DD202306A5 - METHOD OF OBTAINING CELLULASE AND APPARATUS FOR IMPLEMENTING THE PROCESS - Google Patents

Abstract

In the method, a suspension of cellulosic starting material in aqueous medium is inoculated with the microorganisms, i. Culture submersed in fermenters developed and the solids are separated from the Cellulasehaeltigen solution. To d. Process continuous, safe u. Without being able to inhibit production by elaborate preparatory work on the fermenters, the inoculated culture is progressively developed in a plurality of main fermentors connected in series, whereby the suspension is continuously passed through all main fermentation stages in all main fermenters by maintaining a constant filling level in all main fermenters and wherein, from stage to stage, the concentration of cellulase increases and the cellulose concentration decreases. In the plant are arranged in succession: a Aufschlagbehaelter (2) equipped with Rührwerk, at least one equipped with agitator sterilization container (10), which is preceded by a mixer (7) with a supply (8) for sterilization, a Vorfermentationseinrichtung (11, 12, 13), a Hauptfermentationseinrichtung, which has a plurality of fermenters (14), and other means for separating the solids from the cellulase retardant solution, which advantageously Klaerbehaelter (16) and centrifuge (17), optionally an ultrafiltration system (29) for concentrating the cellulase-containing solution and a container (32) for pulping the cellulase.

Description

Berlin, 27.8.1982

AP C 12 N / 238 376/2 60 585/18

Process for the recovery of cellulase and plant for carrying out the process

Field of application of the invention

The invention relates to a process for the production of cellulase by culturing cellulase-producing microorganisms on cellulose-containing! Culture medium in the presence of nutrient salts, wherein a suspension of crushed cellulose-containing starting material in aqueous solution inoculated with the microorganisms, the culture submerged, preferably under aeration, developed in fermenters :, the solids are separated from the culture and the remaining cellulase-containing solution is optionally concentrated, and a facility for carrying out this process.

Characteristic of the known technical solutions

There is already known a method of this kind, wherein the development of the culture in a plurality of juxtaposed and. takes place together connected by lines fermenters. In this so-called "merry-go-round" system, the first fermenter is charged to 80 % of its total volume with cellulose-containing suspension and inoculum, and a fermentation is carried out for about 25 hours. Then, 10 % of the fermented product is fed as an inoculum to the second fermenter, which is filled up with cellulose-containing suspension. Then, the second fermenter is operated for 25 hours, and thereafter 10 % of the obtained fermentate is again supplied to the third fermenter, etc. In this system, sequential inoculation, development and actual production are carried out in each of the fermenters.

3QÄÜ £ 1,982 * 03i767

The disadvantage of this process, in which the growth, adaptation and production phases take place successively in each fermenter, is that a time-consuming and labor-intensive preparation of each individual fermenter, namely sterilization, inoculation, culturing, must be carried out, thereby reducing the production yield becomes. Also, a significant cost is associated with such a production system.

Cellulase is an enzyme which can be used to hydrolyze cellulose-containing starting material, whereby the cellulose is degraded to monosaccharides

It is known that various microorganisms, such as aerobic and anaerobic bacteria, produce actinomycetes and fungi cellulase, with important members of the genus Trichoderma belonging. In particular, Trichoderma reesei strains have been found to be suitable.

The cellulases excreted from the various microorganisms into the substrate differ from each other in their composition, u.zw. with regard to C-1, C-x enzymes and ß-glucosidase. One speaks of so-called incomplete and complete cellulases, i. those which cellulose only up to certain fragments, and those which can degrade cellulose to monosaccharides.

However, so far cellulase has mostly been produced by microbiological means, whereby the growth of microorganisms in surface cultures, e.g. on wheat bran, to which cellulose powder was occasionally added, with ventilation. Such a method is known as Koji method (Biotechnology and Bioengineering, Symposium Report 5.6 / T9 7§7, John

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iViley & Sons).

Disadvantages of this emersen production are that you can only work intermittently, that expensive excipients, such as peptone, protein hydrolysates or emulsifiers and trace elements must be used, that the work-up by leaching the surface cultures is complicated and tedious and that the space required for such plants large is.

Object of the invention

The aim of the invention is to provide an improved process for the production of cellulase, with which the disadvantages of the known processes are avoided.

Explanation of the essence of the invention

The invention has for its object to provide a method which can be carried out continuously, wherein in each fermenter a certain wachuras or production phase can be easily and safely adjusted while maintaining optimal conditions, and wherein the ventilation while avoiding infection of the Substrates by foreign organisms is easier to carry out.

According to the invention, this object is achieved in a method of the type defined in the introduction in that the development of the inoculated culture is carried out in stages in a plurality of main fermenters connected one behind the other, the suspension being maintained in all the main fermenters by all main fermenters while maintaining a constant filling level.

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fermentation stages is carried out continuously and wherein the cellulase concentration increases from step to step and the cellulose concentration decreases.

The cellulose-containing starting material serves as a carbon source for the microorganisms. The nutrient solution is prepared from commercially available mineral fertilizers. As a fuel source for the process according to the invention, in addition to urea and urea derivatives, for example

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Ammonium-containing nitrogen fertilizers, such as ammonium sulfate, chloride and phosphate, - in question. Phosphorus fertilizers such as superphosphate, double superphosphate, triple phosphate and hyperphosphate are suitable as phosphorous sources. On the addition of common trace elements can be omitted, since these in the ordinary tap water used to prepare the nutrient solution and in the added fertilizers as. Accompanying substances are included

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Since most cellulase producing microorganisms require aerobic conditions, the culture medium is preferably aerated.

Appropriately, the cellulosic starting material with nutrient salts and fresh water, preferably ozonated fresh water, added and the suspension is mechanically opened and sterilized before being inoculated.

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For sterilization, steam, ozone or a biolide such as pentachlorophenol sodium can be used.

In each fermenter, the optimal conditions for the respective development phase of the culture with regard to pH, temperature, oxygen content, · consistency and nutrient medium can be adjusted.

The pH is gradually suitably varied gradually between 5.5 to 6 and 3.5 in the course of the development of the culture in the main fermenters arranged one behind the other, the pH being adjusted in particular with ammonia. Ammonia can also be utilized by the microorganisms as nitrogen carriers. 35

According to an advantageous embodiment of the invention

In accordance with the method, the inoculation of the suspension takes place outside the main fermentation stages in pre-fermenters ", the inoculum obtained in the pre-fermentation being supplied to the first main fermenter only at the beginning of the continuous production process.

The pre-fermentation for culturing the microorganisms can also be carried out in several stages, the volumetric ratios of the pre-fermenters in the individual stages being in each case about 1:10. If the pre-fermentation is carried out in three stages, for example, the volumes of the pre-fermenter and of the first main fermenter preferably behave to each other as 1: 20: 200: 5000.

A Vorfermentation proved to enrich the cells of the microorganism as particularly favorable. The main fermenters can have approximately the same volume.

In. Mesophilic microorganisms is suitably maintained in the pre-fermentation and in the Hauptfermentationsstufen a temperature of 25 to 50 C. The initial concentration of cellulose-containing! Material in the suspension is preferably maintained at 1 to 10% mass.

According to a further preferred embodiment of the process according to the invention, after separation of the solids from the cellulase-containing solution of the last main fermentation stage, the solids are hydrolyzed and the hydrolyzate is added as nutrient salt solution to the starting mixture. Thus, an aging of the mycelium is constantly counteracted, u.zw. in that from the first main fermenter constantly new mycelium is led into the next stages. By recycling the hydrolyzate

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important growth substances are reintroduced into the process.

The cellulase-containing solution from the last one becomes advantageous. Hauptfermentationsstufe concentrated in terms of their cellulase content by ultrafiltration and precipitated the cellulase from the concentrate by means of polar solvents. As the polar solvent, for example, acetone is suitable. In this way, cellulase preparations are obtained in dry form.

According to a particularly advantageous embodiment, the culture filtrate withdrawn from the main fermenters is concentrated with respect to its cellulase content by ultrafiltration and the permeate. In fact, as the concentration of cellulase increases, the rate of cellulase formation slows down, so that a continuous removal of cellulase is beneficial to the process, and the recirculated permeate still contains nutrients for the microorganisms.

A plant according to the invention is characterized in that arranged one after the other are: a whipping container equipped with agitator for producing an aqueous suspension containing cellulose and nutrient salts, at least one sterilizing container equipped with stirrers, which is preceded by a mixer with a supply for sterilizing agent, a pre-fermentation device which advantageously has a plurality of pre-fermenters having a capacity increased by about 10 times compared to the preceding fermenter, and a main fermenting device, which advantageously has a plurality of fermenters, wherein the pre-and main fermenter feed devices for acidic

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fabric-containing gases, mixing or mixing organs, venting devices, temperature and pH control devices and possibly foam breakers, and further means for separating the solids from the cellulase-containing solution, which advantageously comprise clarifier and centrifuge, optionally an ultrafiltration unit for concentrating the cellulase-containing solution and a container for precipitating the cellulase.

Embodiment Example

The invention will be explained in more detail below by way of example. The enclosed drawing shows:

A process scheme of an embodiment of the system according to the invention.

Pulp or cellulösehältiges material is first dry pre-ground and then introduced through the feed 1 in a Aufschlagbehälter 2. Nutrient salts are added via the feed 3. Fresh water passes through the line 4, in which an ozonizer 5 is installed for sterilizing the water, in the impact vessel, wherein a suspension is prepared by means of a stirrer. From the impact container, the suspension is fed to a mill in which defibration and fibrillation of the material take place. Subsequently, the suspension passes into a mixer 7, in which it is mixed with a sterilizing agent from the feed 8. The mixer is equipped with deaerators 9 and agitator equipped sterilizing container 10, in which the suspension is sterilized during a predetermined residence time.

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The sterilized suspension is then pumped to pre-fermenters or to main fermenters. The fermentation unit consists in the illustrated embodiment

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three Vorfermentern 11, 12, 13 and four each designated 14, one behind the other main fermenters. All fermenters are equipped with stirrers or mixers, as well as temperature and pH control units and anti-foam systems. Furthermore, each venting with sterile air are provided. The exhaust air from the fermenters is also filtered.

The main fermenters are driven at a constant filling level. The pre-fermenter 11 is added via the feed 15, the inoculum. All fermenters are connected to each other via sterilizable lines, the connections between the main fermenters being arranged so as to permit both discontinuous and continuous operation. Should it be necessary due to a reduced growth rate of the microorganism, then further main fermenters can be switched on in basically the same way.

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In continuous operation is from the last fermenter the entire contents, namely biomass and Enzymlö-) solution, withdrawn and transferred to a settling tank sixteenth Here is a pre-separation of enzyme-containing solution and biomass, such as mushroom mycelia. The biomass can be recycled wholly or partially to increase the cell concentration in the main fermenter 14. Another part of the biomass and the pre-clarified enzyme solution are separated in a separator, such as a centrifuge 17, in solid fractions and KuIturfiltrat, and the filtrate is passed into a equipped with stirring and cooling storage container 18. The separated biomass can then be removed from the plant for any further use, but it can also be used in

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treated form can be recycled by the feeder 19 to the impact container 2 as Nährsalzträger. The. accumulating biomass is protein-containing and can therefore as. Feed additive, as a substrate additive for other microbiological processes but also be used as a nitrogenous fertilizer. However, it is preferable to subject the biomass to prehydrolysis in an autoclave 20 with steam and acid feeds. The resulting hydrolyzate solution is separated from the remaining solid, for example by means of a centrifuge 21, and recycled as nutrient salt solution via line 22. By recycling the biomass in an unchanged plasmolysed form or recycling the hydrolyzate it is not necessary to use additional auxiliaries, such as peptone, protein hydrolysates or emulsifiers, such as polyoxyethylene derivatives of sorbitan anhydrides, which are partially esterified with fatty acid. The over the discharge 23. From the plant removed residual solids can for. Extraction of heat energy to be burned.

In the illustrated embodiment of the invention. An ultrafiltration plant 24 is further provided, by means of which the Kulturfiltr'at optionally withdrawn from the individual fermenters 14 via the manifold 25 can be concentrated in terms of its cellulase content. The enzyme concentrate is combined via the line 2 6 with the culture filtrate from the clarifier 16 and fed to the centrifuge 17, since there may still be living cells in the concentrate, which must be separated before the storage container 18. The permeate is added back to the fermenters 14 through line 27. By doing so, the cell concentration in the fermenters 14 increases, and at the same time, the production of cellulase is accelerated.

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The enzyme solution in the storage container 18 is withdrawn via line 28 and can either continue to be used directly or fed to an ultrafiltration unit 29. The concentrate from (29) can be supplied as such via line 30 to its place of use or mixed in a equipped with a stirrer and feed line 31 for a precipitant container 32 with precipitant. The concentration of the enzyme solution may alternatively also be e.g. be made by thin film evaporation. The precipitated enzyme, after being separated by means of a centrifuge 33 or a filter, is transferred to a drying plant 34.

The solution from the centrifuge 33 is worked up together with the condensed vapors from the drying plant 34 in a distillation unit 35. The recovered precipitant is withdrawn via the supply line 31, the aqueous residue is fed together with the permeate from the ultrafiltration unit 29 via the line 3 6 again the Aufschlagbehälter 2, whereby the need for fresh water is considerably reduced. The drying plant 34 can be designed, for example, as a spray evaporation or thin-film evaporation plant. Im.Anschluss to the drying, the adjustment of the activity of the enzyme dry preparation prepared by the addition of inert material or lactose in a mixer 37. Dry preparations prepared according to the invention have full Cellulasealctivität on.

Example:

30 kg of pulp, 1.5 kg of mycelium from Trichoderma reesei from the current production, 0.7 kg of KH ₂ PO _{4 are placed} in the impact vessel of the plant described above

35- and 1.5 kg (NH ₄ J ₂ SO ₄ introduced and the mixture by addition of ozonated water to a volume of

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1000- · 1 set. After defibrillation, fibrillation and sterilization of the suspension for 20 minutes at 120 C, this approach is the first. Main fermenter with. fresh mushroom mass of Trichoderma reesei has been added from Prefermenter 13.

The residence time in the main fermentors connected in series is in each case 36 hours, the pH values are:

first main fermenter: 5.5 - 6 second. Main fermenter: 3.5 third main fermenter: 3.5 fourth main fermenter: 4,8, the temperature profile is the following:

first main fermenter: 30 ° C

second main fermenter: 29 ° C

third and fourth main fermenter: 28 ° C, supplied air volumes: first main fermenter: 0.2 1/1 substance min

second main fermenter: 0.25 1/1 substance min

third and fourth main fermenter: 0.35 1/1 substance min

The solids content in the fermenters drops from 3% to 2.9%, 1.8% and finally 1%.

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In the first main fermenter is present as a solid cellulose, in the fourth main fermenter is only more mushroom mycelium present, since the cellulose is consumed by cell growth.

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The final (fourth) main fermenter is then followed by 1,000 1 of FPU (filter unit units, formerly FPD activity units, by N. Toyama, Advances in Enzymes Hydrolysis of Cellulose and Related Materials, ET Reese, Ed., Pergamon Press, London 19 63, page 235)) 'per ml corresponding to 5.6 mg protein / ml. The GE-

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total solution thus contains 2.8 × 10 FPU (5.6 kg protein, and 9.6 kg Trichoderma mycelium excess.

For concentration of the enzyme solution, the ultrafiltration was used. A plastic membrane with an exclusion limit of 10,000 was used. This resulted in a concentrate with a volume of 50 1 and a content of 56 χ 10 FPU. This concentrate was treated at room temperature with 100 1 acetone and then the precipitated enzyme was removed by centrifugation and dried in a stream of air. The result was 5 kg of protein in the form of a cheese.

in the form of a white powder containing 2.5 χ 10 FPU Cellu-

Claims (2)

27.8.1982 AP C 12 N / 238 376/2 - 13 * - 60 585/18 Claim for invention 1. A method for obtaining cellulase by culturing cellulase-producing microorganisms on cellulose-containing! Culture medium in the presence of nutrient salts, wherein a suspension of the comminuted cellulosic starting material in aqueous solution with the microorganisms inoculated »the culture subraers, preferably with aeration, developed in fermenters, the solids are separated from the culture and the remaining cellulase-containing solution is optionally concentrated, characterized in that the development of the inoculated culture is carried out stepwise in a plurality of main fermenters connected one behind the other, the suspension being continuously carried out by maintaining all the main fermenters in a constant level in all major fermentation stages and increasing the cellulase concentration from step to step and decreasing the cellulose concentration » 2. The method according to item 1, characterized in that the pH is maintained in the course of development of the culture in the successively arranged main fermenter between 5.5 to 6 and 3.5. 3. The method according to item 2, characterized in that the pH is adjusted with ammonia. 4. The method according to points 1 to 3, characterized in that the inoculation of the suspension takes place outside the main fermentation stages in Vorfermentern, wherein the inoculum obtained in the Vorfermentation the first 08/27/1982 AP C 12 N / 238 375/2 60 585/18 Main fermenter only at the beginning of continuous. Production process is fed » 5 »Method according to points 1 to 4, characterized in that in the pre-fermentation and in the main fermentation stages a temperature of 25 to 50 ⁰ C is maintained. 6. The method according to points 1 to 5, characterized in that the starting concentration of cellulose-containing material in the suspension is maintained at 1 to 10 % by mass 7. The method according to points 1 to 6, characterized in that after separation of the solids from the cellulase-containing solution of the last Hauptfermentations «stage hydrolyzed the solids and the
Hydrolyzate is added as a nutrient solution to the starting mixture » 8. The method according to item 7, characterized in that the cellulase-containing solution by ultrafiltration concentrated in terms of their cellulase content and the cellulase is precipitated from the concentrate by means of polar solvents. 9 »method according to the points 1 to 8, characterized in that from the main fermenter withdrawn KuI turfiltrat concentrated in terms of its cellulase content by ultrafiltration and the permeate is recycled to the fermenters. 08/27/1982 AP C 12 N / 238 37S / 2 50 585/13 ΙΟ. Plant for implementation Inlll ! ' ^{9e), by arranging} in succession: an agitator equipped with agitator (2) for the preparation of an _{aqueous suspension} containing oil and nutritive salts ens of a _mlt agitators equipped sterilization container (10) _{t where M ± Qin echer} -rxlxsxer- leadership '- * - i! TorΓ I l ^Or _h ^{far WimSeinriChtm3} lT ^'Wel0he advantageous ne plurality of Vorfer.entern _{(1 1; 12; 13, asc.} Each one was about 10 times that of the preceding one, the latter being fermented L lit a V ^ 'T'''^'H ° ^ ° ™ »^ tion device di" nhhr »hl _{(solid 14} f the di of solid _(14, which wove _d advantages and Hauptfer ^ enter Zufuhrungseinrichtungen for oxygenated. gases "clock _{b". "techorB.} ^, _Ept. u "r: H ^{n; iChtUn9Sn} · ^« «facilities _{f0 'r} Te _mper ature and pH value and possibly up Schauabrecher ^{ithl e} ITrTTZ ⁱ ^ 9 * "-» Disconnect the solids from the Cellul _ase häl _{term Läsung,} advantageous K _lä rbehäUer (I _6, and centrifuge ^ IZ fa-en, e _{g ge} appropriate, a UItrafiltrationsa la e ₍₂₉₎ 2 Tz ^^: ^ T ^ll the cellulase. For this 1 page drawings