Francesco Guarino

Antibiotic resistance spread potential in urban wastewater effluents disinfected by UV/H2O2 process.

Urban wastewater treatment plants (UWTPs) are among the main hotspots of antibiotic resistance (AR) spread into the environment and the role of conventional and new disinfection processes as possible barrier to minimise the risk for AR transfer is presently under investigation. Accordingly, the aim of this work was to evaluate the effect of an advanced oxidation process (AOP) (specifically UV/H2O2) on AR transfer potential. UV/H2O2 disinfection experiments were carried out on real wastewater samples to evaluate the: i) inactivation of total coliforms, Escherichia coli and antibiotic resistant E. coli as well as ii) possible removal of target antibiotic resistance genes (ARGs) (namely, blaTEM, qnrS and tetW). In particular, DNA was extracted from both antibiotic resistant E. coli bacterial cells (intracellular DNA), grown on selective culture media, and the whole water suspension (total DNA) collected at different treatment times. Polymerase chain reaction (PCR) assay was performed to detect the absence/presence of the selected ARGs. Real Time quantitative Polymerase Chain Reaction (qPCR) was used to quantify the investigated ARGs in terms of copiesmL(-1). In spite of the bacterial inactivation and a decrease of ARGs in intracellular DNA after 60min treatment, UV/H2O2 process was not effective in ARGs removal from water suspension (total DNA). Particularly, an increase up to 3.7×10(3)copiesmL(-1) (p>0.05) of blaTEM gene was observed in total DNA after 240min treatment, while no difference (p>0.05) was found for qnrS gene between the initial (5.1×10(4)copiesmL(-1)) and the final sample (4.3×10(4)copiesmL(-1)). On the base of the achieved results, the investigated disinfection process may not be effective in minimising AR spread potential into the environment. The death of bacterial cells, which results in DNA release in the treated water, may pose a risk for AR transfer to other bacteria present in the receiving water body.

β-lactams resistance gene quantification in an antibiotic resistant Escherichia coli water suspension treated by advanced oxidation with UV/H2O2

Water is one of the most important habitats and route for the spread of antibiotic resistance (AR) in the environment and disinfection processes can be a potential barrier to minimise this risk. In this study the effect of UV/H2O2 process on the potential of AR transfer was investigated through cultivation methods vs (polymerase chain reaction) PCR based methods. blaTEM was selected as target antibiotic resistance gene (ARG) and was quantified by qPCR in the survived colonies and the whole suspension (total DNA). The detection limit of residual antibiotic resistant Escherichia coli (E. coli) colonies (5CFUmL-1) was reached after 240min treatment, but blaTEM gene was still present in total DNA after 300min (2.8×106 copies mL-1), and no effect was observed in DNA extracted from cell cultures (3.8×108 copies mL-1 after 90min). Accordingly, the investigated disinfection process may select for unaffected ARGs, therefore contributing to the potential transfer of AR in the environment.

Global networks for invasion science: benefits, challenges and guidelines

Much has been done to address the challenges of biological invasions, but fundamental questions (e.g., which species invade? Which habitats are invaded? How can invasions be effectively managed?) still need to be answered before the spread and impact of alien taxa can be effectively managed. Questions on the role of biogeography (e.g., how does biogeography influence ecosystem susceptibility, resistance and resilience against invasion?) have the greatest potential to address this goal by increasing our capacity to understand and accurately predict invasions at local, continental and global scales. This paper proposes a framework for the development of ‘Global Networks for Invasion Science’ to help generate approaches to address these critical and fundamentally biogeographic questions. We define global networks on the basis of their focus on research questions at the global scale, collection of primary data, use of standardized protocols and metrics, and commitment to long-term global data. Global networks are critical for the future of invasion science because of their potential to extend beyond the capacity of individual partners to identify global priorities for research agendas and coordinate data collection over space and time, assess risks and emerging trends, understand the complex influences of biogeography on mechanisms of invasion, predict the future of invasion dynamics, and use these new insights to improve the efficiency and effectiveness of evidence-based management techniques. While the pace and scale of global change continues to escalate, strategic and collaborative global networks offer a powerful approach to inform responses to the threats posed by biological invasions.

Epigenetic Diversity of Clonal White Poplar (Populus alba L.) Populations: Could Methylation Support the Success of Vegetative Reproduction Strategy?

The widespread poplar populations of Sardinia are vegetatively propagated and live in different natural environments forming large monoclonal stands. The main goals of the present study were: i) to investigate/measure the epigenetic diversity of the poplar populations by determining their DNA methylation status; ii) to assess if and how methylation status influences population clustering; iii) to shed light on the changes that occur in the epigenome of ramets of the same poplar clone. To these purposes, 83 white poplar trees were sampled at different locations on the island of Sardinia. Methylation sensitive amplified polymorphism analysis was carried out on the genomic DNA extracted from leaves at the same juvenile stage. The study showed that the genetic biodiversity of poplars is quite limited but it is counterbalanced by epigenetic inter-population molecular variability. The comparison between MspI and HpaII DNA fragmentation profiles revealed that environmental conditions strongly influence hemi-methylation of the inner cytosine. The variable epigenetic status of Sardinian white poplars revealed a decreased number of population clusters. Landscape genetics analyses clearly demonstrated that ramets of the same clone were differentially methylated in relation to their geographic position. Therefore, our data support the notion that studies on plant biodiversity should no longer be restricted to genetic aspects, especially in the case of vegetatively propagated plant species.

Assessment of perchlorate-reducing bacteria in a highly polluted river.

A 1-year monitoring experiment of the Sarno River basin was conducted during 2008 to evaluate the overall quality of the water over time and to compare the results with those obtained previously. The physico-chemical and microbiological characteristics of the water course had not changed appreciably with respect to previous determinations, thus emphasizing the major contribution of untreated urban wastewater to the overall pollution of the river. Moreover, attention was paid to the perchlorate ion, one of the so-called emerging contaminants, which is widespread in natural environments and is known to have adverse effects on the human thyroid gland. Over the entire monitoring program, we did not find appreciable levels of perchlorate, although the particular environmental condition could support its development. Thus, a dedicated study was designed to assess the presence of bacteria that can reasonably reduce perchlorate levels. By enrichment and molecular procedures, we identified α- and β-Proteobacteria strains, classified by 16S rDNA sequences as Dechlorospirillum sp. and Dechlorosoma sp., respectively. Further physiologic characterization and the presence of the alpha subunit gene (pcrA) of the perchlorate reductase in both strains confirmed the presence in the river of viable and active perchlorate dissimilatory bacteria.

Use of Zea mays L. in phytoremediation of trichloroethylene

Trichloroethylene (TCE) is a chlorinated aliphatic organic compound often detected as pollutant in soils and ground water. “Green technologies” based on phytoremediation were proven to be effective to reclaim organic pollutants (e.g. TCE) and heavy metals from different environmental matrices. In this work, we use Zea mays L. for the removal of high TCE concentrations from medium cultures. In particular, we investigated a sealed bioreactor where the growth medium was contaminated with an increasing amount of TCE, in the range 55–280 mg/L; the removal capability of the maize plants was assessed by means of GC-MS and LC-MS analyses. An accurate mass balance of the system revealed that the plants were able to remove and metabolise TCE with an efficiency up to 20 %, depending on the total amount of TCE delivered in the bioreactor. Morphometric data showed that the growth of Z. mays is not significantly affected by the presence of the pollutant up to a concentration of 280 mg/L, while plants show significant alterations at higher TCE concentrations until the growth is completely inhibited for [TCE] ≃ 2000 mg/L. Finally, the presence of several TCE metabolites, including dichloroacetic and trichloroacetic acids, was detected in the roots and in the aerial part of the plants, revealing that Z. mays follows the green liver metabolic model. These results encourage further studies for the employment of this plant species in phytoremediation processes of soils and waters contaminated by TCE and, potentially, by many other chlorinated solvents.

Genetic and biochemical characterization of rhizobacterial strains and their potential use in combination with chelants for assisted phytoremediation

Copper and zinc are essential micronutrients in plants but, at high concentrations, they are toxic. Assisted phytoremediation is an emerging “green” technology that aims to improve the efficiency of tolerant species to remove metals from soils through the use of chelants or microorganisms. Rhizobacteria can promote plant growth and tolerance and also affect the mobility, bioavailability, and complexation of metals. A pot experiment was conducted to evaluate the phytoremediation effectiveness of sunflowers cultivated in a Cu- and Zn-spiked soil, in the presence or absence of bacterial consortium and/or chelants. The consortium was constituted of two Stenotrophomonas maltophilia strains and one of Agrobacterium sp. These strains were previously isolated from the rhizosphere of maize plants cultivated on a metal-polluted soil and here molecularly and biochemically characterized. Results showed that the consortium improved sunflower growth and biomass production on the spiked soils. Sunflowers accumulated large amounts of metals in their roots and leaves; however, neither the bacterial consortium nor the chelants, singularly added to pots, influenced significantly Cu and Zn plant uptake. Furthermore, the consecutive soil amendment with the EDTA and bacterial consortium determined a consistent accumulation of metals in sunflowers, and it might be an alternative strategy to limit the use of EDTA and its associated environmental risks in phytoremediation.

In vitro propagation of Leucocroton havanensis Borhidi (Euphorbiaceae): A rare serpentine-endemic species of Cuba

Abstract Leucocroton havanensis Borhidi is an endemic plant species of Cuba able to hyperaccumulate nickel. In order to sustain the conservation of this species, an efficient protocol for its micropropagation, via axillary bud proliferation, is described. We placed apical segments from aseptic seedlings on basal medium supplemented with indole-3-acetic acid (IAA) and 6-benzylaminopurine (BAP) or thidiazuron (TDZ), individually or in combination. On a medium containing 0.5 mg L−1 IAA and 1.0 mg L−1 BAP explants (65.5%) developed axillary buds. Nevertheless, combinations of 0.5 mg L−1 IAA with 0.1 mg L−1 TDZ was the most effective treatment producing the highest number of buds per explant (30.3); while the control treatment, without growth regulators, produced no buds at all. Transfer of buds to medium supplemented with indole-3-butyric acid, indicated that 0.25 mg L−1 is the amount of hormone required to generate roots on young buds (100%). In order to assess DNA variations in micropropagated plants, an Amplified Fragment Length Polymorphism analysis was performed and no genetic variation was detected. This study demonstrates that a high multiplication rate can be obtained by means of the reported protocol, and that plantlets can be readily hardened (96% survival) in a greenhouse by transplanting them on serpentine soil.

Reclamation of Cr-contaminated or Cu-contaminated agricultural soils using sunflower and chelants

Chromium (Cr) and copper (Cu) are pollutants with a strong environmental impact. “Green biotechnology” as phytoremediation represents a sustainability opportunity for soil reclamation. In this study, we evaluated the possibility to reclaim agricultural soils located in the Solofrana valley, contaminated by Cr or Cu. Chromium contamination derives by repeated flooding events of Solofrana rivers containing Cr because of leather tanning plants, while Cu soil pollution was due to the use of Cu-rich pesticides in agriculture. Both metals showed a very low bioavailability. In order to perform an assisted phytoremediation of polluted fields, we carried out a preliminary ex situ experimentation testing for the first time sunflowers (cv. Pretor) and chelants (ethylenediaminetetraacetic acid (EDTA) and/or ethylene diamine disuccinate (EDDS)), useful when metal bioavailability is low. No symptoms of toxicity were observed in sunflowers grown on both soils, while biomass was improved when EDDS was added. Cr and Cu bioavailability was only slightly enhanced by chelants at the end of the treatments. Both Cr and Cu were mainly accumulated in the roots; moreover, Cu was also translocated to the aboveground organs in the presence of EDTA. The ex situ experimentation demonstrated that assisted phytoremediation is a very slow process not useful in the case of persistent pollution.

Genetic characterization, micropropagation, and potential use for arsenic phytoremediation of Dittrichia viscosa (L.) Greuter

In the last decade, many scientists have focused their attention on the search for new plant species that can offer improved capacities to reclaim polluted soils and waters via phytoremediation. In this study, seed batches from three natural populations of Dittrichia viscosa, harvested in rural, urban, and industrial areas of central and southern Italy, were used to: (i) evaluate the genetic and morphological diversity of the populations; (ii) develop an efficient protocol for in-vitro propagation from seedling microcuttings; (iii) achieve optimal acclimatization of micropropagated plants to greenhouse conditions; (iv) test the response to arsenic (As) soil contamination of micropropagated plants. The genetic biodiversity study, based on Random Amplification of Polymorphic DNA (RAPD), as well as the morphometric analysis of 20 seedlings from each population revealed some degree of differentiation among populations. Based on these data, the most biodiverse plants from the three populations (10 lines each) were clonally multiplied by micropropagation using microcuttings of in-vitro grown seedlings. Three culture media were tested and Mureshige and Skoog medium was chosen for both seedling growth and micropropagation. The micropropagated plants responded well to greenhouse conditions and over 95% survived the acclimatization phase. Four clones were tested for their capacity to grow on soil spiked with NaAsO2 and to absorb and accumulate the metalloid. All clones tolerated up to 1.0mg As. At the end of the trial (five weeks), As was detectable only in leaves of As-treated plants and concentration varied significantly among clones. The amount of As present in plants (leaves) corresponded to ca. 0.10-1.7% of the amount supplied. However, As was no longer detectable in soil suggesting that the metalloid was taken up, translocated and probably phytovolatilized.