WO1997048816A1

WO1997048816A1 - Method for improving the product yield in fermentation processes

Info

Publication number: WO1997048816A1
Application number: PCT/US1997/010639
Authority: WO
Inventors: Hubert Herrmann; Reinhard Schroder
Original assignee: Genencor International, Inc.
Priority date: 1996-06-20
Filing date: 1997-06-20
Publication date: 1997-12-24
Also published as: DE19624624A1; AU3403597A

Abstract

A method is described for improving the product yield in fermentation processes. In this fermentation method according to the invention, a chemically modified carbohydrate source is used as the C-source-substrate for utilization by the microorganisms in otherwise conventional fermentations, in order to boost the yield of the biological products or valuable substances produced in these fermentation processes by means of microorganisms. This enables the yield of products or valuable substances produced by fermentation to be increased easily without requiring expensive modification of an existing fermentation process in terms of apparatus or analytical technique, or with respect to the existing measurement and control technology. The fermentation method according to the invention for achieving an increased yield is suitable for the production of diverse valuable substances, especially enzymes, and for the use of diverse microorganisms.

Description

METHOD FOR IMPROVING THE PRODUCT YIELD IN FERMENTATION PROCESSES

INTRODUCTION

The invention concerns the improvement of fermentation processes in order to increase the yield of the biological products or valuable substances produced by means of microorganisms in these fermentation processes.

The fermentation of microorganisms is used, for example, to produce biological products or valuable substances, such as proteins, enzymes, or other valuable chemical substances, including those having pharmaceutical action. For this, the microorganisms require nutrient media for both the intracellular and the extracellular formation of the products or valuable substances, which generally contain a so-called carbon source ("C-source"), a nitrogen source ("N-source"), and so-called supplements, such as salts and vitamins. The N-source can be low- molecular- weight chemical substances such as ammonium salts or urea, but also high-molecular- weight nitrogen-containing substrates, such as proteins of natural origin, e.g., soybean meal, cottonseed proteins, cereal proteins, proteins of animal origin such as fish meal, etc. In particular, N-sources of vegetable origin, besides the protein content, can have more or less high starch content and thus serve as the C-source at the same time. In the state of the art, carbohydrates are used as C- sources, primarily mono-, di-, tri- and oligosaccharides, but also polymeric carbohydrates like cereals, potato starches, tapioca starches, or yam root starches. Certain C-sources, such as glucose, fructose, dextrin, are very easily metabolized by the microorganisms and therefore result in an explosive multiplication of the microorganisms, accompanied by rapid oxygen depletion in the fermentation nutrient solution. The oxygen depletion entails a rearrangement of the microorganism metabolism, so that the desired fermentation products are no longer produced, or are produced only in insufficient amount. In the worst case, the microorganisms die because of lack of oxygen. Furthermore, glucose and other mono- or disaccharides in the microorganism cell very often hinder the intracellular induction of enzymes for the secretion of a desirable valuable substance, enzymes for starch breakdown (e.g. , amylase), or other enzymes. Thus, as a result, the desired production of the biological product or valuable substance is often reduced to low quantities, or is even partly or totally stopped by the microorganisms.

Within the state of the art, there has been no lack of attempts to circumvent the foregoing difficulties. For example, attempts have been made to employ C- sources of higher molecular weight, such as the already-mentioned carbohydrates or starches. Essentially, all vegetable starch sources can be used as potential C- sources. Often, the starch products used for this purpose are not isolated from the plants, but instead the vegetable raw materials used for production of starch, such as ground-up fruit or the storage organs of plants, like root nodes or rhizomes, are used as is. Such polymeric C-sources usually involve the meal of cereals, such as wheat meal or corn meal, potato meal or tapioca meal of various grain sizes. However, the meals and starches are very often subjected to further enzymatic treatment before the fermentation, in order to lower the viscosity of the fermentation medium. On the one hand, this should reduce the tendency of the starches to gel in water, thus ensuring that the fermentation medium can be easily agitated, and on the other hand it intensifies the transport of oxygen and carbon dioxide essential for the fermentation of the microorganisms. As a rule, therefore, the polymeric C-sources like starch or starch-containing meals cannot be used without pretreatment, since they would otherwise result in highly viscous suspensions or solutions and possibly even form gels. In order to eliminate the resulting oxygen and mixing problems, the polymeric carbohydrate sources are therefore generally broken down with enzymes (such as amylases or glucoamylases) to such an extent that the proper degree of viscosity is obtained in the fermentation medium, while avoiding a disadvantageous repression of the substrate by low-molecular carbohydrate fragments.

Furthermore, it is known in the state of the art that the yield of valuable substances in biological processes, such as the production of enzymes, is closely related to the ratio of the C-source and N-source in the fermentation medium. Given a knowledge of this optimal ratio, one can then boost the yield of enzymes or other valuable substances by specifically increasing the quantity of substrates such as starch or starch-containing meals in the fermentation medium. Yet there is a natural limit to increasing the quantity of available substrate in that aqueous fermentation media can only take up the dissolved or suspended raw materials to a manageable limiting content. Beyond this limiting content, further increasing of the yield by increasing the quantity of C-substrate is not possible, because again highly viscous fermentation media and deficient material transport would result - even with prior enzymatic breakdown of the C-sources. The resulting reduced growth of the microorganisms would also make the yield of valuable substances, such as enzymes, extremely low. In order to overcome these difficulties, it has been attempted within the state of the art to add C-sources (such as glucose or enzymatically decomposed starch) afterwards, so that the concentration level in the fermentation medium is kept low and the fermentation process can be stabilized. However, the proper dosage over the course of the fermentation process demands a complicated technology. Costly measurement and control equipment is required, and the analysis expense for the fermentation process, e.g., the monitoring of the uptake of substrate by the microorganisms, also increases greatly. The apparatus expense also increases, since additional receptacles are needed. Furthermore, the process becomes more expensive through the frequent addition of raw material, since the raw materials must be sterilized in batches or continuously, and despite these steps the risk of contamination with foreign microbes is still substantially higher.

Therefore, the objective was to provide a simple and economical fermentation method in which the yield of substrate is improved with respect to the C-source, without having to modify an already existing fermentation technology with respect to apparatus, measurement and control equipment, or analysis expense. At the same time, the fermentation method should result in an increased yield of the biological products or valuable substances produced in the fermentation process, such as the enzymes produced. Surprisingly, it has been found that the purpose can be achieved by the use of chemically modified carbohydrate sources. Accordingly, the invention concerns a method of fermentation of microorganisms for the production of biological 4 products or valuable substances, in which one employs a nutrient medium for use by the microorganisms in the fermentation, which contains a chemically modified carbohydrate source, in particular, a chemically modified oligomeric or polymeric carbohydrate source as the C-source substrate. The chemically modified carbohydrate source can be based on any conventional carbohydrate-containing substrates which can be modified in familiar chemical ways. Such basic carbohydrates are, therefore, mono-, di-, tri- and oligosaccharides and, in particular, polymeric carbohydrates, whereby essentially all vegetable starch sources come into consideration; for example, meals of corn, rice, cereals, potatoes, tapioca or yam root, as well as the starches obtained from these plants after chemical modification, can be used within the context of the invention. The chemically modified carbohydrate source used in the method of the invention can be used in the fermentation process either as the sole carbohydrate source or to replace or supplement only a desired portion of a conventional nonmodified carbohydrate source. The possibility also exists of adding the carbohydrate source modified according to the invention, particularly derivatized starch, either at the same time as the nonmodified carbohydrate source or at a suitable later time, in batches or continuously in the fermenter.

The chemical modification in this case occurs at the free hydroxy-groups of the glucose building blocks of the particular carbohydrate source. These hydroxy- groups allow, for example, etherifications, esterifications, oxidations and cross- linking with bifunctional or polyfunctional organic compounds. Examples are: for etherifications, hydroxyethylation with ethylene oxide; for esterifications, acetylation, e.g., with acetylchloride or acetanhydride; or for cross-linking, reaction with epichlorohydrin or phosphoric acid chlorides. Even small degrees of modification are enough to achieve the effects of the invention by substantially increasing the yield of valuable substances or enzymes. Generally speaking, the degree of modification is understood to be the number of chemically modified hydroxy-groups per glucose unit of the carbohydrate source in mol/mol. The degree of modification is advisedly 0.005-4 mol/mol, and in particular 0.01-1 mol/mol. In this context, the practitioner can determine the advisable degree of modification for the particular fermentation process with a little experimentation. 5

Suitable chemically modified carbohydrate sources which can be used in the fermentation method of the invention are therefore, in particular, those whose carbohydrate source is chemically modified and/or derivatized. Preferably, this carbohydrate source serving as the C-source substrate is derivatized by esterification or etherification or chemically transformed by oxidation or cross- linking. Preferred chemically modified carbohydrate sources for this are characterized in that they are derivatized by esterification, in particular, by acetylation of free carbohydrate hydroxy-groups. Especially favorable increased yield in the production of enzymes is accomplished by the use of esterified, especially acetylated starch, with even relatively low degrees of derivatization

(degrees of acetylation) being sufficient. The degree of esterification of the esterified or acetylated carbohydrate sources, preferably the degree of esterification or acetylation of starch used according to the invention, can be determined by saponifying a certain quantity of a specified esterified or acetylated carbohydrate source with a likewise specific excess of alkaline base, such as sodium hydroxide, and then quantitatively determining the unused quantity of the base. The degree of substitution (DS), i.e., the degree of esterification or acetylation, can then be calculated from the molar quantity of consumed base as the number of ester or acetyl groups per glucose unit of the particular esterified or acetylated carbohydrate source. In preferred embodiments of the invention, the degree of esterification or degree of acetylation, determined by saponification of the esterified carbohydrate source as the number of substitution groups per glucose unit (DS), is 0.005-4 mol/mol, and in particular 0.01-1 mol/mol. Especially preferred are esterification or acetylation degrees of 0.02-0.15 mol/mol. In an especially preferred embodiment of the fermentation method according to the invention, a chemically modified carbohydrate source is used, based on an (isolated) starch or a natural starch source, and in particular, on pure starch or starch-containing meal. One possible chemically derivatized, especially esterified or acetylated starch, is acetyl starch. Examples are the tapioca starch acetyl ester with a degree of substitution DS of 0.025-0.032 mol/mol, which is commercially available under the name "FARAZYM T^R" (e.g., that distributed by the AVEBE Co. of Germany, Meerbusch; produced by Avebe B.A., Foxhol, NL), 6 or a corresponding acetylated potato starch ("FARAZYM 76^R"), available from Avebe.

The method according to the invention enables a better utilization of substrate by simple means when using chemically modified carbohydrate sources (C-sources), in order to achieve an increased yield of valuable substances produced by fermentation, such as biological products like enzymes. Furthermore, the method according to the invention offers the advantage that it can be used in existing fermentation plants without changing the technology or the apparatus or analysis expense, or the monitoring and metering expense. Difficult dosing technologies are avoided. Another advantage of the invention is that it can be used for any kind of fermentation, and thus allows the use of advantageous chemically- modified carbohydrate sources for a number of microorganisms conventionally used in fermentation. Therefore, the method according to the invention has diverse uses, especially for microorganisms of the fungus and bacteria families, especially microorganisms which are used in production of enzymes. Examples of possible microorganisms are the genus Aspergillus, Rhizopus, Trichoderma, Penicillium and Bacillus. Special examples for the genus Bacillus are, in particular, Bacillus licheniformis, Bacillus subtiiis, Bacillus alcalophilus, Bacillus lentus, Bacillus amyloliquefaciens, which have been sufficiently described in the technology of enzyme production. The advantages of the invention can also be achieved with optimized, e.g., genetically altered microorganisms, especially those which are used on an industrial scale for the production of enzymes.

The following examples should further explain the invention, without limiting its scope. Examples

Example 1:

Fermentation for production of a highly alkaline protease

Under the standard conditions for the production of a highly alkaline protease of the type Subtilisin 309, comparison fermentation was carried out using as the C-source, on the one hand, potato starch and native tapioca starch (not the standard according to the invention) and, on the other hand, acetylated tapioca starch at pH values of 6.0 or 7.0, by means of a Bacillus alcalophilus strain HA1 (described in the published EP-A 415 296), optimized by conventional mutation and genetic alteration. After this, the yield of protease activity achieved was determined in DU/ml by conventional techniques. By "DU" is meant the enzymatic activity in Delft Units, with 1,000 DU corresponding to the proteolytic activity producing an extinction difference (1 cm light path; 275 nm; compared to blind sample test) of 0.4000 in a volume of 1 ml of a 2-W/W-% enzyme solution after breakdown of casein.

The native and chemically modified starches used had the following characteristics:

Potato starch:

Moisture content 15 to 20 wt. % pH value 6 to 8

(30 mg/g solution in distilled water)

Solubility < 10² g/l

Colony count < 50,000 CFU/g ^*)

Screen analysis < 1 mm 100 wt. %

(10 min. mechanical sifting through a 1 mm screen)

Tapioca starch, native:

Moisture content 11 to 14 wt. % pH value 6 to 8

(30 mg/g solution in distilled water)

Ash (measured as dry substance) ≤. 2 mg/g

Colony count 50,000 CFU/g ^*)

Screen analysis < 1 mm 100 wt. %

(10 min. mechanical sifting through a 1 mm screen)

[^*) CFU = Colony-forming units] Tapioca starch, acetylated:

Moisture content 110 to 140 wt. %

(5 g, dried at

130°C for 90 min.) pH value 5 to 7

(30 mg/g solution in distilled water)

Degree of substitution (DS) 0.025 to 0.032 mol/mol

Screen analysis < 1 mm 100 wt. %

(10 min. mechanical sifting through a 1 mm screen)

The conventional starches used here, potato starch and tapioca starch (native or acetylated), can be obtained, for example, from the firm AVEBE Germany (in Meerbusch).

The following results were found for the fermentation with the above- indicated starches or starch derivatives with respect to the particular maximum achieved yield of activity (for a fermentation time of 72 hours):

^*) Activity yield for the particular pH value, standardized to the activity yield when using nonderivatized potato starch (Experiment No. 1.1 and Experiment No. 1.3) as 100%.

The acetylated tapioca starch used according to the invention revealed a striking improvement in activity formation, which lies clearly above the result obtained with the standard potato starch and also with the native tapioca starch.

The method can also be advantageously used for other fermentations, e.g., for production of enzymes such as proteases, lipases, amylases, cellulases, glucosidases, amyloglucosidases, etc., and shall be further explained in the following example. Example 2:

Fermentation to produce an alkaline protease

Under the standard conditions for production of an alkaline protease of type BPN', a comparison fermentation was carried out using potato starch (as the standard, like in Example 1, not according to the invention) as the C-source, on the one hand, and, on the other hand, acetylated tapioca starch, in two fermentation experiments with two different Bacillus licheniformis strains, after which the achieved activity yield in protease in DU/ml was determined in conventional way (see Example 1 for the unit 1 DU/ml). The potato starch described more closely in Example 1 (as the standard) and tapioca starch acetylated according to the invention were used.

The following results were found for the fermentation with these starches or starch derivatives with respect to the particular maximum achieved yield of activity (for fermentation times of 77 h and 72h for potato starch, compared to a fermentation time of only 56 h for acetylated tapioca starch):

Activity yield for the respective strain, standardized to the activity yield when using nonderivatized potato starch (Experiment No. 2.1 and Experiment No. 2.3) as 100% . The fermentation duration is given in h = hours. The acetylated tapioca starch used according to the invention revealed a striking improvement in activity formation, which lies clearly above the result obtained with the standard potato starch, by analogy with Example 1 , while at the same time advantageously shortening the fermentation time when using acetylated tapioca starch as the C-source substrate.

Claims

WHAT IS CLAIMED IS:

1. Method for the fermentation of microorganisms in order to produce biological products or valuable substances, characterized in that one uses a nutrient medium in the fermentation for utilization by the microorganisms that contains, as the C-source substrate, a chemically modified carbohydrate source, in particular, an oligomeric or polymeric carbohydrate source.

2. Fermentation method according to Claim 1, characterized in that the chemically modified carbohydrate source is a chemically transformed and/or derivatized carbohydrate source.

3. Fermentation method according to Claim 2, characterized in that the carbohydrate source serving as the C-source substrate is derivatized by esterification or etherization or chemically transformed by oxidation or cross- linking.

4. Fermentation method according to Claim 1, characterized in that the chemically modified carbohydrate source is based on an (isolated) starch or a natural starch source, in particular, pure starch or starch-containing meal.

5. Fermentation method according to Claim 3, characterized in that the chemically modified carbohydrate source is derivatized by esterification, especially by acetylation of free carbohydrate hydroxy-groups.

6. Fermentation method according to Claim 5, characterized in that the degree of esterification, especially the degree of acetylation, determined by saponification of the esterified carbohydrate source as the number of substitution groups per glucose unit (DS), amounts to 0.005-4 mol/mol, in particular, 0.01-1 mol/mol.