Method for producing ethanol from xylose-containing substance

A method for fermenting xylose-containing substance, such as a lignocellulosic degradation product into ethanol. Yeast of the species Pichia stipitis and/or Pichia segobiensis and/or Candida shehatae are utilized for the fermentation.

This invention relates to a method for producing ethanol from a 
xylose-containing substance, comprising fermenting said substance with a 
yeast. Such methods have been disclosed recently in the U.S. Pat. Nos. 
4,359,534 and 4,368,268, wherein the fermentation utilizes the yeast 
Pachysolen tannophilus and yeast mutants from the strain Candida sp. The 
obejct of the present invention is to provide a method for fermenting 
xylose in a high yield. 
The yeasts for use in the invention are Pichia stipitis, Pichia segobiensis 
and Candida shehatae. The P. stipitis type strain CBS 5773 (NRRL Y-7124, 
T) was originally isolated from an insect larvae and was designated by 
Pignal. The standard description of P. stipitis, made by Kreger-van Rij, 
1970 (The Yeasts-A Taxonomic Study (Lodder, J. ed.) pp. 533-535, 
North-Holland Publishing Company-Amsterdam, London) is as follows: 
Growth in malt extract: After 3 days at 25.degree. C. the cells are 
spherical to oval (2-7.5).times.2.5-7.5) .mu.m; single or in pairs. A 
sediment is formed. After one month at 17.degree. C. a sediment and, 
occasionally, a ring are present. 
Growth on malt agar: After 3 days at 25.degree. C. the cells are spherical 
to short-oval, (2.5-4.5).times.(2.5-6) .mu.m; single or in pairs. 
Pseudomycelial cells may occur up to 15 .mu.m long. After one month at 
17.degree. C. the streak culture is cream-colored, occasionally with a 
reddish tinge, soft, smooth or delicately wrinkled in the middle, and 
semiglossy. The edge is fringed with pseudomycelium. 
Slide cultures on potato- and corn meal agar: Pseudomycelium is abundantly 
formed. It is more or less branched and consists of long pseudomycelial 
cells with small blastospores. 
Formation of ascospores: Conjugation between mother cell and bud or between 
two single cells precedes ascus formation. The cells may form 
protuberances of various lengths. The spores are hat-shaped; two are 
formed per ascus. They are easily liberated from the ascus. Spores were 
observed in the three strains studied on YM-, Difco malt extract- and corn 
meal agar. 
Fermentation: 
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Glucose + (slow) 
Trehalose + (very weak) or - 
Galactose + (slow) 
Lactose - 
Sucrose - Raffinose - 
Maltose + (slow) 
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Assimilation of carbon compounds: 
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Glucose + D-Ribose + 
Galactose + L-Rhamnose + 
L-Sorbose - Ethanol + 
Sucrose + Glycerol + 
Maltose + Erythritol + 
Cellobiose + Ribitol + 
Trehalose + Galactitol - 
Lactose + D-Mannitol + 
Melibiose - D-Glucitol + 
Raffinose - .alpha.-Methyl-D-glucoside + 
Melezitose + Salicin + 
Inulin - DL-Lactic acid + 
Soluble starch + Succinic acid + 
D-Xylose + Citric acid + 
L-Arabinose + Inositol - 
D-Arabinose - 
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Splitting of arbutin: Positive 
Assimilation of potassium nitrate: Negative 
Growth in vitamin-free medium: Negative or very weakly positive. 
Growth on 50% (w/w) glucose-yeast extract agar: Negative 
Growth at 37.degree. C.: Positive. 
In addition to the above-mentioned fermentable substrates it has 
unexpectedly been found that P. stipitis also ferments D-xylose. P. 
segobiensis is described in the following reference: J. Santa Maria and G. 
G. Aser, An. Inst. Nac. Invest. Agrarias, Ser. General 5 (1977) 45-50. It 
has been found that this yeast ferments D-xylose to ethanol to a degree 
comparable to that of P. stipitis. All P. stipitis strains tested--CBS 
5773, CBS 5774, CBS 5775, CBS 5776, CBS 6054, and P. segobiensis strain 
CBS 6857--share this characteristic. All these strains are therefore 
contemplated for use in the disclosed process. Also the tentatively 
(Lodder: The Yeasts (1970) p. 535, 1046, 1047) imperfect form of P. 
stipitis (Candida shehatae) ferments D-xylose to ethanol and is therefore 
also contemplated for use in the disclosed process. 
All D-xylose-containing substrates are suitable in the disclosed process 
provided they do not contain any constituents which are severely 
inhibitory to the process. 
Since available glucose also will be fermented to ethanol, hydrolyzed 
cellulose and hydrolyzed hemicellulose or mixtures thereof are 
particularly suited as substrates in the disclosed process. Hence, as raw 
material for the process could serve any lignocellulose material 
containing cellulose and hemicellulose such as wood, grass, straw, bagasse 
etc. Also suited as substrates are waste fluids, such as spent sulphite 
liquor, containing D-xylose, besides other sugars if any. It is understood 
that the predominant monosaccharide found in hydrolyzed hemicellulose is 
D-xylose. 
The disclosed process involves the fermentation in an aqueous medium of 
D-xylose, and D-glucose if present, to ethanol. The chemical and physical 
conditions of the medium must otherwise be as to maintain cell viability, 
as known by a person skilled in the art. 
When cell growth is required, the ethanol concentration should not exceed 
45 g/l. At 30 g/l, the growth rate is considerably retarded. Ethanol yield 
is reduced at ethanol concentrations at or above 30 g/l. 
P. stipitis grows well at 28.degree.-32.degree. C. Fermentation is 
supported in the temperature range 15.degree.-40.degree. C. The highest 
rate is observed between 30.degree. C. and 37.degree. C., with 
32.degree.-34.degree. C. being optimum for the ethanol production rate. 
Growth of P. stipitis CBS 5773 occurs in the pH interval 3-7. pH 5 results 
in a slightly better growth than do pH 4 and pH 6. Ethanol production rate 
is good between pH 3 and pH 8, with pH 6 being about maximum. Since it is 
desirable to perform fermentation at the lowest possible pH in order to 
minimize the risk of infection, it should be observed, that at pH 4 the 
production rate is more than 90% of maximum and the growth rate is also 
satisfactory at this pH. 
Ethanol production proceeds in an anaerobic medium. The ethanol yield of 
anaerobic fermentation is approximately equal to that of a fermentation at 
a limited air supply, although the fermentation rate is somewhat lower. 
In a typical batch-type fermentation a cell suspension of P. stipitis 
obtained from a preculture preferably in exponential growth phase, is 
supplied with D-xylose. The conditions are adjusted and maintained within 
the range defined above. Ethanol production is thereby initiated and will 
be continued at the rate governed by the actual cell concentration and 
conditions otherwise prevailing. Provided the conditions are maintained 
within the range defined above and the cells are kept viable, ethanol 
production will not discontinue until the D-xylose is depleted. 
In U.S. Pat. No. 4,359,534 a process is described where the yeast 
Pachysolen tannophilus is used to ferment a D-xylose-containing substance. 
It is reported that aeration is a prerequisite for enhanced ethanol 
production. The highest yield reported in this patent is 0.34 g ethanol/g 
D-xylose. In U.S. Pat. No. 4,368,268 a similar process is described where 
a mutant strain of Candida sp., XF 217 is used to ferment D-xylose to 
ethanol. It is reported that oxygen must be available for enhanced ethanol 
production from D-xylose. The highest yield reported in this patent is 
that demonstrated in FIGS. 1 and 2 showing aerobic fermentation from which 
a yield of 0.42 g ethanol/g D-xylose may be estimated. 
A small amount of air (oxygen) is necessary for cell growth. The ethanol 
yield in aerobic fermentation using P. stipitis with a limited amount of 
air is about the same as in anaerobic fermentation. This makes possible a 
fermentation process where conditions favourable for growth and efficient 
fermentation can be met simultaneously. The effect of a small amount of 
oxygen is thus twofold; it makes the necessary cell growth possible and it 
increases the specific ethanol productivity of the cells. 
The highest yield obtained in a single fermentation experiment is 0.46 g 
ethanol/g D-xylose. It is our conviction that 3/2 molecules of ethanol are 
formed for each molecule of D-xylose. 
This corresponds to a maximum theoretical yield of 0.46 g ethanol/g 
D-xylose consumed. Hence, the yield above is 100%. Usually, the yield is 
slightly lower, 0,43-0,45 g/g (=93-98% of maximum).