The most important piece of evidence of the presence of stabilised enzymes in soil derives from studies on the nitrogen (N) distribution in soil carried out in the 1960s and 1970s and based on the use of acid hydrolysis of soil (Bremner 1965; see also Chap. 2). Amino acids are the main identifiable organic N compounds in soil hydrolysates, where they can range from 30 to 45% of total soil N (Stevenson 1986; Schulten and Schnitzer 1998). However, net N mineralisation, i.e. the amount of N made available to plants, accounts for 2% of the total N, and it corresponds to 100-200 kg N per hectare, a normal rate of N fertilisation (Stevenson 1986). An intriguing question is how this organic N is slowly mineralised. A better understanding of the state of proteins and protein-colloid complexes in soil can clarify mechanisms of the N mineralisation. By considering that the percentage of total N in microbial biomass averages to 4% (Jenkinson 1988) and that acid hydrolysis breaks peptide bonds, it is reasonable to hypothesise that most of the amino acid N in soil hydrolysates derives from extracellular protein N stabilised in soil by soil colloids. The percentage of total N present as amino acid N (85%) determined by 15N NMR was even greater than that found after acid hydrolysis of soil (Knicker et al. 1993; Schulten and Schnitzer 1998). However, direct analysis by 15N NMR spectrometry is difficult because natural 15N abundance is very low (0.366%; see Chap. 2).
The visualisation by scanning electron microscopy of soil sections prepared by ultracytochemical tests has allowed the detection of enzymes such as acid phosphatase, succinic dehydrogenase, peroxidase, and catalase in root and microbial cells and acid phosphatase in fragments of microbial membranes as small as 7 X 20 nm (Foster and Martin 1981; Foster 1985; Ladd et al. 1996). Unfortunately, the presence of electron-dense soil components, such as minerals, or soil components, such as humic molecules, which aspecifically react with the counterstainer OsO4,causedproblemsfor the detection of enzymes adsorbed by clay minerals or englobated by hu-mic complexes. These problems were partially overcome by using electron probe microanalysis and proper controls, where the enzyme was inhibited or the substrate not added (Ladd et al. 1996). By using this approach no phosphatase activity was found on quartz particles while enzyme activity associated to microbial cells was detectable, and there was also little evidence for the presence of phosphatase activity associated to clays (Ladd et al. 1993).
However, it is well established that the contents of organic matter and microbial biomass depend on the clay content of soil (Ladd et al. 1996). Already Jenny in 1941 concluded that the clays retard the decomposition of organic matter by comparing soils differing in clay content but with similar climate, vegetation and management regimes. The addition of 14C-labelled simple (such as glucose) or complex (vegetable remains) substrates to soils with varying clay content has led to the understanding that the clays do not affect the prime degradation of the substrate but the successive stabilisation of the microbial degradation products and the synthesised microbial biomass (Ladd et al. 1996). Differences in the dynamics of C and N turnover were assessed by studies based on fractionation procedures of soils treated with 14C- and 15N-labelled compounds. Extracellular N organic compounds produced by microorganisms were preferentially associated with clay particles during early periods of microbial degradation of 14C-and 15N-labelled wheat straw or 14C-labelled glucose added with 15N-NH+ (Ladd et al. 1996). As shown in Chap. 7, the bibliography on the properties of protein-clay complexes prepared in the laboratory is extensive.
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