CA1145277A - Fermentable acid hydrolyzates and fermentation process - Google Patents
Fermentable acid hydrolyzates and fermentation processInfo
- Publication number
- CA1145277A CA1145277A CA000361776A CA361776A CA1145277A CA 1145277 A CA1145277 A CA 1145277A CA 000361776 A CA000361776 A CA 000361776A CA 361776 A CA361776 A CA 361776A CA 1145277 A CA1145277 A CA 1145277A
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- hydrolyzate
- acid
- glucose
- fermentation
- steam
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
ABSTRACT OF THE DISCLOSURE
Method of preconditioning acid hydrolyzates derived from lignocellulosic materials such as sawdust or newspaper and preconditioned acid hydrolyzates are provided. The precondi-tioning negates the effect of substances which tend to inhibit fermentation and comprises a series of steps including steam-stripping, calcium oxide treatment at a pH of 10 to 10.5, ad-justing the pH to about 6 to 7 with a mineral acid and especial-ly phosphoric acid and concentrating the hydrolyzate solution to a glucose concentration of less than 150 grams per liter.
Glucose contained in such preconditioned hydrolyzates is readily fermentable to ethyl alcohol,in theoretical yield, after fer-mentation for as short a period as 1 to 2 hours.
Method of preconditioning acid hydrolyzates derived from lignocellulosic materials such as sawdust or newspaper and preconditioned acid hydrolyzates are provided. The precondi-tioning negates the effect of substances which tend to inhibit fermentation and comprises a series of steps including steam-stripping, calcium oxide treatment at a pH of 10 to 10.5, ad-justing the pH to about 6 to 7 with a mineral acid and especial-ly phosphoric acid and concentrating the hydrolyzate solution to a glucose concentration of less than 150 grams per liter.
Glucose contained in such preconditioned hydrolyzates is readily fermentable to ethyl alcohol,in theoretical yield, after fer-mentation for as short a period as 1 to 2 hours.
Description
~ g~ ~ ~
_ckground of the Invention Traditionally, used wood, paper and agricultural by-products, such as sawdust, woodwaste, corncobs, straw, sugar cane bagasse, newspaper and the like have been regarded essen-tially as waste materials, and have been disposed of through incineration or by other, similarly unproductive, means. It is well known that the lignocellulosic constituents of such materials can be hydrolyzed to produce more valuable products which in turn can be converted into additional and different valuable products; however, such operations are in limited use, due largely to the relatively low returns on investment which they have been capable of generating. The capital expenditures required to design and construct the facilit,ies for carrying out such recovery operations tend to be significant, thus demanding that relatively high conversion rates be attainable in order to justify the expense invo:Lved.
In U.S. Patent No. 4,201,596 issued May 6, 1980 to John A. Church et al entitled "Continuous Process For Cellulose Saccharification" and commonly assigned herewith, there is described a method and apparatus for saccharification of cellu-losic products in which the cellulosic constituents of typi-cal waste products may be converted into glucose, furfural and xylose. Such a process conveniently, rapidly and econom-ically provides by acid cellulose hydrolysis, a hydrolyzate which may be used as the raw material for the production of more valuable products. For e~ample, as disclosed in U.S.
Patent No. 4,201,596, such hydrolyzates may be used as the raw fermentable substance in a process for converting sugar into ethyl alcohol.
~hile it has long been known that cellulosic hydro-lyzate solutions ma~ be made fermentable for the production of alcohols, prior procedures have been feasible only on a small -- 1 -- ~ "~., ~s~
laboratory scale and have not significantly developed beyond this stage. Among the principal conditions contributing to this state of the art has been the inordinately slow rates of reaction with extremely low yield and the lack of predicta-bility of conditions that would pernit fermen~ation with any given hydrolyzate. Additionally, the economic considerations inherent in the chemical conversion of sugar to alcohol have been a limiting factor. For example, it is only theoretically possible to obtain one unit of alcohol from every two units of sugar present in the raw material. Losses in sugar content of the raw material through process conditions, mechanical processing, etc., serve to even further decrease the low yields that it has been possible to obtain.
Cellulosic extractives and their decomposition pro-~ ducts as fermentable raw materials have been particularly ; enigmatic to approach because of the wide variety of factors, many of which are unknown, that adversely affect and, in many cases, prevent the fermentation process. One factor that has long been recognized in the art as significantly retarding the development of a feasible fçrmentation process has been the presence of materials in the hydrolyzate that act as toxins or fermentation inhibitors. However, the toxins present in any given hydrolyzate may vary considerably depending on its processing history, its source, etc. Moreover, the problem is further compounded by the fact that even after the particu-lar toxins have been identified in a given hydrolyzate, their action under any given set of conditions has been largely un-predictable and fermentation has been difficult even under special conditions. Various wor~ers in the art have suggested that 1hese difficulties may be dependent on any number of factors including processing temperatures, pH of the media, the presence or absence of oxygen, the concentration and type
_ckground of the Invention Traditionally, used wood, paper and agricultural by-products, such as sawdust, woodwaste, corncobs, straw, sugar cane bagasse, newspaper and the like have been regarded essen-tially as waste materials, and have been disposed of through incineration or by other, similarly unproductive, means. It is well known that the lignocellulosic constituents of such materials can be hydrolyzed to produce more valuable products which in turn can be converted into additional and different valuable products; however, such operations are in limited use, due largely to the relatively low returns on investment which they have been capable of generating. The capital expenditures required to design and construct the facilit,ies for carrying out such recovery operations tend to be significant, thus demanding that relatively high conversion rates be attainable in order to justify the expense invo:Lved.
In U.S. Patent No. 4,201,596 issued May 6, 1980 to John A. Church et al entitled "Continuous Process For Cellulose Saccharification" and commonly assigned herewith, there is described a method and apparatus for saccharification of cellu-losic products in which the cellulosic constituents of typi-cal waste products may be converted into glucose, furfural and xylose. Such a process conveniently, rapidly and econom-ically provides by acid cellulose hydrolysis, a hydrolyzate which may be used as the raw material for the production of more valuable products. For e~ample, as disclosed in U.S.
Patent No. 4,201,596, such hydrolyzates may be used as the raw fermentable substance in a process for converting sugar into ethyl alcohol.
~hile it has long been known that cellulosic hydro-lyzate solutions ma~ be made fermentable for the production of alcohols, prior procedures have been feasible only on a small -- 1 -- ~ "~., ~s~
laboratory scale and have not significantly developed beyond this stage. Among the principal conditions contributing to this state of the art has been the inordinately slow rates of reaction with extremely low yield and the lack of predicta-bility of conditions that would pernit fermen~ation with any given hydrolyzate. Additionally, the economic considerations inherent in the chemical conversion of sugar to alcohol have been a limiting factor. For example, it is only theoretically possible to obtain one unit of alcohol from every two units of sugar present in the raw material. Losses in sugar content of the raw material through process conditions, mechanical processing, etc., serve to even further decrease the low yields that it has been possible to obtain.
Cellulosic extractives and their decomposition pro-~ ducts as fermentable raw materials have been particularly ; enigmatic to approach because of the wide variety of factors, many of which are unknown, that adversely affect and, in many cases, prevent the fermentation process. One factor that has long been recognized in the art as significantly retarding the development of a feasible fçrmentation process has been the presence of materials in the hydrolyzate that act as toxins or fermentation inhibitors. However, the toxins present in any given hydrolyzate may vary considerably depending on its processing history, its source, etc. Moreover, the problem is further compounded by the fact that even after the particu-lar toxins have been identified in a given hydrolyzate, their action under any given set of conditions has been largely un-predictable and fermentation has been difficult even under special conditions. Various wor~ers in the art have suggested that 1hese difficulties may be dependent on any number of factors including processing temperatures, pH of the media, the presence or absence of oxygen, the concentration and type
- 2 -5~77 of toxin substance, the ratio of yeast cells to toxin sub-stances, the physiological condition of the yeast cells, the wide ~ariation in the toxicity of various substances on the metabolism of the particular yeast, the oxidation-reduction potential developed duriny reaction, and many other factors.
Discussions of the various difficulties of fermentation and general factors influencing fermentation are found in many sources in the literature.
Cumulative discussions are given by Harris et al in "Fermentation of Douglas Fir Hydrolyzates by S. cerevisiae"
and Leonard et al, "Fermentation of Wood Sugars to Ethyl Alcohol"; Industrial and Engineering Chemistry, Vol. 38, pp.
896 to 904, (1946) and Vol. 37, pp. 390 to 397, (1946), respec-tively.
Other workers in the art include Eklund et at, "Acid Hydrolysis of Sunflower Seed Husks for Production of Single !~ Cell Protein", European ~ournal of Applied Microbiology, Vol. 2, pp. 143-152 (1976) who disclose a method of hydrolyzing sun-flower seed husks and degradation of the resulting hydrolyzates to produce protein.
German Patent 676,967 to Scholler (1939) describes a method for clarifying xylose worts obtained by acid hydrolysis of cellulose-containing substances for feed purposes or yeast production by precipitating calcium phosphate and calcium sulfate after heating to 65 to 100 together with centrifuging and conducting the wort over oxidized metal fillings or large surface area materials while the wort is at a pH of 4 to 7.5, adding malt sprouts to the thus clari~ied wort and stirring for several hours.
U.S. Ratent No. 2,203,360 dated June ~, 1940 to Partansky discloses a method ~or improvin~ the fermentation characteristics of acid wood hydrolyzates by treating the hydrolyzate with lime to adjust the pH to between 9 and 10, aging for 1 to 2 days, reducing the pH with sulfuric acid to pH 5, purifying the solution with activated charcoal, diluting the solution to contain 40-70~ by volume of hydrolyzate, inocu-lating the solution with yeast culture and fermenting for 2 days.
The prior art, as represented by the methods dis-cussed above, is illustrative of the absence of a feasible com-mercial process for fermentation of acid hydrolyzates to alco-hol~ due to inordinately slow reaction times and low yields and/or the lack of direction for obtaining the samPO A method for readily and efficiently producing alcohol by fermentation of sugaxs present in wood and wood-byproducts is a particularly timely and significant development :in view of eurrent interest in alcohol as a potential energy source available from renew-able raw materials.
A primary object of this invention is to provide a process for fermentation of sugars present in acid hydrolyzates derived from lignocellulosic materlals.
Another object of the invention is to provide such a process in which reaction times are relatively short, in whieh fermentation may be effected at relatively high sugar concentrations and in which control mechanisms are established which permit predietability, reproduction of results with con-sistency and production of end products of high value~
Another object of this invention is to provide a process in which the acid hydrolyzate of cellulosic waste materials may be converted into ethyl alcohol.
The ~ccomplishment of these and other objects will be apparent from the descrip-tion of the invention which follows:
Summary_of the Inventlon The foregoing and related objects of this invention 7~7 are attained in a method for preconditioning acid hydrolyzates derived from lignocellulosic materials, to negate the effect of substances tending to inhibit the fermentation of such hydrolyzates and to a process for the production of ethyl al~ohol from glucose contained in such preconditioned acid hydrolyzates. The hydrolyzate is preconditioned to remove and/or reduce or otherwise negate the effect of inhibitory substances to a level whereby the hydrolyzate may be readily fermented to ethyl alcohol in substantially theoretical yield.
The preconditioning method broadly comprises the steps of: (1) subjecting the hydrolyzate to steam to remove furfural and other steam-volatile substances therefrom;
(2) adding sufficient calcium oxide to the steam-stripped hydrolyzate, at room temperature, to adjust the pH to between about 10 and 10.5, maintaining the resulting mixture at said pH for about 1 to 3 hours and separating the hydrolyzate from the resultant precipitate; (3) adding sufficient amounts of a mineral acid to adjust the p~ of said hydrolyzate to about 5 to 7; and (4) adjusting the concentration of said hydroly-zate to a glucose concentration of less than about 150 grams pe~ liter to provide a solution fermentable to ethyl alcohol.
The fermentation process broadly comprises the steps of: (1) preconditioning an acid hydrolyzate to negate the effect of substances tending to inhibit the fermentation thereof by subjecting the hydrolyzate to the preconditioning method described hereinabove; (2) inoculating the precondi-tioned hydrolyzate with yeast inoculum comprising from about 0.7 to about 7 dry weight percent of yeast cells per 100 grams per liter of glucose in the hydrolyzate; (3) fermenting the inoculated ` 30 hydrolyzate at a pH of 5 to 7 for a period sufficient to con-vert glucose to ethyl alcohol; and (4) recovering ethyl alcohol from the fermentation mixture.
~! ~
~5~7~1~
In a preferred embodiment, yeast cells are recovered, reconcentrated and recycled to a subsequent fermentation medium comprising preconditioned, concentrated hydrolyzate.
General Disclosure The process of this invention utilizes a combination of steps and conditions which are interrelated and interdepen-dent for the successful achievement of the objectives of the invention. This interrelationship will best be seen from the following description of the effect or function of each parti-0 cular sequence within the context of the total process.Hydrolyzate Raw Material The invention may be successfully realized with any hydrolyzate derived from the acid hydrolysis of lignocellu-losic material. Such lignocellulosic material may be selected from a wide variety of materials including wood and paper and particularly used paper and wood by-products such as sawdust, wood waste, straw, sugar cane bagasse, rice hulls, newspaper and the like. Such materials may be hydrolyzed in the presence of an acid catalyst by methods well known in the art to pro-vide a suitable hydrolyzate raw material for use in the pro-cess.
The hydrolyzate raw material provided will vary in sugar content and other components depending on the conditions under which it has been produced. This can be best understood by a consideration of the chemistry involved in acid hydroly-sis stated for the sake of illustration in simplified terms.
When cellulosic material is heated with dilute aqueous acid, glycosidic bonds which connect individual anhydroglucose units to one another in the cellulose molecule are cleaved by acid catalysis and one molecule of water adds to each anhydroglucose unit to fornl one molecule of glucose as illustrated by the idealized equation:
7~ .
H-~
(C6 ~llo s)n ~ n 2 ~ ~ 6 12 6 Glucose is inherently unstable in hot acid solutions and can lose three molecules of water to yield 5-hydroxymethylfurfural (HMF) ac-oxding to the equation:
H+
6 12 6 ~ ~ C6 H6 3 + 3H2O
HMF in turn is unstable and can add two molecules of water to yield levulinic acid and formic acid:
H~
C6 H6 3 + 2H2 ~ ~ C5 8 3 Other very complex reactions also occur in which it is believed HMF condenses into dark insoluble residues known as humins.
Lignin breakdown products such as vanillin or other aromatic compounds may also be present. Additionally, the bonds of the hemicellulose molecule are cleaved to produce free molecules of xylose from xylan. Cex~ain reaction conditions will favor formation of glucose or xylose and accompanying decomposition ; products.
An especially preferred method and apparatus for producing suitable acid hydrolyzate raw materials for use in this invention is that disclosed and claimed in U.S. Patent No. 4,201,596 entitled "Continuous Process for Cellulose Saccharification" referred to hereinabove. ~s disclosed therein, whereas the xylan conversion to xylose occurs at relatively low temperatures, the cellulose conversion to glu-cose best occurs under more severe conditions. It has not been possible to produce maximum amounts of both xylose and glucose in a one phase method due to the fact that the 5~77 xylose dehydrates to furfural under the conditions which most efficiently effect the conversion of cellulose to glucose.
The method of U.S. Patent No. 4,201,596 defines the conditions which favor production of glucose/furfural and which minimize degradation of glucose to HMF and levulinic acid. Since, as discussed and illustrated further hereinbelow, furfural, HMF
and levulinic acid are each toxins to the fermentation organ~
ism, the present invention most preferably utilizes a hydro-lyzate raw material obtained under such conditions that mini-mize, to the extent possible, the presence of such toxin sub-stances.
Such conditions in general provide for acid hydro-lysis, in the presence of steam, of cellulose feedstock having a solids c~ntent of from about 20 to 45 weight percent at temperatures within the range of about 190 to 225 C and pres-sures of about 200 to 400 psi with residence times in a reac-tion zone of about 1 to 10 minutes. In the reaction mass, the optimum amount of water after steam injection is about 75 to 80 weight percent and virtually any strong mineral acid can be employed to catalyze the hydrolysis reactant, sulfuric acid being normally the acid employed in amounts of about 1 to 3 percent based on the total weight of the reaction mass.
In this preferred process for production of hydrolyzate raw material, the reaction mass will be subjected to an abrupt pressure reduction whereby a fraction of the hydrolyzate vaporizes and may be recovered. This fraction will normally comprise furfural and acetic acid.
It will be understood that the above description is for purposes of illustration of the preferred mode of obtain-ing an acid hydrolyzate that is especially suitable for usein the present invention. The method of the invention may utilize acid hydrolyzates from any source since it is a feature o~ the invention that the preconditioning method will serve to reduce or remove certain of the toxin materials to - tolerable levels and/or to otherwise negate the effect of such materials without substantially adversely affecting the glucose present in the hydrolyzate.
Such hydrolyzate raw materials will in general, how-ever, comprise glucose, furfural, 5-hydroxymethylfurfural, acetic acid, formic acid, and levulinic acid and will have a p~ of less than about 1.5 and preferably of about 0.5.
Preconditioning of the Hydrolyzate The hydrolyzate raw material as received is a con-glomerate of chemical substances. Many of such substances act as inhibitory agents or toxins to the yeast while many of such substances are unknown in identity and effect. It is possible that the presence of such substances, known or un-known, may exert a cumulative affect on the fermentation mech-ism or yeast culture. It is also possible that some of the substances may be combining synergistically to inhibit either the particular yeast organism or other mechanism involved in the fermentation. Thus, while there is necessarily a degree of uncertainty as to exactly how the objectives of this in-vention are realized, it is believed that the preconditioning method renders the hydrolyzate fermentable either through removal of toxin substances or through conversion of at least a portion of such substances to non-toxin forms.
Several materials that are known toxins have been found to be present in the acid hydrolyzates derived from the lignocellulosic materials utilized herein. Their effect has been quantified to enable elimination or at least mini-mization of the same. The effect of such substances may beseen from the results of the following experiments in which an anaerobic culture of S. uvarum was employed at about 0.7 - g _ ~` .
7~
dry weight percent cell concentration with acid hydrolyzates under the conditions indicated and employing identical inocu-lum and fermentation media. Glucose sugar determination was made using a Beckman Glucose Analyzer.
Where fermentation was achieved or attempted, the steam-stripped, CaO pre-treated hydrolyzate was neutralized with HCl as the neutralizing agent.
Cell concentration as referred to herein is deter-mined by optical densit~ or dry weight measurements.
1. Acetic and Formic Acids Both acids are toxins to the alcohol-producing yeast. Acetic acid is present in the hydrolyzate in concentra-tions of about 3 to 4 g/l while formic acid is present in amounts of about 8 to 9 g/l. The toxic effect of these acids can be negated by conducting the fermentation at a pH of abcut 5 to 7, preferably about 5.5 to 6.5. Fermenting below about pH 5 to 6 does not negate the effect of the toxins while fermenting above pH 7 is unfavorable for ethanol production.
The parameters may best be illustrated by the results from the Eollowing experiments in which a newspaper hydrolyzate was preconditioned in accordance with the invention and analyzed ! for Eormic and acetic acids. A control solution of pure glu-cose was also provided. Identical nutrients in identical amounts were added to each of the solutions. Each of the solutions was inoculated with 0.7~ yeast cells and allowed to ferment for 18 hours. The results were as indicated in Table 1.
T~LE I
Fermentation of ~lucose in Presence of Formic and _ Acetic Acids at pH 4.0 and pH 7~0 g/l pH 4.0 pH 7.0 -A. Control Experiment Initial Glucose 40.0 40.0 Added Formic Acid 9.0 9,0 Added Acetic Acid 4.0 4.0 Final Glucose 41.0 0 Final Ethanol 0 18.5 % ~ield (based on Glucose) 0 46.3 B. Newspaper Hydrolyzate Initial Glucose 36.4 39.8 Contains Formic Acid3.0 3.0 Contains ~cetic Acid3.0 3.0 Final Glucose 36.4 0 Final Ethanol 0 18.9 Yield o 47.5%
2. 5-Hydroxymethylfurfural_(HMF) HMF is a strong inhibitor of yeast growth. However, this material can be destroyed or degraded by CaO treatment at room temperature at a pH o~ about 10 to about 10.5 without adversely affecting the glucose. The pH range is believed to be critical herein since at a pH below about 10, the effect of HMF is not negated while at a pH above about 10.5, the su~ar product is unstable. It has been discovered that CaO
treatment of the hydrolyzates at pH 10 to 10.5 results in rapid depletion of HMF during the first two hours and levels off after that period. For example, it was observed that approximately 63~ of the HMF is removed in 1 hour at pH 10.5.
At pH 10.25, approximately 2 hours is required to remove the same amount of HMF while at pH 10.75, some glucose is concomi-tantly destroyed. There~ore, in the preferred embodiment of the invention, hydrolyzate is treated with sufficient CaO, at ro~m temperatu~e and with stirring, to maintain the pH at about 10.5 for about 1 to l.S hours.
,~ .
27~
The effect of the HMF on the yeast may be seen from the following experiments in which yeast growth as a function of HMF concentration in the hydrolyzate was determined at a pH within the fermentation range of 6 to 7. The doubling time is that time it takes for a cell to reproduce itself and was determined by optical density measurements.
H~F, g/l Doubling Time, hours Growth Rate, h-0 4.6 0.15
Discussions of the various difficulties of fermentation and general factors influencing fermentation are found in many sources in the literature.
Cumulative discussions are given by Harris et al in "Fermentation of Douglas Fir Hydrolyzates by S. cerevisiae"
and Leonard et al, "Fermentation of Wood Sugars to Ethyl Alcohol"; Industrial and Engineering Chemistry, Vol. 38, pp.
896 to 904, (1946) and Vol. 37, pp. 390 to 397, (1946), respec-tively.
Other workers in the art include Eklund et at, "Acid Hydrolysis of Sunflower Seed Husks for Production of Single !~ Cell Protein", European ~ournal of Applied Microbiology, Vol. 2, pp. 143-152 (1976) who disclose a method of hydrolyzing sun-flower seed husks and degradation of the resulting hydrolyzates to produce protein.
German Patent 676,967 to Scholler (1939) describes a method for clarifying xylose worts obtained by acid hydrolysis of cellulose-containing substances for feed purposes or yeast production by precipitating calcium phosphate and calcium sulfate after heating to 65 to 100 together with centrifuging and conducting the wort over oxidized metal fillings or large surface area materials while the wort is at a pH of 4 to 7.5, adding malt sprouts to the thus clari~ied wort and stirring for several hours.
U.S. Ratent No. 2,203,360 dated June ~, 1940 to Partansky discloses a method ~or improvin~ the fermentation characteristics of acid wood hydrolyzates by treating the hydrolyzate with lime to adjust the pH to between 9 and 10, aging for 1 to 2 days, reducing the pH with sulfuric acid to pH 5, purifying the solution with activated charcoal, diluting the solution to contain 40-70~ by volume of hydrolyzate, inocu-lating the solution with yeast culture and fermenting for 2 days.
The prior art, as represented by the methods dis-cussed above, is illustrative of the absence of a feasible com-mercial process for fermentation of acid hydrolyzates to alco-hol~ due to inordinately slow reaction times and low yields and/or the lack of direction for obtaining the samPO A method for readily and efficiently producing alcohol by fermentation of sugaxs present in wood and wood-byproducts is a particularly timely and significant development :in view of eurrent interest in alcohol as a potential energy source available from renew-able raw materials.
A primary object of this invention is to provide a process for fermentation of sugars present in acid hydrolyzates derived from lignocellulosic materlals.
Another object of the invention is to provide such a process in which reaction times are relatively short, in whieh fermentation may be effected at relatively high sugar concentrations and in which control mechanisms are established which permit predietability, reproduction of results with con-sistency and production of end products of high value~
Another object of this invention is to provide a process in which the acid hydrolyzate of cellulosic waste materials may be converted into ethyl alcohol.
The ~ccomplishment of these and other objects will be apparent from the descrip-tion of the invention which follows:
Summary_of the Inventlon The foregoing and related objects of this invention 7~7 are attained in a method for preconditioning acid hydrolyzates derived from lignocellulosic materials, to negate the effect of substances tending to inhibit the fermentation of such hydrolyzates and to a process for the production of ethyl al~ohol from glucose contained in such preconditioned acid hydrolyzates. The hydrolyzate is preconditioned to remove and/or reduce or otherwise negate the effect of inhibitory substances to a level whereby the hydrolyzate may be readily fermented to ethyl alcohol in substantially theoretical yield.
The preconditioning method broadly comprises the steps of: (1) subjecting the hydrolyzate to steam to remove furfural and other steam-volatile substances therefrom;
(2) adding sufficient calcium oxide to the steam-stripped hydrolyzate, at room temperature, to adjust the pH to between about 10 and 10.5, maintaining the resulting mixture at said pH for about 1 to 3 hours and separating the hydrolyzate from the resultant precipitate; (3) adding sufficient amounts of a mineral acid to adjust the p~ of said hydrolyzate to about 5 to 7; and (4) adjusting the concentration of said hydroly-zate to a glucose concentration of less than about 150 grams pe~ liter to provide a solution fermentable to ethyl alcohol.
The fermentation process broadly comprises the steps of: (1) preconditioning an acid hydrolyzate to negate the effect of substances tending to inhibit the fermentation thereof by subjecting the hydrolyzate to the preconditioning method described hereinabove; (2) inoculating the precondi-tioned hydrolyzate with yeast inoculum comprising from about 0.7 to about 7 dry weight percent of yeast cells per 100 grams per liter of glucose in the hydrolyzate; (3) fermenting the inoculated ` 30 hydrolyzate at a pH of 5 to 7 for a period sufficient to con-vert glucose to ethyl alcohol; and (4) recovering ethyl alcohol from the fermentation mixture.
~! ~
~5~7~1~
In a preferred embodiment, yeast cells are recovered, reconcentrated and recycled to a subsequent fermentation medium comprising preconditioned, concentrated hydrolyzate.
General Disclosure The process of this invention utilizes a combination of steps and conditions which are interrelated and interdepen-dent for the successful achievement of the objectives of the invention. This interrelationship will best be seen from the following description of the effect or function of each parti-0 cular sequence within the context of the total process.Hydrolyzate Raw Material The invention may be successfully realized with any hydrolyzate derived from the acid hydrolysis of lignocellu-losic material. Such lignocellulosic material may be selected from a wide variety of materials including wood and paper and particularly used paper and wood by-products such as sawdust, wood waste, straw, sugar cane bagasse, rice hulls, newspaper and the like. Such materials may be hydrolyzed in the presence of an acid catalyst by methods well known in the art to pro-vide a suitable hydrolyzate raw material for use in the pro-cess.
The hydrolyzate raw material provided will vary in sugar content and other components depending on the conditions under which it has been produced. This can be best understood by a consideration of the chemistry involved in acid hydroly-sis stated for the sake of illustration in simplified terms.
When cellulosic material is heated with dilute aqueous acid, glycosidic bonds which connect individual anhydroglucose units to one another in the cellulose molecule are cleaved by acid catalysis and one molecule of water adds to each anhydroglucose unit to fornl one molecule of glucose as illustrated by the idealized equation:
7~ .
H-~
(C6 ~llo s)n ~ n 2 ~ ~ 6 12 6 Glucose is inherently unstable in hot acid solutions and can lose three molecules of water to yield 5-hydroxymethylfurfural (HMF) ac-oxding to the equation:
H+
6 12 6 ~ ~ C6 H6 3 + 3H2O
HMF in turn is unstable and can add two molecules of water to yield levulinic acid and formic acid:
H~
C6 H6 3 + 2H2 ~ ~ C5 8 3 Other very complex reactions also occur in which it is believed HMF condenses into dark insoluble residues known as humins.
Lignin breakdown products such as vanillin or other aromatic compounds may also be present. Additionally, the bonds of the hemicellulose molecule are cleaved to produce free molecules of xylose from xylan. Cex~ain reaction conditions will favor formation of glucose or xylose and accompanying decomposition ; products.
An especially preferred method and apparatus for producing suitable acid hydrolyzate raw materials for use in this invention is that disclosed and claimed in U.S. Patent No. 4,201,596 entitled "Continuous Process for Cellulose Saccharification" referred to hereinabove. ~s disclosed therein, whereas the xylan conversion to xylose occurs at relatively low temperatures, the cellulose conversion to glu-cose best occurs under more severe conditions. It has not been possible to produce maximum amounts of both xylose and glucose in a one phase method due to the fact that the 5~77 xylose dehydrates to furfural under the conditions which most efficiently effect the conversion of cellulose to glucose.
The method of U.S. Patent No. 4,201,596 defines the conditions which favor production of glucose/furfural and which minimize degradation of glucose to HMF and levulinic acid. Since, as discussed and illustrated further hereinbelow, furfural, HMF
and levulinic acid are each toxins to the fermentation organ~
ism, the present invention most preferably utilizes a hydro-lyzate raw material obtained under such conditions that mini-mize, to the extent possible, the presence of such toxin sub-stances.
Such conditions in general provide for acid hydro-lysis, in the presence of steam, of cellulose feedstock having a solids c~ntent of from about 20 to 45 weight percent at temperatures within the range of about 190 to 225 C and pres-sures of about 200 to 400 psi with residence times in a reac-tion zone of about 1 to 10 minutes. In the reaction mass, the optimum amount of water after steam injection is about 75 to 80 weight percent and virtually any strong mineral acid can be employed to catalyze the hydrolysis reactant, sulfuric acid being normally the acid employed in amounts of about 1 to 3 percent based on the total weight of the reaction mass.
In this preferred process for production of hydrolyzate raw material, the reaction mass will be subjected to an abrupt pressure reduction whereby a fraction of the hydrolyzate vaporizes and may be recovered. This fraction will normally comprise furfural and acetic acid.
It will be understood that the above description is for purposes of illustration of the preferred mode of obtain-ing an acid hydrolyzate that is especially suitable for usein the present invention. The method of the invention may utilize acid hydrolyzates from any source since it is a feature o~ the invention that the preconditioning method will serve to reduce or remove certain of the toxin materials to - tolerable levels and/or to otherwise negate the effect of such materials without substantially adversely affecting the glucose present in the hydrolyzate.
Such hydrolyzate raw materials will in general, how-ever, comprise glucose, furfural, 5-hydroxymethylfurfural, acetic acid, formic acid, and levulinic acid and will have a p~ of less than about 1.5 and preferably of about 0.5.
Preconditioning of the Hydrolyzate The hydrolyzate raw material as received is a con-glomerate of chemical substances. Many of such substances act as inhibitory agents or toxins to the yeast while many of such substances are unknown in identity and effect. It is possible that the presence of such substances, known or un-known, may exert a cumulative affect on the fermentation mech-ism or yeast culture. It is also possible that some of the substances may be combining synergistically to inhibit either the particular yeast organism or other mechanism involved in the fermentation. Thus, while there is necessarily a degree of uncertainty as to exactly how the objectives of this in-vention are realized, it is believed that the preconditioning method renders the hydrolyzate fermentable either through removal of toxin substances or through conversion of at least a portion of such substances to non-toxin forms.
Several materials that are known toxins have been found to be present in the acid hydrolyzates derived from the lignocellulosic materials utilized herein. Their effect has been quantified to enable elimination or at least mini-mization of the same. The effect of such substances may beseen from the results of the following experiments in which an anaerobic culture of S. uvarum was employed at about 0.7 - g _ ~` .
7~
dry weight percent cell concentration with acid hydrolyzates under the conditions indicated and employing identical inocu-lum and fermentation media. Glucose sugar determination was made using a Beckman Glucose Analyzer.
Where fermentation was achieved or attempted, the steam-stripped, CaO pre-treated hydrolyzate was neutralized with HCl as the neutralizing agent.
Cell concentration as referred to herein is deter-mined by optical densit~ or dry weight measurements.
1. Acetic and Formic Acids Both acids are toxins to the alcohol-producing yeast. Acetic acid is present in the hydrolyzate in concentra-tions of about 3 to 4 g/l while formic acid is present in amounts of about 8 to 9 g/l. The toxic effect of these acids can be negated by conducting the fermentation at a pH of abcut 5 to 7, preferably about 5.5 to 6.5. Fermenting below about pH 5 to 6 does not negate the effect of the toxins while fermenting above pH 7 is unfavorable for ethanol production.
The parameters may best be illustrated by the results from the Eollowing experiments in which a newspaper hydrolyzate was preconditioned in accordance with the invention and analyzed ! for Eormic and acetic acids. A control solution of pure glu-cose was also provided. Identical nutrients in identical amounts were added to each of the solutions. Each of the solutions was inoculated with 0.7~ yeast cells and allowed to ferment for 18 hours. The results were as indicated in Table 1.
T~LE I
Fermentation of ~lucose in Presence of Formic and _ Acetic Acids at pH 4.0 and pH 7~0 g/l pH 4.0 pH 7.0 -A. Control Experiment Initial Glucose 40.0 40.0 Added Formic Acid 9.0 9,0 Added Acetic Acid 4.0 4.0 Final Glucose 41.0 0 Final Ethanol 0 18.5 % ~ield (based on Glucose) 0 46.3 B. Newspaper Hydrolyzate Initial Glucose 36.4 39.8 Contains Formic Acid3.0 3.0 Contains ~cetic Acid3.0 3.0 Final Glucose 36.4 0 Final Ethanol 0 18.9 Yield o 47.5%
2. 5-Hydroxymethylfurfural_(HMF) HMF is a strong inhibitor of yeast growth. However, this material can be destroyed or degraded by CaO treatment at room temperature at a pH o~ about 10 to about 10.5 without adversely affecting the glucose. The pH range is believed to be critical herein since at a pH below about 10, the effect of HMF is not negated while at a pH above about 10.5, the su~ar product is unstable. It has been discovered that CaO
treatment of the hydrolyzates at pH 10 to 10.5 results in rapid depletion of HMF during the first two hours and levels off after that period. For example, it was observed that approximately 63~ of the HMF is removed in 1 hour at pH 10.5.
At pH 10.25, approximately 2 hours is required to remove the same amount of HMF while at pH 10.75, some glucose is concomi-tantly destroyed. There~ore, in the preferred embodiment of the invention, hydrolyzate is treated with sufficient CaO, at ro~m temperatu~e and with stirring, to maintain the pH at about 10.5 for about 1 to l.S hours.
,~ .
27~
The effect of the HMF on the yeast may be seen from the following experiments in which yeast growth as a function of HMF concentration in the hydrolyzate was determined at a pH within the fermentation range of 6 to 7. The doubling time is that time it takes for a cell to reproduce itself and was determined by optical density measurements.
H~F, g/l Doubling Time, hours Growth Rate, h-0 4.6 0.15
3 9.7 0.071 11.7 0.059 7 13.6 0.051 Growth Rate = ln 2 Doubling Time 3. Levulinic Acid Levulinic acid inhibits yeast growth at concentra-tions of 10 g/l or greater at pH 6-7.
Levulinic Doubling Growth Acid, g/l Time, h. Rate, h-~
0 4.6 0.150 7.0 0.099 20.4 0.034 18.7 0.37 22 0.32 No sequence in the preconditioning step is believed to negate the effect of this toxin material. Fermentations are obtained, it is believed, because of the removal or nega-tion of the effect of other toxins present which may have a commulative or synergistic effect with the levulinic acid.
- Additionally, as discussed further hereinbelow, levulinic-insensitive yeast also provide an alternative means for further negating the effect of this toxin.
Levulinic Doubling Growth Acid, g/l Time, h. Rate, h-~
0 4.6 0.150 7.0 0.099 20.4 0.034 18.7 0.37 22 0.32 No sequence in the preconditioning step is believed to negate the effect of this toxin material. Fermentations are obtained, it is believed, because of the removal or nega-tion of the effect of other toxins present which may have a commulative or synergistic effect with the levulinic acid.
- Additionally, as discussed further hereinbelow, levulinic-insensitive yeast also provide an alternative means for further negating the effect of this toxin.
4. Furfural Furfural is toxic at concentrations in excess of
5.0 g/l. At concentrations between about 3 to 5 g/l, it has been found to markedly inhibit yeast growth.
~4~7~
FurfuralDoubling Growth Rate, g~l _Time, hours (h) h-l 0 4.6 0.150 2.0 4.2 0.165 4.0 7.4 0.094 5.0 - O
Furfural is readil~ eliminated from the hydrolyzate either b~ steam-stripping or calcium oxide treatment or both.
The effect of steam-stripping and CaO pretr~atment may be illustrated by the following experiments in which hydro-lyzate at pH of about 6.8 was admixed with an anaerobic culture of Candida utilis yeast at about 0.7% dry weight cell concen-tration in a shake flask and observed for fermentation of glucose after 16 hours. The results were as tabulated below in Table II.
TABLE II
A. Effect of Steam-Stripping CaO
Treated Hydrolyzate Glucose after Glucose Furfural HMF 16 h growth Treatment g~l g/l g/l g/l A. None 44 10.0 4~6 44 `~ B. CaO 44 5.0 1.6 44 C. Steam-stripping of B 44 1.2 2.2 0 D. Adding furfural to C 44 4.2 2.2 ~ 0 B. Effect of CaO Treatment on Steam-Stripped Hydrolyzate Glucose after Glucose Furfural HMF 16 h growth Treatment _ g/l g/l g/l g/l A. None 44 10 4.6 44 B. Steam-stripping 42 1.2 4.4 42 C. CaO treatment of B 41 0.1 0.1 0 r It will be seen from the above, that neither CaO
30 treatment alone nor stripping with steam alone is effective to render the h~drol~zate fermentable although both treatments reduce the furfural content. It was found that addition of furfural to CaO treated and steam-stripped hydrolyzate to a concentration nearly equal to that after CaO treatment but before steam-stripping did not inhibit the growth. This indi-cated that the steam-stripping may be removing some additional unknown inhibiting material.
It will be seen from the above that the steps of (1) steam-stripping; (2) treating with CaO at a pH of 10 to 10.5;
and (3) fermentation at pH 5 to 7 are critical to the success-ful operation of the preconditioning stage of the process.
Various additional steps may be interjected between the essential steps of the preconditioning method, if desired.
Thus, in the preferred mode of the invention, the hydrolyzate having a pH of about 0.5 is partially neutralized with lime-stone or ammonium hydroxide to a pH of about 4 prior to steam-stripping. While the hydrolyzate may be steam-stripped either at low pH as received or at pH 4 after partial neutralization, partial neutralization prior to steam-stripping is desirable to reduce corrosion of equipment. This step necessitates an additional filtration step to remove precipitated material and may be omitted in the event equipment is employed that is not readily corroded or whenever corrosion is not a signifi-cant concern. Where the hydrolyzate is neutralized to pH 4 employing calcium carbonate, etc., the resultant precipitate may be incinerated after recovery from the hydrolyzate to provide the fuel for generation of the steam for the process.
Normally, prior to incineration, the filter cake will be wash-ed to remove and recover sugars, with the washings being added to the hydrolyzate filtrate.
Neutralizing the hydrolyzate with ammonium hydroxide has the added advantage of supplying a nutrient which the fermentation microorganism can use for its growth while at the same time raising the pH of the hydrolyzate to preventcorrosion.
.,~, Steam-stripping is accomplished preferably by in-jecting steam into the hydrolyzate in an amount sufficient to maintain the hydrolyzate at a temperature of about 95 to 105C. Conveniently, the hydrolyzate may be passed through a countercurrent extractor to remove steam-volatiles. In this technique, steam is introduced at the bottom of the column and the hydrolyzate is introduced at the top and collected in a vessel at the bottom of the column. Steam-volatile toxins are removed in the steam which is condensed and collected in a separate vessel.
After steam-stripping, the hydrolyzate is treated with sufficient CaO to maintain the pH between about 10 and 10.5 for a period of about 1 to 3 hours at room temperature after which the precipitate is removed by any convenient means including filtration, centrifugation, etc.
The hydrolyzate, after neutralization with a mineral acid and removal of the resultant precipitate, is fermentable at this stage, the rapidity of the reaction having been found to be dependent on the concentration of the sugar solution~
the particular yeast strain employea, the yeast cell concen-tration and the mineral acid used to neutralize the condition-ed hydrolyzate.
Effect of Mineral Acid The hydrolyzate is neutralized to a pH of about 5 to 7, and preferably 5.5 to 6.5 after CaO treatment employing a mineral acid, e.g. hydrochloric acid, sulfuric acid, phos-phoric acid, etc. Phosphoric acid is especially preferred for several reasons. Neutralizing with hydrochloric or sulfuric acid results in a turbid solution which is of no consequence in batch fermentation. However, in a continuous culture fer-mentation, yeast cells must be recycled from the effluent stream back to the fermentor and must be reconcentrated prior ;`' ii2~
to such recycling. The use of phosphoric acid as the neutral-izing acid results in clarification of the hydrolyzate and ther~by enhances reconcentration and recycling of the yeast cells. Even more significantly, as discussed further herein-below, it has been discovered that the use of phosphoric acid results in more rapid fermentation and when employed in combin-ation with a height concentration of yeast cells, results in extremely rapid fermentation rates with concentrated hydroly-zates making it possible to realize theoretical yields after fermentation for as short a period as 1 to 3 hours.
Effect of Concentration of Hydrolyzate It was discovered that preconditioning of the hydro-lyzate by CaO pretreatment, steam-stripping and addition of mineral acid, e.g~ HCl neutralization to pH 6 to 7, lead to fermentable solutions that became progressively more difficult to ferment as the glucose concentration was increased over 50 g/l. It appears that concentration of the solution also increases the level of other inhibitory substances in the hydrolyzate beyond the tolerable level. To counteract this inhibition of concentrated solutions, different yeast and yeast concentrations were evaluated as discussed further here-inbelow. However, to illustrate the effect of the concentra-tion step, typical results obtained with anaerobically propa-gated Candida utilis at a cell concentration of 0.7~ with sawdust hydrolyzate neutralized with HCl and fermented for about 16 hours may be seen from results of the following experiments.
27~
TreatmentGlucoseGlucose after Growth g~
. None 44.0 44.0 B. CaO and stripping 44.0 C. Concentrating B 100.0 100.0 D. Diluting C to70.0 49.5 E. Diluting C to60.0 0 F. Diluting C to50.0 0 Attempts to ferment hydrolyzate solutions concentrated to 150 g/l or grea-ter have not been successful even when em-ploying the means discussed below.
Effect of Yeast Strain, Cell Concentration and Neutralizing Acid ' ' The above-results were obtained in shake flasks using Candida utilis as the yeast culture and HCl as the neutraliz-ing acid.
Other yeast strains were tested and evaluated for effect on the fermentablity of the preconditioned hydrolyzate when neutralized with HCl.
S. uvarum at 0.7~ cells at dry weight was observed to ferment 100 g/l hydrolyzate in about 98 hours and 50 g/l hydrolyzate solutions in about 19 hours while S. cerevisiae (Baker's yeast3 fermented 100 g/l hydrolyzate in about 42 hours to a 50.8 g/l ethanol concentration. A levulinic acid-insensitive strain of S. -~varum was produced and isolated by exposure of the cells to levulinic acid in chemostat culture.
This strain at 0.7% concentration was found to ferment 100 g/l hydrolyzate in about 50 hours resulting in 49.3 g/l ethanol.
It was then discovered that use of phosphoric acid as the neutralizing acid had a definite positive effect on the rate of fermentation of the concentrated hydrolyzates.
Thus, phosphoric acid treated hydrolyzates at 100 g/1, when fermented with either the parent strain S. uvarum or with the levulinic acid-insensitive strain of S. uvarum, both at 0.7%
cell concentration, resulted in yields o~ 47.7 g/l ethanol in ~-~
.
~52~
15.5 hours while Baker's yeast at the same concentration re-sulted in 46.5 g/l ethanol in 11.5 hours.
Even more rapid fermentation to theoretical yield is possible when using higher yeast cell concentrations as will be illustrated by the results obtained and listed below in Table III.
TABLE III
Effect of Cell Concentration on 100 g/l Phosphoric Acid-Neutralized Sawdust Hydrolyzate-Baker's Yeast Cell Dry Weight Time Required Ethànol % Hours Yield, %
0.7 20 50.0 1.5 12 48.1 2.0 8 50.6 : 2.5 5.6 50.2 3.0 3.0 46.8 5.0 1.5 47.8 7.0 1.25 48.4 The interrelationship and interdependence of the various steps of the preconditioning stage of the process as well as the effect o~ the particular neutralizing acid and yeast cell con-centration may be readily appreciated from a consideration of the above experiments.
he function of the CaO treatment to effectively remove or degrade HMF in periods as short as 1 to 3 hours, the effect of neutralizing to pH 5 to 7, the effect of the con-centration of the hydrolyzate and yeast cells and the achieve-men~ of extremely rapid reaction rates of concentrated hydro-lyzates when neutralized with phosphoric acid are each signifi-cant factors that are unexpected and appear to serve a vital function in the context of the overall process.
Provision_of Fermentation Medium Following the preconditioning method, the hydroly-zate is ready for fermentation by either a batch or continuous culture fermentation process under aerobic or anaerobic cell ~527~
propagation conditions.
Inoculum o~ the various yeast strains may be develop-ed by any method well known in the art. Any yeast may be em-ployed that is capable of growth in the fermentation medium.
As discussed above, satisfactory results have been obtained with members of the genus Saccharomyces, such as S. uvanl , S. uvarum modified to be levulinic-insensitive, S. cerevisiae (Baker's yeast), etc. with Baker's yeast being especially pre-ferred. Satisfactory results have also been obtained with C utilis at glucose concentrations up'to about 60 g/l. This particular yeast has not been found to be effective at higher glucose concentrations.
Suitable microbial growth nutrients may be added to the hydrolyzate and to the inoculum development medium as desired including phosphorous and nitrogen in ihe form of phosphate, ammonium, urea, etc. When phosphoric acid is the neutralizing acid, phosphorous nutrient is added during the neutralization step. Additionally, when phosphoric acid is employed as the neutralizing acid, fermentation may be realiz-ed by addition of urea as the sole additive nutrient source.Partial neutralization with ammonium hydroxide prior to steam-stripping also adds nitrogen as a nutrient source. Other mineral salts, trace elements, vitamins, etc. including ammon-ium sulfate, magnesium sulfate, sodium chloride, calcium chloride, potassium phosphate, biotin, folic ac'id, inositol, niacin, p-aminobenzoic acid, riboflavin, thiamine, urea, etc.
may be added to the hydrolyzate as growth nutrients, as desir-ed.
In a preferred embodiment, inoculum for batch fer-mentation is deyeloped by inoculating a loopful of cells froma slant on ~ medium containin~ about 2.0% glucose, 1.0%
peptone, and 0.3~ yeast extract (hereafter rcferred to as t~
YPG mediu~). Medium thus inoculated is incubated with sha~ing for 24 hours at 32C after which it is transferred into 900 milliliters or additional YPG medium, incubated with shaking for 6 to ~ hours and transferred to a fermentor containing 9 liters of ID medium comprising 90 g/l glucose, 7.65 g/l yeast extract, 1.19 g/l ammonium chloride, 0.01 g/l magnesium ; sulfate, 0.05 g/l calcium chloride, and 0 2 mls./l of GE 60 AF, an antifoam agent (available from General Electric Co~). Cells are aerobically propagated at p~ 6-7 under 1,000 rpm agitation and 1 vvm air flow for 16 to 20 hours after which the cells may be recovered by centrifugation or equivalent means and employed in the desired concentration to inoculate the hydro-lyzate.
Cells may also be developed from a continuous culture whereby cells are separated from ethanol product removed from the fermentor. In this step, recovered cells are reconcen-trated and recycled under conditions that preserve the metabo-lic state of the cell, the volume in the fermentor and the constant value of the cell concentration in the fermentor.
Cell separation from the ethanol product may be accomplished by various means including gravity settling, centrifugation, ultrafiltration, etc. Preferably, the cells are recovered and recycled through the use of dual output streams emanating from the fermentor. For example, ethanol and yeast cells are metered out in a first output stream at a rate determined by a sensor in the fermentor which determines when the cell con-centration has exceeded a desired upper limit. A second out-put stream removes ethanol and yeast cells to a cell recycler comprising a suitable membrane r for example a microporous 30 filter as employed in ultrafiltration, which retains the cells but allows the ethanol and unmetabolized medium to penetrate permitting recovery of substantially cell-free ethanol. The 2~7~7 membrane permits continuous cell concentration from which cellsmay be recycled to the fermentor as needed as fresh precondi-tioned hydrolyzate streams are fed for fermentation.
The following illustration will serve to illustrate a batch fermentation in accordance with the invention.
Illustration of a Preferred Embodiment Acid hydrolyzate having a pH of about 0.5 was pro-duced from sawdust by acid hydrolysis in the presence of steam and sulfuric acid in a reaction zone maintained at a tempera-ture of about 190 to 225C under pressure of about 200 to - 400 psi. The hydrolyzate contained about 50.2 g/l glucose, 8.9 g/l furfural, 3.6 g/l hydroxymethylfurural, 6.5 g/l levulinic acid, 9.7 g/l acetic acid and 4.8 g/l formic acid.
The hydrolyzate was partially neutralized with suffi-cient ammonium hydroxide to a pH of about 4 after which the resultant precipitate was removed and the partially neutra-lized stream fed to a countercurrent extractor where it was subjected to steam at a rate of 4 l/h during which steam volatile materials including furfural were removed and collect-ed. 14 g/1 of CaO was added to the steam-stripped hydrolyzate material to adjust the pH to about 10.5, the mixture was stirred at room temperature and maintained at pH 10.5 for about 1 hour after which the resultant precipitate was remov-ed. The hydrolyzate was neutralized to pH 5.5 to 6.5 with 1.5 to 3.0 ml/l of phosphoric acid and the resultant precipi-tate was removed. The neutralized hydrolyzate was then con-centrated to a glucose concentration of about 100 g/l by heating at 35C under vacuum of 28 inches Hg using a continu-ous evaporator.
After cooling to room temperature, 0.1% urea was added to the concentrated hydrolyzate which was next fed to the fermentor together with a sufEicient amount of preformed inoculum comprising Baker's yeast aerobically propagated on an agar slant in ~PG medium to give a dry cell concentration of about 3 to 3.5 weight percent.
The mixture was anaerobically fermented for about 1.5 to 2 hours after which about 50 g/l of ethanol was recover-ed.
Yeast cells were recovered by centrifugation and transferred to a subsequent fermentation batch. Satisfactory results were obtained for several transfers.
It will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention and that the invention is not limited to the preferred embodiments that have been described and illustrated hereinabove.
`:
~4~7~
FurfuralDoubling Growth Rate, g~l _Time, hours (h) h-l 0 4.6 0.150 2.0 4.2 0.165 4.0 7.4 0.094 5.0 - O
Furfural is readil~ eliminated from the hydrolyzate either b~ steam-stripping or calcium oxide treatment or both.
The effect of steam-stripping and CaO pretr~atment may be illustrated by the following experiments in which hydro-lyzate at pH of about 6.8 was admixed with an anaerobic culture of Candida utilis yeast at about 0.7% dry weight cell concen-tration in a shake flask and observed for fermentation of glucose after 16 hours. The results were as tabulated below in Table II.
TABLE II
A. Effect of Steam-Stripping CaO
Treated Hydrolyzate Glucose after Glucose Furfural HMF 16 h growth Treatment g~l g/l g/l g/l A. None 44 10.0 4~6 44 `~ B. CaO 44 5.0 1.6 44 C. Steam-stripping of B 44 1.2 2.2 0 D. Adding furfural to C 44 4.2 2.2 ~ 0 B. Effect of CaO Treatment on Steam-Stripped Hydrolyzate Glucose after Glucose Furfural HMF 16 h growth Treatment _ g/l g/l g/l g/l A. None 44 10 4.6 44 B. Steam-stripping 42 1.2 4.4 42 C. CaO treatment of B 41 0.1 0.1 0 r It will be seen from the above, that neither CaO
30 treatment alone nor stripping with steam alone is effective to render the h~drol~zate fermentable although both treatments reduce the furfural content. It was found that addition of furfural to CaO treated and steam-stripped hydrolyzate to a concentration nearly equal to that after CaO treatment but before steam-stripping did not inhibit the growth. This indi-cated that the steam-stripping may be removing some additional unknown inhibiting material.
It will be seen from the above that the steps of (1) steam-stripping; (2) treating with CaO at a pH of 10 to 10.5;
and (3) fermentation at pH 5 to 7 are critical to the success-ful operation of the preconditioning stage of the process.
Various additional steps may be interjected between the essential steps of the preconditioning method, if desired.
Thus, in the preferred mode of the invention, the hydrolyzate having a pH of about 0.5 is partially neutralized with lime-stone or ammonium hydroxide to a pH of about 4 prior to steam-stripping. While the hydrolyzate may be steam-stripped either at low pH as received or at pH 4 after partial neutralization, partial neutralization prior to steam-stripping is desirable to reduce corrosion of equipment. This step necessitates an additional filtration step to remove precipitated material and may be omitted in the event equipment is employed that is not readily corroded or whenever corrosion is not a signifi-cant concern. Where the hydrolyzate is neutralized to pH 4 employing calcium carbonate, etc., the resultant precipitate may be incinerated after recovery from the hydrolyzate to provide the fuel for generation of the steam for the process.
Normally, prior to incineration, the filter cake will be wash-ed to remove and recover sugars, with the washings being added to the hydrolyzate filtrate.
Neutralizing the hydrolyzate with ammonium hydroxide has the added advantage of supplying a nutrient which the fermentation microorganism can use for its growth while at the same time raising the pH of the hydrolyzate to preventcorrosion.
.,~, Steam-stripping is accomplished preferably by in-jecting steam into the hydrolyzate in an amount sufficient to maintain the hydrolyzate at a temperature of about 95 to 105C. Conveniently, the hydrolyzate may be passed through a countercurrent extractor to remove steam-volatiles. In this technique, steam is introduced at the bottom of the column and the hydrolyzate is introduced at the top and collected in a vessel at the bottom of the column. Steam-volatile toxins are removed in the steam which is condensed and collected in a separate vessel.
After steam-stripping, the hydrolyzate is treated with sufficient CaO to maintain the pH between about 10 and 10.5 for a period of about 1 to 3 hours at room temperature after which the precipitate is removed by any convenient means including filtration, centrifugation, etc.
The hydrolyzate, after neutralization with a mineral acid and removal of the resultant precipitate, is fermentable at this stage, the rapidity of the reaction having been found to be dependent on the concentration of the sugar solution~
the particular yeast strain employea, the yeast cell concen-tration and the mineral acid used to neutralize the condition-ed hydrolyzate.
Effect of Mineral Acid The hydrolyzate is neutralized to a pH of about 5 to 7, and preferably 5.5 to 6.5 after CaO treatment employing a mineral acid, e.g. hydrochloric acid, sulfuric acid, phos-phoric acid, etc. Phosphoric acid is especially preferred for several reasons. Neutralizing with hydrochloric or sulfuric acid results in a turbid solution which is of no consequence in batch fermentation. However, in a continuous culture fer-mentation, yeast cells must be recycled from the effluent stream back to the fermentor and must be reconcentrated prior ;`' ii2~
to such recycling. The use of phosphoric acid as the neutral-izing acid results in clarification of the hydrolyzate and ther~by enhances reconcentration and recycling of the yeast cells. Even more significantly, as discussed further herein-below, it has been discovered that the use of phosphoric acid results in more rapid fermentation and when employed in combin-ation with a height concentration of yeast cells, results in extremely rapid fermentation rates with concentrated hydroly-zates making it possible to realize theoretical yields after fermentation for as short a period as 1 to 3 hours.
Effect of Concentration of Hydrolyzate It was discovered that preconditioning of the hydro-lyzate by CaO pretreatment, steam-stripping and addition of mineral acid, e.g~ HCl neutralization to pH 6 to 7, lead to fermentable solutions that became progressively more difficult to ferment as the glucose concentration was increased over 50 g/l. It appears that concentration of the solution also increases the level of other inhibitory substances in the hydrolyzate beyond the tolerable level. To counteract this inhibition of concentrated solutions, different yeast and yeast concentrations were evaluated as discussed further here-inbelow. However, to illustrate the effect of the concentra-tion step, typical results obtained with anaerobically propa-gated Candida utilis at a cell concentration of 0.7~ with sawdust hydrolyzate neutralized with HCl and fermented for about 16 hours may be seen from results of the following experiments.
27~
TreatmentGlucoseGlucose after Growth g~
. None 44.0 44.0 B. CaO and stripping 44.0 C. Concentrating B 100.0 100.0 D. Diluting C to70.0 49.5 E. Diluting C to60.0 0 F. Diluting C to50.0 0 Attempts to ferment hydrolyzate solutions concentrated to 150 g/l or grea-ter have not been successful even when em-ploying the means discussed below.
Effect of Yeast Strain, Cell Concentration and Neutralizing Acid ' ' The above-results were obtained in shake flasks using Candida utilis as the yeast culture and HCl as the neutraliz-ing acid.
Other yeast strains were tested and evaluated for effect on the fermentablity of the preconditioned hydrolyzate when neutralized with HCl.
S. uvarum at 0.7~ cells at dry weight was observed to ferment 100 g/l hydrolyzate in about 98 hours and 50 g/l hydrolyzate solutions in about 19 hours while S. cerevisiae (Baker's yeast3 fermented 100 g/l hydrolyzate in about 42 hours to a 50.8 g/l ethanol concentration. A levulinic acid-insensitive strain of S. -~varum was produced and isolated by exposure of the cells to levulinic acid in chemostat culture.
This strain at 0.7% concentration was found to ferment 100 g/l hydrolyzate in about 50 hours resulting in 49.3 g/l ethanol.
It was then discovered that use of phosphoric acid as the neutralizing acid had a definite positive effect on the rate of fermentation of the concentrated hydrolyzates.
Thus, phosphoric acid treated hydrolyzates at 100 g/1, when fermented with either the parent strain S. uvarum or with the levulinic acid-insensitive strain of S. uvarum, both at 0.7%
cell concentration, resulted in yields o~ 47.7 g/l ethanol in ~-~
.
~52~
15.5 hours while Baker's yeast at the same concentration re-sulted in 46.5 g/l ethanol in 11.5 hours.
Even more rapid fermentation to theoretical yield is possible when using higher yeast cell concentrations as will be illustrated by the results obtained and listed below in Table III.
TABLE III
Effect of Cell Concentration on 100 g/l Phosphoric Acid-Neutralized Sawdust Hydrolyzate-Baker's Yeast Cell Dry Weight Time Required Ethànol % Hours Yield, %
0.7 20 50.0 1.5 12 48.1 2.0 8 50.6 : 2.5 5.6 50.2 3.0 3.0 46.8 5.0 1.5 47.8 7.0 1.25 48.4 The interrelationship and interdependence of the various steps of the preconditioning stage of the process as well as the effect o~ the particular neutralizing acid and yeast cell con-centration may be readily appreciated from a consideration of the above experiments.
he function of the CaO treatment to effectively remove or degrade HMF in periods as short as 1 to 3 hours, the effect of neutralizing to pH 5 to 7, the effect of the con-centration of the hydrolyzate and yeast cells and the achieve-men~ of extremely rapid reaction rates of concentrated hydro-lyzates when neutralized with phosphoric acid are each signifi-cant factors that are unexpected and appear to serve a vital function in the context of the overall process.
Provision_of Fermentation Medium Following the preconditioning method, the hydroly-zate is ready for fermentation by either a batch or continuous culture fermentation process under aerobic or anaerobic cell ~527~
propagation conditions.
Inoculum o~ the various yeast strains may be develop-ed by any method well known in the art. Any yeast may be em-ployed that is capable of growth in the fermentation medium.
As discussed above, satisfactory results have been obtained with members of the genus Saccharomyces, such as S. uvanl , S. uvarum modified to be levulinic-insensitive, S. cerevisiae (Baker's yeast), etc. with Baker's yeast being especially pre-ferred. Satisfactory results have also been obtained with C utilis at glucose concentrations up'to about 60 g/l. This particular yeast has not been found to be effective at higher glucose concentrations.
Suitable microbial growth nutrients may be added to the hydrolyzate and to the inoculum development medium as desired including phosphorous and nitrogen in ihe form of phosphate, ammonium, urea, etc. When phosphoric acid is the neutralizing acid, phosphorous nutrient is added during the neutralization step. Additionally, when phosphoric acid is employed as the neutralizing acid, fermentation may be realiz-ed by addition of urea as the sole additive nutrient source.Partial neutralization with ammonium hydroxide prior to steam-stripping also adds nitrogen as a nutrient source. Other mineral salts, trace elements, vitamins, etc. including ammon-ium sulfate, magnesium sulfate, sodium chloride, calcium chloride, potassium phosphate, biotin, folic ac'id, inositol, niacin, p-aminobenzoic acid, riboflavin, thiamine, urea, etc.
may be added to the hydrolyzate as growth nutrients, as desir-ed.
In a preferred embodiment, inoculum for batch fer-mentation is deyeloped by inoculating a loopful of cells froma slant on ~ medium containin~ about 2.0% glucose, 1.0%
peptone, and 0.3~ yeast extract (hereafter rcferred to as t~
YPG mediu~). Medium thus inoculated is incubated with sha~ing for 24 hours at 32C after which it is transferred into 900 milliliters or additional YPG medium, incubated with shaking for 6 to ~ hours and transferred to a fermentor containing 9 liters of ID medium comprising 90 g/l glucose, 7.65 g/l yeast extract, 1.19 g/l ammonium chloride, 0.01 g/l magnesium ; sulfate, 0.05 g/l calcium chloride, and 0 2 mls./l of GE 60 AF, an antifoam agent (available from General Electric Co~). Cells are aerobically propagated at p~ 6-7 under 1,000 rpm agitation and 1 vvm air flow for 16 to 20 hours after which the cells may be recovered by centrifugation or equivalent means and employed in the desired concentration to inoculate the hydro-lyzate.
Cells may also be developed from a continuous culture whereby cells are separated from ethanol product removed from the fermentor. In this step, recovered cells are reconcen-trated and recycled under conditions that preserve the metabo-lic state of the cell, the volume in the fermentor and the constant value of the cell concentration in the fermentor.
Cell separation from the ethanol product may be accomplished by various means including gravity settling, centrifugation, ultrafiltration, etc. Preferably, the cells are recovered and recycled through the use of dual output streams emanating from the fermentor. For example, ethanol and yeast cells are metered out in a first output stream at a rate determined by a sensor in the fermentor which determines when the cell con-centration has exceeded a desired upper limit. A second out-put stream removes ethanol and yeast cells to a cell recycler comprising a suitable membrane r for example a microporous 30 filter as employed in ultrafiltration, which retains the cells but allows the ethanol and unmetabolized medium to penetrate permitting recovery of substantially cell-free ethanol. The 2~7~7 membrane permits continuous cell concentration from which cellsmay be recycled to the fermentor as needed as fresh precondi-tioned hydrolyzate streams are fed for fermentation.
The following illustration will serve to illustrate a batch fermentation in accordance with the invention.
Illustration of a Preferred Embodiment Acid hydrolyzate having a pH of about 0.5 was pro-duced from sawdust by acid hydrolysis in the presence of steam and sulfuric acid in a reaction zone maintained at a tempera-ture of about 190 to 225C under pressure of about 200 to - 400 psi. The hydrolyzate contained about 50.2 g/l glucose, 8.9 g/l furfural, 3.6 g/l hydroxymethylfurural, 6.5 g/l levulinic acid, 9.7 g/l acetic acid and 4.8 g/l formic acid.
The hydrolyzate was partially neutralized with suffi-cient ammonium hydroxide to a pH of about 4 after which the resultant precipitate was removed and the partially neutra-lized stream fed to a countercurrent extractor where it was subjected to steam at a rate of 4 l/h during which steam volatile materials including furfural were removed and collect-ed. 14 g/1 of CaO was added to the steam-stripped hydrolyzate material to adjust the pH to about 10.5, the mixture was stirred at room temperature and maintained at pH 10.5 for about 1 hour after which the resultant precipitate was remov-ed. The hydrolyzate was neutralized to pH 5.5 to 6.5 with 1.5 to 3.0 ml/l of phosphoric acid and the resultant precipi-tate was removed. The neutralized hydrolyzate was then con-centrated to a glucose concentration of about 100 g/l by heating at 35C under vacuum of 28 inches Hg using a continu-ous evaporator.
After cooling to room temperature, 0.1% urea was added to the concentrated hydrolyzate which was next fed to the fermentor together with a sufEicient amount of preformed inoculum comprising Baker's yeast aerobically propagated on an agar slant in ~PG medium to give a dry cell concentration of about 3 to 3.5 weight percent.
The mixture was anaerobically fermented for about 1.5 to 2 hours after which about 50 g/l of ethanol was recover-ed.
Yeast cells were recovered by centrifugation and transferred to a subsequent fermentation batch. Satisfactory results were obtained for several transfers.
It will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention and that the invention is not limited to the preferred embodiments that have been described and illustrated hereinabove.
`:
Claims (24)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for preconditioning acid hydrolyzates derived from lignocellulosic materials comprising glucose and substances which tend to inhibit the fermentation thereof, con-sisting essentially of the steps of: (1) subjecting said acid hydrolyzate to steam to remove steam-volatile substances there-from; (2) adding sufficient calcium oxide to said steam-stripped hydrolyzate to adjust the pH to between 10 and about 10.5 and maintaining said mixture at said pH for about 1 to 3 hours;
(3) adding sufficient amounts of a mineral acid to adjust the pH of said hydrolyzate to about 5 to 7; and (4) adjusting the concentration of said hydrolyzate to a glucose concentration of less than 150 grams per liter to provide a solution fermen-table to ethyl alcohol, with the proviso that when said concen-tration is greater than 50 grams per liter, the mineral acid employed in step (3) is phosphoric acid.
(3) adding sufficient amounts of a mineral acid to adjust the pH of said hydrolyzate to about 5 to 7; and (4) adjusting the concentration of said hydrolyzate to a glucose concentration of less than 150 grams per liter to provide a solution fermen-table to ethyl alcohol, with the proviso that when said concen-tration is greater than 50 grams per liter, the mineral acid employed in step (3) is phosphoric acid.
2. A method as claimed in claim 1, wherein said acid hydrolyzate provided in step (1) has a pH within the range of 0.5 to 1.5 and is derived from sawdust or newspaper.
3. A method as claimed in claim 2, wherein prior to subjecting the hydrolyzate to steam, the hydrolyzate is partial-ly neutralized to a pH of about 4 with a sufficient amount of calcium carbonate or ammonium hydroxide and the resulting pre-cipitate is separated therefrom.
4. A method as claimed in claim 3, wherein the min-eral acid employed in step (3) is phosphoric acid.
5. A method as claimed in claim 1, wherein said hydrolyzate is concentrated in step (4) to a glucose concentra-tion of at least 100 grams per liter but less than 150 grams per liter.
6. A method as claimed in claim 5, wherein acid hydrolyzate is concentrated by heating to about 35°C under a vacuum of about 28 to 30 inches Hg.
7. A method for preconditioning acid hydrolyzates derived from lignocellulosic materials to negate the effect of substances tending to inhibit the fermentation thereof, con-sisting essentially of the steps of: (1) providing an acid hydrolyzate comprising glucose, furfural, 5-hydroxymethylfur-fural, acetic acid and formic acid having a pH of about 0.5 to 1.5; (2) partially neutralizing said hydrolyzate to a pH of about 4 with a sufficient amount of ammonium hydroxide; (3) subjecting the partially neutralized hydrolyzate to steam to remove a major proportion of furfural and other steam-volatile substances therefrom; (4) adding sufficient calcium oxide to the steam-stripped hydrolyzate to adjust the pH to about 10.5 and maintaining said pH at room temperature for about 1 hour to degrade 5-hydroxymethylfurfural; (5) adjusting the pH of the hydrolyzate to about 5 to 7 with a sufficient amount of phos-phoric acid and separating said hydrolyzate from the precipitate thus produced; and (6) concentrating the hydrolyzate to a glu-cose concentration of at least about 100 grams per liter but less than 150 grams per liter under conditions that minizmize degradation of the glucose to provide a solution fermentable to ethyl alcohol.
8. A process as claimed in claim 7, wherein said hydrolyzate is derived from sawdust or newspaper.
9. A process as claimed in claim 7, wherein said hydrolyzate is concentrated in step (6) by heating to a tempera-ture of about 35°C under a vacuum of about 28 to 30 inches Hg.
10. A process for the production of ethyl alcohol from glucose contained in an acid hydrolyzate derived from lignocellulosic materials, comprising the steps of: (1) precon-ditioning said acid hydrolyzate to negate the effect of sub-stances tending to inhibit the fermentation thereof by subject-ing said hydrolyzate to the method of claim 1; (2) inoculating the preconditioned hydrolyzate with yeast inoculum developed from a strain that is capable of growth in the hydrolyzate fermentation medium; (3) permitting-said inoculated hydrolyzate to ferment at a pH of about 5 to 7 for a period sufficient to convert glucose to ethyl alcohol;
and (4) recovering ethyl alcohol from the fermentation medium.
and (4) recovering ethyl alcohol from the fermentation medium.
11. A process as claimed in claim 10, wherein said yeast is Baker's yeast.
12. A process as claimed in claim 11, wherein yeast cells are recovered from the fermentation mixture and recycled to a subsequent fermentation medium.
13. A process for the production of ethyl alcohol from glucose contained in an acid hydrolyzate from lignocellulosic materials, comprising the steps of: (1) providing an acid hydro-lyzate comprising glucose, furfural and 5-hydroxymethylfurfural and having a pH of about 0.5 to 1.5; (2) preconditioning said hydrolyzate to negate the effect of substances tending to inhibit the fermentation thereof by subjecting said hydrolyzate to the method of claim 4; (3) inoculating the preconditioned hydroly-zate with yeast inoculum developed from a strain that is capable of growth in the hydrolyzate fermentation medium; (4) permitting said inoculated hydrolyzate to ferment at a pH of 5.5 to 7 for a period sufficient to convert glucose to ethyl alcohol; and (5) recovering ethyl alcohol from the fermentation medium.
14. A process for the production of ethyl alcohol from glucose contained in an acid hydrolyzate derived from lignocell-ulosic materials, comprising the steps of: (1) providing an acid hydrolyzate comprising glucose, furfural and 5-hydroxymethyl-furfural; (2) preconditioning said hydrolyzate to negate the effect of substances tending to inhibit the fermentation there-of by subjecting said hydrolyzate to the steps of (a) steam-stripping furfural and other steam volatile substances there-from; (b) adding sufficient calcium oxide to said steam-strip-ping hydrolyzate, at room temperature to adjust the pH to about 10.5 and maintaining said solution at said pH for a period of about 1 hour and separating the resulting precipitate from said hydrolyzate; (c) adjusting the pH of said hydrolyzate to about 5 to 7 with phosphoric acid and separating the resultant precipitate; and (d) adjusting the concentration of the neutra-lized hydrolyzate to a glucose concentration of at least about 100 grams per liter but less than 150 grams per liter; (3) in-oculating said preconditioned hydrolyzate with Baker's yeast inoculum comprising from about 0.7 to about 7 dry weight percent of yeast cells per 100 grams per liter of glucose in said hydro-lyzate; (4) fermenting said inoculated hydrolyzate at a pH of 5 to 7 for 20 to 1.5 hours to convert glucose to substantially quantitative amounts of ethyl alcohol; and (5) recovering ethyl alcohol from the fermentation mixture.
15. A process as claimed in claim 14, wherein said hydrolyzate is derived from newspaper or sawdust.
16. A process as claimed in claim 14, wherein said hydrolyzate is adjusted to a pH of about 4 with ammonium hydroxide prior to said preconditioning step.
17. A process as claimed in claim 14, wherein urea is added to said hydrolyzate prior to said inoculation step.
18. A process as claimed in claim 14, wherein the inoculum added in step (3) comprises from about 3 to 3.5 dry weight percent of yeast cells per 100 grams per liter of glu-cose in said hydrolyzate.
19. A process as claimed in claim 18, wherein said fermentation is complete in about 2 to 3 hours.
20. A process for the production of ethyl alcohol from glucose contained in an acid hydrolyzate derived from a lignocellulosic material which comprises the steps of: (1) introducing into a fermentor an acid hydrolyzate that has been preconditioned to negate the effect of substances tending to inhibit the fermentation thereof by the method of claim 1, said fermentor containing a preformed yeast culture capable of growth in said hydrolyzate medium; (2) substantially continu-ously maintaining said yeast culture in said fermentor at a concentration of about 0.7 to about 7 dry weight percent cells per 100 grams per liter of glucose in said fermentor; (3) per-mitting said hydrolyzate to ferment at a pH of 5 to 7 for a period sufficient to convert glucose to ethyl alcohol; (4) re-moving an ethyl alcohol stream from the fermentor; (5) separat-ing yeast cells from said ethyl alcohol stream; and (6) recon-centrating said yeast cells and recycling the concentrated cells to said yeast culture maintained in said fermentor.
21. A method as claimed in claim 1, wherein said hydrolyzate is separated from precipitate formed in step (2).
22. A method as claimed in claim 1, wherein said hydrolyzate is separated from precipitate formed in step (3).
23. A method as claimed in claim 1, wherein said hydrolyzate is separated from precipitate formed in steps (2) and (3).
24. A method for preconditioning acid hydrolyzates derived from lignocellulosic materials comprising glucose and substances which tend to inhibit the fermentation thereof, con-sisting essentially of the steps of: (1) providing said acid hydrolyzate having a pH of about 0.5 to 1.5; (2) subjecting said acid hydrolyzate to steam to remove steam-volatile substances therefrom; (3) partially neutralizing said hydroly-zate to a pH of about 4 with a sufficient amount of calcium carbonate; (4) adding sufficient calcium oxide to said steam-stripped hydrolyzate to adjust the pH to about 10.5 and main-taining said mixture at said pH for about 1 to 3 hours; (5) adjusting the pH of the hydrolyzate to about 5 to 7 with a sufficient amount of phosphoric acid and separating said hydro-lyzate from the precipitate thus produced; and (6) concentrat-ing the hydrolyzate to a glucose concentration of at least about 100 grams per liter but less than 150 grams per liter under conditions that minimize degradation of the glucose to provide a solution fermentable to ethyl alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000361776A CA1145277A (en) | 1980-10-08 | 1980-10-08 | Fermentable acid hydrolyzates and fermentation process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000361776A CA1145277A (en) | 1980-10-08 | 1980-10-08 | Fermentable acid hydrolyzates and fermentation process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1145277A true CA1145277A (en) | 1983-04-26 |
Family
ID=4118087
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000361776A Expired CA1145277A (en) | 1980-10-08 | 1980-10-08 | Fermentable acid hydrolyzates and fermentation process |
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| CA (1) | CA1145277A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112111423A (en) * | 2020-09-02 | 2020-12-22 | 内蒙古阜丰生物科技有限公司 | Method for preparing xanthan gum through fermentation |
-
1980
- 1980-10-08 CA CA000361776A patent/CA1145277A/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112111423A (en) * | 2020-09-02 | 2020-12-22 | 内蒙古阜丰生物科技有限公司 | Method for preparing xanthan gum through fermentation |
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