CA1257554A - Conversion of carbohydrates to alcohol with certain yeasts - Google Patents

Abstract

TITLE
CONVERSION OF CARBOHYDRATES TO
ALCOHOL WITH CERTAIN YEASTS

ABSTRACT OF THE DISCLOSURE
Certain yeasts do not need to be presented with simple sugars to produce ethanol; they are also capable of fermenting complex carbo-hydrates under selected conditions. This invention gives methods of producing ethanol using selected yeasts, particularly Schwanniomyces alluvius, on these substrates. Other commercially valuable byproducts include the yeast cells themselves and the carbohydrate-digesting enzymes.

Description

~.2S7554 This application is a divisional of application Serial Number 449,565 filed March 14, 1984.
This inventlon is in the field of yeast fermentations particu-larly to produce ethanol.
Description of the Prior Art Conversion of starches to sugars, as in the production of high fructose corn syrup, using a variety of methods, often lnvolving enzymes that can tolerate hlgh temperatures, and the conversion of sugars to al-cohol are well known. The production of sugars by malting grain has been prac~ised for millenia. Conversion of complex carbohydrate material by microorganis~s to end-products that include alcohol and other deriva-tives, such as enzyme mixtures and cells or single-cell protein, iB
known. Traditional conversion of starch to ~thanol by Saccharomyces cerevisiae requires two very expensive treatments: liquefaction and saccharification. Direct starch fermentation not requiring these treat-ments could be competitive with petrochemical processes in the production of industrial alcohol. Canadian Patent 1,119,538, to H. and F. Muller, describes a process in which a mash is subjected to acid or enzyme de-gradation to give fermentable sugars. I. Spencer-Martins and N. van Uden in European J. Appl. Microbiol. 4, 29-35 (1977) surveyed 81 starch-assim-ilating yeasts, representing 59 species and varieties to study their capacity to directly convert starch into single cell protein. K. Oteng-Gyang, G. Moulin and P. Galzy, in Acta Microbiol. Acad. Sci. Hung. 27, p. 155-159, 1980, studied the influence of amylase excretion on biomass production ~y various genera of amylotytic yeasts. H.J. Phaff in "The Yeasts", J. Lodder, ed., pp. 756-766, 1970, Amsterdam: North-Holland, classifies the genus Schwanniomyces and gives a list of substrates that species of the genus will ferment. Previously, in 1960, ~.J. Phaff, M.W.
Miller and W.B. Cooke, Antonie van Leeuwenhoek J. Microbiol. Serol. 26, pp 182-188, described the new species Schwanniomyces alluvius. A. Nasim, F. Moranelli, V.E. Seligy, M. Yaguchi, E.R. Stephen and A.P. James, at the Third Bloenergy R. and D. Seminar held in Ottawa in March 1981, pp.
140-143, outlined the screening of 17 yeasts for their ability to grow on starch and excrete amylolytic activity into the medium. Some of the properties of this extracellular amylolytic enzyme activity were also reported at the 25th Annual Meeting of the Canadian Federation of Bio-logical Societies held at McGill University in Montreal on June 14-19, 3 2575Sd~

1981 by F. Moranelll, G.B. Calleja, R.H. Lau and A. Nasim. G.B. Calleja, S. Levy-Rick, A. Nasim and F. Moranelli, at a meeting entitled "The Mole-cular Biology of Yeast", held at Cold Spring ~1arbor, New York, on August 11-16, 1981, reported on the growth conditions of Schwanniomyces alluvius required for the excretion of amylolytic activity into the growth medium.
G.B. Calle~a, F. Moranelli, A. Nasim, P.D. Duck and S. Levy at the VILth International Specialized Symposium on Yeast, Valencia, September 1981, Session I, Poster No. 13, described ways of monitoring populatioo growth and extracellular amylolytic activity in Schwanniomyces species, and C.Y.
Lusena, ~. Moranelli and G.8. Calleja, at the same symposium, Session II, Poster No. 17, describe progress in the study of secretion and excretion of amylolytic activities in Schwanniomyces alluvius. The practical pro-blem of exploiting these genera and species of yéast to produce alcohol with by-product enzymes, cells and, by extension, single cell protein, was not discussed. In particular, no indication was given on how to pro-duce ethanol in good yields.
Summary of the Invention The present invention relates to the production OL ethanol and enzymes as well as yeast cells and, by extension, single cell protein.
It describes a method of producing ethanol from carbohydrate material using carbohydrate-digesting, enzyme-secreting yeast which comprises:
Sa) culturing aerobically on a limited supply of the carbohydrate material to allow the yeast cells to multiply and to allow said cells to secrete appropriate carbohydrate-digesting enzymes into the medium;
(b) incubating anaerobically, the same cells and medium with added carbohydrate material to convert the added material to ethanol and;
(c) recovering a product comprislng ethanol and, as byproducts, cells and enzymes.
Schwannlomyces alluvius and S. castelli are two species of yeaat that can be used in the method outlined above and the carbohydrate material is at least one of spoiled grain, root crops, wood-derived mate-rials, starch, glycogen, inulin, dextrins, whey, lactose, cellobiose, pentose sugars, and other carbohydrate oligomers in which the number of monomer units is ten or less. If starch is employed, it is at least one of potato starch, wheat starch, rice starch, cassava starch and corn starch.

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A bioreactor can be used. A bioreactor for converslon of car-bohydrate comprising a vessel containing a porous support material and, immobilized on the support, yeast cells from the genus Schwanniomyces, said support being calcium alginate gel, said gel having a concentration of between 2% and 4% by weLght of calcium alginate in water and, prefer-ably being cross-llDked w1th polyethyleneimine.
Description of the Inventlon Thi~ invention concerns methods of producing ethanol, carbo-hydrate-digesting enzymes and yeast cells from carbohydrate material using a carbohydrate-digesting, enzyme secreting yeast, selected to yield high proportions of ethanol from carbohydrate material. A number of yeasts show promise and it is intended that the methods outlined here, using as example the genus Schwanniomyces, apply to other genera and species such as those given in the prior art references of SpencerMartins et al and Oteng Gyang et al. Schwanniomyces alluvlus (especially strain ATCC 26074) and S. castelli (especially strain ATCC 26077) T~ere studied to develop a basic routlne for the improved utilization of carbohydrate material. The invention is essentially a two-stPp method consisting of (a) culturing the selected yeast aerobically on a limited supply of the carbohydrate material to multiply the yeast cells and to allow said cells to secrete carbohydrate-digesting enzymes into the medium and (b) incu-bating anaerobically the cells and medium from (a) with added carbohy-drate material to convert the added carbohydrate material to ethanol.
These steps are followed by step (c) which concerns recovering a product comprising ethanol, carbohydrate-digesting enzymes and yeast cells.
In passing, it is worth noting that these species of yeast can be used to manufacture enzymes by either stopping step (a) or bleeding off some of the non-cellular material, replacing it with fresh material as required.
The enzymes produced during step (a), outlined above, comprise at least one of: alpha amylase, glucoamylase with strong 1-6 debranching activity, and glucoamylase with weak 1-6 debranching activity. The car-bohydrate-digesting enzymes may comprise of all three of (a) alpha amylase~ (b) glucoamylase with strong 1-6 debranching activity, and ~Z5755D~

(c) glucoamylase with weak 1-6 debranching acitivity. The alpha amylase ( ~1,4-glucan-4-glucanohydrolase) has an Enzyme Commission Number 3.2.1.1 and the two glucoamylases (exo 1,4-~-D-glucosidase) have the Enzyme Com-mission Number 3.2.1.3. Alpha amylases convert amylose into glucose and maltose, and amylopectin into glucose, maltose, isomaltose and several low molecular weight dextrins. The glucoamylases convert amylose and amylopectins, the constituents of starch, into glucose. The debranching activity ensures the complete conversion of starch into fermentable sugars. An absence of debranching activity would mean that amylopectlns would only be partly converted into fermentable sugars, branched dextrins remaining.
We have observed that cells of Schwanniomyces do not grow in anaerobic conditions on the minimal medium that allows them to grow in aeroblc conditions. In addition, cells cultured under anaerobic condi-tions do not excrete amylases and no increase ln the cell population isobserved. We have found that these properties can be e~ploited by first allowing the cell population to increase to stationary phase and secrete appropriate amylases in the presence of air and complex carbohydrates, the anaerobic process of fermentation can be isolated from the int~r-fering substrate-consuming processes of growth and excretion of extra-cellular enzymes, and yields of ethanol increased.
Example (a) Preparation of cells and enzymes - Cells of Schwanniomyces alluvius ATCC 26074 were grown aerobically to stationary phase in Wickerham's yeast nitrogen base without amino acids (Difco), containing 2% (w/v) of soluble potato starch (Anachemia) as sole carbon source, at 30C. The pH
was maintained at 5.5. by automatlc additions of KOH. Under these condi-tions, the generation time was about 90 min.; extracellular amylolytic activity per cell increased tenfold at the end of the logasithmic growth and was maximal during stationary pbase. The pH limits are 4.5 and 6.0 and in this step, the pH is preferably between 5.0 and 5.5.
The rate of aeration used was sufficient to maintain the dls-solved oxygen concentration between 80~ and 100~ of saturation. The cells will grow and produce enzymes as long as the rate of aeration is sufficient to maintain the dissolved oxygen concentration at above 10~ of saturation.

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(b) Anaerobic fermentation - To 5 ml of the stationary phase culture obtained from step (a), (10 cells ml 1), an equal volume of a 5~ 501u-tion of glucose or starch waY added. The final substrate concent}ation was 2.5%. The fermenting cultures, in 15 ml tightly capped conical tubes, were incubated at 25C, undisturbed, except at the time of sampl-lng. Periodically, 1 ml 6amples were withdrawn and the cells pelleted by centrifigation. The amount of ethanol in the cell-free fermentation broth was determined by gas chromatography (stainless steel column, 100/200 mesh, 10% F.F.A.P.) (1% H3PO4, Chromosorb ~ W-AW).
Table 1 shows that fermentation of a complex carbohydrate, such as starch, lagged only slightly behind that of glucose. In both cases, the conversion to ethanol was better than 95% of the theoretical yield.
It should be added that the organism is essentially homofermentative, the main products of fermentation being ethanol and carbon dloxide. The theoretical yield using glucose as a substrate was taken to be 51.14Z of the 2.5% glucose added, ~hich ls equivalent to an ethanol concentration of 1.28~. For starch, 56.83% of 2.5% i8 equivalent to a theoretical yleld of 1.42% ethanol.
The time taken to complete the preparation of cells and enzymes (step a) is less than 12 hours and, preferably, is less than six hours.
The time taken to complete the fermentation step (b) is related to the ratio of amount of carbohydrate material to the number of yeast cells emerging from step (a) but is less than 72 hours and, preferably, is less than 40 hours. As can be seen from Table 1, fermentation is essentially complete by 45.5 hours.
After fermentation, recovery consists of separating the yeast cells from the ethanol enzyme mixture using at least one of centrifuga-tion, decantation and filtration. The enzymes are separated from the ethanol and concentrated by pressure filtration, said enzymes being then separated from each other by chromatography, the ethanol being recovered by at least one of distillation and reverse osmosis.
A ~ethod of purification and characterization of an extracell-ular amylolytic enzyme of S. alluvius was outlined by F. Moranelli et al at the 24th Annual Meeting of the Canadian Federation of Biological Societies noted in the prior art above.

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Table 1 Efficiency of conversion of glucose or starch to ethanol by Schwanniomyces alluvius .
Time Control Glucose Soluble Starch (h) _ % EtOH % EtO~I% Conversion % EtOH~ Conversion O O O O O O
0.015 1.05 82.0 0.96 67.6 45.5 0.019 1.21 94.5 1.34 94.4 72 0.024 1.24 96.9 1.41 99.3 _ ._ _ _ _ _ _ The amounts of ethanol produced from glucose or starch have been corrected for the small amount produced by cells in water (control).
A Bioreactor for Conversion of Carbohydrate A small laboratory vertical glass bioreactor was made and test-ed using species of Schwanniomyces. The vessel contained a porous support material and, immob$1ized on the support, yeast cells from the genus Schwanniomyces. For example, the cells can be immobilized in cal-cium alginate at a concentration of between 2% and 4%, preferably near to

2~ to allow easy inflow of substrate and outflow of enzymes and metabo-lites (there is a trade-off of porosity and strength of the gel).
Strength and resistance to such groups as phosphates is increased with the use of polycationic substances, such as polyethyleneimine, which cross-link with the calcium alginate. The bioreactor design allowed upward and/or downward flow of substrate and the substrate can be aerated separately or the gas flow directed through the bioreactor ltself. The bioreactor was jacketed for temperature control. A single bioreactor can be used for one-step reactions, and two or more bioreactors as a two- or multistage reaction system. This is of advantage in systems where the reaction is perEormed in two or more stages. Each individual stage can be controlled separately and the conditionR opt~mized to the requirements of the reaction performed.

Claims (2)

1. A bioreactor for conversion of carbohydrate comprising: a vessel containing a porous support material and, immobilized on the support, yeast cells from the genus Schwanniomyces, said cells being immobilized in calcium alginate gel, said gel having a concentration of between 2% and 4% by weight of calcium alginate in water.

2. The bioreactor of claim 1 in which the calcium alginate gel is cross-linked with polyethyleneimine.