Brunello Ceccanti

Soil enzymology: classical and molecular approaches

It is still problematic to use enzyme activities as indicators of soil functions because: (1) enzyme assays determine potential and not real enzyme activities; (2) the meaning of measured enzyme activities is not known; (3) the assumption that a single enzyme activity is an indicator of nutrient dynamics in soil neglects that the many enzyme activities are involved in such dynamic processes; (4) spatio-temporal variations in natural environments are not always considered when measuring enzyme activities; and (5) many direct and indirect effects make difficult the interpretation of the response of the enzyme activity to perturbations, changes in the soil management, changes in the plant cover of soil, etc. This is the first review discussing the links between enzyme-encoding genes and the relative enzyme activity of soil. By combining measurements of enzyme activity in soil with expression (transcriptomics and proteomics) of genes, encoding the relative enzymes may contribute to understanding the mode and timing of microbial communities’ responses to substrate availability and persistence and stabilization of enzymes in the soil.

Kinetic parameters of dehydrogenase in the assessment of the response of soil to vermicompost and inorganic fertilisers

Abstract Kinetic parameters (Vmax and Km) of dehydrogenase activity were determined in order to assess the metabolic response of a soil about 1 year after organic and mineral treatments. The soil was planted with maize (Zea mays) and treated with the following fertilisers: organic (vermicompost; VC), mineral (ammonium phosphate and urea), and an organo-mineral mixture. Vmax, which represents a measurement of the quantity of enzyme, markedly increased in organic and organo-mineral treatments, indicating that the addition of organic matter caused an increase in dehydrogenase in the active microbial biomass. Km, representing enzyme-substrate affinity and/or different sources of the enzymes, was similar in VC-treated soil and control soil, while it doubled in organo-mineral and mineral treatments. These results suggest that the use of VC did not alter the enzyme-substrate affinity, while mineral fertiliser reduced this affinity or changed the composition and activity of soil microbiota. A positive correlation was found between Vmax, the metabolic index (dehydrogenase/water-soluble carbon ratio), and the soil organic matter content. The kinetic constants of dehydrogenase activity and the metabolic index may be considered valid parameters to monitor the evolution of microbiological activity in soil.

Fractionation of humus-urease complexes

Abstract Some techniques commonly used for enzyme purification were unsuitable to purify urease extracted by pyrophosphate from soil. An all-or-none behaviour seemed to be a prominent feature of such experiments but in some instances slight increases of specific activity were observed. The most effective purification of soil urease was achieved by exhaustively ultrafiltrating the soil extract against 0.1 m pyrophosphate at pH 7.1, separating the retained material into fractions of mol. wt. higher (AI) and lower (AII) than 105, followed by gel chromatography. Increases in total activities were observed both after ultrafiltration and gel chromatography. Ultra-filtration increased the total activity of the extracted urease by about 8%. The specific activity of fraction AI increased four fold, that of fraction AII by more than three fold. Fractions obtained by gel chromatography accounted in toto for only 13.5% of the organic-C of the soil extract. Total urease activity increased by 45.6%. Specific activities increased to 6.9–18 times that of the soil extract.

Hydrolases extracted from soil: Their properties and activities

Abstract The pH optima and stabilities of some hydrolases extracted by pyrophosphate from two different soils were determined for phosphatase, urease, casein and benzoylargininamide-hydrolysing proteases. Hydrolases activities of soil extracts were more resistant to thermal denaturation than their respective commercial preparations. Extracted phosphatases exhibited a marked stability to temperature and to proteolysis. By exhaustively ultrafiltering soil extracts against 0.1 M pyrophosphate at pH 7.1 fractions of mol. wt higher and lower than 100,000 were obtained. Ultrafiltration increased the total activities of extracted ureases and phosphatases while those of casein and benzoylargininamide-hydrolysing proteases were decreased. Both on an organic C and an N basis, specific activities of ureases and phosphatases increased after ultrafiltration.

Use of 0·1 m pyrophosphate to extract urease from a podzol

Abstract Sodium pyrophosphatc (0·1 m ) at pH 7.1 and 37°C extracted a significant fraction of urease from a podzol. Maximum extraction values were obtained after 18 h. The yields of soil organic matter and urease activity during the extraction show a different pattern: the extraction of non-specific organic matter precedes and may facilitate the following extraction of an active urease organo-complex. The urease extracted by pyrophosphate is about 30 40 per cent of the total urease activity, as shown by plotting the urease activity against the population changes of ureolytic microorganisms, both in the original and extracted soil. The number of ureolytic microorganisms is unaffected by pyrophosphate, and the extracted urease is assumed to be extracellular.

Stability and kinetic properties of humus-urease complexes

Abstract We have determined the kinetics and stabilities of various humus-urease preparations, extracted and purified from soil (Ceccanti, Nannipieri, Cervelli and Sequi, 1977). Km, values of five different fractions in phosphate buffer ranged from 8 to 40 mM and were similar to those of urease extracted from various other sources. However, the optimal pH for urease activity of the soil fractions ranged from 7.5 to 8.8, in contrast to a pH optimum of about 7.0 for urease from sources other than soil. The shift in the optimal pH towards the alkaline region may be due to the influence of the negative charge of the humus matrix localized around the enzyme. Urease organo-complexes of mol. wt. 105 were even more resistant, due probably to the occurrence of molecular arrangements similar to that proposed by Burns, El-Sayed and McLaren (1972a) and Burns, Pukite and McLaren (1972b). Thus although each of the five urease-active organic matter fractions exhibited different characteristics, they could be grouped into two distinct classes, according to mol. wt. and resistance to thermal denaturation and proteolysis.

“In situ” vermicomposting of biological sludges and impacts on soil quality

A laboratory experiment was carried out to study soil quality amelioration through “in situ” vermicomposting of biological sludges. The experiment dealt with the stabilization, through the action of worms (Eisenia fetida), of five mixtures containing aerobic and anaerobic biological sludges spread on the soil surface. The results showed that by increasing the percentage of anaerobic sludge in the mixtures, the number of worms which left the sludge and chose the soil as their habitat increased. The chemico-structural changes of the sludges left on the soil surface by worms were evaluated through the technique of pyrolysis-gas chromatography, which showed that the degrees of mineralization and humification of organic matter were dependent on the composition of the sludge mixtures. When the amount of aerobic sludge in the mixtures was higher than 50%, a stimulation of soil microbial metabolism occurred, as demonstrated by the index of metabolic potential (defined by dehydrogenase/water soluble carbon ratio). All treatments increased the percentage of soil total shrinkage area, mostly due to the formation of cracks of small–medium size (<1000 μm), which represent a favourable site for microbiological and biochemical processes in the soil. A positive statistical correlation between soil dehydrogenase activity, C and N substrates, and cracks of small–medium size was found.

Anaerobic Digestion of Olive Oil Mill Effluents: Evaluation of Wastewater Organic Load and Phytotoxicity Reduction

Olive oil mill effluents (OME) are of great concern worldwide dueto their role as pollutants. This work studies, on a laboratoryscale, the possibility of reducing the polluting load of olivewastewaters (organic, acid and polyphenolic) through chemical-physical processes (sedimentation-filtration) followedby a biological treatment (anaerobic digestion). This is in orderto produce biogas and obtain an effluent suitable for applicationto the soil. The anaerobic process was followed by determiningchemical-physical parameters (pH, COD, N-NH3, polyphenols).The extent of anaerobic digestion was evaluated throughmeasurements of biogas and volatile fatty acids and the microbialmetabolism was examined through dehydrogenase activity. Finally,tests of plant germination and growth were carried out usingdigested waters to determine if they could be used in agriculture.The study demonstrated that anaerobic digestion of OME firstreduced the organic load by 78-89% and the content of polyphenolsby 33-43%, and secondly produced biogas (mean value of methane83-85%). Phytotoxicity tests carried out on Lepidiumsativum seeds showed that the anaerobic treatment considerablyreduced the phyto-toxic character of OME.

Effects of petroleum contamination on soil microbial numbers, metabolic activity and urease activity.

The influence of petroleum contamination on soil microbial activities was investigated in 13 soil samples from sites around an injection water well (Iw-1, 2, 3, 4) (total petroleum hydrocarbons (TPH): 7.5-78 mg kg(-1)), an oil production well (Op-1, 2, 3, 4, 5) (TPH: 149-1110 mg kg(-1)), and an oil spill accident well (Os-1, 2, 3, 4) (TPH: 4500-34600 mg kg(-1)). The growth rate constant (μ) of glucose stimulated organisms, determined by microcalorimetry, was higher in Iw soil samples than in Op and Os samples. Total cultivable bacteria and fungi and urease activity also decreased with increasing concentration of TPH. Total heat produced demonstrated that TPH at concentrations less than about 1 g kg(-1) soil stimulated anaerobic respiration. A positive correlation between TPH and soil organic matter (OM) and stimulation of fungi-bacteria-urease at low TPH doses suggested that TPH is bound to soil OM and slowly metabolized in Iw soils during OM consumption. These methods can be used to evaluate the potential of polluted soils to carry out self-bioremediation by metabolizing TPH.

Humic substances to reduce salt effect on plant germination and growth

Soluble humic substances and saline solutions (NaCl solution or compost water extract) were tested in laboratory experiments of fertigation. The saline solutions were used at two levels of electrical conductivity (EC) (0.5 and 4 mS cm− 1); humic substances (HS) were solubilized in distilled water at two concentrations as humic carbon (HC) (30 and 60 mg L− 1). A mixture of NaCl solution with an EC of 4 mS cm− 1 and humic substances at 60 mg L− 1 as HC was also used to evaluate if HS can reduce the effect of NaCl on Lepidium sativum germination and Maize growth. Germination tests carried out without soil, showed that the best treatment was with HS. Growth tests, using an English soil seeded with Maize, presented the best result when the mixture of saline solution–humic substances was used; while the worst plant performance was obtained with NaCl solution alone. In addition, the treatments with NaCl solution alone caused, in the treated soil, a high release of C and N in soluble forms which could cause environmental pollution problems. Finally, a stimulation of soil metabolic potential occurred using HS at low concentration as HC.