CA2063490A1 - Extracellular preparation of high molecular weight homopolysaccharides and the use thereof, and the fungal strains therefor - Google Patents

Abstract

O.Z. 0960/02014 Abstract of the Disclosure: A process for the extra-cellular preparation of homopolysaccharides of molecular weight 5 - 25 million with exclusively .beta.-1,3-D-glucopyra-nose units in the main chain, each third of which is .beta.-1,6-glycosidically linked to another glucose unit, which comprises culturing microorganisms in the form of at least one of the fungal strains DSM 6318, DSM 6319 and DSM 6320 in a nutrient medium with aeration and agitation at from 15 to 40°C, then separating the culture solution from the biomass and isolating the resulting water-soluble homopolysaccharide therefrom in a conventional manner, and the said fungal strains and the use of the polysaccharides are described.

Description

O.Z. 0960/02014 The extracellular preparation of high molecular weiqht homopolysaccharides and the use ther ofi and the funaal strains therefor The present invention relates to novel micro-organisms and to an improved process for the preparation of high molecular weight uncharged homopolysaccharides (PS) with the aid of these microorganisms.
Non-ionic biopolymers have found various indus-trial uses. Besides use, for example, in the foodstuff, cosmetic and pharmaceutical industries, a particularly important area of use is of high molecular weight PS for secondary and tertiary oil extraction. One PS type which meets particularly well the requirements to be met by polymers in tertiary petroleum extraction (especially high visco~ity with minimal dependence on the salt content, and heat re3istance, a~ well as low adsorption to the rock and high pore-penetrating ability of the aqueous solutions) is composed of a main chain of ~-1,3-linked glucopyranose units, each third of which is ~-1,6-glycosidically linked to another gluco~e unit (as side chain~. The microbial production of compounds of this type with the aid of filamentous fungi is described, for example, in US 3 301 848 and EP-A-271 907. The produc~s obtained as described in the US patent have, however, relatively low molecular weights, and the cultures described in the EP-A are unstable when attemp~s are made to use them in a continuous process, ie. PS production decreases rapidly in favor of fungal growth. According to the thesi~ of S. Munzer (TU Braunschweig 1989), Schizo-phyllum commune A~CC 38548 produces, in submerged cul-ture, considerable amounts of the abovementioned PS, but it has emerged that thi~ fungal strain also undergoes rapid morphological change, with a great reduction in PS
production, on continuous culture.
Niederpruem et al. describe, in Sabouraudia 15 (1977) 283 - 295, the microbial synthesis of a ~-1,3-, ~-1,6-glucan, ie. a PS of the s~ructure described above, by - 2 - O.Z. 0960/02014 cultivation of various mono- and diXaryotic Schiæophyllum commune strains. It has emerged in this connection that all the dikaryotes producP considerably less PS than their monokaryotic parent mycelia added together, even when the dikaryote was d~rived from two monokaryotes both of which were intrinsically very good PS producers.
Similar productivities have also been found by Wang and Miles, Physiol. Plant 17 (1964) 573 - 588. However, even the highest of the very wide range of productivities found are unsatisfactory for practical purposes. Neither publication describes a continuous process.
It is an object of the present invention to find a microorganism which not only produces large amounts of the abovementioned PS but al~o remains stable in continu-ous culture, ie. neither it nor it~ productivity undergo a deleterious change.
We have found that this object is achieved by the three fungal strains DSM 6318, 6319 and 6320 deposited at the Deutsche Sammlung von Mikroorganismen Und Zell-kulturen GmbH, Braunschweig, and by the process claimed in claim 2. For this purpose, initially the two monokaryotic parent mycelia of the dikaryote S. commune ATCC 38548 were i~olated, specifically by protoplast formation and subsequent regeneration of the dikaryotic mycelium of the said strain. It was cultured as follows:
Preculture:
500 ml Erlenmeyer flasks with baffles, which contain 100 ml of medium, were used for the preculture.
A piece of my~elium from an agar slant culture was used as inoculum. The incubation lasted 5 to 6 day~ at 27C on a rotary shaker at 100 rpm.
Subsequently the re3ulting inhomogeneous culture broth was homogenized for 20 to 30 sec with an Ultra-Turrax at 20,000 rpm under sterile conditions (Ultra-Turrax type TP 18-10 and shaft 18 from IKA, Staufen Lm Breisgau).

2~63~9Q

- 3 - O.Z. 0960/02014 Main culture:
The main cultures were carried out in 1 1 Erlen-meyer flasks without baffles containing 250 ml of medium.
The flasks were inoculated with 5~ (VtV) of the homogen-ized preculture and incubated at 27C on a rotary shakerat 100 rpm.
The media for the preculture and main culture had the following composition:
Glucose H2O 33 g~l techn. yeast extract 3 g/l KH2PO4 1 g/ 1 MgSO4 7 H2O 0.5 g/l For protoplast formation, the mycelium from the main culture, after cultivation for 48 h, was filtered through nylon gauze with a mesh width of 120 ~m and washed several times with protoplast-formation buffer (0.05 ~ maleic acid/NaOH buffer, pH 5.8 + 0.5 M MgSO4 as osmotic agent). Subsequently 30 ml of sterile-filtered protoplast-formation buffer in which 300 mg of Novozym 234 (supplied by Novo Industrie, Bagsvaerd, Denmark) were dissolved were added to 15 g of wet mycelium in a sterile glass beaker. This was followed by incubation for 2 to 3 h on a shaker at 100 rpm, during which the protoplasts were released.
After the end of the incubation, the protopla~t~
were separated from the other cell debris by filtration through a cotton plug. It was possible to remove the lytic enzyme solution from the protoplasts by gentle centrifugation (40 min at 1500 g) followed by resuspension in protoplast-formation buffer.
For regeneration, a protoplast suspension waq ad~usted to a defined number of protoplasts using 10 ml of a liquid regeneration agar medium at about 45C. The protoplasts suspended in the agar medium were then poured onto prepared, osmotically stabilized agar plates in which the bottom and top agars had the same composition.
The plates were then incubated at 27C for 5 to 8 days, _ 4 _ o.z. 0960/02014 and the colonies were then examined under the microscope.
The bottom and top agars (regeneration agar medium) had the following composition:
glucose H2O 33 g/l techn. yeast extract 0.5 g/l peptone 2 g/l KH2PO4 0.46 g/l R2HPO4 1 g/l MgSO4 7 H2O 122 g/l as osmotic agent agar 10 g/l Once the regenerated mycelia had reached a diameter of about 5 mm they were examined under the microscope for clamp connection formation. Mycelia whose septa had no clamp connections were identified as monokaryotes, singled out by subcultivation and underwent strain maintenance. 13 monokaryotes (= protoclones) were isolated in this way from three protoplast-formation mixtures. Hereinafter the term protoclone is always used when referring to monokaryote~ obtained by protoplast formation from a dikaryote.
Since the initial dikaryote contained only two genetically different nuclei, it was possible to assign the 13 isolated monokaryotes according to their genome to group A (protoclone A) or B (protoclone B) on the ba~is of crossing experiments and by screening of the protein spectra.
Comparison of submerged cultures of the two monokaryotic parent mycelia protoclone~ A and B with the initial dikaryote S. commune ATCC 38548 revealed that, whereas the dikaryote produced 10 g of PS/l in 120 h, the two monokaryotes synthecized Rcarcely detectable amounts in the same period. This re ult shows that the finding of Niederpruem et al~, that the monokaryotic parent mycelia produced more PS than the resulting dikaryote, has no general validity.
The two protoclones were cros~ed with various other monokaryotic S. commune strains. These crosses 206349~
- 5 - O.z. 0960/02014 resulted in dikaryotes which had a higher productivity than the initial dikaryote S. commune ATCC 38548, as illustrated by the following Example3.

Crossing of the protoclone B strain with the monokaryotic strain S. commune ATCC 36481 results in S. commune DSM

The crossing was carried out in Petri dishes inoculated in the center with a piece of mycelium from each of the fungal strains at a distance of about 1 cm.
The composition of the agar medium corresponded to the complex medium for the submerged cultivation (see above) with the addition of 15 g of agar/l. The incubation was carried out at room temperature and under daylight. As soon as the first fruiting bodies were visible (after about 10 to 14 days), a type culture of the novel dikaryote wa~ set up.
Subsequently the novel dikaryote DSM 6320 under-went submerged culture in 1 1 Erlenmeyer flasks (see abo~e for cultivation conditions). The results of this cultivation are compared with those of some of the fungal strains most suitable for thi~ purpose, including the initial dikaryote S. commune ATCC 38548, in Table 1.

Crossing of the protoclone A strain with the monokaryotic strain S. commune ATCC 36481 ~ DSM 6319 The crossing and subsequent submerged cultivation of the dikaryote S. commune ATCC 36481 x protoclone A
were carried out a~ in Example 1. The results of the submer~ed cultivation are shown in Table 1.

Crossing of the protoclone B strain with the monokaryotic strain S. commune ATCC 26891 ~ D5M 6318 The crossing and subsequent submerged cultivation of the dikaryote S. commune ATCC 26891 x protoclone B
were carried out as in Example 1. The results of the submerged cultivation are shown in Table 1.

2063~Q
- 6 - O.Z. 0960/02014 Comparison of polysaccharide production by various filamentous fungi on submerged cultivation in complex medium in 1 1 Erlenmeyer fla~ks. The parameter~ were determined when cultivation was stopped (after 120 h) E~ample DWM (g/l) PS (g/l) Yps/S

1 (S. commune DSM 6320) 2.5 12.0 0.4 2 (S. commune DSM 6319) 4.5 11.0 0.36 3 (S. commune DSM 6318) 4.1 11.5 0.38 S. commune ATCC 38548 3.9 10.0 0.35 Fungal strain DSM 3887 - 8.7 0.29 Fungal strain DSM 3891 - 4.0 0.16 Fungal strain DSM 3892 - 9.0 0.27 * cf. EP-A-271 907 DWM = dry weight of mycelium YPS/S = product yield index (g of PS/g of substrate consumed) In contrast to the results of Niederpruem et al., 20 that only monokaryotic mycelia produce substantial amounts of PS, the crossings between the protoclones and other compatible monokaryotes have resulted in dikaryotes which prove to be excellent PS producers. It is particu-larly noteworthy that not only do all three novel strains produce more PS in the same cultivation time than some of the microorganism4 hitherto di~closed as mo t ~uitable, but they also (in contrast thereto) undergo no morpho-logical changes on continuous cultivation and, in parti-cular, show no change in productivity, moreover, as is evident from Example 4, especially Table~ 2 and 3.
The valuable PS described in the introduction are obtained using the novel fungal strain3 in the conven-tional proces~ as described in, for example, EP-A-271 907. For this, the said fungal strains are cultured at about 15-40C in a nutrient m~dium with aeration and agitation, preferably continuously, and ~ubsequently heated at 70-90, preferably 75-85, C, expediently for ~ 7 ~ O-Z- 0960/020~4 6 3 ~ ~ O
2-30, preferably 5-lS, min, a~ soon as the fungal strains have fully grown, and then the culture solution is separated from the biomass, ~nd the produced PS is isolated therefrom with a purity in the range from 0.03 to 0.1% by weight of residual pLotein, for example by evaporation or spray drying of the solution. The continuous process can expediently be carried out as follows:
After incubation in a 30 1 bioreactor for 50 h, a change LS made to continuous dosage of the medium. The dosage of the medium takes place with a peristaltic pump from a sterilizable stirred vessel with an effective volume of 200 1. The control of the level in the bio-reactor is carried out by conductivity probes located on the surface of the liquid in th~ bioreactor and, on contact, actuate a peristaltic pump to con~ey culture suspension out of the reactor through an open outlet (vapor-treated T-piece). The limits, specific for the microorganism, of the throughput D, the ratio of the volume of medium flowing in and the effective volume of the bioreactor, are fixed at the lower end by maintenance metabolism and at the upper end by the maximum growth rate.
The fungal strains are provided with conventional ~5 nutrient media supplying the sources of utilizable nitrogen and carbon and the inorganic ion necessary for growth. The upper limit of concentration of the nutrient medium is established when the solid/liquid/gas three-phase system becomes too viscous to stir. In batchwise cultivation, culturing i5 expediently ~topped when the residual concentration of the ~ource of carbon is of the order of 0.1% of the weight of the culture broth. This particularly applies when the fungal strain produces 1,3-and/or 1,6-glucanase which would degrade the produced PS.
This gluGanase production start~ about 2 days after the glucose supplied to the fungus has been consumed.
The non-ionic PS products have a viscosity in the 2~ 34~

- 8 - O.Z. 0960/02~14 range from 50 to 190 mPa.s at a shear rate ~ _ 0.3 5-1 at 40C determined on 0.3 g of PS dissolved in 1000 ml of water of high salinity (containin~ up to 100 g/l alkali metal ions and up to 30 g/l alkaline earth metal ions).
This viscosity remains virtually constant at 60C in the air for 1 year or more. With exclusion of oxygen this then applies up to 90C. The viscosity of the novel non-ionic PS products varies little at concentrations in the range from 0.1 to 1 g/l at about 20 - 60C and with a shear rate in the range from about ~ = 3.0 s-1 and above, so that it can be regarded as constant for practical purposes. Even after a solution containing 0.3 g/l of the PS obtainable according to the invention has been heated in an autoclave at neutral pH and 120C` under 1 bar superatmospheric pressure for one hour, the viscosity after cooling to 20C and replacement of the evaporated water i~ virtually unaffected at shear rate~ in the range from 0.1 to 300 s-1.
The fungal ~trains can be used Lmmobilized on a carrier substance, preferably a polyurethane foam, in a conventional manner, and this Lmmobilizate can be used to produce the PS semicontinuously or completely continu-ously.
The PS obtainable according to the invention are outstandingly suitable for u~e as dispersants or emulsi-fiers (alone or in combination with anionic and/or non-ionic surfactants), as pseudoplastic thickening agents, as gel formers, as humectants and for improving the adhesion of materials to solid surfaces. The u~e as thickening agents especially relates to use in drilling muds, for secondary and tertiary flooding of petroleum deposits and generally for reducing frictional resi~tance in flowing, especially turbulent aqueous liquids in order to reduce the pressure drop. Examples of uses as disper-sants are in agricultural chemicals such a~ fungicidesand pesticide~, and in human and animal foods.
The filtration properties of the PS produced ~063~90 _ 3 - O.Z. 0960/0201~
according to the invention, which indicate the inject-ability into porous media such as petroleum-bearing rocks, are outstanding: for a sample of 0.3 g of PS/l of water of high salinity containing up to 100 g/l alkali metal ions and up to 30 g/l alkaline earth metal ions on pressure filtration under one bar superatmospheric pressure through a 3 ~m membrane filter with 200 ml of solution they are in the range from 0.5 to 3.0 min sheared for 5 sec and in the range from 0.8 to 180 min unsheared, or through a 1.2 ~m membrane filter in the range from 1.6 to 5 min sheared for 5 sec and in the range from 4.0 to 300 min unsheared.

Cultivation conditions for the three novel Schizophyllum commune strains DSM 6318 to 6320:
The preculturing and batch culturing were carried out in the following medium:
glucose: 30 g 1~
techn. yeast extract: 3 g 1~1 MgSO4: 0.5 g 1 KH2PO4: 1 g 1~1 After cultivation for about 50 h in a 30 1 bioreactor equipped with 3 paddle mixers with inclined blades with a diameter ràtio of D = 0.64, continuous operation was started as described above.
Since the productivity of the microorganism depends on the homogeneity of the culture broth, when the average diameter of the cell agglomerates exceeds about 1 mm the culture broth is recycled by a gear pump (Verder type V 150.12). The homogeneity of the culture broth during the continuous fermentation is sufficient when there are 4 cycles per volume exchange and per gram of bioma~s produced per liter. This number of shear cycles was determined empirically and represents a compromise between a high cell surface area, extensive detachment of glucan from the cell wall and little cell damage~
The pH was not controlled. The continuously 2063~90 - 10 - O.Z. 0960/02014 produced culture broth is worked up batchwise as des-cribed in EP-A-271 907.
Feed concentrations for the continuous process:
glucose: 0.80 g 1~1 h~
techn. yeast extract: 0~08 g 1-1 h-MgSO4: 0.01 g KH2PO4: 0.02 g 1-1 h-Cultivation conditions The following Tables show the stationary biomass and PS concentrations for S. commune DS~ 6320 during continuous fermentation as a function of the cultivation time with the following process parameters (which also apply to the preculturing and batch culturing):
Temperature: 27C
Aeration rate: 0.08 vvm (volume/volume minute) Stirring speed: 150 min~~
Throughput: D = 0.072 h~

Time [days] X1 [g 11] PS [g 1-1]
2 0.97 5.4 8 1.10 5.4 14 1.05 5.5 18 1.02 5.4 31 0.98 5.4 X = dry bioma95 At a throughput of D = 0.04 h~1 and a stirring speed of 100 min~1, with the conditions otherwise the same, the analytical results are as follows:

20634~

~ O.Z. 0960/02014 Time [days] X [g 1-1] PS [g 1lJ
-0.41 4.1 11 0.39 4.0 23 0.38 3.9 43 0.41 4.2 0.39 4.2 6a 0.42 4.1 0.41 4.1 The differences in throughput are unimportant because the system i8 stable in both cases (in equili-brium). However, it is possible that altering the aera-tion rate (stirring speed) has an effect.
Physical data on the PS produced by S. commune DSM 6318, 6319 and 6320 under the stated cultivation conditions:
DSM 6318 MW = 14.8 x 105 g/mol DS~ 6319 MN = 12.2 x 106 gtmol DSM 6320 MW = 17.8 x 106 g/mol The molecular weights MW were determined from the Staudinger index: the Mark-Houwink equation relate~ the average molecular weigh~ MW of the PS to tha easily mea~urable Staudinger index t~], as follows:
[~7] = K x MWa K = 4.45 x 10 7 MW = ([~]/K)l/~ a = 1.49 Heat-resistances The vi~cosity found at a product concentration of 3.3 g l-1, a shear rate of 0.3 s-1 and at 40C was 152 mPa. 5 . The viscosity did not change when a product sample was ~tored in the air at 80C for ten days~
pH stability:
The vi~cosity, measured under the conditions stated above, ic independent of the pH in a wide range, as shown in Figure 1. Only when the pH exceeds 12.2 is there, owing to a change in conformation, a brief rise followed by a drastic fall in viscosity.
The PS produced by S. commune DSM 6318 and 6319 - 12 - O.Z. 0960/02014 have approximately the same physical data. The molecular weights measured by the abovementioned method are in the range from 5 to 25, preferably 12 to 25, million. DSM
6320 is particularly preferred because this strain has the highest productivity.
The fungal strains are "fully grown" when they have entirely consumed, or nearly so, the available nutrient solution. It is possible in principle to dis-pense with heating the culture medium after the fungal strains are fully grown, if it is certain that the PS
solution does not contain a single organism after removal of the biomass.
The PS of the three novel fungal strains have identical structures and are equivalent in molecular weight, and thus in all their properties, to the best of those hitherto disclosed. The invention lies in the provi~ion of the novel Schizophyllum commune strains DSM
6318 to 6320 which produce these high-value PS in rela-tively large amounts in a continuou~ process, which was not hitherto possible.

Claims (6)

1. The fungal strains DSM 6318, DSM 6319 and DSM 6320.

2. A process for the extracellular preparation of homopolysaccharides of molecular weight 5 - 25 million with exclusively .beta.-1,3-D-glucopyranose units in the main chain, each third of which is .beta.-1,6-glycosidically linked to another glucose unit, which comprises culturing microorganisms in the form of at least one of the fungal strains DSM 6318, DSM 6319 and DSM 6320 in a nutrient medium with aeration and agitation at from 15 to 40°C, then separating the culture solution from the biomass and isolating the resulting water-soluble homopolysaccharide therefrom in a conventional manner.

3. A process as claimed in claim 2, wherein an aqueous solution of starch, hemicellulose, glucose or sucrose is employed as nutrient medium in a concentration such that the culture suspension in the solid/liquid/gas 3-phase system can be mixed in every stage of growth.

4. A process as claimed in claim 2, wherein the cultivation of fungal strains whose .beta.-1,3- or .beta.-1,6-glucanase or both are positive is stopped at a residual concentration of carbon substrate of about 0.1% by weight.

5. A process as claimed in claim 2, which is carried out continuously or semicontinuously.

6. A process as claimed in claim 5, wherein the fungal strains are immobilized by conventional methods on a carrier substance before use.