Microorganisms (bacteria, yeast and fungi) may have a direct action on metal mobility through biosorption, bioaccumulation or resistance/
detoxification processes (Fig. 1). In addition, they may influence the environment by producing compounds from metabolic reactions such as acids or chelating agents such as siderophores. In this part, some examples of microbial interaction mechanisms are presented including biosorption, metabolism by-product complexation and indirect metal use for microbial life, bioaccumulation and resistance/detoxification systems. Indirect influences of microorganisms on the speciation of heavy metals and/or radionuclides are also presented.
Biosorption
Biosorption is a physico-chemical mechanism including sorption, surface complexation, ion exchange and entrapment, which is relevant for living and dead biomass as well as derived products. Biosorption can be considered as the first step in the microorganism-metal interaction. It encompasses the uptake of metals by the whole biomass (living or dead) through physico-chemical mechanisms such as sorption, surface complexation, ion exchange or surface precipitation. These mechanisms take place on the cell wall (Shumate and Strandberg 1985) which is a rigid
100 BIOREMEDIATION OF AQUATIC & TERRESTRIAL ECOSYSTEMS
layer around the cell (Fig. 1) and they have fast kinetics. One dominant factor affecting the capacity of the microbial cell wall to "trap" the metal ion is the composition of this outer layer. For a better understanding of the biosorption mechanisms, the cell wall structure of microorganisms will be briefly and simply presented.
Cell wall structure
In the prokaryotic world (bacteria), the wall is composed by a peptidoglycan structure bound to a techoic acid (Gram-positive bacteria) or to a lipopolysaccharide (Gram-negative bacteria). These two groups are differentiated using a coloration reaction. The cell wall of Gram-positive bacteria is 50 to 150 nm thick and mainly consists of 40 to 90 % peptidoglycan. It is a rigid, porous, amorphous material, made of linear chains of the disaccharide N-acetylglucosamine-b-1,4-N-acetylmuramic acid. The cell wall of Gram-negative bacteria appears to be somewhat thinner, usually 30 to 80 nm thick, and only 10 % of the material is made up of peptidoglycan (Remacle 1990). The cell wall composition of archaebacteria differs from the eubacteria by the lack of muramic acid and peptidoglycan.
The cells of many bacteria groups are often covered by an additional surface layer non-covalently associated with the cell wall. This structure, called the S-layer, is usually composed of regular arrays of homogeneous polypeptides and sometimes of carbohydrates.
Figure 1. Microorganisms / metal relationships (adapted from Gadd and White 1993).
In the eukaryotic world (fungi and yeast), the cell wall is made up of various polysaccharides arranged in a multilaminate microfibrillar structure. Ultrastructural studies reveal two phases: (i) an outer layer cons-tituted of glucans, mannans or galactans and (ii) an inner microfibrillar layer. The crystalline properties of this latter are given by the parallel arrangement of chitin or sometimes of cellulose chains or, in some yeasts, of non-cellulosic glucan chains. There is a continuous transition between these two layers (Remacle 1990).
Cell wall characteristics and biosorption
A large variety of chemical microenvironments is present on the bacterial surface (Table 2). These include phosphate, carboxyl, hydroxyl and amino functional groups, among others. Various methods have been investigated to identify the bacterial surface functional groups involved in metal uptake.
A first approach consisted of performing metal binding studies on extracted cell wall polymers, such as peptidoglycan and teichoic acid, to determine the types of cell wall components responsible for metal binding (Beveridge and Fyfe 1985). In addition, selective chemical modifications of the various functional groups were carried out to assess their contribution to the metal uptake (Beveridge and Murray 1980, Doyle et al. 1980). The major incon-venience in the use of this kind of technique is the rather heavy experimental protocol, which does not allow the study of intact cells for adsorption investigations. The potentiometric titration technique provides a simple and efficient method to measure and determine the different functional groups available to bind metallic ions. Consequently, the use of this method is interesting for the surface characterization of algae, fungi (Deneux-Mustin et al. 1994, Schiewer and Wong 2000), and bacteria (Van Table 2. Functional groups of microbial complexing compounds (Birch and Bachofen 1990).
Basic Acidic
- NH2 amino - CO2H carboxylic
= NH imino - SO3H sulphonic
- N = tertiary acyclic or - PO(OH)2 phosphonic heterocyclic nitrogen
= CO carbonyl - OH enolic, phenolic
- O - ether = N - OH oxime
- OH alcohol - SH thioenolic and thiophenolic
- S - thioether
- PR2 substituted phosphine
102 BIOREMEDIATION OF AQUATIC & TERRESTRIAL ECOSYSTEMS der Wal et al. 1997, Texier et al. 1999). For example, Fein et al. (1997) have characterized the acid/base properties of the cell wall of Bacillus subtilis and have shown three distinct types of surface organic acid functional groups with pKa of 4.82, 6.9 and 9.4. These various values are generally attributed to carboxyl, phosphate and hydroxyl moieties respectively.
Furthermore, various spectroscopic methods, including IR spectroscopy, XANES spectroscopy (X-ray absorption near-edge structure), EXAFS spectroscopy (extended X-ray absorption fine structure) and NMR spectroscopy amongst many others, can provide information about the chemical environment of the sorbed metallic ions on biological material.
Until recently, the emphasis has been placed on the use of such spectroscopic methods to characterize the surfaces of algae (Kiefer et al.
1997), bacteria (Schweiger 1991), fungi (Sarret et al. 1998) and plant cells (Tiemann et al. 1999, Salt et al. 1999). Drake et al. (1997), Texier et al. (2000) and Markai et al. (2003) have investigated the binding of europium to a biomaterial derived respectively from the plant Datura innoxia, from Pseudomonas aeruginosa and from Bacillus subtilis. They characterized the functional groups answerable for metal ion uptake with the help of laser-induced spectrofluorometry. A simultaneous determination of emission wavelength and fluorescence lifetime provided two-dimensional information about fluorescing ions. These spectroscopic approaches have confirmed many times that the fixation occurs with the free functional groups present in the cell wall layer of the microorganisms. For Gram-negative bacteria, the functional groups are, for example, present in the lipopolysaccharide of the outer layer and in the peptidoglycan and for Gram-positive bacteria in the techoic acid. Mullen et al. (1989) indicated, after electronic microscopy studies, that lanthanum was accumulated at the surface of P. aeruginosa inducing crystalline precipitation.
Biosorption capacities
Biosorption capacities of microorganisms for metal ions generally depend on the metal concentration, the pH of the solution, the contact time, the ionic strength and the presence of competitive ions in the solution. Significant differences were observed in the uptake capacities of gadolinium ions by the various microorganisms used and no general relationship was applicable to all microbial species. These differences could be related to the nature, the structure and the composition of the cell wall layers and the specific surface developed by the sorbents in suspension. Morley and Gadd (1995) concluded for fungal biomass that the different cell wall polymers have various functional groups and differing charge distributions and therefore different metal-binding capacities and affinities. Schiewer and
Wong (2000) described a decrease in the biosorption capacities in relation to the algae species. Furthermore, the physiological stage of the bacteria seems to be important in the case of Mycobacterium smegmatis (Andrès et al.
2000) This observation could be explained by the fact that cell starvation leads to a modification in the composition of the cell wall layer. Penumarti and Khuller (1983) measured effectively an increase in the total amount of mannosides with the age of culture from Mycobacterium smegmatis. These observations could be correlated with the variation in the composition of the macromolecular compounds or in their quantity at the microbial surface and with the growth conditions. Daughney et al. (2001) have shown that the number of functional groups present at the cell surface, their pKa values and, related to these, the electronegativity of the cells wall could be changed according to the physiological state of the bacteria. Various authors (Volesky 1994, Andrès et al. 2000, Goyal et al. 2003) have shown that biosorption on bacterial, fungal and yeast biomass is a function of the growth medium composition and the culture age of the cells. McEldowney and Fletcher (1986) concluded that the macromolecular compounds of bacterial surfaces varied in quantity and in composition with the growth conditions and the growth rate.