The Fermentation of Beet Sugar Syrup to Produce Bioethanol

Kenneth A. Leiper1, Cornelia Schlee1, Ian Tebble2 and Graham G. Stewart1,3

ABSTRACT

J. Inst. Brew. 112(2), 122–133, 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 sub-strates 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 con-ditions. 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 in-vestigated as a way of reducing water usage and effluent dis-posal. 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, fermen-tation, yeast.

INTRODUCTION Bioethanol can be defined as ethanol produced by fer-

menting sugars extracted from agricultural crops, or by-products using micro-organisms (normally yeast) to pro-duce ethanol, which is then recovered by distillation. The most common use for bioethanol is as a motor fuel substi-tute or supplement. Production of bioethanol accounts for the vast majority of ethanol manufacture. Worldwide pro-duction in 2003 was approximately 30,000 million litres3, 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 co-product from sugar refining (molasses is the liquid residue remaining after the extraction of sugar from cane or beet)

or from sugar cane grown especially for ethanol produc-tion. 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 sucrose8. 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 fa-vourable, 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 fer-mentation 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 back-setting 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 sav-ing 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, how-ever, must be used with care, it is only a partial solution to water usage and cannot be used indefinitely as the con-stituents of the liquid will become progressively concen-trated 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 & Ingledew1. None of the workers found back-setting to have an effect on ethanol production despite the increasing concentration of a range of metabolites. De-spite this, the process is still regarded with suspicion in many quarters.

1 International Centre for Brewing and Distilling, Heriot-Watt Uni-versity, 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

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In this work, backsetting was found to cause no reduc-tion in ethanol production, provided the level of glycerol was observed and taken into account. Initial work investi-gated 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 back-setted 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 dis-tilling strain Safdistil B-28, a gift from Fermentis, Lille, France. Before use, the yeast was slurried in warm water (~30°C) 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 micro-scope at ×400 magnification. Viability was determined us-ing methylene blue staining according to the IOB method 9.1.3.2 7. These last two measurements were used to calcu-late 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 ap-proximately 1/4 with warm water (~40°C) to give original gravities of 1088 (22°P) or 1096 (24°P), pH was buffered from approximately 9 to 5.6 by the addition of hydro-chloric 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 calculat-ing digital gravity meter (Anton Paar). For the fermenta-tions 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 32°C 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 con-tent 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 fermenta-tions 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 ICBD’s 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 30°C by being passed through the brewery’s wort cooling system and was then pumped into a fermentation vessel. Following pitching, the fermenta-tions 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 deter-mination was as previously described.

Distillation

The ethanol produced during fermentation was recov-ered 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 con-taining approximately 40% (v/v) ethanol. The spirit frac-tion, or “Low Wines” (the term used in the Scotch whisky industry to describe spirit from an initial pot still distilla-tion) was analysed for ethanol content by gravity and vola-tile content by GC. The spent wash fraction was retained for use in backsetting.

The pilot scale fermentations were distilled in the ICBD’s 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.

Original FAN Sugars (g/L)

Sample gravity pH (mg/L) Glucose Fructose Sucrose

Laboratory scale – control 1087.1 5.66 207.4 0.08 — 240.76 Laboratory scale – OG 1088 with 100% backset 1094.5 5.63 281.4 0.69 4.73 260.33 Laboratory scale – OG 1088 with 50% backset 1092.2 5.61 229.1 0.55 1.52 230.28 Laboratory scale – OG 1096 with 100% backset 1102.9 5.65 314.5 1.03 4.45 255.55 Laboratory scale – OG 1096 with 50% backset 1100.4 5.65 245.5 0.41 2.28 277.86 Pilot scale – control 1086.8 5.60 205.2 0.33 0.15 256.54 Pilot scale – OG 1088 with 30% backset 1089.6 5.61 249.6 0.30 0.07 248.34

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Sugar levels

Levels of glucose, fructose, sucrose, maltose and mal-totriose 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

Dionex PAD electrochemical detector (all Dionex Corpo-ration, 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 glyc-erol. This appeared as an unknown peak on the chromato-graphs and was identified by running potential chemicals

Fig. 1. Specific gravity during laboratory (2 L) and pilot scale (2 hL) fermentations. � =Laboratory scale – control. � = Laboratory scale – OG 1088 with 100% backset. = Labo-ratory scale – OG 1088 with 50% backset. � = Laboratory scale – OG 1096 with 100% back-set. = Laboratory scale – OG 1096 with 50% backset. � = Pilot scale – control. � = Pilotscale – OG 1088 with 30% backset.

Fig. 2. pH levels during laboratory (2 L) and pilot scale (2 hL) fermentations. � = Laboratoryscale – 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.

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on the system until a match was found, quantitative analy-sis 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 al-cohols in the low wines were measured using Headspace GC. The system consisted of a Hewlett Packard 5890 Se-ries II GC with a split-splitless injector (Hewlett Packard

Fig. 4. Yeast viability during laboratory (2 L) and pilot scale (2 hL) fermentations. � = Labo-ratory scale – control. � = Laboratory scale – OG 1088 with 100% backset. = Labora-tory scale – OG 1088 with 50% backset. � = Laboratory scale – OG 1096 with 100% back-set. = Laboratory scale – OG 1096 with 50% backset. � = Pilot scale – control. � = Pilotscale – OG 1088 with 30% backset.

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. = Labo-ratory 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.

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Ltd, Stockport, England) with a FID, a Chrompack CP-Wax-57-CB column (0.23 mm × 60 m) (Chrompack Inter-national 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 us-ing a confocal microscope to investigate the effects of distillation. To gauge the effect on cell walls, cells were

stained for trehalose with concanavalin A fluorescein-conjugate (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 fol-lowing RNAse digestion according to the methods of Hut-ter4, and Hutter and Eipel5.

The samples were examined using a DM-IRE2 Con-focal Fluorescence Microscope with a Laser Scanning Confocal Microscope Attachment (Leica Microsystems, Wetzlar, Germany). Both stains underwent excitation at a

Fig. 5. Free amino nitrogen during laboratory (2 L) and pilot scale (2 hL) fermentations. � = Labora-tory scale – control. � = Laboratory scale – OG 1088 with 100% backset. = Laboratory scale –OG 1088 with 50% backset. � = Laboratory scale – OG 1096 with 100% backset. = Laboratoryscale – 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.

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wavelength of 488 nm, Con-A-fluorescein emitted detect-able light at 500–542 nm and propidium iodide at 618–710 nm. The data were digitized to produce a photo-graphic 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. 1–5. 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) indi-cating 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 fructose2.

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 de-tected. 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 in-creased 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.

Final Residual FAN Ethanol Residual sugars (g/L)

Sample gravity gravity pH (mg/L) (% v/v) Glucose Fructose Sucrose

Laboratory scale – control 990.0 1007.3 3.67 46.0 13.2 0.42 5.63 — Laboratory scale – OG 1088 with 100% backset 994.1 1011.6 4.75 75.8 13.9 0.91 0.04 0.04 Laboratory scale – OG 1088 with 50% backset 991.1 1008.5 4.14 36.3 13.4 0.76 0.35 — Laboratory scale – OG 1096 with 100% backset 993.7 1012.9 4.89 106.0 14.9 1.23 2.27 — Laboratory scale – OG 1096 with 50% backset 993.2 1012.3 4.39 57.3 14.8 0.88 11.70 — Spent wash from laboratory scale – control — 1010.1 3.61 83.7 — 0.62 6.01 — Spent wash from laboratory scale – OG 1088 with

100% backset

—

1016.7

4.53

155.7

—

1.81

0.04

0.05 Spent wash from laboratory scale – OG 1088 with

50% backset

—

1012.3

3.94

104.3

—

1.19

0.21

— Spent wash from laboratory scale – OG 1096 with

100% backset

—

1017.9

4.59

145.5

—

1.59

1.93

0.08 Spent wash from laboratory scale – OG 1096 with

50% backset

—

1016.6

4.13

116.6

—

1.16

12.46

0.05 Pilot scale – control 988.0 1005.2 3.74 55.8 13.3 0.68 — — Pilot scale – OG 1088 with 30% backset 992.6 1009.3 4.04 113.6 13.5 0.92 3.52 0.05 Spent wash from pilot scale – control — 1012.7 3.80 129.5 — 1.66 — — Spent wash from pilot scale – OG 1088 with

30% backset

—

1021.1

3.87

207.1

—

1.64

7.11

0.11

TABLE III. Volatiles in low wines produced from laboratory and pilot scale fermentations (mg/L).

Laboratory scale Pilot scale

Compound

Control

OG 1088 with 100%

backset

OG 1088 with 50% backset

OG 1096 with 100%

backset

OG 1096 with 50% backset

Control

OG 1088 with 30% backset

Acetaldehyde 184.43 304.89 250.84 307.12 318.01 214.3 198.6 Acetone — 2.81 3.16 4.52 — 5.9 8.5 Ethyl acetate 86.34 52.04 46.56 53.79 65.29 30.2 17.1 Isobutyl acetate — — — — — — — Ethyl butyrate 12.38 6.72 3.33 9.14 5.79 1.3 1.2 Isoamyl acetate 0.82 1.08 1.02 1.14 0.89 0.5 0.2 Ethyl hexanoate — 0.72 0.51 0.94 1.18 0.2 — Ethyl octanoate — — — — — — — Propan-1-ol 61.04 66.54 56.85 91.65 67.48 47.0 42.1 Isobutanol 254.34 317.67 335.73 355.84 340.86 157.8 234.5 2-Methyl butanol 359.10 372.32 393.14 422.69 404.52 176.9 238.3 3-Methyl butanol 426.39 542.37 596.07 497.96 532.18 353.7 277.8 Ethanol (% v/v) 41.1 42.4 43.0 50.8 47.0 24.2 27.6

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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 in-creased 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 1–5. 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

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 pro-duction provided the original gravities are increased to compensate for glycerol. The higher residual gravities and the gravity of the spent wash samples indicate the ac-cumulation of glycerol. Levels of volatiles in the low wines samples were generally higher than observed in the

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

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

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control with the exception of acetaldehyde and ethyl ace-tate (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 in-creased to 200 L scale. The control fermentation was set up as shown in Table I, fermentation data are shown in Figs. 1–5. Compared to the laboratory scale control fer-mentation, 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, up-take 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 vola-tiles. 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 cli-mate). 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. 1–5, gravity fell faster than the control initially, al-though 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 attenua-tion 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 fol-lowing 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 fer-mentations 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 re-duced from 9 × 107 cells /mL, but not by too much. The rate of 6 × 107 cells /mL is the lowest that could be

Fig. 9. Specific gravity during fermentation of syrup inoculated at a range of pitching rates. Pitchingrates: � = 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.

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achieved without wasting fermentable extract. Regarding Fig. 9, it can be seen that the 9 × 107 cells /mL fermenta-tion 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 encour-aging, 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 9–6 × 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 here8. Thus, the potential for reducing the pitching rate in back-setted 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

to the control fermentations. At the time, it was consid-ered 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 be-cause 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 distilla-tion 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 re-sults to be recalculated, in this way the viability at 24 h “increased” from 46.8% to 98.9%.

TABLE IV. Influence of yeast pitching rate on fermentation (no backset added).

Pitching rate

Final gravity

Residual gravity

pH

Ethanol (% v/v)

9 × 107 cells /mL 988.9 1006.1 3.83 12.4 8 × 107 cells /mL 988.6 1006.1 3.83 12.4 7 × 107 cells /mL 988.2 1006.2 3.82 12.2 6 × 107 cells /mL 988.7 1006.5 3.67 12.3 5 × 107 cells /mL 988.5 1006.4 3.68 11.9 4 × 107 cells /mL 993.1 1010.1 3.33 11.2 3 × 107 cells /mL 998.6 1014.9 3.17 10.8

Fig. 10. Specific gravity during fermentation of syrup with 30% backset inoculated at a range ofpitching 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.

TABLE V. Influence of yeast pitching rate on fermentation (30% back-set added).

Pitching rate

Final gravity

Residual gravity

pH

Ethanol (% v/v)

9 × 107 cells /mL 991.1 1008.5 4.30 13.3 8 × 107 cells /mL 991.1 1008.6 4.30 13.5 7 × 107 cells /mL 990.1 1008.4 4.30 13.4 6 × 107 cells /mL 990.8 1008.4 4.28 13.6 5 × 107 cells /mL 990.7 1008.4 4.28 13.6 4 × 107 cells /mL 990.7 1008.3 4.26 13.8 3 × 107 cells /mL 990.8 1008.6 4.26 13.6 2 × 107 cells /mL 991.3 1009.0 4.28 13.6

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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 dis-tillation it could be that they will remain throughout a sub-sequent 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.

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.

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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 con-tained 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 be-fore 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 ob-served probably originates from both the destroyed cells and leakage from other cells that although damaged, ap-pear 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 mul-tiple 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),

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 stain-ing as this would show if the cell nucleus was still present, if the nucleus was no longer present it is a reasonable as-sumption 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 dis-tillation and persist into spent wash as empty shells.

CONCLUSIONS This work has confirmed that the production of bio-

ethanol 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 pos-sible. This gives the benefits of reduced water usage, re-duced 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 back-setted fermentations. Levels of glycerol will build up with each cycle of backsetting, this will affect the original

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.

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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 viabil-ity. Ideally, an alternative method of yeast monitoring should be employed such as one using electrical capaci-tance. Lastly, backsetting must be used with caution to avoid build-up of undesirable metabolites. Provided rea-sonable levels are used, such as the 30% rate used in this work, accumulation of toxins can be avoided.

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, 1158–1163.

2. D’Amore, T., Russell, I. and Stewart, G.G., The effect of carbo-hydrate adjuncts on brewer’s wort fermentation by Saccharo-myces uvarum (carlsbergensis). J. Inst. Brew., 1989, 95, 333–336.

3. Fulton, L., Driving ahead – biofuels for transport around the world. Renewable Energy World, 2004, 7, 180–189.

4. Hutter, K.-J., Die DNS- und Proteinsynthese von Saccharomyces cerevisiae in unterschiedlichen Wachstumsphasen. Brauwissen-schaft, 1987, 31, 71–74.

5. Hutter, K.-J. and Eipel, H.E., DNA determination of yeast by flow cytometry. FEMS Microbiology Letters, 1978, 3, 35–38. Provided these conditions are followed, the production

of bioethanol from sugar beet syrup is technically feasible in the United Kingdom, combining straightforward fer-mentation and distillation processes, using equipment that is currently available.

6. Hutter, K.-J., Kliemt, C., Nitzsche, F. and Wiessler, M., Biomoni-toring der Betriebshefen in Praxi mit Fluoreszenzoptischen ver-fahren. IX. Mitteilung: Trehalose – Stressprotektant der Sac-charomyces – Hefen. Brauwissenschaft, 2003, 56, 121–125.

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. 89–96.

ACKNOWLEDGEMENTS

The authors would like to thank British Sugar plc for permis-sion to publish this work. Thanks are also extended to Graham McKernan for assistance in the pilot brewery and Jim Mac-Kinlay for assistance with the GC and HPLC analysis. (Manuscript accepted for publication June 2006)

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