Journal of Applied Phycology 9: 249–253, 1997. 249c 1997 Kluwer Academic Publishers. Printed in Belgium.

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

G. Zulpa de Caire1;�, M. Storni de Cano1, M. C. Zaccaro de Mule1, R. M. Palma2 &K. Colombo1

1 Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires(UBA), Intendente Guiraldes 2620 (1428) Buenos Aires, Argentina2 Catedra de Edafologıa, Facultad de Agronomıa, Universidad de Buenos Aires (UBA). Av. San Martın 4453(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 cyanobac-terial mass proliferation were evaluated in a greenhouse experiment. By day 180 the exopolysaccharide increasedsoluble C by 100%, microbial activity by 366% and the amount of water-stable aggregates larger than 250�m by 12times. Inoculation with living cyanobacterial mass increased at the end of 365 days oxidizable C by 11%, solubleC by 66%, microbial activity by 73% and aggregates larger than 250 �m by 66%. A slimy film 3–5 mm thick,with N. muscorum predominating, covered all the surface of inoculated soils. The higher soil aggregate stabilityproduced by both treatments is a consequence of increased microbial activity and concentrating the soil polysac-charide. The high percentage of clays favours the creation of firm and long-lasting slime-mineral joints. Additionof isolated exopolysaccharide produces a faster and higher increase in soil aggregate stability than cyanobacterialmass inoculation.

Introduction

Studies on the mechanisms for soil aggregation leavemany gaps in knowledge. There is a mechanical com-ponent, since filaments or rhizoids form a networkaround soil particles, and a chemical component due toextracellular polysaccharides or polypeptides adheringto particles. Cyanobacteria that form a sheath or a slimewith exopolysaccharides around their cells are suit-able for the latter process (Schulten, 1985). Studies onextracellular polysaccharides from cyanobacteria havedealt largely with chemical composition (e.g Drews& Weckesser, 1982; Nakagawa et al., 1987; Panoffet al., 1988) and ultrastructural descriptions of extra-cellular layers formed by the accumulation of varioustypes of polymeric substances around cell walls andtheir protective role (Potts, 1994). Sudo et al. (1995)isolated a cyanobacterium that produces large quanti-

ties of exopolysaccharide and studied the influence ofculture medium, particurlarly its NaCI concentration,on production of the exopolysaccharide.

Only a few studies have been made on the effectsof cyanobacterial mass inoculation on soil aggregation.Oscillatoria prolifica and Nostoc commune increasedwater stability of aggregates when they were grownseparately on Peoria loess soil (Bailey et al., 1973). Theeffects of N. muscorum mass on soil physical, chemicaland biological properties indicates the possible bene-fits of cyanobacteria as soil inoculants (Roger & Burns,1994). The increase in soil aggregate stability is prob-ably due to the extracellular substances produced byN. muscorum, mainly to the exopolysaccharide (Canoet al., 1997). However, there is apparently no literatureon the effects of isolated N. muscorum exopolysaccha-ride on soil aggregates stability.

Article: japh487 GSB: Pips nr 142689 BIO2KAP

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The aim of this research was to establish whetherthe addition to soil of exopolysaccharide isolated frommedium in which N. muscorum had grown exerts aneffect comparable to that produced by inoculation andproliferation of N. muscorum biomass.

Material and methods

Growth of biomass

Nostoc muscorum Ag. was isolated from mud (pH6.7) between plants in rice fields in Argentina, andheld in our culture collection. It was chosen for itsgrowth on saline soil after tests with a range of speciescultured in our laboratory. Strain No. 50 was madeaxenic by irradiation with UV light (F68132-A germi-cide lamp 253.7 nm,General Electric, USA) and showsno obvious morphological changes as a result of thistreatment. It was cultured on Allen and Stanier (1968)modified culture medium (without sodium nitrate) in2-L Erlenmeyer flasks and incubated under fluorescentlight 45 �mol photon m�2s�1(LI-COR, inc. QuantumRadiometer Photometer, mod LI-185B USA).

Cyanobacterial mass (exponential phase) was sepa-rated by centrifugation and about 12 g L�1 fresh weightbiomass was obtained.

Isolation of slime material (exopolysaccharide)

Exopolysaccharide was isolated from the medium (sta-tionary phase culture) by the Nakagawa et al. (1987)method in a ratio fresh weight: volume of 30 g L�1.The final solution (pH 7.2) was concentrated approxi-mately 5-fold by evaporation at 35� C.

Design of experiment

In order to establish the effect of biomass andexopolysaccharideon the structure, oxidizable and sol-uble carbon contents and microbial activity of the soil,treatments 1 and 2 were carried out.

Treatment 1. Inoculation with biomass: non-sterilesoil samples were dried, sieved through a 2 mm-pore diameter sieve and finally saturated with distilledwater; 160 g of soil were dispensed in each of 20 plasticboxes (13 � 8 � 5) cm. The boxes were placed under45 �mol photon m�2s�1 irradiance, 12-h photoperiod,and kept at 25� 1�C; 10 boxes containing the soil sam-ple were sown with 6 g fresh weight of cyanobacterialmass. The remaining 10 boxes were kept as controls.

The moisture content was kept constant by gravimetryduring the experiment.

Analytical determinations on soil samples were car-ried out at the initial stage of the research (= day 0)and then repeated after a year (day 365). Macro- andmicroscopic observations of soil samples were madeafter 365 days in order to study the flora.

Treatment 2. Inoculation with exoplysaccharide:20 boxes were prepared as in treatment 1. Ten weresown with 20 mL exopolysaccharide preparation andthe rest were kept as controls. The boxes were kept inthe dark for 180 days with constant moisture contentthroughout the experiment as in treatment 1. Analyticaldeterminations of soil samples were carried out at theinitial stage of the research and then repeated after 180days (day 180).

Soil analytical determinations

A saline sodic soil Typic Natralboll (United StatesDepartment of Agriculture, 1994) with clay silt loamtexture from Pila, Province of Buenos Aires, was col-lected from 0–15 cm depth. Oxidizable carbon wasstudied with Walkley and Black technique. The David-son et al. (1987) technique was used for soluble car-bon determinations; this fraction quantifies the carbonavailable for microorganisms. Microbial activity wasstudied by arginine ammonification (Alef & Kleiner,1987).

Soil aggregate stability, defined as the resistance ofaggregates to degradation during wetting and physicaldisruption, was assessed by wet sifting, in accordancewith the method developed by Grieve (1979). Wet sam-ples were placed at the top of a nested set of sieves, theuppermost one having a 2000-�m mesh (size 10), top-ping ones with 1000-�m mesh (size 18), 500-�m mesh(size 35) and 250-�m mesh (size 60). The sieves weresubmerged in water and mechanically lifted 8 cm perrevolution by a 15 revolution min�1 motor during 20min. The soil sample in each sieve was dried at 105� Cfor 24 h and then weighed. The Bouyucos technique(1962) without the addition of dispersant agent, wasused to evaluate the percentages of 50 �m and 2 �maggregates. All soil samples were assayed in triplicate.

Statistical methodology: Analysis of variance wasperformed for all data, using the completely ran-domised experimental design. The homogeneity of themean squares of the experimental error was assessedby the Bartlett test. Duncan’s test was used for thecomparison of means, at level of significance of 0.05‘Statistics’ PC program (Steel & Torrie, 1985).

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Figure 1. Percentage of aggregates in soil after 365 days of cyanobac-terial mass proliferation. Values represent mean (n=3) – – Biomassof Nostoc muscorum, –B – control

Results

Macro and microscopic observation of soil samplesafter 365 days of N. muscorum proliferation

A film 3–5 mm thick, with N. muscorum predomi-nating, covered all the surface in boxes where thisorganism had been sown. There were many soil parti-cles adhered to the film due to the glueing propertiesof the slime material produced by the cyanobacteri-um. Cyanobacteria such as Chroococcus sp., Aphan-othece stagnina and several Oscillatoriaceae were alsopresent. Bacteria were visually abundant, but diatomsand fungi less so; bryophytes were scarce. In con-trol boxes, the film also covered all the surface, butwas only 2 mm thick; there were abundant bryophytes,some diatoms and fungal hyphae, fewer bacteria andplant residues. The cyanobacteria included Chroococ-cus sp., Oscillatoria sp. and Phormidium sp..

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

Soil analytical determinations

The influence of N. muscorum biomass andexopolysaccharide isolated from culture medium onoxidizable C, soluble C and microbial activity of thesoil is shown in Table 1. After 365 days of N. mus-corum proliferation there was a significant increase ofthe studied variables. Inoculation with exopolysaccha-ride increased the microbial activity after 180 days.Exopolysaccharide produced a 100% rise in solubleC directly available for the microflora, whose activ-ity increased about 3.5-fold. Changes in the quan-tity of different size aggregates due to the presenceof N. muscorum biomass and exopolysaccharide aregiven in Figures 1 and 2. After 365 days, the soilinoculated with biomass showed an increase of 66%in water-stable aggregates larger than 250 �m and adecrease of 91% of aggregates smaller than 2 �m (Fig-ure 1). Exopolysaccharide produced aggregates largerthan 250 �m 12 times the control value and a decreaseof 85% in aggregates smaller than 2 �m (Figure 2). Inorder to attribute the effect on soil aggregation entirely

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Table 1. Effect of Nostoc muscorum biomass and exopolysaccharide inoculation on oxidable C, soluble C and microbialactivity of soil. Values represent mean (n=3) � S.D. Different letters show significant statistical differences, p<0.05.

Parameters Biomass Exopolysaccharide

Day 0 Day 365 Day 180

Control Control Treated soil Control Treated soil

Oxidizable C (%) 1.84 � 0.07 1.85 � 0.05b 2.05 � 0.10a – –

Soluble C (mg C 1000 g�1) 18.55 � 2.44 30.90 � 2.76b 50.92 � 4.06a 20.45 � 1.68a 40.89 � 3.85a

Microbiological activity 3.78 � 0.29 4.28 � 0.90b 7.40 � 1.29a 3.48 � 0.51b 16.25 � 1.66a

(�g NH4-N g�1h�1)

to the added exopolysaccharide, experiments were car-ried out in the dark to avoid growth of the indigenousphotoautotrophic flora.

Discussion

The strain of Nostoc muscorum introduced into asaline-sodic soil exerted similar glueing properties asobserved previously (Halperin, 1969; Shulten, 1985)for cyanobacterial slime material formed by indige-nous cyanobacterial microflora.After 365 days of inoc-ulation with N. muscorum the increase in oxidizable Cwas at similar level to that after 180 days (Cano et al.,1997). This increase of 11% is due to the survival andproliferation of biomass, although part of it has under-gone death and cell lysis. Exopolysaccharide increasesthe soil organic matter content as a consequence of thesugar derived from the abundant slime mainly secretedby N. muscorum in addition to the polymers producedby other microorganisms in the soil, as shown by thecontrols.

The percentage of soluble C rise due to biomassproliferation decreased with time: 80% for 180 days(Cano et al., 1997) and 66% for 365 days.This decreasecan be due to the original microflora restoring the com-munity balance altered by the massive inoculation of N.muscorum. However the control value after 365 daysproduced by the indigenous microbial population (30.9mg C kg1) did not match the value of the inoculatedsoil after 180 days (36.6 mg C kg1: Cano et al., 1997),confirming the positive influence of N. muscorum.

The higher soluble C in the soil resulting from bio-mass or exopolysaccharide indicated higher microbialactivity, verified by arginine ammonification. Van Ges-tel et al. (1996) state that clay particles and organic col-loids ensure microbial growth and survival in soils bytheir capacity to buffer the nutrient supply to microor-ganisms closely associated with their surfaces; they

determine in this way the spatial distribution of micro-bial biomass in the soil structure. The amendment withN. muscorum mass or exopolysaccharides producedby it showed effects that support this opinion. Ourresults agree with those of Kandeler and Murer (1993),who in field experiments relate higher stability of soilaggregates with larger microbial biomass – and/or itsby-products – due to its enzymatic activity, which min-eralise high molecar weight compounds. Field experi-ments (Shulten, 1985) have shown a similar influencedue to naturally occurring cyanobacteria. Organic soilinoculants such as green manure, farmyard manure orpeat (Gerzabek et al., 1995) also increased soil aggre-gate stability in a similar way to N. muscorum.

Addition of exopolysaccharide produces a fasterand higher increase in the quantity of aggregates>250�m either by direct glueing of the particles orby increasing activity of the microflora, which in turnproduces more exopolysaccharide, with the result-ing amplified effect. However, cyanobacteria prolif-erating in soil have a long-term influence, eventual-ly requiring only sporadic reinforcements, whereasexopolysaccharide needs to be added more frequently.When exopolysaccharide was used, microbial activi-ty increased 3.5-fold due to the immediate availabilityof carbohydrates for their heterotrophic growth; thisincrease in microbial activity promotes a consequentrise in the aggregation of soil particles.

Acknowledgements

The authors thank Dr J. Wright for supervision of thetranslation.

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