GB2178437A - Improved process for the preparation of polysaccharides by fermentation - Google Patents

Abstract

Polysaccharides are prepared by the fungal fermentation of an aqueous solution of a sugar having an initial pH of 2 to 3; NH4<+> ions are introduced into the medium so that it contains 10<-3> to 10<-2> ions per litre throughout the fermentation, and fermentation is continued substantially without the formation of oxalate. The process is applicable to a range of fungi and is illustrated by the use of a species of Sclerotium rolfsii.

Description

SPECIFICATION Improved process for the preparation of polysaccharides by fermentation The invention relates to improvements in the production of polysaccharides by microbiological means. It relates more particularly to the preparation of polysaccharides comprising polymers of glucopyranose, by the fungal fermentation of sugars. The improvements according to the invention allow preparation by means of moulds of such polymers and in particular those in which the glucopyranose groups are connected by > 1,6 and/or , > 1,3 bonds, with good yields and practically without the simultaneous formation of oxalic acid.

Various polysaccharides at present have industrial uses, certain of which are of considerable importance. Thus they are employed in aqueous cosmetic compositions, in paints, in preparations comprising various suspensions, in animal feeds, as hypocholesterol agents etc. A very important use is the utilisation of polysaccharides in prospecting muds in petroleum wells and in the assisted recovery of crude oil. A type of polysaccharide, which has proved particularly interesting for these latter uses, comprises chains of polymers formed by units of D-glucose connected by p-1,3 bonds and also carrying D-glucose groups attached to the chain by 1,6 bonds.

These compounds have given rise to numerous investigations, the results of which can be found among others in US Patent 3301848, which describes the fermentation of various sugars by means of filamentous micro-organisms, such as Sclerotium rolfsii, Sclerotium glucanicum, Corticium rolfsii, Sclerotinia gladoli, Stromatinia narcissi etc. The scleroglucanes obtained have degrees of polymerisation of about 180 to 1600 and undergo chemical transformations by the substitution of the -OH with other functional groups. From a medium comprising 30 g of saccharose per litre, containing 0.5 to 3 g/l of each of the salts NaNO3, K2HPO4, MgSO4, KCI and yeast autolysate, as well as 0.01 g/l of FeSO4, in several days of fermentation at 28"C, 8.2 g of sclero glucane per litre is obtained.This process is industrially viable, but in addition to the polysaccharide oxalic acid is found, the presence of which is undesirable for all uses and, in particular, in the assisted recovery of crude petroleum, where precipitation of calcium oxalate blocks the pores in the rock. It is thus necessary to eliminate the oxalate from the polysaccharide solution obtained by fermentation and this increases the manufacturing costs.

MAXWELL and BATEMAN, in "Phytopathology" - 58, No. 10, (1968), pages 1351-1354, have shown that, in the fermentations in question, it is possible to reduce considerably the formation of oxalic acid by lowering the initial pH of the culture medium to 2.3 (page 1353, Table 3); the authors have not measured the concentration of scleroglucane during their experiments, however. Experiments on which the present invention is based show that a simple reduction of the initial pH below 2.5 does not allow the concentration of oxalic acid to be reduced sufficiently and it more negatively influences the production of scleroglucane.The same experiments confirm the observation of the foregoing authors that replacement of part of the glucose by glycerol yields a significant reduction in the oxalic acid content. (Glycerol is, not a good source of carbon, however, and this increases the cost of the process). In fact, neither of the two means proposed in the prior art can provide a truly industrial application and the problem of the presence of the oxalic acid still remains.

The present invention has the object of considerably reducing or even completely suppressing the formation of oxalic acid during the fermentation of sugars for the production of polysaccharides by the micro-organisms cited in US patent 3301848. In particular, it allows the preparation of scleroglucanes of the desired degree of polymerisation by a microbiological process similar to that of the US patent cited above, with an hourly production per unit volume of culture medium as good as or even better, the product being practically free from oxalates.

The improvement according to the invention of the microbiological process described above utilizes the knowledge of the prior art that the initial pH of the culture medium of the mould is low, particularly from 2 to 3.

However, it is characterised in that NH4 ions are introduced into the medium in such a manner that, throughout the period of fermentation, it contains 1 x 10-3 to 11 x 10-3 M/1 and preferably 3 x 10-3 to 6 x 10-3 ion-grams NH4+ per litre.

Due to this characteristic, disappearance of the oxalic acid does not cause a drop in production of the polysaccharide.

The ammonium ions can be provided in the form of a salt such as NH4CI, NH4NO3, (NH4)2SO4, (NH4)2HPO4, (NH4)2SO3 etc.; the preferred form is NH4CI.

In view of the consumption of the NH4+ ions during the fermentation, a preferred form of the invention consists in introducing into the medium an adequate quantity of ammonium salt initially and adding further portions every few hours, for example at every 6, 8, 10 or 12 hours, in order to maintain the level indicated above.

Another preferred form consists in introducing continuously into the medium the proportion of NH4+ to compensate for the ammonium consumed, according to the rate of fermentation.

While the various special conditions indicated above are applicable to the nutrient media of the prior art, to reduce considerably the oxalic acid content of the fermentation product, still better results are obtained when utilising media modified according to a particular feature of the invention. It has in fact been established that the use of a medium where all the components are completely known and defined chemical species, as opposed to the prior art media employing complex natural extracts, such as yeast autolysate or corn steep liquor, gives better results within the scope of the process of the invention. A preferred feature of the invention thus consists in employing the following medium for the fermentation, in which the concentrations are expressed in grams per litre.

In general Preferred Sugar, particularly glucose 20 to 50 30 to 40 K2HPO4 . ............ . 0.1 to 1.4 0.6 to 0.8 NH4NO3 .... . .............. 0.13 to 0.94 0.2 to 0.9 KCI .............. ........................................ 0 to 0.25 0.1 to 0.2 MgSO4.7H2O ... 0.02 to 0.3 0.15 to 0.25 FeSO4.7H2O ... . .......... .......... ......... 0.001-0.005 0.003-0.004 MnSO4.H2O .... ......... .......... . . 0.001-0.004 0.002-0.003 ZnSO4.7H2O . . .......... ........... . ....... 0.001-0.005 0.003-0.004 Thiamine .. ..... .. ............. ...................... . 10-2-10-5 5 x 10-3-10-4 N H4+ ions per litre .... . ................ 0.001.0100 0.0025-0.06 As in the prior art, the process according to the invention can be carried out at various temperatures, particularly between 15 and 35 C, but preferably between 26 and 30 C. As the fermentation is aerobic, the medium is aerated with 0.1 to 1 volume of air per volume of medium and per minute and preferably with 0.3 to 0.6. It is necessary to agitate the medium at increasing speeds (between 150 and 700 revs/minute), depending upon the degree to which it becomes viscous.

The invention is illustrated non-limitatively by examples of fermentation with the fungus Sclerotium rolfsii (ATCC strain 15206), in 20-litre fermenters (total volume) at 28 C, with a speed of agitation progressively increasing as a function of the viscosity of the medium. In each case, an inoculum is first prepared by 48 to 72 hours of preliminary fermentation of the desired medium with the fungus, in a 2-litre fermenter or preferably in several vials agitated togetherjust before inoculation. Then 1200 ml of inoculum is introduced into 13.8 litres of the medium prepared in the 20-litre fermenter and the fermentation is continued for 55 to 72 hours. A fraction of the homogeneous liquid material is removed from the fermenter to determine the sum of the mycelium + polysaccharide, called "native", by precipitation with alcohol.Filtration of another fraction of the liquid mass to separate the mycelium and the polysaccharide followed by precipitation of the filtrate (also with alcohol) gives the quantity of scleroglucane formed. On the other hand, oxalic acid in the supernatant liquid is measured after centrifugation of a fraction of the fermentation must by chromatrography in the liquid or gaseous phase and also by means of an enzymatic probe constituted by an electrode sensitive to oxygen, surrounded by a protein membrane containing oxalate oxydase.

Thus, the concentration of sugar not consumed during the fermentation is determined.

Example 1 The fermentation is conducted in the manner described in US Patent 3301848, in 20-litre fermenters or in agitated 0.5-litre vials, but with the following complex medium (the contents are given in g/l of water): glucose 35 corn steep liquor 4 (maize extract) . NaNO3 1.6 . KH2PO4 0.8 MgSO4.7H2O 1.2 . vitamin B 1 5x10-3 initial pH 4.5.

The results are given in the summary Table at the end of the Examples, for comparison with those of the invention.

Example 2 Operation was as in Example 1, but the pH of the medium was first adjusted to 2.5.

Example 3 The medium of Example 2 at pH 2.5 was provided with 3 g/l of NH4CI, or 5.6 x 10-2 mole/1 of NH4+, all the other conditions being the same as in Example 1.

Example 4 The mode of operation in general remained the same, but the culture medium of the foregoing Examples was replaced with that of the invention (g/1): Glucose 35 . K2HPO4 0.7 .NH4NO3 0.88 . KCl 0.15 MgSO4.7H2O 0.20 FeSO4.7H2O 3.66 x 10-3 MnSO4.H2O 2.68 x 10-3 ZnSO4.7H2O 3.56 x 10-3 Thiamine hydrochloride 10-4 pH=2.5NH4+ = 11 x 10-3 mole/1 (results after Example 6) Example 5 The medium of initial pH 4.5 was the same as that of Example 1 except for the mineral nitrogen, ammonium nitrate at 0.74 g replacing the sodium nitrate.Also, 0.3 g/l of NH4CI was added each time the concentration in N H4+ ions in the medium fell below 3 x 10-3 mole per litre, which occurred three or four times during the entire duration of the fermentation.

Example 6 The medium of initial pH 2.5 was the same as Example 4, but 0.3 g/l of NH4CI was added to it each time the concentration in NH4+ ions fell below 3 x 10-3 mole/1.

Summary of Results TABLE 1 Comparison in agitated vials Example Medium Native (g/l) Oxalic Acid Scleroglucane (pure) 1 Known art pH 4.5 12.3 7.3 1.5 2 Known art pH 2.5 11.4 6.2 0.7 3 Known art pH 2.5 + 3g/l NH4CI 7.4 2.2 0 4 New pH 2.5 12.7 8.7 0.5 TABLE 2 Comparison in 20-1 fermenters Example Medium Native Scleroglucane Oxalic Acid (pure) (in g/l) 1 Known art pH 4.5 16.1 12.5 3.2 5 Known art pH 4.5 + NH4Cl/during 17.3 11.3 0.5 fermentation 4 New pH 2.5 20.7 13.5 0.4 6 NewpH 2.5 + NH4CI 19.6 13.7 < N 0.03 Comparison of the results in vials of Example 1 with those of Example 2 shows that the reduction of the pH according to the known art is not sufficient to reduce the formation of oxalic acid to the level of traces.Example 3 indicates that a high concentration of ammonium ions allows complete suppression of the production of oxalic acid. However, such a concentration also produces a considerable reduction in the production of scleroglucane.

There is a median level, however, (as regards the concentration of NH4+ ions in the fermentation medium) which inhibits the production of oxalic acid without having any negative influence on the yield of polysaccharide. This is shown better in the comparison in the 20-1 fermenters.

Example 4 using vials shows that the production of scleroglucane is at least as good with the new medium and the formation of oxalic acid is significantly reduced.

The final concentration in polysaccharide is generally higher in the 20-1 fermenters (where the parameters of agitation and aeration are better controlled) than in vials. Comparison of the results in the 20-litre fermenters of Example 1 with those of Example 5 shows that the addition of ammonium ions during the fermentation produces a considerable reduction, but not total supression, in the production of oxalic acid, with the medium of the prior art. The controlled addition of NH4CI during fermentation with the new medium allows the total suppression of the production of oxalic acid, without reducing the yield of scleroglucane. This is based on the median level mentioned above.

Claims (12)

CLAIMS:

1. A process of preparation of a polysaccharide substantially without the formation of oxalate, by the fungal fermentation of an aqueous solution of a sugar having an initial pH in the range from 2 to 3, in which NH4+ ions are introduced into the fermentation medium so that, throughout the fermentation, it contains from 1 x 10-3 to 11 10-3 ion-grams of NH4+ per litre.

2. A process according to claim 1, in which the NH4+ ions are provided in an amount from 3 x 10-3 to 6 x 10-3 ion-grams per litre.

3. A process according to claim 1 or 2, in which the NH4+ ions are provided by an ammonium salt of an inorganic acid.

4. A process according to claim 3, in which the ammonium salt is the hydrochloride, nitrate, sulphate or phosphate.

5. A process according to any preceding claim, in which NH4+ ions are added to the medium at different times during the fermentation.

6. A process according to any of claims 1 to 4, in which NH4+ ions are added to the medium by continuously injecting a solution of an ammonium salt.

7. A process according to any preceding claim, in which all oligoelements and vitamins contained in the fermentation medium are chemically-defined compounds.

8. A process according to any preceding claim, in which the fermentation medium contains in g/i the following compounds: Sugar, particularly glucose 20 to 50 K2HPO4 0.1 to 1.4 NH4NO, 0.13 to 0.94 KCI 0 to 0.25 MgSO4.7H2O 0.02 to 0.3 FeSO4.7H2O 0.001 - 0.005 MnSO4.H2O 0.001 - 0.004 ZnSO4.7H, 0.001 - 0.005 Thiamine 10-2-10-5 NH4+ ions per litre 0.001-0.0100.

9. A process according to claim 8, in which the fermentation medium contains in g/l the following: Sugar, particularly glucose 30 to 40 K2HPO4 0.6 to 0.8 NH4NO3 0.2 to 0.9 KCI 0.1 to 0.2 MgSO4.7H2O 0.15 to 0.25 FeSO4.7H2O 0.003 - 0.004 MnSO4.H2O 0.002 - 0.003 ZnSO4.7H2O 0.003 - 0.004 Thiamine 5 x 10-3 - 10-4 NH4+ ions per litre 0.0025 - 0.06.

10. A process of preparation of a polysaccharide, substantially as described with reference to any of the foregoing Examples 4, 5 and 6.

11. A polysaccharide, when made by a process according to any preceding claim.

12. A composition of matter, useful for instance in petroleum drilling or crude oil recovery, containing a polysaccharide according to claim 11.