One-Time Addition of Nano-TiO2 Triggers Short-Term Responses in Benthic Bacterial Communities in Artificial Streams.

Nano-TiO2 is an engineered nanomaterial whose production and use are increasing rapidly. Hence, aquatic habitats are at risk for nano-TiO2 contamination due to potential inputs from urban and suburban runoff and domestic wastewater. Nano-TiO2 has been shown to be toxic to a wide range of aquatic organisms, but little is known about the effects of nano-TiO2 on benthic microbial communities. This study used artificial stream mesocosms to assess the effects of a single addition of nano-TiO2 (P25 at a final concentration of 1 mg l(-1)) on the abundance, activity, and community composition of sediment-associated bacterial communities. The addition of nano-TiO2 resulted in a rapid (within 1 day) decrease in bacterial abundance in artificial stream sediments, but bacterial abundance returned to control levels within 3 weeks. Pyrosequencing of partial 16S rRNA genes did not indicate any significant changes in the relative abundance of any bacterial taxa with nano-TiO2 treatment, indicating that nano-TiO2 was toxic to a broad range of bacterial taxa and that recovery of the bacterial communities was not driven by changes in community composition. Addition of nano-TiO2 also resulted in short-term increases in respiration rates and denitrification enzyme activity, with both returning to control levels within 3 weeks. The results of this study demonstrate that single-pulse additions of nano-TiO2 to aquatic habitats have the potential to significantly affect the abundance and activity of benthic microbial communities and suggest that interactions of TiO2 nanoparticles with environmental matrices may limit the duration of their toxicity.

Combined Toxicity of Nano-ZnO and Nano-TiO2: From Single- to Multinanomaterial Systems.

Previous studies on the toxicity of engineered nanomaterials (ENMs) have been primarily based on testing individual ENMs, so little is known about the interactions and combined toxicity of multiple ENMs. In this study the toxicity of chemically stable nano-TiO2 and soluble nano-ZnO was investigated individually and in combination, by monitoring bacterial cell membrane integrity and ATP levels in a natural aqueous medium (Lake Michigan water). Both nano-TiO2 and nano-ZnO damage bacterial cell membranes under simulated solar irradiation (SSI), but their phototoxicity is not additive. Nano-ZnO at 1 mg/L, for example, surprisingly eliminates the damaging effect of nano-TiO2 at 10 mg/L. This phenomenon does not correlate with reactive oxygen species production, but is explained by a reduced extent of bacteria/nano-TiO2 contact in the presence of both nano-ZnO and dissolved zinc. The presence of nano-ZnO also exerts a significant decrease in bacterial ATP levels both under SSI and in the dark, a stress effect not captured by measuring bacterial cell membrane integrity. This inhibitory effect of nano-ZnO, however, is reduced somewhat by nano-TiO2 due to the adsorption of Zn(2+). Therefore, our results reveal that nanoparticle interactions and surface complexation reactions alter the original toxicity of individual nanoparticles and that comprehensive assessments of potential ENM toxicity in the environment require careful integration of complex physicochemical interactions between ENMs and various biological responses.

Comparing Acute Effects of a Nano-TiO2 Pigment on Cosmopolitan Freshwater Phototrophic Microbes Using High-Throughput Screening.

Production of titanium-dioxide nanomaterials (nano-TiO2) is increasing, leading to potential risks associated with unintended release of these materials into aquatic ecosystems. We investigated the acute effects of nano-TiO2 on metabolic activity and viability of algae and cyanobacteria using high-throughput screening. The responses of three diatoms (Surirella angusta, Cocconeis placentula, Achnanthidium lanceolatum), one green alga (Scenedesmus quadricauda), and three cyanobacteria (Microcystis aeruginosa, Gloeocapsa sp., Synechococcus cedrorum) to short-term exposure (15 to 60 min) to a common nano-TiO2 pigment (PW6; average crystallite size 81.5 nm) with simulated solar illumination were assessed. Five concentrations of nano-TiO2 (0.5, 2.5, 5, 10, and 25 mg L-1) were tested and a fluorescent reporter (fluorescein diacetate) was used to assess metabolic activity. Algae were sensitive to nano-TiO2, with all showing decreased metabolic activity after 30-min exposure to the lowest tested concentration. Microscopic observation of algae revealed increased abundance of dead cells with nano-TiO2 exposure. Cyanobacteria were less sensitive to nano-TiO2 than algae, with Gloeocapsa showing no significant decrease in activity with nano-TiO2 exposure and Synechococcus showing an increase in activity. These results suggest that nanomaterial contamination has the potential to alter the distribution of phototrophic microbial taxa within freshwater ecosystems. The higher resistance of cyanobacteria could have significant implications as cyanobacteria represent a less nutritious food source for higher trophic levels and some cyanobacteria can produce toxins and contribute to harmful algal blooms.

Acute effects of TiO2 nanomaterials on the viability and taxonomic composition of aquatic bacterial communities assessed via high-throughput screening and next generation sequencing.

The nanotechnology industry is growing rapidly, leading to concerns about the potential ecological consequences of the release of engineered nanomaterials (ENMs) to the environment. One challenge of assessing the ecological risks of ENMs is the incredible diversity of ENMs currently available and the rapid pace at which new ENMs are being developed. High-throughput screening (HTS) is a popular approach to assessing ENM cytotoxicity that offers the opportunity to rapidly test in parallel a wide range of ENMs at multiple concentrations. However, current HTS approaches generally test one cell type at a time, which limits their ability to predict responses of complex microbial communities. In this study toxicity screening via a HTS platform was used in combination with next generation sequencing (NGS) to assess responses of bacterial communities from two aquatic habitats, Lake Michigan (LM) and the Chicago River (CR), to short-term exposure in their native waters to several commercial TiO2 nanomaterials under simulated solar irradiation. Results demonstrate that bacterial communities from LM and CR differed in their sensitivity to nano-TiO2, with the community from CR being more resistant. NGS analysis revealed that the composition of the bacterial communities from LM and CR were significantly altered by exposure to nano-TiO2, including decreases in overall bacterial diversity, decreases in the relative abundance of Actinomycetales, Sphingobacteriales, Limnohabitans, and Flavobacterium, and a significant increase in Limnobacter. These results suggest that the release of nano-TiO2 to the environment has the potential to alter the composition of aquatic bacterial communities, which could have implications for the stability and function of aquatic ecosystems. The novel combination of HTS and NGS described in this study represents a major advance over current methods for assessing ENM ecotoxicity because the relative toxicities of multiple ENMs to thousands of naturally occurring bacterial species can be assessed simultaneously under environmentally relevant conditions.

Common freshwater bacteria vary in their responses to short-term exposure to nano-TiO2.

Nanostructured titania (nano-TiO2) is an engineered nanomaterial that can be cytotoxic primarily as a result of its ability to generate reactive oxygen species when illuminated. Production of nano-TiO2 has increased rapidly over the last decade, leading to concerns about its release into aquatic environments. To address the possible ecological impacts of nano-TiO2, the authors used high-throughput screening to assess the responses of 4 bacteria representative of genera common in freshwater to short-term exposure (1-2 h) in 2 natural aqueous media (stream water and lake water) to 2 widely used TiO2 products, pigment white 6 (PW6) and P25. Under simulated solar illumination PW6 and P25 reduced the abundance of viable Bacillus subtilis and Aeromonas hydrophila, confirming the cytotoxicity of nano-TiO2 . In contrast, PW6 and P25 stimulated growth of Arthrobacter sp. and Klebsiella sp., which the authors hypothesize was driven by oxidation of organic matter in these natural waters into more labile compounds. This hypothesis is supported by data demonstrating PW6 photo-oxidation of organic matter in stream water, which subsequently supported enhanced bacterial growth. The results indicate that bacterial responses to nano-TiO2 can be species-specific, suggesting that nano-TiO2 may alter bacterial community composition and function. Finally, the results indicate that bacterial responses to nano-TiO2 are influenced by the water matrix, emphasizing the importance of assessing bacterial responses to nanomaterials in natural environmental media.

Effects of material morphology on the phototoxicity of nano-TiO2 to bacteria.

Nanostructured titania (nano-TiO2) is produced in diverse shapes, but it remains largely unknown how tuning the morphology of nano-TiO2 may alter its toxicity. Herein, we show that material morphology plays a critical role in regulating the phototoxicity of nano-TiO2 to bacteria. Low-dimensional nano-TiO2, including nanotubes, nanorods, and nanosheets, were synthesized hydrothermally, and their effects on the bacterial viability of Escherichia coli and Aeromonas hydrophila were compared to spherical nanostructures (anatase nanospheres and P25). Results reveal that TiO2 nanotubes and nanosheets are less phototoxic than their rod- and sphere-shape counterparts under simulated solar irradiation. None of the tested nano-TiO2 shows toxicity in the dark. In contrast to their diminished phototoxicity, however, TiO2 nanotubes and nanosheets exhibit comparable or even higher photoactivity than other nanostructures. Observations by scanning transmission electron microscopy suggest that material morphology influences nano-TiO2 phototoxicity by governing how nano-TiO2 particles align at the bacterial cell surface. Overall, when comparing materials with different morphologies and dimensionality, nano-TiO2 phototoxicity is not a simple function of photocatalytic reactivity or ROS production. Instead, we propose that the evaluation of nano-TiO2 phototoxicity encompasses a three-pronged approach, involving the intrinsic photoactivity, aggregation of nano-TiO2, and the nano-TiO2/bacteria surface interactions.

Cytotoxicity of commercial nano-TiO2 to Escherichia coli assessed by high-throughput screening: effects of environmental factors.

The extensive use of nano-TiO2 in industry has led to growing concerns about its potential environmental impacts. However, negligible toxicity is commonly reported under insufficient illumination and artificial solution conditions in the literature, which rarely includes discussion of the regulating role of environmental factors. Herein, we report the results of a high-throughput screening assay to evaluate the acute cytotoxicity of six commercial nano-TiO2 materials to Escherichia coli (E. coli) using Lake Michigan water as a model for aquatic surface environments. In particular, we investigate the specific effects of illumination wavelength and natural organic matter (NOM) content. Under simulated solar irradiation, four anatase-based nano-TiO2 materials including Pigment White 6 exhibit significant bacterial toxicity (2 h-IC50 value of 2.7-9.1 mg/L), with toxicity thresholds much lower than previously reported. Negligible toxicity is caused either by pure-phase rutile or under dark condition. Formation of nano-TiO2 aggregates well beyond nano-scale does not eliminate their toxic effect, but photoactivity dominates over the primary size and extent of aggregation in determining the acute cytotoxicity of nano-TiO2. Under visible light irradiation (UVA&B blocked) the antibacterial activity of nano-TiO2 is essentially erased, whereas removing only UVB wavelengths slightly mitigates the toxicity. Suwannee River fulvic acid, acting as a natural dispersant, reverses the extent of nano-TiO2 aggregation, but also reduces its bacterial cytotoxicity. These results demonstrate that despite particle aggregation, the short-term cytotoxicity of nano-TiO2 is predominantly attributed to its phototoxicity, emphasizing the importance of illumination conditions in toxicological screening of photoactive nanomaterials. In the natural aquatic environment, however, this acute toxicity may be mitigated by the attenuation of UV irradiation and increased NOM concentration in the water column.

Taxonomic and ecological studies of actinomycetes from Vietnam: isolation and genus-level diversity.

Actinomycetes were isolated from 109 soil and 93 leaf-litter samples collected at five sites in Vietnam between 2005 and 2008 using the rehydration-centrifugation (RC) method, sodium dodecyl sulfate-yeast extract dilution method, dry-heating method and oil-separation method in conjunction with humic acid-vitamin agar as an isolation medium. A total of 1882 strains were identified as Vietnamese (VN)-actinomycetes including 1080 (57%) streptomycetes (the genus Streptomyces isolates) and 802 (43%) non-streptomycetes. The 16S ribosomal RNA gene sequences of the VN-actinomycetes were analyzed using BLAST searches. The results showed that these isolates belonged to 53 genera distributed among 21 families. Approximately 90% of these strains were members of three families: Streptomycetaceae (1087 strains, 58%); Micromonosporaceae (516 strains, 27%); and Streptosporangiaceae (89 strains, 5%). Motile actinomycetes of the genera Actinoplanes, Kineosporia and Cryptosporangium, which have quite common morphological characteristics, were frequently isolated from leaf-litter samples using the RC method. It is possible that these three genera acquired common properties during a process of convergent evolution. By contrast, strains belonging to the suborder Streptosporangineae were exclusively isolated from soils. A comparison of the sampling sites revealed no significant difference in taxonomic diversity between these sites. Among the non-streptomycetes, 156 strains (19%) were considered as new taxa distributed into 21 genera belonging to 12 families. Interestingly, the isolation of actinomycetes from leaf-litter samples using the RC method proved to be the most efficient way to isolate new actinomycetes in Vietnam, especially the Micromonosporaceae species.

Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids.

In this study, the prevalence and types of transferable antibiotic resistance plasmids in piggery manure were investigated. Samples from manure storage tanks of 15 farms in Germany were analysed, representing diverse sizes of herds, meat or piglet production. Antibiotic resistance plasmids from manure bacteria were captured in gfp-tagged rifampicin-resistant Escherichia coli and characterized. The occurrence of plasmid types was also detected in total community DNA by PCR and hybridization. A total of 228 transconjugants were captured from 15 manures using selective media supplemented with amoxicillin, sulfadiazine or tetracycline. The restriction patterns of 81 plasmids representing different antibiotic resistance patterns or different samples clustered into seven groups. Replicon probing revealed that 28 of the plasmids belonged to IncN, one to IncW, 13 to IncP-1 and 19 to the recently discovered pHHV216-like plasmids. The amoxicillin resistance gene bla-TEM was detected on 44 plasmids, and sulphonamide resistance genes sul1, sul2 and/or sul3 on 68 plasmids. Hybridization of replicon-specific sequences amplified from community DNA revealed that IncP-1 and pHHV216-like plasmids were detected in all manures, while IncN and IncW ones were less frequent. This study showed that 'field-scale' piggery manure is a reservoir of broad-host range plasmids conferring multiple antibiotic resistance genes.

Short-term effects of amoxicillin on bacterial communities in manured soil.

Antibiotic-resistant bacteria, nutrients and antibiotics that enter the soil by means of manure may enhance the proportion of bacteria displaying antibiotic resistance among soil bacteria and may affect bacterial community structure and function. To investigate the effect of manure and amoxicillin added to manure on soil bacterial communities, microcosm experiments were performed with two soil types and the following treatments: (1) nontreated, (2) manure-treated, (3) treated with manure supplemented with 10 mg amoxicillin kg(-1) soil and (4) treated with manure supplemented with 100 mg amoxicillin kg(-1) soil, with four replicates per treatment. Manure significantly increased the total CFU count and the amoxicillin-resistant CFU count of both soil types. However, only the soil with a history of manure treatment showed a significant increase in the relative number of amoxicillin-resistant bacteria as a result of amoxicillin amendment. The majority of plasmids exogenously isolated from soil originated from soil treated with amoxicillin-supplemented manure. All 16 characterized plasmids carried the bla-TEM gene, and 10 of them belonged to the IncN group. The bla-TEM gene was detected in DNA directly extracted from soil by dot-blot hybridization of PCR amplicons and showed an increased abundance in soil samples treated with manure. Molecular fingerprint analysis of 16S rRNA gene fragments amplified from soil DNA revealed significant effects of manure and amoxicillin on the bacterial community of both soils.