Continuous fermentation process

A fermentable sugar feed containing fermentable sugar oligomers is continuously converted by fermentation to dilute aqueous ethanol ("beer") in a series of agitated fermentation vessels employing at least two strains of yeast, one of which provides a relatively high rate of conversion of fermentable sugar to ethanol and the other of which provides a relatively high rate of conversion of fermentable sugar oligomer to ethanol.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to processes for the manufacture of ethanol by 
continuous fermentation. 
2. Description of the Prior Art 
With the ever-increasing depletion of economically recoverable petroleum 
reserves, the production of ethanol from vegetative sources as a partial 
or complete replacement for conventional fossil-based liquid fuels becomes 
more attractive. The substitution of ethanol for at least a portion of 
petroleum based fuels is particularly critical for developing economies 
where proven domestic petroleum reserves are limited. In some areas, the 
economic and technical feasibility of using a 90% unleaded gasoline-10% 
anhydrous ethanol blend ("gasohol") has shown encouraging results. 
According to a recent study, gasohol powered automobiles have averaged a 
5% reduction in fuel compared to unleaded gasoline powered vehicles and 
have emitted one-third less carbon monoxide than the latter. In addition 
to offering promise as a practical and efficient fuel, biomass-derived 
ethanol in large quantities and at a competitive price has the potential 
in some areas for replacing certain petroleum-based chemical feedstocks. 
Thus, for example, ethanol can be catalytically dehydrated to ethylene, 
one of the most important of all chemical raw materials both in terms of 
quantity and versatility. 
The various operations in processes for obtaining ethanol from such 
recurring sources as cellulose, cane sugar, amylaceous grains and tubers, 
e.g., the separation of starch granules from non-carbohydrate plant matter 
and other extraneous substances, the chemical and/or enzymatic hydrolysis 
of starch to fermentable sugar (liquefaction and saccharification), the 
fermentation of sugar to a dilute solution of ethanol ("beer") and the 
recovery of anhydrous ethanol by distillation, have been modified in 
numerous ways to achieve improvements in product yield, production rates 
and so forth. For ethanol to realize its vast potential as a partial or 
total substitute for petroleum fuels or as a substitute chemical 
feedstock, it is necessary that the manufacturing process be as efficient 
in the use of raw materials as possible so as to maximize the amount of 
product produced per unit of carbohydrate feed and thereby enhance the 
standing of the ethanol as an economically viable replacement for 
petroleum based raw materials. To date, however, relatively little concern 
has been given to optimizing the manufacture of ethanol from biomass and 
consequently, little effort has been made to eliminate or minimize the 
small but significant raw material losses which take place in each of the 
discrete operations involved in the manufacture of ethanol from vegetative 
sources. 
Processes for the continuous fermentation of sugars to provide alcohol are 
well known (viz., U.S. Pat. Nos. 2,155,134; 2,371,208; 2,967,107; 
3,015,612; 3,078,166; 3,093,548; 3,177,005; 3,201,328; 3,207,605; 
3,207,606; 3,219,319; 3,234,026; 3,413,124; 3,528,889; 3,575,813; 
3,591,454; 3,705,841; 3,737,323; and 3,940,492; "Process Design and 
Economic Studies of Alternative Fermentation Methods for the Production of 
Ethanol", Cysewski, et al. Biotechnology and Bioengineering, Vol. XX, pp. 
1421-1444 (1978)). In a typical continuous fermentation process, a stream 
of sterile sugar liquor and a quantity of yeast cells are introduced into 
the first of a battery of fermentation vessels wherein initial 
fermentation takes place, generally under conditions favoring rapid cell 
growth. The partial fermentate admixed with yeast cells is continuously 
withdrawn from the first fermentation vessels wherein fermentation is 
carried out under conditions favoring the rapid conversion of sugar to 
ethanol. The yeast in the last fermentation vessel can be recovered by 
suitable means, e.g., centrifugation or settlement, and recycled. In the 
various known processes for obtaining fermentable sugar from starch by 
hydrolysis of the latter, particularly those which employ acid as the 
hydrolyzing agent, some of the product fermentable sugar will undergo 
chemical modification to provide oligomers, for example, dimers and/or 
trimers, which are not readily converted to ethanol using yeasts commonly 
encountered in the brewing industry. The oligomers which have resisted 
conversion to ethanol and are therefore present in the product "beer" 
stream at the conclusion of fermentation are recovered in the distillation 
bottoms during the process of concentrating the ethanol. Up until now, it 
has been necessary to re-hydrolyze the recovered oligomers to fermentable 
sugar and recycle the sugar re-hydrolysate to fermentation when maximum 
total utilization of raw material for the production of ethanol is 
desired. However, the additional capital investment needed to provide a 
plant having the capability to recover, rehydrolyze and recycle the 
oligomers, and the increased operational complexity and consumption of 
energy which this capability necessarily entails, have to date militated 
against the general adoption of the foregoing procedures. Thus, an 
otherwise valuable raw material for ethanol production, saccharide 
oligomer, is either being routinely discarded or diverted to uses other 
than ethanol production. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, an aqueous solution of 
fermentable sugar is continuously subjected to fermentation in a series of 
fermentation vessels employing at least two strains of yeast for the 
fermentation, one of which provides a relatively high rate of conversion 
of fermentable sugar to ethanol and the other of which provides a 
relatively high rate of conversion of oligomers of fermentable sugar to 
ethanol. 
The process herein also contemplates the adjustment of temperature and/or 
pH in each fermentation vessel as required to maintain optimum 
fermentation activity therein. 
The aqueous ethanol or "beer" containing as much as about 12 weight percent 
ethanol which is obtained by the foregoing process can be concentrated 
employing any of the known and conventional techniques and is 
advantageously concentrated by the anhydrous distillation process 
disclosed in commonly assigned copending U.S. pat. application Ser. No. 
043,189, filed May 29, 1979, now U.S. Pat. No. 4,256,541, issued Mar. 17, 
1981. The stillage effluent obtained from the rectifying column employed 
in the aforesaid anhydrous distillation process contains soluble proteins 
and amino acids of the original beer feed and provides an excellent source 
of nutrient for yeast employed in the fermentation process herein. 
The use of two or more yeast organisms in the aforestated manner results in 
a significantly greater degree of direct carbohydrate conversion to 
ethanol compared to that provided by the use of a single strain of yeast 
in the current practice. As such, the fermentation process of this 
invention is particularly well suited for the production of ethanol which 
is price competitive with ethanol produced from non-vegetative sources. 
The term "fermentable sugar" shall be understood to refer to a single 
fermentable sugar such as glucose (dextrose), fructose, maltose or sucrose 
but more commonly will be applicable to these and similar fermentable 
saccharides in admixture. The terms "fermentable sugar oligomer" and 
"saccharide oligomer" shall be understood to refer to partially 
repolymerized fermentable sugar, i.e., carbohydrate polymer segments built 
up from two to five units of fructose, maltose, sucrose, etc, or from 
three to five units in the case of glucose.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Referring to the drawing, a sterile aqueous solution of fermentable sugar 
from any source containing from about 10 to about 40 weight percent sugar, 
and preferably from about 15 to about 25 weight percent sugar, is taken 
from vessel 50 which can be a storage vessel or a saccharification vessel 
in which the sugar is obtained by the hydrolysis of liquefied starch, and 
is delivered by pump 51 through line 52 to a first temperature regulated, 
agitated fermentation vessel 53 provided with pH control and means for 
introducing nutrients and the small amounts of oxygen conventionally 
employed for maintaining proper yeast metabolism during fermentation. In 
the event the sugar solution contains more than 20 weight percent sugar, 
it is preferable to dilute the solution to about this level of sugar, 
advantageously with the nitrogen-rich stillage obtained from an ethanol 
distillation unit. The use of stillage when available possesses the 
two-fold advantage of recycling nitrogen to the fermentation system which 
would otherwise be lost upon concentration of the ethanol during 
distillation, and reducing process water consumption by avoiding water 
build-up in the still bottoms. In addition to sugar, the foregoing 
solution may also contain significant amounts of partial starch 
hydrolysates (e.g., up to about 40 weight percent of the total 
carbohydrate present) which can be saccharified to fermentable sugar under 
the influence of the saccharifying enzyme produced by the fermenting yeast 
and/or added saccharifying enzyme. A pumpable slurry of ethanol producing 
yeast organisms free of contaminating organisms is conveyed from yeast 
storage tank 54 by pump 55 through lines 56 and 57 into fermentation 
vessel 53. The yeast selected for introduction in fermentation vessel 53 
is one which provides relatively high rates of ethanol production acting 
upon a substrate of fermentable sugar. Many yeasts which perform in this 
manner are known in the brewing industry. Saccharomyces cerevisiae is one 
such yeast and is especially preferred for use herein. The yeast present 
in fermentation vessels 53 and 67 can be present at a level of from about 
2 to about 8 weight percent of the fermentation medium (based on dry 
weight of yeast) and preferably is present at from about 3 to about 6 
weight percent. Once continuous fermentation has started and a steady 
state has been achieved, excess yeast will form which must continuously or 
periodically be withdrawn from the fermentation vessel together with the 
debris of dead yeast cells. The temperature of each fermentation vessel is 
advantageously regulated at a level which favors maximum ethanol 
production, i.e., generally from about 68.degree. F. to about 104.degree. 
F. and preferably from about 86.degree. F. to about 99.degree. F. The pH 
of each fermentation vessels is similarly regulated and can range from 
about 3.5 to about 5.5 and preferable from about 4.0 to 4.6. Dilute 
ethanol produced in fermentation vessel 53 containing a portion of the 
yeast cells therein is conveyed by pump 58 through line 59 to yeast 
separator/recovery unit 60 which separates substantially all of the yeast 
cells from the aqueous ethanol stream. Unit 60 can be a micro-filtration 
device, centrifuge, etc. Since fermentation is exothermic, a portion of 
the fermentation medium passing through line 59 is diverted through line 
61 into cooler 62 and returned to fermentation vessel 53. The yeast cells 
recovered in unit 60 are conveyed as a pumpable slurry or "cream" 
containing from about 5 to about 30 weight percent dry yeast and 
preferably from about 10 to 25 weight percent dry yeast by pump 63 through 
lines 64 and 57 into fermentation vessel 53. The ethanol-containing 
fermentation medium thus freed of the yeast cells is delivered by pump 65 
through line 66 into fermentation vessel 67 which is essentially similar 
to fermentation vessel 53. A pumpable slurry of ethanol-producing yeast 
organisms essentially free of contaminating organisms is conveyed from 
yeast storage tank 68 by pump 69 through lines 70 and 71 into fermentation 
vessel 67. The yeast selected for introduction in fermentation vessel 67 
is one which is especially effective for converting saccharide oligomers 
to ethanol, several of which are known in the art. Saccharomyces 
carlsbergensis and Saccharomyces cerevisiae var. ellipsoideus are 
illustrative of yeasts which can be advantageously used in fermentation 
vessel 67. The dilute aqueous ethanol (approximately 10 to 12 weight 
percent ethanol) containing yeast cells is withdrawn from fermentation 
vessel 67 and conveyed by pump 72 through line 73 to yeast 
separator/recovery unit 74. A portion of the fermentation medium passing 
through line 73 is diverted through line 75 into cooler 76 and returned to 
fermentation vessel 67. The yeast cells recovered in unit 74 are conveyed 
as a pumpable slurry (similar in fluid characteristics to the yeast slurry 
recovered from unit 60) by pump 77 through lines 78 and 71 to fermentation 
vessel 67. The cell-free ethanol solution from yeast separator/recovery 
unit 74 is delivered by pump 79 through line 80 directly to an ethanol 
concentration unit, e.g., anhydrous distillation apparatus, and/or to a 
storage facility. It is also within the scope of this invention to employ 
both types of yeast herein in such fermentation vessel with only one yeast 
separation/recovery unit (receiving the fermentation medium from the last 
fermentation vessel in the series) being provided. Metabolically evolved 
carbon dioxide gas containing ethanol is conveyed from each of 
fermentation vessels 53 and 67 through common line 81 and by means of 
blower 82 is introduced into the bottom of ethanol absorption unit 83. 
Water at ambient temperature entering the top of the absorption unit 
through line 84 and flowing downwardly, absorbs substantially all of the 
ethanol vapor rising through the unit. The aqueous solution of ethanol 
withdrawn from the base of ethanol absorption unit 83 through line 85 is 
conveyed to line 80 where it is combined with the bulk of the flow from 
the last fermenter. Vent gases are discharged from ethanol absorption unit 
83 through atmospheric vent line 86.