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Article

Waste Bread as Raw Material for Ethanol Production: Effect of Mash Preparation Methods on Fermentation Efficiency

by
Maria Balcerek
*,
Urszula Dziekońska-Kubczak
,
Katarzyna Pielech-Przybylska
,
Anna Oleszczak
,
Magdalena Koń
and
Andrea Maria Patelski
Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Wolczanska 171/173, 90-530 Lodz, Poland
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(20), 9565; https://doi.org/10.3390/app14209565
Submission received: 24 September 2024 / Revised: 15 October 2024 / Accepted: 18 October 2024 / Published: 20 October 2024
(This article belongs to the Special Issue Bioprocessing and Fermentation Technology for Biomass Conversion)
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Abstract

:
The issue of managing waste bread is a global concern, with significant environmental and the economic implications. The utilisation of waste bread for bioethanol production, employing energy-saving technology, could prevent these consequences and reduce the consumption of traditionally used fossil fuels. The objective of this study was to evaluate the influence of the type of waste bread (wheat and wheat–rye sourdough) and the mash preparation method on the results of alcoholic fermentation and the concentration of selected congeners in the distillates. The highest fermentation efficiency (96% of theoretical) was achieved for both types of bread through the utilisation of the pressureless starch liberation method combined with simultaneous saccharification and fermentation. The separate saccharification of starch resulted in lower process efficiencies (from 85.75 to 88.60% of theoretical). The application of the native starch hydrolysis method (without starch activation) for the fermentation of wheat bread-based mashes exhibited a higher efficiency (87.85% of the theoretical) than that observed for the wheat–rye bread-based mash sample (83.74% of theoretical). All of the obtained spirit distillates exhibited a low concentration of methanol (≤300 mg/L alcohol 100% v/v) and comply with the requirements of the EU regulation for ethyl alcohol of agricultural origin (rectified spirit).

1. Introduction

One of the challenges facing the modern world is reduction of food waste and the implementation of effective waste management strategies. Approximately one-third of the food produced globally is wasted, resulting in the loss of valuable resources, such as energy, fresh water and land. In turn, poor waste management contributes to an increase in greenhouse gas emissions, including carbon dioxide, which is responsible for increasing global warming [1].
The issue of food waste is also noticeable in the baking industry. Given the widespread consumption of bread, its global production has increased to nearly 100 million tons annually [2], resulting in the generation of excessive amounts of bakery waste. It is estimated that 7–10% of bakery waste is generated annually throughout the supply chain. This is a serious problem in numerous European countries, where the volume of bread represents over 53.6% of global production [3]. For instance, approximately 290,000 tonnes of bakery waste is generated in the UK annually, contributing to an increase in food waste and significant economic and environmental losses. In Sweden, the quantity of bakery waste produced on an annual basis is considerable, reaching approximately 29,870 tonnes in households, and close to 80,410 tonnes across the entire supply chain from producer to consumer [4]. In turn, in the United States, about 20% of bread is wasted in households and 12% by retailers. A similar situation can be observed in Germany, where approximately 14% of bakery waste is generated during production process itself [1]. The formation of a large amount of bakery waste is also a serious problem in Poland, where every year about 170,000 tons of bakery waste are produced [5].
The generation of bakery waste can be attributed to two primary causes: overproduction and the failure to sell bakery products after their expiry date. Rapid staling and spoilage of bakery products also contribute to the generation of large amounts of bakery waste [6]. Furthermore, the utilisation of contaminated or defective intermediate products in the production of finished goods contributes to the overall volume of waste [4]. The increasing amount of bakery waste requires implementation of appropriate management strategies, which are also driven by legislations and the principles of the circular economy [3]. The presence of many valuable compounds, including carbohydrates (mainly starch), proteins and fats in bakery waste offers substantial potential for its utilisation in biotechnological processes [7,8,9].
One potential method for managing bread waste is its utilisation in anaerobic fermentation processes, which can result in the production of biogas. The resulting biogas can then be transformed into heat and electricity, which will ultimately contribute to a reduction in the demand for energy from non-renewable sources. It is noteworthy that approximately 1% of the substrates used in agricultural biogas plants are derived from bakery waste. The production of approximately 536 m3 of biogas from 1 Mg dry matter (DM) of bread waste is a notable outcome [10]. Compared to other substrates, the pre-treatment of bakery waste is conducted under relatively mild conditions, resulting in sugars that are highly pure and devoid of reaction inhibitors. It can therefore be concluded that the obtained hydrolysates are suitable as fermentation substrates for the production of biochemicals and biofuels, including bioethanol, lactic acid, succinic acid and others. The incorporation of more sustainable production methods based on biorefining processes, such as the utilisation of bakery waste, into production processes may prove beneficial for many companies in the food industry from a financial standpoint. Moreover, products generated through biotechnological processes may potentially offer a competitive alternative to those produced through chemical methods [11].
Of the various types of biofuel, ethanol is regarded as the most prevalent, offering an alternative to fossil fuels. Furthermore, this particular fuel is employed as a fundamental ingredient in the chemical and pharmaceutical industries, along with the production of alcoholic beverages [12]. The most widely utilised raw materials for the production of bioethanol are starch-based materials (wheat, corn), sugar-based materials (sugar beet, sugar cane), and tuber plants (potatoes, cassava). In recent years, the utilisation of these raw materials has been a significant source of concern in the context of the intensifying global food crisis. Moreover, the use of foodstuffs as raw materials for ethanol production may potentially lead to an increase in food prices [13]. To prevent this, inedible raw materials in the form of waste biomass are employed. However, a significant argument against the use of waste biomass in the form of lignocellulosic materials is the necessity for pre-treatment, which requires the utilisation of non-ecological chemical reagents. It is also worth noting that the ethanol yield from lignocellulosic biomass is considerably lower than that from starch feedstocks.
Given the aforementioned data, which indicate that bread waste is the most commonly wasted foodstuff and that it contains a high starch content of more than 70% on a dry weight basis, it is evident that bioethanol production through ethanol fermentation represents an effective strategy for recycling bread waste, as has been increasingly documented in the scientific literature [7]. Mihajlovski et al. [14] conducted a study on the optimisation of ethanol production by response surface methodology (RSM) using waste bread hydrolysate as a raw material. The effect of fermentation time (24–72 h) and waste brewer’s yeast inoculum (1–4%) on ethanol production was studied. The hydrolysis of starch was performed using enzymes derived from the Hymenobacter sp. CKS3 bacterial strain. The resulting hydrolysate obtained from bread waste contained 19.89 g of reducing sugars per 1 L. The findings indicated that the optimal conditions were 48.6 h of fermentation and 2.85% of inoculum. Under these conditions, the maximum ethanol concentration achieved was 2.06% v/v. In turn, Narisetty et al. [15] used bread waste (BW) as a substrate for ethanol production by Saccharomyces cerevisiae KL17 yeast strain. The acidic and enzymatic saccharification of BW resulted in the release of 75 and 97.9 g/L of glucose, representing a theoretical yield of 73.5 and 95.9%, respectively. In the fed-batch mode of the fermentation process, the maximum ethanol concentration of 114.9 g/L and a productivity of 3.2 g/L/h were achieved after fermentation of hydrolysates obtained by treating BW with Dextrozyme Peak enzyme preparation with glucoamylase and pullulanase activities. Pietrzak and Kawa-Rygielska [16] investigated the possibility of using granular starch hydrolysing enzyme (GSHE) for the processing of wheat–rye bread waste for ethanol production. In addition, pre-treatment methods, i.e., enzymatic prehydrolysis, microwave irradiation and sonication were employed to enhance the yield of starch hydrolysis and fermentation. These methods were then compared with separate hydrolysis and fermentation (SHF). Due to the high water binding capacity of the raw material, fermentations were carried out at a substrate loading of 150 g/kg mash. Only during the enzymatic pre-treatment and the SHF process was the raw material preliminary liquefied so that its higher concentrations could be applied. The fermentation dynamics exhibited a similar pattern across all variants studied. The fermentation of the untreated bread waste resulted in an ethanol yield of 80.00% (354.36 g/kg raw material). Pre-treatment of the raw material improved the ethanol yield by ca. 3–8%. In another study, Kawa-Rygielska et al. [17] investigated the potential of employing the edible filamentous fungi Neurospora intermedia and Aspergillus oryzae for the production of bioethanol and high-protein biomass through the cultivation of these fungi on enzymatically liquefied bread waste. The fermentation of the hydrolysate, containing 150 g/L solids, by N. intermedia resulted in an ethanol titre of 32.2 g/L and a biomass yield of 19.2 g/L, with an approximate protein content of 45%. To utilise the residual fermentable sugars, the liquid medium after the first fermentation and distillation processes was fermented again by the aforementioned two fungal strains, resulting in the production of additional ethanol and biomass. The authors observed that A. oryzae showed better performance in the production of biomass, while the other strain demonstrated a higher ethanol yield. The final products’ yield ranged from 0.29 to 0.32 g EtOH/g and from 0.20 to 0.22 g biomass/g bread waste, depending on the strain employed in the second fermentation.
Despite numerous reports on the potential use of waste bread in the production of ethanol, there is a lack of information on the feasibility of employing the methods and techniques used in the processing of starchy raw materials in the distilleries for the efficient production of ethanol from bakery waste, given the physicochemical changes that occur in bread, such as starch retrogradation and the subsequent staling of bread. The objective of this study was to assess the impact of the type of waste bread (wheat and wheat–rye sourdough) and the method of mash preparation, typically employed in distilleries for starchy raw materials processing, on the results of alcoholic fermentation and the concentrations of selected congeners in the obtained distillates.

2. Materials and Methods

2.1. Raw Materials

Two types of waste bread, namely wheat bread and wheat–rye sourdough bread, obtained from a local bakery were used for the research as the dominant types of bakery waste [18]. These products had reached the end of their shelf life. The raw materials were stored in a closed container at room temperature until required for use.

2.2. Enzymatic Preparations

The following amylolytic and supportive enzyme preparations (Novozymes A/S, Bagsværd, Denmark) were used during the preparation of the mashes in accordance with the pressureless liberation of starch (PLS) method:
  • Termamyl SC (α-amylase from Bacillus stearothermophilus, EC 3.2.1.1),
  • SAN Extra (glucan 1,4-α-glucosidase from Aspergillus niger, EC 3.2.1.3),
  • Viscoferm® (a multienzyme complex containing non-starch-degrading enzymes: cellulase, EC 3.2.1.4; xylanase (endo-1,4-), EC 3.2.1.8; and β-glucanase (endo-1,3(4)-), EC 3.2.1.6),
  • Neutrase® (neutral protease from Bacillus amyloliquefaciens, EC.3.4.24.28).
The following amylolytic enzyme preparations (DuPont™ Genencor® Science, Wilmington, DE, USA) were used during the preparation of the mashes in accordance with the native starch hydrolysis (NSH) method:
  • GC 626 (acid α-amylase from Trichoderma reesei, EC 3.2.1.1),
  • Stargen 002® (a blend of α-amylase from Aspergillus kawachi expressed in Trichoderma reesei, EC 3.2.1.1, and glucoamylase from Trichoderma reesei, EC 3.2.1.3).
  • As supportive preparations, the aforementioned Viscoferm® and Neutrase® were applied.

2.3. Yeast

The fermentation process was conducted using SaftSpiritTM HG-1 dry distillery yeast (S. cerevisiae) (Fermentis by Lesaffre, Marcq-en-Barœul, France), which has been designed to produce a high ethanol concentration with a broad range of applications across various process conditions (pH range 3.5–6.0, temperature range 33–37 °C) and with diverse substrates [19].

2.4. Preparation and Fermentation of Sweet Mashes

Two methods were employed for the preparation of sweet mashes: pressureless liberation of starch (PLS) and native starch hydrolysis (NSH). The PLS process was performed in two variants, with separate hydrolysis and fermentation (SHF) and with simultaneous saccharification and fermentation (SSF), as presented in Figure 1. The native starch hydrolysis (NSH) process was also performed in two variants, i.e., without starch ‘activation’ (NSH-N/A) and with starch ‘activation’ (NSH-A), as presented in Figure 2. All mashes were prepared in duplicate.
Prior to inoculation of the bread-based mashes (each in the volume of 3 L), an appropriate amount of yeast (0.5 g per 1 L of mash) was hydrated and disinfected (15 min incubation of cells suspended in a 25% w/w H2SO4 solution, pH 2.5, at room temperature). The yeast cream (without neutralisation) was added to the mashes, along with (NH4)2HPO4 as a source of nutrients, which were then mixed carefully. Fermentation was conducted in 4 L glass bottles. The anaerobic conditions necessary for fermentation were maintained by sealing the bottles with an airlock filled with paraffin oil.

2.5. Distillation

The distillation of ethanol from the mashes was performed using a simple distillation unit. The resulting distillates, with an ethanol content of 20–23% v/v, were then strengthened to approximately 43% v/v in a distillation apparatus equipped with a dephlegmator and cooler.

2.6. Analytical Methods

The bread analysis entailed the determination of moisture (using a Radwag WPS-30S Moisture Analyzer, Radwag, Poland); protein content (N × 6.25), using the Kjeldahl method; and sugar content, using the HPLC technique [20]. The analysis of mashes (sweet and fermented) included the determination of pH (pH-meter HandyLab, SI Analytics, Germany), extract content (total in sweet mash and apparent in fermented mash), using a hydrometer with a scale in % w/w [21], and content of sugars, ethanol as well as other fermentation by-products through HPLC [20].
The analysis of distillates was conducted according to the methods recommended in distilleries and comprised the quantification of methanol, aldehydes and acidity.
The methanol content in spirit distillates was determined using a colorimetric method [22], which involves the oxidation of methyl alcohol to formaldehyde, followed by a reaction with Schiff’s reagent. The colouration obtained was measured spectrophotometrically at a wavelength of 610 nm. The colour obtained was then compared with the colours of standard solutions containing known quantities of methanol. The concentration of methanol was expressed in grams per 1 L of 100% v/v alcohol.
The content of aldehydes was determined using a colorimetric method [23] based on the reaction of aldehydes with Schiff’s reagent. The concentration of the aldehydes was calculated from the calibration curve prepared on the aldehyde standards and expressed in grams of acetaldehyde per 1 L of 100% v/v alcohol.
The acidity was determined by the titrimetric method [24], which involved diluting the tested sample with an equal amount of carbon dioxide-free water, followed by titration with a standard sodium hydroxide solution against phenolphthalein as an indicator. The results were expressed in grams of acetic acid per 1 L of 100% v/v alcohol.

2.7. Calculations

The results of the HPLC analysis were used to calculate the concentration of total reducing sugars and total sugars (after acid hydrolysis of starch or dextrin), both in the raw materials and obtained mashes. The calculations were performed using a conversion coefficient to glucose (maltotriose × 1.07; maltose × 1.05) and expressed in g glucose/100 g raw material and in g glucose/L mash, respectively. Subsequently, the starch or dextrin content was calculated as the difference between the total sugars and reducing sugars, with the conversion coefficient into starch/dextrin (0.9) taken into account. This was expressed in g/100 g raw material, and in g/L mash, respectively.
The intake of sugars was calculated as the ratio of sugars utilised during fermentation to their initial content in the mash and expressed as a percentage. The real fermentation efficiency was expressed as the ratio of the ethanol yield (obtained in the experiments) to the theoretical ethanol yield (calculated using the ethanol fermentation equation).

2.8. Statistical Analysis

All experiments were performed in triplicate. A one-way and two-way analysis of variance ANOVA with a significance level of 0.05 was employed to compare the bread’s composition and to examine the effect of applied variables (i.e., bread type and mash preparation method) on the composition of mashes before and after fermentation, intake of sugars, fermentation efficiency, as well as the concentration of volatile fermentation by-products in the resulting distillates. A Tukey’s multiple comparison post-hoc test was conducted when significant differences were identified. All calculations were performed using XLSTAT 2024.1.0 software (Lumivero, Denver, CO, USA).

3. Results and Discussion

3.1. The Chemical Composition of Raw Materials

The basic indicator of the storability of cereal grains-based products, which influences the risk of the growth of undesirable microorganisms (including moulds), is moisture content. It is generally accepted that moisture content below approximately 15% (wet basis) is safe, due to the minimal metabolic activity that occurs at these levels [25,26]. The analysis of the tested waste bread showed the higher moisture content, particularly in the wheat bread. This indicates that the long-term storage of this raw material, especially in rooms with elevated humidity, may potentially facilitate the development of undesirable microorganisms. The waste bread used in this study was stored in an environment with a low humidity level (approximately 40%), and no indications of microbiological contamination, such as mould growth, were observed.
One of the most important factors to be considered in the evaluation of raw materials for ethanol production is the sugar content. Cereal grain-based products, such as bread, are rich in starch [27]. The tested waste wheat bread contained starch content of 71.70 ±1.71 g/100 g dry mass, which was consistent with the data reported by Mesta-Corral et al. [28]. In turn, the starch content of the tested wheat–rye bread was higher (77.16 ±4.98 g/100 g dry mass) than that of the wheat bread, due in part to the lower moisture content (see Table 1).
During technological processes, fermentable carbohydrates are produced as a result of the enzymatic degradation of starch [29]. The obtained results show the presence of minimal quantities of reducing sugars in both tested types of bread, with values ranging from 2.02 ± 0.31 g/100 g dry mass in wheat–rye bread to 3.03 ± 0.20 g/100 g dry mass in wheat bread.
The protein content of tested bread waste ranges from 7.08 ± 0.01 g/100 g dry mass in wheat bread to 8.18 ± 0.05 g/100 g dry mass in wheat–rye bread. Following the hydrolysis of proteins, the resulting peptides and amino acids facilitate yeast growth and fermentation [30].
Cereal grains used for flour production contain various amounts of non-starch polysaccharides (NSPs), which are predominantly composed of arabinoxylans (pentosans), β-glucans and cellulose [31]. Arabinoxylan is defined as a type of hemicellulose that is found in the cell walls of cereal endosperm. It consists of a linear backbone of xylose residues with arabinose units attached [32]. The quantity of arabinoxylans (AXs) is less in wheat (6–8%) than in rye (8.9%) [33], which has been confirmed by the lower concentration of pentose sugars in wheat bread in comparison to wheat–rye bread (Table 1).
In the tested wheat and wheat–rye bread, the presence of succinic acid, lactic acid, acetic acid and glycerol was shown. These compounds are produced by lactic acid bacteria and yeasts of the Saccharomyces genus, which are present in bakery sourdough. The amount of fermentation by-products depends on the type of flour, the active microorganisms present in the sourdough and the fermentation temperature [34].

3.2. Chemical Characteristic of Mashes and Fermentation Results

The chemical composition of the prepared distillery mashes varied according to the type of bread used and the method of mash preparation. The obtained results are presented in Table 2.
The pH of mashes prepared from wheat bread was found to be 5.1, while those prepared from wheat–rye bread had a pH of 4.5. This difference can be attributed to the higher concentration of lactic and acetic acids present in the used raw material (see Table 1). Due to the fact that the optimal pH range for yeast growth can vary from pH 4.00 to 6.00 [35], the pH of the prepared bread-based mashes was not adjusted in order to limit the number of additional treatments and reagents used.
Despite the same proportions of the raw material and water employed in the preparation of mashes by all of the aforementioned methods, statistically significant differences (p ≤ 0.05) in the extract content of the obtained mashes were observed. The PLS-SHF method resulted in the preparation of wheat and wheat–rye bread-based sweet mashes with a higher extract content (p ≤ 0.05) than those that were prepared using the PLS-SSF method. This may be attributed to the release of greater quantities of sugars into the medium during the separate saccharification stage, as evidenced by the total reducing sugars content in the obtained mashes (Table 2). It is notable that the highest extract content was observed in samples prepared using the NSH method, which involved native starch hydrolysis. It is possible that the heterogeneity of the bread portions and the progressive drying process may have contributed to the observed differences in the extract content.
The fermentable sugars present in sweet mashes prepared by all used methods, i.e., PLS-SSF, PLS-SHF, NSH-A and NSH-N/A consisted mainly of glucose at concentrations ranging from 77.28 ± 1.62 g/L (wheat–rye bread, PLS-SSF) to 147.81 ± 0.51 g/L (wheat–rye bread, NSH-A). However, significantly higher (p ≤ 0.05) glucose concentrations were determined in the mashes in which separate hydrolysis/saccharification of dextrins was carried out than in the samples saccharified during fermentation (SSF). Furthermore, relatively high concentrations of maltose (ranging from 33.79 ± 1.70 to 35.10 ± 3.74 g/L) were also determined in the mashes prepared from both types of bread using the PLS-SSF method. This is due to the fact that the conditions of separate hydrolysis at a temperature of 65 °C favour the hydrolysis of dextrins into monosaccharides. Additionally, all mashes contained small amounts of maltotriose, with the majority of these concentrations remaining below 1 g/L. The concentrations of reducing sugars in the obtained mashes (from both types of bread) were similar to those determined in the rye-grain mashes previously studied by us using the PLS method [36]. Kawa-Rygielska et al. [17], who carried out starch hydrolysis of bread residues using a commercial α-amylase preparation, determined dextrin as the most abundant carbohydrate in the bread hydrolysate (73.3% of the total). In turn, sugars fermentable by S. cerevisiae were present in the amount of 14.63 ± 0.43 g maltose/L and 5.52 ± 0.03 g glucose/L, representing only 15.5% of the available carbohydrates. The authors concluded that the use of a saccharifying enzyme would be recommended to improve the efficiency of starch hydrolysis from waste bread.
In our study, an interesting phenomenon was observed in the case of mashes prepared by the native starch hydrolysis method, both without and with starch ‘activation’. Among the reducing sugars, glucose was the most abundant (between 139.01 ± 1.13 and 147.81 ± 0.51 g/L). In turn, the concentrations of other sugars, especially of maltose, were significantly lower in these samples (p ≤ 0.05) than in the mashes prepared by both the PLS-SSF and PLS-SHF methods (Table 2). This confirms the high hydrolytic activity of the enzyme preparations used for native starch hydrolysis.
With regard to the content of dextrins in sweet mashes prepared using the PLS method, no beneficial effect of separate starch saccharification before fermentation (SHF) on their content in the obtained mashes was observed in relation to the samples prepared according to the simultaneous saccharification and fermentation (SSF) method. The dextrin content in the mashes prepared from wheat bread did not exhibit a statistically significant difference (p ≤ 0.05) between the SHF and SFF methods. Conversely, the SHF method resulted in a higher dextrin content in the mashes made from wheat–rye bread than the SSF method. It is likely that the high concentrations of liberated fermentable sugars may have caused product inhibition of the activity of the enzymes responsible for catalysing starch hydrolysis [37].
The native starch hydrolysis method without starch ‘activation’ resulted in the highest level of dextrin being found in the wheat bread-based mash. An application of initial ‘activation’ of starch during the preparation of wheat–rye bread-based mashes significantly improved the initial degree of starch saccharification (p < 0.05), which in turn resulted in a decrease in dextrin concentration in comparison to the samples without starch ‘activation’ (p ≤ 0.05) (Table 2). It is due to the disruption of the bonds between the glucose molecules in the starch chain that occurs during the activation process [38]. The highest concentration of dextrin (68.50 ± 0.09 g/L) was found in the wheat–rye-based mash prepared using the NSH-N/A method. It may be supposed that one of the probable reasons of lower results of starch hydrolysis is, apart from the lack of its ‘activation’, the ongoing starch retrogradation, which initially involves the rapid recrystallisation of amylose molecules and is followed by a slow recrystallisation of amylopectin molecules [39]. Moreover, wheat starch exhibits a lower degree of retrogradation in comparison to rye due to the presence of higher levels of phosphorus (phospholipids) [40], which provides an explanation for the observed differences in the degree of starch hydrolysis in the wheat and wheat–rye bread-based mashes.
The chemical analysis of fermented mashes entailed the determination of pH, apparent extract, as well as the concentration of ethanol, reducing sugars (glucose, maltose, and maltotriose) and dextrins. Additionally, the concentration of other sugars (xylose, arabinose) as well as organic acids and glycerol was determined (see Table 3).
During the process of fermentation, yeast secretes H+ ions, which causes a decline in pH levels within the medium. The pH value of the waste wheat bread-based mashes after process completion decreased from 5.1 up to 4.3, whereas for wheat–rye bread-based mashes, the pH value decreased from 4.5 up to 4.1. These findings are consistent with the data presented in the literature and confirm the correct duration of fermentation [41].
In distilleries, the parameter used to assess the degree of fermentation is apparent extract, which is measured in the presence of ethanol. In the case of well-fermented distillery mashes with an initial extract of approximately 18% w/w, the apparent extract should not exceed (1.0–1.5)% w/w [42]. After completion of the fermentation process, the apparent extract of the tested mashes ranged from 0.63 ± 0.02 to 1.11 ± 0.06% w/w, with the tendency towards higher values observed in mashes prepared by the PLS-SHF method in comparison to those prepared by the PLS-SSF and NSH methods.
The ethanol concentration of the mashes prepared by both the PLS-SSF and PLS-SHF methods did not exhibit a statistically significant difference (p ≥ 0.05), with values ranging from 70.95 ± 1.02 to 72.54 ± 1.64 g/L. Furthermore, no differences were observed when the type of bread used was taken into account. The highest concentration of ethanol was determined in the mashes prepared via the native starch hydrolysis method, with values ranging from 80.95 ± 2.11 to 85.68 ± 0.02 g/L. No statistically significant impact of the starch ‘activation’ process was observed (p ≥ 0.05). Kawa-Rygielska et al. [17], during fermentation of bread-waste medium (with a solids concentration of 150 g/L) by fungi Neurospora intermedia, found significantly lower ethanol yield of 32.2 g/L. In turn, the conversion of waste bread to ethanol by S. cerevisiae with the application of granular starch hydrolysing enzyme at the same substrate concentration (150 g/L) resulted in an ethanol yield of 55 g/L [16]. The aforementioned results of ethanol production are significantly lower than those obtained in the present study. It is noteworthy that the ethanol content after fermentation of waste bread-based mashes tested in this study, irrespective of the type of bread and the method of mash preparation, was comparable to the values obtained after processing traditional starchy raw materials, such as rye, barley and cereal malts, to ethanol [36,37,38,43].
In the majority of fermentation trials, low concentrations of reducing sugars were identified, namely maltotriose (from 0.02 ± 0.00 to 0.21 ± 0.00 g/L), maltose (from 0.14 ± 0.02 to 0.90 ± 0.01 g/L) and glucose (from 0.01 ± 0.00 to 0.08 ± 0.02 g/L), which indicates their high utilisation during fermentation. It is noteworthy that only in samples of wheat bread mashes prepared by the PLS-SHF and NSH-N/A methods higher concentrations of glucose were determined, amounting to 3.79 ± 0.42 g/L and 2.07 ± 0.15 g/L, respectively (see Table 3). In the aforementioned PLS-SHF sample, a relatively high concentration of lactic acid (2.61 ± 0.14 g/L) in the mash after fermentation was recorded, which may indicate the development of undesirable microorganisms, such as lactic acid bacteria, during a separate saccharification step [44].
With regard to the non-hydrolysed dextrins present in mashes upon completion of fermentation, an interesting phenomenon was observed. The concentration of these compounds was found to be relatively high (ranging from 1.87 ± 0.56 to 2.52 ± 0.58 g/L) in mashes prepared from both wheat and wheat–rye bread using the PLS-SSF and PLS-SHF methods, in comparison to the samples prepared using the native starch hydrolysis method. Moreover, pentose sugars, namely xylose and arabinose, were also identified in trace amounts in all of the fermented mashes. The yeast of the S. cerevisiae genus used in this study metabolizes pentoses, but without converting them into ethanol [45].
All samples after fermentation contained organic acid, i.e., succinic acid, lactic acid, formic acid, and acetic acid. These acids are regarded as fermentation by-products [46]. Succinic acid is an intermediate product of the tricarboxylic acid (TCA) cycle and constitutes one of the end products of the anaerobic metabolism of yeast [47]. Moreover, all tested mashes exhibited relatively elevated concentrations of glycerol, which is one of the products of yeast metabolism. The primary function of this compound is to provide protection for yeast against environmental stressors [48].
In order to evaluate the results of fermentation process, the degree of sugar utilisation and the efficiency of ethanol biosynthesis (expressed as a percentage of the theoretical amount) were determined. The utilisation of sugars was found to be high in all fermentation trials, regardless of the type of bread used and the method of mash preparation. The degree of sugar utilisation ranged from 96.03 ± 0.48% to 99.17 ± 0.50% (see Figure 3). The lowest value of this indicator was determined for the wheat bread-based mash prepared by the PLS-SHF method, which contained relatively high concentration of unutilised glucose, as mentioned above (see Table 3).
A comparison of the fermentation efficiency revealed that the PLS-SSF method was the optimal method for processing both types of bread. The fermentation efficiency for these mashes was found to be at the same level of 96.1 ± 0.48% of the theoretical. The application of the PLS-SHF method for the preparation of the mashes did not result in an increase in fermentation efficiency. In fact, the process efficiencies were lower than those observed in the PLS-SSF method (from 85.75 ± 0.43 to 88.60 ± 0.44%). With regard to the NSH-N/A method, the fermentation of wheat bread-based mashes exhibited a higher efficiency, with a value of 87.85 ± 0.43% of the theoretical, than that observed for the wheat–rye bread-based mash sample (83.74 ± 0.43% of theoretical). It may be hypothesised that the reason for the lower ethanol yields obtained from wheat–rye bread is the more intensive phenomenon of starch retrogradation. The resulting starch complexes hinder the hydrolysis process, creating a barrier to the action of amylolytic enzymes [39]. Despite some differences between the process indicators depending on the type of processed waste bread and the method of the mash preparation, it can be stated that these indicators are comparable to those obtained during the processing of grain raw materials commonly used in the distilling industry for the production of ethanol [36,37,38,43].
A brief economic analysis of the process carried out in the framework of our study, comparing it to traditional methods, indicates that this solution is economically attractive and in line with the principles of the circular economy. The cost of raw materials represents approximately 50% of the total production costs when traditional starchy raw materials (maize and rye) are used for the production of spirit. The utilisation of waste bread for ethanol production appears to be advantageous for both the distillery and the bakery. It is possible to achieve a significant increase in profitability by reducing the cost of raw materials for a distillery by up to ten times that of grain cereals. For bakeries, the net cost of the operation is zero, and in addition, waste management costs are eliminated [49].
In turn, when assessing the pressureless methods of mash preparation (PLS, NSH) used for the processing of waste bread, it is important to consider their competitive solutions in relation to pressure-thermal methods that rely on the use of pressure devices (i.e., steamers). These pressureless methods offer a cost-effective alternative, as they do not require specialized or expensive equipment. The elimination of the processing of the raw material at high temperatures (approximately 150 °C) in favour of starch liquefaction carried out at a temperature of approximately 90 °C results in thermal energy savings of up to 50%. Nevertheless, the consumption of electricity during the grinding of the raw material processed using the PLS method may result in a total energy saving of 20–34% in comparison to the pressure-thermal methods [50]. Additionally, the implementation of simultaneous saccharification and fermentation (SSF) methods increases thermal energy savings, as the distinct step of separate saccharification at a temperature of approximately 65 °C is eliminated. This solution also ensures a high fermentation efficiency of 96% of the theoretical efficiency. An alternative and promising solution to the PLS-SSF strategy, which allows for the maximum reduction of investment costs and energy savings, with slightly lower process efficiency (up to about 88% of theoretical yield), might be the method of native starch hydrolysis (NSH). It should be noted, however, that the critical point of pressureless methods of processing starchy raw materials, especially of native starch hydrolysis (NSH), is the risk of microbiological contamination. This can be effectively minimised by using protection with hop α-acids preparations dedicated for the distilling industry [51]. Nevertheless, in order to fully assess the feasibility of the process, other technical and economic factors throughout the entire value chain should also be taken into consideration. These include fluctuations in the composition of bakery waste, the collection of waste bread, and the methods of ethanol recovery and purification [52].

3.3. Chemical Composition of the Obtained Distillates

During the fermentation process, yeast produces ethanol and carbon dioxide, which facilitate the synthesis of alcohols, esters, and organic acids. The chemical composition of the obtained agricultural distillates was evaluated, revealing a differential effect of the type of bread used and the method of sweet mash preparation (see Table 4).
The undesirable compound present in spirit distillates is methanol, which is generated through the hydrolysis of methylated pectins present in plants and fruits, which may also occur during the backing process. Pielech-Przybylska et al. [51] observed a higher concentration of methanol in the spirits obtained from starchy raw materials-based mashes prepared by the pressure-thermal method (approx. 150 °C) than with the pressureless method (90 °C). While EU Regulation No. 2019/787 [53] defines acceptable concentrations of methanol in ethyl alcohol of agricultural origin, wine spirits and fruit spirits, no limits are set for the content of this compound in distillates of agricultural origin. It should be noted, however, that all the obtained distillates comply with the requirements set out in the regulation, which stipulates that the maximum permitted methanol content in ethyl alcohol of agricultural origin (rectified spirit) shall be 30 g/hL absolute alcohol (equivalent to 300 mg/L).
Aldehydes present in spirits are intermediates in the two-step decarboxylation of alpha-keto acids to alcohols, as well as in the synthesis and oxidation of alcohols. These volatiles are often found to have a negative effect on the quality of spirits. Their concentration depends on the quality of the raw materials, their chemical composition, the conditions of the technological processes and microbial contamination [54]. According to the Polish Standard [55], the concentration of aldehydes, expressed as acetaldehyde, in agricultural distillates should not exceed 100 mg/L alcohol 100% v/v, while the EU regulation [53] does not set any limits for the acetaldehyde content in agricultural distillates. The concentrations of aldehydes (expressed as acetaldehyde) in the obtained bread-based distillates exceeded the recommended limit, and ranged from 0.120 ± 0.01 to 0.233 ± 0.02 g acetaldehyde/L alcohol 100% v/v. The average content of these compounds was higher (p ≤ 0.05) in the distillates obtained from wheat–rye bread than from wheat bread. In turn when assessing the mash preparation methods, the highest levels of this compound were found in the samples of distillates obtained from mashes prepared by the PLS-SHF method. The application of the PLS-SSF, NSH-N/A and NSH-A methods resulted in lower concentrations of aldehydes in the distillates.
In accordance with the aforementioned regulation [55], the acidity of agricultural distillates, expressed in grams of acetic acid per litre of alcohol 100% v/v, should not exceed 80 mg/L for rye- and potato-based spirits, and 0.2 g/L for the so-called ‘mixed spirits’. The obtained bread-based spirit distillates were characterised with acidity levels exceeding the recommended limit. Both the type of bread and the method of mash preparation were identified as influencing factors in this regard. The highest acidity (0.940 ± 0.04 g acetic acid/L alcohol 100% v/v) was observed in the distillate obtained from wheat bread processed by the PLS-SHF method. Additionally, the distillate from wheat–rye bread processed by the NSH-A method exhibited relatively high acidity, with a value of 0.850 ± 0.02 g acetic acid/L alcohol 100% v/v. The lowest acidity was observed in samples of both wheat and wheat–rye bread processed by the PLS-SSF method, with values ranging from 0.583 ± 0.04 to 0.620 ± 0.01 g acetic acid/L alcohol 100% v/v (p ≤0.05). Acetic acid is formed during the Maillard reaction as the result of the degradation of Amadori products [56]. Those processes occur during the baking of bread. Furthermore, the presence of acetic acid in the distillates may also be attributed to the fermentation process, resulting from the metabolic activity of yeast and other microorganisms [51]. Depending on the intended use of the obtained spirit distillate, there may be a need to eliminate volatile congeners. In the production of a purified spirit, a multiple distillation process utilising a multi-column continuous system is typically employed in order to achieve the desired concentration of alcohol and the level of congeners. Following the primary distillation, which in industrial conditions consists of a stripping and pre-rectifying (strengthening) column, the spirit may undergo processing through an additional multicolumn rectifying system. This system typically comprises three to five columns, including a heads column for the elimination of congeners such as aldehydes and acids, a main rectification column for purified spirit recovery, a demethylising column and a fusel oil recovery column [57,58]. The main product, namely purified/rectified spirit, may be used for many biotechnological processes and for the production of spirit beverages. By-products may be utilised in the preparation of cost-effective solvents [59]. Moreover, fusel oils can serve as a source of supply energy for processing plants and as additives in petroleum-based fuels [60].

4. Conclusions

The paper presents the results of an assessment of the suitability of waste, wheat and wheat–rye sourdough bread for use in ethanol production. The assessment considers both the efficiency of the process and the quality of the resulting spirit distillate. The following methods were employed for the preparation of the mashes and their fermentation: pressureless starch liberation combined with separate hydrolysis and fermentation (PLS-SHF), pressureless starch liberation combined with simultaneous saccharification and fermentation (PLS-SSF) and native starch hydrolysis without preliminary starch ‘activation’ (NSH-N/A) or with starch ‘activation’ (NSH-A) at a temperature not exceeding the starch gelatinisation temperature.
The intake of sugars was within the range of 96–99%, while fermentation efficiency reached a level between 83 and 96% of the theoretical efficiency. The observed differences in these indicators were dependent on the method of preparing the mashes, but the type of bread used had no significant effect on the final ethanol yield.
At the planning stage of the experiments, the method based on native starch hydrolysis appeared to offer a promising alternative to the traditional temperature-assisted treatment combined with two-step enzymatic hydrolysis. However, the results of the experiments demonstrated that the fermentation efficiency following native starch hydrolysis (NSH) was statistically significantly lower than that observed for the pressureless starch liberation (PLS) method. The application of the separate hydrolysis and fermentation (SHF) method resulted in a prolonged technological procedure, yet the process efficiency was not significantly improved.
Summarising the results and observations described above, it can be concluded that the optimal solution in terms of time, energy consumption and fermentation efficiency for both types of bread is the utilisation of the pressureless starch release method combined with simultaneous saccharification and fermentation (PLS-SSF) for the preparation of mashes.

Author Contributions

Conceptualization, M.B. methodology, M.B.; software, K.P-P.; validation, K.P.-P.; formal analysis, M.B. and U.D.-K.; investigation, A.O. and M.K.; resources, M.B.; data curation, M.B. and K.P.-P.; writing—original draft preparation, M.B.; writing—review and editing, U.D.-K.; visualization, A.M.P.; supervision, U.D.-K.; project administration, M.B. and A.M.P.; funding acquisition, M.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 09565 g001
Figure 1. Schematic representation of mash preparation by the PLS method and fermentation thereof.
Figure 1. Schematic representation of mash preparation by the PLS method and fermentation thereof.
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Figure 2. Schematic representation of mash preparation by the NSH method and fermentation thereof.
Figure 2. Schematic representation of mash preparation by the NSH method and fermentation thereof.
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Figure 3. Fermentation indices of waste bread-based mashes. Different letters indicate significant differences (p < 0.05) between mean values of intake of sugars (lowercase letters) and fermentation efficiency (capital letters).
Figure 3. Fermentation indices of waste bread-based mashes. Different letters indicate significant differences (p < 0.05) between mean values of intake of sugars (lowercase letters) and fermentation efficiency (capital letters).
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Table 1. Chemical composition of tested raw materials.
Table 1. Chemical composition of tested raw materials.
ParametersWheat BreadWheat–Rye Bread
Content [g/100 g Dry Mass]
MeanSDMeanSD
Moisture *24.35 b0.1519.80 a0.37
Total protein (Nx6.25)7.08 b0.018.18 a0.05
Total reducing sugars expressed as glucose, incl.:3.03 a0.202.02 b0.31
Maltotriose0.20 a0.010.21 a0.01
Maltose0.22 a0.010.12 b0.01
Glucose2.58 a0.241.66 b0.20
Starch 71.70 b1.7177.16 a4.98
Xylose0.16 b0.030.49 a0.02
Arabinose0.11 b0.040.27 a0.00
Succinic acid0.04 a 0.010.04 a0.01
Lactic acid0.05 b0.01 0.10 a0.01
Acetic acid0.04 b0.010.07 a0.01
Glycerol0.04 b0.010.14 a0.01
* g/100 g; the results expressed as mean values ± SD (n = 3); mean values with different letters (a, b) within the same row are significantly different (ANOVA, p < 0.05).
Table 2. Chemical composition of the sweet mashes.
Table 2. Chemical composition of the sweet mashes.
ParameterWheat Bread-Based MashesWheat–Rye Bread-Based Mashes
PLS-SSFPLS-SHFNSH-N/APLS-SSFPLS-SHFNSH-N/ANSH-A
MeanSDMeanSDMeanSDMeanSDMeanSDMeanSDMeanSD
Extract [% w/w]17.60 c0.2818.93 b0.1720.00 a0.1016.40 d0.2118.00 c0.1020.00 a0.1020.00 a0.10
Total reducing
sugars expressed as glucose [g/L], incl.:		120.44 d1.58135.25 c1.22145.92 a0.12113.43 e1.67102.49 f1.20140.34 b0.17148.24 a0.13
Maltotriose0.95 b0.175.85 a0.050.13 de0.020.60 c0.060.32 d0.020.11 e0.000.12 e0.00
Maltose35.10 a3.748.12 b0.110.29 c0.0233.79 a1.700.88 c0.041.15 c0.010.29 c0.01
Glucose82.57 e5.91120.45 c3.67145.48 ab1.8577.29 e1.62101.23 d2.34139.01 b1.13147.81 a0.51
Dextrin [g/L]22.71 f0.2123.97 f0.2568.50 a0.0930.84 e1.1051.53 b0.4645.11 c0.5738.02 d0.34
The results expressed as mean values ± SD (n = 3); mean values with different letters (a, b, c, etc.) within the same row are significantly different (ANOVA, p < 0.05).
Table 3. Chemical composition of the mashes after completion of fermentation.
Table 3. Chemical composition of the mashes after completion of fermentation.
ParameterWheat Bread-Based MashesWheat–Rye Bread-Based Mashes
PLS-SSFPLS-SHFNSH-N/APLS-SSFPLS-SHFNSH-N/ANSH-A
MeanSDMeanSDMeanSDMeanSDMeanSDMeanSDMeanSD
Apparent extract
[% w/w]		0.71 bc0.071.11 a0.060.70 bc0.010.63 c0.020.78 b0.010.76 b0.020.75 b0.02
Ethanol [g/L]71.42 b1.3270.95 b1.0285.68 a0.0272.50 b2.5372.54 b1.6481.52 a2.2580.95 a2.11
Total reducing
sugars expressed as glucose [g/L], incl.:		0.32 c0.084.34 a0.350.90 b0.050.30 c0.010.23 c0.010.98 b0.011.09 b0.03
Maltotriose0.16 ab0.060.21 a0.070.07 bc0.000.09 bc0.000.02 c0.000.07 bc0.000.07 bc0.00
Maltose0.14 d0.010.31 c0.050.72 b0.090.18 cd0.030.14 d0.020.80 ab0.070.90 a0.01
Glucose0.01 c0.003.79 a0.422.07 b0.150.02 c0.010.05 c0.010.08 c0.020.07 c0.02
Dextrin [g/L]1.94 a0.091.87 a0.560.72 b0.122.52 a0.581.89 a0.130.75 b0.020.44 b0.08
Other compounds [g/L]:
Xylose0.16 c0.010.10 cd0.020.16 c0.010.37 a0.020.03 d0.010.27 b0.050.36 a0.05
Arabinose0.02 c0.000.03 c0.000.01 c0.000.01 c0.000.10 a0.020.06 b0.010.03 c0.00
Citric acid0.25 a0.010.22 a0.010.21 a0.020.01 c0.000.09 b0.010.07 bc0.050.12 b0.03
Succinic acid0.92 c0.111.01 bc0.031.09 bc0.081.24 ab0.021.05 bc0.021.47 a0.151.53 a0.22
Lactic acid0.62 cd0.082.61 a0.140.50 d0.050.81 c0.211.14 b0.050.46 d0.050.39 d0.05
Formic acid0.23 a0.010.05 b0.010.06 b0.020.09 b0.010.07 b0.030.06 b0.010.05 b0.01
Acetic acid0.09 cd0.020.24 ab0.050.31 a0.080.07 d0.020.28 ab0.040.22 abc0.060.15 bcd0.03
Glycerol7.23 b0.357.01 b0.167.97 a0.137.10 b0.096.71 b0.558.25 a0.038.36 a0.02
The results expressed as mean values ± SD (n = 3); mean values with different letters (a, b, c, etc.) within the same row are significantly different (ANOVA, p < 0.05).
Table 4. Chemical composition of the waste bread-based spirit distillates.
Table 4. Chemical composition of the waste bread-based spirit distillates.
Parameter
[g/L alcohol
100% v/v]		Wheat Bread-Based MashesWheat–Rye Bread-Based Mashes
PLS-SSFPLS-SHFNSH-N/APLS-SSFPLS-SHFNSH-N/ANSH-A
MeanSDMeanSDMeanSDMeanSDMeanSDMeanSDMeanSD
Methanol 0.200 b0.040.240 b0.030.200 b0.030.150 b0.020.300 a0.080.210 b0.020.190 b0.03
Aldehydes
as acetaldehyde		0.170 c0.010.120 d0.010.160 c0.020.220 a0.020.180 bc0.010.233 a0.020.210 ab0.01
Acidity
as acetic acid		0.620 d0.010.940 a0.040.723 c0.050.583 d0.040.620 d0.010.710 c0.010.850 b0.02
The results expressed as mean values ± SD (n = 3); mean values with different letters (a, b, c, etc.) within the same row are significantly different (ANOVA, p < 0.05).
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Balcerek, M.; Dziekońska-Kubczak, U.; Pielech-Przybylska, K.; Oleszczak, A.; Koń, M.; Patelski, A.M. Waste Bread as Raw Material for Ethanol Production: Effect of Mash Preparation Methods on Fermentation Efficiency. Appl. Sci. 2024, 14, 9565. https://doi.org/10.3390/app14209565

AMA Style

Balcerek M, Dziekońska-Kubczak U, Pielech-Przybylska K, Oleszczak A, Koń M, Patelski AM. Waste Bread as Raw Material for Ethanol Production: Effect of Mash Preparation Methods on Fermentation Efficiency. Applied Sciences. 2024; 14(20):9565. https://doi.org/10.3390/app14209565

Chicago/Turabian Style

Balcerek, Maria, Urszula Dziekońska-Kubczak, Katarzyna Pielech-Przybylska, Anna Oleszczak, Magdalena Koń, and Andrea Maria Patelski. 2024. "Waste Bread as Raw Material for Ethanol Production: Effect of Mash Preparation Methods on Fermentation Efficiency" Applied Sciences 14, no. 20: 9565. https://doi.org/10.3390/app14209565

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