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Beet Sugar Bioethanol Fermentation

Beet Sugar Bioethanol Fermentation

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The Fermentation of Beet Sugar Syrupto Produce Bioethanol
Kenneth A. Leiper
, Cornelia Schlee
, Ian Tebble
and Graham G. Stewart
J. Inst. Brew. 112(2), 122–133, 2006
Fermentation of sugar or starch-containing substrates by yeast toproduce ethanol for use as a liquid fuel has been an acceptedtechnology 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 substratesare now being considered, choice of these depends on local con-ditions. This paper presents findings from work carried out onsyrup from sugar beet, an ideal crop for cultivation in the UnitedKingdom and parts of Europe. Fermentation of this substratewas 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 productionprovided compensation was made for increases in gravity causedby glycerol levels. Backsetting was also found to be beneficial toyeast growth. As yeast remain in the fermented substrate, theeffect of distillation on yeast cells was also investigated. It wasfound that dead yeast cells are present in backset and thus persistinto subsequent fermentations. This can cause difficulties inviability measurement if the methylene blue method is used.
Key words:
Backsetting, beet sugar syrup, cell walls, fermen-tation, yeast.
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. Themost common use for bioethanol is as a motor fuel substi-tute or supplement. Production of bioethanol accounts forthe vast majority of ethanol manufacture. Worldwide pro-duction in 2003 was approximately 30,000 million litres
,dwarfing the output of potable ethanol – approximately4,000 million litres.The bulk of bioethanol production is in Brazil and theUSA. 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 residueremaining 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 othercountries depends on the local agricultural environmentand economics.If substrates containing starch are used, they must firstbe subjected to heat and enzyme treatments to convert thestarch to fermentable sugars, thus adding to productioncosts. Molasses and other beet extracts do not requiresuch treatment as the sugar content is almost all in theform of sucrose
. This is readily split into glucose andfructose in the initial stage of fermentation by the enzymeinvertase, located in the periplasmic space between theyeast cell wall and cell membrane. The only preparationrequired with molasses is dilution to a suitable originalgravity and pH buffering.This paper presents data from fermentations carried outusing syrup extracted from sugar beet (this came from anearly stage in the sugar manufacturing process and had nosugar removed from it). If economic conditions were fa-vourable, the production of bioethanol could become aviable proposition in the United Kingdom. If this situationdevelops, sugar beet would be a suitable substrate. Initialproduction would use syrup currently available fromsugar refining, with the possibility of dedicated crops inthe future.Initial work at the laboratory scale indicated that fer-mentation of beet sugar syrups to produce ethanol waseasy to conduct. As the reduction of production costswould be vital for bioethanol production, the use of back-setting was investigated. This process makes use of spentmedia, also known as spent wash, stillage or vinasse, fromdistillation to dilute subsequent batches of syrup thus sav-ing water and waste water treatment charges.This process has been used successfully in whisk(e)yand grain neutral spirit production in Scotland and NorthAmerica for many years. More recently, it has found arole in the production of bioethanol. The process, how-ever, must be used with care, it is only a partial solution towater usage and cannot be used indefinitely as the con-stituents of the liquid will become progressively concen-trated causing problems associated with viscosity andbuild-up of toxins. The material must be discarded atsome point.Backsetting has been investigated by many workers atvarying levels of spent wash usage, and has been reviewedby Chin & Ingledew
. None of the workers found back-setting to have an effect on ethanol production despite theincreasing concentration of a range of metabolites. De-spite this, the process is still regarded with suspicion inmany quarters.
International Centre for Brewing and Distilling, Heriot-Watt Uni-versity, Riccarton, Edinburgh, EH14 4AS, Scotland.
British Sugar plc, Oundle Road, Peterborough, Cambridgeshire,PE2 9QU, England.
Corresponding author. E-mail:G.G.Stewart@hw.ac.uk 
Publication no. G-2006-0629-427© 2006 The Institute of Brewing & Distilling
In this work, backsetting was found to cause no reduc-tion in ethanol production, provided the level of glycerolwas observed and taken into account. Initial work investi-gated backsetting at 50% and 100%, but later pilot scalework used backset at 30%, as this level was considered tobe realistic on an industrial scale. Both normal and back-setted fermentations were successfully scaled up to pilotscale volumes. It had been assumed that yeast cells wouldbe completely destroyed during distillation, but this wasfound not to be the case.
Beet sugar syrup was supplied in liquid form by BritishSugar.
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 occasionalstirring. Cell number in the slurries was determined usingan Improved Neubauer Haemocytometer and a micro-scope at ×400 magnification. Viability was determined us-ing methylene blue staining according to the IOB method9.1.3.2
. These last two measurements were used to calcu-late the volumes of yeast slurry required to be added tothe fermentations. Other yeast strains were screened forpossible use, but all were found to be less effective thanthe above strain (data not shown).
Laboratory scale fermentations were carried out with1.5 L syrup in 2 L bottles. The syrup was diluted by ap-proximately 1/4 with warm water (~40°C) to give originalgravities of 1088 (22°P) or 1096 (24°P), pH was bufferedfrom approximately 9 to 5.6 by the addition of hydro-chloric acid and the use of a pH 210 microprocessor pHmeter (Hanna Instruments), gravity was adjusted by theaddition 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 wasreplaced with spent wash. Following the addition of yeast,normally at a pitching rate of 9 × 10
cells/mL, the bottleswere incubated in a shaking incubator at 150 rpm at 32°Cfor up to 70 h. During fermentation, gravity, pH, cellnumber and viability were measured using the methodspreviously described. Samples were retained for FAN andsugar analyses. Following fermentation, final ethanol con-tent was determined by distillation according to the IOBmethod 8.5.1
. The residues from the distillations wereretained and analysed for FAN and sugars. The fermenta-tions for determining optimal pitching rates were carriedout in 500 mL flasks containing 250 mL syrup. Thesewere fermented for up to 96 h.The pilot scale fermentations were conducted in theICBD’s 2 hL brewery. Syrup and water (or spent wash)were mixed in the mashing-in equipment followed byfurther mixing in the mash mixer vessel. Here pH andgravity were adjusted if required as described previously,with the pH meter probe placed in the vessel. No attemptwas made to control levels of oxygen, it was assumed thatsufficient oxygen would be picked up during mixing. Thesyrup was cooled to 30°C by being passed through thebrewery’s wort cooling system and was then pumped intoa fermentation vessel. Following pitching, the fermenta-tions were left for up to 70 h. The vessels were fitted witha cooling system, but this was not used, the temperaturewas allowed to “free rise”. Sampling and ethanol deter-mination was as previously described.
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 acontinuous still (which would have been more realistic)was not possible. The laboratory scale fermentations weredistilled in an apparatus consisting of a 500 mL flask, afoaming chamber, a lyne arm and a condenser (Kiko,Osaka, Japan). Wash (330 mL) was placed in the flask andheated with a Bunsen flame. The foaming chamber waslargely redundant as no foaming occurred. The distillationwas 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 whiskyindustry 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 retainedfor use in backsetting.The pilot scale fermentations were distilled in theICBD’s pilot distillery in 20 L batches in a glass pot still.The distillations were conducted until the spirit comingfrom the still contained less than 1% v/v ethanol. This is alonger distillation than that carried out on the laboratoryscale stills, so the low wines contained a lower level of ethanol. Low wines and spent wash samples were analysedas described previously.
Analysis of unfermented beet syrups used in laboratory (2 L) and pilot scale (2 hL) fermentations.
Original FANSugars (g/L)Sample gravity pH (mg/L) Glucose Fructose Sucrose
Laboratory scale control 1087.1 5.66 207.4 0.08 240.76Laboratory scale OG 1088 with 100% backset 1094.5 5.63 281.4 0.69 4.73 260.33Laboratory scale OG 1088 with 50% backset 1092.2 5.61 229.1 0.55 1.52 230.28Laboratory scale OG 1096 with 100% backset 1102.9 5.65 314.5 1.03 4.45 255.55Laboratory scale OG 1096 with 50% backset 1100.4 5.65 245.5 0.41 2.28 277.86Pilot scale control 1086.8 5.60 205.2 0.33 0.15 256.54Pilot scale OG 1088 with 30% backset 1089.6 5.61 249.6 0.30 0.07 248.34
 VOL. 112, NO. 2, 2006
Sugar levels
Levels of glucose, fructose, sucrose, maltose and mal-totriose were measured by HPLC. The system consistedof a Dionex Carbopak PA-100 guard column (4 mm × 50mm), a Dionex PA-100 column (4 mm × 250 mm) and aDionex PAD electrochemical detector (all Dionex Corpo-ration, Sunnyvale, California, USA). The eluent used was500 mM NaOH and cellobiose was used as the internalstandard. 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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