Microbial Diversity of Bacteria Involved in Biomineralization Processes in Mine-Impacted Freshwaters.

In order to increase the knowledge about geo-bio interactions in extreme metal-polluted mine waters, we combined microbiological, mineralogical, and geochemical analyses to study the indigenous sulfate-reducing bacteria (SRB) involved in the heavy metal (HM) biomineralization processes occurring in Iglesiente and Arburese districts (SW Sardinia, Italy). Anaerobic cultures from sediments of two different mining-affected streams of this regional framework were enriched and analyzed by 16S rRNA next-generation sequencing (NGS) technique, showing sequences closely related to SRB classified in taxa typical of environments with high concentrations of metals (Desulfovibrionaceae, Desulfosporosinus). Nevertheless, the most abundant genera found in our samples did not belong to the traditional SRB groups (i.e., Rahnella, Acinetobacter). The bio-precipitation process mediated by these selected cultures was assessed by anaerobic batch tests performed with polluted river water showing a dramatic (more than 97%) Zn decrease. Scanning electron microscopy (SEM) analysis revealed the occurrence of Zn sulfide with tubular morphology, suggesting a bacteria-mediated bio-precipitation. The inocula represent two distinct communities of microorganisms, each adapted to peculiar environmental conditions. However, both the communities were able to use pollutants in their metabolism and tolerating HMs by detoxification mechanisms. The Zn precipitation mediated by the different enriched cultures suggests that SRB inocula selected in this study have great potentialities for the development of biotechnological techniques to reduce contaminant dispersion and for metal recovery.

Phenol biodegradation by plant growth promoting bacterium, S. odorifera: kinetic modeling and process optimization.

One of the main organic pollutants that could result from industrial products and chemical transformations is phenol. In the current study, the kinetics of Serratia odorifera, which was isolated from arable soil, was studied by growing it on broth minimal medium spiked with phenol as only carbon source and energy. The newly isolated plant growth-promoting bacterium (PGPB), S. odorifera, was used for the first time for phenol biodegradation. The growth kinetics parameters (phenol-dependent) including maximum specific growth rate (µmax), half-saturation coefficient (Ks), and the Haldane's growth kinetics inhibition coefficient (Ki), were tested via Haldane inhibition model and resulted on the 0.469 (h -1), 26.6 (mgL-1), and 292 (mgL-1), respectively. The sum of squared error (SSR) of 4.89 × 10-3 was fitted to the experimental data by Haldane equation. The results of phenol biodegradation were fitted into the modified Gombertz model. The increase of phenol concentrations led to increases in both the rate of phenol biodegradation and lagging time. The optimal phenol biodegradation and bacterial growth obtained by S. odorifera, were at 28 °C incubation temperature and a pH of 7.0. The pathway of phenol biodegradation by S. odorifera was proposed in the current study to provide a new insight into synchronization of phenol biodegradation and plant growth-promoting bacteria. This may play an important role in remediation of phenol-contaminated soil besides promoting the plant growth, thus lessening the plant stress.

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.

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 Sørensen's 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.

Evaluation of triticale as energy crop in Italy.

The promotion of renewable energy represents a target of the European 2020 strategy for economical growth and sustainable competitiveness. Cereals are considered a promising biomass producing crop in temperate regions of Europe to be used for both fuel alcohol and biogas production. Among cereals, triticale represents a good candidate for this kind of application, showing a number of advantages such as high grain yield even in marginal environments, tolerance to drought, tolerance to more acid soils, lower production costs and lower susceptibility to biotic stresses. The aim of this study was to compare yield and quality of eight triticale lines grown in marginal areas in a two-year experiment. Italian variety, Magistral, and a bread wheat variety (EW9) were selected for comparison. Data from fields, chemical analyses and preliminary results from fermentation are reported.

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.

Comparative analysis of the effects of locally used herbicides and their active ingredients on a wild-type wine Saccharomyces cerevisiae strain.

Herbicides are released to the environment with potential ecotoxicological risks for mammals. Yeast is a good model to elucidate toxicity mechanisms. We investigated how three commercial herbicides (Proper Energy, Pointer, and Silglif) and their active ingredients (respectively, fenoxaprop-P-ethyl, tribenuron methyl, and glyphosate) can affect biological activities of an oenological Saccharomyces cerevisiae strain, which may be resident on grape vineyards of the same geographical areas where herbicides are used. The use of commercial grade herbicides employed in Italy allowed us to reproduce the same conditions applied in crops; at the same time, assaying pure single active compounds made it possible to compare the effects obtained with commercial formulations. Interestingly, we found that while pure active compounds affect cell growth and metabolism at a lower extent, commercial preparations have a significant major negative influence on yeast biology.

Investigating heavy metal resistance, bioaccumulation and metabolic profile of a metallophile microbial consortium native to an abandoned mine.

Contaminated sites represent new ecological niches where historical pollution has originated an unusual microbial biodiversity. The knowledge of these microorganisms contributes to the discovery of new pathways and metabolic networks and may offer potential solutions for damaged areas. In the present work seven microbial consortia have been isolated from an abandoned mine of blend and galena (Ingurtosu, Italy) through a selection for resistance to zinc (tested up to 40 mM in solution). All the consortia were able to accumulate zinc and the best accumulator, named Ing5, has been studied for the following characteristics: resistance and accumulation of Zn, Cd, Hg, bioaccumulation mechanisms of Zn, and influence of Zn and Cd on the metabolic profile. The results indicate that the consortium Ing5 bears resistance systems for Cd and Hg as well as Zn and that, for some of the 5 isolates belonging to Ing5, the resistance thresholds are higher in consortium than in pure culture. The prevalent mechanism for zinc accumulation can be reasonably considered to be metabolism-dependent, inducible and regulated by metal concentrations. The study on the metabolic profile, carried out by the Biolog system, shows that Zn exerts a very low influence on the metabolic profile and that this influence can also be positive; Cd has a stronger negative influence but that, despite this, the consortium is able to maintain a wide metabolic potential in the presence of heavy metals. These features of Ing5 make it a good candidate for biotechnological applications and for further investigation of the degradation of organic pollutants in the presence of metals.

A rhizospheric Burkholderia cepacia complex population: genotypic and phenotypic diversity of Burkholderia cenocepacia and Burkholderia ambifaria.

The Burkholderia cepacia'complex' (Bcc) presently comprises nine species and genomovars. In order to acquire a better comprehension of the species and genomovar distribution and of the genetic diversity among environmental Bcc bacteria, a natural population of 60 bacterial isolates recovered from the rhizosphere of maize and belonging to the Bcc has been characterised to assess the exact taxonomic position, the genetic polymorphism and the metabolic profiles of isolates. The identification of the different species and genomovars was accomplished by a combination of techniques including sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and recA-based restriction fragment length polymorphism analyses. The genetic diversity among Bcc isolates was analysed by means of the random amplified polymorphic DNA and amplified fragment length polymorphism techniques; the analysis of molecular variance method was applied to estimate the genetic differences among the various species and genomovars identified within the bacterial population. Metabolic profiles based on carbon source utilisation were obtained by means of the Biolog GN assay and analysed by means of cluster analysis. Forty-four strains were identified as B. ambifaria, 11 as B. cenocepacia recA lineage III-B, four as B. pyrrocinia, and one as B. cepacia genomovar I. Marked genetic differences were observed between B. cenocepacia and B. ambifaria, whereas limited differences were found between B. pyrrocinia and B. ambifaria and between B. pyrrocinia and B. cenocepacia. No significant differences (P>0.05) were observed between the mean genetic distances of isolates belonging to B. cenocepacia, B. ambifaria, and B. pyrrocinia. Phenotypic analyses revealed that all isolates tested were able to utilise more than 75% of substrates. The highest variability in the number of utilised substrates was found among B. cenocepacia isolates, whereas the lowest was found among B. ambifaria isolates. Cluster analysis of metabolic profiles revealed pronounced differences between B. cenocepacia and B. ambifaria; in contrast, B. pyrrocinia could not be clearly separated either from B. cenocepacia or from B. ambifaria.