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

Studies on the mechanisms for soil aggregation leave
many gaps in knowledge. There is a mechanical com-
ponent, since filaments 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 suit-
able 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 extra-
cellular 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 quanti-
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 prolifica 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 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 by
N. muscorum, mainly to the exopolysaccharide (Cano
et al., 1997). However, there is apparently no literature
on the effects of isolated N. muscorum exopolysaccha-
ride on soil aggregates stability.

*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 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.
Material and methods
Growth of biomass
Nostoc muscorum Ag. was isolated from mud (pH
6.7) between plants in rice fields 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 germi-
cide 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)
modified culture medium (without sodium nitrate) in
2-L Erlenmeyer flasks and incubated under fluorescent
light 45 mol photon m 2 s 1 (LI-COR, inc. Quantum
Radiometer Photometer, mod LI-185B USA).
Cyanobacterial mass (exponential phase) was sepa-
rated by centrifugation and about 12 g L 1 fresh weight
biomass 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 and
exopolysaccharide on 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-sterile
soil samples were dried, sieved through a 2 mm-
pore diameter sieve and finally 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 sam-
ple were sown with 6 g fresh weight of cyanobacterial
mass. The remaining 10 boxes were kept as controls.
The moisture content was kept constant by gravimetry
during 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- and
microscopic observations of soil samples were made
after 365 days in order to study the flora.
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 col-
lected from 0–15 cm depth. Oxidizable carbon was
studied with Walkley and Black technique. The David-
son et al. (1987) technique was used for soluble car-
bon determinations; this fraction quantifies the carbon
available for microorganisms. Microbial activity was
studied by arginine ammonification (Alef & Kleiner,
1987).
Soil aggregate stability, defined 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 sam-
ples were placed at the top of a nested set of sieves, the
uppermost 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 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 ran-
domised experimental design. The homogeneity of the
mean squares of the experimental error was assessed
by the Bartlett test. Duncan’s test was used for the
comparison of means, at level of significance of 0.05
‘Statistics’ PC program (Steel & Torrie, 1985).
japh487.tex; 22/09/1997; 11:47; v.7; p.2

Figure 1. Percentage of aggregates in soil after 365 days of cyanobac-
terial mass proliferation. Values represent mean (n=3) – – Biomass
of Nostoc muscorum, –B – control
Results
Macro and microscopic observation of soil samples
after 365 days of N. muscorum proliferation
A film 3–5 mm thick, with N. muscorum predomi-
nating, covered all the surface in boxes where this
organism had been sown. There were many soil parti-
cles adhered to the film due to the glueing properties
of the slime material produced by the cyanobacteri-
um. Cyanobacteria such as Chroococcus sp., Aphan-
othece stagnina and several Oscillatoriaceae were also
present. Bacteria were visually abundant, but diatoms
and fungi less so; bryophytes were scarce. In con-
trol boxes, the film 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 Chroococ-
cus sp., Oscillatoria sp. and Phormidium sp..
251
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 influence 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. mus-
corum proliferation there was a significant increase of
the studied variables. Inoculation with exopolysaccha-
ride increased the microbial activity after 180 days.
Exopolysaccharide produced a 100% rise in soluble
C 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 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 (Fig-
ure 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
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
Day 0 Day 365
Control Control
Oxidizable C (%) 1.84  0.07 1.85  0.05b
Soluble C (mg C 1000 g 1) 18.55  2.44 30.90  2.76b
Microbiological activity 3.78  0.29 4.28  0.90b
(g NH4 -N g 1 h 1 )
to the added exopolysaccharide, experiments were car-
ried out in the dark to avoid growth of the indigenous
photoautotrophic flora.
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 indige-
nous cyanobacterial microflora. After 365 days of inoc-
ulation 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 under-
gone 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 microflora restoring the com-
munity 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),
confirming the positive influence of N. muscorum.
The higher soluble C in the soil resulting from bio-
mass or exopolysaccharide indicated higher microbial
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
Exopolysaccharide
Day 180
Treated soil Control Treated soil
2.05  0.10a – –
50.92  4.06a 20.45  1.68a 40.89  3.85a
7.40  1.29a 3.48  0.51b 16.25  1.66a
determine in this way the spatial distribution of micro-
bial 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 field experiments relate higher stability of soil
aggregates with larger microbial biomass – and/or its
by-products – due to its enzymatic activity, which min-
eralise high molecar weight compounds. Field experi-
ments (Shulten, 1985) have shown a similar influence
due to naturally occurring cyanobacteria. Organic soil
inoculants such as green manure, farmyard manure or
peat (Gerzabek et al., 1995) also increased soil aggre-
gate 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 microflora, which in turn
produces 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, whereas
exopolysaccharide needs to be added more frequently.
When exopolysaccharide was used, microbial activi-
ty 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.
japh487.tex; 22/09/1997; 11:47; v.7; p.4

References
Alef K, Kleiner D (1987) Applicability of arginine ammonification
as indicator of microbial activity in different soils. Biol. Fert.
Soils 5: 148–151.
Allen MM, Stanier RY (1968) Growth and division of some unicel-
lular blue-green algae. J. gen Microbiol 51: 199–202.
Bailey D, Mazurak AP, Rosowski JR (1973) Aggregation of soil
particles by algae. J. Phycol. 9: 99–101.
Barclay WR, Lewin RA (1985) Microalgal polysaccharide produc-
tion for the conditioning of agricultural soils. Plant and Soil 88:
159–169.
Bouyucos GJ (1962) Hydrometer method improved for making part
the size analysis of soils. Agron. J. 54: 464–465.
Cano MS de, Mulé MCZ de, Caire GZ de, Palma RM, Colombo
K (1997) Aggregation of soil particles by Nostoc muscorum Ag.
(Cyanobacteria). Phyton 60 (1/2): 35–40.
Davidson EA, Galloway LF, Strand MK (1987) Assessing available
carbon: comparison of techniques across selected forest soils.
Comm. Soil Sci. Plant Anal. 18: 45–64.
Drews G, Weckesser J (1982) Function, structure and composition
of cell walls and external layers. In Carr NG, Whitton BA (eds)
The Biology of Cyanobacteria. Blackwell, Oxford. 688 pp., pp.
333–357.
Gerzabek MH, Kirchmann H, Pichlmayer F (1995) Response of soil
aggregate stability to manure amendments in the Ultuna long-
term soil organic matter experiment. Pflanzenernähr. Bodenk,
158: 257–260.
Grieve IC (1979) Soil aggregate stability test for the geomorphlogist.
Br. Geomorph. Res. Gr. 25: 1–28.
Halperin DR de (1969) Biodermas algales y su papel en la consoli-
dación de los agregados del suelo. Algal crusts and their role in
soil aggregates consolidation. Physis 29: 37–48.
253
Kandeler E, Murer E (1993) Aggregate stability and soil microbial
processes in a soil with different cultivation. Geoderma 56: 503–
513.
Nakagawa M, Takamura Y, Yagi O (1987) Isolation and characteriza-
tion of the slime from a cyanobacterium, Microcystis aeruginosa
K-3A Agric. biol. Chem. 51: 329–337.
Panoff JM, Priem B, Morvan H, Joset F (1988) Sulphated
exopolysaccharides produced by two unicellular strains of cyan-
bacteria, Synechocystis PCC6803 and 6714. Arch. Microbiol.
150: 558–563.
Potts M (1994) Desiccation tolerance of prokaryotes. Microbiol. Rev
58: 755–805.
Rogers SL, Burns RG (1994) Changes in aggregate stability, nutrient
status, indigenous microbial populations and seedling emergence,
following inoculation of soil with Nostoc muscorum. Biol. Fertil.
Soils 18: 209–215.
Schulten JA (1985) Soil aggregation by cryptogams of a sand prairie.
Amer. J. Bot. 72: 1657–1661.
Steel RGD, Torrie JH (1985) Principles and Procedures of Statistics,
2nd edn. Mc Graw-Hill Inc. 622 pp.
Sudo H, Burgess JG, Tamemasa H, Nakamura N, Matsunaga T
(1995) Sulfated exopolysaccharide production by the halophilic
cyanobacterium Aphanocapsa halophytica. Curr. Microbiol 30:
219–222.
United States Department of Agriculture (1994) Key to Soil Taxon-
omy, (6th edn), 306 pp.
Van Gestel M, Merck R, Vlassak K (1996) Spatial distribution of
microbial biomass in microaggregates of a silty-loam soil and the
relation with resistance of microorganisms to soil drying. Soil.
Biol. Biochem. 28: 503–510.
japh487.tex; 22/09/1997; 11:47; v.7; p.5