Journal of Applied Phycology 9: 249253, 1997.

c 1997 Kluwer Academic Publishers. Printed in Belgium.

249

Exopolysaccharide of Nostoc muscorum (Cyanobacteria) in the aggregation of soil particles

G. Zulpa de Caire1; , M. Storni de Cano1 , M. C. Zaccaro de Mul 1 , R. M. Palma2 & e K. Colombo1
1

Departamento de Ciencias Biol gicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires o (UBA), Intendente G iraldes 2620 (1428) Buenos Aires, Argentina u 2 C tedra de Edafologa, Facultad de Agronoma, Universidad de Buenos Aires (UBA). Av. San Martn 4453 a (1417) Buenos Aires, Argentina ( Author for correspondence; phone/fax + 54-1-782-0620 or 782-0582)
Received 11 November 1996; revised 23 May 1997; accepted 30 May 1997

Key words: Nostoc muscorum, cyanobacteria, exopolysaccharide, soil aggregation, soil inoculation

Abstract The effects on a saline-sodic soil of exopolysaccaride isolated from Nostoc muscorum or the addition of a cyanobacterial mass proliferation were evaluated in a greenhouse experiment. By day 180 the exopolysaccharide increased soluble C by 100%, microbial activity by 366% and the amount of water-stable aggregates larger than 250 m by 12 times. Inoculation with living cyanobacterial mass increased at the end of 365 days oxidizable C by 11%, soluble C by 66%, microbial activity by 73% and aggregates larger than 250 m by 66%. A slimy lm 35 mm thick, with N. muscorum predominating, covered all the surface of inoculated soils. The higher soil aggregate stability produced by both treatments is a consequence of increased microbial activity and concentrating the soil polysaccharide. The high percentage of clays favours the creation of rm and long-lasting slime-mineral joints. Addition of isolated exopolysaccharide produces a faster and higher increase in soil aggregate stability than cyanobacterial mass inoculation. Introduction Studies on the mechanisms for soil aggregation leave many gaps in knowledge. There is a mechanical component, since laments or rhizoids form a network around soil particles, and a chemical component due to extracellular polysaccharides or polypeptides adhering to particles. Cyanobacteria that form a sheath or a slime with exopolysaccharides around their cells are suitable for the latter process (Schulten, 1985). Studies on extracellular polysaccharides from cyanobacteria have dealt largely with chemical composition (e.g Drews & Weckesser, 1982; Nakagawa et al., 1987; Panoff et al., 1988) and ultrastructural descriptions of extracellular layers formed by the accumulation of various types of polymeric substances around cell walls and their protective role (Potts, 1994). Sudo et al. (1995) isolated a cyanobacterium that produces large quantities of exopolysaccharide and studied the inuence of culture medium, particurlarly its NaCI concentration, on production of the exopolysaccharide. Only a few studies have been made on the effects of cyanobacterial mass inoculation on soil aggregation. Oscillatoria prolica and Nostoc commune increased water stability of aggregates when they were grown separately on Peoria loess soil (Bailey et al., 1973). The effects of N. muscorum mass on soil physical, chemical and biological properties indicates the possible benets of cyanobacteria as soil inoculants (Roger & Burns, 1994). The increase in soil aggregate stability is probably due to the extracellular substances produced by N. muscorum, mainly to the exopolysaccharide (Cano et al., 1997). However, there is apparently no literature on the effects of isolated N. muscorum exopolysaccharide on soil aggregates stability.

250 The aim of this research was to establish whether the addition to soil of exopolysaccharide isolated from medium in which N. muscorum had grown exerts an effect comparable to that produced by inoculation and proliferation of N. muscorum biomass. The moisture content was kept constant by gravimetry during the experiment. Analytical determinations on soil samples were carried out at the initial stage of the research (= day 0) and then repeated after a year (day 365). Macro- and microscopic observations of soil samples were made after 365 days in order to study the ora. Treatment 2. Inoculation with exoplysaccharide: 20 boxes were prepared as in treatment 1. Ten were sown with 20 mL exopolysaccharide preparation and the rest were kept as controls. The boxes were kept in the dark for 180 days with constant moisture content throughout the experiment as in treatment 1. Analytical determinations of soil samples were carried out at the initial stage of the research and then repeated after 180 days (day 180). Soil analytical determinations A saline sodic soil Typic Natralboll (United States Department of Agriculture, 1994) with clay silt loam texture from Pila, Province of Buenos Aires, was collected from 015 cm depth. Oxidizable carbon was studied with Walkley and Black technique. The Davidson et al. (1987) technique was used for soluble carbon determinations; this fraction quanties the carbon available for microorganisms. Microbial activity was studied by arginine ammonication (Alef & Kleiner, 1987). Soil aggregate stability, dened as the resistance of aggregates to degradation during wetting and physical disruption, was assessed by wet sifting, in accordance with the method developed by Grieve (1979). Wet samples were placed at the top of a nested set of sieves, the uppermost one having a 2000-m mesh (size 10), topping ones with 1000-m mesh (size 18), 500-m mesh (size 35) and 250-m mesh (size 60). The sieves were submerged in water and mechanically lifted 8 cm per revolution by a 15 revolution min 1 motor during 20 min. The soil sample in each sieve was dried at 105 C for 24 h and then weighed. The Bouyucos technique (1962) without the addition of dispersant agent, was used to evaluate the percentages of 50 m and 2 m aggregates. All soil samples were assayed in triplicate. Statistical methodology: Analysis of variance was performed for all data, using the completely randomised experimental design. The homogeneity of the mean squares of the experimental error was assessed by the Bartlett test. Duncans test was used for the comparison of means, at level of signicance of 0.05 Statistics PC program (Steel & Torrie, 1985).

Material and methods Growth of biomass Nostoc muscorum Ag. was isolated from mud (pH 6.7) between plants in rice elds in Argentina, and held in our culture collection. It was chosen for its growth on saline soil after tests with a range of species cultured in our laboratory. Strain No. 50 was made axenic by irradiation with UV light (F68132-A germicide lamp 253.7 nm, General Electric, USA) and shows no obvious morphological changes as a result of this treatment. It was cultured on Allen and Stanier (1968) modied culture medium (without sodium nitrate) in 2-L Erlenmeyer asks and incubated under uorescent light 45 mol photon m 2 s 1 (LI-COR, inc. Quantum Radiometer Photometer, mod LI-185B USA). Cyanobacterial mass (exponential phase) was separated by centrifugation and about 12 g L 1 fresh weight biomass was obtained. Isolation of slime material (exopolysaccharide) Exopolysaccharide was isolated from the medium (stationary phase culture) by the Nakagawa et al. (1987) method in a ratio fresh weight: volume of 30 g L 1 . The nal solution (pH 7.2) was concentrated approximately 5-fold by evaporation at 35 C. Design of experiment In order to establish the effect of biomass and exopolysaccharide on the structure, oxidizable and soluble carbon contents and microbial activity of the soil, treatments 1 and 2 were carried out. Treatment 1. Inoculation with biomass: non-sterile soil samples were dried, sieved through a 2 mmpore diameter sieve and nally saturated with distilled water; 160 g of soil were dispensed in each of 20 plastic boxes (13  8  5) cm. The boxes were placed under 45 mol photon m 2 s 1 irradiance, 12-h photoperiod, and kept at 25  1 C; 10 boxes containing the soil sample were sown with 6 g fresh weight of cyanobacterial mass. The remaining 10 boxes were kept as controls.

251

Figure 1. Percentage of aggregates in soil after 365 days of cyanobacterial mass proliferation. Values represent mean (n=3) Biomass of Nostoc muscorum, B control

Figure 2. Percentage of aggregates in soil after 180 days of exopolysaccharide addition. Values represent mean (n=3) exopolysaccharide of Nostoc muscorum, B control

Soil analytical determinations The inuence of N. muscorum biomass and exopolysaccharide isolated from culture medium on oxidizable C, soluble C and microbial activity of the soil is shown in Table 1. After 365 days of N. muscorum proliferation there was a signicant increase of the studied variables. Inoculation with exopolysaccharide increased the microbial activity after 180 days. Exopolysaccharide produced a 100% rise in soluble C directly available for the microora, whose activity increased about 3.5-fold. Changes in the quantity of different size aggregates due to the presence of N. muscorum biomass and exopolysaccharide are given in Figures 1 and 2. After 365 days, the soil inoculated with biomass showed an increase of 66% in water-stable aggregates larger than 250 m and a decrease of 91% of aggregates smaller than 2 m (Figure 1). Exopolysaccharide produced aggregates larger than 250 m 12 times the control value and a decrease of 85% in aggregates smaller than 2 m (Figure 2). In order to attribute the effect on soil aggregation entirely

Results Macro and microscopic observation of soil samples after 365 days of N. muscorum proliferation A lm 35 mm thick, with N. muscorum predominating, covered all the surface in boxes where this organism had been sown. There were many soil particles adhered to the lm due to the glueing properties of the slime material produced by the cyanobacterium. Cyanobacteria such as Chroococcus sp., Aphanothece stagnina and several Oscillatoriaceae were also present. Bacteria were visually abundant, but diatoms and fungi less so; bryophytes were scarce. In control boxes, the lm also covered all the surface, but was only 2 mm thick; there were abundant bryophytes, some diatoms and fungal hyphae, fewer bacteria and plant residues. The cyanobacteria included Chroococcus sp., Oscillatoria sp. and Phormidium sp..

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Table 1. Effect of Nostoc muscorum biomass and exopolysaccharide inoculation on oxidable C, soluble C and microbial activity of soil. Values represent mean (n=3)  S.D. Different letters show signicant statistical differences, p<0.05. Parameters Day 0 Control Oxidizable C (%) Soluble C (mg C 1000 g Microbiological activity (g NH4 -N g 1 h 1 ) 1.84  0.07 18.55  2.44 3.78  0.29 Biomass Day 365 Control 1.85  0.05b 30.90  2.76b 4.28  0.90b Exopolysaccharide Day 180 Control Treated soil 20.45  1.68a 3.48  0.51b 40.89  3.85a 16.25  1.66a

Treated soil 2.05  0.10a 50.92  4.06a 7.40  1.29a

1)

to the added exopolysaccharide, experiments were carried out in the dark to avoid growth of the indigenous photoautotrophic ora.

Discussion The strain of Nostoc muscorum introduced into a saline-sodic soil exerted similar glueing properties as observed previously (Halperin, 1969; Shulten, 1985) for cyanobacterial slime material formed by indigenous cyanobacterial microora. After 365 days of inoculation with N. muscorum the increase in oxidizable C was at similar level to that after 180 days (Cano et al., 1997). This increase of 11% is due to the survival and proliferation of biomass, although part of it has undergone death and cell lysis. Exopolysaccharide increases the soil organic matter content as a consequence of the sugar derived from the abundant slime mainly secreted by N. muscorum in addition to the polymers produced by other microorganisms in the soil, as shown by the controls. The percentage of soluble C rise due to biomass proliferation decreased with time: 80% for 180 days (Cano et al., 1997) and 66% for 365 days. This decrease can be due to the original microora restoring the community balance altered by the massive inoculation of N. muscorum. However the control value after 365 days produced by the indigenous microbial population (30.9 mg C kg1 ) did not match the value of the inoculated soil after 180 days (36.6 mg C kg1 : Cano et al., 1997), conrming the positive inuence of N. muscorum. The higher soluble C in the soil resulting from biomass or exopolysaccharide indicated higher microbial activity, veried by arginine ammonication. Van Gestel et al. (1996) state that clay particles and organic colloids ensure microbial growth and survival in soils by their capacity to buffer the nutrient supply to microorganisms closely associated with their surfaces; they

determine in this way the spatial distribution of microbial biomass in the soil structure. The amendment with N. muscorum mass or exopolysaccharides produced by it showed effects that support this opinion. Our results agree with those of Kandeler and Murer (1993), who in eld experiments relate higher stability of soil aggregates with larger microbial biomass and/or its by-products due to its enzymatic activity, which mineralise high molecar weight compounds. Field experiments (Shulten, 1985) have shown a similar inuence due to naturally occurring cyanobacteria. Organic soil inoculants such as green manure, farmyard manure or peat (Gerzabek et al., 1995) also increased soil aggregate stability in a similar way to N. muscorum. Addition of exopolysaccharide produces a faster and higher increase in the quantity of aggregates >250m either by direct glueing of the particles or by increasing activity of the microora, which in turn produces more exopolysaccharide, with the resulting amplied effect. However, cyanobacteria proliferating in soil have a long-term inuence, eventually requiring only sporadic reinforcements, whereas exopolysaccharide needs to be added more frequently. When exopolysaccharide was used, microbial activity increased 3.5-fold due to the immediate availability of carbohydrates for their heterotrophic growth; this increase in microbial activity promotes a consequent rise in the aggregation of soil particles.

Acknowledgements The authors thank Dr J. Wright for supervision of the translation.

253 References
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