Evaluation of ethanol fermentation parameters ... - Wiley Online Library

Research article Received: 7 February 2014

Institute of Brewing & Distilling

Revised: 9 May 2014

Accepted: 13 July 2014

Published online in Wiley Online Library: 29 September 2014

(wileyonlinelibrary.com) DOI 10.1002/jib.181

Evaluation of ethanol fermentation parameters for bioethanol production from sugar beet pulp and juice Małgorzata Gumienna,* Katarzyna Szambelan, Henryk Jeleń and Zbigniew Czarnecki The aim of this study was to investigate the efficient utilization of sugar beet pulp, as well as raw, concentrated raw and thick sugar beet juice, for bioethanol production. Different fermentation conditions were examined. The influence of raw material pre-treatment (pasteurization or sterilization), type of batch culture process (stationary or shaken) as well as the type of Saccharomyces cerevisiae yeast preparation on the yields of the process were studied. Moreover, the fermentation process effectiveness was examined in connection with the quality of the obtained distillates. Sterilization, stationary batch culture and Safdistil C-70 yeast preparation were identified as the most profitable factors for sugar beet pulp fermentation, providing a high fermentation efficiency and ethanol yield (87.7% of theoretical ethanol yield). Concentrated raw beet juice resulted in a value of 94.2% of theoretical yield, and thick juice a 92.6% yield. The results suggest that bioethanol production from sugar beet pulp and sugar beet juice has promise as an alternative fuel. The raw spirits obtained from the sugar beet juice were characterized as having the lowest quantity of volatile by-products. Copyright © 2014 The Institute of Brewing & Distilling Keywords: sugar beet; raw beet juice; thick beet juice; bioethanol; volatile by-products

Introduction

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* Correspondence to: Małgorzata Gumienna, Poznan University of Life Sciences, Institute of Food Technology of Plant Origin, Wojska Polskiego 31, 60-624 Poznan, Poland. E-mail: [email protected] Poznan University of Life Sciences, Institute of Food Technology of Plant Origin, Wojska Polskiego 31, 60-624 Poznan, Poland

Copyright © 2014 The Institute of Brewing & Distilling

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Biofuels have expanded in the last few decades and are of great interest globally. This is out of necessity, as the availability of fuels sourced from mines is rapidly decreasing. Bioethanol as a renewable energy source is an especially promising alternative. There is the option of using bioethanol as a mixture with petrol or by itself as an alternative for traditional fuels. Biofuels present on the market are first-generation biofuels, mainly bioethanol produced during an ethanol fermentation and biodiesel from rape seed oil esterification. The first generation biofuels were characterized by a decrease of 32–71% in carbon dioxide emissions. That is why the European Commission will substitute a minimum of 20% of conventional fossil fuels with alternative biofuels for the transport sector by 2020, with an intermediate goal of 5.75% set for 2010 (1–3). Bioethanol can be applied directly as neat alcohol or blended with petrol in dedicated engines. Bioethanol has already been applied on a large scale as a fuel in Brazil, the USA, Canada and some European countries (4). The EU produced 5.4 million tons of bioethanol in 2012, being the fourth largest producer of the fuel in the world. In recent years, the number of entrepreneurs producing bioethanol in Poland has remained constant at 13–14 manufacturers, who produced 167,802 tons of bioethanol in 2012. In the past two years, a decrease in Polish bioethanol sales to foreign entities has been observed, indicating that domestic bioethanol almost entirely meets the demand of the internal market (5). Bioethanol, from a global perspective, is produced from different initial starting materials, which are classified into three main types: sugars, starches and cellulose. Sugar materials, such as sugar cane, sugar beet and molasses can be converted into ethanol directly. Starchy materials, such as corn, wheat and

triticale must first be hydrolysed to fermentable sugars. Lignocellulosic materials, such as agriculture residues and wood, must be converted into sugars by the action of mineral acids. Cellulose hydrolysis is much slower than starch hydrolysis and requires a greater amount of processing to make the sugar available for the microorganisms to use in a fermentation process, as compared with the enzymatic degradation of other sugars as a pre-fermentation process (6,7). Industrial ethanol production has been performed using various starchy materials such as corn, triticale, wheat, rye, potatoes, cassava root and corn stover. Molasses and sugar beets have also been used as a feedstock for bioethanol production. These two materials do not require a complicated pre-treatment process, as the sugar content is almost all in the form of sucrose, which can be directly used for fermentation (8,9). Sugar beet is the only raw material used for sugar production in Poland (10). However, there is now an additional possibility to use it as a feedstock for bioethanol production. The sugar beet crop utilizes 3% of Poland’s total cultivation area. There are high cultivation costs such as pesticides, sowing and harvest, but sugar beets stand out against other agricultural raw materials in terms of productivity and yield. The average yield of sugar beets, in Poland in 2012, amounted to 58.2 t ha 1, while in the EU the average yield was 73.5 t ha 1. Poland’s participation in the production of sugar beet in the European Union amounted

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to 9.8% (11,12). Sugar beet and its intermediate products are materials that are suitable for bioethanol synthesis as far as their handling is concerned. Unlike starchy raw materials, in the case of sugar beet there is no necessity to apply any additional technological operations, such as chemical or enzymatic hydrolysis, which are energy- and cost-consuming (8). Compiling beneficial conditions for the bioethanol fermentation process, such as temperature, fermentation time, pH of the medium or wort concentration of dry solids (% w/w), can account for high ethanol yield. The selection of proper yeast strains capable of synthesizing ethanol from high-concentration media (% w/w) is especially important. The Saccharomyces cerevisiae strain chosen is widely used as a microbial producer in bioconversion processes and is suitable for the production of ethanol from sugar materials (6,13,14). Bioethanol production in the sugar industry concerns not only raw and thick beet juice, but also sugar beet roots. Raw juice contains about 15–20% of dry solids. Sugars constitute 85–90% of the dry matter. Thin juice can be used directly for fermentation, with the addition of nutrients, thus making this material a very profitable option for ethanol production. The only disadvantages of raw juice are low storability and easy decomposition by the action of microorganisms. That is why the thin juice is often submitted to evaporation, to enforce a high sugar concentration, in order to reduce volume and inhibit microbial growth. Thick juice is an intermediate product; however it is a one with a higher price, mostly owing to evaporation process costs (14). To produce bioethanol from sugar beet and its intermediate products in a profitable manner, the sugar industry must be restructured. The modernization of existing sugar-refineries and the development of biorefineries would allow for increased technological competitiveness and an increase in production capacity (15). In this study, the focus was on the effective utilization of sugar beet pulp and beet juices for bioethanol production and their batch culture with free S. cerevisiae cells depending on different fermentation parameters.

fermentation. The sugar beet pulp was pasteurized using a boiling water bath for 15 min or sterilized at 121°C for 15 min. The juices were sterilized at 121°C for 15 min before fermentation. The fermentation media were supplemented with 0.4 g L 1 of (NH4)2HPO4. Enzyme preparation OptimashTMVR (xylanase and cellulase) was applied 15 min before yeast inoculation, in an amount according to the recommendation of the manufacturer (Genencor International) S. cerevisiae dried yeast was re-hydrated prior to inoculation and added to fermentation media at 0.5 g L 1. Fermentation proceeded for 72 h at 30°C for the stationary process and for the sugar beet pulp, with shaking (at an agitation rate of 200 rpm). Analytical methods The dry matter of the sugar beet pulp was determined using a two-step drying method. The dry matter of the juices was determined using a refractometric method (16). A polarimetric method was used to determine sucrose content in the experimental material (16). The amount of reducing sugars was estimated according to Miller (17). Ethanol was determined by an areometric method after distillation. The ethanol yield was expressed as ethanol % v/v, calculated as a percentage of the theoretical ethanol yield, and as L of ethanol obtained from 100 kg sugar beet root. The composition and purity of the obtained raw distillates were analysed using a gas chromatograph method on a Hewlett Packard HP chromatograph, using a Supelcowax-10 column (60 m × 0.53 mm × 1.0 μm) and a free induction decay detector. Statistical analysis All experiments were carried out and analysed in triplicate. The results were tested statistically by analysis of variance (ANOVA) , using Statistica 6.0 (α = 0.05).

Materials and methods

Results and discussion

Sugar beet

Raw material characteristics

Sugar beet roots and thick sugar beet juice were obtained from a domestic sugar factory in Opalenica Poland. Raw juice was produced using a laboratory-scale process and a manual press. Concentrated raw juice was produced using an evaporation process (Buchi Rotavapor R-215, 50°C, 0.008 MPa, 50 rpm).

Table 1 presents the composition of the substrates. The dry matter content in the sugar beet root was 24.1%. The root contained 17.1% sucrose and 1.6 mg mL 1 reducing sugars. The composition was characteristic for this type of raw material (18). The raw, concentrated raw and thick beet juices were characterized by their dry matter content (25.0, 60.0 and 69.0%, respectively). The sucrose content was ~23.9, 58.0 and 67.7% for the raw, concentrated raw and thick juice, with the values being higher than those reported by Henke et al. (19) who reported 16.4 and 65.0% of dry matter, and 14.8 and 63.4% sucrose for the raw and thick juices, respectively. Raw juice had a concentration of reducing sugars of 9.7 mg mL 1 and the concentrated raw juice content contained 53.4 mg mL 1. The higher content of reducing sugars is a result of the enhanced temperature conditions (50°C) during raw juice evaporation, where partial sucrose inversion can occur and cause an increase in reducing sugars. In the thick juice, only 2.8 mg mL 1 of reducing sugars was detected. The pH value determined for sugar beet pulp was 6.8 and for raw juice and 6.7 for concentrated raw juice. Thick juice was characterized by a higher pH at 9.2, an effect of the juice defecation and saturation processes.

Yeast Dried alcohol yeasts (S. cerevisiae) were used in the experiments. Ethanol Red and Safdistil C-70 (Lesaffre, Fermentis, France) preparations were used for the sugar beet pulp fermentations and Safdistil C-70 for the juice fermentations. Ethanol fermentation process

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Sugar beet roots were treated with water in a proportion of 1:0.75, 1:1 and 1:1.25. Fermentation media with a dry solids concentration of 20% and 25% w/w were prepared from the beet juices by dilution with distilled water. Fermentations were carried out in Erlenmeyer flasks containing the fermentation medium (200 g for sugar beet pulp and 200 mL for juices). The pH of the media was adjusted to 5.5 before

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J. Inst. Brew. 2014; 120: 543–549

Sugar beet for bioethanol production


Table 1. Composition of raw materials Component

Dry matter

pH

Reducing substances 1

(%) Sugar beet root pulp Raw juice Concentrated raw juice Thick juice

24.1 ± 2.2 25.0 ± 0.1 60.0 ± 0.1 69.0 ± 0.1

6.8 ± 0.3 6.7 ± 0.6 6.7 ± 0.7 9.2 ± 1.6

Determining the most favourable conditions for the fermentation of sugar beet pulp The general process of sugar beet ethanol fermentation is well known, but it is very important to select the appropriate process variables, in order to determine their values for an optimal process. The research presented in this paper comprises: (a) the selection of the most effective thermal medium pre-treatment; (b) the evaluation of conditions for cultivation (shaken or stationary process); and (c) selection of the S. cerevisiae yeast preparation (Safdistil C-70 or Ethanol Red) for the intensification of the alcohol fermentation process. First, the influence of the thermal pre-treatment method (pasteurization or sterilization) of the fermentation material on the efficiency of ethanol yield was determined. For this purpose the Ethanol Red yeast was utilized. Prior to the fermentation process, the sugar beet pulp was thermally treated to inactivate non-desired microorganisms. The first applied variant of the treatment was pasteurization in a boiling water bath for 15 min, whereas the second variant was sterilization at 121°C for 15 min. The milder conditions of pasteurization minimized sucrose losses but did not result in complete sterilization of

Sucrose

(mg mL )

(%)

(% dry matter)

1.6 ± 0.5 9.7 ± 1.0 53.4 ± 2.9 2.8 ± 1.0

17.1 ± 0.7 23.9 ± 0.4 58.0 ± 0.6 67.7 ± 0.2

71.2 ± 0.9 95.6 ± 0.7 97.6 ± 0.8 98.1 ± 0.5

fermentation media, leaving non-desired microorganisms, which consequently can cause lower ethanol efficiency. In the presented experiments, the ethanol yield from the pasteurized sugar beet pulp after fermentation with S. cerevisiae (Ethanol Red) was between 70.5 and 77.6% of the theoretical ethanol yield (Table 2). When total sterilization of the pulp was applied, a significantly (p < 0.05) higher ethanol yield, ranging from 76.1 to 83.3% of the theoretical ethanol yield, was obtained (Table 2). Experimental alcohol fermentations of sugar beet pulp were conducted under shaken and stationary conditions. Significant (p < 0.05) differences between the cultivation methods was demonstrated (Table 2). The shaken culture, in spite of better fermentation substrate dispersion in the medium, did not cause improvement of fermentation efficiency rates, although it might have created non-favourable aerobic conditions. In such a case, yeast cells could start to produce biomass intensively. Ethanol fermentation of sugar beet pulp conducted under stationary conditions gave higher yield rates of 77.6% and 83.3% of the theoretical ethanol yield, for pasteurized and sterilized media, respectively (Table 2). Thus the stationary culture process was selected for further experiments.

Table 2. Ethanol yield from sugar beet pulp fermentation with Saccharomyces cerevisiae (Ethanol Red; 30°C, 72 h) Ethanol production Concentration (%w/w dry solids) Pasteurization Stationary culture 10.7 8.9 7.7 Shaken culture 10.0 8.0 6.6 Sterilization Stationary culture 9.7 8.1 6.9 Shaken culture 9.7 8.1 6.9

Stillage

pH after fermentation

(% v/ v)

(L /100 kg sugar beet)

(percentage theoretical yield)

Reducing substances (mg mL 1)

Dry matter (%)

4.2 ± 0.1 4.2 ± 0.1 4.1 ± 0.1

7.0 ± 0.3 7.1 ± 0.1 7.1 ± 0.1

9.3 ± 0.3 9.5 ± 0.2 9.5 ± 0.2

76.5 ± 0.4 77.6 ± 0.2 77.6 ± 0.2

2.7 ± 0.1 1.9 ± 0.1 2.1 ± 0.1

5.4 ± 0.1 4.4 ± 0.1 3.8 ± 0.1

3.9 ± 0.1 3.8 ± 0.1 3.9 ± 0.1

4.3 ± 0.1 4.4 ± 0.1 4.6 ± 0.1

5.7 ± 0.2 5.8 ± 0.1 6.1 ± 0.2

70.5 ± 0.2 71.3 ± 0.2 75.4 ± 0.2

4.4 ± 0.1 4.9 ± 0.1 3.5 ± 0.1

3.37 ± 0.1 3.56 ± 0.1 3.13 ± 0.2

4.0 ± 0.1 4.0 ± 0.1 3.9 ± 0.1

6.7 ± 0.1 6.9 ± 0.1 6.9 ± 0.1

8.9 ± 0.2 9.2 ± 0.2 9.2 ± 0.2

80.9 ± 0.2 83.3 ± 0.2 83.3 ± 0.2

2.5 ± 0.1 1.6 ± 0.1 2.5 ± 0.1

5.7 ± 0.1 6.1 ± 0.2 5.8 ± 0.1

4.1 ± 0.1 3.9 ± 0.2 3.8 ± 0.1

6.3 ± 0.1 6.6 ± 0.6 6.7 ± 0.1

8.4 ± 0.2 8.8 ± 0.6 8.9 ± 0.2

76.1 ± 0.2 79.7 ± 0.9 80.9 ± 0.2

2.9 ± 0.1 3.9 ± 0.1 3.0 ± 0.1

5.2 ± 0.2 5.8 ± 0.2 5.7 ± 0.1

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Concentration (% w/w dry solids) calculated as sucrose content in the raw material.


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Next, sugar beet pulp was fermented using the stationary process with two different S. cerevisiae yeast preparations: Safdistil C-70 and Ethanol Red. On the basis of conducted research it was stated that Safdistil C-70 application increased ethanol yield significantly (p < 0.05) , as compared with the application of Ethanol Red. Safdistil C-70 gave an ethanol yield of 87.7% of the theoretical ethanol yield (Table 3). The application of Ethanol Red for sugar beet pulp fermentation decreased the efficiency to 83.3% of theoretical ethanol yield as a maximum (Table 2). Similar effects were observed by Patrascu et al. (9), who obtained maximum ethanol productivity from molasses by using Safdistil C-70, in contrast to other strains used. This part of the experiment suggests that the Safdistil C-70 preparation (Lesaffre) is very suitable for the sugar beet fermentation process. Influence of the fermentation medium type on ethanol yield Ethanol fermentation efficiency depends heavily on the raw materials used, in particular on sugar content and availability. During the processing of the raw materials to intermediate products, many physicochemical changes occur, which can positively or negatively influence ethanol fermentation yield. Ethanol fermentations were conducted using sugar beet pulp and its intermediate products: the raw, the concentrated raw and the thick juice. The most favourable conditions for the fermentation process in sugar beet pulp were examined. The results suggest the use of a Safdistil C-70 yeast preparation, sterilization of the medium and a stationary culture for sugar beet intermediate product fermentation. Taking into account the results of ethanol production from raw juice and beet pulp fermented under identical conditions (Safdistil C-70, fermentation time of 72 h, sterilized medium, stationary culture), the difference was found to be statistically significant (p < 0.05): 87.7–78.3% theoretical ethanol yield for sugar beet pulp and raw juice, respectively (Tables 3 and 4). Comparable ethanol efficiency for sugar beet pulp (at a level of 0.11 L kg 1) was described by Icöz et al. (20). Prolonging the fermentation time up to 96 h for the raw beet juice gave a higher ethanol yield (Table 4). The data on the sugar beet intermediate products showed that a statistically significant (p < 0.05) higher ethanol yield could be obtained from the concentrated raw juice and the thick juice, rather than just from the raw juice fermentation (Table 4). However, results obtained did not show significant (p > 0.05) differences between ethanol yield from the concentrated raw juice (83.5–94.2% of the theoretical ethanol yield) and the thick juice (83.3–92.6% of the theoretical ethanol yield). It can be concluded that the content of the non-sugar compounds (such as proteins, fats, and organic acids present in concentrated raw juice) did not influence the ethanol yield unfavourably. These compounds may constitute a source of nutrition that is beneficial to the

growth of the yeast. Utilizing concentrated raw juice as a medium for ethanol production seems to be a good solution from a technological and economical point of view. Both the concentrated raw juice and the thick juice gave higher ethanol yield values than those obtained from the sugar beet pulp. Sugar beet roots can be stored for a period of several months; however, a thin juice cannot be stored. Therefore it is necessary to prepare a concentrate of the juice. Concentrated raw juice is economical to produce since there is no need to use any energy-absorptive and expensive purifying processes. It was observed that enabling the raw juice to thicken, despite physicochemical changes occurring in the concentrated juice, did not influence the fermentation ability of this product unfavourably. As was the case with raw juice, prolonging the fermentation time to 96 h increased ethanol efficiency, both for the concentrated and the thick juice. The content of reducing sugars in concentrated juice increased to 53.4 mg mL 1, owing to partial sucrose inversion during the raw juice concentration process. Such initial simple sugars levels are profitable for the fermentation process, because invert sugar is more easily taken up by the yeast and does not inhibit yeast growth. Additionally, another advantage of concentrated raw juice is its higher microbiological stability, resulting from the high sugar concentration, which accounts for its longer storage suitability. Influence of the fermentation medium dry solids concentration (% w/w) and fermentation time on ethanol yield To prepare the fermentation medium, sugar beet pulp was diluted with distilled water in proportions of 1:0.75; 1:1 and 1:1.25. The empirically counted concentration of dry solids (% w/w) ranged from 6 to 14% w/w in particular trials. A correlation between the pulp medium’s dry solid concentration (% w/w) and ethanol yield was observed (Tables 2 and 3). Sugar beet pulp fermentations, for both S. cerevisiae preparations, confirmed that a higher fermentation media concentration of dry solids (% w/w) resulted in a statistically significant (p < 0.05) lower ethanol yield. This has also been stated by Balcerek and Pielech-Przybylska (21). Higher sugar content in a fermentation media causes an increase in the osmotic pressure, which can reduce the fermentation ability of the yeast. Fermentation media from raw juice, concentrated raw juice and thick juice were characterized by concentrations of 20–25% w/w. Fermentations lasting for 72 h showed no significant differences (p > 0.05), although there were differences in ethanol yield depending on the media’s dry solids concentration (% w/w; Table 4). With an initial dry solids concentration of 20% w/w in thick juice, the ethanol yield was at the level 89.5%, whereas with the concentration of 25% w/w, it decreased to 83.3% of the theoretical ethanol yield. In the case of concentrated raw juice the values

Table 3. Ethanol yield from sterilized sugar beet pulp fermentation with S. cerevisiae (Safdistil C-70; 30°C, 72 h, stationary culture) Ethanol production Concentration (%w/w dry solids)

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9.8 7.8 6.5

Stillage

pH after fermentation

(% v/v)

(L/100 kg sugar beet)

(percentage theoretical yield)

Reducing substances (mg mL 1)

Dry matter (%)

4.2 ± 0.4 4.1 ± 0.1 4.0 ± 0.1

7.4 ± 0.3 7.7 ± 0.4 7.8 ± 0.1

9.9 ± 0.2 10.3 ± 0.4 10.4 ± 0.1

83.2 ± 0.2 86.6 ± 0.5 87.7 ± 0.1

1.7 ± 0.1 2.0 ± 0.1 1.8 ± 0.1

5.4 ± 0.1 5.7 ± 0.1 5.9 ± 0.2



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Table 4. Ethanol yield from sterilized sugar beet intermediate products, fermentation with S. cerevisiae (Safdistil C-70; 30°C, stationary culture) Ethanol production Fermentation time (h) Raw juice 72 96

Concentration pH after (%w/w dry solids) fermentation 20 25 20 25

Concentrated raw juice 72 20 25 96 20 25 Thick juice 72 20 25 96 20 25

(% v/v)

(L/100 L (percentage theoretical Reducing Dry matter medium) yield) substances (mg mL 1) (%)

4.5 ± 0.1 4.4 ± 0.1 4.5 ± 0.1 4.4 ± 0.1

10.2 ± 0.2 12.2 ± 0.4 11.0 ± 0.2 12.2 ± 0.2

10.2 ± 0.2 12.2 ± 0.4 11.0 ± 0.2 12.2 ± 0.2

78.3 ± 0.5 74.9 ± 0.7 84.4 ± 0.5 74.9 ± 0.3

2.1 ± 0.4 31.6 ± 0.2 3.9 ± 0.2 8.7 ± 0.6

1.0 ± 0.1 5.5 ± 0.1 1.0 ± 0.1 1.0 ± 0.1

4.1 ± 0.1 4.1 ± 0.1 4.1 ± 0.1 4.1 ± 0.1

10.8 ± 0.1 11.0 ± 0.2 11.4 ± 0.4 11.4 ± 0.2

10.8 ± 0.1 11.0 ± 0.2 11.4 ± 0.4 11.4 ± 0.2

87.5 ± 0.1 83.5 ± 0.5 94.2 ± 0.5 86.5 ± 0.5

1.5 ± 0.4 50.0 ± 0.2 0.9 ± 0.1 44.6 ± 0.6

1.0 ± 0.1 3.0 ± 0.1 1.5 ± 0.1 2.0 ± 0.1

4.2 ± 0.5 4.1 ± 0.1 4.2 ± 0.1 4.1 ± 0.1

8.7 ± 0.3 8.7 ± 0.3 9.6 ± 0.1 9.6 ± 0.1 9.0 ± 0.1 9.0 ± 0.1 10.1 ± 0.1 10.1 ± 0.1

89.5 ± 0.5 83.3 ± 0.1 92.6 ± 0.1 87.3 ± 0.3

95.7 ± 0.9 197.5 ± 1.4 78.8 ± 0.7 134.1 ± 1.1

4.5 ± 0.1 12.0 ± 0.1 1.0 ± 0.1 3.0 ± 0.1

were 87.5 and 83.5%, respectively, and for raw juice, 78.3 and 74.9% of the theoretical ethanol yield. The 72 h fermentation times were sufficient for sucrose fermentation. Considering the still high sugar content in the stillage (Table 4), the fermentation times were prolonged to 96 h. Prolonging the fermentation time for intermediate sugar beet products allowed for the remaining sugar content to decrease and the ethanol yield average to increase by 8.4%. Furthermore, for the 96 h process, significant (p < 0.05) differences in fermentation yield between the 20 and 25% w/w media were demonstrated (Table 4). The fermentation medium, whose concentration of dry solids was at a level of 25% w/w, was characterized by lower ethanol production. This might have been due to the higher osmotic pressure, as well as a lack of nutrients that could have affected the fermentation activity of the yeast. Influence of the fermentation medium type on the composition and purity of the obtained raw spirits

of 8.1% w/w, whereas the lowest content of volatile by-products (2.53 g L 1 100% spirit) was found in distillates from concentrated raw juice fermentation (Table 5). Aldehydes, esters and methanol were detected as the compounds with higher volatile properties than ethanol. These compounds go to the distillate in the first distillation stage. The second group of volatile by-products that were in the distillate were the higher alcohols; these moved to the distillate at the end of distillation. The aldehyde content (Table 5) in the distillates obtained from the sugar beet pulp and thick juice fermentation exceeded values given in the Polish Standards for agriculture raw spirit from molasses (0.3 g L 1 100% spirit) (22). The requirements for raw spirits are crucial when the spirit is intended for consumption. As regards as the spirit for other needs, such as bioethanol, the quality of the obtained spirits was in agreement with the Polish and EU Standards (22,23). In the case of distillates obtained from raw juice and concentrated raw juice fermentation, the aldehyde content, 0.238 and 0.235 g L 1 100% spirit, respectively, was lower and compatible with the standard. The content of aldehydes may depend on factors connected with technological processes. A higher aldehyde content in the thick juice could be the result of raw juice saturation. Aldehyde precursors can be created during saturation and defecation, because of the highly alkaline environment and temperature promoting this process (24). Higher alcohols constituted the largest contamination of the distillate (Table 5). Higher alcohols are formed mostly from the amino acids in the raw materials, but a small part is also formed as a result of carbohydrate metabolism by the yeast. Saturation of the thick juice contributed to a reduction of higher alcohols in the distillate. The research conducted on thick juice confirmed the lowest content of higher alcohols in distillates from thick juice fermentation (2.057 g L 1). Polish standards do not standardize the content of higher alcohols for distillates from molasses. Methanol (also not standardized for a molasses distillate), formed from pectins contained in sugar beet pulp, was detected



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The quality of obtained raw spirits was analysed using a GC method, which detects the volatile compounds that are produced in addition to ethanol. The distillates that were examined were from the sugar beet pulp fermentations using the most favourable conditions (sterilization, Safdistil C-70 yeast preparation, 30°C, 72 h, stationary culture, and 20% w/w). During the fermentation of plant-based raw materials using yeast, different chemical substances in addition to ethanol as the main product are produced. The differences in distillates’ quality demonstrate that the final quantity and composition of fermentation by-products depend on raw material quality and fermentation conditions. The research showed that the distillates from sugar beet pulp fermentation were characterized by double the amount of byproduct content compared with the distillates from the juice fermentations (Table 5). The highest content of by-products (5.81 g L 1 100% spirit) was noticed in the distillates obtained from sugar beet pulp fermentation with a dry solids concentration

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Stillage

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Table 5. Ethanol content and by-product content (g L 1 100% spirit) of sugar beet and its intermediate products. Raw distillates from the ethanol fermentation (sterilization treatment, Safdistil C-70, 30°C, 72 h, stationary culture) Constituents Aldehydes Esters Higher alcohols Methanol Ethanol, percentage of total compounds

Sugar beet pulp, Sugar beet pulp, Sugar beet pulp, 7.5% w/w 8.1% w/w 10.5% w/w 0.413 ± 0.038 0.330 ± 0.037 4.758 ± 0.122 0.150 ± 0.015 99.29 ± 0.07

0.524 ± 0.046 0.386 ± 0.016 4.783 ± 0.117 0.119 ± 0.009 99.27 ± 0.05

0.701 ± 0.027 0.321 ± 0.018 4.065 ± 0.133 0.178 ± 0.012 99.34 ± 0.08

Raw juice, 20% w/w

Concentrated raw juice, 20% w/w

Thick juice, 20% w/w

0.238 ± 0.011 0.111 ± 0.009 2.603 ± 0.102 0.041 ± 0.004 99.62 ± 0.07

0.235 ± 0.014 0.077 ± 0.010 2.196 ± 0.126 0.025 ± 0.005 99.67 ± 0.11

0.401 ± 0.030 0.103 ± 0.011 2.057 ± 0.113 0.017 ± 0.003 99.67 ± 0.09

The content of volatile acids in all samples, counted as acetic acid, was determined in trace amounts. The concentrations of pulp and juices are given as percentage w/w dry solids. at the highest amount of 0.178 g L 1 100% spirit after fermentation of 10.5% w/w sugar beet pulp. It is worth noting that the methanol content in the distillates obtained from the juice fermentation was 10 times lower, owing to the low content of pectins remaining in the separated pulp, which must be taken into consideration (Table 5). On the basis of the GC analysis, it was concluded that the best intermediate sugar beet products, considering the low quantity of distillate by-products formed during fermentation, were the raw and the concentrated raw juices.

Conclusions

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Research confirmed that bioethanol production from sugar beet pulp, as well as the raw, the concentrated raw and the thick beet juices is promising as an alternative fuel source. The sterilization process applied for the thermal treatment of raw material allowed for the elimination of microbiological contaminants and an increase in ethanol yield. Stationary cultivation enabled better ethanol yield values (between 3 and 9%) as compared with shaken cultivation. It was observed that the type of fermentation medium had a statistically significant (p < 0.05) influence on the fermentation process efficiency. Fermentation media with a dry solids concentration of 25% w/w exhibited substrate inhibition. The highest ethanol yield was achieved from the sterilized concentrated raw beet juice (94.2% of the theoretical ethanol yield) with S. cerevisiae at 30°C with a stationary culture and a dry solids medium concentration of 20% w/w. It was concluded that there was no need for the purification of the concentrated raw juice, which provided comparable or even better ethanol yields than the thick juice from the sugar factory, subjected to a purification process. Elimination of the purification step can further influence the reduction of ethanol production costs. The research demonstrated that sugar beet juices are raw materials that are more efficient and easier to process than sugar beet pulp as far as ethanol production is concerned. The GC analysis indicated that the raw spirits obtained from sugar beet juice fermentations contained lower quantities of volatile by-products compared with the sugar beet pulp fermentation. The quality of the resulting alcohol was compatible with the European Fuel Ethanol Standards. The efficient utilization of this research should be correlated with the improvement of sugar beet root growth efficiency, as it is a promising raw material for the production of biofuels.


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