Interaction of Microorganisms with Soil Colloids Observed by X-Ray Microscopy

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1 Interaction of Microorganisms with Soil Colloids Observed by X-Ray Microscopy Galina Machulla 1, Jürgen Thieme 2, Jürgen Niemeyer 3 1 Institut für Bodenkunde und Pflanzenernährung, Martin-Luther-Universität, Weidenplan 14, D Halle, Germany 2 Forschungseinrichtung Röntgenphysik, Georg-August-Universität, Geiststrasse 11, D Göttingen, Germany 3 Fachbereich VI - Geowissenschaften, Abteilung Bodenkunde, Universität Trier, D Trier, Germany Abstract. In an X-ray laboratory study the interaction of bacteria with a sterile montmorillonite suspension was studied. It was found that the inoculation of the sterile montmorillonite suspension with a culture of soil bacteria resulted in adhesion and aggregation of montmorillonite platelets on the surface of bacterium cells. The platelets appear to be stuck in bacterial slime in a typical edge-to-face association, parallel to each other. The adhesion is due to the extracellular polysaccharides produced by the soil bacteria. 1 Introduction Soils build complex environments that generally contain large amounts of microorganisms. The viability, activity and mobility of bacteria and other microbes in soil depend strongly on the extent to which they are attached to the surfaces of organic and inorganic soil particles. Adhesion of microorganisms to mineral soil colloids may lead to aggregation of soil mineral components, which improves soil structure and its stability [1]. This, in turn, may increase again the biological activity and soil productivity. In view of the fact that microorganisms excrete extracellular polymer substances, the production of microbial substances is assumed to be the major mechanism by which bacteria and fungi contribute to aggregation processes. This is the material that first makes contact between a cell and a surface and which can cause an irreversible adhesion [2]. It is hypothesized that there are four main types of adhesion between microorganisms and soil solids such as mineral particles. These particles can either be larger 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 hydrophobic interactions [4]. Many polysaccharides can adsorb several particles simultaneously, and thus bind and flocculate them. Phenomena of building mineral colloidal flocculates, as well as the microbial biodegradation of some xenobiotics, can be considered as basic features in the pro-

2 II - 22 G. Machulla et al. cess of soil and water decontamination [5]. Therefore, studies about the interaction of microorganisms with mineral colloids are fundamental for the science of microbial ecology and of great practical importance for the disciplines of agronomy and plant pathology. The development of a new technique X-ray microscopy has made it possible to study visually the role of bacteria in building colloidal flocculates as well as the mode of their association. Due to a much shorter wavelength, X-ray microscopy provides higher resolution than optical microscopy. Most importantly, X-ray microscopy has the potential for imaging hydrated specimen with high resolution. Moreover, the preparation of samples is simple and the biotic-abiotical system can be studied without distortion. 2 Methods and Materials The method most generally used in laboratory experiments to observe the interaction of microorganisms with mineral colloids applies liquid systems. This method was also used for the present study. The microbes observed in our investigations represent a species of bacterium Bacillus megatherium on one hand and a mixed culture of several soil bacteria which are common in most native German soils on the other. The cell length of Bacillus megatherium is about 4 µm and in pure cultures bacterial chains could be observed. The culture of soil bacteria includes cells of different size and shape. To obtain the bacterial suspension and to study the aggregating ability of soil bacteria, we cultured Bac. megatherium in a standard nutrient medium consisting of 2 g glycerine, 0.25 g peptone, 0.1 g K 2 HPO 4, g CaCO 3, 0.3 g NaCl and 0.25 g MgSO 4 per liter of distilled water. The culture was grown for 72 h at a temperature of 28 C, and after the incubation a 0.1% Na-montmorillonite (Wyoming montmorillonite) 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% of the original medium) and in the original (100%) nutrient medium after the addition of the Na-montmorillonite (1g per liter). Test tubes containing 2 ml of this bacteriamontmorillonite mixture were incubated at 28 C for 48 h. At the end of the incubation period the mode of interaction between microorganisms and clay particles was determined with X-ray microscopy. 3 Results In Figures 1 and 2 the mode of cell-montmorillonite association and the influence of the medium concentration on the delimitation of Wyoming montmorillonite in a liquid system is shown. It can be seen (Fig. 1, top) that the swollen montmorillonite aggregates disperse into tactoids (or quasicrystals), which consist of several packs of crystallites. In general, the crystallites are associated in a subparallel manner (faceto-face) and have to be considered as interactions of clay particles with micro-

3 Interaction of Microorganisms with Soil Colloids II - 23 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 the very small clay particles (Fig. 2, top). It was observed (see Fig. 1, bottom) that the crystallites are in an edge-to-face association with the bacterial cells. The very small particles, 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 in the surroundings. Large quantities of polysaccharide secretion are seen as several microns thick shadows around the microbial bodies. The extracellular polysaccharide slime and its ability to bind clay particles has also been observed by means 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 microbes obtain these nutrients through biological weathering processes. These processes are of a biochemical nature and result from the secretion of acid metabolites. For phvalues smaller than 5, it has been reported that mineral destruction by complexation or dissolution takes place [6]. This biochemical weathering leads to a destruction of clay quasicrystals, which fall apart into thinner subparticles (Fig. 2, bottom). In the X-ray micrograph, fully dispersed montmorillonite tactoids in the 1.0% nutrient solution can be observed. This 1.0% solution appears to be the optimal microhabitat for an active mixed culture, since it destroys clay mineral tactoids completely. Soil bacteria 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 both nutrient solutions (0.1% and 100%) the montmorillonite suspension keeps therefore its original appearance. It thus seems possible that mineral destruction by soil microbes occurs in particular, whenever the microbe population begins to starve after there 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 extracellular polymer substances in microsystems, as well as the affected mineral aggregates and particles. 2. The polysaccharide secretion is noticed as a white shadow that surrounds the cells. 3. The inoculation of the sterile montmorillonite suspension with bacteria resulted in the adhesion and aggregation of montmorillonite platelets on the surface of the cells. The platelets appear to be attached in the bacterial slime in a typical edgeto-face association with a parallel orientation to each other. This result seems to be due to the extra-cellular polysaccharides produced by the soil bacteria. 4. By secretion of organic polymers and by physicochemical action, microorganisms can change the organization and physical characteristics of the media in which they live.

4 II - 24 G. Machulla et al. Fig. 1. The interaction of soil microbes with colloidal particles: (top) montmorillonite aggregate 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.

5 Interaction of Microorganisms with Soil Colloids II - 25 Fig. 2. The interaction of soil microbes with colloidal particles: (top) small clay particles adsorption 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 clay crystallites in an edge- to-face manner.

6 II - 26 G. Machulla et al. Fig. 3. Microbe - clay mineral system in the 0.1% (top) and 100% (bottom) nutrient solutions.

7 Interaction of Microorganisms with Soil Colloids II - 27 References 1 Emerson, W.W., R.C. Foster, and J.M. Oades in Interactions of Soil Minerals with Natural Organics and Microbes (Huang and Schnitzer; SSSA, Inc., Madison, Wisconsin, 1986), pp Robert, M. and C. Chenu in Soil Biochemistry, Vol.7 (Stotzky and Bollag; Marcel Dekker, Inc., NewYork, Basel, Hong Kong, 1992) pp 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 Capone, D. G. and J. E. Bauer in Environmental Microbiology (Mitchell; Wiley- Liss, Inc., New York,1992), pp Robert, M. and J Berthelin in Interactions of Soil Minerals with Natural Organics and Microbes (Huang and Schnitzer; SSSA, Inc., Madison, Wisconsin, 1986), pp