Humic-enzyme complexes

Contact: Grazia Masciandaro (Unit of Pisa)
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The aim of this research is to investigate the chemico-structural properties of soil organic matter and the interaction between humic substances and extracellular enzymes (humus-enzyme complexes). Enzymes that bind clay minerals and/or humic substance are more stable to some environmental stresses than free enzymes and can persist longer than microbial cells that produced them. Humic-bound enzymes represent a sink of biochemical energy and slow-release nutrients capable to sustain the ecosystem functionality even in stressed situations; they are considered the last barrier against irreversible soil degradation (desertification) and are co-responsible of soil resilience. The maintenance of an equilibrium is necessary between the “transient” part of the organic matter, which can be used by microorganisms and is an useful energy source to maintain the cycle of nutrients, and the “stable” humic part, which represent the “cement” of the soil mineral particles. The biochemical properties of humic matter can be investigated by means of the isoelectric focusing and the ratio between the “transient” and the humic “stable” fractions through gas-chromatographic pyrolysis.


Extraction of stabilised enzymes and their purification are necessary actions to study better the origin, location and persistence of the extracellular enzymes in soil. Extracted humus-enzyme complexes can be fractionated according to the following three steps:

  1. sodium pyrophosphate extraction (pH 7.1) of humic matter;
  2. ultrafiltration (UF) of the organic extracts by molecular weight cut-off membranes (mol wt >104);
  3. analytical isoelectric focusing technique (IEF). IEF can isolate humic-enzyme complexeson the basis of their isoelectric point (pI), which depends on the net surface charges, by preserving enzyme activity and molecular structure of the complexes ( Fig.1 -a and Fig.1 -b ).

Humo-enzyme complexes are possible candidates for assessing soil ecosystem quality under:

  • natural conditions (supporting microbial activity and contributing to soil biological fertility);
  • stressed conditions (seeming to be the necessary condition for soil resilience);
  • restored conditions (indicating potential reaction of soil to regeneration practices).


In addition, the study of biochemical and chemical-structural properties of organic matter represent an useful tool to investigate the characteristics of different matrices, such as sludges and sediments as well as soil; moreover, it is possible to highlight the effects of the stabilization of organic matter with respect to pollutants, such as heavy metals and toxic organic compounds, reducing their bioavailability for the environment. Changes in the chemical-structural properties of sludges treated in a reed bed system have been evaluated through the technique of Pyrolysis-Gas Chromatography (Py-GC). The mineralization index (pyrrole/furfural, O/N) showed a significant decrease, while the humification index (benzene/toluene, B/E3) increased over time (Fig.2); these results confirmed the effectiveness of biostabilization process occurring in reed beds system as the labile organic matter is degraded the more stable one (with humic characteristics) is being formed during the time.