The Fermentation of Beet Sugar Syrup to Produce Bioethanol

Kenneth A. Leiper 1, Cornelia Schlee 1, Ian Tebble 2 and Graham G. Stewart 1,3
ABSTRACT

J. Inst. Brew. 112(2), 122133, 2006 Fermentation of sugar or starch-containing substrates by yeast to produce ethanol for use as a liquid fuel has been an accepted technology for many years. Currently, the most popular substrates are sugar cane molasses and starch from maize or wheat. Interest in renewable liquid fuels is growing and other substrates are now being considered, choice of these depends on local conditions. This paper presents findings from work carried out on syrup from sugar beet, an ideal crop for cultivation in the United Kingdom and parts of Europe. Fermentation of this substrate was found to be successful. The process of backsetting was investigated as a way of reducing water usage and effluent disposal. This was found to have no effect on ethanol production provided compensation was made for increases in gravity caused by glycerol levels. Backsetting was also found to be beneficial to yeast growth. As yeast remain in the fermented substrate, the effect of distillation on yeast cells was also investigated. It was found that dead yeast cells are present in backset and thus persist into subsequent fermentations. This can cause difficulties in viability measurement if the methylene blue method is used. Key words: Backsetting, beet sugar syrup, cell walls, fermentation, yeast.

INTRODUCTION
Bioethanol can be defined as ethanol produced by fermenting sugars extracted from agricultural crops, or byproducts using micro-organisms (normally yeast) to produce ethanol, which is then recovered by distillation. The most common use for bioethanol is as a motor fuel substitute or supplement. Production of bioethanol accounts for the vast majority of ethanol manufacture. Worldwide production in 2003 was approximately 30,000 million litres 3, dwarfing the output of potable ethanol approximately 4,000 million litres. The bulk of bioethanol production is in Brazil and the USA. The most common substrate in the USA is maize (corn) starch, while in Brazil sugar cane molasses is used. They can either make use of molasses produced as a coproduct from sugar refining (molasses is the liquid residue remaining after the extraction of sugar from cane or beet)
1 International

Centre for Brewing and Distilling, Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS, Scotland. 2 British Sugar plc, Oundle Road, Peterborough, Cambridgeshire, PE2 9QU, England. 3 Corresponding author. E-mail: G.G.Stewart@hw.ac.uk
Publication no. G-2006-0629-427 2006 The Institute of Brewing & Distilling

or from sugar cane grown especially for ethanol production. The choice of suitable agricultural crops in other countries depends on the local agricultural environment and economics. If substrates containing starch are used, they must first be subjected to heat and enzyme treatments to convert the starch to fermentable sugars, thus adding to production costs. Molasses and other beet extracts do not require such treatment as the sugar content is almost all in the form of sucrose 8. This is readily split into glucose and fructose in the initial stage of fermentation by the enzyme invertase, located in the periplasmic space between the yeast cell wall and cell membrane. The only preparation required with molasses is dilution to a suitable original gravity and pH buffering. This paper presents data from fermentations carried out using syrup extracted from sugar beet (this came from an early stage in the sugar manufacturing process and had no sugar removed from it). If economic conditions were favourable, the production of bioethanol could become a viable proposition in the United Kingdom. If this situation develops, sugar beet would be a suitable substrate. Initial production would use syrup currently available from sugar refining, with the possibility of dedicated crops in the future. Initial work at the laboratory scale indicated that fermentation of beet sugar syrups to produce ethanol was easy to conduct. As the reduction of production costs would be vital for bioethanol production, the use of backsetting was investigated. This process makes use of spent media, also known as spent wash, stillage or vinasse, from distillation to dilute subsequent batches of syrup thus saving water and waste water treatment charges. This process has been used successfully in whisk(e)y and grain neutral spirit production in Scotland and North America for many years. More recently, it has found a role in the production of bioethanol. The process, however, must be used with care, it is only a partial solution to water usage and cannot be used indefinitely as the constituents of the liquid will become progressively concentrated causing problems associated with viscosity and build-up of toxins. The material must be discarded at some point. Backsetting has been investigated by many workers at varying levels of spent wash usage, and has been reviewed by Chin & Ingledew 1. None of the workers found backsetting to have an effect on ethanol production despite the increasing concentration of a range of metabolites. Despite this, the process is still regarded with suspicion in many quarters.

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In this work, backsetting was found to cause no reduction in ethanol production, provided the level of glycerol was observed and taken into account. Initial work investigated backsetting at 50% and 100%, but later pilot scale work used backset at 30%, as this level was considered to be realistic on an industrial scale. Both normal and backsetted fermentations were successfully scaled up to pilot scale volumes. It had been assumed that yeast cells would be completely destroyed during distillation, but this was found not to be the case.

MATERIALS AND METHODS

Syrup Beet sugar syrup was supplied in liquid form by British Sugar. Yeast A dried yeast culture was used for this work, a distilling strain Safdistil B-28, a gift from Fermentis, Lille, France. Before use, the yeast was slurried in warm water (~30C) at 1 g/10 mL and left for 2 h with occasional stirring. Cell number in the slurries was determined using an Improved Neubauer Haemocytometer and a microscope at 400 magnification. Viability was determined using methylene blue staining according to the IOB method 9.1.3.2 7. These last two measurements were used to calculate the volumes of yeast slurry required to be added to the fermentations. Other yeast strains were screened for possible use, but all were found to be less effective than the above strain (data not shown). Fermentations Laboratory scale fermentations were carried out with 1.5 L syrup in 2 L bottles. The syrup was diluted by approximately 1/4 with warm water (~40C) to give original gravities of 1088 (22P) or 1096 (24P), pH was buffered from approximately 9 to 5.6 by the addition of hydrochloric acid and the use of a pH 210 microprocessor pH meter (Hanna Instruments), gravity was adjusted by the addition of water or syrup and the use a DMA46 calculating digital gravity meter (Anton Paar). For the fermentations with backset, some or all of the dilution water was replaced with spent wash. Following the addition of yeast, normally at a pitching rate of 9 107 cells /mL, the bottles were incubated in a shaking incubator at 150 rpm at 32C for up to 70 h. During fermentation, gravity, pH, cell number and viability were measured using the methods

previously described. Samples were retained for FAN and sugar analyses. Following fermentation, final ethanol content was determined by distillation according to the IOB method 8.5.1 7. The residues from the distillations were retained and analysed for FAN and sugars. The fermentations for determining optimal pitching rates were carried out in 500 mL flasks containing 250 mL syrup. These were fermented for up to 96 h. The pilot scale fermentations were conducted in the ICBDs 2 hL brewery. Syrup and water (or spent wash) were mixed in the mashing-in equipment followed by further mixing in the mash mixer vessel. Here pH and gravity were adjusted if required as described previously, with the pH meter probe placed in the vessel. No attempt was made to control levels of oxygen, it was assumed that sufficient oxygen would be picked up during mixing. The syrup was cooled to 30C by being passed through the brewerys wort cooling system and was then pumped into a fermentation vessel. Following pitching, the fermentations were left for up to 70 h. The vessels were fitted with a cooling system, but this was not used, the temperature was allowed to free rise. Sampling and ethanol determination was as previously described. Distillation The ethanol produced during fermentation was recovered by distillation, with the yeast remaining in the liquid. Pot stills were used, as small scale distillation using a continuous still (which would have been more realistic) was not possible. The laboratory scale fermentations were distilled in an apparatus consisting of a 500 mL flask, a foaming chamber, a lyne arm and a condenser (Kiko, Osaka, Japan). Wash (330 mL) was placed in the flask and heated with a Bunsen flame. The foaming chamber was largely redundant as no foaming occurred. The distillation was conducted until 110 mL spirit was collected, this containing approximately 40% (v/v) ethanol. The spirit fraction, or Low Wines (the term used in the Scotch whisky industry to describe spirit from an initial pot still distillation) was analysed for ethanol content by gravity and volatile content by GC. The spent wash fraction was retained for use in backsetting. The pilot scale fermentations were distilled in the ICBDs pilot distillery in 20 L batches in a glass pot still. The distillations were conducted until the spirit coming from the still contained less than 1% v/v ethanol. This is a longer distillation than that carried out on the laboratory scale stills, so the low wines contained a lower level of ethanol. Low wines and spent wash samples were analysed as described previously.

TABLE I. Analysis of unfermented beet syrups used in laboratory (2 L) and pilot scale (2 hL) fermentations. Sample Laboratory scale control Laboratory scale OG 1088 with 100% backset Laboratory scale OG 1088 with 50% backset Laboratory scale OG 1096 with 100% backset Laboratory scale OG 1096 with 50% backset Pilot scale control Pilot scale OG 1088 with 30% backset Original gravity 1087.1 1094.5 1092.2 1102.9 1100.4 1086.8 1089.6 pH 5.66 5.63 5.61 5.65 5.65 5.60 5.61 FAN (mg/L) 207.4 281.4 229.1 314.5 245.5 205.2 249.6 Sugars (g/L) Glucose 0.08 0.69 0.55 1.03 0.41 0.33 0.30 Fructose 4.73 1.52 4.45 2.28 0.15 0.07 Sucrose 240.76 260.33 230.28 255.55 277.86 256.54 248.34

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Fig. 1. Specific gravity during laboratory (2 L) and pilot scale (2 hL) fermentations. = Laboratory scale control. = Laboratory scale OG 1088 with 100% backset. = Laboratory scale OG 1088 with 50% backset. = Laboratory scale OG 1096 with 100% backset. = Laboratory scale OG 1096 with 50% backset. = Pilot scale control. = Pilot scale OG 1088 with 30% backset.

Fig. 2. pH levels during laboratory (2 L) and pilot scale (2 hL) fermentations. = Laboratory scale control. = Laboratory scale OG 1088 with 100% backset. = Laboratory scale OG 1088 with 50% backset. = Laboratory scale OG 1096 with 100% backset. = Laboratory scale OG 1096 with 50% backset. = Pilot scale control. = Pilot scale OG 1088 with 30% backset.

Sugar levels Levels of glucose, fructose, sucrose, maltose and maltotriose were measured by HPLC. The system consisted of a Dionex Carbopak PA-100 guard column (4 mm 50 mm), a Dionex PA-100 column (4 mm 250 mm) and a
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Dionex PAD electrochemical detector (all Dionex Corporation, Sunnyvale, California, USA). The eluent used was 500 mM NaOH and cellobiose was used as the internal standard. This system also detected the presence of glycerol. This appeared as an unknown peak on the chromatographs and was identified by running potential chemicals

Fig. 3. Total cell number during laboratory (2 L) and pilot scale (2 hL) fermentations. = Laboratory scale control. = Laboratory scale OG 1088 with 100% backset. = Laboratory scale OG 1088 with 50% backset. = Laboratory scale OG 1096 with 100% backset. = Laboratory scale OG 1096 with 50% backset. = Pilot scale control. = Pilot scale OG 1088 with 30% backset.

Fig. 4. Yeast viability during laboratory (2 L) and pilot scale (2 hL) fermentations. = Laboratory scale control. = Laboratory scale OG 1088 with 100% backset. = Laboratory scale OG 1088 with 50% backset. = Laboratory scale OG 1096 with 100% backset. = Laboratory scale OG 1096 with 50% backset. = Pilot scale control. = Pilot scale OG 1088 with 30% backset.

on the system until a match was found, quantitative analysis was not attempted. Free amino nitrogen Free Amino Nitrogen (FAN) was measured according to the IOB method 8.3 7.

Volatile concentrations Levels of acetaldehyde, acetone, esters and higher alcohols in the low wines were measured using Headspace GC. The system consisted of a Hewlett Packard 5890 Series II GC with a split-splitless injector (Hewlett Packard
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Fig. 5. Free amino nitrogen during laboratory (2 L) and pilot scale (2 hL) fermentations. = Laboratory scale control. = Laboratory scale OG 1088 with 100% backset. = Laboratory scale OG 1088 with 50% backset. = Laboratory scale OG 1096 with 100% backset. = Laboratory scale OG 1096 with 50% backset. = Pilot scale control. = Pilot scale OG 1088 with 30% backset.

Fig. 6. Sugar uptake during laboratory (2 L) scale fermentations. = Sucrose. = Glucose. = Fructose.

Ltd, Stockport, England) with a FID, a Chrompack CPWax-57-CB column (0.23 mm 60 m) (Chrompack International BV) and a Hewlett Packard 19395A autosampler with a 1 mL injection loop. The internal standard used was 3-heptanone. Confocal microscopy Samples of healthy and boiled yeast were examined using a confocal microscope to investigate the effects of distillation. To gauge the effect on cell walls, cells were
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stained for trehalose with concanavalin A fluoresceinconjugate (Con-A-fluorescein) according to the method of Hutter et al. 6 To gauge the effect on cell contents, cells were stained for DNA content with propidium iodide following RNAse digestion according to the methods of Hutter 4, and Hutter and Eipel 5. The samples were examined using a DM-IRE2 Confocal Fluorescence Microscope with a Laser Scanning Confocal Microscope Attachment (Leica Microsystems, Wetzlar, Germany). Both stains underwent excitation at a

wavelength of 488 nm, Con-A-fluorescein emitted detectable light at 500542 nm and propidium iodide at 618 710 nm. The data were digitized to produce a photographic record.

RESULTS AND DISCUSSION

Laboratory scale fermentations The control fermentations were set up as detailed in Table I and were conducted for 70 h. Specific gravity, pH, total cell number, cell viability and FAN are shown in Figs. 15. Gravity fell steadily reaching full attenuation by 70 h (Fig. 1), pH fell from 5.66 to 3.80 by 20 h and remained stable thereafter (Fig. 2), cell number increased from 9 to approximately 20 107 cells /mL by 20 h and remained stable thereafter (Fig. 3), viability remained above 80% until 44 h (Fig. 4) and FAN fell rapidly until 20 h followed by a slight increase until 70 h (Fig. 5) indicating a degree of secretion or cell autolysis. Sugar uptake is shown in Fig. 6, it can be seen that all the sucrose was hydrolysed into glucose and fructose by 20 h. The glucose was taken up preferentially with none remaining after 44 h, uptake of fructose took place until 70 h. This pattern has

also been observed with some brewing yeast strains, where glucose is taken up preferentially due to the yeast sugar transport system having a higher affinity for glucose than fructose 2. The control fermentations produced an average ethanol level of 13.2% (v/v). Data from the fermented molasses are shown in Table II. The final gravity was 990 and the residual gravity was 1007.3. This was found to be caused mainly by glycerol, but some residual fructose was detected. The fermented syrups were distilled to give spirit (low wines) and spent wash. Data on the spent wash are shown in Table II, gravity, FAN and sugar levels all increased in comparison to the residual gravity. This is due to the concentrating effect of distillation and extraction of sugars and amino acids from yeast cells during boiling. Data on the low wines are shown in Table III, it can be seen that the spirit had low levels of volatiles other than ethanol, they also had clean aromas. Most of the volatiles present are higher alcohols principally 2 and 3 methyl butanol (amyl and iso amyl alcohol) but also propanol (propan-1-ol) and isobutanol (2-methyl propanol). Levels of acetaldehyde and ethyl acetate are significant, levels of other esters are low. The ethanol content of this spirit was 41.1% (v/v).

TABLE II. Analysis of fermented beet syrups and spent wash samples from laboratory (2 L) and pilot scale (2 hL) fermentations. Sample Laboratory scale control Laboratory scale OG 1088 with 100% backset Laboratory scale OG 1088 with 50% backset Laboratory scale OG 1096 with 100% backset Laboratory scale OG 1096 with 50% backset Spent wash from laboratory scale control Spent wash from laboratory scale OG 1088 with 100% backset Spent wash from laboratory scale OG 1088 with 50% backset Spent wash from laboratory scale OG 1096 with 100% backset Spent wash from laboratory scale OG 1096 with 50% backset Pilot scale control Pilot scale OG 1088 with 30% backset Spent wash from pilot scale control Spent wash from pilot scale OG 1088 with 30% backset Final gravity 990.0 994.1 991.1 993.7 993.2 988.0 992.6 Residual gravity 1007.3 1011.6 1008.5 1012.9 1012.3 1010.1 1016.7 1012.3 1017.9 1016.6 1005.2 1009.3 1012.7 1021.1 pH 3.67 4.75 4.14 4.89 4.39 3.61 4.53 3.94 4.59 4.13 3.74 4.04 3.80 3.87 FAN (mg/L) 46.0 75.8 36.3 106.0 57.3 83.7 155.7 104.3 145.5 116.6 55.8 113.6 129.5 207.1 Ethanol (% v/v) 13.2 13.9 13.4 14.9 14.8 13.3 13.5 Residual sugars (g/L) Glucose 0.42 0.91 0.76 1.23 0.88 0.62 1.81 1.19 1.59 1.16 0.68 0.92 1.66 1.64 Fructose 5.63 0.04 0.35 2.27 11.70 6.01 0.04 0.21 1.93 12.46 3.52 7.11 Sucrose 0.04 0.05 0.08 0.05 0.05 0.11

TABLE III. Volatiles in low wines produced from laboratory and pilot scale fermentations (mg/L). Laboratory scale OG 1088 with 100% backset 304.89 2.81 52.04 6.72 1.08 0.72 66.54 317.67 372.32 542.37 42.4 OG 1088 with 50% backset 250.84 3.16 46.56 3.33 1.02 0.51 56.85 335.73 393.14 596.07 43.0 OG 1096 with 100% backset 307.12 4.52 53.79 9.14 1.14 0.94 91.65 355.84 422.69 497.96 50.8 OG 1096 with 50% backset 318.01 65.29 5.79 0.89 1.18 67.48 340.86 404.52 532.18 47.0 Pilot scale OG 1088 with 30% backset 198.6 8.5 17.1 1.2 0.2 42.1 234.5 238.3 277.8 27.6

Compound Acetaldehyde Acetone Ethyl acetate Isobutyl acetate Ethyl butyrate Isoamyl acetate Ethyl hexanoate Ethyl octanoate Propan-1-ol Isobutanol 2-Methyl butanol 3-Methyl butanol Ethanol (% v/v)

Control 184.43 86.34 12.38 0.82 61.04 254.34 359.10 426.39 41.1

Control 214.3 5.9 30.2 1.3 0.5 0.2 47.0 157.8 176.9 353.7 24.2

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Fig. 7. Sugar uptake during pilot scale (2 hL) control fermentations. = Sucrose. = Glucose. = Fructose.

= Fructose.

Fig. 8. Sugar uptake in pilot scale (2 hL) fermentations with 30% backset.= Sucrose. = Glucose.

Fermentations containing backset were set up with original gravities of 1088 and 1096, diluted with 100 or 50% spent wash as shown in Table I. Gravities were increased to compensate for the glycerol content of the spent wash. The increase in FAN and sugar levels due to the varying amounts of backset used can be seen. Specific gravity, pH, total cell number, cell viability and FAN are shown in Figs 15. Gravities initially fell faster than in the control fermentations, but ended at similar levels (Fig. 1), pH levels fell less far than observed in the controls (Fig. 2), cell numbers apparently increased much more than observed in the controls, up to 55 107 cells /mL (Fig. 3), viability in contrast, was much lower (Fig. 4) and uptake
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of FAN followed a similar pattern to the controls (Fig. 5). Sugar uptake in all four fermentations followed a similar pattern to that observed in the controls (data not shown). Alcohol levels, final gravities and data on the spent wash samples are shown in Table II. The low levels of residual sugars showed that full attenuation was achieved in all the fermentations. The high ethanol results show that the use of backsetting has no effect on ethanol production provided the original gravities are increased to compensate for glycerol. The higher residual gravities and the gravity of the spent wash samples indicate the accumulation of glycerol. Levels of volatiles in the low wines samples were generally higher than observed in the

Fig. 9. Specific gravity during fermentation of syrup inoculated at a range of pitching rates. Pitching rates: = 9 107 cells /mL; = 8 107 cells /mL; = 7 107 cells /mL; = 6 107 cells /mL; = 5 107 cells /mL; = 4 107 cells /mL; = 3 107 cells /mL.

control with the exception of acetaldehyde and ethyl acetate (Table III). All four spirits had roughly the same levels of volatiles but the spirits from the fermentations with original gravities of 1096 contained more ethanol. These fermentations show that backsetting is a viable proposition for the production of bioethanol. However, the apparent effect on yeast viability is a concern. Pilot scale fermentations These fermentations were carried out to ascertain if the laboratory scale fermentations could be successfully increased to 200 L scale. The control fermentation was set up as shown in Table I, fermentation data are shown in Figs. 15. Compared to the laboratory scale control fermentation, the gravity in the pilot control fermentation fell more slowly (Fig. 1), pH levels were similar (Fig. 2), cell numbers were higher (Fig. 3), cell viability remained high throughout the fermentation (Fig. 4) and uptake of FAN was similar (Fig. 5). Sugar uptake is shown in Fig. 7, uptake of glucose and fructose was slower than observed in the laboratory scale fermentations (Fig. 6). The control fermentation attenuated fully and produced 13.3% (v/v) ethanol (Table II), similar to the 13.2% (v/v) obtained in the laboratory scale work. Volatile levels in the low wines were lower than in the spirits from the laboratory scale fermentations (Table III) due to the different distillation methods used, however, the relative proportions were similar. Differences may also have been due to differences in oxygenation during fermentation and the presence of copper in the larger stills which would remove some volatiles. The spirit again had a clean aroma. The absence of temperature control in the larger vessels did not cause any problems with overheating, thus the expense of installing a temperature control system in a bioethanol plant can be avoided (at least in areas with a Northern European climate). This work indicated that scaling up is not a problem.

A fermentation diluted with 30% backset was set up as detailed in Table I. The increase in FAN due to the use of spent wash can be seen. Fermentation data are shown in Figs. 15, gravity fell faster than the control initially, although the final gravities were similar (Fig. 1), pH did not fall as much as the control (Fig. 2), cell numbers were similar (Fig. 3) but viability was much lower (Fig. 4) and uptake of FAN followed a similar pattern (Fig. 5). Sugar uptake is shown in Figure 8, uptake in the period up to 48 h is faster than in the control (Fig. 7), but full attenuation does not occur until 72 h. This fermentation produced 13.5% (v/v) ethanol, volatile levels in the low wines were similar to the control. This work indicated that backsetting causes no problems at this larger scale. Pitching rates One of the concerns regarding the fermentations was the high pitching rate. The rate of 9.0 107 cells /mL was used to ensure full attenuation. Early work was carried out at a rate of 3.0 107 cells /mL but this was increased following poor results (data not shown). The use of large amounts of yeast would be a cost issue in an industrial plant, so it was hoped that the higher levels of nutrients present in backsetted fermentations would permit the pitching rate to be reduced. To test this, laboratory scale fermentations were set up using media from the pilot scale fermentations pitched with differing amounts of yeast. The first series of fermentations was from the control fermentation, these were pitched at rates of 9, 8, 7, 6, 5, 4 and 3 107 cells /mL. The specific gravities of these fermentations are shown in Fig. 9 and data on the fermented molasses are shown in Table IV. It can be seen that the amount of yeast could be reduced from 9 107 cells /mL, but not by too much. The rate of 6 107 cells /mL is the lowest that could be
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Fig. 10. Specific gravity during fermentation of syrup with 30% backset inoculated at a range of pitching rates. Pitching rates: = 9 107 cells /mL; = 8 107 cells /mL; = 7 107 cells /mL. = 6 107 cells /mL; = 5 107 cells /mL; = 4 107 cells /mL; = 3 107 cells /mL; = 2 107 cells /mL.

achieved without wasting fermentable extract. Regarding Fig. 9, it can be seen that the 9 107 cells /mL fermentation reached attenuation by 48 h. Fermentation speed must be taken into account when calculating the cost of yeast, a fast fermentation may be worth paying for. A similar experiment was carried out using media from the 30% backset fermentation with an additional flask pitched at 2 107 cells /mL. The specific gravities of these fermentations are shown in Fig. 10 and the fermentation data are shown in Table V. These results are most encouraging, even the low pitching rate of 2 107 cells /mL was a success. This suggests that the amount of yeast could be substantially reduced. In addition, the fermentations pitched with 96 107 cells/mL reached attenuation by 48 h. Cane molasses are known to be deficient in nitrogen and this is also the case with the beet molasses used here 8. Thus, the potential for reducing the pitching rate in backsetted fermentations definitely exists. Cell numbers One of the features of all the backsetted fermentations carried out in this study was the effect on cell number and viability. In the laboratory scale work (Figs. 3 and 4) the cell number increased while viability decreased compared
TABLE IV. Influence of yeast pitching rate on fermentation (no backset added). Pitching rate 9 107 cells /mL 8 107 cells /mL 7 107 cells /mL 6 107 cells /mL 5 107 cells /mL 4 107 cells /mL 3 107 cells /mL Final gravity 988.9 988.6 988.2 988.7 988.5 993.1 998.6 Residual gravity 1006.1 1006.1 1006.2 1006.5 1006.4 1010.1 1014.9 pH 3.83 3.83 3.82 3.67 3.68 3.33 3.17 Ethanol (% v/v) 12.4 12.4 12.2 12.3 11.9 11.2 10.8

to the control fermentations. At the time, it was considered that the cell growth was caused by higher levels of nutrients being available and the lower viability was caused by the presence of toxins. This opinion changed when the pilot scale work was carried out. This was because the spent wash from the control fermentations was examined microscopically (out of general curiosity) and was found to contain large numbers of whole yeast cells. These cells were dead of course, but it was still a surprise to find them as it had been assumed that they would have been destroyed during the lengthy and vigorous distillation process. The fermentation vessel containing the 30% backset fermentation was sampled before pitching and the number of cells counted. This was found to be 15.35 107 cells / mL. To see how these extra dead cells interfered with the estimation of viability, the initial count was subtracted from the total and dead cell counts taken at 24 h. This reduced the total cell count from 29.10 107 to 13.75 107 cells /mL and reduced the dead cell count from 15.50 107 to 0.15 107 cells /mL This allowed the viability results to be recalculated, in this way the viability at 24 h increased from 46.8% to 98.9%.
TABLE V. Influence of yeast pitching rate on fermentation (30% backset added). Pitching rate 9 107 cells /mL 8 107 cells /mL 7 107 cells /mL 6 107 cells /mL 5 107 cells /mL 4 107 cells /mL 3 107 cells /mL 2 107 cells /mL Final gravity 991.1 991.1 990.1 990.8 990.7 990.7 990.8 991.3 Residual gravity 1008.5 1008.6 1008.4 1008.4 1008.4 1008.3 1008.6 1009.0 pH 4.30 4.30 4.30 4.28 4.28 4.26 4.26 4.28 Ethanol (% v/v) 13.3 13.5 13.4 13.6 13.6 13.8 13.6 13.6

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Fig. 11. Total cell number during pilot scale (2 hL) fermentations showing recalculation of the 30% backset cell count to remove interference of cells from the backset. = Control fermentation. = Fermentation with 30% backset. = Fermentation with 30% backset minus the dead cells from the backset.

Fig. 12. Yeast viability during pilot scale (2 hL) fermentations showing recalculation of the 30% backset viability to remove interference of cells from the backset. = Control fermentation. = Fermentation with 30% backset. = Fermentation with 30% backset minus the dead cells from the backset.

This is only an estimation as there is no way of telling with the method used which dead cells in the 24 h samples came from the backset and which came from the pitching yeast. However, as some yeast cells remain intact after distillation it could be that they will remain throughout a subsequent fermentation, although the action of proteinases secreted by live yeast cells could destroy dead yeast cells. To illustrate the effect of removing the extra cells from the cell counts, the cell number and viability graphs from

the 30% backset fermentation have been redrawn in Figs. 11 and 12. It can be seen that there was hardly any cell growth during the fermentations, while viability was higher than that seen in the control up to 48 h. This is only an estimation, but it helps explain why the backsetted fermentations performed so well, by showing that the low viabilities observed are a distraction. Thus the information on the backsetted fermentations shown in Figs. 3 and 4 is not giving an accurate illustration of the actual situation.
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Fig. 13. Healthy cells with no stain visualised in A, visualised for trehalose in B and for DNA in C. Boiled yeast (after distillation) with no stain visualized in D, visualised for trehalose in E and for DNA in F.

So how many of the yeast present in the wash at the end of fermentation persist into the spent wash used for backsetting? The cell count in the unpitched fermentation vessel was 15.35 107 cells /mL, the spent wash was used at 30% so this means that the original spent wash contained 51.17 107 cells /mL, this was used to dilute the molasses by 1/4 so this increases the count to 68.22 107 cells /mL. The distillation process caused approximately a two times concentration of the wash, so the cell count in the wash would be approximately 34.11 107 cells /mL. The cell count at 72 h in the control fermentation (just before distillation) was 23.15 107 cells /mL although some yeast would have sedimented to the bottom of the vessel. These figures are only estimates, but it would appear that the vast majority of the yeast cells present at the end of fermentation pass through distillation intact and only a few are destroyed. The increase in FAN and sugars observed probably originates from both the destroyed cells and leakage from other cells that although damaged, appear intact under the microscope. To confirm this, samples of healthy and boiled cells were examined under a confocal microscope. This method allows a more detailed examination of yeast than normal light microscopy. Cells can be stained with a range of fluorescent dyes specific to cellular components and these can be visualised individually by varying the wavelength of the light beam used. This avoids the staining of multiple samples. The cells were stained with dyes specific to trehalose (a component of the cell wall) and for DNA and photographs are shown in Fig. 13. Healthy and boiled cells are shown in a) and d) with neither stain activated. The same cells visualised for trehalose are shown in b) and e),
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it can be seen that the photographs are similar indicating that the distillation process had little effect on the cells walls. In contrast, the cells visualised for DNA shown in c) and f) show a distinct difference. DNA can be seen in all of the cells in the healthy sample, but in only one of the cells in the boiled sample. DNA was chosen for staining as this would show if the cell nucleus was still present, if the nucleus was no longer present it is a reasonable assumption that the cytoplasm and the other cell organelles will have been lost too. The results indicate that most of the yeast cells lose their intracellular contents during distillation and persist into spent wash as empty shells.

CONCLUSIONS
This work has confirmed that the production of bioethanol from sugar beet syrup is technically possible in a Northern European setting. With the use of a suitable yeast strain at a high pitching rate, fermentation is rapid and can be carried out in a fermentation vessel without temperature control. The use of spent wash in place of dilution water is possible. This gives the benefits of reduced water usage, reduced waste water purification costs, easier mixing with syrup if used warm, lower use of acids for pH buffering, and increased levels of nutrients. The last benefit is of particular interest as this should permit the pitching rate of backsetted fermentations to be reduced. Care must be taken with three characteristics of backsetted fermentations. Levels of glycerol will build up with each cycle of backsetting, this will affect the original

gravity of subsequent fermentations, so gravities must be proportionally increased to compensate for this. Secondly, if the haemocytometer method is used to monitor yeast growth and viability in backsetted fermentations, this will be interfered with by dead yeast shells from the spent wash resulting in falsely high cell counts and low viability. Ideally, an alternative method of yeast monitoring should be employed such as one using electrical capacitance. Lastly, backsetting must be used with caution to avoid build-up of undesirable metabolites. Provided reasonable levels are used, such as the 30% rate used in this work, accumulation of toxins can be avoided. Provided these conditions are followed, the production of bioethanol from sugar beet syrup is technically feasible in the United Kingdom, combining straightforward fermentation and distillation processes, using equipment that is currently available.
ACKNOWLEDGEMENTS

REFERENCES 1. Chin, P.M. and Ingledew, W.M., Effect of recycled laboratory backset on fermentation of wheat molasses. J. Agric. Food Chem., 1993, 41, 11581163. 2. DAmore, T., Russell, I. and Stewart, G.G., The effect of carbohydrate adjuncts on brewers wort fermentation by Saccharomyces uvarum (carlsbergensis). J. Inst. Brew., 1989, 95, 333336. 3. Fulton, L., Driving ahead biofuels for transport around the world. Renewable Energy World, 2004, 7, 180189. 4. Hutter, K.-J., Die DNS- und Proteinsynthese von Saccharomyces cerevisiae in unterschiedlichen Wachstumsphasen. Brauwissenschaft, 1987, 31, 7174. 5. Hutter, K.-J. and Eipel, H.E., DNA determination of yeast by flow cytometry. FEMS Microbiology Letters, 1978, 3, 3538. 6. Hutter, K.-J., Kliemt, C., Nitzsche, F. and Wiessler, M., Biomonitoring der Betriebshefen in Praxi mit Fluoreszenzoptischen verfahren. IX. Mitteilung: Trehalose Stressprotektant der Saccharomyces Hefen. Brauwissenschaft, 2003, 56, 121125. 7. IOB Methods of Analysis, Vol. 1 Analytical, Institute of Brewing: London, 1997. 8. Murtagh, J.E., Molasses as a feedstock for alcohol production. In: The Alcohol Textbook, 3rd ed., K.A. Jacques, T.P. Lyons and D.R. Kelsall, Eds., Nottingham University Press: Nottingham, 1999, pp. 8996.

The authors would like to thank British Sugar plc for permission to publish this work. Thanks are also extended to Graham McKernan for assistance in the pilot brewery and Jim MacKinlay for assistance with the GC and HPLC analysis.

(Manuscript accepted for publication June 2006)

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