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Journal of Applied Phycology 9: 249–253, 1997.

249
c 1997 Kluwer Academic Publishers. Printed in Belgium.

Exopolysaccharide of Nostoc muscorum (Cyanobacteria) in the aggregation


of soil particles

G. Zulpa de Caire1; , M. Storni de Cano1 , M. C. Zaccaro de Mulé1 , R. M. Palma2 &


K. Colombo1
1
Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires
(UBA), Intendente Güiraldes 2620 (1428) Buenos Aires, Argentina
2
Cátedra de Edafologı́a, Facultad de Agronomı́a, Universidad de Buenos Aires (UBA). Av. San Martı́n 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 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 film 3–5 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 polysac-
charide. The high percentage of clays favours the creation of firm 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 ties of exopolysaccharide and studied the influence of


culture medium, particurlarly its NaCI concentration,
Studies on the mechanisms for soil aggregation leave on production of the exopolysaccharide.
many gaps in knowledge. There is a mechanical com- Only a few studies have been made on the effects
ponent, since filaments or rhizoids form a network of cyanobacterial mass inoculation on soil aggregation.
around soil particles, and a chemical component due to Oscillatoria prolifica and Nostoc commune increased
extracellular polysaccharides or polypeptides adhering water stability of aggregates when they were grown
to particles. Cyanobacteria that form a sheath or a slime separately on Peoria loess soil (Bailey et al., 1973). The
with exopolysaccharides around their cells are suit- effects of N. muscorum mass on soil physical, chemical
able for the latter process (Schulten, 1985). Studies on and biological properties indicates the possible bene-
extracellular polysaccharides from cyanobacteria have fits of cyanobacteria as soil inoculants (Roger & Burns,
dealt largely with chemical composition (e.g Drews 1994). The increase in soil aggregate stability is prob-
& Weckesser, 1982; Nakagawa et al., 1987; Panoff ably due to the extracellular substances produced by
et al., 1988) and ultrastructural descriptions of extra- N. muscorum, mainly to the exopolysaccharide (Cano
cellular layers formed by the accumulation of various et al., 1997). However, there is apparently no literature
types of polymeric substances around cell walls and on the effects of isolated N. muscorum exopolysaccha-
their protective role (Potts, 1994). Sudo et al. (1995) ride on soil aggregates stability.
isolated a cyanobacterium that produces large quanti-

*142689
Article: japh487 GSB: Pips nr 142689 BIO2KAP
japh487.tex; 22/09/1997; 11:47; v.7; p.1
250

The aim of this research was to establish whether The moisture content was kept constant by gravimetry
the addition to soil of exopolysaccharide isolated from during the experiment.
medium in which N. muscorum had grown exerts an Analytical determinations on soil samples were car-
effect comparable to that produced by inoculation and ried out at the initial stage of the research (= day 0)
proliferation of N. muscorum biomass. 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 flora.
Material and methods Treatment 2. Inoculation with exoplysaccharide:
20 boxes were prepared as in treatment 1. Ten were
Growth of biomass sown with 20 mL exopolysaccharide preparation and
the rest were kept as controls. The boxes were kept in
Nostoc muscorum Ag. was isolated from mud (pH the dark for 180 days with constant moisture content
6.7) between plants in rice fields in Argentina, and throughout the experiment as in treatment 1. Analytical
held in our culture collection. It was chosen for its determinations of soil samples were carried out at the
growth on saline soil after tests with a range of species initial stage of the research and then repeated after 180
cultured in our laboratory. Strain No. 50 was made days (day 180).
axenic by irradiation with UV light (F68132-A germi-
cide lamp 253.7 nm, General Electric, USA) and shows Soil analytical determinations
no obvious morphological changes as a result of this
treatment. It was cultured on Allen and Stanier (1968) A saline sodic soil Typic Natralboll (United States
modified culture medium (without sodium nitrate) in Department of Agriculture, 1994) with clay silt loam
2-L Erlenmeyer flasks and incubated under fluorescent texture from Pila, Province of Buenos Aires, was col-
light 45 mol photon m 2 s 1 (LI-COR, inc. Quantum lected from 0–15 cm depth. Oxidizable carbon was
Radiometer Photometer, mod LI-185B USA). studied with Walkley and Black technique. The David-
Cyanobacterial mass (exponential phase) was sepa- son et al. (1987) technique was used for soluble car-
rated by centrifugation and about 12 g L 1 fresh weight bon determinations; this fraction quantifies the carbon
biomass was obtained. available for microorganisms. Microbial activity was
studied by arginine ammonification (Alef & Kleiner,
Isolation of slime material (exopolysaccharide) 1987).
Soil aggregate stability, defined as the resistance of
Exopolysaccharide was isolated from the medium (sta- aggregates to degradation during wetting and physical
tionary phase culture) by the Nakagawa et al. (1987) disruption, was assessed by wet sifting, in accordance
method in a ratio fresh weight: volume of 30 g L 1 . with the method developed by Grieve (1979). Wet sam-
The final solution (pH 7.2) was concentrated approxi- ples were placed at the top of a nested set of sieves, the
mately 5-fold by evaporation at 35 C. uppermost one having a 2000-m mesh (size 10), top-
ping ones with 1000-m mesh (size 18), 500-m mesh
Design of experiment (size 35) and 250-m mesh (size 60). The sieves were
submerged in water and mechanically lifted 8 cm per
In order to establish the effect of biomass and revolution by a 15 revolution min 1 motor during 20
exopolysaccharide on the structure, oxidizable and sol- min. The soil sample in each sieve was dried at 105 C
uble carbon contents and microbial activity of the soil, for 24 h and then weighed. The Bouyucos technique
treatments 1 and 2 were carried out. (1962) without the addition of dispersant agent, was
Treatment 1. Inoculation with biomass: non-sterile used to evaluate the percentages of 50 m and 2 m
soil samples were dried, sieved through a 2 mm- aggregates. All soil samples were assayed in triplicate.
pore diameter sieve and finally saturated with distilled Statistical methodology: Analysis of variance was
water; 160 g of soil were dispensed in each of 20 plastic performed for all data, using the completely ran-
boxes (13  8  5) cm. The boxes were placed under domised experimental design. The homogeneity of the
45 mol photon m 2 s 1 irradiance, 12-h photoperiod, mean squares of the experimental error was assessed
and kept at 25  1 C; 10 boxes containing the soil sam- by the Bartlett test. Duncan’s test was used for the
ple were sown with 6 g fresh weight of cyanobacterial comparison of means, at level of significance of 0.05
mass. The remaining 10 boxes were kept as controls. ‘Statistics’ PC program (Steel & Torrie, 1985).

japh487.tex; 22/09/1997; 11:47; v.7; p.2


251

Figure 1. Percentage of aggregates in soil after 365 days of cyanobac- Figure 2. Percentage of aggregates in soil after 180 days of
terial mass proliferation. Values represent mean (n=3) – – Biomass exopolysaccharide addition. Values represent mean (n=3) – –
of Nostoc muscorum, –B – control exopolysaccharide of Nostoc muscorum, –B – control

Soil analytical determinations

The influence of N. muscorum biomass and


Results exopolysaccharide isolated from culture medium on
oxidizable C, soluble C and microbial activity of the
Macro and microscopic observation of soil samples soil is shown in Table 1. After 365 days of N. mus-
after 365 days of N. muscorum proliferation corum proliferation there was a significant increase of
the studied variables. Inoculation with exopolysaccha-
A film 3–5 mm thick, with N. muscorum predomi- ride increased the microbial activity after 180 days.
nating, covered all the surface in boxes where this Exopolysaccharide produced a 100% rise in soluble
organism had been sown. There were many soil parti- C directly available for the microflora, whose activ-
cles adhered to the film due to the glueing properties ity increased about 3.5-fold. Changes in the quan-
of the slime material produced by the cyanobacteri- tity of different size aggregates due to the presence
um. Cyanobacteria such as Chroococcus sp., Aphan- of N. muscorum biomass and exopolysaccharide are
othece stagnina and several Oscillatoriaceae were also given in Figures 1 and 2. After 365 days, the soil
present. Bacteria were visually abundant, but diatoms inoculated with biomass showed an increase of 66%
and fungi less so; bryophytes were scarce. In con- in water-stable aggregates larger than 250 m and a
trol boxes, the film also covered all the surface, but decrease of 91% of aggregates smaller than 2 m (Fig-
was only 2 mm thick; there were abundant bryophytes, ure 1). Exopolysaccharide produced aggregates larger
some diatoms and fungal hyphae, fewer bacteria and than 250 m 12 times the control value and a decrease
plant residues. The cyanobacteria included Chroococ- of 85% in aggregates smaller than 2 m (Figure 2). In
cus sp., Oscillatoria sp. and Phormidium sp.. order to attribute the effect on soil aggregation entirely

japh487.tex; 22/09/1997; 11:47; v.7; p.3


252
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 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 1 h 1 )

to the added exopolysaccharide, experiments were car- determine in this way the spatial distribution of micro-
ried out in the dark to avoid growth of the indigenous bial biomass in the soil structure. The amendment with
photoautotrophic flora. N. muscorum mass or exopolysaccharides produced
by it showed effects that support this opinion. Our
results agree with those of Kandeler and Murer (1993),
Discussion who in field experiments relate higher stability of soil
aggregates with larger microbial biomass – and/or its
The strain of Nostoc muscorum introduced into a by-products – due to its enzymatic activity, which min-
saline-sodic soil exerted similar glueing properties as eralise high molecar weight compounds. Field experi-
observed previously (Halperin, 1969; Shulten, 1985) ments (Shulten, 1985) have shown a similar influence
for cyanobacterial slime material formed by indige- due to naturally occurring cyanobacteria. Organic soil
nous cyanobacterial microflora. After 365 days of inoc- inoculants such as green manure, farmyard manure or
ulation with N. muscorum the increase in oxidizable C peat (Gerzabek et al., 1995) also increased soil aggre-
was at similar level to that after 180 days (Cano et al., gate stability in a similar way to N. muscorum.
1997). This increase of 11% is due to the survival and Addition of exopolysaccharide produces a faster
proliferation of biomass, although part of it has under- and higher increase in the quantity of aggregates
gone death and cell lysis. Exopolysaccharide increases >250m either by direct glueing of the particles or
the soil organic matter content as a consequence of the by increasing activity of the microflora, which in turn
sugar derived from the abundant slime mainly secreted produces more exopolysaccharide, with the result-
by N. muscorum in addition to the polymers produced ing amplified effect. However, cyanobacteria prolif-
by other microorganisms in the soil, as shown by the erating in soil have a long-term influence, eventual-
controls. ly requiring only sporadic reinforcements, whereas
The percentage of soluble C rise due to biomass exopolysaccharide needs to be added more frequently.
proliferation decreased with time: 80% for 180 days When exopolysaccharide was used, microbial activi-
(Cano et al., 1997) and 66% for 365 days. This decrease ty increased 3.5-fold due to the immediate availability
can be due to the original microflora restoring the com- of carbohydrates for their heterotrophic growth; this
munity balance altered by the massive inoculation of N. increase in microbial activity promotes a consequent
muscorum. However the control value after 365 days rise in the aggregation of soil particles.
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), Acknowledgements
confirming the positive influence of N. muscorum.
The higher soluble C in the soil resulting from bio- The authors thank Dr J. Wright for supervision of the
mass or exopolysaccharide indicated higher microbial translation.
activity, 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 by
their capacity to buffer the nutrient supply to microor-
ganisms closely associated with their surfaces; they

japh487.tex; 22/09/1997; 11:47; v.7; p.4


253

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