GB2082189A - Production of microbial polysaccharides - Google Patents

Abstract

This invention relates to a process for the production of a polysaccharide wherein a microorganism species which produces polysaccharide (preferably in the stationary phase of the growth cycle) is supported on a porous, particulate inert support, the pore size being greater than 0.5 mu m, to form an immobilised cell system, aqueous nutrient medium is passed through the immobilised system, and polysaccharide-containing medium is withdrawn from the system. The invention provides also for the use of the polysaccharide in the displacement of fluid from subsurface formations, and an immobilised cell system for use in the foregoing process.

Description

SPECIFICATION Production of microbial polysaccharides This invention relates to the use of microbial cells in an immobilised system, and in particular to the production of a polysaccharide from immobilised microorganisms.

The use of immobilisation technique has become well established in biochemical transformations using enzymes, and more recently this technique has been applied also to whole microbial cells.

However, hitherto such immobilised cell technology has been applied primarily to the production of relatively iow molecular weight organic molecules, such as aspartic acid, tryptophan and aminopenicillanic acid, and has not been applied to the production of higher molecular weight materials such as the polysaccharides elaborated by a variety of slime-forming microorganisms. It would be expected-thaf the molecular size and viscosity characteristics of such products would tend to impair the effectiveness of an immobilised system and indeeed no success was achieved in attempts to produce an immobilised system by gei entrapment of the cells. However, it has not surprisingly been found that polysaccharides can be produced from cells immobilised on a porous, particulate support, provided that the support particles have a defined pore size.

Accordingly, the present invention provides a process for the production of a polysaccharide wherein a microorganism species which produces a polysaccharide is supported on a porous, particulate inert support, the pore size being greater than 0.5,us, to form an immobilised cell system, aqueous nutrient medium is passed through the immobilised system, and polysaccharide-containing medium is withdrawn from the system.

This process is generally applicable to a variety of microorganisms which produce polysaccharides. It can be applied with those microorganisms where polysaccharide production is associated with growth-and multiplication, such as the xanthan-producing organism Xanthomonas campestris, but in such cases the organism growth results in release of free cells from the support into the medium and thereby loses some of the advantages of the invention. The process is particularly useful with microorganisms which produce polysaccharides in the stationary phase of the growth cycle (i.e., the production is not growth associated). Examples of such microorganisms are certain slimeforming species of the genera Pseudomonas, Rhizobium, Alcaligenes and Agrobacterium, especially Pseudomonas spp.NCIB 11 592 and 11264, Rhizobium meliloti, Alcaligenes faecalis var. myxogenes, Agrobacterium radiobacter, Agrobacterium tumefaciens and Agrobacterium Thizogenes. All these specific organisms elaborate a particular polysaccharide containing glucose, and, for each 7 moles of glucose, 0.9-1.2 moles of galactose and 0.65 to 1.1 moles of pyruvate together with 02 moles of succinate and acetate, whose characteristics and use in oil recovery processes are described in detail in our copending UK Patent Application number 8016832. That application also describes the detailed characteristics of the novel strain NCIB 11592 of Pseudomonas sp.

The support material should naturallyvbe chemically and biologically inert under the conditions of use, and suitable materials are inorganic oxides such as silica, alumina, zirconia, magnesia and mixtures thereof. The effective retention of the microbial cells on the support has been found to be dependent on the selection of the size of the pores in the support particles, and as indicated above this should be greater than 0.5 m. Generally, the best results are obtained when the pore size is at least 1 ym, and the preferred pore sizes are from 2 um to 30 jum. The size of the support particles is less critical than pore size for acceptable operation, and in practice is determined partially by the operational conditions under which the immobilised system will be employed, e.g. whether retained in a fixed bed or used as a slurry. In general the particle size should be less than 1 mm, and for operation in a slurry reactor it has been found that best results are normally attained when using particles within the size range of 100 to 600,1cm, and preferably 150--300 ym.

The location of the cells onto the support may be carried out by a vanety of techniques, but two procedures have been found to be generally suitable. In one technique the microbial cells are cultivated by established methods in a fermenter so as to yield a paste of the required cells which is mixed with the support particles. The mixture is then subjected to a vacuum and the vacuum released so that the atmospheric pressure forces the paste into the pores. The coated particles are then allowed to age for a short period (several hours), after which the surplus cells are washed away. An alternative, and preferred, technique is that of growth in situ, in which the particulate carrier is suspended in a suitable growth medium, which is then inoculated with the desired organism.The inoculated, support containing growth medium is then-incubated under appropriate conditions to provide for growth of the organism, and after a suitable period free cells are removed by washing to leave the cell-containing immobilised system. In a variation of this technique, fresh growth medium containing an assimilable source of carbon and nitrogen is continuously fed into the reactor during immobilisation to provide suitable conditions for attachment. When applying the growth in situ technique it is often found that higher and more stable cell loadings are obtained by the use of high levels of nutrient in the growth medium.

The aqueous nutrient medium should provide a substrate for the production of polysaccharide by the microorganism. In the case of those microorganisms which produce polysaccharide in the stationary phase of their growth cycle, the nutrient medium is preferably one which, whilst providing the necessary substrate, does not provide conditions conducive to growth and multiplication of the microorganism.

Such non-growth conditions can be maintained by the use of a medium deficient in one or more of the components essential to cell growth, eg., sulphate, phosphate or preferably, nitrogen, or by other well established means. The aqueous nutrient medium will normally contain an assimilable source of carbon together with small amounts of inorganic ions. The source of carbon is suitably a carbohydrate, conveniently glucose, and is preferably employed in a concentration between 0.1 and 10% by weight, normally 12% w/v. The temperature and pH at which polysaccharide is most effectively produced will naturally vary according to the organism, and in the case of Pseudomonas sp. NCIB 11592 the temperature is preferably between 200 and 350C, and the pH preferably between 6 and 9.During production of polysaccharide the aqueous nutrient medium will normally be fed continuously into the immobilised system at the same rate as the polysaccharide-containing outlet medium is withdrawn therefrom; that outlet medium can be used per se or may be treated according to known procedures to extract the polysaccharide therefrom.

One of the major benefits of using the immobilisation technique of this invention is that it facilitates separation of the desired polysaccharide from the cells, since the latter are located on the support particles which are readily retained in the fermentation vessel by well established devices such as filter mesh or weirs. Additionally, the immobilisation procedure offers the possibility of prolonging the productive lifetime of the cells, ie., the period during which they will continue to elaborate the polysaccharide without any significant growth, but this lifetime is inevitably finite, and is not in all cases significantly longer than that of a free cell system.However, when the productivity of the supported cells drops to below an acceptable level it can be easily and virtually completely restored by repeating the growth phase (ie., reintroducing a complete growth medium containing all necessary nutrients) for a suitable period, suitably under washout conditions which remove any free cells released from the support, and then reverting once again to the supply of a nutrient medium deficient in, for example, nitrogen.

One important use of the polysaccharides produced by this invention is in enhanced oil recovery (as described in more detail in our copending UK Patent Application 8016832), and hence the invention includes also a process in which the polysaccharide-containing medium, after any necessary concentration adjustment and/or incorporation of additional compounds, is injected into a fluid-bearing permeable subsurface formation. In this application it is particularly important for the polysaccharide solution to be free from cell bodies, and an advantage of the process of this invention is that it simplifies the production of a cell-free polysaccharide solution.Using conventional processes it is necessary to separate the microbial cells from the viscous solution of polysaccharide, but in the process of this invention the cells are immobilised on the support and hence the resultant polysaccharide solution is substantially free from such cells.

The invention is illustrated in the following Examples.

EXAMPLE I A. Preparation of immobilised system 1) Vacuum loading Pseudomonas sp. NCIB 11 592 was continuously cultivated in a Chemap LF-7 fermentation vessel having a liquid volume of 41. The culture temperature was maintained at 280C and the pH controlled at 6.8 by the automatic addition of 2N alkali solution (1 N NaOH + 1 N KOH). Air was sparged into the fermenter at 0.5 1 mien~1 and the culture was agitated by a turbine impeller revolving at 1000 rpm. Fresh sterile medium in two streams was pumped into the fermenter continuously and culture broth withdrawn by a weir to maintain a constant working volume in the reactor.The flow rates and composition of the streams were as follows: STREAM 1. Flow Rate: 300 ml h-'.

Mineral salts medium as defined below containing also 20 gl-1 glucose and 10 mM H3PO4,

Concentration Component gl-1 mM IlM MgSO4.7H2O 0.493 2.0 CaCl2.2H2O 0.147 1.0 FeSO4.7H2O 55.6 x 10-3 200 MnSO4.7H2O 4.46 x 10-3 20 ZnSO4.7H2O 5.74 x 10-3 20 CuSO4.5H2O 4.99 x 10-3 20 CoCI2. 6H2O 2.37 x 10-3 10 H3BO3 0.61 x 10-2 10 Na2MoO4.2H2O 2.41 x 10-3 10 KI 1.66 x 10-3 10 STREAM 2.Flow Rate: 60-120 ml h-1.

(NH4)2SO4 21.14 gl-1 A paste of the cells prepared from the broth by centrifugation and containing 1 00 g cells (dry weight) per litre was mixed with the chosen support particles. This mixture was then evacuated by water'pump and the vacuum released after 3-5 minutes. This procedure was repeated 4 to 5 times and the mixture was aged at 40C for 2 h or 12 hours before repeated washing with a phosphate buffer solution to remove excess cells. The cell loading attained was estimated either by respiration studies using an oxygen electrode, or by measuring the concentration of protein using the method of Lowrey et al., and was expressed as mg dry weight of cells per gram of support material. These tests were carried out with a variety of support materials.The results of all these tests are summarized in Table 1 below. TABLE 1 Support Material

Pore Slze Particle Slze Cell Loading Deslgnetion Composition ( m) ( m) Aging Time (mg. dry wt./gm support) Fractosll 6000 Sillca 0.5 63-125 2 0.1 " 10000 " 1.0 " 2 4.4 " 26000 " 2.5 " 2 3.3 " 25000 " 2.5 " 12 21/28 " 5000 " 0.5 " 12 5.83 Norton 06519 Alumina 3-13 150-250 2 10.7 " " " " 12 10.66 " " " 300-600 12 11.5 " " " > 600 12 8.0 Norton 06482 Silica/Alumina " 150-300 2 11.2 Norton SA 6373 Alumina 0.1 150-600 2 zero Norton SA 5559 " 2-4 300-600 2 zero Norton SA 5221 " 10-30 N.A. 12 10.1 Norton SA 6564 Zlrconla 6-60 N.A. 12 9.4 Norton SA 6525 Alumina 3.2 N.A. 12 9.1 Norton SA 5231 " 1-2/15-100 N.A. 12 4.9 Norton SA 6605 " 5 N.A. 12 10.8 Carborundum SAEH84533 " 0.3 N.A. 2 zero Harshaw AL-183-1P " N.A. 500 2 3.3 " " " " 12 8 2) Loading by growth in situ Pseudomonas sp. NCIB 11 592 was cultivated in a Biotec fermenter having a working volume of 2.5 1 in the presence of 5-10% w/v of alumina (Norton 06519) particles using a gas flow rate of 400 ml/min df air or-200 ml/min oxygen and an agitator speed of 200 rpm, giving a dissolved oxygen tension'greater than 60% air saturation. The temperature was 30 C, the pH was controlled at 7.0 by the automatic addition of 2N alkali solution, and the growth medium comprised the mineral salts medium as defined above, containing also 20 g/l glucose. 2.11 g/l (NH4)2SO4, 0.680 g/l KH2PO4 and 0.709 g/l Na2HPO4.

Immediately before batch growth finished (about 20 hours), reactor conditions were changed to a gas flow rate of 600 ml/min air (or 300 ml/min oxygen) and continuous culture using the given medium at a flow rate in excess of 1 I/h. At this high dilution rate the cells already attached to the carrier were retained and grew further in the carrier, while the free, or loosely secured, cells were washed out. Cells were cultivated in this manner until no further increase in cell loading was observed (48 hours), and the cell loading determined as in A(1). The results are set out in Table 2 below.

TABLE 2

Lcading $mg g-1) Time from start of RUN 1 RUN 2 continuous culture (h) 72 gl-1 carrier conc. 260 91-l carrier conc.

0 4.0 3.5 20 10.5 12.0 40 17.4 n.d.

60 17.2 20 80 1 20 B. Production of biopolymer 1 ) Vacuum immobilisation and batch production Cells of Pseudomonas sp. NCIB 11592, Pseudomonas sp. NClB 11264 and Agrobacterium radiobacter NCIB 9042 were immobilised onto alumina, (Norton 06519) as described previously. 10 g of each biocataylst, after washing, were placed in shake flasks containing 100 ml of 10 mM phosphate buffer at pH 7.4 and 10 g glucose. The flaSks were shaken at 200 rpm and 3O0C with the following results: TABLE 3

Loading 1 Polymer concentration Strain (mg 9z) after 140 h (gl-1) Pseudomonas sp. NCIB 11592 9.6 4.0 Pseudomonas sp. NCIB 11264 11.0 4.5 Agrobacterium radiobacter NICB 9042 8.5 2.0 ,Notes: ' mg cells (dry wt)/g support 2) Vacuum immobilisation and continuous production Cells of Pseudomonas sp. NCIB 11592 were immobilised onto alumina (Norton 06519) as described previously, to give 9.7 mg cells (dry weight)/g support. 180 g of the biocatalyst were placed in a 4 litre Biotec fermentation vessel containing 2.8 1 of medium comprising the mineral salts medium described previously and also 20 g/l of glucose and 10 mM H3PO4. The temperature was maintained at 300C and the pH controlled at 7.0 by the automatic addition of 2N alkali solution.Air was sparged into the reactor at 400 ml/min and the suspension was agitated by an impeller rotating at 200 rpm. Fresh medium was pumped into the reactor at 225 ml h-' and polysaccharide solution was withdrawn by a weir, incorporating a settler, to maintain a constant working volume and constant biocatalyst hold-up in the reactor. The polysaccharide concentration in the outlet stream (as measured by reaction with anthrone) is shown in the following table:

TABLE 4 Run Time (hours) Polysaccharlde Conoentration (gl-1) 20 0.16 42 0.13 63 0.18 114 0.20 139 0.21 3) lmmobllisation by growth in situ and continuous production Pseudomonas sp.NCIB 11592 was immobilised onto 180 g of alumina, (Norton 06519), according to the procedures in Example A2. Following immobilisation, fresh salts medium (nitrogen free) containing also 20 g/l glucose and 10 mM H3PO4 was pumped into the reactor at 225 ml h-' and polysaccharide solution was withdrawn as in Example B2. After initial loss of cells, the loading of cells on the support during polysaccharide concentration in the outlet stream is shown in the following table:

TABLE 5 Run Time (hours) (following immobillsation) Polysaccharide Concentration (gl-1) 26 0.73 42 0.51 63 0.45 85 0.45 4) Growth in situ at different nutrient levels and continuous production Pseudomonas sp.NCIB 11592 was immobilised onto alumina (Norton 06519) according to the procedures in Example A2, except that the growth medium contained the following mineral salt concentrationst Component Concn. mg/l Na2HPO4 3000 KH2PO4 3000 MgSO4.7H2O 200 FeCl3.6H2O 33.4 CaCI2 s 2HzO 16.0 ZnSO4.7H2O 0.36 CuSO4. 5H2O 0.32 MnSO4.4H2O 0.30 CoCla .6H2O 0.36 H1BO4 0.20 Na2MoO4.2H2O 0.60 together with (NH4)2SO4 and glucose in the concentrations of either 0.3 g/l and 10 g/l (I) or 3.0 g/l and 20 g/l (II) respectively. The time of loading growth was 90 hours for I and 70 hours for II.The resulting immobilised cell systems were then supplied with nutrient medium which differed from the growth medium only in the omission of the ammonium sulphate (this modification being referred to as nitrogenfree medium), the polysaccharide solution withdrawn, and the product quotient (g.polymer/g. fixed cells/hr) and productivity (in terms of mg polysaccharide/g. cell-bearing support/hr) determined. The results are set out in Table 6 below, from which it will be apparent that the higher nutrient levels lead to significant improvements in cell loadings and the production parameters.

TABLE 6

Cell Loading (mg/g) Product Quotient Productivity Time (hrs) I II I II 0 11.04 53.2 0.007 0.002 0.074 0.107 16.4 8.77 - 0.051 - 0.449 17.5 - 26.1 - 0.022 - 0.570 40.5 7.4 - 0.065 - 0.483 - 53.1 - 13.2 - 0.045 - 0.593 65.0 6.55 - 0.048 - 0.268 78.1 - 11.2 - 0.085 - 0.728 89.3 - 11.97 - 0.039 - 0.467 99.9 4.45 - 0.040 - 0.178 113.2. - 9.64 - 0.034 - 0.330 126.5 4.13 - 0.021 - 0.086 136.5 4.11 - 0.028 - 0.113 137.3 - 10.59 - 0.034 - 0.360 161.0 3.98 - 0.015 - 0.059 161.6 - 6.6 - 0.029 - 0.251 183.9 5.84 - 0.012 - 0.065 185.4 - 8.96 - 0.034 - 0.301 242.2 3.12 - 0.030 - 0.092 246.3 - 9.4 - 0.019 - 0.174 EXAMPLE II Regeneration of polysaccharide productivity Pseudomonas sp. NCIB 11592 was immobilised onto alumina (Norton 06519) following the procedure of Example B4, with the higher nutrient levels and a loading time of 76 hours. This immobilised cell system was then supplied at a dilution rate of 0.08hr-1 (being the flow of medium divided by the volume of liquid in the reactor) with nitrogen-free (but otherwise similar) medium to initiate a polysaccharide production phase, which was operated for 208 hours. By this time the polysaccharide product quotient had dropped to a low level, and the nitrogen-free medium was replaced by the complete, nitrogen-containing growth medium at a dilution rate of 0.4hr-1 for a period of 96 hours in order to regenerate the polysaccharide producing cell activity. After that regenerative phase, the production phase was resumed by reintroducing the nitrogen-free medium at a dilution rate of 0.08hr-1. The cell loadings and polysaccharide product quotlent were determined throughout this expariment and are set out below in Table 7. from which it will be apparent that the regeneration procedure effectively restores the polysaccharide productivity of the immqbilised system.

TABLE 7

Time (hrs) Cell Loading (mg/g) Product Quotient 0 35.0 0.001 16.25 20.4 0.010 39.43 14.0 0.034 74.34 9.17 0.030 96.2 9.19 0.016 111 8.6 0.02 135 8.9 0.011 159 6.98 0.007 183 6.8 0.005 208 6.8 0.010 240 21.7 262 38.2 Regeneration 281 36.9 Phase 302 35.7 331 /0 35.0 0.007 16.7 22.1 0.021 41.8 ; 16.97 0.046 77.4 14.5 0.063 100.8 15.3 0.043 114.8 12.64 0.049 142 13.9 0.039 EXAMPLE Ill To demoristrate the results obtained from an organism which requires growth to produce polymer, a paste of Xanthomonas campestris ATCC 13951 was loaded by growth in situ onto three different supports. Each system (10 g) was then placed into 100 ml of a 100 mM tris buffer at pH 7.4 containing 1% glucose and the polymer production determined using procedures similar to those of B(1) above.

The results are shown in the table below.

TABLE 8

Polymer concentration Support Material Cell Loading (mg/g) after 140 hr (gel'') Norton alumina, batch 06519 6.8 1.4 Norton silica/alumina, batch 06482 7.2 0.3 Fractosil 25000 (Merck, Darmstadt) 8.8 0.7 It will be seen that polymer is generated, although the rate is lower than that achieved with Pseudomonas sp. NCIB 11 592.

Comparative Example Alternative immobilisation techniques.

The following Examples show that the immobilisation techniques of this invention are necessary for efficient polysaccharide production, since alternative conventional techniques are unsuitable.

1) Immobilisation by covalent attachment.

Suitable supports were activated by the following coupling reagents: 1 -ethyl-3-(3t- dimethylaminopropyl)carbodiimide, glutaraldehyde, quinone and N-hydroxy succinimide. Activated supports were mixed with a cell paste of Pseudomonas sp. NCIB 11592 for several hours to effect immobilisation. In all cases there was either no significant binding of cells or the cells that did bind lost activity for polysaccharide synthesis.

2) Immobilisation by gel entrapment.

Techniques for the immobilisation of cells in gels are well documented in the literature. Cells of Pseudomonas sp. NCIB 11 592 were immóbilised by entrapment in the following gels: agar, polyacrylamide, calcium alginate. In all cases the cells were successfully immobilised and retained the ability to respire in the presence of glucose solution. However, no polymer was released into solution. It is expected that the pores in the gels are too small to allow the export of the polymer from the particles.

Claims (10)

1. Process for the production of a polysaccharide wherein a microorganism species which produces a polysaccharide is supported on a porous, particulate inert support. the pore size being greater than 0.5,am, to form an immobilised cell system, aqueous nutrient medium is passed through the immobilised system, and polysaccharide-containing medium is withdrawn from the system.

2. Process as claimed in claim 1, wherein the microorganism is a species which produces the polysaccharide in the stationary phase of the growth cycle.

3. Process as claimed in claim 2 wherein the microorganism is a slime-forming species of the genera Pseudomonas, Rhizobium, Alcaligenes or Agrobacterium.

4. Process as claimed in any one of the preceding claims wherein the support is an inorganic oxide.

5. Process as claimed in any one of the preceding claims wherein the pore size is from 2 to 30 1tom.

6. Process as claimed in any one of the preceding claims wherein the mean particle diameter is from 100 to 600 m.

7. Process as claimed in any one of the preceding claims, wherein the location of the microorganisms onto the support is effected by growing the microorganisms in the presence of the support and of a growth medium containing an assimilable source of both carbon and nitrogen together with essential minerals.

8. Process as claimed in any one of the preceding claims wherein the aqueous nutrient medium contains insufficient nitrogen to promote active growth, and the polysaccharide production stage is followed by a regenerative growth stage wherein the immobilised cell system is supplied with a complete growth medium containing assimilable sources of carbon and nitrogen, after which the growth medium is replaced by the nitrogen-limited nutrient medium to revert to the production stage.

9. Process as claimed in any one of the preceding claims. wherein the polysaccharide-containing medium, after any necessary Concentration adjustment '..4.;br incorporation of additional compounds. is injected into a fluid-bearing permeable subsurface formation.

10. An immobilised cell system suitable for use in the process of any one of the preceding claims, which comprises a polysaccharide-producing microorganism supported on a porous, particulate inert support having a pore size greater than 0.5 m.