CA1266245A - Rapid process for the conversion of xylose to ethanol - Google Patents

Abstract

TITLE
RAPID PROCESS FOR THE CONVERSION OF XYLOSE TO ETHANOL

INVENTORS
Gode B. Calleja Sue Levy-Rick George Mahmourides John Labelle Henry Schneider ABSTRACT OF DISCLOSURE
This invention concerns a method for producing ethanol from a D-xylose-containing substrate which comprises (a) culturing selected yeast cells for several generations aerobically in the presence of D-xylose to condition the cells to D-xylose as a major carbon source, subsequently harvesting such cells when they are in a physiological state where ethanol productivity is at or near a maximum and (b) inoculating a D-xylose-containing medium with cells conditioned to D-xylose-containing medium and allowing fermentation to proceed under conditions where the availability of oxygen is low.
The conditioned cells can be recycled through step (b) many times.

Description

Background o~ the Invention ___ _ _. __ This invention concerns an improved method of producing ethanol frorn D--xylose and D-xylose-containing feedstocks. Direct fermer1tation of D-xylose to ethanol has been shown by Cong in U.S.
Patent 4,368,268 of January 11, 1983, by Kurtzman et al in U.S.
Patent ll,359,53ll of NoveMber 16, 1982 and Schneider et al i.n U.S.
Patent 4,ll77,569 of October 16, 198ll. These prior art processes have the drawbacks of being relatively slow and/or giving a relatively low yield.
It i3 des,irable that a process for the fermentation of D-xylose to ethanol be rapid, give a high yield and be nutritionally simple, pref`erably not requiring nutrient addition for fermentation to occur. Minimization of loss of substrate to cell growth and the ability to recyc].e cells repeatedly is also desirable.
Sumrnary of the Invention This .invention concerns a method for producing ethanol from a D-xy1.o~e-contaLning sub3tr.lte comprLsing:
(cl) (I) CU]t;llrin~ c01.1.~3 of se1.ectod yeast specLes ~or several ~cnerat.Lol1s aerob.lcal:l.y in the presencQ of D-xylose as su~stantJ.ally the sole source of sugar to condition said cells to D-xylose as a major carbon source;
(ii) harvesting said cells when they are in a physiological state wherein ethanol productivity is about maximal; and (b) inoculating a D-xylose containing medium with cells conditioned to D-xylose as a major carbon source and fermenting fermentable sugars contained therein under conditions deficient in oxygen such that ce:Lls yield ethanol.
The cells resulting from step (b) can be obtained from step (a)(ii) or recycled through step (b) many times and, when the efficiency of ethanol conversion of such cel].s falls they can be recycled through step (a).

Description of the Drawing3 Figure 1 shows a graph of fermentation ability and cell density against time in Schwanniomyces occidentalis. Figure 2 show3 the accumulation of alcohol in the fermentation of 5% D-xylose by Pichia stipitis. Figure 3 shows that cells harvested in the physiological state where ethanol productivity is at or near a maximum can be recycled in a fermentation step without loss of productivity. Figure 4 shows that cells of Pichia stipitis when grown appropriately can efficiently produce ethanol from mixtures containing D-glucose as well as D-xylose.
Detailed Description of the Invention The process has two stages. In the first, the cells are grown aerobically. In the second, they are used for ~ermentation.
Once the cells have been suitably grown in the first stage, they can be recycled repeatedly in the second, or fermentation step.
The stages have four critical aspects or requirements.
(1) In the course of the first stage, the yeasts are grown to a particular physiological state or age in a medium where D-xylose is the sole or preponderant source of sugar. Prior to growing the cells to the desired physiological state, they are grown aerobically for many generations in a medium where D-xylose is the ~ole source of sugar. The physiological state desired is that the cells are at approximately the time of initiation of the last doubling of cell mass in a culture growing aerobioally on D-xylose as the sole sugar source. The use of aerobically grown cells in this state is a critical embodiment of the invention.

(2) After the cells are grown they are suitably concentrated.

(3) The concentrated cells are placed in the medium containing the sugars to be fermented. The culture is then placed in a vessel where only relatively small amounts of oxygen are allowed access during the ~ermentation. A suitable amount of oxygen under the conditions used for demonstration purposes is that present initially in the air-saturated medium and in the head space of the containing vessel prior to sealing the vessel. A more general approach is to allow entry o~ suitably small amounts of oxygen or air during the fermentation.

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6~45 (Il) Selected yea~sts are required for the process. Not all that grow on D-xylose pro~iuce suitable yields and rates of ethanol production.
Species that runction particularly well are listed in Table 1.

Candida fennica CBS 6087 Candida insectorum ATCC 22940 Candlda _shiwadae ATCC 22018 Candida silvanorum ATCC 22942 Candida steatolytica ATCC 18822 Debaryomyces polymorphus (cantarelli) ATCC 24172 Pichia ~ itis CBS 5773 Schwanniomyces occidentalis ATCC 26077 ____ When the cel.~.9 are grown aeroblca]ly ln the flr~t :~t~l~e, alr has contlnuolls ac~e3~ to the culture. Durlng culturing, the medium i9 also agitated to ensure dlstribution of air through the medium and to keep the cells in suspension. Suitable conditions use 10-20 ml of medium in a 125-ml Erlenmeyer flask shàken at 200 rpm on a gyratory shaker Wittl a radius of 0.28 inches.
The medium used to grow cells in the first stage contains, in additLon to D-xylose, nutrients required for growth of yeast. The nutrient can be supplied by a commercial synthetic medium: yeast nitrogen base without amino acids (Difco). Other media ; 25 are suitable provided the D-xylose is the sole or preponderant sugar present. Sodium phosphate may be added to assist in minimizing pH
changes. The pH on inoculation is 5.5 but other values that are suitable for yeast growth can be used. Concentrations of D-xylose that can be used range up to approximately 200 g/L. Temperature of growth is 30C, but variations over the range where yeasts can grow are acceptable.
Prior to beginning the fermentation step, the aerobically grown cells are removed from the medium in which they ; were grown. Centrifugatlon can be employed ~or the separation.

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The cel~s are t~1en resusperlded in the medium to be used for fermentation at an appropriate density.
In some of the exarnples illustrating the invention, the cells are washed in the medium to be used for fermentation prior to their final resuspension in this medLum. The washing step is not a part of the invention. It is used in some examples to avoid carry-over of medium froln the growth phase, which might otherwise obscure some of the trends or results to be demonstrated.
The medium used in the fermentation step can contain D~xylose as the sole sugar. It can also be a mixture of sugars such as those found ln hydrolyzates of hemicellulose. A feature of hemicc~llulose hydr-olyzates i'3 that they contain D-glucose in additlon to l)-xy]ose.
Wlth respect to components o~ t~1e mediurn other than sugar, the composition can be the same as that used for growth.
However, in some instances, as specified below, water plus D-xylose suffices. Conditions of pH and temperature are the same as for growth.
F`or the ferlnentation step proper, the culture is placed in vessels whLch are then sealed so that alr does not have continuous access. Plastic centrifuge tubes with tightly fittin~ caps are suitable. Sultable conditlons employ 0.5 ml of culture in a 1.5 ml tube. The alr available to these cultures is that dissolved in the medium plus that in the volume of the tube not occupied by liquid.
The vessels are normally opened only after the fermentation has been completed. }lowever, opening the tubes periodically for sampling purposes, and then sealing again, does not have an appreciable e~fect on the results, as far as demonstration of trends is concerned.
The amount of air that enters tile culture is important since amounts that are too low or too hi~h cause lower yields. An exalnp1e when too 1ittle air is provlded is that when the 1.5 ml tu~,e ls cornpletely rllled Wit}1 culture. In this lnstarlce, ylelds are always lower by approximately 10%. An example of excess air is that wnen approximately 10 ml of culture is shaken vigorously in a flask that is loosely capped to allow ready access of air. In this i.~

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12~ 4,5 instance, ethanol is not detected in the medium.
Tlle fermentation pathway of D-xylose i3 not known. It is as.sumed that the pathway in the absence of continuous aeration results in th~ followirlg con~Jersion.
; 5 CsHI 005 ~ 5 C2HsOH + 5 C02 (3) (3) (D-xylose) (ethanol) Tl1e theoretical conver.si~n (100% of theoretical) according to this rCl(ltlOrl i9 0.5~ g i:'l,h,lllOI/~ xylost,~ IlSe(i. IJnderl the (-on(ittlon3 ~ f`l(lJ yi~ v~ rl t)l)~llrl~ t ~ r~ (>r~ Jl V.:ll IJ(`.
In ad(iition to providing high ylelds, the conditlons specified result in high rates of production. Yields equivalent to theoretical have been obtained from 10% D-xylose in 17 hours. With 2% D-xylose, yields exceeding 80% of theoretical have been obtained in 6 hours.
E _ ple 1_ Physiologica] Stat_ of Cells Crowth of ceLls to a particular physiological state or age i9 important to the process sLnce ethanol productivity in the fermentation step varies dramatical1y with the physiological state or àge. An example i3 shown in Flgllre 1 for Schwanniomyces occidentalis. As the cu1ture grows, when aliquots are removed and subjected to fermentation conditions, ethanol productivity passes through a maximum. Notably, productivity peaks when ce]ls are removed from the growing culture at a point approximating that where the last doubling of cell mass begins.
For the example in Figure 1, the medium used for growth contained 10% D-xy]ose, 0.67~ ycast nitrogen base (Difco [TM]), 0.4 M
sodlllm pho.sp~late. The pll was adju.~ted lnitially to 5.5. Crowth was followeti turbklimetrlcally and is expressed as Klett units.
Fermentation employed 0.5 ml alLquots of aerobically grown culture ln 1.5 ml centrifuge tubes. The cells were washed once in the medium to be used before final resuspension to their original cell density.
The medium used contained 10~ D-xylose, 3.35% yeast nitrogen base (Difco) plus 0.4 M sodium phosphate. The pH was adjusted initially to 5.5.

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Temperature for growti1 arid f~Ment~tior1 was 30~C. Ethanol concentration was measured after 19 hours of incubation and ethanol productivity wa~s expressed as grams of ethanol produced per 10 mLs of medium per gram unit weight; of cells.
The plrticlilar tlrne at whLch the rnaximllm of ethanol productlvity ocellrs vati(>s wltll flctor~ that de1errnine the rate of gr OWtil Or yea:~ts Irl gcnerlL. These factors lnc1ude the concentratlon of D-xylose, the nature and concentration of ot~ler components of the medium the pH and the temperature. The time will also depend on the particular yeast being grown. Because of the large number of factors that can influence the time for maximum productivity for purposes of optimization, this time will have to be determined specifically for each yeast and set of growth conditions. The examples which follow are intended to demonstrate particular points and are not intended to define optimal conditions.
Example 2 Effects of Cell Density on Rate on Yield of Ethanol Production In the fermentation step both the rate and the yield are influenced by the density of cells. Appropriate densities must therefore be used to obtain suitable performance. Table 2 illustrates the effects of increasing cell density using Schwanniomyces occidentalis. As the cells are concentrated over and above the value in the growth medium, the concentration of ethanol that appears in the medium after 17 hours increases. With a biomass concentratlon flve tLmes that in the growth medlurn the ethanol conocntration found, 4.98~ (5x) corre~pond~ to 97.1l% of theoretlcal.
Th~ ceLL3 u~ed for the exarnple were growrl for 53 ~ours in 10~ D-xylose containing 3.35% yeast nitrogen base (Difco) and 0.4 M sodium phosphate, pll 5.5. The fermentation medium was water plus 10g D-xylose. The cells were washed once in the fermentation medium prior to flnal resuspension.;

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Cell Den~ity and Ethanol Production Rate and Yield in Schwanniomyces occidenta1js 5 Cell Biomass Per cent ethanol formed in 17 hours 0.5x 1.45 1.0x 2.19 2.0x 4.06 5.0x 4.98 Example 3 Rate and Yield of Ethanol Production .
An additional example o~ the high rates and yields that are achievable i9 shown in Figure 2 using 5% D-xylose and Pichia stipitis. Within seven hours, the concentration of ethanol obtained was 2% (w/v), or 78% of theoretical. Longer terms fermentation, 24 hours, resulted in 2.48% (w/v) ethanol or 97% of theoretical. The cells used had been grown aerobically for 20 hours in 5% D-xylose, 3.35% yeast nitrogen base (Difco), 0.4 molar sodium phosphate, pH 5.5 in a 20 ml volume of medium contained in a 1 25-ml Erlenmeyer flask.
For the fermentation step, the cells were concentrated 8.5 fold and placed in 5% D-xylose, 0.67% yeast nitrogen base 0.4 molar sodium phosphate, pH 5.5.
Exam~le 4 Recyclability of Cells Cells harVested at the physiological state where ethanol productivity 1s at or near its maximum can be recycled many times in the fermentation step without 1099 of productivity. An example for 32 successive daily cycles is shown~in~Figure 3 for~five~different yeasts (crosses = Schwannio=yces~oocidentalis;~opèn circles~=~ Candida silvanorum; solid triangles~=~Pichia~stipitis;;open oircles = C.
ishiwadae;~ open~invèrted triangles~- C.~fenni~ca)~
The;cells~were~grown~in 5%~xylose,~0.4~M dibasic sodium~phosphate, pH 5.5 at 30~C~ror~bwo~tr~ansfers~after having been prevlously grown for~many~transfers~in~-1% xylose~ Inoculum = 1% or ;3g~o~.lo~ml~intQ~lo ml~o~5%~xylose.~
Fermentation conditions~employed 0.97~ D-xylose in 0.67~ yeast nitrogen base, ~0~.~2 M~sodium~phosphatej~ pH~5.5. ~Culture volumes~were 0.7 m1s~and were~ke~pt~in~sterile~1~.5~=1 p1ast1c ~;:66~

centrifuge tubes that were tightly ~ealed. The tubes were shaken at 120 stro~es per minute for the first two cycles and 200 strokes per rninute subsequently. The recycling interval was 20-30 hours.
The maxirnum theoretical yield of ethanol expected from the concentration of D-xylose used in the fermentation step is 0.4 w/v. The experimerltal yield centers around 0.4~ w/v or 87~ of theoretical. This example is intended to illustrate recyclabllity, and not to indicate or specify sampling interval3 to maxirnize yield.
Up to 50 cycles have been used with 1% w/v D-xylose, without deleterious e~fects on rate or yield. With 5% w/v D-xylose, 50 cycles have been used without affecting yields.
When relatively low concentrations of xylose are used (l-2%), nutrients nece3sary for growth need not be added to the medium used in the fermentation step when recycling of cells i9 employed. Water is satisfactory. However, nutrients are necessary for long term recyclabillty (more than 5 recycles) with higher concerltratlon~ o~` xyl.o.se.
Example ~
SuKclr Mlxtures Ce~Ll3 ~rown on D-xylose can efficLently produce ethanol from mlxture3 containing D-gluco3e as well as D-xylose. An example with Pichia stipitis is shown in Figure 4 for a series of mlxtures of D-glucose and D-xylo~e. The concentration of ethanol obtained is 2~
w/v arld greater, An ethànol concentration of 2% w/v correspondS to 78% of theoretioal. Growth of the cell9 on D-xylose open circles is essential to obtain high ethanol yields from the mixtures. When D-gluco3e grown cells are used (solid circles), yields of ethanol after 72 hour~ of fermentation decrease as the propor-tion of D-xylose increases.
The cells used were grown in either 5% D-xylose or 5%
D-glucose in .67~ yeast nitrogen base~, 0.4 molar sodium phosphate, pH
5.5.~ The inocula for the D-glucose and D-xylose cultures were obtained from cultures transferred repeatedly on D-glucose and D-xylose, respectively. ~The cells were harvested after l9 hours of growth and washed once in the medium to be used subsequently for ~: :: : :

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fermentation. They were then resuspended in this medium at a density equal to nine times that when they were harvested from the growth medium. The mediurn used for fermentation contained in addition to D-xylose, 0.67% yeast nitrogen base, 0.4 molar sodium phosphate, pH
5.5.

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Claims (9)

Claims:

1. A method for producing ethanol from a D-xylose-containing substrate comprising:
(a) (i) culturing cells of selected yeast species for several generations aerobically in the presence of D-xylose as substantially the sole source of sugar to condition said cells to D-xylose as a major carbon source;
(ii) harvesting said cells when they are in a physiological state wherein ethanol producton is about maximal;
and (b) inoculating D-xylose-containing medium with cells conditioned to D-xylose as a major carbon source and fermenting said medium under conditions deficient in oxygen such that said cells yield ethanol.

2. The method of claim 1 with the additional step of recycling cells resulting from step (b) back through step (b).

3. The method of claim 1 wherein the cells used in step (b) comprise ones derived from step (a) (ii).

4. The method of claim 1 with the additional step of recycling cells resulting from step (b) to the start of step (a)(1).

5. The method of claim 1 wherein the yeast cells are selected from the group consisting of Candida fennica CBS 6087 Candida insectorum ATCC 22940 Candida ishiwadae ATCC 22018 Candida silvanorum ATCC 22942 Candida steatolytica ATCC 18822 Debaryomyces polymorphus (cantarelli) ATCC 24172 Pichia stipitis CBS 5773 Schwanniomyces occidentalis ATCC 26077 Claims: (continued)

6. The method of claim 1 wherein step (a) (ii) comprises harvesting the cells at approximately the time of initiation of the last possible doubling of cell mass in the medium provided for said cells.

7. The method of claim 1 wherein step (a) (i) comprises culturing yeast cells from the group consisting of Candida fennica CBS 6087 Candida insectorum ATCC 22940 Candida ishiwadae ATCC 22018 Candida silvanorum ATCC 22942 Candida steatolytica ATCC 18822 Debaryomyces polymorphus (cantarelli) ATCC 24172 Pichia stipitis CBS 5773 Schwanniomyces occidentalis ATCC 26077 for several generations aerobically in the presence of D-xylose as a major carbon source;
and step (a) (ii) comprises harvesting the cells at approximately the time of initiation of the last possible doubling of cell mass in the medium provided for said cells.

8. The method of claim 1 step (b) wherein, some air is allowed access to the culture only at the beginning of the step.

9, The method of claim 1 step (b) wherein some air or oxygen is allowed access to the culture during the fermentation at a low rate sufficient to allow an improved fermentation yield to be obtained.