Interaction of Microorganisms with Soil ColloidsObserved by X-Ray Microscopy

Galina Machulla1, Jürgen Thieme2, Jürgen Niemeyer3

1 Institut für Bodenkunde und Pflanzenernährung, Martin-Luther-Universität,Weidenplan 14, D-06108 Halle, Germany

2 Forschungseinrichtung Röntgenphysik, Georg-August-Universität, Geiststrasse 11,D-37073 Göttingen, Germany

3 Fachbereich VI - Geowissenschaften, Abteilung Bodenkunde, Universität Trier,D-54286 Trier, Germany

Abstract. In an X-ray laboratory study the interaction of bacteria with a sterilemontmorillonite suspension was studied. It was found that the inoculation ofthe sterile montmorillonite suspension with a culture of soil bacteria resultedin adhesion and aggregation of montmorillonite platelets on the surface ofbacterium cells. The platelets appear to be stuck in bacterial slime in a typicaledge-to-face association, parallel to each other. The adhesion is due to theextracellular polysaccharides produced by the soil bacteria.

1 Introduction

Soils build complex environments that generally contain large amounts ofmicroorganisms. The viability, activity and mobility of bacteria and other microbesin soil depend strongly on the extent to which they are attached to the surfaces oforganic and inorganic soil particles. Adhesion of microorganisms to mineral soilcolloids may lead to aggregation of soil mineral components, which improves soilstructure and its stability [1]. This, in turn, may increase again the biological activityand soil productivity.

In view of the fact that microorganisms excrete extracellular polymer sub-stances, the production of microbial substances is assumed to be the majormechanism by which bacteria and fungi contribute to aggregation processes. This isthe material that first makes contact between a cell and a surface and which cancause an irreversible adhesion [2].

It is hypothesized that there are four main types of adhesion between micro-organisms and soil solids such as mineral particles. These particles can either be lar-ger than microorganisms, of equal size, or smaller, as in the case of clay particles[3]. The mechanisms involved in this adhesion are of a physicochemical nature.They include van der Waals, electrostatic, hydrogen-bonding as well as hydrophobicinteractions [4]. Many polysaccharides can adsorb several particles simultaneously,and thus bind and flocculate them.

Phenomena of building mineral colloidal flocculates, as well as the microbialbiodegradation of some xenobiotics, can be considered as basic features in the pro-

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cess of soil and water decontamination [5]. Therefore, studies about the interaction ofmicroorganisms with mineral colloids are fundamental for the science of microbialecology and of great practical importance for the disciplines of agronomy and plantpathology.

The development of a new technique – X-ray microscopy – has made it possibleto study visually the role of bacteria in building colloidal flocculates as well as themode of their association. Due to a much shorter wavelength, X-ray microscopyprovides higher resolution than optical microscopy. Most importantly, X-raymicroscopy has the potential for imaging hydrated specimen with high resolution.Moreover, the preparation of samples is simple and the biotic-abiotical system can bestudied without distortion.

2 Methods and Materials

The method most generally used in laboratory experiments to observe the interactionof microorganisms with mineral colloids applies liquid systems. This method wasalso used for the present study. The microbes observed in our investigationsrepresent a species of bacterium Bacillus megatherium on one hand and a mixedculture of several soil bacteria which are common in most native German soils on theother. The cell length of Bacillus megatherium is about 4 µm and in pure culturesbacterial chains could be observed. The culture of soil bacteria includes cells ofdifferent size and shape.

To obtain the bacterial suspension and to study the aggregating ability of soilbacteria, we cultured Bac. megatherium in a standard nutrient medium consisting of2 g glycerine, 0.25 g peptone, 0.1 g K2HPO4, 0.004 g CaCO3, 0.3 g NaCl and 0.25 gMgSO4 per liter of distilled water. The culture was grown for 72 h at a temperatureof 28 °C, and after the incubation a 0.1% Na-montmorillonite (Wyoming mont-morillonite) suspension was inoculated with this microbial culture at a 1 : 1 - ratio.Subsequently this suspension was studied by X-ray microscopy.

The mixed culture of soil bacteria was maintained in a diluted (0.1% or 1.0% ofthe original medium) and in the original (100%) nutrient medium after the additionof the Na-montmorillonite (1g per liter). Test tubes containing 2 ml of this bacteria-montmorillonite mixture were incubated at 28 °C for 48 h. At the end of the incu-bation period the mode of interaction between microorganisms and clay particles wasdetermined with X-ray microscopy.

3 Results

In Figures 1 and 2 the mode of cell-montmorillonite association and the influence ofthe medium concentration on the delimitation of Wyoming montmorillonite in aliquid system is shown. It can be seen (Fig. 1, top) that the swollen montmorilloniteaggregates disperse into tactoids (or quasicrystals), which consist of several packs ofcrystallites. In general, the crystallites are associated in a subparallel manner (face-to-face) and have to be considered as interactions of clay particles with micro-

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organisms. After the culture of Bac. Megatherium was added to the clay suspension,a close look was taken at the clay platelets (or crystallites, Fig. 1, bottom) and at thevery small clay particles (Fig. 2, top). It was observed (see Fig. 1, bottom) that thecrystallites are in an edge-to-face association with the bacterial cells. The very smallparticles, however, are located on the surface of the microbial cells and in between(Fig. 2, top). In these location the concentration of microbial slime is higher then inthe surroundings. Large quantities of polysaccharide secretion are seen as severalmicrons thick “shadows“ around the microbial bodies. The extracellularpolysaccharide slime and its ability to bind clay particles has also been observed bymeans of ultra-thin section and low-temperature scanning electron microscopy [1, 2].

Clay particles in soil are a main source of nutrients for microbes. The microbesobtain these nutrients through biological weathering processes. These processes areof a biochemical nature and result from the secretion of acid metabolites. For pH-values smaller than 5, it has been reported that mineral destruction by complexationor dissolution takes place [6]. This biochemical weathering leads to a destruction ofclay quasicrystals, which fall apart into thinner subparticles (Fig. 2, bottom). In theX-ray micrograph, fully dispersed montmorillonite tactoids in the 1.0% nutrientsolution can be observed. This 1.0% solution appears to be the optimal microhabitatfor an active mixed culture, since it destroys clay mineral tactoids completely. Soilbacteria cultured in the 0.1% solution, however, appear to be inactive (Fig. 3, top),because of the extremely low nutrient concentration, whereas in the case of the 100%solution (Fig. 3, bottom) they find sufficient nutrients in the solution. In bothnutrient solutions (0.1% and 100%) the montmorillonite suspension keeps thereforeits original appearance. It thus seems possible that mineral destruction by soilmicrobes occurs in particular, whenever the microbe population begins to starve afterthere is a rapid drop of nutrient concentration in the soil solution.

4 Conclusions

1. X-ray microscopy allows the direct visualization of bacteria in soil, their extra-cellular polymer substances in microsystems, as well as the affected mineralaggregates and particles.

2. The polysaccharide secretion is noticed as a white „shadow“ that surrounds thecells.

3. The inoculation of the sterile montmorillonite suspension with bacteria resultedin the adhesion and aggregation of montmorillonite platelets on the surface of thecells. The platelets appear to be attached in the bacterial slime in a typical edge-to-face association with a parallel orientation to each other. This result seems tobe due to the extra-cellular polysaccharides produced by the soil bacteria.

4. By secretion of organic polymers and by physicochemical action, microorganismscan change the organization and physical characteristics of the media in whichthey live.

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Fig. 1. The interaction of soil microbes with colloidal particles: (top) montmorilloniteaggregate dispersion; (bottom) polysaccharide secretion and crystallite adhesion caused by

Bacillus megatherium.T - tactoid, C - crystallite, P - small clay particles, B - bacterial cell associated with clay

crystallites in an edge- to-face manner.

Interaction of Microorganisms with Soil Colloids II - 25

Fig. 2. The interaction of soil microbes with colloidal particles: (top) small clay particlesadsorption in polysaccharide slime; (bottom) tactoids destruction in a 1.0% nutrient solution.

T - tactoid, C - crystallite, P - small clay particles, B - bacterial cell associated with claycrystallites in an edge- to-face manner.

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Fig. 3. Microbe - clay mineral system in the 0.1% (top) and 100% (bottom) nutrient solutions.

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References

1 Emerson, W.W., R.C. Foster, and J.M. Oades in Interactions of Soil Mineralswith Natural Organics and Microbes (Huang and Schnitzer; SSSA, Inc.,Madison, Wisconsin, 1986), pp. 521 - 548.

2 Robert, M. and C. Chenu in Soil Biochemistry, Vol.7 (Stotzky and Bollag;Marcel Dekker, Inc., NewYork, Basel, Hong Kong, 1992) pp. 333-360.

3 Hattori, T., Microbial Life in the Soil (Marcel Dekker, Inc.; New York, 1973),235 p.

4 Zvyagintsev, D.G., Pochva i microorganizmy/Soil and Microorganisms(University of Moscow; Moscow, 1987) p. 35.

5 Capone, D. G. and J. E. Bauer in Environmental Microbiology (Mitchell; Wiley-Liss, Inc., New York,1992), pp. 191-238.

6 Robert, M. and J Berthelin in Interactions of Soil Minerals with NaturalOrganics and Microbes (Huang and Schnitzer; SSSA, Inc., Madison,Wisconsin, 1986), pp. 453-495.