L ) with-out

major changes.

Variant Y

Table 2Ethanol yield and production rate at specified time intervals of fermentation of waste wheat-rye bread with GSHE (Ymicrowavepretreatment,IVultrasonicpretreatment,VSHFprocess).

6.06.5 g L ). The lac-tic acid content in fermentation media was similar in all

The improvement in fermentation dynamics, as described by CO2 emission

from fermentation media, for SHF and enzymatically prehydrolyzed waste
bread was probably caused by hydrolysis of proteins present in raw material
to short-chained peptides and amino acids, which are essential for proper
yeast metabolism [7] and increased amount of fermentable sugars in
comparison to other variants. Previous study on the utilization of waste bread
for ethanol production proved that application of Ceremix 6X MG enzyme
blend, prior to a-amylase aided liquefaction, improved the dynamics and
ethanol yield of separate hydrolysis and fermen-tation process [12]. Balcerek
and Pielech-Przybylska [22] also proved that addition of proteases increase
the ethanol formation rate in the native starch hydrolysis and fermentation
process, espe-cially in its early stages. The increase in reducing sugars
concentra-tion in GSHE aided fermentations in the early stages of
fermentation was typical for the simultaneous saccharification and
fermentation process. This was observed earlier [23,32] and was caused by
higher rate of enzymatic hydrolysis of sugars than utilization of them by yeast.
It was observed that not all dextrins in the fermentation media were
hydrolyzed and fermented by the end of fermentation. This could be caused
by enzyme inactivation by high concentration of ethanol or decrease in the pH
of the media.

256

W. Pietrzak, J. Kawa-Rygielska / Fuel 134 (2014) 250256

3.3. Final effects and kinetics of

fermentation of waste bread using GSHE
as affected by pretreatment method
It was observed that application of all
pretreatment methods used in present study
increased the final ethanol yield in comparison
to
unpretreated
waste
bread
fermentation (Table 2). For enzy-matically
and microwave pretreated waste bread
fermentation with GSHE and SHF process
the yield of the process was the high-est, in
the case of other variants lower ethanol yields
were achieved, but still they were very high
(above 80% of theoretical). The highest
values of ethanol productivity were achieved
in all studied samples within the first day of
fermentations. Between the second and third
day of fermentation the changes in the yields
and productivity for all studied variants of
experiment did not change significantly what
suggested that these processes could be
shortened to 48 h.
Previous studies dealing with utilization of
waste bread into ethanol reported similar or
even lower ethanol yields. Ebrahimi et al. [11]
studied the separate hydrolysis and
fermentation of waste wheat bread at a raw
1

material loading of 350 g kg . They obtained

overall ethanol yield of 350 g per kilogram of
bread dry matter and the alcohol
concentration in the fermentation media
1

about 100 g L . Kawa-Rygielska et al. [12]

reported that prehydro-lysis of waste wheatrye bread with Ceremix 6X MG preparation
increase the ethanol yield from mashes at
320 g kg

waste wheat-rye bread loading

1

from 352.4 to 366.0 g kg in comparison to

control. In present study the ethanol yield
from substrate (sug-ars in raw material) unit
was also very high without high-temper-ature
liquefaction and separate saccharification,
however the raw material loadings were
relatively low so the impact of substrate and
product inhibition on yeast cells was minor. In
industrial application higher raw material
loadings would be more profitable because if
the higher ethanol concentration is achieved
the less energy is needed to distill it [33].

4. Conclusions
Present study proved that waste wheatrye bread is a high eth-anol yielding material
in direct conversion to ethanol process using
1

GSHE resulting in ca. 354 g kg

of raw
material. Moreover applica-tion of various raw
material pretreatment methods (enzymatic
prehydrolysis, sonification and microwave
irradiation) further increased the ethanol yield
1

by ca. 1235 g kg . However the stud-ies

were conducted at low raw material loading

due to water hold-ing capacity of bread what
resulted in solid-like consistence of the media
at higher loadings. The solution to this seems
to be the enzy-matic prehydrolysis which
causes preliminary liquefaction of raw
material.

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