Carmen Fajardo

Assessing the impact of zero-valent iron (ZVI) nanotechnology on soil microbial structure and functionality: a molecular approach.

In this work, nanoscale zero-valent iron (NZVI) particles have been used as an immobilisation strategy to reduce Pb and Zn availability and mobility in polluted soils. The application of NZVI to two soil microcosms (MPb and MZn) at a dose of 34 mg g(-1) soil efficiently immobilised Pb (25%) and zinc (20%). Exposure to NZVI had little impact on the microbial cellular viability and biological activity in the soils. Three bacterial genes (narG, nirS and gyrA) were used as treatment-related biomarkers. These biomarkers ruled out a broad bactericidal effect on the bulk soil microbial community. A transcriptome analysis of the genes did not reveal any changes in their expression ratios after the NZVI treatment: 1.6 (narG), 0.8 (nirS) and 0.7 (gyrA) in the MPb microcosm and 0.6 (narG), 1.2 (nirS) and 0.5 (gyrA) in the MZn microcosm. However, significant changes in the structure and composition of the soil bacteria population were detected by fluorescence in situ hybridisation. Thus, our results showed that NZVI toxicity could be highly dose and species dependent, and the effective applicability of the proposed molecular approach in assessing the impact of this immobilisation strategy on soil microbial population.

Transcriptional and proteomic stress responses of a soil bacterium Bacillus cereus to nanosized zero-valent iron (nZVI) particles

Nanosized zero valent iron (nZVI) is emerging as an option for treating contaminated soil and groundwater even though the potentially toxic impact exerted by nZVI on soil microorganisms remains uncertain. In this work, we focus on nanotoxicological studies performed in vitro using commercial nZVI and one common soil bacterium (Bacillus cereus). Results showed a negative impact of nZVI on B. cereus growth capability, consistent with the entrance of cells in an early sporulation stage, observed by TEM. Despite no changes at the transcriptional level are detected in genes of particular relevance in cellular activity (narG, nirS, pykA, gyrA and katB), the proteomic approach used highlights differentially expressed proteins in B. cereus under nZVI exposure. We demonstrate that proteins involved in oxidative stress-response and tricarboxilic acid cycle (TCA) modulation are overexpressed; moreover proteins involved in motility and wall biosynthesis are repressed. Our results enable to detect a molecular-level response as early warning signal, providing new insight into first line defense response of a soil bacterium after nZVI exposure.

The role of a groundwater bacterial community in the degradation of the herbicide terbuthylazine

A bacterial community in an aquifer contaminated by s-triazines was studied. Groundwater microcosms were treated with terbuthylazine at a concentration of 100 microg L(-1) and degradation of the herbicide was assessed. The bacterial community structure (abundance and phylogenetic composition) and function (carbon production and cell viability) were analysed. The bacterial community was able to degrade the terbuthylazine; in particular, Betaproteobacteria were involved in the herbicide biotransformation. Identification of some bacterial isolates by PCR amplification of the 16S rRNA gene revealed the presence of two Betaproteobacteria species able to degrade the herbicide: Advenella incenata and Janthinobacterium lividum. PCR detection of the genes encoding s-triazine-degrading enzymes indicated the presence of the atzA and atzB genes in A. incenata and the atzB and atzC genes in J. lividum. The nucleotide sequences of the PCR fragments of the atz genes from these strains were 100% identical to the homologous genes of the Pseudomonas sp. strain ADP. In conclusion, the results show the potential for the use of a natural attenuation strategy in the treatment of aquifers polluted with the terbuthylazine. The two bacteria isolated could facilitate the implementation of effective bioremediation protocols, especially in the case of the significant amounts of herbicide that can be found in groundwater as a result of accidental spills.

Integrating classical and molecular approaches to evaluate the impact of nanosized zero-valent iron (nZVI) on soil organisms.

Nanosized zero-valent iron (nZVI) is a new option for the remediation of contaminated soil and groundwater, but the effect of nZVI on soil biota is mostly unknown. In this work, nanotoxicological studies were performed in vitro and in two different standard soils to assess the effect of nZVI on autochthonous soil organisms by integrating classical and molecular analysis. Standardised ecotoxicity testing methods using Caenorhabditis elegans were applied in vitro and in soil experiments and changes in microbial biodiversity and biomarker gene expression were used to assess the responses of the microbial community to nZVI. The classical tests conducted in soil ruled out a toxic impact of nZVI on the soil nematode C. elegans in the test soils. The molecular analysis applied to soil microorganisms, however, revealed significant changes in the expression of the proposed biomarkers of exposure. These changes were related not only to the nZVI treatment but also to the soil characteristics, highlighting the importance of considering the soil matrix on a case by case basis. Furthermore, due to the temporal shift between transcriptional responses and the development of the corresponding phenotype, the molecular approach could anticipate adverse effects on environmental biota.

A new fluorescent oligonucleotide probe for in situ detection of s-triazine-degrading Rhodococcus wratislaviensis in contaminated groundwater and soil samples.

A bacterial strain (FPA1) capable of using terbuthylazine, simazine, atrazine, 2-hydroxysimazine, deethylatrazine, isopropylamine or ethylamine as its sole carbon source was isolated from a shallow aquifer chronically contaminated with s-triazine herbicides. Based on its 16S rDNA sequence analysis, the strain FPA1 was identified as Rhodococcus wratislaviensis. The disappearance time of 50% of the initial terbuthylazine concentration in the presence of this strain (DT(50)) was 62days. This strain was also able to mineralise the [U-ring (14)C] triazine-ring, albeit at a slow rate. A 16S rRNA target oligonucleotide probe (RhLu) was designed, and the FISH protocol was optimised, in order to detect R. wratislaviensis in s-triazine-contaminated sites. The RhLu probe gave a positive signal (expressed as % of total DAPI-positive cells) in both the groundwater (2.19+/-0.41%) and soil (2.10+/-0.96%) samples analysed. Using the RhLu probe, R. wratislaviensis can be readily detected, and its population dynamics can be easily monitored, in soil and in water ecosystems contaminated with s-triazine. To the best of our knowledge, this is the first report showing the isolation, from groundwater, of a bacterial strain able to degrade s-triazines.

Residual impact of aged nZVI on heavy metal-polluted soils.

In the present study, the residual toxicity and impact of aged nZVI after a leaching experiment on heavy metal (Pb, Zn) polluted soils was evaluated. No negative effects on physico-chemical soil properties were observed after aged nZVI exposure. The application of nZVI to soil produced a significant increase in Fe availability. The impact on soil biodiversity was assessed by fluorescence in situ hybridization (FISH). A significant effect of nZVI application on microbial structure has been recorded in the Pb-polluted soil nZVI-treated. Soil bacteria molecular response, evaluated by RT-qPCR using exposure biomarkers (pykA, katB) showed a decrease in the cellular activity (pykA) due to enhanced intracellular oxidative stress (katB). Moreover, ecotoxicological standardised test on Caenorhabditis elegans (C. elegans) showed a decrease in the growth endpoint in the Pb-polluted soil, and particularly in the nZVI-treated. A different pattern has been observed in Zn-polluted soils: no changes in soil biodiversity, an increase in biological activity and a significant decrease of Zn toxicity on C. elegans growth were observed after aged nZVI exposure. The results reported indicated that the pollutant and its nZVI interaction should be considered to design soil nanoremediation strategies to immobilise heavy metals.

Impact of Ag and Al2O3 nanoparticles on soil organisms: In vitro and soil experiments

In vitro analyses were conducted to assess the impact of Al2O3 and Ag nanoparticles on two common soil bacteria, Bacillus cereus and Pseudomonas stutzeri. Al2O3 nanoparticles did not show significant toxicity at any dose or time assayed, whereas exposure to 5 mg L(-1) Ag nanoparticles for 48 h caused bactericidal effects. Moreover, alterations at the morphological level were observed by transmission electron microscopy (TEM); Ag but not Al2O3 nanoparticles evoked the entrance of B. cereus cells in an early sporulation stage and both nanoparticles penetrated P. stutzeri cells. At the molecular level, a dramatic increase (8.2-fold) in katB gene expression was found in P. stutzeri following Al2O3 nanoparticles exposure, indicative of an oxidative stress-defence system enhancement in this bacterium. In the microcosm experiment, using two different natural soils, Al2O3 or Ag nanoparticles did not affect the Caenorhabditis elegans toxicity endpoints growth, survival, or reproduction. However, differences in microbial phylogenetic compositions were detected by fluorescence in situ hybridization (FISH). The use of katB- and pykA-based sequences showed that the microbial transcriptional response to nanoparticle exposure decreased, suggesting a decrease in cellular activity. These changes were attributable to both the nanoparticles treatment and soil characteristics, highlighting the importance of considering the soil matrix on a case by case basis.

Assessment of s-Triazine Catabolic Potential in Soil Bacterial Isolates Applying atz Genes as Functional Biomarkers

Fluorescence in situ hybridization (FISH) technique and qPCR analyses, targeting atz genes, were applied to detect the presence of simazine-degrading bacteria in an agricultural soil with a history of herbicide application. atzB-targeted bacteria detected by FISH represented 5% of total soil bacteria with potential capability to metabolize the herbicide. The soil natural attenuation capacity was confirmed in soil microcosms by measuring simazine degradation. Moreover, four bacterial strains were isolated from the soil and identified as Acinetobacter lwoffii, Pseudomonas putida, Rhizobium sp. and Pseudomonas sp. The isolates were able to grow using different s-triazine compounds and related metabolites as the sole carbon source. Growth parameters in presence of simazine were calculated using the Gompertz model. Rhizobium sp. showed the highest simazine degradation (71.2%) and mineralization (38.7%) rates, whereas the lowest values were found to A. lwoffii—50.4% of degradation and 22.4% of mineralization. Results from qPCR analyses of atzA, atzB and atzC genes revealed their presence in Rhizobium sp. and A. lwoffii, being atzB and atzC the most abundant functional genes. Rhizobium sp. showed a higher amount of the three biomarkers compared to A. lwoffii: the atzA, atzB and atzC gene copy number per microlitre were, respectively, 101, 102 and 103-fold higher in the former. Therefore the proposed molecular approaches based on the use of atz genes as biomarkers can be considered as useful tools to evaluate the presence and potential capability of degrading-s-triazines soil microorganisms.

Potential risk of acute toxicity induced by AgI cloud seeding on soil and freshwater biota

Silver iodide is one of the most common nucleating materials used in cloud seeding. Previous cloud seeding studies have concluded that AgI is not practically bioavailable in the environment but instead remains in soils and sediments such that the free Ag amounts are likely too low to induce a toxicological effect. However, none of these studies has considered the continued use of this practice on the same geographical areas and thus the potential cumulative effect of environmental AgI. The aim of this study is to assess the risk of acute toxicity caused by AgI exposure under laboratory conditions at the concentration expected in the environment after repeated treatments on selected soil and aquatic biota. To achieve the aims, the viability of soil bacteria Bacillus cereus and Pseudomonas stutzeri and the survival of the nematode Caenorhabditis elegans exposed to different silver iodide concentrations have been evaluated. Freshwater green algae Dictyosphaerium chlorelloides and cyanobacteria Microcystis aeruginosa were exposed to silver iodide in culture medium, and their cell viability and photosynthetic activity were evaluated. Additionally, BOD5 exertion and the Microtox® toxicity test were included in the battery of toxicological assays. Both tests exhibited a moderate AgI adverse effect at the highest concentration (12.5µM) tested. However, AgI concentrations below 2.5µM increased BOD5. Although no impact on the growth and survival endpoints in the soil worm C. elegans was recorded after AgI exposures, a moderate decrease in cell viability was found for both of the assessed soil bacterial strains at the studied concentrations. Comparison between the studied species showed that the cyanobacteria were more sensitive than green algae. Exposure to AgI at 0.43μM, the reference value used in monitoring environmental impact, induced a significant decrease in photosynthetic activity that is primarily associated with the respiration (80% inhibition) and, to a lesser extent, the net photosynthesis (40% inhibition) in both strains of phytoplankton and a moderate decrease in soil bacteria viability. These results suggest that AgI from cloud seeding may moderately affect biota living in both terrestrial and aquatic ecosystems if cloud seeding is repeatedly applied in a specific area and large amounts of seeding materials accumulate in the environment.