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Ethanol production from bread residues

Fatemeh Ebrahimia, Morteza Khanahmadib,, Shapoor Roodpeymaa, Mohammad J. Taherzadehc
a

Chemical Engineering Department, Isfahan University of Technology, Isfahan, Iran Agricultural Engineering Research Department, Isfahan Center for the Research of Agricultural Science & Natural Resources, Isfahan, Iran c School of Engineering, University of Boras, SE-50190 Boras, Sweden
b

art i cle info

Article history: Received 11 July 2006 Received in revised form 18 October 2007 Accepted 23 October 2007 Keywords: Bread residues Ethanol Hydrolysis a-Amylase Starch Amyloglucosidase

ab st rac t
Bread residues were converted into a suitable fermentation feed via a two-step starch hydrolysis using amylolytic enzymes. Wheat our hydrolysis was also carried out at the same conditions for comparison. For the rst stage, namely liquefaction, effects of temperature (5085 1C) and substrate concentration (20% and 35%) were investigated. The 3-h liquefaction of the 20% bread suspension made 70% of initial dry matter soluble regardless of the temperature. The liquefaction of the 35% bread suspension had to be carried out by a fed-batch method due to the pasty behavior of the suspension. It resulted in a 65% dissolution of the suspended bread at 85 1C. Saccharication of the latter product led to a fermentation feedstock having a dextrose equivalent (DE) of more than 95 and almost 80% dissolution of the initial dry matter. The prepared feedstock was then cultivated using Saccharomyces cerevisiae, which resulted in an overall yield of 350 g ethanol per kg of initial bread dry matter. Staling of the bread for a week had no effect on liquefaction, saccharication and ethanol yield. & 2007 Elsevier Ltd. All rights reserved.

1.

Introduction

Wheat bread is probably the most popular part of the meals in Iran and some other countries. More than 10 million tons of wheat are annually converted into bread in Iran, where more than 10% of the bread becomes residue because of various reasons such as poor quality of wheat, deciencies in baking technology, storage and distribution systems. While part of the non-bread residues ends in municipal solid wastes, a major portion of it is collected and used as animal feed. However, a few percent of the bread residues is moldy and its implementation as animal feed is a gate for the entrance of mycotoxins into the human food chain, and raises severe health problems [13]. Hence, alternative handling methods should be investigated for taking care of such huge amounts of bread residues. Starch is the main constituent of the bread dry weight. Since starch is a generic fermentation feed, for e.g. ethanol in
Corresponding author. Tel.: +98 311 7760061; fax: +98 311 7757022.

industry [46], the bread residues could be considered as possible fermentation feed for different products. Despite the fact that the chemical composition of bread is almost similar to that of its original our, the processes of hydrolyzing our and bread to fermentation feedstock may differ due to structural changes resulting from dough production and baking. The gluten network that develops in dough may protect some parts of starch chains from enzyme attack [7,8]. Millard reactions may also compound a part of sugars with amino acids. On the other hand, gelatinization of starch during baking may alter its hydrolysis temperature dependence. Moreover, some part of starch is already hydrolyzed by ours native enzymes during the leavening process. These differences favor an investigation of the possibility of using bread residues as a general feedstock for fermentation processes, which might result in different processes in comparison to using our for this purpose.

E-mail address: khanahmadi@yahoo.com (M. Khanahmadi). 0961-9534/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2007.10.007

Please cite this article as: Ebrahimi F, Khanahmadi M, Roodpeyma S, Taherzadeh MJ. Ethanol production from bread residues. Biomass and Bioenergy (2007), doi:10.1016/j.biombioe.2007.10.007

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Ethanol is nowadays the most important product of biotechnology in terms of volume and the market [9]. Its market grew from less than 1 109 liters in 1975 to more than 39 109 liters in 2006 [10], where fuel ethanol has been the main driving force. The same trend is expected to follow during the next several years [10]. Ethanol production from grains such as corn and wheat is an established process in industry. However, we detected no report on ethanol production from bread residues. The present work aimed to examine two key stages, liquefaction and saccharication, in conversion of bread residues into a generic fermentation feed. Liquefaction processes for bread residues and wheat our were compared, and the effects of temperature and initial solids percentage were investigated. Then, saccharication of the liqueed products was examined. Suitability of the saccharied bread for fermentation was then tested by fermenting this material to ethanol.

suspension was then adjusted to 4.5 using 10 M sulfuric acid. The saccharication experiments were performed in 1-l asks by addition of 0.8 ml 1000 g1 DM amyloglucosidase, located in a shaker incubator at 60 1C. The samples of either liquefying or saccharifying solutions were withdrawn at appropriate time intervals and stored at 20 1C for analyses.

2.4.

Cultivation by high cell density

The saccharication product was diluted enough to obtain a glucose concentration of 250 g l1, centrifuged and its pH was adjusted to 5.0 using sulfuric acid. It was then inoculated with 20 g l1 S. cerevisiae and cultivated anaerobically in 250-ml conical asks at 30 1C and 150 rpm as previously mentioned [11].

2.5.

Analytical methods

2.
2.1.

Materials and methods

Enzymes and yeast

A thermostable a-amylase (Thermamyl 120L) and a fungal amyloglucosidase (AMG 300L) were provided by Novozyme A/S (Denmark). Saccharomyces cerevisiae used for ethanol production was commercial dried bakers yeast produced by Kolarmayeh Co. in Iran.

2.2.

Raw materials

The sugar of the samples was quantied using the DNS method [12]. This measurement of the sugar concentration was used to calculate dextrose equivalent. The initial starch content of bread or our dry matter was assumed to be 80% [13]. The centrifugation precipitants of the samples were washed twice using distilled water, dried in oven at 80 1C and weighed to determine remaining insoluble parts of the material. The samples of the fermentation were analyzed by an HPLC (Jasco International Co., Tokyo, Japan). The glucose, ethanol and glycerol contents of the samples were determined using ion exchange Aminex column HPX-87 H (Bio-Rad, USA) at 60 1C with a refractive index detector. The eluent was 5 mM H2SO4 at a ow rate of 0.6 ml min1.

A commercial at-wheat bread and wheat our were obtained from a local bakery shop in Isfahan, Iran. The bread was dried in ambient conditions and partly powdered by a laboratory hammer mill. The powdered bread was then sizeclassied by a mesh sieve, and mixed to obtain a size distribution identical to that of our. Both bread powder and wheat our were stored at 20 1C until use. The moisture of the bread powder and our was measured by overnight drying in an oven at 80 1C. Stale bread was prepared by storing fresh bread in a refrigerator for a week prior to drying.

3.
3.1.

Results
Liquefaction

2.3.

Liquefaction and saccharication

Liquefaction experiments were performed in a 1-l balloon, equipped with an adjustable mechanical overhead stirrer and a water bath to control the temperature. The experiments were initiated with 400 g 20% w/w suspension of the bread powders or our in water. The our or bread powder suspensions having more than 20% solids had a pasty behavior and a uniform mixing was not possible. This problem was dealt with using a fed-batch method, where the liquefaction started at lower dry substance concentration and additional bread powder or our was added gradually to obtain 20% suspension in a 10-min period of time. The pH was adjusted to 6 using caustic soda solution. The liquefaction was carried out by adding 0.8 ml 1000 g1 DM a-amylase, while the suspension was mixed vigorously. The liquefaction was ended by freezing the suspension. The pH of the liqueed

Liquefaction of 20% w/w suspensions of bread powders and wheat our was carried out at identical conditions at 50 and 75 1C, and the results are presented in Fig. 1. Despite the fact that at least 98% of at-bread dry matter is composed of wheat our, the hydrolysis of the bread and our seem to have several considerable differences. While initially soluble solids (mainly sugars) of our were negligible, about 15% of the bread powder solids were soluble. It resulted in 17% initially non-dissolved materials and 34% initial DE of the powder bread liquefaction (Fig. 1). Furthermore, the bread powder liquefaction was less inuenced by temperature than that of the our (Fig. 1). A 3-h hydrolysis decreased the bread powder suspension non-dissolved solids to 6% at both 50 and 75 1C, while the increase of liquefaction temperature from 50 to 75 1C caused a sharp increase in hydrolysis rate of our suspension especially at initial stages of liquefaction. The results of experiments for both our and bread at 85 1C and 35% w/w nal total solids are presented in Fig. 2. At these conditions, 65% of either bread powders or wheat our was dissolved. Final attained DEs were reduced by using 35% solids, especially for the our, compared with the experiments using 20% solid concentration (cf. Figs. 1 and 2).

Please cite this article as: Ebrahimi F, Khanahmadi M, Roodpeyma S, Taherzadeh MJ. Ethanol production from bread residues. Biomass and Bioenergy (2007), doi:10.1016/j.biombioe.2007.10.007

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50 T=50C 40 DE (%) 30 20 10 0 20 15 10 5 0 0 50 100 Time (min) 150 0 50 100 Time (min) 150 T=50C T=75C T=75C

Fig. 1 The effect of temperature on the time course of reducing sugars concentration (DE) and percentage of non-dissolved solids during the liquefaction process of 20% (w/w) suspensions of wheat our (m) and bread powder (&).

Non dissolved solids (%)

Despite the adverse effects of increasing the solid contents, the achieved DE value of around 15 was appropriate for the liquefaction product to be used as saccharication feed.

3.4.

Staling effect

3.2.

Saccharication

The liquefaction of stale breads at 35% solids and 85 1C followed by saccharication resulted in a similar yield and time course as the fresh breads (data not shown).

The trends of hydrolysis progress during saccharication of a liqueed 35% w/w bread powder suspension using two different concentrations of amyloglucosidase are presented in Fig. 3. Conversion of the liqueed bread starch into glucose was practically completed, with DE 495 being achieved. Furthermore, the dissolution proceeded during the saccharication stage, leaving 20% non-dissolved dry matter at the end of the process (data not shown).

4.

Discussion

3.3.

Fermentation to ethanol

The hydrolyzed bread was cultivated using high cell density of S. cerevisiae in order to check for produced feedstock applicability. The results are presented in Fig. 4. The saccharied bread solution was rst diluted to give an initial glucose concentration of 250 g l1. It was then inoculated using an initial concentration of 20 g l1 dry yeast. This resulted in complete assimilation of glucose within 10 h, giving nal ethanol and glycerol concentrations of 100 and 15 g l1, respectively. It corresponds to ethanol and glycerol yields of 0.40 and 0.06 g g1 of the glucose, respectively. Combining these data with the hydrolysis results points to an ethanol yield of 0.35 g g1 of the initial dry bread (Table 1).

Despite the fact that at least 98% of at-bread dry matter is composed of wheat our, the hydrolysis of the bread and our seem to have several considerable differences. The leavening process makes some parts of the biopolymers already soluble [14]. Furthermore, the baking process partially gelatinizes the starch granules. On the other hand, our starch granules are not gelatinized at 50 1C while they are almost fully gelatinized at 75 1C [15]. This fact seems to be the reason for more enhanced hydrolysis of bread compared with our at 50 1C (Fig. 1), in which both slightly higher DE and much lower nondissolved solids were attained for the bread. However, the situation is reversed at 75 1C in which both DE and solid dissolution are much higher for the our. It seems that enhancement in starch gelatinization and increase in amylase activity due to increase in temperature did not increase the degree of solids dissolution. It may result from the fact that the gluten network developed by the kneading process may act as a barrier and protect some parts of the starch polymer from being attacked by amylases. However, increased amylase activity accelerated further hydrolysis of dissolved oligosaccharides and resulted in a higher DE (Fig. 1).

Please cite this article as: Ebrahimi F, Khanahmadi M, Roodpeyma S, Taherzadeh MJ. Ethanol production from bread residues. Biomass and Bioenergy (2007), doi:10.1016/j.biombioe.2007.10.007

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50

100

80

40
60

30 DE (%)

DE 40 20 0

20

10

20

40 Time (h)

60

80

0 35 30 Non dissolved solids (%) 25 20 15 10 5 0 0 50 100 Time (min)

Fig. 2 The time course of reducing sugars concentration and percentage of non-dissolved solids during the liquefaction process of 35% (w/w) suspensions of wheat our (m) and bread powder (&) at 85 1C.

Fig. 3 The effect of amyloglucosidase dosages of 1 ( ) and 0.2 (B) ml of amyloglucosidase solution per kg of bread powder starch on trend of changes in reducing sugars concentration during the saccharication of 35% (w/w) bread powder suspension.

300

250

glucose ethanol glycerol

Concentration (gl-1)

200

150

150

200

100

50

0 0 5 10 Time (h) 15 20 25

As a conclusion, the liquefaction knowledge developed for starch and our is not fully extendable to the bread residues. The overall saccharication behavior of the bread residues was similar to the established starch enzymatic saccharication procedure, in which hydrolysis is fast at initial steps but gradually slows down as a result of the produced glucoseinhibiting effect and the diminishing of reaction substrate. The quantitative results are also in agreement with the data for suggested commercial amyloglucosidase dosages [16,17]. Overall conversion efciencies depicted in Table 1 show the feasibility of producing a suitable fermentation feedstock from bread residues. Despite the fact that kneading, leavening and baking processes result in some differences in the enzymatic hydrolysis of bread and wheat our, it seems that after some renements, the technology developed for conversion of wheat to generic feedstock is applicable also for that of bread residues.

Fig. 4 The prole of glucose consumption and metabolites production in anaerobic cultivation of bread powder hydrolyzate at 30 1C using 20 g l1 dry bakers yeast.

Table 1 Overall results of liquefaction at 85 1C, saccharication at 60 1C and fermentation at 30 1C of 35% ( w/w) bread solution Step Target parameter Results (g g1)
0.65 0.80 0.35

Liquefaction Saccharication Fermentation

Dissolved solids/initial dry bread Glucose/initial dry bread Ethanol/initial dry bread

Please cite this article as: Ebrahimi F, Khanahmadi M, Roodpeyma S, Taherzadeh MJ. Ethanol production from bread residues. Biomass and Bioenergy (2007), doi:10.1016/j.biombioe.2007.10.007

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It should be recalled that commercial bread residues is not a homogeneous raw material and that different degrees of mold growth, staling and baking conditions plus the presence of impurities cause differences from batch to batch or even from piece to piece. A few percent of bread residues batches is usually visibly molded. Mold growth seems to be accompanied by consumption of valuable substrates and release of heat-resistant mycotoxins and other metabolites that pollute the feedstock. Detailed effects of such heterogeneity and the ways to handle them need further research.

Acknowledgment
This work was nancially supported by Isfahan University of Technology, Isfahan Center for the Research of Agricultural Science & Natural Resources and University of Boras.
R E F E R E N C E S

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Glossary
DE: Dextrose equivalent, i.e. the percentage of glucoseglucose bonds in starch which is hydrolyzed. The concentration of hydrolyzed bonds is measured by the DNS method. DM: Dry matter of the sample measured by weighing it after evaporation of its water in oven.

Please cite this article as: Ebrahimi F, Khanahmadi M, Roodpeyma S, Taherzadeh MJ. Ethanol production from bread residues. Biomass and Bioenergy (2007), doi:10.1016/j.biombioe.2007.10.007