WO2006048503A1

WO2006048503A1 - Ethanol production

Info

Publication number: WO2006048503A1
Application number: PCT/FI2005/000475
Authority: WO
Inventors: Harri Miettinen; Jarmo Luhtala
Original assignee: Kemira Oyj
Current assignee: Kemira Oyj
Priority date: 2004-11-08
Filing date: 2005-11-08
Publication date: 2006-05-11
Anticipated expiration: 2007-05-08
Also published as: FI20041430L; FI20041430A7; FI20041430A0

Abstract

The application describes a method for ethanol production, in which method a mixture containing fermentable sugars is fermented by means of a yeast. According to the invention, formic acid, propionic acid, sorbic acid, or benzoic acid, or at least one salt of such an acid, or a mixture thereof is added to the mixture before or during the fermentation in order to promote the fermentation reaction and increase the ethanol yield.

Description

ETHANOL PRODUCTION

Field of the Invention

This invention relates to ethanol production by means of fermentative conversion of fermentable sugars. The sugars may be derived from suitable carbohydrate sources.

Description of the Prior Art

In recent years, considerable attention has been paid to the conversion of biomass to chemicals and liquid fuels. Biomass offers the potential to replace non-renewable fossil fu¬ els with fuels derived from vegetative carbon-containing sources such as grains, tubers, sugar cane and similar materials. The carbohydrates contained in the aforementioned mate¬ rials are commonly pretreated, e.g. hydrolyzed, to fermentable sugars which are subse- quently converted to ethanol by fermentation. Fermentation processes accounted for 83 % of total production of ethanol in the U.S., Western Europe and Japan in 1997, by 2001 fer¬ mentation increased to 90 %. Overall demand for ethanol for fuel consumption is expected to amount to 10 x 10⁹ litres in 2005. Due to the importance of ethanol as an alternative bio¬ compatible fuel and growing consumption of ethanol worldwide significant efforts have been made to increase the industrial fermentation and distillation process efficiency. These include, e.g.,

- continuous fermentation process

- heating and simultaneous distillation of ethanol

- bacterial fermentation - genetically transformed microorganisms to increase the yield of ethanol

- addition ofantibiotics to fermentation medium

- immobilization of yeast or microorganisms

- use of zeolite or similar sorbent for absorption of ethanol in fermentor

- pervaporation (using membrane) process. However, most of the proposed means are expensive to implement and their large scale commercial practicality has yet to be established.

Alkali treatment followed by pH adjustment to 5.5 by acid has been used when fer¬ menting lignocellulose hydrolysates with Saccharomyces cerevisae to ethanol (Persson Per et al., Swed. Journal of Agricultural and Food Chemistry (2002), 50(19), 5318-5325). It was suggested that the positive effects of alkali treatment may have possible stimulatory effects on the fermenting organism. It has also been found that low levels of acetic acid stimulate yeast ethanol productivity from hydrolysed cellulose (Lawford Hugh G., Rous- seau Joyce D., Applied Biochemistry and Biotechnology, Vol. 105-108, 2003, 457-469).

Summary of the Invention

Now it has been found that implementing small amounts of formic acid, propionic acid, sorbic acid, or benzoic acid increase the yield of ethanol from fermentation and ac¬ celerate at the same time the process. Thus, the same fermentation extent using these acid additives can be achieved in a shorter time as compared to conventional fermentation without additives, or the fermentation can be carried out with better total yield. Both achievements are significant from the prospective of industrial production of ethanol, and fuel ethanol, especially.

The invention is defined in the independent claims. Some preferable embodiments are defined in the dependent claims.

Detailed Description of the Invention

In accordance with the invention, a mixture is prepared from a material containing fer¬ mentable sugars and an acid composition comprising at least one of formic acid, propionic acid, sorbic acid, or benzoic acid, or at least one salt of such an acid, or a mixture thereof in a liquid medium. The amount of the acid is high enough to promote the fermentation but too low to inhibit it. Suitable acid concentration is from 0.5 to 40 mM, especially from 1 to 20 mM, more especially from 2 to 15 mM from the volume of the mixture before adding the microorganism.

The observation that these acids promote the fermentation was unexpected. Namely, it is well known that such acids inhibit yeast growth, and in fact all these acids have been used as cereal preservatives. Especially formic acid is a well known preservative.

The acid may be added also in the form of salts, such as alkali metal (e.g. Na, K) or ammonium, or alkali earth metal (e.g. Ca, Mg) salts. Also partially neutralized acids, as well as mixtures of salts and acids, may be used. The use of salts may be advantageous since it decreases corrosion risks. The mixtures of formic acid and propionic acid are ex¬ amples of preferable mixtures. These mixtures may be also partly neutralized especially by ammonium ions.

The invention may be used for fermenting sugars from corn (maize), cereal, especially barley, wheat or oat grains, tubers, or sugar cane.

The acid composition is preferably added when preparing the fermentation mixture. It may be added to biomass materials also in some preceding step. The acid may be added especially to the source materials which contain fermentable sugars or from which such materials are produced. E.g., if cereal is used as the raw material, the acid may be added to the grains in harvesting, storing or processing stage, such as malting step. The acid compo¬ sition may be added also during the fermentation.

The biomass may be pretreated before the fermentation, e.g. hydrolyzed, in order to convert poorly fermentable sugars to better fermentable ones. Especially it has been found that maltose or maltotriose may be poorly fermentable in the presence of the acids. How- ever, these sugars may be converted to better fermentable ones, such as glucose, before the fermentation. Accordingly, it may be advantageous to convert completely poorly ferment¬ able sugars to better fermentable ones. The conversion can be accomplished by hydrolysis using suitable enzymes, such as amyloglucosidase, or α-amylase.

It has been found that the acids both increase the yield of ethanol and accelerate the fermentation reaction. Thus, in accordance with the invention, higher ethanol yield can be achieved, or same ethanol yield can be attained in a shorter time or both. Thus, the ethanol production can be optimized in a suitable manner. For instance, common fermentation time for a batch in distilleries is 24 hours, whereby the ethanol yield is typically 98 %. Now yields of about 98.7 to 99.7 % may be obtained using the same fermentation time. The fermenting microorganisms are suitable yeasts, such as Saccharomyces cerevisiae.

The yeast maybe e.g. distiller's yeast, brewer's yeast or baker's yeast.

Fermentable sugars may be obtained directly from certain plants, such as sugar beet, sugar cane, or fruits. Fermentable sugars may be also obtained from different carbohy¬ drates, especially from starches. This may be achieved by suitable pretreatment process, e.g. by enzymatic or chemical hydrolysis.

Grain crops that contain sugars and starches are often easily perishable so that special means, such as drying, is needed for storing and transportation. E.g. drying highly in¬ creases energy consumption of the process, thus worsening substantially the economy of ethanol production for fuel use. In accordance with the invention it is now possible to add acid to the crop in order to preserve it. The same acid then acts as a promoter later in the fermentation process.

The fermentation process may be batch-type or continuous.

The following examples further illustrate the invention.

Example 1.

A barley wort corresponding to brewer's wort was made without adding any hop. The composition of the wort is shown in Table 1. Because the zinc concentration was only 0.07 mg/L, 0.15 mg/L of zinc was added as zinc sulphate.

Table 1. Composition of wort

Parameter Unit Value Method

Glucose g/L 87.5 HPLC

Fructose g/L 5 HPLC

Sucrose g/L 2.4 HPLC

Maltose g/L 52.3 HPLC

Maltotriose g/L 15.4 HPLC

Total fermentable sugars g/L 162.6 HPLC

Free amino nitrogen mg/L 284 EBC 4.10

Zinc mg/L 0.07 + 0.15 AAS

Calcium mg/L 43 AAS

Magnesium mg/L 120 AAS

Density g/ml 1.077 Anton Paar pH fresh (autoclaved) 5.20 (4.8)

Three worts were prepared and to two of them carboxylic acid was added so that they contained, respectively, 30 mM propionic acid and 13 mM formic acid. One control con¬ tained no acid. AU worts were with pH 4.8. 400 g of each wort were placed into fermenta¬ tion bottles equipped with a glycerol-containing bubble trap for CO₂ escape and a magnetic stirrer. Fermentation was started by adding 10.0 g of a 20 %(w/w) distiller's yeast strain (Saccharomyces cerevisiae) slurry to give an initial yeast concentration of 5.1 g/L (99.5 % viability). The bottles were weighed immediately after yeast addition and incubated with stirring at 30.5 ⁰C. Fermentation progress was followed by mass loss. The fermentations were stopped at 67 h. At the end of the fermentations, the entire contents of each bottle were centrifuged. The superaatants were used for analyses of ethanol by distillation (Ana- lytica-EBC, Verlag Hans Carl Getranke-Fachverlag, Nϋrnberg, 1998, 9.2.1). The relative ethanol yields were calculated on the basis of the stoichiometric amount of sugars. Results are shown in Table 2.

Table 2. Ethanol yields

1 2 3 Control Propionic acid 30 mM Formic acid 13 mM

Ethanol yield/% 89.5 89.9 89.6

Example 2.

Because maltotriose and maltose were not fermented in the presence of the acids in Example 1, the wort was treated with amyloglucosidase (1.0 g Quest AG to 3 L wort; 7 h at 45 °C) before the fermentation. The sugar composition of the treated wort is shown in Table 3. The increase (1.1 g/L) in fructose from untreated wort showed that most sucrose (2.2 g/L) was hydrolyzed by the Quest AG.

Table 3. Sugar composition of Quest AG-treated wort

Sugar Cone, g/L

Glucose 164

Maltose 5.2

Maltotriose 4.3

Fructose 6.1

Sucrose 0.2

A series of six fermentations was performed, following the procedure described in Ex- ample 1, using the Quest AG-treated wort and lower acid concentrations. Bottles 4 and 5 were controls with no added acid. Bottles 6 to 9 contained, respectively, 10.6 mM propi¬ onic acid, 13.9 mM formic acid, 4.1 mM formic acid and 1.0 g/L ammonium formate- propionate (AmmForProp; 50.9 % of formic acid, 30 % of ammonium formate, 18.3 % of propionic acid, 0.8% of water). They were pitched with distiller's yeast (99.8 % viability) at 30.5 ⁰C (as in Example 1). The fermentations were stopped and analyzed at 38.5 h. The supernatants were used for analyses of ethanol and other compounds. The pellets were washed with water and weighed to give the fresh yeast mass. Portions of the resuspended pellets (in water) were dried overnight at 105 °C to give the dry yeast mass. The amount of ethanol found by distillation agreed well with that calculated from the mass loss, i.e. total CO₂ production.

All fermentations containing acids or their mixture were faster than the duplicate con¬ trols without acids. All fermentations were at least 98 % complete in 24 h (calculated as mass loss at 24 h/mass loss at 38.5 h). Ethanol contents at 38.5 h were determined by distillation and by HPLC and were also calculated from the mass losses by assuming that molar productions of ethanol and carbon dioxide were equal. HPLC ethanol results were 2 % higher than the distillation results and ethanol calculated from mass loss was 0-2 % higher than that found by distillation. The good agreement between these three methods of analysis suggests that the differences in ethanol between different bottles are reliable.

Final ethanol yield was higher for all the fermentations with acids than for the controls. The increases were 0.8 to 2.0 % for formic and propionic acids and 0.1 % for AmForProp. The mass losses indicate that if the fermentations had been stopped at 24 h, larger increases in ethanol would have been observed. Stoichiometric ethanol yields were calculated by assuming all the glucose and fructose consumed are converted into ethanol.

Yields of fresh yeast mass (6.6 to 8.3 g per bottle) were highest for the bottles with no added acid. This is consistent with the acids inhibiting yeast growth and diverting glucose into ethanol production. However, differences in dry yeast mass were much smaller. Glyc- erol was determined by HPLC and enzymatically. HPLC results were higher by 7 to 35 %. Both analyses showed higher glycerol in the bottles without acid. Glycerol is produced by fermenting yeast to balance excess NADH production associated with cell growth. Results are shown in Table 4. Table 4. Analysis of final (38.5 h) products from fermentation

4 5 6 7 8 9

Con¬ Con¬ Propionic Formic Formic AmForPro trol trol 10.6 niM 13.9 mM 4.I mM 1.0 g/L

Initial wort volume (ml) 371.1 370.9 372.3 371.9 370.6 371.8

Initial glucose+fructose (g) 63.1 63.1 63.3 63.3 63.0 63.2

Final pH 3.96 3.97 3.85 3.71 3.91 3.67

PRODUCTS

Fresh yeast mass (g) 8.25 8.11 7.08 6.67 7.91 6.86

Dry yeast mass (g) 2.29 2.15 1.93 2.15 2.20 2.20

Residual sugars (HPLC)

Glucose (g/L) 0.3 0.3 0.3 0.8 0.3 1.1

Fructose (g/L) 0.2 0.2 0.1 3.0 0.2 3.2

Maltose (g/L) 6.3 6.3 6.2 6.2 6.2 6.2

Maltotriose (g/L) 4.4 4.4 4.5 4.5 4.5 4.5

Ethanol (w%)

EtOH from distillation 7.88 7.88 8.04 7.94 7.96 7.89

EtOH from HPLC 8.02 8.02 8.24 8.11 8.11 8.07

EtOH calc. from mass loss 7.93 7.91 8.12 7.97 8.09 7.95

EtOH calc. from stoich. sugar 8.22 8.22 8.27 7.49 8.23 7.43

EtOH found/stoich. (%) 95.9 95.9 97.2 106.2 96.7 106.1

Other compounds (g/L)

Glycerol (HPLC) 5.4 5.7 3.2 4.0 4.9 3.8

Glycerol (enzymatic) 4.94 4.94 3.0 3.4 4.1 2.82

Sum ofvolatiles 0.41 0.43 0.38 0.35 0.40 0.33

The decreased glycerol levels are consistent with the increased ethanol yields. Thus, these results are consistent with more yeast growth in the acid-free bottles as suggested by the fresh yeast measurements. The formation of less yeast and less glycerol in the presence of organic acids is consistent with higher ethanol yields in these bottles.

Volatiles (higher alcohols, esters and acetaldehyde) were determined by GC. Total volatiles (0.35 to 0.43 g/L) were lower for the fermentations containing added organic ac- ids, but the changes were small.

Formic acid, propionic acid or AmForProp increased fermentation rates and final etha¬ nol yields. The yield increases after 38.5 h of fermentation were 0.8 to 2.0 % for formic and propionic acids and 0.1 % for ArnForProp. The fermentations were continued to 38.5 h to ensure they had reached completion.

In a distillery, the fermentations would have been stopped at 24 h when the controls were 98.3 % complete and the fermentation with the carboxylic acids were 99.0 to 99.7 % complete as indicated in table 5 by the fermentation extent. This was calculated as

100*(mass loss at 24 h)/(mass loss at 38.5 h) . A clearly higher completion was reached at

24 h using the carboxylic acids as the reaction rate of fermentation was accelerated.

Similarly, at 24 h all fermentations with the carboxylic acids gave better yields com¬ pared to controls. The relative ethanol yield corresponding to the mass loss and the total CO₂ production showed an increase of 1.9 to 2.9 % as indicated in table 5. The relative ethanol yield is calculated as 100*(mass loss)/(average mass loss of tubes 4 and 5).

Table 5. Calculated extent of fermentation and relative ethanol yields at 24 h

4 5 6 7 8 9

Control Control Propionic Formic Formic AmForrop acid 11 acid 14 acid 4 1.1 g/L mM mM mM

Fermentation 98.3 98.3 98.7 99.3 99.0 99.7 extent

Relative etha¬ 100.2 99.8 102.9 101.9 102.6 101.9 nol yield

Claims

1. A method for ethanol production, in which method a material containing ferment¬ able sugars in a liquid medium is fermented by means of a yeast, characterized in that, for promoting the fermentation, an acid composition comprising at least one of formic acid, propionic acid, sorbic acid, and benzoic acid, or at least one salt of such an acid, or a mix¬ ture thereof, is added to said material to be fermented before or during the fermentation.

2. A method of claim 1, wherein the amount of acid is from 0.5 to 40 niM.

3. A method of claim 2, wherein the amount of acid is from 1 to 20 mM.

4. A method of claim 3, wherein the amount of acid is from 2 to 15 mM.

5. A method of any previous claim, wherein the acid is formic acid or propionic acid.

6. A method of any claim 5, wherein the acid is formic acid.

7. A method of any previous claim, wherein the fermentable sugars comprise sugars obtained by a sugar producing pretreatment from carbohydrates, especially from starches.

8. A method of claim 7, wherein the petreatment is hydrolysis.

9. A method of claim 8, wherein hydrolysis is enzymatic hydrolysis, such as amy- loglucosidase, or α-amylase hydrolysis

10. A method of claim 8 or 9, wherein the fermentable sugars comprise sugars ob¬ tained by hydrolyzing maltose or maltotriose.

11. A method of any of claims 7 to 10, wherein the carbohydrates comprise carbohy¬ drates from corn (maize), cereal such as barley, wheat or oat grains, tubers, or sugar cane.

12. Use of a method of any previous claim in ethanol production, such as in fuel etha¬ nol production.

13. Use of formic acid, propionic acid, sorbic acid, or benzoic acid, or at least one salt of such an acid, or a mixture thereof for promoting the fermentation method of fermentable sugars to produce ethanol, in which method a mash containing fermentable sugars is fer¬ mented by means of yeast.

14. Use of claim 13 for accelerating the speed of the fermentation reaction and/or in¬ creasing the yield of ethanol.

15. A method for promoting the fermentation method of fermentable sugars to produce ethanol, in which method a material containing fermentable sugars is fermented by means of yeast, characterized in that acid comprising at least one of formic acid, propionic acid, sorbic acid, or benzoic acid, or at least one salt of such an acid, or a mixture thereof is added to the material.