Dissolved iron released from nanoscale zero-valent iron (nZVI) activates the defense system in bacterium Pseudomonas putida, leading to high tolerance to oxidative stress.

Nanoscale zero-valent iron (nZVI) has increasingly been applied to remediate aquifers polluted by organochlorines or heavy metals. As a result, bacteria in the vicinity of remediate action can be stressed by surplus iron released from nZVI. However, the understanding of the iron stress defense pathways during this process is currently incomplete. Therefore, we aimed to elucidate the physiological and transcriptomic response of the bacterium, Pseudomonas putida NCTC 10936, to 100 mg/L of nZVI and 44.5 µg/L of dissolved iron obtained from nZVI suspension. Cell viability was neither affected by nZVI nor dissolved iron, although the dissolved iron caused stress that altered the cell physiology and caused the generation of smaller cells, whereas cells were elongated in the presence of nZVI. Transcriptomic analysis confirmed the observed stronger physiological effect caused by dissolved iron (in total 3839 differentially expressed genes [DEGs]) than by nZVI (945 DEGs). Dissolved iron (but not nZVI) activated genes involved in oxidative stress-related pathways, antioxidant activity, carbohydrate and energy metabolism, but downregulated genes associated with flagellar assembly proteins and two-component systems involved in sensing external stimuli. As a result, bacteria very effectively faced oxidative insults and cell viability was not affected.

The effect of low-pH concrete on microbial community development in bentonite suspensions as a model for microbial activity prediction in future nuclear waste repository.

Concrete as an important component of an engineered barrier system in deep geological repositories (DGR) for radioactive waste may come into contact with bentonite, or other clays, rich in indigenous microorganisms, with potentially harmful impacts on barrier integrity. Our study aimed to assess the effect of a concrete environment on indigenous bentonite and groundwater microbial communities as these particular conditions will select for the potentially harmful microorganisms to the concrete in the future DGR. The two-month experiment under anoxic conditions consisted of crushed, aged, low-pH concrete, Czech Ca-Mg bentonite, and anoxic groundwater, with control samples without concrete or with sterile groundwater. The microbial diversity and proliferation were estimated by qPCR and 16S rRNA gene amplicon sequencing. The presence of concrete had a strong effect on microbial diversity and reduced the increase in total microbial biomass, though low-pH concrete harbored indigenous bacteria. The growth of sulfate reducers was also limited in concrete samples. Several genera, such as Massilia, Citrifermentans, and Lacunisphaera, dominant in bentonite controls, were suppressed in concrete-containing samples. In contrast, genera such as Bacillus, Dethiobacter and Anaerosolibacter specifically proliferated in the presence of concrete. Genera such as Thermincola or Pseudomonas exhibited high versatility and proliferated well under both conditions. Because several of the detected bacterial genera are known to affect concrete integrity, further long-term studies are needed to estimate the effect of bentonite and groundwater microorganisms on concrete stability in future DGR.

Miscanthus x giganteus role in phytodegradation and changes in bacterial community of soil contaminated by petroleum industry.

The second generation energy crop Miscanthus x giganteus (Mxg) was cultivated in pots with mixtures of clean and petroleum industry contaminated soil affected by petroleum, Pb, Zn contamination and high salinity. The survival rate reached 100%, nevertheless the biomass parameters were negatively affected even in the lowest proportion of contaminated soil. In the lowest contamination, where the plant grew still quite successfully, C10-C40 degradation was significantly enhanced compared to the unplanted control with degradation of 58 ± 14%. The plant contribution to aliphatics degradation was significantly correlated with biomass, thus it was negligible in higher contamination. A similar pattern was documented in development of the soil bacterial community. The shift in community structure after Mxg cultivation was observed mainly in the soil with the lowest contaminant proportion, though an increase of bacterial diversity in the miscanthus rhizosphere was observed in all cases. Relative abundance of Actinobacteria was reduced on behalf of several less abundant phyla (Verrucomicrobia, Bacterioides, Acidobacteria). The majority of genera identified as potential petroleum degraders (Pseudomonas, Shinella, Altererythrobacter, Azospirillum, Mesorhizobium, Dyella) were more abundant in contaminated soil with miscanthus, suggesting that Mxg could be a promising crop for phytomanagement of petroleum contaminated soils but salt phytotoxicity needs to be mitigated first.

Anaerobic microbial corrosion of carbon steel under conditions relevant for deep geological repository of nuclear waste.

We examined microbial corrosion of carbon steel in synthetic bentonite pore water inoculated with natural underground water containing microorganisms over a period of 780-days under sterile and anaerobic conditions. Corrosion behaviour was determined using the mass loss method, SEM-EDS analysis and Raman spectroscopy, while qualitative and quantitative changes in the microbial community were analysed using molecular-biological tools (16S rDNA amplicon sequencing and qPCR analysis, respectively). Corrosion rates were significantly higher in the biotic environment (compared with an abiotic environment), with significant localisation of corrosion attacks of up to 1 mm arising within 12-months. Nitrate reducing bacteria, such as Pseudomonas, Brevundimonas and Methyloversatilis, dominated the microbial consortium, the high abundance of Methyloversatilis correlating with periods of highest localised corrosion penetrations, suggesting that this bacterium plays an important role in microbially influenced corrosion. Our results indicate that nitrate-reducing bacteria could represent a potential threat to waste canisters under nuclear repository conditions.

Effect of Restriction of Fluoroquinolone Antibiotics on Clostridioides difficile Infections in the University Hospital Hradec Králové.

Clostridioides difficile is the most common pathogen responsible for hospital-acquired diarrhea. This complication of antibiotic treatment mainly endangers the health of elder patients. Preventing the development of C. difficile infections (CDI) is still a challenge that needs to be addressed. In our study, the results of 872 C. difficile positive stool samples were used to describe the epidemiological situation affected by a change in the prescription of fluoroquinolone antibiotics. In a total, 93 of strains were typed by polymerase chain reaction (PCR) and capillary gel electrophoresis. Between years 2014 and 2018 the decline in the fluoroquinolones consumption was 69.3 defined daily dose (DDD) per 1000 patient-days (from 103.3 to 34.0), in same period CDI incidence declined by 1.3 cases per 10,000 patient-bed days (from 5.6 to 4.3). Results of epidemiologic and statistical analysis shows that decline in fluoroquinolones consumption has significant influence on CDI incidence and prevalence of hypervirulent strains. In the University Hospital Hradec Králové properly managed antibiotic stewardship policy has reduced CDI incidence by 23.2% and lowered rate of hypervirulent ribotypes 001 and 176.

Discovering the potential of an nZVI-biochar composite as a material for the nanobioremediation of chlorinated solvents in groundwater: Degradation efficiency and effect on resident microorganisms.

Abiotic and biotic remediation of chlorinated ethenes (CEs) in groundwater from a real contaminated site was studied using biochar-based composites containing nanoscale zero-valent iron (nZVI/BC) and natural resident microbes/specific CE degraders supported by a whey addition. The material represented by the biochar matrix decorated by isolated iron nanoparticles or their aggregates, along with the added whey, was capable of a stepwise dechlorination of CEs. The tested materials (nZVI/BC and BC) were able to decrease the original TCE concentration by 99% in 30 days. Nevertheless, regarding the transformation products, it was clear that biotic as well as abiotic transformation mechanisms were involved in the transformation process when nonchlorinated volatiles (i.e., methane, ethane, ethene, and acetylene) were detected after the application of nZVI/BC and nZVI/BC with whey. The whey addition caused a massive increase in bacterial biomass in the groundwater samples (monitored by 16S rRNA sequencing and qPCR) that corresponded with the transformation of trichloro- and dichloro-CEs, and this process was accompanied by the formation of less chlorinated products. Moreover, the biostimulation step also eliminated the adverse effect caused by nZVI/BC (decrease in microbial biomass after nZVI/BC addition). The nZVI/BC material or its aging products, and probably together with vinyl chloride-respiring bacteria, were able to continue the further reductive dechlorination of dichlorinated CEs into nonhalogenated volatiles. Overall, the results of the present study demonstrate the potential, feasibility, and environmental safety of this nanobioremediation approach.

Application of nanofiber carriers for sampling of microbial biomass from contaminated groundwater.

Sampling of microbial biomass is crucial for understanding and controlling remediation processes ongoing at contaminated sites in general, particularly when molecular genetic analyses are employed. In this study, fiber-based carriers with a nanofiber layer were developed and tested as a method to sample microbial biomass in groundwater for molecular genetic analysis. Nanofiber carriers, varying in the shape and the linear density of nanofibers, were examined throughout a 27-month monitoring period in groundwater contaminated with benzene, toluene, ethylbenzene and xylene isomers (BTEX), and chlorinated ethenes. The effect of carrier shape and nanofiber layer density on the microbial surface colonization and composition of the microbial biofilm was determined using real-time PCR and next-generation sequencing (NGS) analysis. Differences in microbial community composition between nanofiber carriers, groundwater, and soil samples were also analyzed to assess the applicability of carriers for biomass sampling at contaminated sites. The nanofiber carriers showed their applicability as a sampling tool, particularly because of their easy manipulation that facilitates DNA isolation. The majority of taxa (Proteobacteria, Firmicutes, and Bacteroidetes) present on the carrier surfaces were also detected in the groundwater. Moreover, the microbial community on all nanofiber carriers reflected the changes in the chemical composition of groundwater. Although the carrier characteristics (shape, nanofiber layer) did not substantially influence the microbial community on the carrier surface, the circular and planar carriers with a nanofiber layer displayed faster microbial surface colonization. However, the circular carrier was the most suitable for biomass sampling in groundwater because of its high contact area and because it does not require pre-treatment prior to DNA extraction.

Impact of Zero-Valent Iron on Freshwater Bacterioplankton Metabolism as Predicted from 16S rRNA Gene Sequence Libraries.

The application of zero-valent iron particles (ZVI) for the treatment of heavily polluted environment and its biological effects have been studied for at least two decades. Still, information on the impact on bacterial metabolic pathways is lacking. This study describes the effect of microscale and nanoscale ZVI (mZVI and nZVI) on the abundance of different metabolic pathways in freshwater bacterial communities. The metabolic pathways were inferred from metabolism modelling based on 16S rRNA gene sequence data using paprica pipeline. The nZVI changed the abundance of numerous metabolic pathways compared to a less influencing mZVI. We identified the 50 most affected pathways, where 31 were related to degradation, 17 to biosynthesis, and 2 to detoxification. The linkage between pathways was two times higher in nZVI samples compared to mZVI, and was specifically higher considering the arsenate detoxification II pathway. Limnohabitans and Roseiflexus were linked to the same pathways in both nZVI and mZVI. The prediction of metabolic pathways increases our knowledge of the impacts of nZVI and mZVI on freshwater bacterioplankton.

Deciphering indigenous bacteria in compacted bentonite through a novel and efficient DNA extraction method: Insights into biogeochemical processes within the Deep Geological Disposal of nuclear waste concept.

Compacted bentonites are one of the best sealing and backfilling clays considered for use in Deep Geological Repositories of radioactive wastes. However, an in-depth understanding of their behavior after placement in the repository is required, including if the activity of indigenous microorganisms affects safety conditions. Here we provide an optimized phenol:chloroform based protocol that facilitates higher DNA-yields when other methods failed. To demonstrate the efficiency of this method, DNA was extracted from acetate-treated bentonites compacted at 1.5 and 1.7 g/cm3 densities after 24 months anoxic incubation. Among the 16S rRNA gene sequences identified, those most similar to taxa mediating biogeochemical sulfur cycling included sulfur oxidizing (e.g., Thiobacillus, and Sulfurimonas) and sulfate reducing (e.g., Desulfuromonas and Desulfosporosinus) bacteria. In addition, iron-cycling populations included iron oxidizing (e.g., Thiobacillus and Rhodobacter) plus reducing taxa (e.g., Geobacillus). Genera described for their capacity to utilize acetate as a carbon source were also detected such as Delftia and Stenotrophomonas. Lastly, microscopic analyses revealed pores and cracks that could host nanobacteria or spores. This study highlights the potential role of microbial driven biogeochemical processes in compacted bentonites and the effect of high compaction on microbial diversity in Deep Geological Repositories.

Bifidobacterium canis sp. nov., a novel member of the Bifidobacterium pseudolongum phylogenetic group isolated from faeces of a dog (Canis lupus f. familiaris).

A fructose-6-phosphate phosphoketolase-positive strain (GSD1FST) was isolated from a faecal sample of a 3 weeks old German Shepherd dog. The closest related taxa to isolate GSD1FST based on results from the EZBioCloud database were Bifidobacterium animalis subsp. animalis ATCC 25527T, Bifidobacterium animalis subsp. lactis DSM 10140T and Bifidobacterium anseris LMG 30189T, belonging to the Bifidobacterium pseudolongum phylogenetic group. The resulting 16S rRNA gene identities (compared length of 1454 nucleotides) towards these taxa were 97.30, 97.23 and 97.09 %, respectively. The pairwise similarities of strain GSD1FST using argS, atpA, fusA, hsp60, pyrG, rpsC, thrS and xfp gene fragments to all valid representatives of the B. pseudolongum phylogenetic group were in the concatenated range of 83.08-88.34 %. Phylogenomic analysis based on whole-genome methods such as average nucleotide identity revealed that bifidobacterial strain GSD1FST exhibits close phylogenetic relatedness (88.17 %) to Bifidobacetrium cuniculi LMG 10738T. Genotypic characteristics and phylogenetic analyses based on nine molecular markers, as well as genomic and comparative phenotypic analyses, clearly proved that the evaluated strain should be considered as representing a novel species within the B. pseudolongum phylogenetic group named as Bifidobacterium canis sp. nov. (GSD1FST=DSM 105923T=LMG 30345T=CCM 8806T).

Engineered in situ biogeochemical transformation as a secondary treatment following ISCO - A field test.

ISCO using activated sodium persulphate is a widely used technology for treating chlorinated solvent source zones. In sensitive areas, however, high groundwater sulphate concentrations following treatment may be a drawback. In situ biogeochemical transformation, a technology that degrades contaminants via reduced iron minerals formed by microbial activity, offers a potential solution for such sites, the bioreduction of sulphate and production of iron sulphides that abiotically degrade chlorinated ethenes acting as a secondary technology following ISCO. This study assesses this approach in the field using hydrochemical and molecular tools, solid phase analysis and geochemical modelling. Following a neutralisation and bioaugmentation, favourable conditions for iron- and sulphate-reducers were created, resulting in a remarkable increase in their relative abundance. The abundance of dechlorinating bacteria (Dehalococcoides mccartyi, Dehalobacter sp. and Desulfitobacterium spp.) remained low throughout this process. The activity of iron- and sulphate-reducers was further stimulated through application of magnetite plus starch and microiron plus starch, resulting in an increase in ferrous iron concentration (from

Bacterial community composition responds to changes in copepod abundance and alters ecosystem function in an Arctic mesocosm study.

Combining a minimum food web model with Arctic microbial community dynamics, we have suggested that top-down control by copepods can affect the food web down to bacterial consumption of organic carbon. Pursuing this hypothesis further, we used the minimum model to design and analyse a mesocosm experiment, studying the effect of high (+Z) and low (-Z) copepod density on resource allocation, along an organic-C addition gradient. In the Arctic, both effects are plausible due to changes in advection patterns (affecting copepods) and meltwater inputs (affecting carbon). The model predicts a trophic cascade from copepods via ciliates to flagellates, which was confirmed experimentally. Auto- and heterotrophic flagellates affect bacterial growth rate and abundance via competition for mineral nutrients and predation, respectively. In +Z, the model predicts low bacterial abundance and activity, and little response to glucose; as opposed to clear glucose consumption effects in -Z. We observed a more resilient bacterial response to high copepods and demonstrate this was due to changes in bacterial community equitability. Species able to use glucose to improve their competitive and/or defensive properties, became predominant. The observed shift from a SAR11-to a Psychromonodaceae - dominated community suggests the latter was pivotal in this modification of ecosystem function. We argue that this group used glucose to improve its defensive or its competitive abilities (or both). Adding such flexibility in bacterial traits to the model, we show how it creates the observed resilience to top-down manipulations observed in our experiment.

Thermally enhanced in situ bioremediation of groundwater contaminated with chlorinated solvents - A field test.

In situ bioremediation (ISB) using reductive dechlorination is a widely accepted but relatively slow approach compared to other technologies for the treatment of groundwater contaminated by chlorinated ethenes (CVOCs). Due to the known positive kinetic effect on microbial metabolism, thermal enhancement may be a viable means of accelerating ISB. We tested thermally enhanced ISB in aquifers situated in sandy saprolite and underlying fractured granite. The system comprised pumping, heating and subsequent injection of contaminated groundwater aiming at an aquifer temperature of 20-30°C. A fermentable substrate (whey) was injected in separate batches. The test was monitored using hydrochemical and molecular tools (qPCR and NGS). The addition of the substrate and increase in temperature resulted in a rapid increase in the abundance of reductive dechlorinators (e.g., Dehalococcoides mccartyi, Dehalobacter sp. and functional genes vcrA and bvcA) and a strong increase in CVOC degradation. On day 34, the CVOC concentrations decreased by 87% to 96% in groundwater from the wells most affected by the heating and substrate. On day 103, the CVOC concentrations were below the LOQ resulting in degradation half-lives of 5 to 6days. Neither an increase in biomarkers nor a distinct decrease in the CVOC concentrations was observed in a deep well affected by the heating but not by the substrate. NGS analysis detected Chloroflexi dechlorinating genera (Dehalogenimonas and GIF9 and MSBL5 clades) and other genera capable of anaerobic metabolic degradation of CVOCs. Of these, bacteria of the genera Acetobacterium, Desulfomonile, Geobacter, Sulfurospirillum, Methanosarcina and Methanobacterium were stimulated by the substrate and heating. In contrast, groundwater from the deep well (affected by heating only) hosted representatives of aerobic metabolic and aerobic cometabolic CVOC degraders. The test results document that heating of the treated aquifer significantly accelerated the treatment process but only in the case of an abundant substrate.

Stratification of chlorinated ethenes natural attenuation in an alluvial aquifer assessed by hydrochemical and biomolecular tools.

Biomolecular and hydrochemical tools were used to evaluate natural attenuation of chlorinated ethenes in a Quaternary alluvial aquifer located close to a historical source of large-scale tetrachloroethylene (PCE) contamination. Distinct stratification of redox zones was observed, despite the aquifer's small thickness (2.8 m). The uppermost zone of the target aquifer was characterised by oxygen- and nitrate-reducing conditions, with mixed iron- to sulphate-reducing conditions dominant in the lower zone, along with indications of methanogenesis. Natural attenuation of PCE was strongly influenced by redox heterogeneity, while higher levels of PCE degradation coincided with iron- to sulphate reducing conditions. Next generation sequencing of the middle and/or lower zones identified anaerobic bacteria (Firmicutes, Chloroflexi, Actinobacteria and Bacteroidetes) associated with reductive dechlorination. The relative abundance of dechlorinators (Dehalococcoides mccartyi, Dehalobacter sp.) identified by real-time PCR in soil from the lower levels supports the hypothesis that there is a significant potential for reductive dechlorination of PCE. Local conditions were insufficiently reducing for rapid complete dechlorination of PCE to harmless ethene. For reliable assessment of natural attenuation, or when designing monitoring or remedial systems, vertical stratification of key biological and hydrochemical markers should be analysed as standard, even in shallow aquifers.