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Some properties of polysaccharides of microorganisms from degraded straw
Some properties of polysaccharides of microorganisms from degraded straw S. J. C h a p m a n *
Agricultural and Food Research Council Letcombe Laboratory, Wantage, Oxon 0)(12 9JT, UK and J. M. L y n c h ~
Glasshouse Crops Research Institute, Worthing Road, Littlehampton, West Sussex BN17 6LP, UK (Received 23 August 1984)
Extracellular polysachcarides from bacteria and yeasts isolated from decomposed straw contained various proportions of D-galactose, D-glucose, D-mannose, uronic acid, D-xylose, L-fucose and L-rhamnose. Molecular weights of the polymers determined by viscometry and gel filtration were in the range 40 000-1 800 000. All the polysaccharides stabilized aggregates of volcanic ash and most were more effective than the polysaccharide from Lipomyces starkeyi. Effectiveness seemed to be more related to molecular weight than to chemical composition. Keywords: Polysaccharide; straw; soil; ash; Enterobacter cloacae; Pseudomonas fluorescens; Lipomyces starkeyi; Rhodotorula rubra ; Candida humieola
Introduction
Materials and m e t h o d s
Carbohydrates, and particularly microbial polysaccharides. are important in stabilizing soil aggregates. 1 Specific polysaccharides have been shown to increase aggregate stability in model systems but their effectiveness depends on their chemical composition and molecular structure as well as on the nature of the soil to be stabilized and the presence of other components, such as metal cations,z We have described previously how microbial polysaccharide is formed during the decomposition of wheat straw and its ability to increase the aggregate stability of Mount St Helens volcanic ash.3 The latter material has a particle size distribution equivalent to a silt loam but, as it contains no organic matter and therefore has no inherent structure, it provides a very useful model system to study the generation of structure. A particularly relevant measurement of soil structure is the stability of aggregates formed to disruption by shaking in water; this provides an index of the potential for water erosion. In a previous paper,4 the potential for microbial inoculation of straw to stimulate soil aggregation was examined but no advantage was found. We here describe the properties of six polysaccharides produced by bacteria and yeasts isolated from decomposed straw and their aggregating ability. For comparison, we have included the polysaccharide from Lipomyces starkeyi, which has been shown to be active in soil stabilization, s'6
Polysaccharide p r o d u c t i o n Polysaccharide-producing bacteria and yeasts were isolated on peptone dextrose agar from water-logged wheat straw degraded in Kilner jars or in glass columns similar to those described previously.7 Lipomyces starkeyi was originally isolated from a Welsh soil.8 Polysaccharide was obtained from L. starkeyi growing on a glucose-peptone medium as previously described, s Polysaccharide from the straw isolates was similarly obtained except that sodium acetate was not required to precipitate the polymer from alcoholic solution. Also, Enterobacter cloacae strain L1/14 was grown in peptone (3 g 1-1), malt extract (9 g 1-1), D-fructose (9g1-1) medium as capsule production in D-glucose-peptone medium was poor.
*Present address: Macaulay Institute for Soil Craigiebuckler, Aberdeen AB9 2QJ, Scotland. tAuthor to whom correspondence should be addressed. 0141 --0229/85/040161 --03 $03.00 © 1985 Butterworth & Co. (Publishers) Ltd
Research,
C h e m i c a l analysis Neutral sugars were estimated by gas chromatography.3 Uronic acid content was measured by the carbazole method 9 using sodium o-glucuronate as the standard and applying a correction for the interference by hexoses. 1° Physical m e a s u r e m e n t s The intrinsic viscosities of the polysaccharides were measured using a Cannon-Fenske viscometer (size 50) at 20°C with a range of polysaccharide concentrations (0.044g1-1) and distilled water as the standard. Molecular weights were estimated using the Staudinger relationship with a K m value of 5 x 10 -4 (ref. 11) after conversion of the intrinsic viscosities to primary mo1-1 . Molecular weights
Enzyme Microb. Technol., 1985, vol. 7, April
161
Papers
reagent. 11 The value will change with differences in primary and secondary structure.
were also determined by gel chromatography on Sephacryl S-500 eluted with distilled water a n d calibrated using Blue Dextran 2000 and dextrans grade A and C (BDH Biochernicals).
Effect o f aggregation All the polysaccharides produced an increase in the stability of aggregates of volcanic ash (Figure 1). The d o s e response curves showed 'saturation' at 0.05 or 0.1% (w/w) polysaccharide in four cases. E. cloacae C2/4 was most effective; C. humicola, L. starkeyi and E. cloacae L1/14 were the least effective. The mean stability index in the range 0.01 0.2% (w/w) gave a low but significant correlation with the intrinsic viscosity (r = 0.742, p < 0.05). The correlation is similar to that obtained by Geoghegan and Brian 11 for a set of levans and one dextran though the range of molecular weights was very much smaller than used here.
Aggregate stabilization The components of the Mount St Helens volcanic ash used have been described previously.4 Solutions of each polysaccharide at a range of concentrations were stirred with the volcanic ash using 1 ml solution to 4 g ash. This produced aggregates of ash which were air-dried and sieved (500/~m sieve) to remove non-aggregated material. Aggregate stability was measured as previously described. 3 Results
Chemical composition Table 1 shows the neutral sugar and uronic acid content
Discussion
of the polysaccharides from the straw isolates and from L. starkeyL The polysaccharide of Enterobacter cloacae C2/4 was shown to be colanic acid 12 but that of E. cloacae L1/14 did not appear to contain L-fucose. The composition of the L. starkeyi polysaccharide was similar to that reported for this species 13 with the exception that the strain used by us contains D-galactose.
Several mechanisms relating stabilization to composition have been proposed. The abudance of hydroxyl groups in polysaccharides allows for hydrogen-bond formation. Cis-hydroxy groups, such as occur in D-mannose and L-rhamnose, may also have potential for involvement in cationic bridges to the negatively charged clay surfaces. Uronic acids have greater potential for such links. 14 It has been suggested Is that entropy changes resulting from water desorption may be involved. Increasing the molecular weight of polysaccharides also increases the strength of adsorption and aggregate stabilization. 13,16 Despite differences in chemical composition the seven polysaccharides investigated were all similarly effective in aggregate stabilization. Five of the polysaccharides contained D-mannose and one also contained L-rhamnose. Four contained uronic acid but there appears to be no relation to aggregate stabilization. The P. fluorescens polysaccharide, which contained only 12% (w/w) o-mannose, was one of the more effective agents. Of more importance, as illustrated by the correlation with intrinsic viscosity, is
Molecular weight The relatively high intrinsic viscosities indicated that all the polysaccharides are linear, high molecular weight chains (Table 2). The two lowest values, E. cloacae L1/14 and Candida humicola, may have resulted from some degradation during extraction. Upon gel filtration the E. cloacae LI/14 polysaccharide showed a broad band of material while the C. humicola polysaccharide also contained 20% (w/w) low molecular weight ( < 4 0 000) material. Molecular weight estimates by the two methods were in agreement for two of the polysaccharides but differed for the remainder. This is probably due at least in part to inapplicability of the K m value used originally for cellulose in Schweitzer's
Table 1 Proportions (%) of neutral sugars and uronic acid (as D-glucuronic acid) in the extracellular polysaccharide of isolates from straw and Lipomyces starkeyi
L-Rhamnose
Isolate
Enterobacter cloacae (C2/4) E. cloacae ( L1/14) Pseudomonas sp.
L-Fucose
D-Xylose
D-Mannose
29.4 22.2
41.6 12.0 28.6 45.0 53.5
P. fluorescens Rhodotorula rubra Candida humicola Lipomyces starkeyi
55.0
D-Glucose
D-G alactose
Uronic acid
20.8 55.2 24.3 57.4 29.7
34.5 32.5 12.0 30.6 25.8
15.3 12.3
29.1
17.4
16.1
Table 2 Physical characteristics of extracellular polysaccharide of isolates from decomposed straw and Lipomyces starkeyi Molecular weight Isolate
Polysaccharide form
Intrinsic viscosity a (cm 3 g-l)
Enterobacter cloacae (C2/4) E. cloacae (L1/14) Pseudomona$ sp. P. fluorescens Rhodotorula rubra Candida humicola Lipomyces starkeyi
Capsule/slime Capsule Slime Capsule Capsule Capsule Capsule
3500 530 1600 4500 2800 610 1700
+ 240 (18) -+ 4 (14) -+ 60 (15) +- 70 (18) + 110 (18) +_26 (12) +- 140 (10)
a Results expressed as mean -+ standard error of the mean; number of observations in parentheses
162
E n z y m e M i c r o b . T e c h n o l . , 1985, vol. 7, A p r i l
Viscometry
Gel filtration
1 200 000 190000 590 000 1 600 000 1 000 000 200 000 610 000
1 100 000 <40000 1 800 000 1 400 000 1 800 000 1 700 000 1 800 000
Microbial polysaccharides from degraded straw: S. J. Chapman and J. M. Lynch 100
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96 polysaccharide in ash
Figure 1 Aggregate stabilization of Mount St Helens volcanic ash by extracellular polysaccharide from isolates of decomposed straw and from Lipomyces starkeyi. Control stability index of ash (no polysaccharide added) = 35.2%: (a) Enterobacter cloacae C2/4; (b) E. cloacae L1/14; (c) Pseudomonas sp.; (d) P. fluorescens; (el Rhodotorula rubra; (f) Candida humico/a; (g) Lipomyces starkeyii
the molecular weight. This probably reflects the ability to span gaps between clay particles. It is noteworthy that the polysaccharides from five of the straw isolates were superior as stabilizing agents to that from L. starkeyi, which has been shown to be effective in other softs,s'6 Most o f the neutral sugar components detected in the microbial polysaccharide formed during the decomposition o f wheat straw 3 are represented in the polysaccharides o f the straw isolates. It is likely that some o f these are responsible for the ash-stabilizing components o f this microbial polysaccharide. This may prove useful in the inoculation o f straw composts where the aim is to generate soil conditioning properties.
Acknowledgements We thank Mr S. H. T. Harper for supplying the straw isolates, Dr D. Jones for the L. starkeyi culture, the EEC for contract No. RUW-033-UK that gave financial support and the National Collection o f Yeast Cultures (Norwich) and the National Collection o f Industrial Bacteria (Aberdeen) for identification o f microorganisms.
References
1 Lynch, J. M. and Bragg, E. Adv. Soil ScL in press 2 Martin, J. P. Soil Biol. Biochem. 1971, 3, 33-41 3 Chapman, S. J. and Lynch, J. M. J. Appl. Bacteriol. 1984, 56,337-342 4 Lynch, J. M. and Elliott, L. F. Appl. Environ. Microbiol. 1983, 45, 1398-1401 5 Jones, D. and Griffiths, E. Plant Soil 1967, 27, 187-200 6 Lynch, J. M. J. Gen. Microbiol. 1981, 126,371-375 7 Lynch, J. M. and Harper, S. H. T. J. Gen. Mierobiol. 1983, 129, 251-253 8 Brady, B. L. and Jones, D. Trans. Br. Mycol. Soc. 1964, 47,293 9 Bitter, T. and Muir, H. M.Anal. Chem. 1962, 4, 330-334 10 Cheshire, M. Nature and Origin of Carbohydrates in Soils Academic Press, London, 1979 11 Geoghegan, M. J: and Brian, R. C. Biochem. J. 1948, 43, 5-13 12 Grant, W. D., Sutherland, I. W. and Wilkinson, J. F.J. Bacteriol. 1969, 100, 1187-1193 13 Slodki, M. E. and Wickerman, L. J.J. Gen. Microbiol. 1966, 42, 381-385 14 Martin, J. P. and Aldrich, D. G. Proc. Soil ScL Soc. Am. 1955, 19, 50-54 15 Parfitt, R. L. and Greenland, D. J. Proc. Soil ScL Soc. Am. 1970, 34,862-866 16 Greenland, D. J. Soils and Fert. 1965, 28,415-426
Enzyme Microb. Technol., 1985, vol. 7, April
163
Agricultural and Food Research Council Letcombe Laboratory, Wantage, Oxon 0)(12 9JT, UK and J. M. L y n c h ~
Glasshouse Crops Research Institute, Worthing Road, Littlehampton, West Sussex BN17 6LP, UK (Received 23 August 1984)
Extracellular polysachcarides from bacteria and yeasts isolated from decomposed straw contained various proportions of D-galactose, D-glucose, D-mannose, uronic acid, D-xylose, L-fucose and L-rhamnose. Molecular weights of the polymers determined by viscometry and gel filtration were in the range 40 000-1 800 000. All the polysaccharides stabilized aggregates of volcanic ash and most were more effective than the polysaccharide from Lipomyces starkeyi. Effectiveness seemed to be more related to molecular weight than to chemical composition. Keywords: Polysaccharide; straw; soil; ash; Enterobacter cloacae; Pseudomonas fluorescens; Lipomyces starkeyi; Rhodotorula rubra ; Candida humieola
Introduction
Materials and m e t h o d s
Carbohydrates, and particularly microbial polysaccharides. are important in stabilizing soil aggregates. 1 Specific polysaccharides have been shown to increase aggregate stability in model systems but their effectiveness depends on their chemical composition and molecular structure as well as on the nature of the soil to be stabilized and the presence of other components, such as metal cations,z We have described previously how microbial polysaccharide is formed during the decomposition of wheat straw and its ability to increase the aggregate stability of Mount St Helens volcanic ash.3 The latter material has a particle size distribution equivalent to a silt loam but, as it contains no organic matter and therefore has no inherent structure, it provides a very useful model system to study the generation of structure. A particularly relevant measurement of soil structure is the stability of aggregates formed to disruption by shaking in water; this provides an index of the potential for water erosion. In a previous paper,4 the potential for microbial inoculation of straw to stimulate soil aggregation was examined but no advantage was found. We here describe the properties of six polysaccharides produced by bacteria and yeasts isolated from decomposed straw and their aggregating ability. For comparison, we have included the polysaccharide from Lipomyces starkeyi, which has been shown to be active in soil stabilization, s'6
Polysaccharide p r o d u c t i o n Polysaccharide-producing bacteria and yeasts were isolated on peptone dextrose agar from water-logged wheat straw degraded in Kilner jars or in glass columns similar to those described previously.7 Lipomyces starkeyi was originally isolated from a Welsh soil.8 Polysaccharide was obtained from L. starkeyi growing on a glucose-peptone medium as previously described, s Polysaccharide from the straw isolates was similarly obtained except that sodium acetate was not required to precipitate the polymer from alcoholic solution. Also, Enterobacter cloacae strain L1/14 was grown in peptone (3 g 1-1), malt extract (9 g 1-1), D-fructose (9g1-1) medium as capsule production in D-glucose-peptone medium was poor.
*Present address: Macaulay Institute for Soil Craigiebuckler, Aberdeen AB9 2QJ, Scotland. tAuthor to whom correspondence should be addressed. 0141 --0229/85/040161 --03 $03.00 © 1985 Butterworth & Co. (Publishers) Ltd
Research,
C h e m i c a l analysis Neutral sugars were estimated by gas chromatography.3 Uronic acid content was measured by the carbazole method 9 using sodium o-glucuronate as the standard and applying a correction for the interference by hexoses. 1° Physical m e a s u r e m e n t s The intrinsic viscosities of the polysaccharides were measured using a Cannon-Fenske viscometer (size 50) at 20°C with a range of polysaccharide concentrations (0.044g1-1) and distilled water as the standard. Molecular weights were estimated using the Staudinger relationship with a K m value of 5 x 10 -4 (ref. 11) after conversion of the intrinsic viscosities to primary mo1-1 . Molecular weights
Enzyme Microb. Technol., 1985, vol. 7, April
161
Papers
reagent. 11 The value will change with differences in primary and secondary structure.
were also determined by gel chromatography on Sephacryl S-500 eluted with distilled water a n d calibrated using Blue Dextran 2000 and dextrans grade A and C (BDH Biochernicals).
Effect o f aggregation All the polysaccharides produced an increase in the stability of aggregates of volcanic ash (Figure 1). The d o s e response curves showed 'saturation' at 0.05 or 0.1% (w/w) polysaccharide in four cases. E. cloacae C2/4 was most effective; C. humicola, L. starkeyi and E. cloacae L1/14 were the least effective. The mean stability index in the range 0.01 0.2% (w/w) gave a low but significant correlation with the intrinsic viscosity (r = 0.742, p < 0.05). The correlation is similar to that obtained by Geoghegan and Brian 11 for a set of levans and one dextran though the range of molecular weights was very much smaller than used here.
Aggregate stabilization The components of the Mount St Helens volcanic ash used have been described previously.4 Solutions of each polysaccharide at a range of concentrations were stirred with the volcanic ash using 1 ml solution to 4 g ash. This produced aggregates of ash which were air-dried and sieved (500/~m sieve) to remove non-aggregated material. Aggregate stability was measured as previously described. 3 Results
Chemical composition Table 1 shows the neutral sugar and uronic acid content
Discussion
of the polysaccharides from the straw isolates and from L. starkeyL The polysaccharide of Enterobacter cloacae C2/4 was shown to be colanic acid 12 but that of E. cloacae L1/14 did not appear to contain L-fucose. The composition of the L. starkeyi polysaccharide was similar to that reported for this species 13 with the exception that the strain used by us contains D-galactose.
Several mechanisms relating stabilization to composition have been proposed. The abudance of hydroxyl groups in polysaccharides allows for hydrogen-bond formation. Cis-hydroxy groups, such as occur in D-mannose and L-rhamnose, may also have potential for involvement in cationic bridges to the negatively charged clay surfaces. Uronic acids have greater potential for such links. 14 It has been suggested Is that entropy changes resulting from water desorption may be involved. Increasing the molecular weight of polysaccharides also increases the strength of adsorption and aggregate stabilization. 13,16 Despite differences in chemical composition the seven polysaccharides investigated were all similarly effective in aggregate stabilization. Five of the polysaccharides contained D-mannose and one also contained L-rhamnose. Four contained uronic acid but there appears to be no relation to aggregate stabilization. The P. fluorescens polysaccharide, which contained only 12% (w/w) o-mannose, was one of the more effective agents. Of more importance, as illustrated by the correlation with intrinsic viscosity, is
Molecular weight The relatively high intrinsic viscosities indicated that all the polysaccharides are linear, high molecular weight chains (Table 2). The two lowest values, E. cloacae L1/14 and Candida humicola, may have resulted from some degradation during extraction. Upon gel filtration the E. cloacae LI/14 polysaccharide showed a broad band of material while the C. humicola polysaccharide also contained 20% (w/w) low molecular weight ( < 4 0 000) material. Molecular weight estimates by the two methods were in agreement for two of the polysaccharides but differed for the remainder. This is probably due at least in part to inapplicability of the K m value used originally for cellulose in Schweitzer's
Table 1 Proportions (%) of neutral sugars and uronic acid (as D-glucuronic acid) in the extracellular polysaccharide of isolates from straw and Lipomyces starkeyi
L-Rhamnose
Isolate
Enterobacter cloacae (C2/4) E. cloacae ( L1/14) Pseudomonas sp.
L-Fucose
D-Xylose
D-Mannose
29.4 22.2
41.6 12.0 28.6 45.0 53.5
P. fluorescens Rhodotorula rubra Candida humicola Lipomyces starkeyi
55.0
D-Glucose
D-G alactose
Uronic acid
20.8 55.2 24.3 57.4 29.7
34.5 32.5 12.0 30.6 25.8
15.3 12.3
29.1
17.4
16.1
Table 2 Physical characteristics of extracellular polysaccharide of isolates from decomposed straw and Lipomyces starkeyi Molecular weight Isolate
Polysaccharide form
Intrinsic viscosity a (cm 3 g-l)
Enterobacter cloacae (C2/4) E. cloacae (L1/14) Pseudomona$ sp. P. fluorescens Rhodotorula rubra Candida humicola Lipomyces starkeyi
Capsule/slime Capsule Slime Capsule Capsule Capsule Capsule
3500 530 1600 4500 2800 610 1700
+ 240 (18) -+ 4 (14) -+ 60 (15) +- 70 (18) + 110 (18) +_26 (12) +- 140 (10)
a Results expressed as mean -+ standard error of the mean; number of observations in parentheses
162
E n z y m e M i c r o b . T e c h n o l . , 1985, vol. 7, A p r i l
Viscometry
Gel filtration
1 200 000 190000 590 000 1 600 000 1 000 000 200 000 610 000
1 100 000 <40000 1 800 000 1 400 000 1 800 000 1 700 000 1 800 000
Microbial polysaccharides from degraded straw: S. J. Chapman and J. M. Lynch 100
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96 polysaccharide in ash
Figure 1 Aggregate stabilization of Mount St Helens volcanic ash by extracellular polysaccharide from isolates of decomposed straw and from Lipomyces starkeyi. Control stability index of ash (no polysaccharide added) = 35.2%: (a) Enterobacter cloacae C2/4; (b) E. cloacae L1/14; (c) Pseudomonas sp.; (d) P. fluorescens; (el Rhodotorula rubra; (f) Candida humico/a; (g) Lipomyces starkeyii
the molecular weight. This probably reflects the ability to span gaps between clay particles. It is noteworthy that the polysaccharides from five of the straw isolates were superior as stabilizing agents to that from L. starkeyi, which has been shown to be effective in other softs,s'6 Most o f the neutral sugar components detected in the microbial polysaccharide formed during the decomposition o f wheat straw 3 are represented in the polysaccharides o f the straw isolates. It is likely that some o f these are responsible for the ash-stabilizing components o f this microbial polysaccharide. This may prove useful in the inoculation o f straw composts where the aim is to generate soil conditioning properties.
Acknowledgements We thank Mr S. H. T. Harper for supplying the straw isolates, Dr D. Jones for the L. starkeyi culture, the EEC for contract No. RUW-033-UK that gave financial support and the National Collection o f Yeast Cultures (Norwich) and the National Collection o f Industrial Bacteria (Aberdeen) for identification o f microorganisms.
References
1 Lynch, J. M. and Bragg, E. Adv. Soil ScL in press 2 Martin, J. P. Soil Biol. Biochem. 1971, 3, 33-41 3 Chapman, S. J. and Lynch, J. M. J. Appl. Bacteriol. 1984, 56,337-342 4 Lynch, J. M. and Elliott, L. F. Appl. Environ. Microbiol. 1983, 45, 1398-1401 5 Jones, D. and Griffiths, E. Plant Soil 1967, 27, 187-200 6 Lynch, J. M. J. Gen. Microbiol. 1981, 126,371-375 7 Lynch, J. M. and Harper, S. H. T. J. Gen. Mierobiol. 1983, 129, 251-253 8 Brady, B. L. and Jones, D. Trans. Br. Mycol. Soc. 1964, 47,293 9 Bitter, T. and Muir, H. M.Anal. Chem. 1962, 4, 330-334 10 Cheshire, M. Nature and Origin of Carbohydrates in Soils Academic Press, London, 1979 11 Geoghegan, M. J: and Brian, R. C. Biochem. J. 1948, 43, 5-13 12 Grant, W. D., Sutherland, I. W. and Wilkinson, J. F.J. Bacteriol. 1969, 100, 1187-1193 13 Slodki, M. E. and Wickerman, L. J.J. Gen. Microbiol. 1966, 42, 381-385 14 Martin, J. P. and Aldrich, D. G. Proc. Soil ScL Soc. Am. 1955, 19, 50-54 15 Parfitt, R. L. and Greenland, D. J. Proc. Soil ScL Soc. Am. 1970, 34,862-866 16 Greenland, D. J. Soils and Fert. 1965, 28,415-426
Enzyme Microb. Technol., 1985, vol. 7, April
163