Anna Rosa Sprocati

Bioremediation of diesel oil in a co-contaminated soil by bioaugmentation with a microbial formula tailored with native strains selected for heavy metals resistance.

The aim of the work is to assess the feasibility of bioremediation of a soil, containing heavy metals and spiked with diesel oil (DO), through a bioaugmentation strategy based on the use of a microbial formula tailored with selected native strains. The soil originated from the metallurgic area of Bagnoli (Naples, Italy). The formula, named ENEA-LAM, combines ten bacterial strains selected for multiple resistance to heavy metals among the native microbial community. The biodegradation process of diesel oil was assessed in biometer flasks by monitoring the following parameters: DO composition by GC-MS, CO2 evolution rate, microbial load and composition of the community by T-RFLP, physiological profile in Biolog ECOplates and ecotoxicity of the system. The application of this microbial formula allowed to obtain, in the presence of heavy metals, the complete degradation of n-C(12-20), the total disappearance of phenantrene, a 60% reduction of isoprenoids and an overall reduction of about 75% of the total diesel hydrocarbons in 42 days. Concurrently with the increase of metabolic activity at community level and the microbial load, the gradual abatement of the ecotoxicity was observed. The T-RFLP analysis highlighted that most of the ENEA-LAM strains survived and some minor native strains, undetectable in the soil at the beginning of the experiment, developed. Such a bioaugmentation approach allows the newly established microbial community to strike a balance between the introduced and the naturally present microorganisms. The results indicate that the use of a tailored microbial formula may efficiently facilitate and speed up the bioremediation of matrices co-contaminated with hydrocarbons and heavy metals. The study represents the first step for the scale up of the system and should be verified at a larger scale. In this view, this bioaugmentation strategy may contribute to overcome a critical bottleneck of the bioremediation technology.

Assessment of the applicability of a “toolbox” designed for microbially assisted phytoremediation: the case study at Ingurtosu mining site (Italy)

The paper describes the fieldwork at the Italian test site of the abandoned mine of sphalerite and galena in Ingurtosu (Sardinia), with the aim to assess the applicability of a “toolbox” to establish the optimized techniques for remediation of soils contaminated by mining activities. A preliminary characterization—including (hydro)geochemistry, heavy metal concentration and their mobility in soil, bioprospecting for microbiology and botany—provided a data set for the development of a toolbox to deliver a microbially assisted phytoremediation process. Euphorbia pithyusa was selected as an endemic pioneer plant to be associated with a bacterial consortium, established with ten selected native strains, including metal-tolerant bacteria and producers of plant growth factors. The toolbox was firstly assessed in a greenhouse pot experiment. A positive effect of bacterial inoculum on E. pithyusa germination and total plant survival was observed. E. pithyusa showed to be a well-performing metallophyte species, and only inoculated soil retained a microbial activity with a high functional diversity, expanding metabolic affinity also towards root exudates. These results supported the decision to proceed with a field trial, investigating different treatments used singly or in combination: bioaugmentation with bacterial consortia, mycorrhizal fungi and a commercial mineral amendment. Microbial activity in soil, plant physiological parameters and heavy metal content in plants and in soil were monitored. Five months after the beginning, an early assessment of the toolbox under field conditions was carried out. Despite the cold season (October–March), results suggested the following: (1) the field setup as well as the experimental design proved to be effective; (2) plant survival was satisfactory; (3) soil quality was increased and bioaugmentation improved microbial activity, expanding the metabolic competences towards plant interaction (root exudates); and (4) multivariate analysis supported the data provided that the proposed toolbox can be established and the field trial can be carried forward.

Bioprospecting at former mining sites across Europe: microbial and functional diversity in soils

The planetary importance of microbial function requires urgently that our knowledge and our exploitation ability is extended, therefore every occasion of bioprospecting is welcome. In this work, bioprospecting is presented from the perspective of the UMBRELLA project, whose main goal was to develop an integral approach for remediation of soil influenced by mining activity, by using microorganisms in association with plants. Accordingly, this work relies on the cultivable fraction of microbial biodiversity, native to six mining sites across Europe, different for geographical, climatic and geochemical characteristics but similar for suffering from chronic stress. The comparative analysis of the soil functional diversity, resulting from the metabolic profiling at community level (BIOLOG ECOPlates) and confirmed by the multivariate analysis, separates the six soils in two clusters, identifying soils characterised by low functional diversity and low metabolic activity. The microbial biodiversity falls into four major bacterial phyla: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, including a total of 47 genera and 99 species. In each soil, despite harsh conditions, metabolic capacity of nitrogen fixation and plant growth promotion were quite widespread, and most of the strains showed multiple resistances to heavy metals. At species-level, Shannon’s index (alpha diversity) and Sørensens Similarity (beta diversity) indicates the sites are indeed diverse. Multivariate analysis of soil chemical factors and biodiversity identifies for each soil well-discriminating chemical factors and species, supporting the assumption that cultured biodiversity from the six mining sites presents, at phylum level, a convergence correlated to soil factors rather than to geographical factors while, at species level, reflects a remarkable local characterisation.

Microscopic processes ruling the bioavailability of Zn to roots of euphorbia pithyusa L. Pioneer plant

Euphorbia pithyusa L. was used in a plant growth-promoting assisted field trial experiment. To unravel the microscopic processes at the interface, thin slices of E. pithyusa roots were investigated by micro-X-ray fluorescence mapping. Roots and rhizosphere materials were examined by X-ray absorption spectroscopy at the Zn K-edge, X-ray diffraction, and scanning electron microscopy. Results indicate some features common to all the investigated samples. (i) In the rhizosphere of E. pithyusa, Zn was found to exist in different phases. (ii) Si and Al are mainly concentrated in a rim at the epidermis of the roots. (iii) Zn is mostly stored in root epidermis and does not appear to be coordinated to organic molecules but mainly occurs in mineral phases such as Zn silicates. We interpreted that roots of E. pithyusa significantly promote mineral evolution in the rhizosphere. Concomitantly, the plant uses Si and Al extracted by soil minerals to build a biomineralization rim, which can capture Zn. This Zn silicate biomineralization has relevant implications for phytoremediation techniques and for further biotechnology development, which can be better designed and developed after specific knowledge of molecular processes ruling mineral evolution and biomineralization processes has been gained.

Coupled pot and lysimeter experiments assessing plant performance in microbially assisted phytoremediation.

We performed an experiment at pot scale to assess the effect of plant growth-promoting bacteria (PGPB) on the development of five plant species grown on a tailing dam substrate. None of the species even germinated on inoculated unamended tailing material, prompting use of compost amendment. The effect of inoculation on the amended material was to increase soil respiration, and promote elements immobilisation at plant root surface. This was associated with a decrease in the concentrations of elements in the leaching water and an increase of plant biomass, statistically significant in the case of two species: Agrostis capillaris and Festuca rubra. The experiment was repeated at lysimeter scale with the species showing the best development at pot scale, A. capillaris, and the significant total biomass increase as a result of inoculation was confirmed. The patterns of element distribution in plants also changed (the concentrations of metals in the roots of A. capillaris and F. rubra significantly decreased in inoculated treatments, while phosphorus concentration significantly increased in roots of A. capillaris in inoculated treatment at lysimeter scale). Measured variables for plant oxidative stress did not change after inoculations. There were differences of A. capillaris plant–soil system response between experimental scales as a result of different substrate column structure and plant age at the sampling moment. Soil respiration was significantly larger at lysimeter scale than at pot scale. Leachate concentrations of As, Mn and Ni had significantly larger concentrations at lysimeter scale than at pot scale, while Zn concentrations were significantly smaller. Concentrations of several metals were significantly smaller in A. capillaris at lysimeter scale than at pot scale. From an applied perspective, a system A. capillaris—compost—PGPB selected from the rhizosphere of the tailing dam native plants can be an option for the phytostabilisation of tailing dams. Results should be confirmed by investigation at field plot scale.

Plant growth promotion by inoculation with selected bacterial strains versus mineral soil supplements

In the process of remediation of mine sites, the establishment of a vegetation cover is one of the most important tasks. This study tests two different approaches to manipulate soil properties in order to facilitate plant growth. Mine waste from Ingurtosu, Sardinia, Italy rich in silt, clay, and heavy metals like Cd, Cu, and Zn was used in a series of greenhouse experiments. Bacteria with putative beneficial properties for plant growth were isolated from this substrate, propagated and consortia of ten strains were used to inoculate the substrate. Alternatively, sand and volcanic clay were added. On these treated and untreated soils, seeds of Helianthus annuus, of the native Euphorbia pithyusa, and of the grasses Agrostis capillaris, Deschampsia flexuosa and Festuca rubra were germinated, and the growth of the seedlings was monitored. The added bacteria established well under all experimental conditions and reduced the extractability of most metals. In association with H. annuus, E. pithyusa and D. flexuosa bacteria improved microbial activity and functional diversity of the original soil. Their effect on plant growth, however, was ambiguous and usually negative. The addition of sand and volcanic clay, on the other hand, had a positive effect on all plant species except E. pithyusa. Especially the grasses experienced a significant benefit. The effects of a double treatment with both bacteria and sand and volcanic clay were rather negative. It is concluded that the addition of mechanical support has great potential to boost revegetation of mining sites though it is comparatively expensive. The possibilities offered by the inoculation of bacteria, on the other hand, appear rather limited.

A new biogenic, struvite-related phosphate, the ammonium-analog of hazenite,(NH4) NaMg2 (PO4) 2• 14H2O

Abstract A new biogenic, struvite-related phosphate, the ammonium analog of hazenite (AAH), ideally (NH4) NaMg2(PO4)2·14H2O, has been found in cultures containing the bacterial strain Virgibacillus sp.NOT1 (GenBank Accession Number: JX417495.1) isolated from an XVII Century document made of parchment. The chemical composition of AAH, determined from the combination of electron microprobe and X-ray structural analyses, is [(NH4)0.78K0.22]NaMg2(PO4)2·14H2O. Single-crystal X-ray diffraction shows that AAH is orthorhombic with space group Pmnb and unit-cell parameters a = 6.9661(6), b = 25.236(3), c = 11.292(1) Å, and V = 1985.0(3) Å3. Compared with hazenite, the substitution of NH4+ for K+ results in a noticeable increase of the average A-O (A = NH4++K+) bond length and the unit-cell volume for AAH, as also observed for struvite vs. struvite-K. Both infrared and Raman spectra of AAH resemble those of hazenite, as well as struvite. Our study reveals that AAH forms only in cultures with Na-bearing solutions and pH below 10.0. No AAH or hazenite was found in experiments with the K-bearing solutions, suggesting the necessity of a Na-bearing solution for AAH formation.

A new biogenic, struvite-related phosphate, the ammonium-analog of hazenite, (NH4)NaMg2(PO4)2·14H2O Yang Hexiong hyang@u.arizona.edu

Abstract A new biogenic, struvite-related phosphate, the ammonium analog of hazenite (AAH), ideally (NH4) NaMg2(PO4)2·14H2O, has been found in cultures containing the bacterial strain Virgibacillus sp.NOT1 (GenBank Accession Number: JX417495.1) isolated from an XVII Century document made of parchment. The chemical composition of AAH, determined from the combination of electron microprobe and X-ray structural analyses, is [(NH4)0.78K0.22]NaMg2(PO4)2·14H2O. Single-crystal X-ray diffraction shows that AAH is orthorhombic with space group Pmnb and unit-cell parameters a = 6.9661(6), b = 25.236(3), c = 11.292(1) Å, and V = 1985.0(3) Å3. Compared with hazenite, the substitution of NH4+ for K+ results in a noticeable increase of the average A-O (A = NH4++K+) bond length and the unit-cell volume for AAH, as also observed for struvite vs. struvite-K. Both infrared and Raman spectra of AAH resemble those of hazenite, as well as struvite. Our study reveals that AAH forms only in cultures with Na-bearing solutions and pH below 10.0. No AAH or hazenite was found in experiments with the K-bearing solutions, suggesting the necessity of a Na-bearing solution for AAH formation.

A new biogenic, struvite-related phosphate, the ammonium-analog of hazenite, (NH4)NaMg2(PO4)2{middle dot}14H2O

Abstract A new biogenic, struvite-related phosphate, the ammonium analog of hazenite (AAH), ideally (NH4) NaMg2(PO4)2·14H2O, has been found in cultures containing the bacterial strain Virgibacillus sp.NOT1 (GenBank Accession Number: JX417495.1) isolated from an XVII Century document made of parchment. The chemical composition of AAH, determined from the combination of electron microprobe and X-ray structural analyses, is [(NH4)0.78K0.22]NaMg2(PO4)2·14H2O. Single-crystal X-ray diffraction shows that AAH is orthorhombic with space group Pmnb and unit-cell parameters a = 6.9661(6), b = 25.236(3), c = 11.292(1) Å, and V = 1985.0(3) Å3. Compared with hazenite, the substitution of NH4+ for K+ results in a noticeable increase of the average A-O (A = NH4++K+) bond length and the unit-cell volume for AAH, as also observed for struvite vs. struvite-K. Both infrared and Raman spectra of AAH resemble those of hazenite, as well as struvite. Our study reveals that AAH forms only in cultures with Na-bearing solutions and pH below 10.0. No AAH or hazenite was found in experiments with the K-bearing solutions, suggesting the necessity of a Na-bearing solution for AAH formation.

Fluorescence lidar measurements at the archaeological site House of Augustus at Palatino, Rome

Early diagnostics and documentation fulfill an essential role for an effective planning of conservation and restoration of cultural heritage assets. In particular, remote sensing techniques that do not require the use of scaffolds or lifts, such as fluoresence lidar, can provide useful information to obtain an overall assessment of the status of the investigated surfaces and can be exploited to address analytical studies in selected areas. Here we present the results of a joint Italian-Swedish project focused on documenting and recording the status of some sections of the part closed to the public by using fluorescence hyperspectral imaging lidar. The lidar used a tripled-frequency Nd:YAG laser emitting at 355 nm as excitation source and an intensified, gated 512x512-pixel CCD as detector. The lidar had imaging capabilities thanks to a computer-controlled scanning mirror. The fluorescence characteristics of fresco wall paintings were compared to those of fresco fragments found at the same archaeological site and separately examined in the lab using FT-IR and Raman techniques for the identification of pigments. The fluorescence lidar was also used to remotely detect the growth of phototrophic biodeteriogens on the walls. The fluorescence lidar data were compared with results from biological sampling, cultivation and laboratory analysis by molecular techniques.