Long-Term Effects of Multiwalled Carbon Nanotubes and Graphene on Microbial Communities in Dry Soil.

Little is known about the long-term effects of engineered carbonaceous nanomaterials (ECNMs) on soil microbial communities, especially when compared to possible effects of natural or industrial carbonaceous materials. To address these issues, we exposed dry grassland soil for 1 year to 1 mg g(-1) of either natural nanostructured material (biochar), industrial carbon black, three types of multiwalled carbon nanotubes (MWCNTs), or graphene. Soil microbial biomass was assessed by substrate induced respiration and by extractable DNA. Bacterial and fungal communities were examined by terminal restriction fragment length polymorphism (T-RFLP). Microbial activity was assessed by soil basal respiration. At day 0, there was no treatment effect on soil DNA or T-RFLP profiles, indicating negligible interference between the amended materials and the methods for DNA extraction, quantification, and community analysis. After a 1-year exposure, compared to the no amendment control, some treatments reduced soil DNA (e.g., biochar, all three MWCNT types, and graphene; P < 0.05) and altered bacterial communities (e.g., biochar, carbon black, narrow MWCNTs, and graphene); however, there were no significant differences across the amended treatments. These findings suggest that ECNMs may moderately affect dry soil microbial communities but that the effects are similar to those from natural and industrial carbonaceous materials, even after 1-year exposure.

Soybean susceptibility to manufactured nanomaterials with evidence for food quality and soil fertility interruption.

Based on previously published hydroponic plant, planktonic bacterial, and soil microbial community research, manufactured nanomaterial (MNM) environmental buildup could profoundly alter soil-based food crop quality and yield. However, thus far, no single study has at once examined the full implications, as no studies have involved growing plants to full maturity in MNM-contaminated field soil. We have done so for soybean, a major global commodity crop, using farm soil amended with two high-production metal oxide MNMs (nano-CeO(2) and -ZnO). The results provide a clear, but unfortunate, view of what could arise over the long term: (i) for nano-ZnO, component metal was taken up and distributed throughout edible plant tissues; (ii) for nano-CeO(2), plant growth and yield diminished, but also (iii) nitrogen fixation--a major ecosystem service of leguminous crops--was shut down at high nano-CeO(2) concentration. Juxtaposed against widespread land application of wastewater treatment biosolids to food crops, these findings forewarn of agriculturally associated human and environmental risks from the accelerating use of MNMs.

Integrated approach to evaluating the toxicity of novel cysteine-capped silver nanoparticles to Escherichia coli and Pseudomonas aeruginosa.

Because of microbial resistance to conventional antibiotics, there is increasing interest in silver, including silver nanoparticles (nano-Ag), in antimicrobial applications. However, questions remain regarding the relative roles of nano-Ag particles, versus Ag(+) ions released from nano-Ag dissolution, in imparting bacterial toxicity. Here, we developed a novel nano-Ag that, based on its cysteine cap, was expected to dissolve slowly and thus potentially allow for differentiating nanoparticle, versus ionic, effects of Ag. The nano-Ag was systematically tested for its differential toxicity to Escherichia coli and Pseudomonas aeruginosa. Bacterial growth, reactive oxygen species (ROS) generation, particle dissolution, cellular electron transfer activity, and cell membrane damage and potential were evaluated. In minimal growth medium, E. coli and P. aeruginosa growth were slowed at 100 mg L(-1) (0.93 mM) and 5 mg L(-1) (0.046 mM), respectively; P. aeruginosa was completely inhibited at and above 10 mg L(-1) (0.093 mM). For both strains, toxicity was associated with ROS and cell membrane damage. Based on comparisons to AgNO3 exposures, toxicity from nano-Ag was due to Ag(+) ions and not intact nano-Ag, even though nanoparticle dissolution was less than 2% in minimal growth medium. Because of their stability and slow Ag(+) ion release, the cysteine-capped nano-Ag particles here are useful to antimicrobial applications. Additionally, our systematic approach to evaluating toxicity, membrane damage, and ROS generation can be applied with other nanomaterials and bacteria.

Evaluation of exposure concentrations used in assessing manufactured nanomaterial environmental hazards: are they relevant?

Manufactured nanomaterials (MNMs) are increasingly produced and used in consumer goods, yet our knowledge regarding their environmental risks is limited. Environmental risks are assessed by characterizing exposure levels and biological receptor effects. As MNMs have rarely been quantified in environmental samples, our understanding of exposure level is limited. Absent direct measurements, environmental MNM concentrations are estimated from exposure modeling. Hazard, the potential for effects on biological receptors, is measured in the laboratory using a range of administered MNM concentrations. Yet concerns have been raised regarding the "relevancy" of hazard assessments, particularly when the administered MNM concentrations exceed those predicted to occur in the environment. What MNM concentrations are administered in hazard assessments and which are "environmentally relevant"? This review regards MNM concentrations in hazard assessments, from over 600 peer-reviewed articles published between 2008 and 2013. Some administered MNM concentrations overlap with, but many diverge from, predicted environmental concentrations. Other uncertainties influence the environmental relevance of current hazard assessments and exposure models, including test conditions, bioavailable concentrations, mode of action, MNM production volumes, and model validation. Therefore, it may be premature for MNM risk research to sanction information on the basis of concentration "environmental relevance".

Effects of TiO2 and Ag nanoparticles on polyhydroxybutyrate biosynthesis by activated sludge bacteria.

Manufactured nanomaterials (MNMs) are increasingly incorporated into consumer products that are disposed into sewage. In wastewater treatment, MNMs adsorb to activated sludge biomass where they may impact biological wastewater treatment performance, including nutrient removal. Here, we studied MNM effects on bacterial polyhydroxyalkanoate (PHA), specifically polyhydroxybutyrate (PHB), biosynthesis because of its importance to enhanced biological phosphorus (P) removal (EBPR). Activated sludge was sampled from an anoxic selector of a municipal wastewater treatment plant (WWTP), and PHB-containing bacteria were concentrated by density gradient centrifugation. After starvation to decrease intracellular PHB stores, bacteria were nutritionally augmented to promote PHB biosynthesis while being exposed to either MNMs (TiO2 or Ag) or to Ag salts (each at a concentration of 5 mg L(-1)). Cellular PHB concentration and PhyloChip community composition were analyzed. The final bacterial community composition differed from activated sludge, demonstrating that laboratory enrichment was selective. Still, PHB was synthesized to near-activated sludge levels. Ag salts altered final bacterial communities, although MNMs did not. PHB biosynthesis was diminished with Ag (salt or MNMs), indicating the potential for Ag-MNMs to physiologically impact EBPR through the effects of dissolved Ag ions on PHB producers.

Soybean plants modify metal oxide nanoparticle effects on soil bacterial communities.

Engineered nanoparticles (ENPs) are entering agricultural soils through land application of nanocontaining biosolids and agrochemicals. The potential adverse effects of ENPs have been studied on food crops and soil bacterial communities separately; however, how ENPs will affect the interacting plant-soil system remains unknown. To address this, we assessed ENP effects on soil microbial communities in soybean-planted, versus unplanted, mesocosms exposed to different doses of nano-CeO2 (0-1.0 g kg(-1)) or nano-ZnO (0-0.5 g kg(-1)). Nano-CeO2 did not affect soil bacterial communities in unplanted soils, but 0.1 g kg(-1) nano-CeO2 altered soil bacterial communities in planted soils, indicating that plants interactively promote nano-CeO2 effects in soil, possibly due to belowground C shifts since plant growth was impacted. Nano-ZnO at 0.5 g kg(-1) significantly altered soil bacterial communities, increasing some (e.g., Rhizobium and Sphingomonas) but decreasing other (e.g., Ensifer, Rhodospirillaceae, Clostridium, and Azotobacter) operational taxonomic units (OTUs). Fewer OTUs decreased from nano-ZnO exposure in planted (41) versus unplanted (85) soils, suggesting that plants ameliorate nano-ZnO effects. Taken together, plants--potentially through their effects on belowground biogeochemistry--could either promote (i.e., for the 0.1 g kg(-1) nano-CeO2 treatment) or limit (i.e., for the 0.5 g kg(-1) nano-ZnO treatment) ENP effects on soil bacterial communities.

An assessment of fluorescence- and absorbance-based assays to study metal-oxide nanoparticle ROS production and effects on bacterial membranes.

The production and inevitable release of engineered nanoparticles requires rapid approaches to screen for their potential effects in environmental organisms, including bacteria. In bacteria, engineered nanoparticle effects can initiate at the cell membrane, for example by structurally damaging membranes or inhibiting energy transduction. Commercially available fluorescence- and absorbance-based assays could allow for rapidly assaying engineered nanoparticle effects on bacterial membranes, but there are limitations, including that: 1) assays are not currently configured to operate as part of a comprehensive high-throughput screening system, since assay conditions vary widely and formats are mostly high-volume and thus low-throughput, and; 2) engineered nanoparticles can interfere with assay reagents or function, yielding false-negative or -positive outcomes. Here, key assays to study reactive oxygen species (total ROS, and superoxide) production, and impacts on bacterial membrane integrity, membrane potential, and electron transport chain activity, are assessed for their potential use as a comprehensive system to test for nanoparticle effects in bacteria. To address (1), assays are adapted for simultaneous use in 96-well microplates under harmonized conditions. To address (2), a general scheme to test for engineered nanoparticle interferences with assay reagents and function is conceived, and used to study assay interferences by three nanoscale metal-oxides: nano-TiO2 , nano-CeO2 , and nano-ZnO. The results show that the selected assays can be used as a suite, and that nanoparticle interferences, when they occur, can be systematically investigated and often accounted for.

Differential growth of and nanoscale TiO2 accumulation in Tetrahymena thermophila by direct feeding versus trophic transfer from Pseudomonas aeruginosa.

Nanoscale titanium dioxide (TiO2) is increasingly used in consumer goods and is entering waste streams, thereby exposing and potentially affecting environmental microbes. Protozoans could either take up TiO2 directly from water and sediments or acquire TiO2 during bactivory (ingestion of bacteria) of TiO2-encrusted bacteria. Here, the route of exposure of the ciliated protozoan Tetrahymena thermophila to TiO2 was varied and the growth of, and uptake and accumulation of TiO2 by, T. thermophila were measured. While TiO2 did not affect T. thermophila swimming or cellular morphology, direct TiO2 exposure in rich growth medium resulted in a lower population yield. When TiO2 exposure was by bactivory of Pseudomonas aeruginosa, the T. thermophila population yield and growth rate were lower than those that occurred during the bactivory of non-TiO2-encrusted bacteria. Regardless of the feeding mode, T. thermophila cells internalized TiO2 into their food vacuoles. Biomagnification of TiO2 was not observed; this was attributed to the observation that TiO2 appeared to be unable to cross the food vacuole membrane and enter the cytoplasm. Nevertheless, our findings imply that TiO2 could be transferred into higher trophic levels within food webs and that the food web could be affected by the decreased growth rate and yield of organisms near the base of the web.

Potential mechanisms and environmental controls of TiO2 nanoparticle effects on soil bacterial communities.

It has been reported that engineered nanoparticles (ENPs) alter soil bacterial communities, but the underlying mechanisms and environmental controls of such effects remain unknown. Besides direct toxicity, ENPs may indirectly affect soil bacteria by changing soil water availability or other properties. Alternatively, soil water or other environmental factors may mediate ENP effects on soil bacterial communities. To test, we incubated nano-TiO2-amended soils across a range of water potentials for 288 days. Following incubation, the soil water characteristics, organic matter, total carbon, total nitrogen, and respiration upon rewetting (an indicator of bioavailable organic carbon) were measured. Bacterial community shifts were characterized by terminal restriction fragment length polymorphism (T-RFLP). The endpoint soil water holding had been reported previously as not changing with this nano-TiO2 amendment; herein, we also found that some selected soil properties were unaffected by the treatments. However, we found that nano-TiO2 altered the bacterial community composition and reduced diversity. Nano-TiO2-induced community dissimilarities increased but tended to approach a plateau when soils became drier. Taken together, nano-TiO2 effects on soil bacteria appear to be a result of direct toxicity rather than indirectly through nano-TiO2 affecting soil water and organic matter pools. However, such directs effects of nano-TiO2 on soil bacterial communities are mediated by soil water.

Dynamic energy budget approach to modeling mechanisms of CdSe quantum dot toxicity.

A mechanistic model of bacterial growth based on dynamic energy budget (DEB) theory is utilized to investigate mechanisms of toxicity of CdSe quantum dots (QDs). The model of QD toxicity is developed by extending a previously published DEB model of cadmium ion toxicity to include a separate model of QD toxic action. The extension allows for testing whether toxicity from QD exposure can be explained fully by dissolved cadmium exposure only, or if the separate effects of QDs need to be taken into account as well. Two major classes of QD toxicity mechanisms are considered: acclimation expressed through initial retardation of growth, and three separate metabolic effects that can be a result of QDs either reversibly or irreversibly associating with the cell. The model is consistent with the data, and is able to distinguish toxic effects due to QD nano-particles from the effects due to cadmium ions. Results suggest that, in contrast to ionic exposure where required acclimation remains constant as exposure increases, increase of the energy required for acclimation with exposure is the primary toxic effect of QDs. Reactive oxygen species measurements help conclude that increase in energetic cost of maintenance processes such as cellular repair and maintenance of cross-membrane gradients is the most important of the three metabolic effects of QD toxicity.

Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy.

Bacterial biofilms, i.e. surface-associated cells covered in hydrated extracellular polymeric substances (EPS), are often studied with high-resolution electron microscopy (EM). However, conventional desiccation and high vacuum EM protocols collapse EPS matrices which, in turn, deform biofilm appearances. Alternatively, wet-mode environmental scanning electron microscopy (ESEM) is performed under a moderate vacuum and without biofilm drying. If completely untreated, however, EPS is not electron dense and thus is not resolved well in ESEM. Therefore, this study was towards adapting several conventional SEM staining protocols for improved resolution of biofilms and EPS using ESEM. Three different biofilm types were used: 1) Pseudomonas aeruginosa unsaturated biofilms cultured on membranes, 2) P. aeruginosa cultured in moist sand, and 3) mixed community biofilms cultured on substrates in an estuary. Working with the first specimen type, a staining protocol using ruthenium red, glutaraldehyde, osmium tetroxide and lysine was optimized for best topographic resolution. A quantitative image analysis tool that maps relief, newly adopted here for studying biofilms, was used to compare micrographs. When the optimized staining and ESEM protocols were applied to moist sand cultures and aquatic biofilms, the smoothening effect that bacterial biofilms have on rough sand, and the roughening that aquatic biofilms impart on initially smooth coupons, were each quantifiable. This study thus provides transferable staining and ESEM imaging protocols suitable for a wide range of biofilms, plus a novel tool for quantifying biofilm image data.

Damage assessment for soybean cultivated in soil with either CeO2 or ZnO manufactured nanomaterials.

With increasing use, manufactured nanomaterials (MNMs) may enter soils and impact agriculture. Herein, soybean (Glycine max) was grown in soil amended with either nano-CeO2 (0.1, 0.5, or 1.0gkg-1 soil) or nano-ZnO (0.05, 0.1, or 0.5gkg-1 soil). Leaf chlorosis, necrosis, and photosystem II (PSII) quantum efficiency were monitored during plant growth. Seed protein and protein carbonyl, plus leaf chlorophyll, reactive oxygen species (ROS), lipid peroxidation, and genotoxicity were measured for plants at harvest. Neither PSII quantum efficiency, seed protein, nor protein carbonyl indicated negative MNM effects. However, increased ROS, lipid peroxidation, and visible damage, along with decreased total chlorophyll concentrations, were observed for soybean leaves in the nano-CeO2 treatments. These effects correlated to aboveground leaf, pod, and stem production, and to root nodule N2 fixation potential. Soybeans grown in soil amended with nano-ZnO maintained growth, yield, and N2 fixation potential similarly to the controls, without increased leaf ROS or lipid peroxidation. Leaf damage was observed for the nano-ZnO treatments, and genotoxicity appeared for the highest nano-ZnO treatment, but only for one plant. Total chlorophyll concentrations decreased with increasing leaf Zn concentration, which was attributable to zinc complexes-not nano-ZnO-in the leaves. Overall, nano-ZnO and nano-CeO2 amended to soils differentially triggered aboveground soybean leaf stress and damage. However, the consequences of leaf stress and damage to N2 fixation, plant growth, and yield were only observed for nano-CeO2.

Cerium dioxide and zinc oxide nanoparticles alter the nutritional value of soil cultivated soybean plants.

The aim of this study was to determine nutrient elements in soybean (Glycine max) plants cultivated in farm soil amended with nCeO2 at 0-1000 mg kg(-1) and nZnO at 0-500 mg kg(-1). Digested samples were analyzed by ICP-OES/MS. Compared to control, pods from nCeO2 at 1000 mg kg(-1) had significantly less Ca but more P and Cu, while pods from 100 mg kg(-1)nZnO had more Zn, Mn, and Cu. Plants treated with nZnO showed significant correlations among Zn, P, and S in pods with Zn in roots. Correlations among pod Zn/root Zn was r = 0.808 (p ≤ 0.01) and pod P/root P was r = 0.541 (p ≤ 0.05). The correlation among pod S/root S was r = -0.65 (p ≤ 0.01). While nCeO2 treatments exhibited significant correlations between pod Ca/root Ca (r = 0.645, p ≤ 0.05). The data suggest that nCeO2 and nZnO alter the nutritional value of soybean, which could affect the health of plants, humans, and animals.

In situ synchrotron X-ray fluorescence mapping and speciation of CeO2 and ZnO nanoparticles in soil cultivated soybean (Glycine max).

With the increased use of engineered nanomaterials such as ZnO and CeO2 nanoparticles (NPs), these materials will inevitably be released into the environment, with unknown consequences. In addition, the potential storage of these NPs or their biotransformed products in edible/reproductive organs of crop plants can cause them to enter into the food chain and the next plant generation. Few reports thus far have addressed the entire life cycle of plants grown in NP-contaminated soil. Soybean ( Glycine max ) seeds were germinated and grown to full maturity in organic farm soil amended with either ZnO NPs at 500 mg/kg or CeO2 NPs at 1000 mg/kg. At harvest, synchrotron µ-XRF and µ-XANES analyses were performed on soybean tissues, including pods, to determine the forms of Ce and Zn in NP-treated plants. The X-ray absorption spectroscopy studies showed no presence of ZnO NPs within tissues. However, µ-XANES data showed O-bound Zn, in a form resembling Zn-citrate, which could be an important Zn complex in the soybean grains. On the other hand, the synchrotron µ-XANES results showed that Ce remained mostly as CeO2 NPs within the plant. The data also showed that a small percentage of Ce(IV), the oxidation state of Ce in CeO2 NPs, was biotransformed to Ce(III). To our knowledge, this is the first report on the presence of CeO2 and Zn compounds in the reproductive/edible portion of the soybean plant grown in farm soil with CeO2 and ZnO NPs.

Modeling physiological processes that relate toxicant exposure and bacterial population dynamics.

Quantifying effects of toxicant exposure on metabolic processes is crucial to predicting microbial growth patterns in different environments. Mechanistic models, such as those based on Dynamic Energy Budget (DEB) theory, can link physiological processes to microbial growth.Here we expand the DEB framework to include explicit consideration of the role of reactive oxygen species (ROS). Extensions considered are: (i) additional terms in the equation for the "hazard rate" that quantifies mortality risk; (ii) a variable representing environmental degradation; (iii) a mechanistic description of toxic effects linked to increase in ROS production and aging acceleration, and to non-competitive inhibition of transport channels; (iv) a new representation of the "lag time" based on energy required for acclimation. We estimate model parameters using calibrated Pseudomonas aeruginosa optical density growth data for seven levels of cadmium exposure. The model reproduces growth patterns for all treatments with a single common parameter set, and bacterial growth for treatments of up to 150 mg(Cd)/L can be predicted reasonably well using parameters estimated from cadmium treatments of 20 mg(Cd)/L and lower. Our approach is an important step towards connecting levels of biological organization in ecotoxicology. The presented model reveals possible connections between processes that are not obvious from purely empirical considerations, enables validation and hypothesis testing by creating testable predictions, and identifies research required to further develop the theory.

Effects of soluble cadmium salts versus CdSe quantum dots on the growth of planktonic Pseudomonas aeruginosa.

With their increased use, engineered nanomaterials (ENMs) will enterthe environment where they may be altered by bacteria and affect bacterial processes. Metallic ENMs, such as CdSe quantum dots (QDs), are toxic due to the release of dissolved heavy metals, but the effects of cadmium ions versus intact QDs are mostly unknown. Here, planktonic Pseudomonas aeruginosa PG201 bacteria were cultured with similar total cadmium concentrations as either fully dissolved cadmium acetate (Cd(CH3COO)2) or ligand capped CdSe QDs, and cellular morphology, growth parameters, intracellular reactive oxygen species (ROS), along with the metal and metalloid fates were measured. QDs dissolved partially in growth media, but dissolution was less in biotic cultures compared to sterile controls. Dose-dependent growth effects were similar for low concentrations of either cadmium salts or QDs, but effects differed above a concentration threshold of 50 mg/L(total cadmium basis) where (1) the growth of QD-treated cells was more impaired, (2) the membranes of QD-grown cells were damaged, and (3) QD-grown cells contained QD-sized CdSe cytoplasmic inclusions in addition to Se0 and dissolved cadmium. For most concentrations, intracellular ROS were higher for QD-versus cadmium salts-grown bacteria. Taken together, QDs were more toxic to this opportunistic pathogen than cadmium ions, and were affected by cells through QD extracellular stabilization, intracellular enrichment and cell-associated decay.

Enhanced exopolymer production and chromium stabilization in Pseudomonas putida unsaturated biofilms.

Chromium-contaminated soils threaten surface and groundwater quality at many industrial sites. In vadose zones, indigenous bacteria can reduce Cr(VI) to Cr(III), but the subsequent fate of Cr(III) and the roles of bacterial biofilms are relatively unknown. To investigate, we cultured Pseudomonas putida, a model organism for vadose zone bioremediation, as unsaturated biofilms on membranes overlaying iron-deficient solid media either containing molecular dichromate from potassium dichromate (Cr-only treatment) or with deposits of solid, dichromate-coated hematite (Fe+Cr treatment) to simulate vadose zone conditions. Controls included iron-deficient solid medium and an Fe-only treatment using solid hematite deposits. Under iron-deficient conditions, chromium exposure resulted in lower cell yield and lower amounts of cellular protein and carbohydrate, but providing iron in the form of hematite overcame these toxic effects of Cr. For the Cr and Fe+Cr treatments, Cr(VI) was completely reduced to Cr(III) that accumulated on biofilm cells and extracellular polymeric substances (EPSs). Chromium exposure resulted in elevated extracellular carbohydrates, protein, DNA, and EPS sugars that were relatively enriched in N-acetyl-glucosamine, rhamnose, glucose, and mannose. The proportions of EPS protein and carbohydrate relative to intracellular pools suggested Cr toxicity-mediated cell lysis as the origin. However, DNA accumulated extracellularly in amounts far greater than expected from cell lysis, and Cr was liberated when extracted EPS was treated with DNase. These results demonstrate that Cr accumulation in unsaturated biofilms occurs with enzymatic reduction of Cr(VI), cellular lysis, cellular association, and extracellular DNA binding of Cr(III), which altogether can facilitate localized biotic stabilization of Cr in contaminated vadose zones.