PS-7A New Bacterial Heteropolysaccharide

17 P S - 7 — A N e w Bacterial Heteropolysaccharide

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K. S. KANG and W. H. McNEELY Kelco, Division of Merck & Co., Inc., 8225 Aero Drive, San Diego, CA 92123

Dextran p r o d u c t i o n by f e r m e n t a t i o n o r by c e l l f r e e s y n t h e s i s has l o n g been known ( 1 ) . However, p o l y s a c c h a r i d e fermentation o f other types i s a r e l a t i v e l y new f i e l d . The P e o r i a L a b o r a t o r y o f t h e USDA, which c a r r i e d o u t t h e i n i t i a l m i c r o b i a l r e s e a r c h on xanthan gum, a l s o has done development work on p o l y s a c c h a r i d e p r o d u c t i o n by v a r i o u s A r t h r o b a c t e r and yeast s t r a i n s (2). Dextran, xanthan gum, p o l y t r a n , and ZANFLO r e p r e s e n t m i c r o b i a l p o l y s a c c h a r i d e s which a r e now commercially a v a i l a b l e . The most n o t a b l e f e r m e n t a t i o n p o l y s a c c h a r i d e w i t h t h e g r e a t e s t commercial s u c c e s s a t t h i s time i s xanthan gum. T h i s paper i s i n t e n d e d t o r e p o r t on t h e v a r i o u s properties i n relation to possible industrial applic a t i o n s o f a n o v e l p o l y s a c c h a r i d e , PS-7. The m i c r o b i o l o g i c a l and f e r m e n t a t i o n a l a s p e c t s o f t h i s p o l y s a c c h a r i d e are described only b r i e f l y . A preliminary report o f PS-7 as a p o t e n t i a l f o o d a d d i t i v e has been made (3). PS-7 i s produced by a s o i l b a c t e r i u m which was i s o l a t e d from a l o c a l s o i l sample. T h i s organism was i d e n t i f i e d as a s t r a i n o f A z o t o b a c t e r i n d i c u s on t h e b a s i s o f growth c h a r a c t e r i s t i c s , b i o c h e m i c a l and morphological properties. PS-7 i s an e x t r a c e l l u l a r p o l y s a c c h a r i d e produced by means o f an a e r o b i c , submerged f e r m e n t a t i o n . The t y p i c a l f e r m e n t a t i o n medium i s i l l u s t r a t e d i n T a b l e I . The c o n c e n t r a t i o n o f p o t a s s i u m phosphate may be reduced to as low as 0.01% i f t h e pH has been m a i n t a i n e d d u r i n g the f e r m e n t a t i o n , o r d i n a r i l y by u s i n g KOH. By employing an a u t o m a t i c a g i t a t i o n - c o n t r o l l i n g system w i t h an oxygen probe, t h e minimum d i s s o l v e d oxygen c o n c e n t r a t i o n was determined t o be 5-10% d u r i n g the f i r s t 20-25 hours o f t h e f e r m e n t a t i o n . During t h i s f e r m e n t a t i o n time w i t h c o n s t a n t a e r a t i o n r a t e o f 0.25 220

Sandford and Laskin; Extracellular Microbial Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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l i t e r p e r l i t e r o f f e r m e n t a t i o n medium p e r minute, the t i p speed f o r a g i t a t i o n v a r i e d between 60 and 110 meters per minute t o m a i n t a i n the D.O. o f 10%. The v i s c o s i t y o f the f e r m e n t a t i o n l i q u o r s t e a d i l y i n c r e a s e d , and, a t 24 h o u r s , the v i s c o s i t y was a p p r o x i m a t e l y 2000 c p s , as measured by a B r o o k f i e l d v i s c o m e t e r at a r o t a t i o n speed o f 60 rpm and ambient temperature. PS-7 may be r e c o v e r e d from the f e r m e n t a t i o n l i q u o r by a l c o h o l s such as methanol, e t h a n o l and i s o p r o p a n o l , etc., o r by lower a l k y l ketones such as a c e t o n e . The p r e f e r r e d s o l v e n t f o r r e c o v e r y purposes i s IPA. After p r e c i p i t a t i o n , the polymer f i b e r s a r e d r i e d and m i l l e d t o o b t a i n a p a l e , c r e a m - c o l o r e d powder. The c h e m i c a l component a n a l y s e s by paper chroma tography and g a s - l i q u i d chromatography were c a r r i e d out and the r e s u l t s a r e shown i n T a b l e I I . The r e s u l t s i n d i c a t e t h a t PS-7 c o n s i s t s o f g l u c o s e , rhamnose and a u r o n i c a c i d i n an approximate r a t i o o f 6.6:1.5:1.0. I t has an a c e t y l c o n t e n t o f about 8.0-10.0%. Neither the u r o n i c a c i d nor the l i n k a g e s p r e s e n t i n t h i s polymer have been e l u c i d a t e d a t t h i s t i m e . PS-7 has an u n u s u a l l y h i g h v i s c o s i t y , as demon s t r a t e d i n F i g u r e 1. The v i s c o s i t y o f PS-7 i s much g r e a t e r than t h a t o f xanthan gum, w i t h the d i f f e r e n c e becoming s i g n i f i c a n t a t c o n c e n t r a t i o n s as low as 0.15%. T h i s p o i n t i s f u r t h e r i l l u s t r a t e d i n F i g u r e 2. The f e r m e n t a t i o n l i q u o r i n the f l a s k becomes so v i s c o u s at the end o f the f e r m e n t a t i o n time t h a t t h e r e i s l i t t l e f l o w , even i f t h e f l a s k i s h e l d u p s i d e down. F i g u r e 3 i l l u s t r a t e s another important p r o p e r t y of any commercial p o l y s a c c h a r i d e , and t h a t i s i t s v i s c o s i t y response t o temperature. T h i s d a t a shows t h a t PS-7 i s s i m i l a r t o xanthan gum i n t h a t i t s v i s c p s i t y i s s t a b l e over a wide temperature range. The v i s c o s i t y response o f PS-7 t o changes i n pH i s shown i n F i g u r e 4. The v i s c o s i t y o f PS-7 i s almost as s t a b l e as xanthan gum, a l t h o u g h the v i s c o s i t y s t a r t s t o decrease below pH 3 and beyond pH 12. Another i m p o r t a n t c h a r a c t e r i s t i c f o r some a p p l i c a t i o n s i s p s e u d o p l a s t i c i t y . P s e u d o p l a s t i c i t y i s indicated when the v i s c o s i t y d e c r e a s e s as the shear r a t e i s i n creased. T h i s p r o p e r t y o f PS-7 i s d e p i c t e d i n Figure 5. T h i s v i s c o g r a m was o b t a i n e d by u s i n g the Fann v i s c o meter Model 35. I t s h o u l d be noted t h a t t h e c o n c e n t r a t i o n o f PS-7 i s o n l y h a l f the c o n c e n t r a t i o n o f xanthan gum o r a q u a r t e r o f o t h e r polymers such as CMC, HEC, and guar. There i s a remarkable d e c r e a s e i n the v i s c o s i t y of PS-7 as i t i s s h e a r e d , t h e magnitude w e l l e x c e e d i n g t h a t o f xanthan gum. F i g u r e 6 compares v i s c o s i t y and p s e u d o p l a s t i c i t y o f PS-7 t o xanthan gum a t the same


222

EXTRACELLULAR MICROBIAL POLYSACCHARIDES Table I

TYPICAL FERMENTATION MEDIUM THE INGREDIENTS

K2HPO4 MGSOZI

CARBOHYDRATE COMPOSITION

5.0 GRAMS

7 H2SO4

0.1

GRAMS

NH14NO3

0.9

GRAMS

PR0M0S0Y

0.5

GRAMS

GLUCOSE

CHEMICAL COMPONENTS OF PS-7

CONCENTRATION

30.0

URONIC ACID

11%

GLUCOSE

73

GRAMS RHAMN0SE

TAP

WATER

TO 1 LITER

16 100%

ACETYL


9%

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M C N E E L Y

H eteropoly saccharide

Figure i

2,000

S ι,οοο h

TEMPERATURE C O Figure 3.

The effect of heat on the viscosity of PS-7 and xanthan gum solutions


224

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

XANTHAN GUN I

ι

I

ι

I

ι

0

2

H

6

8

10

ι 12

PH Figure 4.

The effect of pH on PS-7 and xanthan gum solutions

S h e a r Rate, r p m

Xanthan G u m 1.0lb/bbl

1

10

100

S h e a r Rate, s e c

1000

- 1

Figure 5


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polymer c o n c e n t r a t i o n . Here, the d i f f e r e n c e s i n b o t h v i s c o s i t y and p s e u d o p l a s t i c i t y between PS-7 and xanthan gum a r e more o u t s t a n d i n g t h a n i n F i g u r e 5. PS-7 i s f u l l y s o l u b l e hot o r c o l d i n d i s t i l l e d water, t a p water, b r i n e water o r sea w a t e r . T h i s f a c t i s i l l u s t r a t e d i n T a b l e I I I . The sea water has a s a l t c o n c e n t r a t i o n o f a p p r o x i m a t e l y 3.5%, and the Permian B r i n e water o f West Texas has a s a l t c o n t e n t o f approxi m a t e l y 26%. The l o w e s t v i s c o s i t y was o b t a i n e d i n d i s t i l l e d water. P o l y s a c c h a r i d e PS-7 i s a l s o c o m p a t i b l e w i t h a wide v a r i e t y of s a l t s . Examples o f t h i s c o m p a t i b i l i t y a r e shown i n T a b l e IV. These c o n c e n t r a t i o n s a r e not n e c e s s a r i l y the l i m i t s f o r PS-7 c o m p a t i b i l i t y . The s t a b i l i t y o f t h e s e s o l u t i o n s was checked a f t e r t h r e e hours s t i r r i n g and 24 hours s t a n d i n g , l o o k i n g f o r p r e c i p i t a t i o n , g e l a t i o n o r changes i n f l o w p r o p e r t i e s . A l l s o l u t i o n s l i s t e d i n t h i s s l i d e were s t a b l e . The s t a b i l i t y o f PS-7 was a l s o s t u d i e d o v e r a one-month p e r i o d i n many o f t h e s e s a l t s , and t h i s d a t a i s shown i n T a b l e V. A d i s t i l l e d water c o n t r o l was carried. As a p r e s e r v a t i v e , formaldehyde was added t o the s o l u t i o n s a t the c o n c e n t r a t i o n o f 200 ppm. The d a t a i n d i c a t e s t h a t PS-7 was s t a b l e i n a l l c a s e s . P o l y s a c c h a r i d e PS-7 i s i n c o m p a t i b l e w i t h c a t i o n i c o r p o l y v a l e n t i o n s a t h i g h pH. This incompatiblity r e s u l t s i n a g e l . S o l u t i o n s o f PS-7 a l s o e x h i b i t some tendency t o g e l i n the p r e s e n c e o f h i g h concentrations o f monovalent s a l t s above a pH o f 10. While t h i s g e l a t i o n i s c o n s i d e r e d an i n c o m p a t i b i l i t y , i t can a l s o be a d e s i r a b l e e f f e c t , as w i l l be shown l a t e r . Solut i o n s o f PS-7 show l i m i t e d s t o r a g e s t a b i l i t y under c o n d i t i o n s o f s t r o n g a c i d i t y or a l k a l i n i t y . The p r o p e r t i e s i l l u s t r a t e d i n t h e p r e v i o u s s l i d e s demonstrate many c h a r a c t e r i s t i c s o f PS-7 which make i t a h i g h l y u s e f u l agent i n o i l w e l l d r i l l i n g muds. D r i l l i n g muds are g e n e r a l l y aqueous f l u i d s which cont a i n s u b s t a n t i a l q u a n t i t i e s o f c l a y s and o t h e r c o l l o i d a l materials. An optimum d r i l l i n g f l u i d would be one which, f i r s t l y , i s f l e x i b l e i n i t s v i s c o s i t y c h a r a c t e r i s t i c s so as t o p r o v i d e s u s p e n s i o n o f s o l i d s w i t h i n the f l u i d , and, s e c o n d l y , would l u b r i c a t e the d r i l l b i t . The h i g h v i s c o s i t y , p s e u d o p l a s t i c i t y and r e l a t i v e i n s e n s i t i v i t y o f the v i s c o s i t y t o temperature i n d i c a t e t h a t the use o f PS-7 i n a d r i l l i n g mud would come c l o s e t o the optimum c h a r a c t e r i s t i c s d i s c u s s e d e a r l i e r . A v e r y s i m p l e , easy and r a p i d way t o t e s t the s u s p e n d i n g a b i l i t y o f a f l u i d was d e v e l o p e d i n our l a b o r a t o r y (4) . The method employs a s t a n d a r d American P e t r o l e u m I n s t i t u t e s a n d - c o n t e n t t u b e . The tube i s



Table I I I

VISCOSITY OF PS-7 IN VARIOUS WATERS OF A 0.5% CONCENTRATION WATER

VISCOSITY

DISTILLED

690 CPS

TAP WATER

820 CPS

SEA WATER

860 CPS

PERMIAN BRINE

720 CPS

(CPS)


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A N D M C N E E L Y


Table IV Final Salt Concn.

Salt

(40%) (25%) (30%) (65%) (22%) (32%) (55%) (36%) (44%) (30%) (7%) (25%) (26%) (16%) (40%) (10%) (1 5%) (20%) (40%) (40%) (37%)

Aluminum Nitrate Aluminum Sulfate Ammonium Chloride Ammonium Nitrate Ammonium Sulfate Calcium Chloride Calcium Nitrate Magnesium Chloride Magnesium Nitrate Magnesium Sulfate Potassium Ferricyanide Potassium Ferrocyanide Sodium Chloride Sodium Dichromate Sodium Nitrate Sodium Phosphate (dibasic) Sodium Sulfate Sodium Sulfite Sodium Thiosnlfate

Table V

S0II1TI0N S T A B I L I T Y OF P S - 7

1%

PS-7

SOLUTION WITH

VISCOSITY ( C P S ) INITIAL AGED 1 MO,

D I S T I L L E D WATER (CONTROL)

1700

1900

SODIUM CHLORIDE ( 1 5 % )

3150

3450

CALCIUM CHLORIDE ( 1 5 % )

3050

3250

ALUMINUM SULFATE ( 1 5 % )

3000

3100

ZINC SULFATE ( 1 5 % )

3100

3200

AMMONIUM CHLORIDE ( 2 3 % )

2850

3100

CUPRIC CHLORIDE ( 1 3 % )

2950

2850

FERROUS SULFATE ( 1 3 % )

3100

2650

MONOSODIUM PHOSPHATE ( 1 3 % )

3050

3550

ZINC CHLORIDE ( 1 3 % )

3000

3350


227

228


100

200

300

400

500

600

Settling Time, sec Figure 7


700

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f i l l e d w i t h t h e f l u i d t o be t e s t e d . Then,1 gram o f 20-35 mesh sand i s p l a c e d i n t o t h e t u b e . The tube i s shaken v i g o r o u s l y t o t h o r o u g h l y d i s p e r s e t h e sand, p l a c e d u p r i g h t , and t i m i n g i s begun. The time t h a t i t t a k e s f o r t h e sand t o r e a c h s e l e c t e d g r a d u a t e d marks a t t h e bottom o f t h e tube i s r e c o r d e d . Figure 7 i s a plot of the s e t t l i n g rates of p a r t i c u l a t e matter i n the d i f f e r e n t f l u i d s . The d a t a i n d i c a t e t h a t PS-7 was by f a r t h e most e f f i c i e n t i n suspending p a r t i c l e s . Guar gum was i n t e r m e d i a t e , and the c e l l u l o s i c s the l e a s t e f f e c t i v e . Figure 8 i s a plot of e f f e c t i v e s e t t l i n g rate vs. concentration f o r the f l u i d s . Here, a g a i n , PS-7 was t h e most e f f e c t i v e , and i t t a k e s approximately h a l f t h e c o n c e n t r a t i o n o f xanthan gum t o produce comparable suspending e f f e c t . D r i l l i n g f l u i d s a r e t y p i c a l l y p r e p a r e d w i t h whate v e r water i s a v a i l a b l e nearby. The good s o l u b i l i t y o f PS-7 makes i t s u i t a b l e t o t h i s a p p l i c a t i o n — e i t h e r the s e a water f o r o f f - s h o r e w e l l s o r f r e s h o r b r i n e water from water w e l l s a t t h e d r i l l i n g s i t e , w h i c h e v e r may be a v a i l a b l e . I n some d r i l l i n g f l u i d s , i t i s des i r a b l e to g e l the f l u i d . P o l y s a c c h a r i d e PS-7 i s a l s o u s e f u l i n t h i s r e s p e c t , as i n c r e a s i n g t h e pH t o 11 causes a d r i l l i n g f l u i d c o n t a i n i n g PS-7 and chromium to g e l . The v i s c o s i t y o f t h e d r i l l i n g f l u i d c a n be a d j u s t e d t o t h e d e s i r e d l e v e l by s i m p l y a l t e r i n g t h e pH and t h e c o n c e n t r a t i o n o f t h e c r o s s - l i n k i n g a g e n t . B e s i d e s t h e o i l w e l l d r i l l i n g a p p l i c a t i o n s , we have e s t a b l i s h e d t h e p o t e n t i a l use o f PS-7 i n o t h e r a p p l i c a t i o n s such as d r i p l e s s water-base l a t e x p a i n t , w a t e r f l o o d i n g systems f o r secondary o i l r e c o v e r y , w a l l j o i n t cement a d h e s i v e s and t e x t i l e p r i n t i n g .

Abstract PS-7 is ananionic heteropolysaccharide produced by a strain of Azotobacter indicus in an aerobic fermentation. PS-7 is composed of glucose, rhamnose, and uronic acid in an approximate weight ratio of 6.6:1.5:1. The polysaccharide has an acetyl content of about 9%. Solutions of PS-7 are characterized by high viscosity and a high degree of pseudoplasticity. The polysaccharide has excellent solubility in sea water and even in brine containing 25% salt. PS-7 exhibits excellent temperature and pH stability and is compatible with a variety of salts. In the presence of Cr , PS-7 gum can be cross-linked at pH 9.0-9.5. These properties indicate that PS-7 w i l l find wide u t i l i t y in a +++


230


variety of applications. Literature Cited (1)

Jeans, Allene, "Dextrans and Pullulans," Extracellular Microbial Polysaccharides of Practical Importance, ACS Symposium series (1976).

(2)

McNeely, William H. and Kang, Kenneth S., "Xanthan and Some Other Biosynthetic Gums," Industrial Gums (2nd Ed.), p. 473, Academic Press, New York (1973).

(3)

Kang, Kenneth S. and Kovacs, Peter, "New Microbial Polysaccharides as Potential Food Additives," IVth International Congress of Food S c i . and Technol., Madrid, Spain (1974).

(4)

Carico, Robert D . , "New Field Test Improves Fluid-Suspension Measurements," O i l and Gas Journal, (1976) 74, (27), p. 81.


PS-7A New Bacterial Heteropolysaccharide

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