CA1329159C - High molecular weight homopolysaccharides, their extracellular preparation and their use, and corresponding fungal strains - Google Patents
High molecular weight homopolysaccharides, their extracellular preparation and their use, and corresponding fungal strainsInfo
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- CA1329159C CA1329159C CA000554284A CA554284A CA1329159C CA 1329159 C CA1329159 C CA 1329159C CA 000554284 A CA000554284 A CA 000554284A CA 554284 A CA554284 A CA 554284A CA 1329159 C CA1329159 C CA 1329159C
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
Abstract of the Disclosure: Nonionic biopolymers in the form of homopolysaccharides having a high specific viscosity are prepared by an extracellular process in which microorganisms in the form of certain fungal strains are cultivated in a nutrient medium with aeration and agi-tation at about 15-40°C, if necessary the culture suspen-sions are then heated, the culture solution is then separated from the cell mass, and the resulting homopoly-saccharide is isolated therefrom with virtually only glucose as a basic monomer component.
Description
~ - 1 ; 1 3~9 1 59 The present invention relates to high molecular weight homopolysaccharides, their extra-cellular preparation and their use, and corresponding fungal strains.
, Water-soluble biopolymers are used in indus-try for various applications. U.S. Patent 3,301,848 issued on January 31, 1967 to the Allsbury Company describes water-soluble, nonionic polysaccharides of defined structure ~ as glucans. These are produced microbially as metabolites J''~: by growing the microorganism sclerotium glucanicum n.sp., NRRL 3006. However, they have a relatively low molecular :, .
weight. The same applies to the polysaccharides described in British Patent 2,178,~37 published on February 11, 1987 in the name of Sociéte Nationale ELF Aquitaine.
; W.L. Griffith and ~.L. Compere, in Dev. Ind.
Microbiol. 19 (1977), 609-617, describe the microbial production of highly viscous glucans by cultivation of the micro-organism Sclerotium rolfsii, ATCC 15206, in a nutrient medium containing inorganic salts, glucose , and a small amount of yeast extract, with a growth period `~ 20 of 2.5 days. The glucans produced exhibit a decrease in solubility in water at pH 1.5-~.
The present invention relates to a process for ~ the extracellular preparation of homopolysaccharides (PS) 'l having a high specific viscosity, the fungal strains which produce these PS, the PS themselves and their use.
It is an object of the present invention to prepare nonionic homopolysaccharides having a hlgh specific viscosity and high heat stability, salt stabill;ty and long-term stability. Stability means that the molecular `n 30 weight and hence the properties do not change significantly under the (possibly simultaneous) influence of elevated temperatures, salts and atmospheric oxygen.
More particularly, the invention relates to the fungal strains DSM 3887, DSM 3888, DSM 3890, DSM 3891 ..
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` and DSM 3892, which are deposited at the Deutsche Sammlung !~ ' fur Mikroorganismen IDSM), Gottingen.
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' ' -- 132915q - 2 - O.Z. 0960/32003 We have found that this object is achieved if microorgan;sms in the form of the fungal strains stated in claims 1 and 2 are cultivated in a nutrient medium with aeration and agitation at from about 15 to 40C and are then heated for from 2 to 30, preferably from S to 15, minutes at from 70 to 90C, preferably from 75 to 85C, if the fungal strains have matured, the culture solution is i then seParated from the cell mass, and the resulting -~ homopolysaccharide is isolated therefrom with a purity o~
-~ 10 greater than 99~ from 0.03 to 0.1~ by weight of residual - protein and with virtually only glucose as a basic monomer component.
rhe conventional nitrogen and hydrogen carbon source ~hich can be assimiLated by the fungal strains, and the ` 15 inorganic ions required for growth, are made available to the fungal strains as a nutrient medium, advantageously in a concentration such that, when the cultivation is terminated, the residual concentration of the carbon source is of the order of 0.1X by weight, based on the total solution. The nonionic PS produced have a viscosity of from 50 to 190 mPa.s at a shear rate D of 0.3 s 1 at 40C, determined with 0.3 9 of PS dissolved in 1,000 ml of highly saline water (containing up to 1ûO g/l of alkali metal ions and up to 30 i~` g/l of alkaline earth metal ions). This viscosity remains virtually constant for 1 year or longer at 60C in the air.
In the absence of oxygen, this even applies at temperatures up to 90C. The viscosity of the nonionic PS produced i according to the invention is influenced only to a small ~-~ extent at a concentration of from 0.1 to 1 g/l at about 20-60C and in the shear range D of about 3 0 s 1 or higher, so that it may be regarded as constant for practic!al purposes. Even after a solution containing 0.3 g/l of the PS obtainabLe according to the invention has been heated for one hour in an autoclave at neutraL pH and at 120C
under 1 bar g3ge pressure, viscosity re~ains virtually unaffected in the shear range D from 0.1 to 300 s 1 after cooling to 20C and supple~enting the evaporated ~ater. The ~' .
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^` 132915~
_ 3 - o.Z. 0960/02003 PS consist of a linear chain of 1,3-bonded ~-D-glucopyranose un;ts subst;tuted to different degrees by 1,6-bonded ~-D-glucopyranosyl groups.
~or use, the fungal strains are advantageously immobilized in a conventional manner on a substrate, pre-~ ferably a polyurethane foa~, and the formation of the PS
is carried out semicontinuously or completely continuously using this immobilized material.
The PS obtainable according to the invention are very useful as viscosity-increasing agents in polymer and ; surfactant flooding of oil deposits, as dispersants or emulsifiers (alone or in combinat;on with anionic and/or nonionic surfactants), as thickeners possessing structural viscosity, as water retention agents and for improvin~ the adhesion of substances to solid surfaces. ~hen employed as thickeners, they are intended in particular for use in drilling muds, for secondary and tertiary flooding of oil deposits and in general for reducing frictional resistance in flo~ing, in particular turbulent, aqueous liquids in order to reduce the pressure loss. Examples of suitable applications as dispersants are in agricultural chemicals, such as fungicides and pesticides, and in the food and animal feed sectors.
The fil~ration properties of the PS produced ac-cording into the invention, as an e~pression of the inject-ability into porous med;a, such as oil-bearing rock, are outstanding. For a sa~ple of 0.3 9 of PS per l of highly sal;ne ~a~er containing up to 100 g/l of alkali metal ions and up to 30 9/~ ~f alkaline e~rth me~al ions and pressure filtration under one bar gage pressure over a 3 ~ me~brane `; filter of 19 cm2, using 200 ml of solu~ion, the relevant properties are from 0~5 to 3.0 ~in in the case of shearing for 5 sec and from 078 to 10 min ~ithout shearing; the cor-responding propert;es for filtration over a 1.2 ~ me~brane filter are from 1.6 ~o 18 min in ~he case of shearing for 5 sec and from 4.0 to 14 min without shearing.
The sus~ension Gulture is grr~n for about iû-1aû, 1 32q 1 5~
- 4-- O.Z. 0960/02003 preferably 50-80, hours.
rhe noveL process gives the PS directly in very pure form. The resiclual protein content is from about 0.03 to Q.1X by weight.
For the purposes of the present invention, water-soluble means that not less than 1, preferably not less than 20, 9 of PS are soluble in a liter of water at Z0C.
In spite of their high molecular weight, the novel PS are water-soluble because they are substituted to a high deg-ree by side chains. They differ from the polysaccharides ~?~,~ described in U.S. Patent 3,301,848 in that, inter alia, they have a substantially higher molecular weight. rheir superior properties are mainly due to this.
The preferably used microorganisms in the form of fungal strains are characterized in the Tables below.
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+ ~, ~ + ~ n 3 a: ~ ~S
-- o L: o ~ m a~ m 1' ~ ~ 3 ,~ ~, . E c V~ ~E -- -- n ~ _ C _ =,~,, s ~ o W . i L L
o Cl 0 ~ O -- ~ ~ C -- I~ 11 11 11 11 ~ ~ L + î ~1 ~ +
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~ 1329159 . - 6 - 0. Z. 0960/02003 ..,.' `~", `, oOo` .
~,' ~, ++++I~ +I~++ I+++++~+I
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~ E ~+++I+ +I+++ I+++++I+I+
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.~,-i Cl + + + + + I + + + + + I ~ + + + + + + + I O
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,,'1 ~ +++++~ +I+~+ I+++++++++ ~. '.
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.Q> ~ ~, ~ ' 1 ~ a~ c .,,~ ~ 0 ~ ~ ~0 0 0 .~ C ~ C C u~ U~ a .~ C~ al (11 111 (~ Z U~ ~_> L
~i .~ ~ J O C (~1 f~3 ~ L-- -- -- -- -- -- C a .~ t e ~v ~ = c = ~ C ~ ~ ~ O ~ u ~ ~ ~ ~ ~ .
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; ~ 1 329 1 59 - 7 - o.Z. 0960/02003 Typical viscometric data for characterizing the polysac-charides AW
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~u ~mPas~187 81 188 51 163 144 n 0.1 -0.150.06 O.t2 0.15 0.13 C~] r~l/g] 6200091000 17000 - - 43000 M~ Cg/mol] 22.5x106 9x1o68X106 - _ 6 ~- n = Zero shear viscosity of an aqueous PS soLut;on ontaining 0.3 g/l ' n = Flow behavior index ~ .~., C~] = Intrinsic viscosity. This gives the space require-` 15 ment of a polymer coil in dilute solution (<=0.5 g/l) ; and is directly related to the molecular ~eight via the Mark-Houwink equation n = K.M~a MW = Molecular weight DP = Degree of poly~erization M~/162 16Z= D-glucose - 1 H2O
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The Examples ~hich follow illustrate the invention.
; EXAMPLE 1 Preparation of a ho~opolysaccharide, type AW 106, using the fungal strain DSM 3889O
Two slant agar cultures of the strain DSM 3A89 ~ere suspended in 3 ml of 0.9X strength sodium chloride solution, and this suspension was used to inoculate ~,~ 500 ml of a nutrient solution containing 1.5 9 of NaN03, 30 0.5 9 ot K2HPO4 3 HzO, 0.25 g of MgS04 . 7 H20, O.Z5 g ot KCl, 1.0 9 of yeas~ extract and 5.0 g o~ glu-cose. The shaken suspension culture ~as cultivated at ~I 27C for 60 hours at pH 4.5. The preculture thus pre-;~ pared ~as used to inoculate a 15 l bioreactor equipped with three six-blade disk stirrers and containing 10 l of a culture neuiun having the folLo~in~ cor,position:
, - 8 - O.Z. 0960lOZ003 NaN03 30 9 K2HP04 3 H2013 g MgS04 . 7 H2O 9 KCl - 5.0 9 ZnSO4 7 H200 03 9 Citric acid . H20 7.0 9 Glucose 300.0 9 Cultivation was carried out for 80 hours at 27C
~ith stirring at 500 rp~ and aeration with 1.67 liters of sterile air per liter of nutrient medium per minute and at an initial pH of 3.5. During the cultivation, the pH
decreased to 2.7. After the end of the cultivation, the residual glucose content was less than 0.1% by ~eight.
The culture suspension was heated at 80C for 30 ninutes, diluted ~ith 50 l of deionized water and cen-trifuged at 15,ûO0 9. The polysaccharide A~ 106 was pre-cipitated from the supernatant liquid with ;sopropanol (volume ratio 1:1), filtered off and washed ~ith 70Z
strength aqueous isopropanol solut;on, and the precipitate ~as resolvatized in 1 l of deioni~ed water and freeze-dried. 120 9 ot polysaccharide A~ 106 were obtain@d. The product contained glucose, as a ~onomer building block, and less than 0.05~ by weight o~ protein. 0.3 9 of AW 106 dissolved in 1,000 ml of formation water tcomposition in g/l: CaCl2 42.6 MgCl~ 10.5, NaCl 138.0, Na2S04 1.2 and Na802 . 4 H2O 0.4) had a viscosity of 137.7 mPa.s at a shear rate of 0.3 s 1 and 40C.
Preparativn of a homopolysaccharide, type AW 110~ using the fungal strain DSM 3887.
Four slant agar cultures of the stra;n DSM 3887 were suspended in 3 ml of O.9X strength sodium chloride ~ solut;on, and this suspension ~as used to inoculate ;l 35 1,000 ~l of a nutrient salt solution containing 1.0 9 of KzHP04 . 3 H2O, 1.5 ~ of KCl, 1~5 9 of N~NO3, 0.5 9 o~
MgSO4 . 7 HzO, 0.5 g gf yeast estrsct and 10.0 9 of glucose.
.
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~ 1 329 1 59 - ~ - O.Z~ 0960/02003 The shaken suspension culture was cultivated at 27C for 54 hours at pH 4.5. This inoculation culture was used to inoculate a 70 l bioreactor equipped ~ith an Intensor~
System fr-om Giovanola, Monthey, Switzerland, and con-taining 50 l of a nutrient medium having the follo~ing ` composition:
NaN03 75 9 ~; KCl 75 9 K2HPo~ 3 H2O
10 MgS04 7 H20 25 9 FeSO4 . 7 H2O2.5 9 Xylose 1500.0 9 Cultivation ~as carried out for 96 hours at 27Cith stirring at 1,Z00 rpm and aeration with 0.5 liter of sterile air per liter of nutri~nt medium per ~inute, at an initial pH of 4.5. After the end of the cultivation, the pH had decreased to 3.0 and the xylose was virtually -~ compLetely consu0ed.
~; The culture medium was heated at 85C, and the 2Q cell mass ~as separated off at this temperature by means of pressure filtration. The clear, highly viscous fil-~ trate was precipitated with iscpropanol and freeze-dried, ;1 as described in Exa~ple 1. 435 9 of the homopolysaccharide type AW 110 ~ere obtained. The product contained exclu-sively glucose as the basic monomer component and had a residual protein content of less than 0.03~ by weight.
-~ Elemental analysis gave the following composition:
Carbon: 44-5~
Hydrogen: ~.25%
; 30 Nitrogen: 0~005~
0.3 g of A~ 110 in 1,000 ml of for0ation ~ater in oil production, having the composition according to ~ ,:
Example 1, had a viscosity of 81.6 0Pa.s at a shear rate ~` of 0.3 s 1 and 40~C.
Preparation of a ho~opolysaccharide, type A~ 114, using q the fungal strain DSM 3888~
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- 10 - O.Z. 0960/02003 T~o s~ant agar cultures of the strain DSM 3888 were suspended in 3 ml of 9% strength sodium chloride solution~ and this suspension ~as used to ;nocuLate 500 ml o~ a nutrient salt solution containing 3.0 9 of yeast extract, 0.75 9 of (NH4~2HP04, 0.5 9 of K2HP04 . H20, -0.25 9 of MgS04 . 7 H20 and 10 9 of glucose. The shaken suspension culture was cultivated at 27C for 96 hours at pH 5.3. The pellets formed ~ere homogenized using an Ultra-Turra~Q stirrer at 20,000 rpm for 10 seconds, and this homogeni~ed innoculation culture was used to inoculate a 15 l bioreactor equipped ~ith three si~-bLade disk stir-rers and containing 10 l of a nutrient medium having the - following composition:
.~ Technical-grade yeast extract 30.0 9 15 K2HP04 . 3 H20 10.0 9 MgS04 . 7 H20 Xylose Z50.0 9 Cultivat;on was carr;ed out for 76 hours at 27C
~;th stirr;ng at 400 rpm and aeration with 0.1 Liter of ~ 20 sterile a;r per l;ter of nutrient mediu~ per minute, at - an initial pH of 5.3. After the end of the cultivation, the pH had decreased to 4.6 and the residual xylose con-tent was 0.1% by weight.
The react;on mixture was heated to 80C, 10 9 of formaldehyde were added, and the predominant amount of the polysaccharide ~as liberated from the resul~ing pel-~i Lets by shearing with an Ultra-Turrax for 10 seconds at Z0,000 rp~. Thereaf~er, the cell ~ateriaL ~as separated off by pressure f;ltrat;on, and the clear f;ltrate was freed from lo~ molecular we;ght substances by ultra--- f;ltration at an exclusion limit of 100,000 Dalton'. The uLtrafiltraee ~a~ prec;pitated w;th isopropanoL and freeze-dried, as descr;bed in Example 1. 7Z g of a ho~o-polysaccharide A~ 114 were ob~a;ned. The product contained excLusively gLucose as ~h~ basic monomer component and had a res;dual protein content of less than 0.05X by we;ght.
Ele-ent~l analysis gs~e the foLLowing co-position:
132~15q - 11 - O.Z~ 0960/02003 Carbon: 44.42%
~- Hydrogen: 6~19%
Nitrogen: 0.01X
.3 9 of A~ 114 dissolved in 1,000 ml of forma-tion water having the composit;on described ;n Example 1 had a viscosity of 188.7 mPa.s at a shear rate of 0 3 5 1 and 40C.
Preparation of a ho~opolysaccharide, type AW 115~ using the fungal strain DSM 3890.
A slant agar culture o~ the strain DSM 3890 was ; suspended ;n 3 ml of 0.9~ strength sodium chloride solu~
tion, and this suspension ~as used to inoculate 100 ~l of a nutrient salt solution conta;n;ng 0.1 g of yeast e~tract, ~.1 9 of K2HP04, 0.5 9 of MgS04 . 7 H20 and 1.0 9 of glucose. The suspension culture ~as cultivated at 25C for 60 hours at pH 5.7, ~h;le shaking on a rotary shaking ~achine at 110 rpm. This preculture was used to inoculate t~o 2 l conical flasks, each of which contained 500 l of nutrient medium having the following composition:
K2HP04 3 ~2 9 MgS04 7 H20 0.25 9 Yeast extract 0.5 9 Glucose 12.0 9 25 After incubation at Z5C on a rotary shaking machine at 110 rpm and at a pH of 5.7, the spec;fied amount of glucose was consumed after 142 hours and a pH
of 3.5 reached. The two batches were combined and dilu-ted ~ith 5 l of deionized water, and ~he cell mass was separated off by centrifu~ing. The clear supernatant liquid ~as concentrated to a polysaccharide content of 4.3 g/l by ultrafiltration at an exclusion limit of 100,000 Dalton~ the lo~ ~olecular weight components simultaneously being separated off. Ethanol was added to the concentrate (volume ratio 1:1), and the prec;pitated polysaccharide A~ 115 was separated off by centrifuging~
~ashed ~ith ~00 ml of 70~ strength ethanol and free2e-~ ~' 1329159 - 12 - O.z. 0960/OZ003 dried. 5.05 9 of polysaccharide AW 115 were obtained.
This product contained exclusively glucose as the mono-mer building block and 0.11% by weight of protein as an accompany-ing substance, the percentages being based on the ; 5 polysaccharide content.
0.3 9 of AW 115 dissolved in 1,000 ml of for~mation ~ater (for composition, see Example 1) had a viscosity of 51 mPa.s at a shear rate of 0~3 s 1 and 40C.
Preparation of a homopolysaccharide, type AW 116~ using the fungal strain DSM 3891.
`~ Starting from a slant agar culture of the fungal strain DSM 3891, the ho~opolysaccharide AW 116 was pro-duced under the sa~e conditions as described in Example 4. However, 50 9 of maltose/l of nutrient medium ~ere used as the C source. The cultivation was terminated after 120 hours, when a pH of 3.6 had been reached and the res;dual content of maltose had decreased to û.1~ by eight.
The mixture was worked up as described in Exa~-ple 4 to gi~e 4.0 9 of homopolysaccharide containing less than 0.05~ by ~eight of proteinsu 0.3 g of A~ 116 dis-solved in 1,000 ~l of formation water having the co~posi-~` tion described in Example 1 had a viscosity of 163.2 mPa.s 25 at a shear rate of 0.3 s 1 and 40C.
Preparation of a homopolysaccharide, type A~ 117, using the fungal strain DSM 3892.
Two slant agar cultures of the strain DSM 3892 ~. .
were suspended in 3 ml of 0.9% strength sodiu~ chloride solution, and this suspension was used to inoculate 500 ~l of a nutrient salt solution containing 0.5 g of yeast extract, 0O5 g of K2HPO4 . 3 H2O, 0.25 9 of ~gS04 . 7 H2O, 0.25 g of citric acid-H20 and 5 9 of suc-}5 rose. The suspension culture was cultivated a~ 25C for 72 hours at pH 5.6, ~hile shaking on a rotary shaking ~achina ae 100 rp~. This preculture ~as used to .' . ~
t 329 1 59 - 13 - O.Z. 0960/02003 inoculate a 15 l bioreactor which was equipped with a guide tube possessing a propeLler stirrer and contained 10 l of nutrient medium having the following composition:
K2HP04 ~3 H20 10.û 9 S MgS0~ . 7 H20 5.0 9 Yeast extract 5.0 Asparagine 12.0 9 Glucose 33U.0 9 Cultivation was carried out for 48 hours at 25C
; 10 ~ith stirring at 6ûO rpm and aeration w;th 1.0 liter of sterile air per liter of nutrient medium per minute, at an initial pH of 5.6. After the end of the cultivation, the pH had decreased to 3.2 and the residual glucose con-- tent was 0.15% by weight.
8 1 of deionized water ~ere added to the culture medium, the mixture ~as heated to 85C and the cell mass was separated off by means of pressure filtration. The clear cultur~ solution was concentrated to a homopoly-saccharide content of 10 g/l by ultrafiltration at an ex-clusion Limit of 100,000 Dalton, and was free2e-dr;ed.
90 9 of homopolysaccharide A~ 117 were obtained.
After hydrolysis of the production in 6 N H2S04 at 90C under an N2 atmosphere, glucose was detected as the only monomer component by thin layer chromatography and by high pressure liquid chromatography.
Elemental analysis gave the follo~ing composition:
Carbon: 44.32X
Hydrogen: 6.21X
Nitrogen: 0.09~.
This gives a residual protein content of 0.56%.
0.3 9 of A~ 117 dissolved in 1,000 ml of forma-tion ~ater according to ExampLe 1 had a viscosity of 144.5 mPa.s at a shear rate of 0.3 s 1 and 40C.
In order to test the suitability of the PS pro-duced by the process of the invention for tertiary oilproduction, the fungal strains stated ;n cLaim 1 ~ere grown in submerse culture, subjected to pressure ~ 1 32q 1 5q `:
- 14 - O.Z. 0963/Q2003 filtration and substantially freed from inorganic ions, residual glucose and proteins by subsequent ultrafiltra-- tion at an exclusion limit of 100,0ûO Dalton. Formalde-- hyde was-added to this stock solution as bactericide and as a stabilizer.
~ The follobing evaluation criteria were checked using these stock solutions:
The viscosity in formation water having the com-;~ position according to Example 1, with a total salinity of 190 g/l.
The polymer concentration was adjusted so that, -~ for industrial use at from 40 to 60C, the viscosities were fro~ 30 to 40 mPa.s at a shear gradien~ D of 0.3 s 1.
~; The ~iscosities at D = ~.3 s 1 and D = 3.0 s 1 were used for characterizat;on.
The data in the Table below shou that 0.3 g/l of the homopolysaccharide solutions produced are sufficient.
It is found that the speci~ic viscosity at 60C is higher, and the temperature dependence smaller, than in the case of the com~ercial products used for compar;son.
- The Table shows that at, for e~ampLe, 40C, 1 g/l of Flocon~ 4800 have to be used, compared with only ; 0.3 g/l of the PS type AW It4 obtainable according to ; the invention, in order to obtain similar viscosities.
Evaluation ot the Table also shows that, owing to its pronounced temperature dependence, the sa~e com-~ercial product cannot be used at a temperature above 60C.
In contrast, the viscosity of the homopolysaccharide obtained by the process of the invention exhibits little te~perature dependence.
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r~ 1 329 1 59 - 15 - O.Z. 0960/02003 Viscosity n tmPa.s] Shear Concent-gradient ration Product ~) T - 25C 40C 60C
`` 5 A~ 114 74 53 44 0.3 0.3 `;~ 30 -26 22 3.0 A~ 117 48 39 27 0.3 0.3 i~ 24 20 16 3.0 ;~ Actigu ~ 20 13 8 0.3 0.3 ; 10 12 9 7 3.0 Actigum 80 50 32 0.3 0.5 : 38 30 2~ 3~0 Flocon~34800 - 42 18 0.3 1.0 .: 15 +) The solutions of the products are each sheared under . the same cond;tions.
The commercial product Actigum has to be used in ~;: an amount of 0.5 g/l in order to obtain a viscosity of 30-4Q mPa.s at D = 0.3 s 1 and at T = 60C. In contrast, `.- 20 only 0.3 g/l of the product prepared by the process of A~ the invention, type A~ 114, has to be used.
:i: Filtration behavior:
;:
As a technical rule, the solutions of the homo-; polysaccharides should flo~ freely, ~ithout causing bLock-.: 25 ages, through a filter having a pore diameter Dp of 1.2 ~m.
200 ~l of the solutions of the homopolysaccharides are filtered through a filter having a diameter of 47 mm, under a pressure of 0.4 bar. The ratio of ~he fLow rate of the first fourth to the fourth fourth is the ~illipore filter ratio ~MPFR). Solutions ~hich can readily be filtered ~; have an MPFR value of less than 106u This filtra~ion behavior can be further improved by subjecting the stock so~utions to shear forces.
; The Table belo~ gives the data for ~he viscosities and the filtration of the PS produced by the process of the inventir,n using the fungal streins according to clail 1.
, 1 ~29 1 59 - - 16 - O.Z. 0960/02003 The types AW: 106, 110, 114, 115~ 116 and 117 correspond to the fungal strains DSM: 388Q, 3887, 3888, 3890, 3891 and 3892.
. S n 0.3 and n 3.0 are the viscosi,ties at shear rates of 0.3 and 3.0 s 1, respectiveLy.
MPfR = millipore filter ratio Dp = pore diameter ~ Conc. = concentration in g/l ,,, 10 The solvent used ~as formation water according to Ex'ample .~ 1.
The shear conditions are:
,- 7,000 rpm x 3 min in an Ultra Turrax circu,lation appara-~ tus from Janke & Kunkel, Staufen~ FederaL RePublic of '~ 15 Germany.
'~ - = Not subjected to shearing , + = Subjected to shearing.
'~ All solutions are filtered ~ith 3 ~m or 5 ~m me~brane ,, filters. The viscosities after filtration are measured ,' 20 at T = 25C and expressed in mPa.s.
~,~
' Homopoly- A~ 106 AW 110 A~ 114 ; saccharide = + - ~ - *
n 0~3 46 57 31 118 74 ,j 25 n 3-0 18 26 18 32 30 MPFR 1.4 1.3 1.2 1.0 1.0 ~, Dp C~ 1. 2 3 1. 2 Conc. ~g/lJ _ 0.3 0~3 - 0'3 ;~ 30 , Homopoly- A~ 115 AW 116 AW 117 saccharide - +
J , _ , _ n 0.3 121 73 181 79 133 48 3.0 30 30 3~ 34 29 24 -MPfR 1.5 1.81.8 1.6 1.16 1.0 ,; Dp C~m] 1.2 1.2 1.2 Conc. [~/l] ~,4 0.3 0.3 ,.
.
.
t 329 1 59 - 17 - O.Z. 0960/02003 The Table shows that types AW 114 and AW 117 can very readily be filtered even without being subjected to shearing. As a result of shear;ng, the v1scosity is pre-ferentiaL-ly reduced at the lower shear gradient of D =
0.3 s 1 This results in a flattening of the viscosity curve, and this measure enables the viscosity to be~
adjusted.
`~ Long-term stability:
Solutions subjected to shearing and filtered for '- 10 use in practice were used to check the long-term stability `~ of the viscosity at T = 40C, 60C and 85C, different solutions stabilized with 200 or 1,000 ppm of formaldehyde being employed in the presence and absence of oxygen (2)-~' 15 Type AW 114 is stable, for example at 60C~ for ;~ 1 year or longer. This result is also found, for example, for type AW 117. These two solutions of the homopoly-saccharides were stabilized with formaldehyde but were not free of oxygen.
An important characteristic for practical use is j the filtration time of the PS obtainable according to the invention. The Table below shows the filtration times in ~inutes using ~embrane filters having pore diameters .~
of 1.2 ~m and 3 ~ and a filter dia~eter of 47 m~. To ,t~ 25 determine the filtration property, 0.3 g/l of PS is dis-~~ solved in formation water according to Example 1 and the solution is filtered over the membrane filters under 1 bar.
` The time, in minutes, required for the filtration of 200 `~ ml is determined.
3~ D;fferent filtration times are obtained. Negative solutions of types A~ 114 and A~ 117 possess good!filtra-tion properties after uLtrafiltration without treatment :,~
f by shearing. For good filtration properties, products ~; A~ 106, AW 110 an~ A~ 116 require treatment by shearing for 5 seconds in a ~aring ~lendor in order to obtain an industrially usable filtration ti~e.
~'l Type A~ 115 requires a longer filtra~ion time.
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- 18 - O.Z. 0960/0Z003 Where a shorter filtration time is important for indus-trial use, a longer shearing time will be required. The preferred types of the homopolysaccharides prepared by the proc~ss of the invention make it possible to prepare S products having the filtration times reyuired for indus-trial use.
Table showing the filtration times at 2 different pore diameters of the membrane filters, in minutes 3 ~m 1.2 ~m 10 Type ~min.] [min.]
AW 106 0.5 18.0 AW 110 3.0 12.0 AW 114 0.8 4.0 AW 116 1.6 1.6 AW 11? _ _ _ 2.5 _ 4.5 Treatment by shearing for 5 seconds in a Waring ~lendor The PS produced by the process of the invention using microorganisms in the form of fungal strains accord-ing to claim 1 have the advantage of good water solubility.
For this reason and because of their rheological proper-, ties~ these products have a wide range of uses in indus-try in the form of solutions~ as well as in the for~ of , shaped materials or of films and filaments.
Other advantages are the heat stability, the long-term stability and the shearing stability of the solutions, as well as their controllable filtration pro-perties.
, Water-soluble biopolymers are used in indus-try for various applications. U.S. Patent 3,301,848 issued on January 31, 1967 to the Allsbury Company describes water-soluble, nonionic polysaccharides of defined structure ~ as glucans. These are produced microbially as metabolites J''~: by growing the microorganism sclerotium glucanicum n.sp., NRRL 3006. However, they have a relatively low molecular :, .
weight. The same applies to the polysaccharides described in British Patent 2,178,~37 published on February 11, 1987 in the name of Sociéte Nationale ELF Aquitaine.
; W.L. Griffith and ~.L. Compere, in Dev. Ind.
Microbiol. 19 (1977), 609-617, describe the microbial production of highly viscous glucans by cultivation of the micro-organism Sclerotium rolfsii, ATCC 15206, in a nutrient medium containing inorganic salts, glucose , and a small amount of yeast extract, with a growth period `~ 20 of 2.5 days. The glucans produced exhibit a decrease in solubility in water at pH 1.5-~.
The present invention relates to a process for ~ the extracellular preparation of homopolysaccharides (PS) 'l having a high specific viscosity, the fungal strains which produce these PS, the PS themselves and their use.
It is an object of the present invention to prepare nonionic homopolysaccharides having a hlgh specific viscosity and high heat stability, salt stabill;ty and long-term stability. Stability means that the molecular `n 30 weight and hence the properties do not change significantly under the (possibly simultaneous) influence of elevated temperatures, salts and atmospheric oxygen.
More particularly, the invention relates to the fungal strains DSM 3887, DSM 3888, DSM 3890, DSM 3891 ..
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' ' -- 132915q - 2 - O.Z. 0960/32003 We have found that this object is achieved if microorgan;sms in the form of the fungal strains stated in claims 1 and 2 are cultivated in a nutrient medium with aeration and agitation at from about 15 to 40C and are then heated for from 2 to 30, preferably from S to 15, minutes at from 70 to 90C, preferably from 75 to 85C, if the fungal strains have matured, the culture solution is i then seParated from the cell mass, and the resulting -~ homopolysaccharide is isolated therefrom with a purity o~
-~ 10 greater than 99~ from 0.03 to 0.1~ by weight of residual - protein and with virtually only glucose as a basic monomer component.
rhe conventional nitrogen and hydrogen carbon source ~hich can be assimiLated by the fungal strains, and the ` 15 inorganic ions required for growth, are made available to the fungal strains as a nutrient medium, advantageously in a concentration such that, when the cultivation is terminated, the residual concentration of the carbon source is of the order of 0.1X by weight, based on the total solution. The nonionic PS produced have a viscosity of from 50 to 190 mPa.s at a shear rate D of 0.3 s 1 at 40C, determined with 0.3 9 of PS dissolved in 1,000 ml of highly saline water (containing up to 1ûO g/l of alkali metal ions and up to 30 i~` g/l of alkaline earth metal ions). This viscosity remains virtually constant for 1 year or longer at 60C in the air.
In the absence of oxygen, this even applies at temperatures up to 90C. The viscosity of the nonionic PS produced i according to the invention is influenced only to a small ~-~ extent at a concentration of from 0.1 to 1 g/l at about 20-60C and in the shear range D of about 3 0 s 1 or higher, so that it may be regarded as constant for practic!al purposes. Even after a solution containing 0.3 g/l of the PS obtainabLe according to the invention has been heated for one hour in an autoclave at neutraL pH and at 120C
under 1 bar g3ge pressure, viscosity re~ains virtually unaffected in the shear range D from 0.1 to 300 s 1 after cooling to 20C and supple~enting the evaporated ~ater. The ~' .
~1 . . .
^` 132915~
_ 3 - o.Z. 0960/02003 PS consist of a linear chain of 1,3-bonded ~-D-glucopyranose un;ts subst;tuted to different degrees by 1,6-bonded ~-D-glucopyranosyl groups.
~or use, the fungal strains are advantageously immobilized in a conventional manner on a substrate, pre-~ ferably a polyurethane foa~, and the formation of the PS
is carried out semicontinuously or completely continuously using this immobilized material.
The PS obtainable according to the invention are very useful as viscosity-increasing agents in polymer and ; surfactant flooding of oil deposits, as dispersants or emulsifiers (alone or in combinat;on with anionic and/or nonionic surfactants), as thickeners possessing structural viscosity, as water retention agents and for improvin~ the adhesion of substances to solid surfaces. ~hen employed as thickeners, they are intended in particular for use in drilling muds, for secondary and tertiary flooding of oil deposits and in general for reducing frictional resistance in flo~ing, in particular turbulent, aqueous liquids in order to reduce the pressure loss. Examples of suitable applications as dispersants are in agricultural chemicals, such as fungicides and pesticides, and in the food and animal feed sectors.
The fil~ration properties of the PS produced ac-cording into the invention, as an e~pression of the inject-ability into porous med;a, such as oil-bearing rock, are outstanding. For a sa~ple of 0.3 9 of PS per l of highly sal;ne ~a~er containing up to 100 g/l of alkali metal ions and up to 30 9/~ ~f alkaline e~rth me~al ions and pressure filtration under one bar gage pressure over a 3 ~ me~brane `; filter of 19 cm2, using 200 ml of solu~ion, the relevant properties are from 0~5 to 3.0 ~in in the case of shearing for 5 sec and from 078 to 10 min ~ithout shearing; the cor-responding propert;es for filtration over a 1.2 ~ me~brane filter are from 1.6 ~o 18 min in ~he case of shearing for 5 sec and from 4.0 to 14 min without shearing.
The sus~ension Gulture is grr~n for about iû-1aû, 1 32q 1 5~
- 4-- O.Z. 0960/02003 preferably 50-80, hours.
rhe noveL process gives the PS directly in very pure form. The resiclual protein content is from about 0.03 to Q.1X by weight.
For the purposes of the present invention, water-soluble means that not less than 1, preferably not less than 20, 9 of PS are soluble in a liter of water at Z0C.
In spite of their high molecular weight, the novel PS are water-soluble because they are substituted to a high deg-ree by side chains. They differ from the polysaccharides ~?~,~ described in U.S. Patent 3,301,848 in that, inter alia, they have a substantially higher molecular weight. rheir superior properties are mainly due to this.
The preferably used microorganisms in the form of fungal strains are characterized in the Tables below.
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; ~ 1 329 1 59 - 7 - o.Z. 0960/02003 Typical viscometric data for characterizing the polysac-charides AW
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~u ~mPas~187 81 188 51 163 144 n 0.1 -0.150.06 O.t2 0.15 0.13 C~] r~l/g] 6200091000 17000 - - 43000 M~ Cg/mol] 22.5x106 9x1o68X106 - _ 6 ~- n = Zero shear viscosity of an aqueous PS soLut;on ontaining 0.3 g/l ' n = Flow behavior index ~ .~., C~] = Intrinsic viscosity. This gives the space require-` 15 ment of a polymer coil in dilute solution (<=0.5 g/l) ; and is directly related to the molecular ~eight via the Mark-Houwink equation n = K.M~a MW = Molecular weight DP = Degree of poly~erization M~/162 16Z= D-glucose - 1 H2O
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The Examples ~hich follow illustrate the invention.
; EXAMPLE 1 Preparation of a ho~opolysaccharide, type AW 106, using the fungal strain DSM 3889O
Two slant agar cultures of the strain DSM 3A89 ~ere suspended in 3 ml of 0.9X strength sodium chloride solution, and this suspension was used to inoculate ~,~ 500 ml of a nutrient solution containing 1.5 9 of NaN03, 30 0.5 9 ot K2HPO4 3 HzO, 0.25 g of MgS04 . 7 H20, O.Z5 g ot KCl, 1.0 9 of yeas~ extract and 5.0 g o~ glu-cose. The shaken suspension culture ~as cultivated at ~I 27C for 60 hours at pH 4.5. The preculture thus pre-;~ pared ~as used to inoculate a 15 l bioreactor equipped with three six-blade disk stirrers and containing 10 l of a culture neuiun having the folLo~in~ cor,position:
, - 8 - O.Z. 0960lOZ003 NaN03 30 9 K2HP04 3 H2013 g MgS04 . 7 H2O 9 KCl - 5.0 9 ZnSO4 7 H200 03 9 Citric acid . H20 7.0 9 Glucose 300.0 9 Cultivation was carried out for 80 hours at 27C
~ith stirring at 500 rp~ and aeration with 1.67 liters of sterile air per liter of nutrient medium per minute and at an initial pH of 3.5. During the cultivation, the pH
decreased to 2.7. After the end of the cultivation, the residual glucose content was less than 0.1% by ~eight.
The culture suspension was heated at 80C for 30 ninutes, diluted ~ith 50 l of deionized water and cen-trifuged at 15,ûO0 9. The polysaccharide A~ 106 was pre-cipitated from the supernatant liquid with ;sopropanol (volume ratio 1:1), filtered off and washed ~ith 70Z
strength aqueous isopropanol solut;on, and the precipitate ~as resolvatized in 1 l of deioni~ed water and freeze-dried. 120 9 ot polysaccharide A~ 106 were obtain@d. The product contained glucose, as a ~onomer building block, and less than 0.05~ by weight o~ protein. 0.3 9 of AW 106 dissolved in 1,000 ml of formation water tcomposition in g/l: CaCl2 42.6 MgCl~ 10.5, NaCl 138.0, Na2S04 1.2 and Na802 . 4 H2O 0.4) had a viscosity of 137.7 mPa.s at a shear rate of 0.3 s 1 and 40C.
Preparativn of a homopolysaccharide, type AW 110~ using the fungal strain DSM 3887.
Four slant agar cultures of the stra;n DSM 3887 were suspended in 3 ml of O.9X strength sodium chloride ~ solut;on, and this suspension ~as used to inoculate ;l 35 1,000 ~l of a nutrient salt solution containing 1.0 9 of KzHP04 . 3 H2O, 1.5 ~ of KCl, 1~5 9 of N~NO3, 0.5 9 o~
MgSO4 . 7 HzO, 0.5 g gf yeast estrsct and 10.0 9 of glucose.
.
:
~ 1 329 1 59 - ~ - O.Z~ 0960/02003 The shaken suspension culture was cultivated at 27C for 54 hours at pH 4.5. This inoculation culture was used to inoculate a 70 l bioreactor equipped ~ith an Intensor~
System fr-om Giovanola, Monthey, Switzerland, and con-taining 50 l of a nutrient medium having the follo~ing ` composition:
NaN03 75 9 ~; KCl 75 9 K2HPo~ 3 H2O
10 MgS04 7 H20 25 9 FeSO4 . 7 H2O2.5 9 Xylose 1500.0 9 Cultivation ~as carried out for 96 hours at 27Cith stirring at 1,Z00 rpm and aeration with 0.5 liter of sterile air per liter of nutri~nt medium per ~inute, at an initial pH of 4.5. After the end of the cultivation, the pH had decreased to 3.0 and the xylose was virtually -~ compLetely consu0ed.
~; The culture medium was heated at 85C, and the 2Q cell mass ~as separated off at this temperature by means of pressure filtration. The clear, highly viscous fil-~ trate was precipitated with iscpropanol and freeze-dried, ;1 as described in Exa~ple 1. 435 9 of the homopolysaccharide type AW 110 ~ere obtained. The product contained exclu-sively glucose as the basic monomer component and had a residual protein content of less than 0.03~ by weight.
-~ Elemental analysis gave the following composition:
Carbon: 44-5~
Hydrogen: ~.25%
; 30 Nitrogen: 0~005~
0.3 g of A~ 110 in 1,000 ml of for0ation ~ater in oil production, having the composition according to ~ ,:
Example 1, had a viscosity of 81.6 0Pa.s at a shear rate ~` of 0.3 s 1 and 40~C.
Preparation of a ho~opolysaccharide, type A~ 114, using q the fungal strain DSM 3888~
, ., ~,', li ;:
- 10 - O.Z. 0960/02003 T~o s~ant agar cultures of the strain DSM 3888 were suspended in 3 ml of 9% strength sodium chloride solution~ and this suspension ~as used to ;nocuLate 500 ml o~ a nutrient salt solution containing 3.0 9 of yeast extract, 0.75 9 of (NH4~2HP04, 0.5 9 of K2HP04 . H20, -0.25 9 of MgS04 . 7 H20 and 10 9 of glucose. The shaken suspension culture was cultivated at 27C for 96 hours at pH 5.3. The pellets formed ~ere homogenized using an Ultra-Turra~Q stirrer at 20,000 rpm for 10 seconds, and this homogeni~ed innoculation culture was used to inoculate a 15 l bioreactor equipped ~ith three si~-bLade disk stir-rers and containing 10 l of a nutrient medium having the - following composition:
.~ Technical-grade yeast extract 30.0 9 15 K2HP04 . 3 H20 10.0 9 MgS04 . 7 H20 Xylose Z50.0 9 Cultivat;on was carr;ed out for 76 hours at 27C
~;th stirr;ng at 400 rpm and aeration with 0.1 Liter of ~ 20 sterile a;r per l;ter of nutrient mediu~ per minute, at - an initial pH of 5.3. After the end of the cultivation, the pH had decreased to 4.6 and the residual xylose con-tent was 0.1% by weight.
The react;on mixture was heated to 80C, 10 9 of formaldehyde were added, and the predominant amount of the polysaccharide ~as liberated from the resul~ing pel-~i Lets by shearing with an Ultra-Turrax for 10 seconds at Z0,000 rp~. Thereaf~er, the cell ~ateriaL ~as separated off by pressure f;ltrat;on, and the clear f;ltrate was freed from lo~ molecular we;ght substances by ultra--- f;ltration at an exclusion limit of 100,000 Dalton'. The uLtrafiltraee ~a~ prec;pitated w;th isopropanoL and freeze-dried, as descr;bed in Example 1. 7Z g of a ho~o-polysaccharide A~ 114 were ob~a;ned. The product contained excLusively gLucose as ~h~ basic monomer component and had a res;dual protein content of less than 0.05X by we;ght.
Ele-ent~l analysis gs~e the foLLowing co-position:
132~15q - 11 - O.Z~ 0960/02003 Carbon: 44.42%
~- Hydrogen: 6~19%
Nitrogen: 0.01X
.3 9 of A~ 114 dissolved in 1,000 ml of forma-tion water having the composit;on described ;n Example 1 had a viscosity of 188.7 mPa.s at a shear rate of 0 3 5 1 and 40C.
Preparation of a ho~opolysaccharide, type AW 115~ using the fungal strain DSM 3890.
A slant agar culture o~ the strain DSM 3890 was ; suspended ;n 3 ml of 0.9~ strength sodium chloride solu~
tion, and this suspension ~as used to inoculate 100 ~l of a nutrient salt solution conta;n;ng 0.1 g of yeast e~tract, ~.1 9 of K2HP04, 0.5 9 of MgS04 . 7 H20 and 1.0 9 of glucose. The suspension culture ~as cultivated at 25C for 60 hours at pH 5.7, ~h;le shaking on a rotary shaking ~achine at 110 rpm. This preculture was used to inoculate t~o 2 l conical flasks, each of which contained 500 l of nutrient medium having the following composition:
K2HP04 3 ~2 9 MgS04 7 H20 0.25 9 Yeast extract 0.5 9 Glucose 12.0 9 25 After incubation at Z5C on a rotary shaking machine at 110 rpm and at a pH of 5.7, the spec;fied amount of glucose was consumed after 142 hours and a pH
of 3.5 reached. The two batches were combined and dilu-ted ~ith 5 l of deionized water, and ~he cell mass was separated off by centrifu~ing. The clear supernatant liquid ~as concentrated to a polysaccharide content of 4.3 g/l by ultrafiltration at an exclusion limit of 100,000 Dalton~ the lo~ ~olecular weight components simultaneously being separated off. Ethanol was added to the concentrate (volume ratio 1:1), and the prec;pitated polysaccharide A~ 115 was separated off by centrifuging~
~ashed ~ith ~00 ml of 70~ strength ethanol and free2e-~ ~' 1329159 - 12 - O.z. 0960/OZ003 dried. 5.05 9 of polysaccharide AW 115 were obtained.
This product contained exclusively glucose as the mono-mer building block and 0.11% by weight of protein as an accompany-ing substance, the percentages being based on the ; 5 polysaccharide content.
0.3 9 of AW 115 dissolved in 1,000 ml of for~mation ~ater (for composition, see Example 1) had a viscosity of 51 mPa.s at a shear rate of 0~3 s 1 and 40C.
Preparation of a homopolysaccharide, type AW 116~ using the fungal strain DSM 3891.
`~ Starting from a slant agar culture of the fungal strain DSM 3891, the ho~opolysaccharide AW 116 was pro-duced under the sa~e conditions as described in Example 4. However, 50 9 of maltose/l of nutrient medium ~ere used as the C source. The cultivation was terminated after 120 hours, when a pH of 3.6 had been reached and the res;dual content of maltose had decreased to û.1~ by eight.
The mixture was worked up as described in Exa~-ple 4 to gi~e 4.0 9 of homopolysaccharide containing less than 0.05~ by ~eight of proteinsu 0.3 g of A~ 116 dis-solved in 1,000 ~l of formation water having the co~posi-~` tion described in Example 1 had a viscosity of 163.2 mPa.s 25 at a shear rate of 0.3 s 1 and 40C.
Preparation of a homopolysaccharide, type A~ 117, using the fungal strain DSM 3892.
Two slant agar cultures of the strain DSM 3892 ~. .
were suspended in 3 ml of 0.9% strength sodiu~ chloride solution, and this suspension was used to inoculate 500 ~l of a nutrient salt solution containing 0.5 g of yeast extract, 0O5 g of K2HPO4 . 3 H2O, 0.25 9 of ~gS04 . 7 H2O, 0.25 g of citric acid-H20 and 5 9 of suc-}5 rose. The suspension culture was cultivated a~ 25C for 72 hours at pH 5.6, ~hile shaking on a rotary shaking ~achina ae 100 rp~. This preculture ~as used to .' . ~
t 329 1 59 - 13 - O.Z. 0960/02003 inoculate a 15 l bioreactor which was equipped with a guide tube possessing a propeLler stirrer and contained 10 l of nutrient medium having the following composition:
K2HP04 ~3 H20 10.û 9 S MgS0~ . 7 H20 5.0 9 Yeast extract 5.0 Asparagine 12.0 9 Glucose 33U.0 9 Cultivation was carried out for 48 hours at 25C
; 10 ~ith stirring at 6ûO rpm and aeration w;th 1.0 liter of sterile air per liter of nutrient medium per minute, at an initial pH of 5.6. After the end of the cultivation, the pH had decreased to 3.2 and the residual glucose con-- tent was 0.15% by weight.
8 1 of deionized water ~ere added to the culture medium, the mixture ~as heated to 85C and the cell mass was separated off by means of pressure filtration. The clear cultur~ solution was concentrated to a homopoly-saccharide content of 10 g/l by ultrafiltration at an ex-clusion Limit of 100,000 Dalton, and was free2e-dr;ed.
90 9 of homopolysaccharide A~ 117 were obtained.
After hydrolysis of the production in 6 N H2S04 at 90C under an N2 atmosphere, glucose was detected as the only monomer component by thin layer chromatography and by high pressure liquid chromatography.
Elemental analysis gave the follo~ing composition:
Carbon: 44.32X
Hydrogen: 6.21X
Nitrogen: 0.09~.
This gives a residual protein content of 0.56%.
0.3 9 of A~ 117 dissolved in 1,000 ml of forma-tion ~ater according to ExampLe 1 had a viscosity of 144.5 mPa.s at a shear rate of 0.3 s 1 and 40C.
In order to test the suitability of the PS pro-duced by the process of the invention for tertiary oilproduction, the fungal strains stated ;n cLaim 1 ~ere grown in submerse culture, subjected to pressure ~ 1 32q 1 5q `:
- 14 - O.Z. 0963/Q2003 filtration and substantially freed from inorganic ions, residual glucose and proteins by subsequent ultrafiltra-- tion at an exclusion limit of 100,0ûO Dalton. Formalde-- hyde was-added to this stock solution as bactericide and as a stabilizer.
~ The follobing evaluation criteria were checked using these stock solutions:
The viscosity in formation water having the com-;~ position according to Example 1, with a total salinity of 190 g/l.
The polymer concentration was adjusted so that, -~ for industrial use at from 40 to 60C, the viscosities were fro~ 30 to 40 mPa.s at a shear gradien~ D of 0.3 s 1.
~; The ~iscosities at D = ~.3 s 1 and D = 3.0 s 1 were used for characterizat;on.
The data in the Table below shou that 0.3 g/l of the homopolysaccharide solutions produced are sufficient.
It is found that the speci~ic viscosity at 60C is higher, and the temperature dependence smaller, than in the case of the com~ercial products used for compar;son.
- The Table shows that at, for e~ampLe, 40C, 1 g/l of Flocon~ 4800 have to be used, compared with only ; 0.3 g/l of the PS type AW It4 obtainable according to ; the invention, in order to obtain similar viscosities.
Evaluation ot the Table also shows that, owing to its pronounced temperature dependence, the sa~e com-~ercial product cannot be used at a temperature above 60C.
In contrast, the viscosity of the homopolysaccharide obtained by the process of the invention exhibits little te~perature dependence.
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'',',~
.' .
r~ 1 329 1 59 - 15 - O.Z. 0960/02003 Viscosity n tmPa.s] Shear Concent-gradient ration Product ~) T - 25C 40C 60C
`` 5 A~ 114 74 53 44 0.3 0.3 `;~ 30 -26 22 3.0 A~ 117 48 39 27 0.3 0.3 i~ 24 20 16 3.0 ;~ Actigu ~ 20 13 8 0.3 0.3 ; 10 12 9 7 3.0 Actigum 80 50 32 0.3 0.5 : 38 30 2~ 3~0 Flocon~34800 - 42 18 0.3 1.0 .: 15 +) The solutions of the products are each sheared under . the same cond;tions.
The commercial product Actigum has to be used in ~;: an amount of 0.5 g/l in order to obtain a viscosity of 30-4Q mPa.s at D = 0.3 s 1 and at T = 60C. In contrast, `.- 20 only 0.3 g/l of the product prepared by the process of A~ the invention, type A~ 114, has to be used.
:i: Filtration behavior:
;:
As a technical rule, the solutions of the homo-; polysaccharides should flo~ freely, ~ithout causing bLock-.: 25 ages, through a filter having a pore diameter Dp of 1.2 ~m.
200 ~l of the solutions of the homopolysaccharides are filtered through a filter having a diameter of 47 mm, under a pressure of 0.4 bar. The ratio of ~he fLow rate of the first fourth to the fourth fourth is the ~illipore filter ratio ~MPFR). Solutions ~hich can readily be filtered ~; have an MPFR value of less than 106u This filtra~ion behavior can be further improved by subjecting the stock so~utions to shear forces.
; The Table belo~ gives the data for ~he viscosities and the filtration of the PS produced by the process of the inventir,n using the fungal streins according to clail 1.
, 1 ~29 1 59 - - 16 - O.Z. 0960/02003 The types AW: 106, 110, 114, 115~ 116 and 117 correspond to the fungal strains DSM: 388Q, 3887, 3888, 3890, 3891 and 3892.
. S n 0.3 and n 3.0 are the viscosi,ties at shear rates of 0.3 and 3.0 s 1, respectiveLy.
MPfR = millipore filter ratio Dp = pore diameter ~ Conc. = concentration in g/l ,,, 10 The solvent used ~as formation water according to Ex'ample .~ 1.
The shear conditions are:
,- 7,000 rpm x 3 min in an Ultra Turrax circu,lation appara-~ tus from Janke & Kunkel, Staufen~ FederaL RePublic of '~ 15 Germany.
'~ - = Not subjected to shearing , + = Subjected to shearing.
'~ All solutions are filtered ~ith 3 ~m or 5 ~m me~brane ,, filters. The viscosities after filtration are measured ,' 20 at T = 25C and expressed in mPa.s.
~,~
' Homopoly- A~ 106 AW 110 A~ 114 ; saccharide = + - ~ - *
n 0~3 46 57 31 118 74 ,j 25 n 3-0 18 26 18 32 30 MPFR 1.4 1.3 1.2 1.0 1.0 ~, Dp C~ 1. 2 3 1. 2 Conc. ~g/lJ _ 0.3 0~3 - 0'3 ;~ 30 , Homopoly- A~ 115 AW 116 AW 117 saccharide - +
J , _ , _ n 0.3 121 73 181 79 133 48 3.0 30 30 3~ 34 29 24 -MPfR 1.5 1.81.8 1.6 1.16 1.0 ,; Dp C~m] 1.2 1.2 1.2 Conc. [~/l] ~,4 0.3 0.3 ,.
.
.
t 329 1 59 - 17 - O.Z. 0960/02003 The Table shows that types AW 114 and AW 117 can very readily be filtered even without being subjected to shearing. As a result of shear;ng, the v1scosity is pre-ferentiaL-ly reduced at the lower shear gradient of D =
0.3 s 1 This results in a flattening of the viscosity curve, and this measure enables the viscosity to be~
adjusted.
`~ Long-term stability:
Solutions subjected to shearing and filtered for '- 10 use in practice were used to check the long-term stability `~ of the viscosity at T = 40C, 60C and 85C, different solutions stabilized with 200 or 1,000 ppm of formaldehyde being employed in the presence and absence of oxygen (2)-~' 15 Type AW 114 is stable, for example at 60C~ for ;~ 1 year or longer. This result is also found, for example, for type AW 117. These two solutions of the homopoly-saccharides were stabilized with formaldehyde but were not free of oxygen.
An important characteristic for practical use is j the filtration time of the PS obtainable according to the invention. The Table below shows the filtration times in ~inutes using ~embrane filters having pore diameters .~
of 1.2 ~m and 3 ~ and a filter dia~eter of 47 m~. To ,t~ 25 determine the filtration property, 0.3 g/l of PS is dis-~~ solved in formation water according to Example 1 and the solution is filtered over the membrane filters under 1 bar.
` The time, in minutes, required for the filtration of 200 `~ ml is determined.
3~ D;fferent filtration times are obtained. Negative solutions of types A~ 114 and A~ 117 possess good!filtra-tion properties after uLtrafiltration without treatment :,~
f by shearing. For good filtration properties, products ~; A~ 106, AW 110 an~ A~ 116 require treatment by shearing for 5 seconds in a ~aring ~lendor in order to obtain an industrially usable filtration ti~e.
~'l Type A~ 115 requires a longer filtra~ion time.
:, : `
'` 1 ~2~ 1 5~
- 18 - O.Z. 0960/0Z003 Where a shorter filtration time is important for indus-trial use, a longer shearing time will be required. The preferred types of the homopolysaccharides prepared by the proc~ss of the invention make it possible to prepare S products having the filtration times reyuired for indus-trial use.
Table showing the filtration times at 2 different pore diameters of the membrane filters, in minutes 3 ~m 1.2 ~m 10 Type ~min.] [min.]
AW 106 0.5 18.0 AW 110 3.0 12.0 AW 114 0.8 4.0 AW 116 1.6 1.6 AW 11? _ _ _ 2.5 _ 4.5 Treatment by shearing for 5 seconds in a Waring ~lendor The PS produced by the process of the invention using microorganisms in the form of fungal strains accord-ing to claim 1 have the advantage of good water solubility.
For this reason and because of their rheological proper-, ties~ these products have a wide range of uses in indus-try in the form of solutions~ as well as in the for~ of , shaped materials or of films and filaments.
Other advantages are the heat stability, the long-term stability and the shearing stability of the solutions, as well as their controllable filtration pro-perties.
Claims (8)
1. The fungal strains DSM 3887, DSM 3888, DSM 3890, DSM 3891 and DSM 3892.
2. A process for the extracellular preparation of a homopolysaccharide having a high specific viscosity, wherein microorganisms in the form of one or more of the fungal strains DSM 3887, DSM 3888, DSM 3890, DSM 3891 and DSM 3892 are cultivated in a nutrient medium with aeration and agitation at from 15 to 40°C, the culture suspension is then heated if the fungal strains have matured, the culture solution is then separated from the cell mass, and the resulting water-soluble homopolysaccharide is isolated therefrom in a conventional manner, with virtually only glucose as a basic monomer component.
3. A process as claimed in claim 2, wherein starch, cellulose, hemicellulose, glucose or sucrose is used as the carbon source, in a concentration such that the culture suspension in the three-phase solid/liquid/gas system is still miscible.
4. A process as claimed in claim 2, wherein, in the case of fungal strains whose 1,4.alpha.- or 1,4.beta.-glucanase is positive or whose 1,4.alpha.- and 1,4.beta.-glucanase are both posi-tive, a carbon source is used in a concentration such that the residual concentration is about 0.1% by weight after termination of the cultivation.
5. A process as claimed in claim 2, wherein the fungal strains are immobilized on a substrate by a con-ventional method, and formation of the homopolysaccharide is carried out semicontinuously or continuously using this immobilized material.
6. A homopolysaccharide obtainable by the process of claim 2.
7. A process for the preparation of an oil-in-water emulsion or an aqueous dispersion, wherein the dispersant used is a homopolysaccharide as obtainable by a process according to claim 2.
8. A process for thickening water, aqueous solutions, emulsions or dispersions, wherein the thickener used is a homopolysaccharide as obtainable by the process of claim 2.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3643467.1 | 1986-12-19 | ||
| DE19863643467 DE3643467A1 (en) | 1986-12-19 | 1986-12-19 | METHOD FOR THE EXTRACELLULAR PRODUCTION OF NONIONIC BIOPOLYMERS AND THE USE THEREOF |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1329159C true CA1329159C (en) | 1994-05-03 |
Family
ID=6316609
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000554284A Expired - Fee Related CA1329159C (en) | 1986-12-19 | 1987-12-14 | High molecular weight homopolysaccharides, their extracellular preparation and their use, and corresponding fungal strains |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0271907B1 (en) |
| AT (1) | ATE74381T1 (en) |
| CA (1) | CA1329159C (en) |
| DE (2) | DE3643467A1 (en) |
| NO (1) | NO169850C (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8852750B2 (en) | 2011-03-29 | 2014-10-07 | Wintershall Holding GmbH | Method for the coating of a cellulose material by using a glucan |
| US9206348B2 (en) | 2011-02-16 | 2015-12-08 | Wintershall Holding GmbH | Process for mineral oil production from mineral oil deposits with high deposit temperature |
| WO2017172707A1 (en) * | 2016-03-28 | 2017-10-05 | Cargill, Incorporated | Soluble & filterable biopolymer solids |
| CN107406865A (en) * | 2014-12-12 | 2017-11-28 | 温特沙尔控股有限公司 | Method for fungal bacterial strain fermentation |
| US10000720B2 (en) | 2014-05-22 | 2018-06-19 | Basf Se | Lubricant compositions containing beta-glucans |
| WO2019059901A1 (en) * | 2017-09-20 | 2019-03-28 | Cargill, Incorporated | Soluble & filterable biopolymer solids |
| US10273514B2 (en) | 2014-12-04 | 2019-04-30 | Wintershall Holding GmbH | Method for preparing an aqueous solution of beta-glucan |
| US10767105B2 (en) | 2015-10-02 | 2020-09-08 | Basf Se | Redispersion of schizophyllan |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2623207B1 (en) * | 1987-11-13 | 1990-05-18 | Inst Francais Du Petrole | PROCESS FOR THE PURIFICATION OF A POLYSACCHARIDE MUST WITH THE PURPOSE OF INCREASING THE FILTRABILITY AND USE OF THE PURIFIED MUST IN ASSISTED OIL RECOVERY |
| AU608713B2 (en) * | 1988-06-16 | 1991-04-11 | Standard Oil Company, The | The production of polysaccharides from filamentous fungi |
| DE4109457A1 (en) * | 1991-03-22 | 1992-11-12 | Wintershall Ag | METHOD FOR THE EXTRACELLULAR PREPARATION OF HIGH-MOLECULAR HOMOPOLYC-CHARIDES AND THEIRIR USE, AND THE CORRESPONDING MUSHROOM STAMPS |
| JPH05199892A (en) * | 1991-09-11 | 1993-08-10 | Shin Etsu Chem Co Ltd | Production of chitosan |
| FR2827192B1 (en) * | 2001-07-13 | 2004-06-04 | Cognis France Sa | PREPARATIONS CONTAINING NON-IONIC SURFACTANTS AS EXTRACTING AGENTS |
| EP2513324B1 (en) | 2009-12-17 | 2016-07-13 | Wintershall Holding GmbH | Method for producing homopolysaccharides |
| DK2675866T3 (en) | 2011-02-16 | 2015-04-07 | Wintershall Holding GmbH | PROCEDURE FOR TRANSPORTING OIL OIL FROM OIL OIL DEPOSITS WITH HIGH DEPOSITION TEMPERATURE |
| BR112013023849A2 (en) | 2011-03-29 | 2019-09-24 | Basf Se E Wintershall Holding Gmbh | method for coating a lamina cellulose-containing material, composition, coated lamina cellulose-containing material, and use of a glucan |
| EP2764069A1 (en) | 2011-10-04 | 2014-08-13 | Wintershall Holding GmbH | Method for extracting petroleum from an underground deposit |
| WO2013171137A1 (en) | 2012-05-16 | 2013-11-21 | Wintershall Holding GmbH | Method for precipitating and re-dissolving beta-glucan |
| EP2900790A1 (en) | 2012-09-27 | 2015-08-05 | Wintershall Holding GmbH | Flowable composition, method for producing the flowable composition and method for fracing a subterranean formation using the flowable composition |
| EP3212677A1 (en) | 2014-10-31 | 2017-09-06 | Wintershall Holding GmbH | Method for concentrating beta-glucans |
| EP3688113A4 (en) * | 2017-09-28 | 2021-06-23 | Cargill, Incorporated | Refined beta-glucans and methods of maintaining filterability of beta-glucan compositions at various salinities |
| EP3549958A1 (en) * | 2018-04-04 | 2019-10-09 | Clariant International Ltd | Process for the purification of complex biocompositions |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5461112A (en) * | 1977-10-24 | 1979-05-17 | Ono Pharmaceut Co Ltd | Oncostatic polysaccharide* its preparation* and oncostatic drugs containing it as an effective component |
| JPS58138392A (en) * | 1982-02-13 | 1983-08-17 | Japan Synthetic Rubber Co Ltd | Beta-d-gulcan |
| JPH0757761B2 (en) * | 1984-03-08 | 1995-06-21 | 株式会社林原生物化学研究所 | β-glucan, production method and use thereof |
-
1986
- 1986-12-19 DE DE19863643467 patent/DE3643467A1/en not_active Withdrawn
-
1987
- 1987-12-14 CA CA000554284A patent/CA1329159C/en not_active Expired - Fee Related
- 1987-12-17 EP EP87118712A patent/EP0271907B1/en not_active Expired - Lifetime
- 1987-12-17 DE DE8787118712T patent/DE3777979D1/en not_active Expired - Fee Related
- 1987-12-17 AT AT87118712T patent/ATE74381T1/en active
- 1987-12-18 NO NO875319A patent/NO169850C/en unknown
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9206348B2 (en) | 2011-02-16 | 2015-12-08 | Wintershall Holding GmbH | Process for mineral oil production from mineral oil deposits with high deposit temperature |
| US8852750B2 (en) | 2011-03-29 | 2014-10-07 | Wintershall Holding GmbH | Method for the coating of a cellulose material by using a glucan |
| US10000720B2 (en) | 2014-05-22 | 2018-06-19 | Basf Se | Lubricant compositions containing beta-glucans |
| US10273514B2 (en) | 2014-12-04 | 2019-04-30 | Wintershall Holding GmbH | Method for preparing an aqueous solution of beta-glucan |
| CN107406865A (en) * | 2014-12-12 | 2017-11-28 | 温特沙尔控股有限公司 | Method for fungal bacterial strain fermentation |
| US10767105B2 (en) | 2015-10-02 | 2020-09-08 | Basf Se | Redispersion of schizophyllan |
| WO2017172707A1 (en) * | 2016-03-28 | 2017-10-05 | Cargill, Incorporated | Soluble & filterable biopolymer solids |
| CN109153911A (en) * | 2016-03-28 | 2019-01-04 | 嘉吉公司 | Solvable and filterable biopolymer solid |
| US20190106618A1 (en) * | 2016-03-28 | 2019-04-11 | Cargill, Incorporated | Soluble & filterable biopolymer solids |
| WO2019059901A1 (en) * | 2017-09-20 | 2019-03-28 | Cargill, Incorporated | Soluble & filterable biopolymer solids |
| CN111263797A (en) * | 2017-09-20 | 2020-06-09 | 嘉吉公司 | Soluble and filterable biopolymer solids |
Also Published As
| Publication number | Publication date |
|---|---|
| ATE74381T1 (en) | 1992-04-15 |
| DE3777979D1 (en) | 1992-05-14 |
| NO875319D0 (en) | 1987-12-18 |
| EP0271907A3 (en) | 1989-03-08 |
| NO169850B (en) | 1992-05-04 |
| DE3643467A1 (en) | 1988-06-30 |
| EP0271907A2 (en) | 1988-06-22 |
| NO169850C (en) | 1992-08-12 |
| NO875319L (en) | 1988-06-20 |
| EP0271907B1 (en) | 1992-04-01 |
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