Laura Giagnoni

Role of Phosphatase Enzymes in Soil

Soil phosphatases, particularly acid and alkaline phosphomonoesterases, have been extensively studied [see reviews by Ramirez-Martinez (Folia Microbiol 13:161–174, 1968); Speir and Ross (Soil phosphatase and sulphatase. Soil enzymes. Academic, London, pp 197–250, 1978); Malcom (Soil Biol Biochem 15:403–408, 1983); and Tabatabai (Soil enzymes. Methods of soil analysis. Part 2. Microbiological and biochemical properties. Soil Science Society of America, Madison, pp 775–833, 1994)] because they mineralise organic phosphorus (P) to inorganic P. The effects of agricultural and forest managements, pollutants and any environmental factor on phosphatase activities of soil cannot be adequately interpreted because the currently available enzyme assays do not discriminate between the contribution of phosphatases associated with active microbial cells and that of extracellular phosphatases stabilised by soil colloids. Despite multiple evidence indicating that phosphatases can be adsorbed by surface-reactive particles such as clay minerals or entrapped by humic materials, the visualisation of the extracellular phosphatases in the soil matrix has never been achieved because ultracytochemical tests combined with electron microscopy cannot locate enzymes in electron-dense minerals such as clays, or in soil components such as humic materials, that react with counterstainers such as OsO4.

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.

Maize lines with different nitrogen use efficiency select bacterial communities with different β-glucosidase-encoding genes and glucosidase activity in the rhizosphere

We studied the molecular diversity of β-glucosidase-encoding genes, microbial biomass, cellulase, N-acetyl-glucosaminidase, β-glucosidase, and β-galactosidase activities in the rhizosphere and bulk soil of two maize lines differing in nitrogen use efficiency (NUE). The maize lines had significant differences in diversity of β-glucosidase-encoding genes in their rhizosphere, and Actinobacteria and Proteobacteria were the dominating phyla in all samples, but representatives of Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Cyanobacteria were also detected. Among the Proteobacteria, β-glucosidase genes from α-, β-, and γ-Proteobacteria were dominant in the rhizosphere of the high NUE maize line, whereas δ-Proteobacteria β-glucosidase genes were dominant in the rhizosphere of the low NUE maize line. The high NUE maize line also showed higher glucosidase activities in the rhizosphere than the low NUE maize line. We concluded that plants with high NUE select bacterial communities in the rhizosphere differing in the diversity of β-glucosidase-encoding genes which likely result in higher C-hydrolyzing enzyme activities. These effects on the diversity of β-glucosidase-encoding genes may influence the C dynamics in the agro-ecosystems.

Enzyme activity and microbial community structure in the rhizosphere of two maize lines differing in N use efficiency

AimsStudy of the changes in soil microbial biomass, enzyme activity and the microbial community structure in the rhizosphere of two contrasting maize lines differing in the nitrogen use efficiency (NUE).MethodsThe Lo5 and T250 inbred maize characterized by high and low NUE, respectively, were grown in rhizoboxes allowing precise sampling of rhizosphere and bulk soil and solution. We also determined microbial biomass, enzyme activities involved in the C, N, P and S cycles, and the microbial community structure using a phylogenetic group specific PCR-DGGE approach in the rhizosphere and bulk soil of both Lo5 and T250 maize lines.ResultsHigh NUE Lo5 maize induced faster inorganic N depletion in the rhizosphere and larger changes in microbial biomass and enzyme activities than the low NUE T250 maize line. The two maize lines induced differences in the studied microbial groups in the rhizosphere, with the larger modifications induced by the high NUE Lo5 maize line.ConclusionsThe Lo5 maize line with higher NUE induced larger changes in soil chemical properties and in the enzyme activity, soil microbial biomass and community structure than the low NUE T250 maize line, probably due to differences in the root exudates of the two maize lines.

Interactions between proteins and humic substances affect protein identification by mass spectrometry

Soil proteomics is facing problems such as low yields of protein extraction from soil and low protein identification rates as compared to theoretical estimates of soil proteome. This work aimed to evaluate the effect of soil-borne humic substances (HS) on the identification of model proteins with different properties, such as myoglobin (Mb), α-glucosidase (αG), and β-glucosidase (βG), by using electrophoretic and ESI- and MALDI-mass spectrometry (MS) methodologies. Results showed that the contact between proteins and HS did not alter protein electrophoretic mobility but led to protein modifications that affected protein identification by MS. The decrease in protein identification parameters was more evident for Mb than for αG and βG, probably due to its lower molecular weight and less complex molecular structure. Analysis of MS data indicated that hydrophobic interactions could be responsible for the observed effects of contact between proteins and HS.

Assessment of Methods for Determining Bioavailability of Trace Elements in Soils: A Review

Abstract Trace element-contaminated soils (TECSs) are one of the consequences of the past industrial development worldwide. Excessive exposure to trace elements (TEs) represents a permanent threat to ecosystems and humans worldwide owing to the capacity of metal(loid)s to cross the cell membranes of living organisms and of human epithelia, and their interference with cell metabolism. Quantification of TE bioavailability in soils is complicated due to the polyphasic and reactive nature of soil constituents. To unravel critical factors controlling soil TE bioavailability and to quantify the ecological toxicity of TECSs, TEs are pivotal for evaluating excessive exposure or deficiencies and controlling the ecological risks. While current knowledge on TE bioavailability and related cumulative consequences is growing, the lack of an integrated use of this concept still hinders its utilization for a more holistic view of ecosystem vulnerability and risks for human health. Bioavailability is not generally included in models for decision making in the appraisal of TECS remediation options. In this review we describe the methods for determining the TE bioavailability and technological developments, gaps in current knowledge, and research needed to better understand how TE bioavailability can be controlled by sustainable TECS management altering key chemical properties, which would allow policy decisions for environmental protection and risk management.

Management with willow short rotation coppice increase the functional gene diversity and functional activity of a heavy metal polluted soil

We studied the microbial functional diversity, biochemical activity, heavy metals (HM) availability and soil toxicity of Cd, Pb and Zn contaminated soils, kept under grassland or short rotation coppice (SRC) to attenuate the risks associated with HM contamination and restore the soil ecological functions. Soil microbial functional diversity was analyzed by the GeoChip, a functional gene microarray containing probes for genes involved in nutrient cycling, metal resistance and stress response. Soil under SRC showed a higher abundance of microbial genes involved in C, N, P and S cycles and resistance to various HM, higher microbial biomass, respiration and enzyme activity rates, and lower HM availability than the grassland soil. The linkages between functional genes of soil microbial communities and soil chemical properties, HM availability and biochemical activity were also investigated. Soil toxicity and N, P and Pb availability were important factors in shaping the microbial functional diversity, as determined by CCA. We concluded that in HM contaminated soils the microbial functional diversity was positively influenced by SRC management through the reduction of HM availability and soil toxicity increase of nutrient cycling. The presented results can be important in predicting the long term environmental sustainability of plant-based soil remediation.

Use of phytoremediated sediments dredged in maritime port as plant nursery growing media

We evaluated the potential of a phytoremediated sediment (TR) dredged from maritime port as peat-free growth substrate for seven ornamental plants, in comparison with an untreated sediment (NT), in a greenhouse experiment. The studied plants were Quercus ilex, Photinia x fraseri, Viburnum tinus, Cistus albidus, Raphiolepis indica, Westringia fruticosa and Teucrium fruticans. Plant growth was monitored for ten months, and the changes in the physico-chemical properties, toxicity, microbial biomass and enzyme activities involved in the C, P and N cycles were also monitored during the plant growth period. The results showed that the studied ornamental plants could grow on both NT and TR sediments, but that the growth was higher on TR sediment. The plant growth induced changes in the sediment chemical functional groups, with clear separation between NT and TR sediments for each of the studied plant. Microbial biomass and enzyme activities significantly increased during the plant growth, more in TR than in NT sediment. Toxicity was detected in NT sediments during the plant growth whereas it was not observed in NT sediments during the whole growth period. We concluded that phytoremediation converted the dredged maritime sediments into suitable substrates for growing ornamental plants, and that the re-use by plant nursery industry can be a sustainable management and valorization for remediated sediments.

Greenhouse gas emissions from a Cu-contaminated soil remediated by in situ stabilization and phytomanaged by a mixed stand of poplar, willows, and false indigo-bush

ABSTRACT Phytomanagement of trace element-contaminated soils can reduce soil toxicity and restore soil ecological functions, including the soil gas exchange with the atmosphere. We studied the emission rate of the greenhouse gases (GHGs) CO2, CH4, and N2O; the potential CH4 oxidation; denitrification enzyme activity (DEA), and glucose mineralization of a Cu-contaminated soil amended with dolomitic limestone and compost, alone or in combination, after a 2-year phytomanagement with a mixed stand of Populus nigra, Salix viminalis, S. caprea, and Amorpha fruticosa. Soil microbial biomass and microbial community composition after analysis of the phospholipid fatty acids (PLFA) profile were determined. Phytomanagement significantly reduced Cu availability and soil toxicity, increased soil microbial biomass and glucose mineralization capacity, changed the composition of soil microbial communities, and increased the CO2 and N2O emission rates and DEA. Despite such increases, microbial communities were evolving toward less GHG emission per unit of microbial biomass than in untreated soils. Overall, the aided phytostabilization option would allow methanotrophic populations to establish in the remediated soils due to decreased soil toxicity and increased nutrient availability.

Past, Present and Future in Soil Enzymology

The bibliography on soil enzymes is extensive as showed by books and many review chapters devoted to the subject. The assays of soil enzymes are generally simple, accurate, sensitive and relatively rapid and for this reasons they have been extensively used to determine the effects of contaminants, changes in management practices and effects of environmental factors and plant cover on soil metabolism. However, the present enzyme assays determine potential rather than real enzyme activities due to the optimal conditions of the assays and they do not discriminate the contribution of extracellular stabilised enzymes from that of intracellular enzyme activities. The determination of the latter is important to evaluate the answer of soil microorganisms to any effect on soil. Methods based on fumigation of soil with chloroform or with the physiological response of soil microorganisms to glucose addition to soil present drawbacks. Presently, enzyme activities are still used to evaluate the response of soil metabolism to any effect not only in arable soils but also in forest soils. However, not always the past bibliography and the limits of the present enzyme assays are considered. A few innovative studies have been carried out. Measurements of enzyme activities have been combined with those on microbial diversity evaluated by molecular techniques. Both synthesis and persistence of phosphomonoesterases have been quantified in studies based on the stimulation of microbial growth by adding easily degradable organic compounds to soil. Metcalfe et al. (Metcalfe AC, Krsek M, Gooday GW, Prosseer JI, Wellington EM (2002) Appl Environ Microbiol 68:5042–5050) covered all events from gene presence, through gene expression and up to the detection of target enzyme in soil. The addition of sludge to a pasture soil increased chitinase activity and the number of actinobacteria but selected actinobacterium-like chitinase sequences. Enzyme assays distinguishing the contribution of extracellular stabilised enzymes from that of intracellular enzyme activities are needed. Future research should increase the number of enzyme activities which can be determined in soil. For example, an accurate assay for determining nuclease activity in soil is not available. It is important to set up accurate methods for extracting intracellular and stabilised extracellular proteins, which are largely prevailing, so as to be able to carry out the proteomic approach in soil. The understanding of microbial synthesis of proteins (functional proteomic) as affected by different environmental conditions can increase our knowledge on the synthesis of enzymes in soil whereas the characterization of proteins protected against microbial degradation by their interactions with surface-reactive particles or their inclusion within humic component (structural proteomic) can give insights on the stabilization of organic N, including enzymes, in soil. The set up of suitable techniques is needed to visualise the location of stabilised enzymes in soil sections by both scanning electron microscopy and transmission electron microscopy. Acid phosphatase activity has been detected in small (7 × 20 nm) fragments of microbial membranes, roots, mycorrhizae, etc. of soil but not in naturally-electron dense soil components (minerals) and in soil components reacting with OsO4 (humus) and this does not permit to localize extracellular enzymes or proteins stabilized by clay minerals or humic materials (Ladd JN, Butler JHA (1966) Aust J Soil Res 4:41–54).