EP0788551A1 - Eine methode für verbesserte rohmaterialverwendung in fermentationsverfahren - Google Patents

Eine methode für verbesserte rohmaterialverwendung in fermentationsverfahren

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Publication number
EP0788551A1
EP0788551A1 EP95938927A EP95938927A EP0788551A1 EP 0788551 A1 EP0788551 A1 EP 0788551A1 EP 95938927 A EP95938927 A EP 95938927A EP 95938927 A EP95938927 A EP 95938927A EP 0788551 A1 EP0788551 A1 EP 0788551A1
Authority
EP
European Patent Office
Prior art keywords
enzyme
maltulose
unfermentable
saccharides
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95938927A
Other languages
English (en)
French (fr)
Inventor
Bertus Noordam
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Danisco US Inc
Original Assignee
Genencor International Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genencor International Inc filed Critical Genencor International Inc
Priority to EP95938927A priority Critical patent/EP0788551A1/de
Publication of EP0788551A1 publication Critical patent/EP0788551A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C12P7/20—Glycerol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid
    • 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

Definitions

  • the present invention relates to the enzymatic hydrolysis of unfermentable carbohydrates into fermentable carbohydrates. More specifically, the invention provides a method to produce fermentable monosaccharides from unfermentable saccharides, present in, for example, liquefied and/or saccharified starch, beet molasses and cane molasses, in order to improve the raw material utilization in fermentation processes such as the fermentative production of ethanol.
  • Yield is a crucial issue in fermentative production processes. Yield is of particular importance in the production of primary metabolites such as ethanol, glycerol and lactic acid due to the low profit margins these products provide. As a result, significant effort has been focused on the improvement of yield to facilitate achieving higher levels of product from the raw materials used. In the field of fermentative ethanol production, product yield has been improved by reducing the amount of byproducts produced and also by improving the utilization of the raw material. For example, yeast recycle systems (yeast re-use) have been used to reduce sugar consumption for yeast biomass production. Additionally, simultaneous saccharification and fermentation to maintain the free glucose level at a minimum, and thereby prevent high infection levels, has been shown to result in improved yields.
  • yeast recycle systems yeast re-use
  • Maltulose is not hydrolysed by commonly used starch processing enzymes such as amyloglucosidase, pullulanase or acid amylase.
  • starch processing enzymes such as amyloglucosidase, pullulanase or acid amylase.
  • concentration of maltulose in the liquefied product can be as high as 2%.
  • Raffinose ⁇ -D-galactosyl(1 ,6)- ⁇ -D-glucose(1,2)- ⁇ -D-fructose. This sugar is found in, among others, beet molasses. Raffinose is also a product of partial hydrolysis of stachyose. Porter et al., disclose hydrolysis of raffinose.
  • Melibiose ⁇ -D-galactosyl(1 ,6)- ⁇ -D-glucose. This disaccharide, found in, among others, beet molasses, is a product of partial hydrolysis of both stachyose and raffinose. Melibiose has been reportedly hydrolyzed by Aspergill ⁇ s niger ⁇ - galactosidase, Kaneko et al., Agric. Biol. Chem., vol 55, no.1, pp 109-115 (1991), and Azotobacter vionelandii exo- ⁇ -galactosidase, Wong, Appl. of Environ. Microb., vol 56, no. 7 pp 2271-2273 (1990).
  • Enzymatic hydrolysis of these unfermentable carbohydrates has thus far not been applied to improve raw material utilization during production of primary metabolites such as ethanol.
  • the utilization of such a process has likely been ignored in the industry due to an expectation in the field that hydrolysis of these unfermentable carbohydrates, prior to fermentation (when the carbohydrate concentrations are high) and using an immobilized enzyme or enzyme mixture, will result in production of large amounts of unfermentable reversion products and, thus, will only make the situation worse.
  • the unfermentable carbohydrate fraction consists of a mixture of carbohydrates and as a consequence requires a mixture of enzymes for hydrolysis. This situation is further complicated when using enzyme mixtures.
  • a fermentative production process is provided for the hydrolysis of unfermentable noncellulose and nonhemicellulose based saccharides using a residual sugar hydrolyzing enzyme preparation capable thereof.
  • the enzyme preparation is applied at a point in the process where the overall carbohydrate concentration is below 20% w/v.
  • enzymes for the hydrolysis of unfermentable saccharides in fermentative production processes are provided.
  • the enzymes are preferably used in mixtures of enzymes and/or are used in an immobilized form.
  • the invention further discloses plants for the fermentative production of ethanol which comprise enzyme reactors for the hydrolysis of noncellulose and nonhemicellulose based unfermentable saccharides.
  • Figure 1A Schematic representation of a "wet milling" ethanol plant.
  • Figure 1 B Schematic representation of a "wet milling” ethanol plant including an enzyme reactor for the hydrolysis of unfermentable saccharides.
  • FIG. 2A Schematic representation of a plant for the batchwise production of ethanol, including an enzyme reactor for the hydrolysis of unfermentable saccharides.
  • FIG 3A HPLC chromatogram of beer from “wet milling” ethanol production.
  • Figure 3B HPLC chromatogram of beer from “wet milling” ethanol production after treatment with ⁇ -galactosidase (SUMIZYME AGS).
  • Figure 3C HPLC chromatogram of beer from "wet milling" ethanol production after treatment with an enzyme cocktail to hydrolyse unfermentable saccharides .
  • Figure 3D HPLC chromatogram of beer from "wet milling" ethanol production after treatment with an enzyme cocktail to hydrolyse unfermentable saccharides, followed by the addition of yeast. 6/13600 PC17US95/13876
  • Figure 4 Chromatogram of gel filtration of ⁇ -galactosidase on Sephacryl 5200 HR OD (at OD 280) vs. elution time.
  • the present invention provides a method to increase the yield of a fermentative production process by increasing the amount of fermentable sugars used as a starting material in such processes through the enzymatic hydrolysis of unfermentable noncellulose and nonhemicellulose based saccharides.
  • the enzyme composition may be derived from the fermentation broth of a microorganism which produces the enzyme.
  • the residual sugar hydrolyzing enzyme of the invention comprises any enzyme capable of hydrolyzing residual sugars present in carbohydrate raw material streams which are unfermentable by sugar fermenting organisms and, especially, in glucose syrup or precursors thereof derived from starch processing streams. Such residual sugar hydrolyzing enzymes will preferably possess as their major activity the hydrolysis of one or more residual sugars.
  • the enzyme composition is derived from a fungal source, more preferably from Aspergillus or Trichoderma.
  • the enzyme composition is derived from A. niger and comprises a maltulose hydrolyzing activity and/or a residual sugar hydrolyzing activity having a molecular weight of approximately 132 kD and 120 kD, respectively, as measured by gel filtration.
  • the enzyme composition includes an enzyme commonly used in preparing sugar stocks for fermentative production processes, e.g., amyloglucosidase, pullulanse or acid amylase, it is preferable to enrich the composition to include more of the residual sugar hydrolyzing enzyme than exists in the natural composition.
  • An "enriched" residual sugar hydrolyzing enzyme preparation is a preparation which is derived from a fermentation broth produced by the fermentation of a naturally occurring microorganism which produces residual sugar hydrolyzing enzyme and which preparation includes a higher concentration of residual sugar hydrolyzing enzyme than would be found naturally due to the fermentation of the microorganism.
  • an enriched residual sugar hydrolyzing enzyme preparation can be prepared by purifying the residual sugar hydrolyzing enzyme from the fermentation broth of a natural or genetically engineered microorganism so as "enrich" the residual sugar hydrolyzing enzyme relative to the removed contaminants.
  • the purified residual sugar hydrolyzing enzyme can be added to a naturally occurring enzyme mixture containing, for example, pullulanase, or acid amylase, in a concentration greater than exists in the naturally occurring fermentation of the organism(s) from which the enzyme mixture is desired.
  • an enriched residual sugar hydrolyzing enzyme preparation may be derived from the fermentation of a genetically modified microorganism which has been subject to recombinant techniques so as to amplify expression of residual sugar hydrolyzing enzyme in a fermentation broth.
  • the enzyme preparation applied in the present invention for the hydrolysis of the noncellulose and nonhemicellulose based saccharides may comprise the enzyme maltulase (described herein in and co-pending Patent Application (Serial No. ,
  • the enzyme preparation is applied at a suitable step during the fermentative production process.
  • the overall carbohydrate concentration is below 20% w/v, more preferably below 10% w/v, and most preferably below 5% w/v, in order to minimize reversion reactions which will result in other and/or new unfermentable sugars.
  • the overall carbohydrate content is preferably low, i.e., below 10% w/v.
  • the addition of the enzyme preparation can be modified so as to be added at a step in the process which is least disruptive of the sugar preparation process and will be most suitable for the optimal conditions under which the enzyme acts.
  • the residual sugar hydrolyzing enzyme is utilized to hydrolyze residual sugars in a liquefied starch solution.
  • the residual sugar hydrolyzing enzyme can be added to the liquefied starch produced by jet liquefaction of starch with ⁇ -amylase.
  • the residual sugar hydrolyzing enzyme can be added simultaneously with glucoamylase in the saccharification step.
  • the residual sugar hydrolyzing enzyme can be added after the liquefied starch has been treated with glucoamylase to further increase the DX value of the saccharified starch or during the actual fermentation process to increase fermentable substrate.
  • isomerized fructose/glucose syrups may be treated with the maltulase enzyme to further increase the concentration of glucose and fructose and reduce maltulose content.
  • residual sugar hydrolyzing enzyme will be modified to best take advantage of the kinetics of the specific enzyme selected. Such process modification is well within the skill in the art.
  • the temperature of the residual sugar hydrolysis step is from about 15° to about 70°C, more preferably from about 20°C to about 60°C; the pH is preferably from about 4-8 and more preferably from about 4.5-7.
  • This embodiment of the invention has proven to be especially successful in the hydrolysis of maltulose and isomaltose present in corn starch derived sugar syrup. It is believed that residual sugar content increases with the increasing pH of the liquefaction step of starch hydrolysis.
  • the isomaltose content isomerases with increasing DS content during saccharification.
  • the present invention will be especially useful in fermentative production processes which involve a starch product which was produced by liquefaction at a pH of between 5-7, or which has a DS content of greater than 20% w/v during saccharification.
  • concentration of residual sugar hydrolyzing enzyme used in a particular process will be dependent on the specific process in use. However, given the disclosure herein, one of ordinary skill in the art would be able to easily ascertain the appropriate concentration. For example, in the case of maltulose hydrolysis in a 20% dry solids sugar solution containing 2% maltulose, maltulose will be present in a quantity of approximately 4 g/kg of d.s. sugar. Thus, where 1 unit equals the hydrolysis of I ⁇ mole of maltulose/minute, 43 U/kg of syrup will be needed to hydrolyze the maltulose in solution in 10 hours.
  • added residual sugar hydrolyzing activity in this case maltulase, is greater than about 10 U/kg sugar d.s., more preferably between 20 and 5000 U/kg sugar d.s., and most preferably between 25 and 1000 U/kg sugar d.s.
  • the term "fermentative production process” is defined as any production process which comprises the culturing of a microorganism to produce a desired product.
  • the present invention is demonstrated with examples from the field of fermentative production of ethanol, it should be appreciated that the invention is not limited to ethanol production, but can also be applied in other fermentative production processes where unfermentable noncellulose and nonhemicellulose based saccharides are present.
  • Possible examples of such processes are the fermentative production of primary metabolites (such as ethanol, and glycerol), organic acids (e.g. lactic acid, acetic acid, succinic acid, etc), amino acids, antibiotics (e.g. penicillin), yeast, biomass to be used as single cell protein, proteins (such as enzymes), vitamins, dyes, and steroids.
  • the term "unfermentable noncellulose and nonhemicellulose based saccharide” refers to any saccharide which does not originate from cellulose or hemicellulose and which is not fermentable.
  • saccharides include isomaltose, maltulose, stachyose, raffinose, and melibiose.
  • fermentable refers to the ability of the microorganism employed in a fermentative production process (e.g. the use of yeast S. cerevisiae to produce ethanol from glucose) to utilize these saccharides.
  • residual sugars means unfermentable sugars present in carbohydrate based fermentation media including isomaltose, maltulose, stachyose, raffinose and melibiose.
  • residual sugars consist mainly of isomaltose and maltulose.
  • a further aspect of the present invention ensures that the method to hydrolyze the unfermentable saccharides is applied in an economically attractive way.
  • the application of the enzyme preparation for the hydrolysis in the unfermentable saccharides in an immobilized form results in a drastic reduction in the amount of enzymes required compared to the use of soluble enzymes.
  • Suitable means of immobilization of enzymes are known in the art and include, e.g., inclusion, attachment, fixation in, to, or on carrier materials.
  • the present invention contemplates incorporation of an immobilized enzyme reactor in ethanol production processes. Process outlines thereof are presented in Figures 1A and 1 B for wet milling continuous ethanol production and in Figures 2A and 2B for batchwise ethanol production.
  • the method of the invention is advantageously used at a step having a low carbohydrate concentration to allow efficient conversion of residual sugars to glucose without the production of reversion products.
  • the invention should also be used at low carbohydrate concentrations, for example, at the end of the fermentation.
  • the present invention is utilized as a separate reactor step during the fermentation process or as a combined step by applying residual sugar hydrolyzing enzyme to the fermenting microorganism vessel.
  • the fermentation broth may be recycled from the fermenter and subjected to the inventive method with the product stream sent back to the fermenter for fermentation of released fermentables.
  • inventive method with the product stream sent back to the fermenter for fermentation of released fermentables.
  • Carbohydrate analysis may be performed by the HPLC method under the following conditions:
  • Support material Sephacryl S 200 HR.
  • the maltulose preparation was diluted 4 times with distilled water. 100 ⁇ l of this solution were mixed with 200 ⁇ l of a fraction having maltulose hydrolyzing activity and
  • the specific activity is expressed as an activity value per mg of protein per minute of reaction time. 1. Determination of the protein content.
  • the protein content is determined using the BCA method with bovine serum albumin as standard. 2.
  • a solution is made of 10 mM p-nitrophenol in 50 mM sodium acetate buffer pH 5.5. This solution is diluted to 240-160-80-40 mM. 1 ml of these solutions is added to 2 ml of
  • Activity definition one Unit of ⁇ -galactosidase is the amount of enzyme which hydrolyses 1 ⁇ ol of p-NPGal/minute under the standard conditions.
  • maltulose solution 100 ⁇ l of maltulose solution is mixed with 200 ⁇ l of enzyme solution and 700 ⁇ l of distilled water. The mixture is incubated at 33°C. Samples were taken at different incubation times and analyzed by HPLC in order to determine the amount of maltulose hydrolysed.
  • ⁇ -galactosidase units ⁇ -gal per mg protein.
  • Maltulose hydrolysing activity ⁇ g maltulose hydrolysed per mg of protein per minute.
  • Residual sugar hydrolysing activity ⁇ g residual sugars hydrolysed per mg of protein per minute.
  • the chromatogram resulting from gel filtration of the ⁇ -galactosidase preparation is presented in Figure 4.
  • the results of assaying of fractions on the presence of ⁇ -galactosidase activity, maltulose hydrolysing activity and residual sugar hydrolysing activity are presented in the following Table 8.
  • the results were used to pool fractions.
  • the pooled fractions and starting material were assayed for specific activity. The results are shown in Table 9.
  • Tables 8 and 9 demonstrate that the maltulose and residual sugar hydrolysing activity are side activities in the ⁇ -galactosidase preparation and are not due to the ⁇ -galactosidase itself. In addition, it appears that the specific activity of both enzymes can be significantly increased by a single purification step.
  • a solution of ⁇ -galactosidase was heat treated for 30 minutes at 65°C. Next, the starting material and the heat treated solution were assayed for specific activity.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
EP95938927A 1994-10-27 1995-10-26 Eine methode für verbesserte rohmaterialverwendung in fermentationsverfahren Withdrawn EP0788551A1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP95938927A EP0788551A1 (de) 1994-10-27 1995-10-26 Eine methode für verbesserte rohmaterialverwendung in fermentationsverfahren

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP94203123 1994-10-27
EP94203123 1994-10-27
PCT/US1995/013876 WO1996013600A1 (en) 1994-10-27 1995-10-26 A method for improved raw material utilization in fermentation processes
EP95938927A EP0788551A1 (de) 1994-10-27 1995-10-26 Eine methode für verbesserte rohmaterialverwendung in fermentationsverfahren

Publications (1)

Publication Number Publication Date
EP0788551A1 true EP0788551A1 (de) 1997-08-13

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EP95938927A Withdrawn EP0788551A1 (de) 1994-10-27 1995-10-26 Eine methode für verbesserte rohmaterialverwendung in fermentationsverfahren

Country Status (5)

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EP (1) EP0788551A1 (de)
AU (1) AU4013295A (de)
CA (1) CA2203811A1 (de)
FI (1) FI971781A (de)
WO (1) WO1996013600A1 (de)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001062947A1 (en) * 2000-02-23 2001-08-30 Novozymes A/S Fermentation with a phytase
AU2002213841A1 (en) * 2000-11-10 2002-05-21 Novozymes A/S Secondary liquefaction of starch in ethanol production
WO2002038786A1 (en) * 2000-11-10 2002-05-16 Novozymes A/S Ethanol process
EP1604019B1 (de) 2003-03-10 2010-01-06 Novozymes A/S Verfahren zur herstellung von alkohol
WO2009049136A2 (en) * 2007-10-12 2009-04-16 Novozymes A/S A process of producing a fermentation product from molasses
JP5463349B2 (ja) * 2008-04-30 2014-04-09 ダニスコ・ユーエス・インク モラセスを用いた効率が向上した発酵法
WO2010086840A2 (en) * 2009-02-02 2010-08-05 Richcore Life Sciences Pvt. A process to enhance ethanol yield from molasses fermentation, by addition of enzymes which convert unfermentable sugars into fermentable sugars
CN105339500A (zh) 2013-06-20 2016-02-17 诺维信公司 具有减少的发泡的发酵工艺
CA3007420A1 (en) * 2015-12-17 2017-06-22 Cargill, Incorporated Sugar transporter-modified yeast strains and methods for bioproduct production
BR112019002238A2 (pt) 2016-08-05 2019-05-14 Cargill, Incorporated polipeptídeo e célula manipulados, e, método de fermentação.

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Publication number Priority date Publication date Assignee Title
DE1919867A1 (de) * 1968-05-08 1970-07-23 Forsch Die Gaerungsindustrie Verfahren zur Verzuckerung und Vergaerung von polysaccharidhaltigen Maischen fuer die Spiritusgewinnung
US3832284A (en) * 1972-03-30 1974-08-27 Agency Ind Science Techn Method for manufacture of alpha-galactosidase by microorganisms
SE7907035L (sv) * 1979-08-23 1981-02-24 Berbel Hegerdal Forfarande for framstellning av flytande brensle ur biologiska ravaror
DE3533352A1 (de) * 1985-09-19 1987-03-19 Sabine Tramm-Werner Biotechnologisches kontinuierliches verfahren zur hydrolyse von kohlenhydraten und die simultane weiterverarbeitung der spaltprodukte durch mikroorganismen
US5231017A (en) * 1991-05-17 1993-07-27 Solvay Enzymes, Inc. Process for producing ethanol

Non-Patent Citations (1)

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Title
See references of WO9613600A1 *

Also Published As

Publication number Publication date
FI971781A0 (fi) 1997-04-25
AU4013295A (en) 1996-05-23
WO1996013600A1 (en) 1996-05-09
CA2203811A1 (en) 1996-05-09
MX9702933A (es) 1997-07-31
FI971781A (fi) 1997-04-25

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