Patent Application
20170362620
The present invention relates to a process for the fermentation of fungal strains which secrete glucans with a β-1,3-glycosidically linked main chain and side groups β-1,6-glycosidically bonded thereto, in a cascade of tanks using high-shear mixers.
In natural occurrences of petroleum, petroleum is present in the voids of porous storage rocks, which are closed to the Earth's surface by impermeable covering layers. The voids may be very fine voids, capillaries, pores or the like. Fine pore necks can have, for example, a diameter of only about 1 m. Apart from petroleum, including proportions of natural gas, a reservoir comprises more or less salt-comprising water.
In petroleum recovery, a distinction is made between primary, secondary and tertiary recovery. In primary recovery, the petroleum flows spontaneously under the reservoir's own pressure through the well to the surface after drilling down to the reservoir. Depending on the reservoir type, it is usually only possible, however, to recover about 5 to 10% of the amount of petroleum present in the reservoir by means of primary recovery; then, the intrinsic pressure is no longer sufficient for recovery. In secondary recovery, the pressure in the reservoir is maintained by injection of water and/or steam, but the petroleum cannot be fully recovered even with this technology. Tertiary petroleum recovery includes processes in which suitable chemicals are used as auxiliaries for oil recovery. These include so-called “polymer flooding”. In polymer flooding, an aqueous solution of a thickener polymer is injected into the petroleum reservoir via the injection wells instead of water. This enables the yield to be increased further compared with the use of water or steam.
Suitable thickening polymers for tertiary petroleum recovery (also known as enhanced oil recovery (EOR)) must meet a number of specific requirements. In addition to sufficient viscosity, the polymers must also be thermally very stable and retain their thickening effect even at high salt concentrations.
A large number of different water-soluble polymers have been proposed for polymer flooding, specifically both synthetic polymers, such as polyacrylamides or copolymers comprising acrylamide and other monomers, and also water-soluble polymers of natural origin.
An important class of polymers of natural origin for polymer flooding is formed by branched homopolysaccharides from glucose. Polysaccharides composed of glucose units are also called glucans. The specified branched homopolysaccharides have a main chain composed of -1,3-linked glucose units, of which, statistically, about each third unit is -1,6-glycosidically linked with a further glucose unit. Aqueous solutions of such branched homopolysaccharides have advantageous physicochemical properties, meaning that they are particularly well suited to polymer flooding.
Particularly important glucans in this context are beta-glucans. Beta-glucans are known well-conserved components of cell walls in several microorganisms, particularly in fungi and yeast (Novak, Endocrine, Metabol & Immune Disorders—Drug Targets (2009), 9: 67-75). Biochemically, beta-glucans are non-cellulosic polymers of beta-glucose linked via glycosidic beta(1-3) bonds exhibiting a certain branching pattern with beta(1-6) bound glucose molecules (Novak, loc cit). A large number of closely related beta-glucans exhibit a similar branching pattern such as schizophyllan, scleroglucan, pendulan, cinerian, laminarin, lentinan and pleuran, all of which exhibit a linear main chain of beta-D-(1-3)-glucopyranosyl units with a single beta-D-glucopyranosyl unit (1-6) linked to a beta-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3 (Novak, loc cit; EP-B1 463540; Stahmann, Appl Environ Microbial (1992), 58: 3347-3354; Kim, Biotechnol Letters (2006), 28: 439-446; Nikitina, Food Technol Biotechnol (2007), 45: 230-237). At least two of said beta-glucans—schizophyllan and scleroglucan—even share an identical structure and differ only slightly in their molecular mass, i.e. in their chain length (Survase, Food Technol Biotechnol (2007), 107-118).
Homopolysaccharides of said structure are secreted by various fungal strains, for example by the filamentously growing basidiomycete Schizophyllum commune, which secretes, during growth, a homopolysaccharide of said structure having a typical molecular weight Mw of about 5 to about 25*106 g/mol (trivial name schizophyllan). Mention is also to be made of homopolysaccharides of said structure secreted by Sclerotium rolfsii (trivial name: scleroglucans).
Processes for producing branched homopolysaccharides from -1,3-linked glucose units by fermentation of fungal strains are known.
EP 0 271 907 A2 and EP 0 504 673 A1 disclose processes and fungal strains for producing branched homopolysaccharides composed of -1,3-linked glucose units in the main chain. Production takes place by discontinuous fermentation of the strains with stirring and aeration. The nutrient medium consists essentially of glucose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate and water. The polymer is secreted by the fungus into the aqueous fermentation broth, and ultimately an aqueous polymer solution is separated off from the biomass-comprising fermentation broth, for example by centrifugation or filtration.
DE 40 12 238 A1 discloses a process for increasing the space-time yield during the production of nonionic biopolymers, in particular of the fungal strains disclosed in EP 0 271 907 A2. To increase the space-time yield, the limitation of oxygen on the one hand, and furthermore the shearing of the cell walls with homogenization of the fermentation broth, and also the avoidance of pellet formation during the cultivation is disclosed. For the shearing, a toothed-wheel pump in a bypass is proposed.
Processes for fermentation of fungal strains are known for example from EP 0 271 907 A2, EP 0 504 673 A1, DE 40 12 238 A1, WO 03/016545 A2.
In particular, EP 0 271 907 A2, EP 0 504 673 A1 and DE 40 12 238 A1 disclose processes for the preparation, i.e. the preparation is effected by batchwise fermentation of the fungus Schizophyllum commune with stirring and aeration. The culture medium substantially comprises glucose, yeast extract, potassium dihydrogen phosphate, magnesium sulfate and water. EP 0 271 907 A2 describes a method for separating the polysaccharide, in which the culture suspension is first centrifuged and the polysaccharide is precipitated from the supernatant with isopropanol. A second method comprises a pressure filtration followed by an ultrafiltration of the solution obtained, without details of the method having been disclosed. “Udo Rau, “Biosynthese, Produktion and Eigenschaften von extrazellulären Pilz-Glucanen”, Habilitationsschrift, Technical University of Brunswick, 1997, pages 70 to 95″ and “Udo Rau, Biopolymers, Editor A. Steinbüchel, Volume 6, pages 63 to 79, WILEY-VCH Publishers, New York, 2002” describe the preparation of schizophyllan by continuous or batchwise fermentation. To prevent pellet formation, the fermentation broth was circulated in an external circuit equipped with a toothed-wheel pump. “GIT Fachzeitung Labor 12/92, pages 1233-1238” describes a continuous preparation of branched beta-1,3-glucans with cell recycling. WO 03/016545 A2 discloses a continuous process for the preparation of scleroglucans using Sclerotium rolfsii.
U.S. Pat. No. 5,010,186 and U.S. Pat. No. 4,873,323 disclose polysaccharide biopolymers having improved filterability that are prepared by acidifying an aqueous polysaccharide composition with nitric acid to a pH value from about 2 to 0.1 and treating said acidified composition at a temperature from about 50° C. to 100° C. for about 5 to 60 minutes.
U.S. Pat. No. 4,667,026 describes an aqueous solution of polysaccharide biopolymers that are heat treated for more than 5 minutes at a pH value ranging from 3.5 to 6.2 to improve the filterability thereof.
It is essential that an aqueous solution comprising at least one beta-glucan that is used for polymer flooding does not comprise any gel particles or other small particles at all. Even a small number of particles having dimensions in the micron range blocks the fine pores in the mineral oil-containing formation and thus at least complicates or even stops the mineral oil production.
Consequently, it is therefore also important that aqueous solutions containing at least one beta-glucan are substantially free of cells and cell fragments because these cells and/or cell fragments otherwise block the mineral oil formation which complicates the extraction of the mineral oil or even makes it impossible. The so-called Filtration Ratio (FR value) can be used for characterizing the quality of aqueous solutions comprising at least one beta-glucan.
In principle the removal of cell fragments, gel particles and other small particles could be improved by using filter membranes with a small pore size. With decreasing pore size, however, the filter membranes increasingly also retain the beta-glucans, in particular the fractions of the beta-glucans that have very high molecular weights. The retention of beta-glucans with a very high molecular weight leads to a loss of beta-glucans and makes the overall process for the production of beta-glucans less economical.
In addition, an aqueous solution of beta-glucans is susceptible to bacterial invasion. As the beta-glucan acts as a nutrient for the bacteria, the beta-glucans are degraded. Upon degradation of the beta-glucans undesirable products from the metabolism of the bacteria are excreted into the aqueous solution such as for example hydrogen sulfide. Due to the formation of unwanted products the aqueous solution containing at least one beta-glucan cannot be used for oil field applications. Overall, even though processes for producing branched homopolysaccharides form -1,3-linked glucose units by fermentation of fungal strains are known from the prior art, there is a problem with the production in that the fungi have a tendency towards pellet formation during growth. If the size of the pellets exceeds a diameter of about 0.3 cm, the fungi in the core of the pellets can no longer be supplied adequately with oxygen. The phenomenon of oxygen limitation arises. This condition then leads to cell death of the fungi and to the formation of undesired byproducts such as ethanol, meaning that the space-time yield during the production of the homopolysaccharides is drastically reduced.
The methods specified in the prior art for preventing the pellet formation of fungi are not adequately reproducible and can therefore not be used for processes which are carried out on an industrial scale, carried out in particular in fermentation tanks larger than 5 m3. There was therefore the need to provide further processes for the fermentation of fungal strains which make it possible to produce homopolysaccharides with a high space-time yield (STY).
One object of the present invention is thus to provide a process for the fermentation of fungal strains which secrete glucans with a β-1,3-glycosidically linked main chain and side groups β-1,6-glycosidically bonded thereto, which makes it possible to produce glucans with a β-1,3-glycosidically linked main chain and side groups β-1,6-glycosidically bonded thereto with a high space-time yield.
The present invention provides a process for the fermentation of fungal strains which secrete glucans with a β-1,3-glycosidically linked main chain and side groups β-1,6-glycosidically bonded thereto according to the subject matter of the independent claim(s). Further embodiments are incorporated in the dependent claims.
According to an exemplary embodiment there is provided a process for the fermentation of fungal strains which secrete glucans with a β-1,3-glycosidically linked main chain and side groups β-1,6-glycosidically bonded thereto, in a cascade of tanks comprising at least a first tank with a first volume and a second tank with a second volume, comprising at least the steps
a) fermentation of the fungal strains in a first aqueous medium in the first tank and the volume of the first aqueous medium, resulting in a first mixture,
b) transfer of the first mixture to the second tank, and
c) fermentation of the fungal strains in the first mixture in a second aqueous medium in the second tank and the volume of the second aqueous medium, resulting in a second mixture, where the proportion of the volume of the first mixture to the volume of the second tank is in the range between ≧0.1% to ≦50% and where the first mixture in step b) is passed through at least one shear mixer or high-shear mixer, the shear mixer or high-shear mixer has a shearing geometry, such that the entire first mixture entirely passes through the shearing geometry of the at least one shear mixer or high-shear mixer.
Thus, it is possible to treat the first mixture by a high shear mixing process in order to keep a size of fungal agglomerates small before a second fermentation step starts. This will significantly increase the efficiency of the fermentation process. The agglomerates grow in size during fermentation. Larger agglomerates have a lower relative fermentation rate, so that it may be desirable keeping the size of the agglomerates lower. By treating the agglomerates with a shearing procedure the size decreases, so that the later fermentation process is more efficient.
As the process for fermentation of glucans with a fermentation step in a first tank and a further fermentation in a second tank includes a transfer from the first tank to the second tank includes a high shear mixing, such that the entire mixture passes through the shearing geometry of the high shear mixer, the shearing result becomes more homogenous than a shearing in a batch process, where the high shear mixer is arranged within the tank. In the latter case some parts of the mixture regularly pass the high shear mixer twice or more, while other parts do not pass the high shear mixer at all. Consequently without a high shear mixing during which the entire mixture passes through the shearing geometry of the high shear mixer a broad spectrum of particle size occurs including very small (twice or more passed) and very large (not passed) particles. The invention however, provides a procedure in which a high shear mixer in a flow process urges the entire mixture to pass the mixer, so that the spectrum of particle size is much smaller, as each part of the mixture passes the high shear mixer geometry. A small spectrum results in a more equalized subsequent fermentation, so that the in line high shear mixer of the invention results in a better, faster and more reliable fermentation result. Therefore, batch mixers, like e.g. an Ultraturax machine for a shearing process, do not result in comparable fermentation results.
Glucans are a class of homopolysaccharides whose monomer building block is exclusively glucose. The glucose molecule can be α-glycosidically or β-glycosidically linked, branched to varying degrees or be linear. Preference is given to glucans selected from the group consisting of cellulose, amylose, dextran, glycogen, lichenin, laminarin from algae, pachyman from tree fungi and yeast glucans with β-1,3 bonding; nigeran, a mycodextran isolated from fungi (α-1,3-glucan, α-1,4-glucan), curdlan (β-1,3-D-glucan), pullulan (α-1,4-bonded and α-1,6-bonded D-glucan) and schizophyllan (β-1,3 main chain, β-1,6 side chain) and pustulan (β-1,6-glucan).
The glucan preferably comprises a main chain composed of β-1,3-glycosidically linked glucose units and side groups composed of glucose units and β-1,6-glycosidically bonded to the main chain. The side groups preferably consist of a single β-1,6-glycosidically bonded glucose unit, with, statistically, each third unit of the main chain being β-1,6-glycosidically bonded with a further glucose unit. Depending on the source and method of isolation, beta-glucans have various degrees of branching and linkages in the side chains.
Generally, in context with the presently claimed invention, the beta-glucan as described herein may be any beta-glucan such as beta-1,4-glucans, beta-1,3-glucans, beta-1,6-glucans and beta-1,3(1,6)-glucans. In one embodiment, the beta-glucan is a polymer consisting of a linear main chain of beta-D-(1-3)-glucopyranosyl units having a single beta-D-glucopyranosyl unit (1-6) linked to a beta-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3. In context with the presently claimed invention, the term “average branching degree about 0.3” means that in average about 3 of 10 beta-D-(1-3)-glucopyranosyl units are (1-6) linked to a single beta-D-glucopyranosyl unit. In this context, the term “about” means that the average branching degree may be within the range from 0.25 to 0.35, preferably from 0.25 to 0.33, more preferably from 0.27 to 0.33, most preferably from 0.3 to 0.33. It may also be 0.3 or 0.33. The average branching degree of a beta-glucan can be determined by methods known in the art, e.g., by periodic oxidation analysis, methylated sugar analysis and NMR (Brigand, Industrial Gums, Academic Press, New York/USA (1993), 461-472).
In the context of the presently claimed invention, the at least one beta-glucan to be produced as described herein is preferably selected from the group consisting of schizophyllan and scleroglucan, particularly preferably the at least one beta-glucan is schizophyllan.
Schizophyllan and scleroglucan can both be referred to as beta-1,3-glucans. Schizophyllan and scleroglucan have an average branching degree between 0.25 and 0.33 (Novak, loc cit, Survase, loc at); for example, scleroglucan and schizophyllan have an average branching degree of 0.3 to 0.33. The polysaccharide chains usually form a three-dimensional structure of triple helices; polymer chains consist of glucose units whose hydroxy groups in 1- and 3-position are beta-linked to form the polymer main chain, and wherein each third glucose unit contains in position 6 a further glucose moiety linked by its hydroxyl function in position 1 (beta-1,3-bonded glucopyranose as the main chain and beta-1,6-bonded glucopyranose as side chains) and has the structural formula, where n is a number in the range from 7000 to 35 000:
wherein n is a number which provides the beta-1,3-glucan component with a weight average molecular weight (Mw) of 5·105 g/mol to 25·106 g/mol, which is determined by GPC (Gel-Permeation-Chromatography).
Fungal strains that secrete such glucans are known to the person skilled in the art. The fungal strains are preferably selected from the group consisting of Schizophyllum commune, Sclerotium rolfsii, Sclerotium glucanicum, Monilinia fructigena, Lentinula edodes and Botrytis cinera. Suitable fungal strains are also mentioned, for example, in EP 0 271 907 A2 and EP 0 504 673 A1. The fungal strains used are particularly preferably Schizophyllum commune or Sclerotium rolfsii and very particularly preferably Schizophyllum commune. This fungal strain secretes a glucan in which, on a main chain composed of β-1,3-glycosidically linked glucose units, each third unit—viewed statistically—of the main chain is β-1,6-glycosidically linked with a further glucose unit; i.e. the glucan is preferably the so-called schizophyllan.
Typical schizophyllans have a weight-average molecular weight Mw of about 5·105 g/mol to 25·106 g/mol.
The fungal strains are fermented in a suitable aqueous medium or nutrient medium. In the course of the fermentation, the fungi secrete the aforementioned class of glucans into the aqueous medium.
Processes for the fermentation of the aforementioned fungal strains are known in principle to the person skilled in the art, for example from EP 0 271 907 A2, EP 0 504 673 A1, DE 40 12 238 A1, WO 03/016545 A2, and “Udo Rau, “Biosynthese, Produktion and Eigenschaften von extrazellulären Pilz-Glucanen [Biosynthesis, production and properties of extracellular fungal glucans]”, Postdoctoral thesis, Technical University of Braunschweig, 1997”. These documents also each describe suitable aqueous media or nutrient media.
The fungal strains are preferably cultivated in an aqueous medium at a temperature in the range from 15° C. to 40° C., particularly preferably in the range from 25 to 30° C., preferably with aeration and agitation, for example using a stirrer.
In order to ensure an efficient process for the fermentation of the fungal strains, the fermentation takes place in a cascade of tanks. In this connection, in a preceding tank, an amount of fungal strains and thus also a volume of the aqueous medium is produced that is adequate to bring about as rapid as possible a fermentation in the subsequent tank.
According to an exemplary embodiment the high-shear mixer is a rotor-stator mixer having a rotor and a stator.
Thus, it is possible to provide an efficient shear process, in particular when transferring the mixture from the first tank to the second tank. A rotor-stator mixer has a high through flow capacity and a reliable shearing characteristic. Further a rotor-stator mixer allows an in-line process, which means that the mixture once passes the shearing geometry and then is sufficiently sheared. According to an exemplary embodiment the rotor-stator mixer(s) include types of for example, toothed-rim dispersers, annular-gap mills and colloid mills.
According to an exemplary embodiment the rotor-stator mixer is a toothed-rim dispersing machine.
Thus, it is possible to provide a reliable shearing geometry. If the agglomerates after the shearing process are too small, or are destroyed during shearing, the fermentation process also may be less efficient. A toothed-rim dispersing machine allows a sufficient shearing without too much destroying the agglomerates.
According to an exemplary embodiment rotor-stator mixers are used which have means for generating cavitation forces. Means of this type may be elevations on the rotor and/or stator side which protrude into the mixing chamber and have at least one face where the normal has a tangential portion, such as, for example, pins, teeth or blades or coaxial rings having radial disposed slits.
According to an exemplary embodiment the rotor-stator mixer has on the rotor side at least one rotational symmetrically disposed toothed rim and/or at least one rotational symmetrically disposed ring having radial slits (space widths). Apparatuses of this type are also referred to as toothed-rim dispersers or toothed-rim dispersing machines. In particular, the rotor-stator mixer has, on both the rotor side and the stator side, at least one rotational symmetrically disposed toothed rim and/or ring with radial slits (space widths), where the toothed rims/rings disposed on the rotor and stator side are arranged coaxially and mutually intermesh to form an annular gap.
According to an exemplary embodiment the rotor-stator mixer has a construction which corresponds to a stand with annular ridges with slits cut therein and a rotor with annular ridges with slits cut therein which are arranged concentrically and which are arranged at a distance such that they intermesh with one another. With this rotor-stator mixer the aqueous media or mixtures are fed into the middle section between the stand/stator and the rotor, while the rotor is left to rotate, so that it presses the aqueous media or mixture through the slit and the gap middle the medium section in the direction of the perimeter.
According to an exemplary embodiment at least one of the rotor and the stator of the rotor-stator mixer has at least two concentric toothed-rims and the other of the rotor and the stator has at least one toothed rim, wherein the at least one toothed-rim of the other of the rotor and the stator concentrically interleaves with the at least two concentric toothed-rims, wherein the first aqueous medium passes through the interleaved toothed-rims.
Thus, it is possible to have a defined flow path through the high shear mixer. The shearing geometry may have a geometry, which allows a well defined shearing process, the result of which are agglomerates having a suitable size distribution.
According to an exemplary embodiment the at least two concentric toothed-rims of one of the rotor and the stator and the at least one toothed rim of the other of the rotor and the stator have an equidistant tooth geometry, wherein the distance between adjacent teeth of the respective outer toothed-rim is larger than the distance between adjacent teeth of the respective inner toothed-rim, wherein the first aqueous medium passes through the interleaved toothed-rims in a direction of ascending teeth distance.
Thus, it is possible to provide a quasi stepped shearing process within the rotor-stator mixer. As all toothed rims of the rotor rotate with the same number of revolutions per minute, the track speed of the radial outer rims is higher than the track speed of the radial inner rims. When providing a larger tooth distance at radial outer rims, the shearing effect may be adapted and a destruction of the agglomerates in particular at the outer rims can be avoided. Further, a clogging effect in the flow path of the high shear mixer can be avoided.
According to an exemplary embodiment the first mixture passes through a gap in radial direction, which gap in a radial direction is formed by the concentrically interleaving at least two concentric toothed-rims of one of the rotor and the stator and the at least one toothed-rim of the other of the rotor and the stator, wherein the gap between an outer diameter of a toothed rim and an inner diameter of a radial outwardly adjacent toothed-rim has a width between 0.2 mm and 2.0 mm, preferably 0.4 mm and 1.2 mm, more preferably between 0.8 mm and 0.9 mm.
Thus, the size of the agglomerates when leaving the shear mixer may be in a particular size range. This size range may lead to a particularly efficient fermentation process in the subsequent tank behind the high shear mixer.
According to an exemplary embodiment the first mixture dwells for between 0.01 s and 0.004 s, preferably between 0.02 and 0.07 s, more preferably 0.01 s+/−0.005 s while passing the least two concentric toothed-rims of one of the rotor and the stator and the at least one toothed-rim of the other of the rotor and the stator.
Thus, the shearing process can be optimized. The longer the mixture remains in the shear mixer the longer the shearing process takes. On the other hand a quick passing may destroy the agglomerates or may lead to an agglomerate size being too large for an efficient fermentation process.
According to an exemplary embodiment edges of teeth along a flow path through the shearing geometry have rounded edges with a radius of at least 0.2 mm, in particular more than 3 mm.
Thus, the agglomerates do not collide with sharp edges and do not underlay a cutting. The rounded edges allow a shearing of the agglomerates and at the same time preserve the agglomerates from unintended cutting within the high shear mixer. Further, it can be avoided that particular agglomerates or parts thereof or other residuals remain in the shearing geometry.
According to an exemplary embodiment the rotor rotates at a speed relative to the stator between 250 and 7200, preferably between ≧1800 and 6000, more preferably between 4000 and 4500 revolutions per minute.
Thus, the shearing process, in particular in view of the above described geometries and dimensions, is efficient and also the subsequent fermentation process is efficient.
According to an exemplary embodiment the rotor rotates at a peripheral/track speed of the outmost toothed rim of between 2 m/s and 60 m/s, preferably between ≧15 m/s and 50 m/s, more preferably between 35 m/s and 45 m/s.
Thus, the maximum force may be kept in a particular range within the high shear mixer. In particular the agglomerates do not suffer from a too high tension treatment possibly leading to a destruction of the agglomerates.
According to an exemplary embodiment the proportion of the volume of the first mixture to the volume of the second tank is in the range between ≧1% to ≦20%.
Thus, the fermentation process can be made more efficient. According to an exemplary embodiment the proportion of the volume of the first mixture to the volume of the second tank is in the range between ≧2.5% to ≦15%.
According to an exemplary embodiment the at least one beta-glucan is selected from the group consisting of Schizophylian and Sclerogluran, wherein the Schizophyllan or Scleroglucan are obtained by fermentation of fungal strains. According to an exemplary embodiment the fungal strains are Schizophyllum commune or Sclerotium rolfsii.
According to an exemplary embodiment the process for the fermentation of fungal strains is carried out in a tank cascade which further comprises a third tank with a third volume, and the process further comprises at least the steps of
d) transferring the second mixture to the third tank, and
e) fermenting the fungal strains in the second mixture in a third aqueous medium in the third tank, wherein the proportion of the second mixture to the volume of the third tank is in the range between ≧0.1% to ≦50%.
Thus a further cascade step can be provided. The entire fermentation process can be improved. Three fermentation steps with two interleaved shearing processes allow a more controlled process for fermentation of fungal stains. The entire process can be sped up and the efficiency can be increased.
According to an exemplary embodiment the second mixture in step d) is passed through at least one high-shear mixer, the high-shear mixer has a shearing geometry, such that the entire second mixture entirely passes through the shearing geometry of the at least one high-shear mixer.
According to an exemplary embodiment the proportion of the second mixture to the volume of the third tank is in the range between ≧1% to ≦20%, in particular in the range between ≧2.5% to ≦15%.
The high-shear mixer is preferably a high-shear mixer selected from the group consisting of rotor-stator mixers and high-pressure homogenizers, as described above.
According to an exemplary embodiment, there is provided a process for the fermentation of fungal strains which secrete glucans with a β-1,3-glycosidically linked main chain and side groups β-1,6-glycosidically linked thereto in a cascade of tanks comprising at least a first tank with a first volume, a second tank with a second volume, a third tank with a third volume and a fourth tank with a fourth volume, comprising at least the steps
a) fermentating the fungal strains in a first aqueous medium in the first tank and the volume of the first aqueous medium resulting in a first mixture,
b) transferring the first mixture to the second tank,
c) fermenting the fungal strains in the first mixture in a second aqueous medium in the second tank and the volume of the second aqueous medium resulting in a second mixture,
d) transferring the second mixture to the third tank,
e) fermenting the fungal strains in the second mixture in a third aqueous medium in the third tank and the volume of the third medium resulting in a third mixture,
f) transferring the third mixture to the fourth tank, and
g) fermenting the fungal strains in the third mixture in a fourth aqueous medium in the fourth tank,
where the proportion of the volume of the first mixture to the volume of the second tank is in the range between ≧0.1% to ≦50%, the proportion of the volume of the second mixture to the volume of the third tank is in the range between ≧0.1% to ≦50%, and the proportion of the volume of the third mixture to the volume of the fourth tank is in the range between ≧0.1% to ≦50%, wherein the first mixture in step b) is passed through at least one high-shear mixer and/or the second mixture in step d) is passed through at least one high-shear mixer and/or the third mixture in step f) is passed through at least one high-shear mixer, wherein at least one of the high shear mixers has a shearing geometry, such that the entire respective mixture entirely passes through the shearing geometry of the at least one high-shear mixer.
According to an exemplary embodiment the proportion of the volume of the first aqueous medium to the volume of the second tank is in the range between ≧1% to ≦20%, particularly in the range between ≧2.5% to ≦15%.
According to an exemplary embodiment the proportion of the volume of the second aqueous medium to the volume of the third tank is in the range between ≧1% to ≦20%, particularly in the range between ≧2.5% to ≦15%.
According to an exemplary embodiment the proportion of the volume of the third aqueous medium to the volume of the fourth tank is in the range between ≧1% to ≦20%, particularly in the range between ≧2.5% to ≦15%.
According to an exemplary embodiment the fermentation is performed such that the concentration of the glucans to be prepared in the aqueous medium at the end of the fermentation process in the last tank of the reactor cascade is at least 3 g/l. The upper limit is not limited in principle. It results from what viscosity can still be handled in the tank used.
According to an exemplary embodiment the high-shear mixers used in the transferring step have an identical or different design in the individual steps. According to an exemplary embodiment the rotor-stator mixers used in the transferring step in the individual steps have an identical or different design.
Rotor-stator mixers may also comprise in principle all dynamic mixer types in which a high-speed rotor, which may be a rotational symmetrical, cooperates with a stator to form one or more processing regions which are essentially in the shape of an annular gap. In these processing regions, the mixing material is subjected to severe shear stresses, while the high level of turbulence which often prevails within the annular gaps likewise promotes the mixing operation.
In a further embodiment, the high-shear mixer is a high-pressure homogenizer. In such a mixer, the aqueous medium is forced through a small opening under high pressure.
Preferably, the high pressure is in the range from 100 bar to 2000 bar, particularly preferably in the range from 200 bar to 1000 bar.
Preferably, the small opening has a diameter in the range from 0.5 to 2.5 cm, particularly preferably in the range from 0.8 to 2.0 cm.
In a further embodiment, the fermentation broth comprising at least one beta-glucan and biomass (fungal cells with or without cell constituents) at the end of the fermentation process is filtered.
Preferably, the content of the fermentation tank after the fermentation is filtered with the use of asymmetrical filter membranes or symmetrical filter membranes.
Alternatively, the fermentation broth is removed continuously or from time to time from the plant via a side stream and an aqueous solution comprising at least one beta-glucan is separated off therefrom by crossflow microfiltration. The remaining aqueous fermentation broth in which the biomass has a higher concentration than beforehand can be at least partly recycled to the fermentation container.
The crossflow microfiltration process is known in principle to the person skilled in the art and is described, for example, in “Melin, Rautenbach, Membranverfahren Springer-Verlag 3rd edition 2007, page 309 to page 366”. Here, “microfiltration” is understood by the person skilled in the art as meaning the removal of particles having a size of from about 0.1 μm to about 10 μm.
In the crossflow filtration, a stream of the liquid to be filtered is applied, for example, by a suitable circulation pump, parallel to the surface of the membrane used as filtration material. A liquid stream therefore continuously flows over the filter membrane, and the formation of deposits on the membrane surface is prevented or at least reduced thereby. In principle, all types of pump are suitable as the pump. Owing to the high viscosity of the medium to be transported, however, in particular positive displacement pumps and very particularly eccentric screw pumps and rotary piston pumps have proven useful.
Preferably, asymmetrical filter membranes or symmetrical tubular membranes are used for the crossflow microfiltration. Asymmetrical filter membranes consist of at least two different layers having different pore size, i.e. of at least one support layer and one separating layer. The support layer is comparatively thick and has comparatively large pores. It imparts the mechanical strength to the filter membrane. At least one separating layer having finer pores than the pores of the support layer is applied to the support layer. For example, mercury porosimetry can be used in a manner known in principle for measuring the pore sizes. Optionally, one or more intermediate layers may also be arranged between the separating layer and the support layer.
The asymmetrical membranes may be, for example, metallic membranes or ceramic membranes. The asymmetrical membranes used are preferably asymmetrical ceramic membranes. Details of asymmetrical ceramic membranes are described, for example, in “Melin, Rautenbach, Membranverfahren, Springer-Verlag, 3rd edition 2007, page 51 to page 52”.
Symmetrical tubular membranes are tubular membranes which have a pore distribution which is essentially constant over the entire cross section of the membrane wall. Symmetrical tubular membranes are known to those skilled in the art and are described, inter alia, in “Melin, Rautenbach, Membranverfahren, Springer-Verlag 3rd edition, 2007 page 20”.
The good quality of the aqueous solution comprising at least one beta-glucan may be evident from the good filtration properties, which are expressed by the low filtration ratio (FR value). In a preferred embodiment, the FR value of the product may be preferably in the range of ≧1.0 to ≦1.8, more preferably in the range of ≧1.0 to ≦1.5, even more preferably in the range of ≧1.0 to ≦1.3.
In another preferred embodiment, the yield of at least one beta-glucan after filtration, i.e. the amount of at least one beta-glucan which can be recovered from the fermentation broth, based on the amount of at least one beta-glucan present in the fermentation broth prior to filtration, is preferably in the range from ≧25% to ≦97%, more preferably in the range from ≦30% to ≧95% and most preferably in the range from ≦50% to ≦93%.
The aqueous solution containing at least one beta-glucan may be further worked up and concentrated in order to obtain the at least one beta-glucan in highly concentrated form. In one embodiment, the aqueous solution comprising at least one beta-glucan can be brought into contact with at least one precipitating agent to obtain at least one precipitated beta-glucan in a solvent mixture comprising water and the at least one precipitating agent. Preferably, the at least one precipitating agent is selected from the group consisting of low boiling liquids, high boiling liquids and mixtures thereof. Examples of low boiling liquids are formates like methyl formate, acyclic ethers like dimethoxymethane, cyclic ethers like tetrahydrofuran, 2-methyl-1,2-dioxalane, carboxylic acid esters like acetic acid ethyl ester, alcohols like methanol, ethanol, isopropanol or propanol, ketones like acetone or methylethylketone, or mixtures of at least two of them. Examples of high boiling liquids are polyethylene glycols having molecular weights preferably in the range of 10 to 200 kD, more preferably in the range of 15 to 120 kD, polypropylene glycols having molecular weights in the range of 5 to 100 kD, more preferably 10 to 30 kD, or mixtures of at least two of them. The at least one precipitating agent is generally added to the aqueous solution comprising at least one beta-glucan, so that the volume ratio of the precipating agent to the aqueous solution is in the range of preferably 0.1:1 to 20:1, more preferably 0.2:1 to 2:1, most preferably 0.2:1 to 1.5:1, in each case based on the total mixture that is obtained.
The at least one precipitated beta-glucan can be separated from the solvent mixture comprising water and the at least one precipitating agent to obtain a precipitated beta-glucan in highly concentrated form. The separation can in general be conducted by any methods known to the skilled artisan, for example, inter alia, centrifugation, sedimentation, flotation and filtration.
The beta-glucan such as schizophyllan which is obtained according to the inventively claimed method may be further modified after filtration and optionally concentration. The beta-glucan such as schizophyllan may be converted by oxidation, enzyme conversion, acid hydrolysis, heat and/or acid dextrinization or shear. The beta-glucan such as schizophyllan can also be chemically, enzymatically or physically modified. Suitable chemical derivatives of schizophyllan include esters, such as the acetate and half esters, such as the succinate, octenyl succinate and tetradecenyl succinate, phosphate derivatives, ethers such as hydroxyalkyl ethers and cationic ethers, or any other derivatives or combinations thereof. Modification may also be chemical crosslinking. Crosslinking agents that are suitable for use herein include phosphorus oxychloride, epichlorohydrin, sodium trimetaphosphate and adipic acid/acetic acid mixed anhydrides.
It should be noted that the above features may also be combined. The combination of the above features may also lead to synergetic effects, even if not explicitly described in detail.
These and other aspects of the present invention will become apparent from and elucidated with reference to the embodiments described hereinafter.
Exemplary embodiments of the present invention will be described in the following with reference to the following drawings.
It should be noted, although not explicitly described, that also a fermentation process can be provided having more than four steps as described above with respect to
Further, it should be noted that in all three embodiments as described above
Further, it should be noted that for the embodiments described with respect to
Additionally, the proportion of the third mixture S43 to the volume of the fourth tank VK44 in the embodiment described with respect to
The Schizophyllum commune strain used is laid open in EP 0 504 673.
Suitable nutrient media for the precultures and main cultures and cultivation conditions can be found for example in the patent EP 504 6073, EP 0 271 907 and “Process and molecular data of branched 1,3-β-D-glucans in comparison with Xanthan, U. Rau, R.-J. Müller, K. Cordes, J. Klein, Bioprocess Engineering, 1990, Volume 5, Issue 2, pp 89-93” and “Udo Rau, “Biosynthese, Produktion und Eigenschaften von extrazellularen Pilz-Glucanen [Biosynthesis, production and properties of extracellular fungal glucans]”, Postdoctoral thesis, Technical University of Braunschweig, 1997″.
Nutrient medium used: 30 g/l glucose, 3 g/l yeast extract, 1 g/l KH2PO4, 0.5 MgSO4*7 H2O
Strain maintenance and cultivation of the biomass are described for example in “Oxygen controlled batch cultivations of Schizophyllum commune for enhanced production of branched β-1,3-glucans, U. Rau, C. Brandt Bioprocess Engineering September 1994, Volume 11, Issue 4, pp 161-165”. The ratio of the volumes upon transfer was about 5%.
All of the tanks of the preculture were operated at a constant speed and gassing rate so that the pO2 was always above 60%. The duration of the precultures was chosen such that the glucose did not drop below 5 g/l.
The main culture was carried out according to the process described in the literature under oxygen-limiting conditions. The procedure for the main culture is described for example in “Oxygen controlled batch cultivations of Schizophyllum commune for enhanced production of branched β-1,3-glucans, U. Rau, C. Brandt Bioprocess Engineering September 1994, Volume 11, Issue 4, pp 161-165”, “Udo Rau, “Biosynthese, Produktion und Eigenschaften von extrazellulären Pilz-Glucanen [Biosynthesis, production and properties of extracellular fungal glucans]”, Postdoctoral thesis, Technical University of Braunschweig, 1997” and “Process and molecular data of branched 1,3-β-D-glucans in comparison with Xanthan, U. Rau, R.-J. Müller, K. Cordes, J. Klein, Bioprocess Engineering, 1990, Volume 5, Issue 2, pp 89-93”,
3. Transfer of the Preculture to the Main Culture with Rotor-Stator Mixer
The increase in volumetric productivity in the main culture through the use of a toothed-wheel pump in the bypass, as described in DE 4012238 A1, could not be recreated. The opposite effect was observed in experiments that the recirculation via a bypass, as described in DE 4012238 A1, significantly reduces the volumetric productivity in the main culture.
Surprisingly, it was found that using a continuously operated rotor-stator mixer when transferring the preculture to the main culture leads to a significant increase in the STY. In this example, a rotor-stator mixer from Cavitron was used, bench instrument CD 1000 equipped with a chamber system, operated at 5-20 l/min, peripheral speed: 3-50 m/s.
The rotor-stator mixer was incorporated into the pipeline of the last tank of the preculture to the main culture tank in the reactor cascade and steam-sterilized prior to insertion in order to permit aseptic operation.
The space-time yield (STY), also called volumetric productivity, was determined by measuring the glucan concentration in a sample taken after a runtime of 72 h using a method described in the literature. The measured concentration divided by the runtime until the sample was taken (72 h) gives the space-time yield. For the purposes of simplification, relative STY are shown. The STY which were achieved without using a high-shear mixer were set as 100%.
Principle of Measurement:
In the determination of the filtration ratio (FR value), the amount of filtrate which runs through a defined filter is determined as a function of time. The FR value is determined according to the following formula (I)
FR=(t190g−t170g)/(t70g−t50g) (I),
where the variables and the equation have the following meaning:
t190g=time in which 190 g of filtrate are obtained,
t170g time in which 170 g of filtrate are obtained,
t70g=time in which 70 g of filtrate are obtained,
t50g=time in which 50 g of filtrate are obtained.
Thus, in each case the time span which is required for in each case 20 g of filtrate to flow through is determined, i.e. at a early time and at a late time in the filtration process, and the quotient is calculated from the two time spans. The larger the FR value, the more greatly is the filtration velocity slowed down with increasing duration of the filtration process. This indicates increasing blockage of the filter, for example by gels or particles.
The FR value is determined by the following method:
a) Sartorius pressure filtration apparatus 16249; filter diameter 47 mm; with 200 ml digestion cylinder (Øi=41 mm)
b) Isopore membrane 1.2 μm; Ø 47 mm; No. RTTP04700 available from Merck Millipore
First, 50 g of a mixture of the glucan solution obtained from the experiments and water is prepared, i.e. in a ratio such that the concentration of the glucan is 1.75 g/l. The mixture is stirred for 10 min and checked visually for homogeneity. If the mixture is still inhomogeneous, further stirring is effected until the mixture is homogeneous. The mixture is then made up to a total amount of 250 g with 200 g of ultrapure water. Thereafter, stirring is effected for at least 1 h for homogenization, after which the pH is adjusted to 6.0 with 0.1 M NaOH and stirring is then effected again for 15 min. The pH of 6.0 is checked again. The final concentration of the glucan in the mixture is 0.35 g/l.
The filtration test is effected at room temperature (T=25° C.) at a pressure of 1.0 bar (compressed air or N2).
By means of the balance, the amount of filtrate is determined as a function of time. The mass indicated in each case can be read visually but of course also automatically and evaluated.
| Number | Date | Country | Kind |
|---|---|---|---|
| 14197750.4 | Dec 2014 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/079004 | 12/8/2015 | WO | 00 |
