Grazia Masciandaro

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.

Isolation by isoelectric focusing of humic-urease complexes from earthworm (Eisenia fetida)-processed sewage sludges

Abstract Vermicomposting is an eco-biotechnological process that transforms energy-rich and complex organic substances into a stabilized humus-like product. In a laboratory experiment, Eisenia fetida (Sav.) earthworms were employed to process putrescible sewage sludges into a high-value biofertilizer, very rich in urease activity and humic-urease complexes (stabilized extracellular enzymes). Extracellular humic-urease complexes were extracted by a single 24-h extraction at 37  °C using neutral pyrophosphate (0.1 M); then, the extracts were dialysed and characterized by means of an analytical isoelectric focusing technique. This technique gave a multiplicity of humic bands enzymatically active, with isoelectric points ranging from 4.8 to 5.6. The results demonstrated that, after an 18-week incubation period, sewage sludge had undergone a biochemical evolution, which caused a doubling of absolute urease activity and a six-fold increase in specific activity (activity with reference to the humic C fraction). The biochemical evolution of the vermicompost was evaluated also from the sharp decrease in pyrophosphate-extractable C and water-soluble C. Stabilization of organic C during vermicomposting and the activity of humic-urease complexes expressed at low pH values are of extreme importance when organic wastes are used in acid soils for biochemical restoration purposes.

“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.

Enzyme activity and C and N pools in soil following application of mulches

A field experiment was carried out to evaluate the effects of some mulching materials on soil biochemical properties that represent a component of soil quality and functionality. Wheat straw, dairy manure compost, and black polyethylene were used in the mulching experiments of a soil seeded with forage maize. Soil samples were taken from the surface (0–10 cm) and subsurface (10–20 cm) layers 1 and 4 mo after treatments were applied. Soil productivity, biochemical and chemical properties were determined. Compost treatment showed the highest agronomic yield due to the release of nutrients for plant nutrition and soil metabolism. Dehydrogenase activity was used as a measurement of overall microbial activity, whereas β-glucosidase and urease hydrolase activities were determined as an expression of C and N cycles. In addition, a ratio between dehydrogenase activity and water soluble carbon (defined as “metabolic potential index”) was calculated, in order to better represent soil metabolic potential. Compost-mu...

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.

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.

Kinetics of phosphatase activity in organic wastes

Abstract The phosphatase activity of different urban wastes, both fresh and composted, were determined in order to establish any differences which might exist between them as regards several kinetic parameters; these parameters being correlated with the degree of evolution of the organic matter contained in the wastes. All the materials studied contained alkaline phosphatase with an optimal activity at pH 9. The sewage sludges also contained neutral phosphatase (optimum pH 6–7). The composts showed the lowest values of phosphatase activity, suggesting that this parameter can be considered a valid index of the evolution of the organic matter contained in the wastes. The values of K m estimated for different substrate concentrations from the four Michaelis-Menten derived lineal equations, revealed the difficulty of establishing relationships between the affinity of the enzyme for the substrate and the nature of the waste or the degree of stability of its organic matter. The values of maximum phosphatase velocity in the four equations used appeared to establish differences between the different types of waste and between the composted and uncomposted products. Values of V max correlated both with ATP content and with the different carbon fractions of the wastes.

Assessment of pollution impact on biological activity and structure of seabed bacterial communities in the Port of Livorno (Italy)

The main objective of this study was to assess the impact of pollution on seabed bacterial diversity, structure and activity in the Port of Livorno. Samples of seabed sediments taken from five selected sites within the port were subjected to chemical analyses, enzymatic activity detection, bacterial count and biomolecular analysis. Five different statistics were used to correlate the level of contamination with the detected biological indicators. The results showed that the port is mainly contaminated by variable levels of petroleum hydrocarbons and heavy metals, which affect the structure and activity of the bacterial population. Irrespective of pollution levels, the bacterial diversity did not diverge significantly among the assessed sites and samples, and no dominance was observed. The type of impact of hydrocarbons and heavy metals was controversial, thus enforcing the supposition that the structure of the bacterial community is mainly driven by the levels of nutrients. The combined use of chemical and biological essays resulted in an in-depth observation and analysis of the existing links between pollution macro-indicators and biological response of seabed bacterial communities.

Almond tree and organic fertilization for soil quality improvement in southern Italy.

The semi-arid Mediterranean region, characterized by long dry periods followed by heavy bursts of rainfall, is particularly prone to soil erosion. The main goal of this study is to evaluate the soil quality under different practices of bio-physical amelioration which involve the soil-plant system (almond trees) and microorganism-manure. This study, carried out in the South of Italy (Basilicata Region- Pantanello farm), considered two types of fertilization (mineral and organic) and three slope gradients (0, 2 and 6%), in order to evaluate the effects of management practices in resisting soil erosion. Chemical (organic carbon and nitrogen), physical (soil shrinkage and bulk density) and biochemical (dehydrogenase activity and hydrolytic enzyme activities) parameters were selected as markers to follow agro-ecological changes with time. The organic treatment affected soil microbiological and physico-chemical properties by increasing soil nutrient availability, microbial activity, and improving soil structure. The consistently higher values of the hydrolytic enzyme activities (β-glucosidase, phosphatase, urease and protease) often observed in the presence of plants and on the 0 and 2% slopes, suggested the stimulation of nutrient cycles by tree roots, which improve the conditions for soil microorganisms in carrying out their metabolic activity. In the 6% slope and, in particular, in the mineral fertilizer treatment, soil metabolism was lower as suggested by the dehydrogenase activity which was 50% lower than that found in the 0 and 2% slopes, this seemed to be related to a slowdown in the nutrient cycling and organic carbon metabolism. However, on this slope, in both mineral and organic treatments, a significant stimulation of hydrolytic enzyme activities and an improvement of soil structure (reduction of bulk density of about 10% and increase in total shrinkage from 20 to 60%) were observed with plants compared to the control soil. The combination of organic fertilization and almond trees resulted effective, also in the highest slope, in mitigating the degradation processes through the improvement of chemico-nutritional, biochemical and physical soil properties.