Microbial aggregation of sand in an embryo dune system

MICROBIAL

AGGREGATION OF SAND IN AN EMBRYO DUNE SYSTEM SUSAN

Department

of Biological

Sciences.

M.

FORSTER

University

of Dundee,

Dundee,

DDI

4HN

U.K.

Summary---The importance of microorganisms in the aggregation of sand in an embryo dune system was examined. Of three main types of aggregates formed, microbial aggregates were found to be more important at stabilizing sand than either root-microbial or debris-microbial aggregates on the beach and at the edge of the dune but roots and their associated microorganisms were more important on the hummock of the dune. The amount of microbial and root-microbial aggregated sand was higher during the winter when the vegetation was dying down and decaying. In the absence of roots, microorganisms. in particular bacteria, play a major role in aggregating sand. The bacteria are well adapted to the unfavourable habitat of the beach as they are motile and tolerant to sea water, being able to grow in a salinity of 3.5::,. Bacteria may play a major role in aggregating and stabilizing sand prior to colonization by higher plants.

Soil structure is important in fertility and plant growth and can be affected by a number of physical and biological factors. Several workers (McCalla, 19.50; Martin et al., 1955; Bond and Harris, 1Y64; Harris et ul., 1966) have found that microorganisms are important in the formation. stabilization and degradation of soil aggregates. Fungal mycelia as aggregating agents have received particular attention (Barratt, 1962). Swaby (1949) showed that soil particles can be bound together mechanically by fungal and actinomycete hyphae but that the resulting aggregates, though fairly stable, only endure until the hyphae are decomposed by other microorganisms‘ Aggregation and aggregate stability are of fundamental importance in soil profile characterization and in determining the agricultural capacity (Bryan, 1969).

Fig. 1. One of the embryo

While there have been many investigations into the part played by higher plants in dune stabilization (Ranwell, 1972) little attention has been paid to the possible role of microorganisms. Obviously, as in mature soils they will play a part in soil formation and this paper reports investigations on aggregation of sand grains by microorganisms in embryo sand dunes.

MATERIALS

AND METHODS

Three embryo dunes were selected on an area of rapidly-forming sand dunes (Nat. Grid Ref. NO/505267) at Tentsmuir National Nature Reserve. Fife. Every month three sites were sampled on each dune (Fig. I). Site 1 was on the beach 1.8 m from the edge of the dune where there were no higher

dunes studied showing the three sampling sites (numbered) 537

538

SUSAN

M. F~KSTEK

plants. Site 2 was at the edge of the dune. at the advancing margins of the Agrop_)~~~r~jcme$orme (A + D Love) A + D Love, colonies. At the top of the dune there was dense colonization by A. juncrifbnnr. While site 3 remained unflooded sites 1 and 2 were periodically inundated at spring tides. The aggregates were divided into three categories according to the agent primarily associated with them viz: (a) microbial aggregates (m-) formed by the action of microorganisms. (b) Root-microbial (rm-) formed by the aggregation of sand around living and senescent roots. (c) Debris-microbial (dm-) formed by the aggregation of sand around fragments of decaying plant material.

“““”“”_.....

Fig. 3. Developmcnt

lso/utior~s

Samples of about 300 g sand were taken by pushing a copper tube 18cm in length x 5 cm dia into the dune to a depth of 18 cm, Four replicate samples were taken from each of the three sampling sites on each dune. Samples were air dried at constant temperature and humidity for 5 days Each sample was sieved and separated into particles >2.0. ~2.0 >1.4, cl.4 ~1.0 and tl.Omm To remove carbon. leaving sand and vegetation ash, the different types of aggregates were combusted at 500°C for 4 h then weighed. Roots and debris were washed to remove sand and combusted to find the respective average ash weights which were then deducted from the total weights to give the weight of sand adhering to the roots and debris. Little difference in weight was found when microbial aggregates were combusted and therefore this was discontinued.

.

from

of root

microhd

aggregates

x 2.5.

root.s

Fungi were isolated from roots of A. ,jwwjfimw (Harley and Waid, 1955). Roots were collected using sterile forceps and cut into I cm segments. These were shaken in an automatic shaker for 2 min and plated onto Czapek Dox agar. The remaining roots were put into a second vial and the process repeated for 6 root washings. The plates were incubated at 26’C for 7 days and the isolated fungi, cultured and identified. RESULTS

Procedures described by Parkinson ef ul. (1971) were followed. Direct isolations were made from microbial aggregates (Waksman, 1916) by plating five aggregates recovered from each site. onto Rose Bengal-Streptomycin agar. The resultant fungal species were subcultured and identified. Isolations were also carried out using a dilution plate technique. Microbial aggregates from each site were shaken on an orbital shaker in sterile distilled water for I h, a dilution series was prepared and plated on to the following selective agar media: Rose Bengal-Streptomycin for fungi; soil extract for bacteria and dextrose nitrate for actinomycetes. The plates were incubated for I4 days then examined and colonies counted. Bacteria isolated from site I were plated on to soil extract agar with the addition of sea water medium giving a salinity of 3.57” to test their ability to grow under saline conditions.

The m-aggregates recovered from the sand generally ranged in size from about I to about 12 mm dia (Fig. 2). Aggregates up to 20mm dia were also recorded. The average size of individual sand grains was 0.25 mm. The smaller aggregates were charactcristically spherical in shape. but larger aggregates were subspherical or flattened. Rm-aggregates developed on areas of the root surface (Fig. 3) where sand grains trapped in the root hairs. mucigel and secretions from the root surface led to the development of discrete spherical aggregates. Alternatively sand grains become attached along the whole length of the root, forming a firm water-stable structure (Fig. 3). Decaying organic debris in the form of leaves, stems and seaweed colonized by bacteria and other microorganisms, has a sticky surface. Sand grains adhere to this, which includes polysaccharides secreted by bacteria, leading to the build up of sand and the formation of discrete spherical aggregates of up to 6 mm (Fig. 4) which aid in the stabilization of sand. ’ The mean weights of m-. rm- and dm-aggregated sand were, measured over a period of a year (Figs 5. 6 and 7). In the beach sand, I.8 m from the edge of the dune, a greater weight of microbial aggregates was found than on the dune itself (Fig. 5.

Fig. 2. Development of microbial aggregates x 2.5.

Fig. 4. Development of debris microbial aggregates x 2.5.

539

Microbial aggregation of sand Microbial Site

aggregotiin Site

I

Site

2

3

4.0

7a

3.0

0 .$ z

2.0

3 1.0 4

i,i..

0

Fig. 5. Seasonal

variation

in the weight

Roo3 4.0

7 s

M, AM JJ

IMJJASONDJFMMAMJJASONDJFM

of microbial

aggregotii Site I

aggregates

ASONDJFM

from three sites on an embryo

Site 2

dune

ite 3

3.0

0

I

i

2.0

: r IX

rL_

~~ MAMJ

Fig. 6. Seasonal

variation

JASONDJ

in the weight

FMMA~JJASON~JFMMAMJ

of root

JASONDJFM

microbial-a~re~tes dune.

from three sites on an embryo

Debris aggregation Site

I

Site

2

site

3

4.0

3.0 T r” 0 2.0

P 1.0 0.8

i

Es 0.2 0

~~,:= MAMJJASONDJFMMAMJJASONDJFMMAMJJASONDJFM

Fig. 7. Seasonal

variation

in the weight

of debris

microbial dune.

site 1). During the winter, aggregation was reduced increasing to a maximum in August. At the edge on the dune (Fig. 1, site 2) a low amount of aggregation was found, with a small peak in August. A similar pattern was found on the hummock of the dune (site 3) with little aggregation throughout the year. By comparison the mean weights of rm-aggregated sand

aggregates

from three sites on an embryo

(Fig. 6) show a very low weight of aggregated sand (site 1). An increase was observed where the A. junceiforme plants had colonized the margin of the dune (site 2) and more aggregation occurred in the presence of established A. jM~ce~o~nz~ plants (site 3). A similar result was obtained for dm-aggregated sand on the beach (Fig. 1, site 1) in the absence of plants, and

SUSAN M. FORSTER

540

Table

I. Mean total weight of aggregated sand (mg.kg-’ .sand) March 1976 to February 1977

Aggregate

types

Microbial Root-microbial Debris-microbial Total

Weight Site 1’ 730 50 140 920

of aggregates Site 22

(mg) Site 3j

490 270 480 I240

400 1240 750 2390

’ Site l-l.8 m from dune. ’ Site Z-At margin of dune. ‘Site 3-Hummock of dune. any debris was blown into the area by wind or deposited by the tide. A greater weight of dm-aggregated sand was found at the edge of the dune (site 2) particularly in the winter when the grasses were senescent. This feature was also observed on the hummock of the dune (site 3) with twice the amount of dm-aggregated sand. More m-aggregation occurred at site 1 than in either of the two sites (Table 1). By comparison a lower weight of rm- and dm-aggregated sand was found in site 1, increasing in site 3. The total weight of aggregated sand indicates that there was a considerable increase from the beach (site 1) to the hummock of the dune (site 3). On the beach (site 1) larger numbers and weights of smaller aggregates, I .&I .3 mm were found than in the other sites. On the dune itself (sites 2 and 3) fewer aggregates were present but they were larger and heavier > 2.0 mm. Further away from the dune, lower down on the beach there was no aggregation of sand or colonization by higher plants and the sand was very unstable. Chdosporium herharum and four Penic~illium spp. were isolated from site 2 and a Phomu sp. and a hyaline mycelial species from site 3 aggregates. Aggre-

gates recovered from site 1, were crushed and examined microscopically (Fig. 8) and this also showed an absence crushed

of fungal aggregates

hyphae from the sand grains. In from site 2 (Fig. 9) the sand grains

were bound together by a few fungal hyphae forming a water-stable aggregate. Aggregates from site 3 (Fig. IO) showed consistent and considerable binding of the sand grains by fungal hyphac, including hyphae of the endomycorrhizal fungus, Glomus .fu.wiculutus. Mycorrhizas are abundant in sand dune plants (Nicolson, 1960) and the importance of the mycorrhizal endophyte GIomus sp. on the direct aggregation of sand has been noted (Koske CT LII., 1975; Sutton and Sheppard, 1976). The spectrum of organisms recovered by serial dilutions followed the same pattern of microbial colonization. At site I. large numbers of bacteria, few fungi and no actinomycetes were isolated. In contrast, in site 2 the influence of the roots (Webley et (II.. 1952; Old and Nicolson. 1975) is correlated with an increase in the number of fungi. the occurrence of large numbers of a@inomycetes, mainly Strrptomyes spp. and large numbers of bacteria. In site 3 where the grasses and their root systems were well established there were relatively few bacteria and actinomycetes and a considerable increase in the number of fungi. Of the 10 genera of bacteria isolated. six were identified as Psrudor~~or~u.s spp. One Psrudomor~us sp. had slime capsular material on the surface which may aid in the formation of aggregates. Two species were identified as Bacil/u.s spp and two were of unknown genera. Most of the genera isolated were motile, Gram-negative rods. apart from one Budus sp. which was Gram-positive and one which was Gramvariable to Gram-negative. One of the unknown genera was also Gram-variable to Gram-negative and was unusual in that it was highly pleiomorphic and had large motile rods up to 5 pm long. The washing of A. juweiforrne roots indicated some

Fig. 8. Crushed aggregates from site I, showing absence of fungal hyphae x 300

Microbial

Fig. 9. Crushed

aggregate

from

aggregation

site 2. showing

a few fungal x 300.

of the main fungal genera present in the embryo dunes associated with the roots. The commonest isolates were, four species of Penicilliurn, Arthrinium sp., Acremonium sp. and Fusarium sp. Most of the bacteria isolated grew in sea water medium and soil extract agar at a salinity of 3.50/, which may be an adaptation to enable them to withstand periodic inundation by the tide.

Fig. IO. Crushed

\.H.“.

I I!5

<>

aggregate

of sand

541

hyphae

adhering

to the sand

grains

DISCUSSION Few fungi are present in the sand surrounding embryo dunes, which is subjected to extremes of temperature and moisture content and is liable to be temporarily exposed to high salinities. However, microorganisms were more important as aggregating agents than either roots or debris and of particular importance in aggregating beach sand.

from site 3. showing numerous fungal fungus (Glomu.s fusciculafus (arrowed)

hyphae, x 300.

including

the mycorrhizal

542

SUSAN M. FORSTER

The increasing stabilization of the dunes may be correlated with the weight of aggregated sand at each site. Least aggregation occurred on the beach, increasing on the margin of the dune to a maximum level on the hummock where the sand was most stable and colonization by A. junceiforme at a maximum. The physical presence of roots and debris in conjunction with the aggregated sand leads to greater stabilization of sand. There was more m-aggregation over t’he three sites than of aggregates associated with roots or debris indicating their importance in the stabili :ation of sand. Rm-aggregates were less important but more important than dm-aggregates. Sand grains may be trapped by mucigel on the root surface leading to greater aggregation during the summer when the roots are actively growing. The association of the actinomycete, Streptomyces with the rhizosphere of A. jum-#orme and other dune grasses was noted by Watson and Williams (1974). They found that it was the commonest genus in embryo dunes. Clough and Sutton (1978) have shown that the mycelium of the mycorrhizdl fungus Glomus was a dominant factor in the aggregation of sand particles. An amorphous deposit containing polysaccharides was present between sand grains and hyphae in association with other microorganisms. The binding effect of the mycelium persists after death of the plant. Hyphae in old aggregates were as effective at binding sand as hyphae associated with living plants. There is strong evidence that soil polysaccharides contribute to soil aggregate stability (Martin, 1971). Bacteria have been shown to cement soil particles together by forming polysaccharide substances (Harris et al., 1963). Many microorganisms produce polysaccharides (Hepper, 1975) and soil organic matter may contain up to 25% of them (Martin, 1971). In their free state polysaccharides are easily degraded by other microorganisms but they appear to be protected from such degradation once they are firmly bound within an aggregate or incorporated into a clay lattice (Skinner, 1976). Stable aggregates are known to form under grass which is probably due to the combined action of plant and microbe (Allison 1968). The numerous fine roots ramify and divide up the intervening soil into small blocks upon which they exert pressure and cause local drying by removing water. Root exudates and sloughed root tissue also support the growth of microorganisms and promote the formation of polysaccharides just where they are needed to stabilize the newly-formed aggregates. It has been shown by Costerton et al. (1978) that bacteria stick to surfaces by means of a mass of tangled fibres of polysaccharides extending from the bacterial surface. This adhesion determines the particular locations of bacteria in most natural environments. One important feature emerging from the present work was the hitherto unsuspected involvement of bacteria in the aggregation and stabilization of sand prior to colonization by higher plants. Any aggregates formed have no apparent binding agent and contain no fungal or actinomycete hyphae and are therefore likely to be formed by bacteria and their associated polysaccharides. It is interesting to note that all the bacteria isolated were motile rods ranging in size from 1 to 5 pm. The motility of the bacteria may be an adaptation to the extreme conditions and as in

of Bacillus sp. the ability to form spores is an added advantage. The production of binding materials of a polysaccharide nature by bacteria would cause sand particles to adhere and build up an aggregate. It is not clear whether the secreted polysaccharides are acting as centres for aggregation in situ or after deposition by bacterial colonies. the case

Acknowledyrments--I

thank Drs K. M. Old and T. H. Nicolson for advice and Mr 1. MacDonald for technical assistance. I am grateful to the Natural Environment Research Council for financial assistance and to the Nature Conservancy Council for access to the nature reserve.

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