practical and theoretical considerations in the production of high concentrations of alcohol by...

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Process Biochemistry" Vol. 31, No. 4, pp. 321-331, 1996 Copyright © 1996 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0032-9592/96 S15.00 + 0.00

Review

Practical and Theoretical Considerations in the Production of High Concentrations of Alcohol by Fermentation

K. C. Thomas,* S. H. Hynes & W. M. Ingledew Department of Applied Microbiology and Food Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A8

(Received 20 June 1995; accepted 6 August 1995)

Theoretical considerations show that very high gravity fermentation technology promotes considerable savings of water and increased plant productivity and there]ore results in reduced fermentation and distillation costs per litre of ethanol. Formulae are derived to permit calculation of the amounts of grain and water required to prepare mashes with predetermined concentrations of dissolved solids and insoluble materials. Practical and theoretical methods are described to determine the quantity of the liquid portion of the mash before and after fermentation. Using these values, the efficiency of mashing and fermentation can be calculated. A theoretical method is also presented to predict the maximum concentration of ethanol in fermented mash. These calculations, unlike those used currently in industry, take into account changes in the weight and volume of mash during fermentation.

INTRODUCTION

Under appropriate environmental and nutritional conditions, Saccharomyces cerevisiae can produce and tolerate high concentrations of ethanol. The dogma that alcohol tolerance varies widely between brewery, distillery, winery and sake yeasts has been shattered (for reviews see Refs 1

*To whom correspondence should be addressed.

and 2). By considering the nutritional require- ments of yeasts, enologists can eliminate most stuck fermentations 3 and brewers can ferment high gravity (HG) and very high gravity (VHG) worts containing high concentrations of dissolved solids. 4 This technology increases plant productivity and results in increased profit margins for the brewer. Fermentation at VHG sugar levels in brewing was originally proposed by Casey and co- workers 5-7 and later defined as preparation and

321

322 K.C. Thomas et al.

fermentation of worts containing 18 or more grams dissolved solids per 100 g (18 ° Plato or more). 4 Traditional (normal gravity) brewing is done with worts having a dissolved solids concentration of l l -12°P while worts of 13-16°P are used for high gravity brewing.

Distillers and fuel alcohol manufacturers normally ferment grain mashes containing - 20- 24 g of dissolved solids per 100 g. Dissolved solids concentrations in this range have been considered 'normal gravity' in these industries. Although VHG fermentation technology has not yet been routinely used for the production of potable distilled alcohol, research from this laboratory has shown that it can be applied to the production of fuel alcohol from a number of grain mashes. 8-15 VHG fermentation technology for fuel alcohol production has been defined as 'the preparation and fermentation to completion of mashes containing 300 or more grams of dissolved solids per litre'. ~° Subsequent to this description and definition of VHG technology in the literature, a patent was issued for the production of alcohol by fermentation of 'high dry solids mash'. ~6 A number of potential advantages of VHG fermentation technology are listed in Table 1.

Several methods have been developed to increase the fermentable sugar content of grain mashes to the 300 or more grams of fermentable

Table 1. Advantages of V H G technology in fuel alcohol product ion

Increased plant capacity or reduct ion in capital costs - - increase in alcohol concent ra t ion to > 18% v/v - - increase in fermentor space due to removal of

insolubles Increased plant efficiency

- - reduct ion in labour costs/litre ethanol - - reduct ion in energy costs/litre ethanol

- - no cooling of insolubles in ferrnentor - - no heating of insolubles in the still - - less water to process in the still - - op t imum ethanol for efficient distillation - - lower solids in the still ( l iquid-l iquid extraction)

- - reduct ion in inputs/ l i tre e thanol - - decreased water usage - - reduct ion in fermentor downt ime - - reduct ion in cleaners and sanitizers

Other advantages - - reduced survival and proliferat ion of contaminat ing

bacteria which reduce ethanol yield - - opportuni t ies for food quality co-products or distillers

grains - - opportuni t ies for harvest of high protein spent yeast

sugar per litre required. These mashes can be fermented to completion provided that dissolved solids do not exceed 390 g per litre and sufficient amounts of nutrients are available for yeast growth.8,10,14,15,17 Such mashes on fermentation have predictably yielded 21-23% ethanol by volume, 9-11 a concentration only rarely reported in the literature. This has been achieved without specially prepared or genetically engineered yeasts and without incremental addition of mash to the fermentor, all fermentable substrates (or their oligosaccharide and dextrin precursors) being present at the start of batch fermentation, Fermentation conditions with respect to substrate concentration, nutrients for yeast growth, yeast inoculation levels, and fermentation temperature have been optimized. 9-11,14,15

VHG fermentation requires a mash with high sugar concentration and a mash consistency that is easy to handle and ferment. As the concentration of dissolved solids of a mash is raised, its viscosity increases and this results in a greater demand for energy during mashing and fermentation. Unless specially treated, most grain mashes with high carbohydrate contents are too viscous for normal handling. Successful application of VHG technology, therefore, depends to a great extent on the preparation of mashes with low viscosity.

In this report we begin by describing methods for the preparation of such mashes from different grains. Then we discuss theoretical considerations important for the production of VHG mashes, present formulae to calculate the amount of grain and water required, and describe practical methods for the estimation of the quantity of the liquid portion of mash before and after fermentation. We also present formulae to predict accurately the concentrations of ethanol in fermented mashes. These formulae, unlike currently used methods in industry, take into account the changes in weight and volume of the liquid portion of the mash.

M E T H O D S OF VHG M A S H PREPARATION

The methods described below for mashing of ground wheat, barley or oats (or a combination thereof) yield mashes which have high dissolved solids concentrations and low viscosity -- all suitable for VHG fermentation.

High alcohol concentration by fermentation 323

Adjustment of the grain to water ratio

Mashing ground grain or starch-enriched fractions obtained by dry milling The objective of mashing is to gelatinize starch and then hydrolyse it to soluble compounds (mainly dextrins) which, on saccharification, are converted to fermentable sugars. Both gelatinization and hydrolysis require water as a reactant, and non-availability of sufficient amounts of water may result in incomplete gelatinization and may even inhibit complete hydrolysis of the gelatinized starch, is Although the simplest method to raise the carbohydrate content of a grain mash is to adjust the water to grain ratio, decreasing this ratio beyond a critical value may result in incomplete conversion of starch to fermentable sugars. Gelatinization and hydrolysis of starch are not major concerns during the preparation of low or normal gravity mashes (160-200 g dissolved solids per litre) where these processes are not limited by availability of water. 8,1°,14,15 In thick mashes (decreased water to grain ratios), the extract yield is usually low but it can be increased with the addition of amylases, glucanases and proteases. ~4,1s,~9 This increased yield appears to result from the freeing of water which has been bound and trapped by mash components such as fi-glucan, pentosans and proteins. The type and amount of grain components that bind and retain water vary with the type of grain, z° While proteins have only minimal effects on extract yield, fi- glucan decreases the extract yield significantly in thick mashes. 2~ The effect of pentosans on extract yield has not been well studied although it is known to be the primary cause of viscosity in some grains. 22 It follows, therefore, that any process that increases water availability would not only facilitate mash preparation by reducing the viscosity but also would provide water that could be used for other reactions such as gelatinization, hydrolysis and sugar dissolution.

The development of viscosity during mashing can be minimized by treating the ground grain-water slurry with a suitable microbial enzyme prior to gelatinization of starch. The choice of enzyme is determined by the nature of the substrate which causes viscosity in the mash. Mashes prepared from oats or barley are very viscous even at a water to grain ratio of 3:1, the ratio normally used in industry to prepare mashes from corn or wheat. In these cases where much of

the viscosity is caused by fl-glucan, crude preparations of fl-glucanase or Biocellulase have been quite effective. 14,1s Proteases also decrease viscosity 23 (and also aid in yeast nutrition 7,1s,23) although not to the same extent as fl-glucanase or Biocellulase. These commercial enzymes undoub- tedly are a mixture of a number of enzymes; they appear to benefit the process.

A pre-mashing procedure for oats and barley involves warming the required amount of water to 45°C and adding powdered fl-glucanase or liquid Biocellulase to the water. Normally 0-2 g or 0.2 ml of the crude but standardized enzyme preparation is used per kilogram of grain. Both these enzyme preparations possess fl-glucanase activity as evidenced by a reduction in the viscosity of a 0.5% solution of purified fl-glucan from 1-034 centistokes (cs) to 0.339 cs within 5 nfin at a concentration of 0-02%. Immediately after the disper- sal of the enzyme, ground grain (a ]particle size range of 20-60 mesh is adequate ~4) is added to the water and the enzyme allowed to react for 30 min, a reaction time sufficient to reduce the viscosity of the prepared mash to a minimum value. Part of the viscosity reduction may have resulted (but more slowly) through the action of fi-glucan 'solubilase' normally present in grain. 24-27

The reduced viscosity of the glucan-hydrolysed mash makes it possible to decrease the water to grain ratio and thus increase the dissolved solids content of the prepared mash. For example, at a water to grain ratio of 3:1, the dissolved solids content of a mash prepared from hulless oats was 205 + 4 g per litre. On decreasing the water to grain ratio to 2:1, the dissolved solids content increased to 269 _+ 5 g per litre. The viscosities of these enzyme-treated mashes were ]low, usually less than 10 Brabender Units (BU) at: 30°C while the mash (water to grain ratio 3:1 ) prepared without hydrolysing fl-glucan had a viscosity of 1180 BU. is The viscosity was even higher (2460 BU) for a mash prepared from hulless barley without the aid of viscosity-reducing enzymes while it was less than 500 BU when fi-glucan was hydrolysed prior to mashing.

The same procedure can also be used to prepare VHG mashes from hulled oats. At a water to grain ratio of 2:1, the dissolved solids content of a hulled oat mash was 240 g per litre while that of hulless oats was 270 g per litre. Since at a given water to grain ratio, mashes prepared from hulled oats are less viscous and have a dissolved solids


content lower than those prepared from hulless oats, water to grain ratios can be reduced even further.

The method of raising the gravity by decreasing the water to grain ratio cannot be applied indiscri- minately to every type of grain. For example, it is difficult to prepare a wheat mash with a water to grain ratio less than 3:1. Much of the viscosity of wheat mash results from grain components such as pentosans, gluten and insoluble materials. The viscosity of wheat mashes can be reduced by adding proteases but this process is very slow and several hours pass before significant reduction in viscosity can be observed. An inverse relationship appears to exist between the thickness of the mash and proteolysis. 23 To reduce the viscosity to a practical level, large amounts of protease must be used or the reaction must be allowed to proceed for a long time before gelatinizing the starch. This will add to the cost, or extend the time required for mashing. In addition, free amino nitrogen liberated through proteolysis may react with sugars (Maillard reaction) during mashing, result- ing in loss of both fermentable sugar and yeast usable nitrogen (unpublished work).

Mashing starch slurries obtained through wet milling The wet milling procedure used for isolation of corn gluten meal, corn gluten feed, corn oil, refined starch and industrial starch (or ethanol by fermentation) is now well describedY The wet milling operation results in a relatively pure starch slurry with - 40% solids. Mashing of such slurries to fermentable sugars is relatively easy and the dissolved solids concentration can be adjusted to VHG levels by controlling the water to slurry ratio. Even at a dissolved solids concentration of 40°P the viscosity of the mash prepared from corn starch slurry is less than 10 BU. Therefore, there are unique and ideal opportunities to carry out VHG fermentation with this concentrated substrate. As starch slurries are deficient in nutrients required for yeast growth, an exogenous supply of such nutrients must be provided. This is especially important when high concentrations of sugars are to be fermented (as in the case of VHG fermentations). Customarily this industry uses light corn- steep-water and backset (thin stillage) in high volumes to provide nutrients (along with recycled process waters to enable reductions in pollution costs, water conservation and energy savings). These additions effectively reduce the sugar con-

centration in the mash and lead to ethanol concentrations considerably less than that might be obtained by VHG fermentation. Careful control of such additions will still allow alcohol concentrations of - 14% v/v to be reached, but production of higher concentrations of alcohol awaits economic and scientific analyses of this process along with the use of concentrated (by evaporation or other means) steepwater and/or stillage or consideration of alternate yeast foods. Dilution of starch slurry must be reduced to profit from the advantages of VHG fermentation. This is but one reason why dry milling is under consideration in new plants constructed by the corn milling industry. 7 In the theoretical calculations described below, VHG fermentation of starch slurry derived through wet milling is not considered since nutrient composition of each additive must be taken into account to calculate the quantity of each additive needed. This aspect is currently under investigation and the results will be reported later.

Double mashing A double mashing procedure for wheat has been developed to raise the fermentable sugar content of mashes to VHG levels. This method is intended primarily for small scale batch plants and laboratory studies where VHG mash from one type of grain is desired but where it is not possible to make it by simply adjusting the water to grain ratio. The double mashing procedure can also be applied to prepare VHG mashes from different grains. The procedure has been successfully applied by VHG Technologies Inc., Saskatoon, in a pilot plant scale study for the production of fuel alcohol from wheat (unpublished data). The underlying principle of double mashing is the preparation of a normal gravity mash, followed by removal of most of the insoluble materials and use of the extract thus obtained (basal mash or single extract) to prepare a second mash (double extract) from the same or a different grain.

To prepare a double wheat mash, one part of ground wheat is dispersed with stirring into 3.7 parts of prewarmed (60°C) water containing calcium chloride at a concentration of 1 mM. The slurry is mixed continuously and a small amount of high-temperature a-amylase added to keep the increase in viscosity to a minimum. We add 3-7 ml of an enzyme preparation [specific activity of 1.14 g starch (hydrolysed) per min per mg protein] for each kg of ground wheat. After 5 min, the


mash temperature is raised to 95-97°C and held for 45 min with continuous stirring. Starch is completely gelatinized and partially liquefied (converted to dextrins) by this heat treatment. A marked decrease in viscosity results through partial hydrolysis of the starch. To complete the liquefaction, an additional 3.7 ml of high temperature a-amylase per kg of wheat is added to the mash after lowering its temperature to 80°C. Liquefaction is complete within 30 min, as indicated by a negative test for starch by iodine. The mash is then filtered. In scale-up trials, the liquid portion of the mash would be extracted with a decanter centrifuge (ideally avoiding the following step by rinsing the grain as it rises up the centrifuge 'beach'). The grain residues are washed with hot (60°C) water (using a volume approximately equal to 40% of that used for making the slurry) and filtered. The mash liquid and the washings are combined (combined extract). The residue is washed again with an equal volume of water and the washings recovered. This last washing is set aside and used to make up the volume lost through evaporation during the preparation of a second mash.

The temperature of the combined extract is adjusted to 60°C and supplemented with calcium chloride at a concentration of 0.3 mM. One part of ground wheat is dispersed in 2.5 parts of the combined liquid and the mashing procedure repeated. High-temperature a-amylase is added in the same proportion (3.7 ml per kg wheat) and the starch gelatinized by raising the temperature to 95-97°C and holding for 45 min. The gelatinized starch is hydrolyzed with high temperature a-amylase as before. The dextrinized double mash thus prepared is then cooled, saccharified (with amyloglucosidase) and fermented with active dry yeast. All or part of the insoluble materials present in the mash may be removed prior to saccharification. The double mash prepared according to the procedure given above has a dissolved solids concentration of about 360 g per litre of the liquid portion of the mash.

Normal gravity oat mash prepared after hydrolysis of fl-glucan has very low viscosity ( < 10 BU) and can serve as an excellent basal mash for preparation of a double mash with other grains such as wheat or barley. 15 Such double mashes can have dissolved solids contents as high as 400-420 g per litre and still have a viscosity less than 500 BU. Since oat mash is very rich in yeast-assimil- able nitrogen (three times the amount present in

wheat mash of equivalent dissolved solids content), 15 nitrogen supplementation of double mashes thus prepared is unnecessary.

A number of advantages accrue from this procedure. Double mashing permits a rise in the fermentable sugar content of the mash allowing an increase in the throughput efficiency of each fermentor and the alcohol plant in general. Because insoluble materials (35-40% of the grain) are removed prior to fermentation, fermentor space is liberated and the amount of carbohydrate fermented per unit time in a plant of fLxed capacity (the throughput rate) increases. In addition, insoluble materials removed prior to fermentation have potential use for human food. The value of these protein/fibre co-products is higher than that of 'distillers grain' which is a residue consisting of insoluble grain components and the: yeast cells (and bacteria) produced during fermentation. Dis- tillers' grains can only be used as animal feed.

Adjuncts to raise sugar content of grain mashes An alternate method of raising dissolved solids concentration of grain mashes to VHG levels is to add adjuncts which contain high concentrations of fermentable sugars. Such adjuncts may be 'homo- logous' (derived from the same source as the basal mash) or 'heterologous' (derived from a different source or sources). When VHG mash from the same type or sometimes even from the same batch of grain is needed (as is sometimes required for laboratory studies), a water soluble adjunct such as freeze-dried grain hydrolysate can be used. Preparation of such an adjunct from wheat has been described previously. 8,1° Here a normal gravity wheat mash with a dissolved solids content not exceeding 200 g per litre is prepared and the insoluble materials from the mash removed first by coarse filtration and then by centrifugation. The supernatant thus obtained is freeze dried and used as the adjunct to raise the dissolved solids content of a normal gravity mash prepared from the same grain. This method may not be practical for industrial application but is very useful in laboratory studies when homogeneity of the grain mash must be maintained. If desired, the adjunct can be prepared from another grain (heterologous adjunct).

Molasses, an adjunct that is readily available in large quantities, has been used to raise the sugar contents of wheat] 2,29 barley and oat mashes. Because molasses also contains large quantities of non-carbohydrate dissolved solids, it can be used

326 K. C. Thomaset al.

only in limited quantities as an adjunct. Excessive amounts of molasses would increase the osmolar- ity of the V H G mash to values too high for yeast growth and would result in stuck fermentation. For best results, only high test molasses should be used and the dissolved solids contributed by molasses in a V H G mash should not be too high. For example, fermentation of a V H G mash containing 350 g dissolved solids per litre may not proceed to completion if more than 30% of the dissolved solids are derived from molasses. 12 Fermentable sugar contents of grain mashes may also be raised by adding adjuncts such as corn syrups, honey or granulated sugar. 29 This method may have a limited application in industry to salvage distressed high sugar products (for example, candy and corn syrups) and convert them into fuel alcohol. This is carried out in specific alcohol plants in the United States.

Where available, sugar cane juice can be used as a basal medium to prepare V H G mash with molasses, wheat ~2 or other grains. Sugar cane juice contains enough nutrients to support yeast growth and to allow fermentation to complete within 48 h at 30°C. However, extra nutrients need to be supplied if a V H G mash prepared from a grain with sugar cane juice as the basal medium is to be fermented to completion.

CALCULATION OF EFFICIENCY OF HIGH ALCOHOL FERMENTATION

The amount of water available in a mash for the dissolution of dextrins and oligosaccharides (products of starch hydrolysis) is the difference between the amount of water present at the start of mashing and the water used for hydrolysis of the starch. The total amount of water at the beginning of mashing is equal to the sum of water added and the moisture contributed by the grain. It can be calculated that 5 5 4 4 2 k g water is needed to prepare 100000 kg of V H G mash (31 g dissolved solids per 100 g in the liquid portion or 3 I°P) from the wheat described earlier. For the same quantity of wheat (44 558 kg), the amount of water required to prepare a normal gravity mash (water to grain ratio of 3 : 1 yielding a mash of 16°P) is 133674kg. The quantity of water saved by adopting VHG fermentation technology is therefore 78232 kg (58.5% of the amount used in a 16°P mash). Not only is this water saved but much less water is required to be separated in the still and a lesser volume of thin stillage (but with higher dissolved solids) will be produced by the plant.

A simple method is needed to calculate the amount of grain and water required to prepare mashes with predetermined concentrations of dissolved solids. To do this the grain must be analysed for its moisture and starch contents and a decision must be made regarding the fraction of insoluble materials that will be left in the mash. Equation (1) was derived (Table 2) to calculate the

We have indicated that adoption of V H G fermentation technology for alcohol production leads to a considerable saving of water. In the model calculations shown below a sample of wheat with the following composition per kg was used: moisture, 0-120 kg; starch, 0.528 kg (60% w/w, on a dry weight basis); and non-starch solids, 0.352 kg. The amount of glucose that can be derived from 0-528 kg of starch is 0-528 x 1.111 = 0"587 kg. (Where the factor 1"111 is derived by taking into account that one molecule of water is used for each glycosidic bond hydrolysed. The factor is numerically equal to the ratio 1 8 0 n / 1 6 2 n + 18, where n is the number of glucose residues in the polymer. When n is 2, as in maltose, the factor is 1-053 but when n is larger, the ratio approaches the value of 1.111.) There- fore, the quantity of water used for hydrolysis of starch in one kilogram of the above wheat is 0"587 - 0.528 = 0"059 kg.

Table 2. Derivation of an equation to calculate the weight of a grain required to make a given weight of mash with a specified dissolved solids concentration

Wt of mash = wt of grain + wt of water added (M = G + W ) Wt starch in grain = wt of grain x kg of starch per kg of grain

(=GS) Wt of glucose obtainable from grain = wt starch in

grain× 1.111 ( = 1.111 GS) Wt of insolubles in mash = wt of grain x kg of non starch

solids per kg grain = GR or G(1 - S- N) Wt of the liquid portion of mash = wt of mash- wt of

insolubles in mash (GR) ( = M - GR ) Concentration of glucose in liquid portion (C)

wt of glucose GSI-lll wt of liquid portion M - GR

MC °n rearranging' "G 1.111S+RC

"This equation is the same as eqn (1) but with F having a value of 1.


amount of grain required to make a given weight of mash containing a predetermined concentration of dissolved solids. The formula takes into account whether all or part of the insoluble grain particles are left in the mash. The amount of water to be added will vary with the moisture and starch contents of the grain.

M C

G - I . 1 l l S + R F C ' (1)

dissolved solids concentration of 31°P is chosen for VHG mash because the mash at this dissolved solids concentration is completely fermentable at 20°C although fermentation time is increased by 20 h. ~° Efficiency is calculated on the basis of throughput rate.

Equation (2) can be rearranged to give eqn (3) to predict the concentration of the dissolved solids (°P) in the liquid portion of a mash made with a chosen water to grain ratio.

where G=kg of grain required; M=weight of mash (fermentor capacity) in k g = G + W; W= kg of water to be added to prepare M kg of mash; C = desired dissolved solids (glucose) in kg per kg of liquid portion of mash; S= kg of starch per kg of moist grain; R = kg of insoluble materials per kg of the grain = ( 1 - S - N); N = kg of moisture per kg of grain; and F= fraction of insolubles left in the mash. Depending on the percentage of insolubles left in the mash, F can have a value from 0 to 1. As an example, removal of 50% of the insolubles from the mash prior to fermentation is equal to using a grain which has half the amount of insoluble materials. This is equal to F-- 0.5 and therefore insoluble material is 0-5 R.

If the dissolved solids concentration is expressed in terms of °P (g of dissolved solids per 100 g of the liquid portion of the mash) then eqn (1) can be modified as:

M°P/100 G - (2)

1.111 S+ RF°P/IO0 "

Table 3 shows the calculated weights of wheat required to prepare 'normal' and very high gravity mashes and to operate a fermentor at its maximum rated capacity -- in this case, 100000 kg. A

op=(1.111SG)100

M - RFG (3)

It can be shown that the liquid portion of a mash prepared with the wheat described earlier and with a water to grain ratio of 3 : 1 would have a dissolved solids concentration of 16°E This assumes that there is no loss of water through evaporation or through absorption by grain components. Generally there is some loss of water and this results in less water being available for the dissolution of sugars. The amount of water lost through evaporation will depend upon the equip- ment and process technology used. The net effect of this is that the concentration of the dissolved solids (°P) increases. Under laboratory batch cooking conditions, the dissolved sofids concentration of a mash prepared with a water to grain (grain with 60% starch on dry wt basis) ratio of 3:1 is about 20°P instead of the theoretical value of 16°P. It can be calculated that when the dissolved solids concentration is 20°P instead of the theoretical value of 16°P, the amount of water available for dissolving sugar is decreased (through an absorption by grain components and evaporation) by as much as 17 875 kg in a mash whose initial weight was 100 000 kg.

Table 3. Calculation of weight of wheat required to prepare 100 000 kg of normal and very high gravity mash with and without insoluble materials

Mash Insolubles Wt of wheat Fermentation Throughput rate Comparative type removed (kg) time (h) c' (kg wheat/h) efficiency

% (%)

NG (16°P) 0 25 000 60 417 100 NG (16°P) 100 27 275 60 455 109 VHG (3 l°P) 0 44 558 801 557 134 VHG (3 l°P) 50 48 349 80 604 145 VHG (3 I°P) 100 52 846 80 661 159

NG = normal gravity; VHG = very high gravity. aBased on a fermentation temperature of 20°C and an inoculation level of 1.0 x 108 yeast cells per g of dissolved solids in the mash?


DETERMINATION OF WEIGHT OF THE LIQUID PORTION OF MASH

The weight of the liquid portion of the mash can be predicted by eqn (4) or eqn (5). To use these equations the starch content of the grain must be known. In industry, however, this is not assessed routinely in a direct manner. Furthermore, if the observed dissolved solids concentration (°P) is less than that calculated by eqn (3) it is an indica- tion that hydrolysis of starch was not complete.

wt in kg of the liquid portion of the mash

1.111SG C (4)

(1.111SG)100 or = °P , (5)

where C, G, S and op have the same definitions given in eqns (1) and (2).

A direct experimental method for the determination of efficiency of mashing is needed. In the brewing industry (where insolubles are removed in the lauter tun and in the hot wort tank) yield of carbohydrate is determined from the volume of extract obtained and its specific gravity. No such separation of extract from insolubles is done during the preparation of grain mashes for fuel alcohol production. The producer then has no way of estimating extract volume or predicting the ethanol yield. The only option available to the fuel alcohol manufacturer is to ferment the mash and determine the ethanol yield by distillation. Even here, a lower than expected ethanol yield may be the result of factors such as incomplete liquefaction and saccharification, insufficient nutrient supplementation, microbial contamination, stuck fermentation or incomplete distillation. With V H G grain mashes, direct determination of total dissolved solids content is even more difficult, as it may require repeated washing of the insoluble mash residues and a dry weight measurement. Complete removal of soluble materials from the residue becomes progressively more difficult as the 'thickness' of the mash increases. We, therefore, describe a simple procedure using a dilution technique to determine the dissolved solids content of grain mashes. An example from an actual run is given in Table 4.

The total weight of a mash is noted. A repre- sentative homogeneous sample (30 g) of the mash

Table 4. Direct determination of the quantity of liquid portion of the mash by the dilution technique (model calculation from an actual run)

Total weight of the ground wheat used = 887 g Moisture in the ground wheat = 12.0% Dry weight of ground wheat used = 780"6 g Total wt of the mash = 2108 g °P of supernatant (undiluted mash) = 28"9 Dissolved solids per g of supernatant

(undiluted mash) = 0.289 g °P of supernatant (diluted mash) = 21"0 Dissolved solids per g supernatant (diluted mash)= 0.210 g If L is the weight of the liquid portion in 30"0 g of undiluted

mash, then 0.289 L-- (L+ 10)0.210 Solving the equation, L = 26-58 Weight of liquid portion of mash =

26"58/2108= 1868 g 30 /

Weight of dissolved solids in the mash =

1 ~ ] 1868 = 540 g

The 540 g of dissolved solids are derived from 887 g of wheat

540 x 100 Dissolved solids as % of wheat used - 60.9%

887

The wheat contained 53.6% starch (wet basis) Expected glucose yield = 53'6 x 1'111 = 59.6%

is centrifuged (10300g for 15 min) and a portion of the supernatant collected. The specific gravity of the supernatant liquid is measured with a digital density meter (DMA-45, Anton Paar, KG, Garz, Austria). The dissolved solids content of the supematant liquid is estimated by converting the specific gravity readings to °P. Another 30 g of the same homogeneous mash is transferred to a centrifuge tube and mixed thoroughly with 10 g of distilled water and then centrifuged. The specific gravity of the supernatant is again determined and the dissolved solids content calculated.

The calculations in Table 4 show that the dissolved solids content of the mash was not less than the expected value. This indicates that mashing was efficient. Additional dissolved solids in the mash may have been derived from non s ta rch grain components.

A general equation to calculate the weight of the liquid portion of the mash from experimental results is given. Note that all values can be easily determined and that the starch value is not used in the calculation.


wt of liquid portion of whole mash

MED B(C-D)" (6)

By combining eqns (4) and (6) the amount of starch that was converted to glucose can be calculated.

S(kg starch hydrolysed to glucose per kg grain)

MEDC = b i l l GB(C-D) ' (7)

where M = total weight of mash in kg; B = weight of the undiluted mash sample (kg) used for the determination of C; E = kg of water added to B kg of undiluted mash to obtain diluted mash; C = concentration of dissolved solids in the supernatant of the undiluted mash (kg/kg); and D = concentration of dissolved solids in the supernatant of the diluted mash (kg/kg).

When starch hydrolysis is complete (as shown by a negative starch test by iodine) the calculated value is an estimate of the starch content per kg grain. In the numerical example given in Table 4 the starch content per kg of wheat was 0.536 kg whereas the value calculated by eqn (7) was 0-548 kg per kg of wheat. The presence of small amounts of dissolved solids derived from non- starch components accounts for the slight over- estimation of starch by the calculated method.

REPORTING ETHANOL YIELD IN TERMS OF CONCENTRATION: A NEW APPROACH

The theoretical yield of ethanol is 0.511 g (or 0.647 ml at 20°C) per g of glucose. This amount can never be realised under fermentation conditions because some of the glucose taken up by the yeast is used for growth (biomass production), cell maintenance, and production of small amounts of byproducts such as glycerol. Nevertheless, 90-95% of glucose is usually converted to ethanol under ideal fermentation conditions. In the fermentation literature, ethanol yields are routinely reported in terms of concentration and not in absolute amounts. This is a reasonable approach when the total volume of the fermented liquid (as in brewing or enology) is known.The ethanol yield can be calculated from the weight (or volume) of the fermented liquid and the concentration of

ethanol in the liquid portion. This method, however, cannot be applied for grain mashes because the weight of insoluble grain residues is considerable and the weight (or volume) of the liquid portion of a fermented mash is not known. The weight of the liquid portion of fermented mash is the difference between the weight of the liquid portion at the start of fermentation and the weight of glucose converted to carbon dioxide (and lost to atmosphere). Some water may be used up for side reactions such as hydrolysis of dextrins and oligosaccharides. Ethanol produced from sugar through fermentation will add to the, total liquid volume. The quantity of the liquid portion can be experimentally determined by a dilution technique similar to the one used for the; determination of weight of the liquid portion of the mash prior to fermentation. Efficiency of fermentation can be expressed in terms of concentration of ethanol only if the measured value can be related to the predicted theoretical concentration. Changes that occur in weight and volume of the liquid portion during the course of fermentation affect ethanol concentration. The following equations are derived by taking the above factors into consideration and they can be used to calculate the maximum theoretical concentration of ethanol in the liquid portion of the fermented mash. Effi- ciency of fermentation can be then calculated by comparing the observed and theoretical values. It is assumed that hydrolysis is complete (i.e. all of the fermentable sugar at the start is glucose), and that no side reactions which use water as a reactant occur. It is also assumed that the total volume of the liquid portion of the mash is equal to the volume of water plus the volume of ethanol produced.

51.1C % ethanol (w/w)= (8)

1 -0.489(7

51.1C % ethanol (w/v)= (9)

1 - 0.353 C

64"7C % ethanol (v/v) = 1 _ 0.353 C, (10)

where C is concentration (w/w) of glucose in the liquid portion at the start of fermentation.

If water is consumed during the course of fermentation, the final liquid volume will be decreased and this will result in higher ethanol

330 K. C. Thomaset al.

concentrations than predicted by eqns (8)-(10). If the mash at the start contains dextrins, water will be used for their hydrolysis. Corrections can be applied to eqns (8)-(10) if the quantity of oligosaccharides and dextrins in the mash is known. Usually the dissolved solids in a mash before saccharification consist of a substantial amount of dextrins and oligosaccharides in addition to glucose. A typical carbohydrate profile of un- saccharified wheat mashes has the following composition: maltotetraose and other soluble dextrins, 46.8%; maltotriose, 17.3%; maltose, 27"9%; and glucose, 8.0% (saccharification is an on going process during the course of fermentation and nearly all of the soluble dextrins and oligosaccharides are converted to fermentable sugars). It can be shown that mashes containing 31 g dissolved solids per 100 g mash and having the same carbohydrate profile as above will have a theoretical ethanol yield of 24.5% (v/v), whereas the ethanol yield will be only 22.5% (v/v) if all of the dissolved solids consisted of glucose only. Another factor that must be considered is the volume change that occurs when ethanol is mixed with water. Since the volume of a mixture of ethanol and water is slightly less than the sum of volumes of components (water can dissolve a small amount of ethanol without increasing the total volume), the concentrations of ethanol will be slightly higher than those predicted by eqns (9) and (10). At an ethanol concentration of 20% (v/v), this change in volume amounts to about 1% (unpublished results).

SUMMARY

Considerable amounts of water can be saved by applying VHG fermentation technology to fuel alcohol production. The saving may be as high as 58.5% when the dissolved solids concentration is raised from 16 to 31°P. Adoption of VHG technology also increases the throughput rate of an alcohol plant without the necessity to increase the plant capacity. The throughput rate can be increased further by removing part or all of the insoluble non-starch materials or by using the starch-enriched fraction for mashing. Alternately, insoluble materials may be removed prior to fermentation. The non-starch solids (mainly protein and fibre) could have greater economic value than distiller's grains because of their potential use in human food. The efficiency of an alcohol plant

can be increased by 1.5 times or more if a VHG mash without insoluble materials is used.

The choice of mashing method depends on the type of grain to be mashed. Enzymes have to be used to prevent the development of viscosity during mashing of barley or oats. Once the viscosity-causing polymers are hydrolysed, VHG mashes from these grains can be prepared simply by adjusting the water to grain ratio. This method (adjusting the water to grain ratio) cannot be applied to every type of grain, but the described double mashing procedure can be used for all types. In this procedure, part of the insoluble material may be removed prior to fermentation. Double mashing is especially useful when two types of grain are to be mashed and fermented in the same plant. An example is the use of oats to prepare a basal mash (first mash) and the use of the extract obtained from it to prepare a second mash (double mashing) using either wheat or barley. Although the simplest method to raise the fermentable sugar content of a grain mash is to add adjuncts, this method only has application at limited industrial sites.

The amount of grain and water required to prepare various mashes can be calculated using the formulae given. One of the difficulties in calculating efficiency of mashing and fermentation is not knowing the weights (or volumes) of the liquid portion of the mash before and after the fermentation. These can be calculated using the formulae given or experimentally determined by the methods described. In addition, an indirect estimation of the starch content of the grain can be made using eqn (7).

In the literature, ethanol concentrations in fermented liquids are used as a measure of ethanol yields. Reporting ethanol yields in terms of concentration is of value only if the total weight or volume of the liquid portion of a mash is known, or the potential maximum ethanol concentration can be predicted. The weight of the liquid portion can be calculated or determined by the methods described earlier. On the other hand, using the equations given, the theoretical maximum concentration of ethanol can be predicted and compared to the measured concentration of ethanol. These equations were derived by taking into account the changes that occur in weight and volume of the liquid portion of a mash during the course of fermentation. In sharp contrast, industry uses a method where the weight of ethanol in 100 g of fermented mash is divided by the weight of the


carbohydrate used per 100 g of the original mash and the result is compared to the Gay-Lussac theoretical value of 0"511. In such a calculation no consideration has been given to the factors out- lined earlier or to the degree of polymerization of carbohydrate at the start of fermentation. We sub- mit that the use of eqns (8), (9) or (10) will lead to more accurate predictions of the potential yield of alcohol during the fermentation of carbohydrate.

ACKNOWLEDGEMENTS

The authors acknowledge the research support of the Westem Grains Research Foundation and the Natural Sciences and Engineering Research Council. Without such support this work and its supporting research would not have been possible.

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