Margarita Martin

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.

Catabolism of 3- and 4-hydroxyphenylacetic acid by Klebsiella pneumoniae

Klebsiella pneumoniae catabolizes both 4-hydroxyphenylacetic acid and 3-hydroxyphenylacetic acid via meta-cleavage of 3,4-dihydroxyphenylacetic acid, ultimately yielding pyruvate and succinate. The organism can synthesize two hydroxylases catalysing 3,4-dihydroxyphenylacetic acid formation, which differ in substrate specificity, cofactor requirement, kinetics and regulation. Five enzymes sequentially involved in the catabolism of 3,4-dihydroxyphenylacetic acid are encoded on a 7 kbp fragment of the K. pneumoniae chromosome that has been isolated in a recombinant plasmid.

Molecular cloning and analysis of the genes encoding the 4-hydroxyphenylacetate hydroxylase from Klebsiella pneumoniae

Abstract The Klebsiella pneumoniae genes encoding the hydroxylase involved in the meta-cleavage pathway of 4-hydroxyphenylacetic acid (4-HPA) were cloned, and the DNA fragment from the region essential for hydroxylase activity was sequenced. K. pneumoniae 4-HPA hydroxylase was composed of two proteins (HpaA and HpaH) with different molecular masses. HpaA seems to be a flavin-containing hydroxylase with a molecular mass of 58,781 Da. HpaH, with a molecular mass of 18,680 Da, seems to be a “helper” protein required for productive hydroxylation of the substrate. The hpa genes were expressed and the hydroxylase was active in Escherichia coli. Comparison of the enzyme with other monooxygenases indicates that K. pneumoniae 4-HPA hydroxylase is a member of a new family of hydroxylases.

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.

Propachlor Removal by Pseudomonas Strain GCH1 in an Immobilized-Cell System

ABSTRACT A bacterial strain capable of growing on propachlor (2-chloro-N-isopropylacetanilide) was isolated from soil by using enrichment and isolation techniques. The strain isolated, designated GCH1, was classified as a member of the genusPseudomonas. Washed-cell suspensions of strain GCH1 accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain GCH1 grew on propachlor with a generation time of 4.2 h and a rate of substrate utilization of 1.75 ± 0.15 μmol h−1. Gene expression did not require induction but was subject to catabolite expression. Acetanilide was a growth substrate with a yield of 0.56 ± 0.02 mg of protein μmol−1. GCH1 strain cells were immobilized by adsorption onto a ceramic support and were used as biocatalysts in an immobilized cell system. Propachlor elimination reached 98%, with a retention time of 3 h and an initial organic load of 0.5 mM propachlor. The viability of immobilized cells increased 34-fold after 120 days of bioreactor operation.

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.

Klebsiella planticola strain DSZ mineralizes simazine: physiological adaptations involved in the process

We examined the ability of a soil bacterium, Klebsiella planticola strain DSZ, to degrade the herbicide simazine (SZ). Strain DSZ is metabolically diverse and grows on a wide range of s-triazine and aromatic compounds. DSZ cells grown in liquid medium with SZ (in 10xa0mM ethanol) as carbon source mineralized 71.6±1.3% of 0.025xa0mM SZ with a yield of 4.6±0.3xa0μg cell dry weightxa0mmol−1 carbon. The metabolites produced by DSZ during SZ degradation included ammeline, cyanuric acid, N-formylurea and urea. We studied the physiological adaptations which allow strain DSZ to metabolize SZ. Using scanning electron microscopy, we detected DSZ cells covering the surfaces of SZ crystals when the herbicide was used at high concentrations (0.1xa0mM). The membrane order observed by FTIR spectroscopy showed membrane activity at low temperature (4°C) to assimilate the herbicide. Membrane fatty acid analysis demonstrated that strain DSZ adapted to grow on SZ by increasing the degree of saturation of membrane lipid fatty acid; and the opposite effect was detected when both SZ and ethanol were used as carbon sources. This confirms the modulator effect of ethanol on membrane fluidity.