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Phenol oxidases (POs) are a group of soil extracellular oxidoreductase enzymes, which are involved in oxidative processes related to nutrient cycling. This class of enzymes has multiple functions at both the organism and ecosystem level and can trigger either positive or negative feedback loops between soil organisms and soil organic matter. The purpose of this study was to evaluate: (i) whether PO activities have a trend different from those of microbial biomass and activity; and (ii) whether PO activities are enhanced or reduced by fertilizer application. Soil samples were taken from plots in a 14-year-old experimental walnut orchard, subjected, since planting, to three fertilizer treatments: organic (compost) or mineral (urea) fertilization at the same rate of nitrogen application (100 kg N ha− 1), or left unfertilized. Soil samples were divided according to sampling depth (0–20 cm and 20–40 cm). Results showed that the application of compost significantly increased C and N pools. qCO2 and DHD/Cext data indicated that the biota at 20–40 cm soil depth was more stressed or mainly composed of microorganisms with low substrate utilization efficiency. Phenol oxidase, tyrosinase, and catechol oxidase activities were significantly promoted in the surface layer by compost addition. In contrast, laccase activity showed a large increase in the deeper soil layer when supplied with mineral N, whereas compost addition led to increased activity in the surface layer. These findings suggest that soil phenol oxidases play a part in the determination of soil functionality, but they need to be investigated in greater depth in order to understand the mechanisms regulating their activities.

none 7 no The aim of the present study was to compare the quantity and the type of carbon (C) stored during the 14-year lifetime of a commercial nectarine orchard ecosystem fertilized with mineral or organic fertilizers. The study was carried out in the Po valley, Italy, in a nectarine orchard of the variety Stark RedGold, grafted on GF677 hybrid peach × almond. Since orchard planting in August 2001, the following treatments were applied in a randomized complete block design with four replicates per block and compared: 1. unfertilized control; 2. mineral fertilization (including P and K at planting and N applied as NO3NH4 yearly at the rate of 70–130 kg ha−1); 3. compost application at a rate of 5 Mg DW ha−1 yr−1; 4. compost application at a rate of 10 Mg DW ha−1 yr−1. Compost was obtained from domestic organic wastes mixed with pruning material from urban ornamental trees and garden management after a 3-month stabilization period. Application of compost at the highest rate increased C in the soil; the amount of C sequesteredwas approximately 60% fromamendment source and 40% fromthe net primary production of trees and grasseswith a net increase of C compared to mineral fertilization. Compost application was found to be a win-win strategy to increase C storage in soil and, at the same time, to promote plant growth and yield to levels similar to those obtained with mineral fertilization. The rate of C application is crucial, indicated by the fact that compost supply at the rate of 10Mg ha−1 yr−1 was the only fertilization strategy of the ones tested that resulted in higher C sequestration. This shows that compost amendment may stimulate an increase in the net primary production of plants. mixed E. Baldi , L. Cavani, A. Margon, M. Quartieri, G. Sorrenti, C. Marzadori, M. Toselli E. Baldi , L. Cavani, A. Margon, M. Quartieri, G. Sorrenti, C. Marzadori, M. Toselli

Among the numerous products so far promoted as effective urease and/or nitrification inhibitors, it is possible to detect a renewed interest in environmentally friendly tools, such as ammonium thiosulphate (ATS, (NH4)2S2O3) which is currently used as fertilizer for N and S nutrition. Among contradictory results accounted in the current literature, there is reported that ammonium thiosulphate (ATS) exerts inhibitory activity at large but unlikely agronomic rates of 2500 - 5000 mg·kg-1 soil. We carried out a novel experiment aimed to: a) verify the inhibitory action of ATS, even when applied in soil at low rates (25 and 100 mg·kg-1 soil as S-ATS), towards urease activity and nitrification in urea treated and not treated soils; b) investigate on the influence of ATS on the soil microbial biomass as it is generally assumed that soil microorganisms are the main agents of urea hydrolysis. For these purposes we selected an arable sandy soil and a grassland sandy-loam soil which are treated with urea or/and ATS. Results obtained from this novel investigation showed that a) ATS significantly decreased urease activity in both soil types and that the exerted inhibitory effect was moderate and short-term; b) ATS retarded the hydrolysis of urea and lowered nitrate production in the urea treated soils; c) the available fraction of iron and manganese in the used soils might be involved in the mechanism of inhibition; d) ATS did not affect the size of soil microbial biomass pool; e) the efficiency of ATS as urease and nitrification inhibitor was more evident in the sandy soil. Because of its properties, ATS may be applicable on many crops without being harmful on the soil microbial pool.

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment.

Protein hydrolyzates (PHs) are a well-known group of plant biostimulants that are obtained by thermal, chemical, enzymatic or mixed hydrolysis from animal or plant sources. PHs are mainly applied to plants with the aim to improve their nutrition level, stimulate metabolism, and increase resistance to abiotic stress. Chemically, the PHs are a mixture of free amino acids, oligo- and polypeptides. Considering the differences in the source materials and hydrolytic processes used for their production it is not surprising that PHs are a very complex and inhomogeneous category of biostimulants. This is a critical point that should be considered by both manufacturers and potential users of such products. With the aim to identify a putative correlation among PHs physic-chemical properties, the different source materials and the hydrolytic process used for their production, 22 PHs available on the market were analyzed for: (i) total amino acids concentration, (ii) free amino acids concentration, (iii) degree of racemization, (iv) degree of hydrolysis and (v) apparent average molecular size. The obtained results confirmed the inhomogeneity PHs. However, the chosen parameters were able to provide information about the above-mentioned correlation. Total amino acids in particular were useful for the identification of the original source material. The degree of racemization was useful for the identification of the type of hydrolysis used during production (chemical or enzymatic). The degree of hydrolysis and free amino acid concentrations were useful for the identification of the intensity of the hydrolytic process. The average molecular size was useful for the identification of polydispersity of the peptide size. In conclusion, the PHs can be adequately characterized only through a multi-analytical approach.

The main aim of our work was to assess whether strontium (Sr) affects soil microbial biomass size and activity, and the involvement of said biomass in the availability process of the metal. In addition, information concerning the distribution and mobility of the stable element within ecosystems may allow the prediction of the behaviour of its radioisotope counterpart, 90Sr. Samples were collected in the surroundings of a strontium mine and characterised for total and diethylene triamine pentaacetic acid (DTPA)-extractable Sr, total organic C (TOC), microbial biomass C (MBC), MBC/TOC ratio and metabolic quotient (qCO2). Moreover, MBC and DTPA-extractable Sr were measured during a 45-day incubation experiment of samples soils amended with maize. Overall, increased levels of total Sr had a negative effect on both TOC and MBC. DTPA-extractable Sr was significantly correlated to MBC/TOC suggesting a possible role of soil microbial biomass in the mobilisation of the element. The synthesis of new microbi...

In this study we evaluated the dynamics of Cr(VI) reduction in contaminated soil amended or not with glucose together with investigation on its effects on soil microbial biomass size and activity. The influence of native or glucose-induced soil microbial biomass on chromate reduction was assessed. During the incubation period, the concentration of Cr(VI) in soil decreased over time whether glucose was added to soil or not, but with different reduction rates. Soil displayed a natural capacity towards chromate reduction, but the addition of glucose increased the reduction rate by both abiotic and biotic mechanisms. Cr(VI) negatively affected both the size and activity of soil microbial biomass. However, our data suggest that glucose is likely to have exerted an indirect role in the increased rate of Cr(VI) reduction, by promoting growth of indigenous microbial biomass.

The interaction between hexavalent chromium Cr(VI), as K(2)CrO(4), and standard humic acids (HAs) in bulk solution was studied using three complementary analytical methods: UV-Visible spectroscopy, X-ray absorption spectroscopy and differential pulse stripping voltammetry. The observed UV-Vis and X-ray absorption spectra showed that, under our experimental conditions, HAs did not induce reduction of Cr(VI) to its trivalent chemical form. The interaction between Cr(VI) and HAs has rather led to the formation of Cr(VI)-HAs micelles via supramolecular chemical processes. The reported results could contribute towards explaining the relative persistence of ecotoxic hexavalent chromium in soils.

Knowledge concerning the location of soil enzymes and their kinetics is necessary for a better understanding of their roles in biogeochemical cycles. A rapid dichloromethane fumigation procedure and a new extraction method giving negligible co-extraction of humic substances were used. Fumigation and consequent cell lysis increased the activity of arylsulfatase, but not that of acid and alkaline phosphomonoesterase, suggesting that only the former enzyme was located both extracellularly and intracellularly. Extracted arylsulfatase displayed a Michaelis–Menten constant (Km) significantly lower than in fumigated soil, which in turn was significantly lower than in unfumigated soil. In contrast, each phosphomonoesterase displayed the same Km in extracts, fumigated and unfumigated soils. These results show that activity of soil phosphomonoesterases is not influenced by any direct interaction with soil and, therefore, are more likely to be extracellular enzymes primarily devoted to biogeochemical cycling than are arylsulfatases.

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.

Extraction of arylsulphatase (aryS), acid (acP) and alkaline phosphomonoesterase (alkP) from six different soils using sodium pyrophosphate (0.14 M, pH 7.1) yielded brown extracts displaying enzymatic activity mostly below detection limit. Tris–HCl (50 mM, pH 7.5) gave an extraction yield, on average, lower than 0.5%, but addition of Triton X-100 or bovine serum albumine (BSA) to Tris buffer increased the extraction yield 2–8 times. When both Triton X-100 and BSA were added to the buffer, the extraction yield was more than additive and reached 2–13% for acP, 2–5% for alkP and 3–6% for aryS, depending on the soil. In addition, these extracts were colourless or at most light yellow, showing that besides the high yield enzymes were extracted along with negligible amounts of humic substances.

The interaction between the non-steroidal anti-inflammatory drug diclofenac and standard humic acids (HAs) in bulk solution was studied using two complementary analytical methods: UV-Visible spectroscopy and square wave voltammetry. The observed UV-Vis spectra and Ip/V curves suggested that, at our experimental conditions, albeit both substances being negatively charged at pH 6.5, interaction between the pharmaceutical and the soil humic acids may led to the formation of diclofenac-humic acids supramolecules. Our results could contribute to give information on the behaviour of diclofenac into the soil environment, thus suggesting its migration as HAs-micelles through the coarse soil profile.