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.

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.

Iron-sulfide-bearing chimneys as potential catalytic energy traps at life's emergence.

The concept that life emerged where alkaline hydrogen-bearing submarine hot springs exhaled into the most ancient acidulous ocean was used as a working hypothesis to investigate the nature of precipitate membranes. Alkaline solutions at 25-70°C and pH between 8 and 12, bearing HS(-)±silicate, were injected slowly into visi-jars containing ferrous chloride to partially simulate the early ocean on this or any other wet and icy, geologically active rocky world. Dependent on pH and sulfide content, fine tubular chimneys and geodal bubbles were generated with semipermeable walls 4-100 µm thick that comprised radial platelets of nanometric mackinawite [FeS]±ferrous hydroxide [∼Fe(OH)(2)], accompanied by silica and, at the higher temperature, greigite [Fe(3)S(4)]. Within the chimney walls, these platelets define a myriad of micropores. The interior walls of the chimneys host iron sulfide framboids, while, in cases where the alkaline solution has a pH>11 or relatively low sulfide content, their exteriors exhibit radial flanges with a spacing of ∼4 µm that comprise microdendrites of ferrous hydroxide. We speculate that this pattern results from outward and inward radial flow through the chimney walls. The outer Fe(OH)(2) flanges perhaps precipitate where the highly alkaline flow meets the ambient ferrous iron-bearing fluid, while the intervening troughs signal where the acidulous iron-bearing solutions could gain access to the sulfidic and alkaline interior of the chimneys, thereby leading to the precipitation of the framboids. Addition of soluble pentameric peptides enhances membrane durability and accentuates the crenulations on the chimney exteriors. These dynamic patterns may have implications for acid-base catalysis and the natural proton motive force acting through the matrix of the porous inorganic membrane. Thus, within such membranes, steep redox and pH gradients would bear across the nanometric platelets and separate the two counter-flowing solutions, a condition that may have led to the onset of an autotrophic metabolism through the reduction of carbon dioxide.

Design, fabrication, and test of a hydrothermal reactor for origin-of-life experiments.

We describe a continuous high-pressure flow reactor designed to simulate the unforced convective interaction of hydrothermal solutions and ocean waters with submarine crust on early Earth-conditions appropriate to those that may have led to the onset of life. The experimental operating conditions are appropriate for investigating kinetic hydrothermal processes in the early history of any sizable wet, rocky planet. Beyond the description of the fabrication, we report an initial experiment that tested the design and investigated the feasibility of sulfide and silica dissolution in alkaline solution from iron sulfide and basaltic rock, and their possible subsequent transport as HS(-) and H(2)SiO(2-)(4) in hot alkaline solutions. Delivery of hydrogen sulfide and dihydrogen silicate ions would have led to the precipitation of ferrous hydroxide, hydroxysilicates, and iron sulfides as integral mineral components of an off-ridge compartmentalized hydrothermal mound in the Hadean. Such a mound could, we contend, have acted as a natural chemical and electrochemical reactor and, ultimately, as the source of all biochemistry on our planet. In the event, we show that an average of ∼1 mM/kg of both sulfide and silica were released throughout, though over 10 mM/kg of HS(-) was recorded for ∼100 minutes in the early stages of the experiment. This alkaline effluent from the reactor was injected into a reservoir of a simulacrum of ferrous iron-bearing "Hadean Ocean" water in an experiment that demonstrated the capacity of such fluids to generate hydrothermal chimneys and a variety of contiguous inorganic microgeode precipitates bearing disseminations of discrete metal sulfides. Comparable natural composite structures may have acted as hatcheries for emergent life in the Hadean.

Dispersion of TiO2 nanoparticle agglomerates by Pseudomonas aeruginosa.

Engineered nanoparticles are increasingly incorporated into consumer products and are emerging as potential environmental contaminants. Upon environmental release, nanoparticles could inhibit bacterial processes, as evidenced by laboratory studies. Less is known regarding bacterial alteration of nanoparticles, including whether bacteria affect physical agglomeration states controlling nanoparticle settling and bioavailability. Here, the effects of an environmental strain of Pseudomonas aeruginosa on TiO2 nanoparticle agglomerates formed in aqueous media are described. Environmental scanning electron microscopy and cryogenic scanning electron microscopy visually demonstrated bacterial dispersion of large agglomerates formed in cell culture medium and in marsh water. For experiments in cell culture medium, quantitative image analysis verified that the degrees of conversion of large agglomerates into small nanoparticle-cell combinations were similar for 12-h-growth and short-term cell contact experiments. Dispersion in cell growth medium was further characterized by size fractionation: for agglomerated TiO2 suspensions in the absence of cells, 81% by mass was retained on a 5-µm-pore-size filter, compared to only 24% retained for biotic treatments. Filtrate cell and agglomerate sizes were characterized by dynamic light scattering, revealing that the average bacterial cell size increased from 1.4 µm to 1.9 µm because of nano-TiO2 biosorption. High-magnification scanning electron micrographs showed that P. aeruginosa dispersed TiO2 agglomerates by preferential biosorption of nanoparticles onto cell surfaces. These results suggest a novel role for bacteria in the environmental transport of engineered nanoparticles, i.e., growth-independent, bacterially mediated size and mass alterations of TiO2 nanoparticle agglomerates.

Bacterial and mineral elements in an arctic biofilm: a correlative study using fluorescence and electron microscopy.

Few simple labeling methods exist for simultaneous fluorescence and electron microscopy of bacteria and biofilms. Here we describe the synthesis, characterization, and application of fluorescent nanoparticle quantum dot (QD) conjugates to target microbial species, including difficult to label Gram-negative strains. These QD conjugates impart contrast for both environmental scanning electron microscopy (ESEM) and fluorescence microscopy, permitting observation of living and fixed bacteria and biofilms. We apply these probes for studying biofilms extracted from perennial cold springs in the Canadian High Arctic, which is a particularly challenging system. In these biofilms, sulfur-metabolizing bacteria live in close association with unusual sulfur mineral formations. Following simple labeling protocols with the QD conjugates, we are able to image these organisms in fully-hydrated samples and visualize their relationship to the sulfur minerals using both ESEM and fluorescence microscopy. We then use scanning transmission electron microscopy to observe precipitated sulfur around individual cells and within the biofilm lattice. All combined, this information sheds light on the possible mechanisms of biofilm and mineral structure formation. These new QD conjugates and techniques are highly transferable to many other microbiological applications, especially those involving Gram-negative bacteria, and can be used for correlated fluorescence and electron microscopy.

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.

Novel sulfur-oxidizing streamers thriving in perennial cold saline springs of the Canadian high Arctic.

The perennial springs at Gypsum Hill (GH) and Colour Peak (CP), situated at nearly 80 degrees N on Axel Heiberg Island in the Canadian high Arctic, are one of the few known examples of cold springs in thick permafrost on Earth. The springs emanate from deep saline aquifers and discharge cold anoxic brines rich in both sulfide and sulfate. Grey-coloured microbial streamers form during the winter months in snow-covered regions of the GH spring run-off channels (-1.3 degrees C to 6.9 degrees C, approximately 7.5% NaCl, 0-20 p.p.m. dissolved sulfide, 1 p.p.m. dissolved oxygen) but disappear during the Arctic summer. Culture- and molecular-based analyses of the 16S rRNA gene (FISH, DGGE and clone libraries) indicated that the streamers were uniquely dominated by chemolithoautotrophic sulfur-oxidizing Thiomicrospira species. The streamers oxidized both sulfide and thiosulfate and fixed CO(2) under in situ conditions and a Thiomicrospira strain isolated from the streamers also actively oxidized sulfide and thiosulfate and fixed CO(2) under cold, saline conditions. Overall, the snow-covered spring channels appear to represent a unique polar saline microhabitat that protects and allows Thiomicrospira streamers to form and flourish via chemolithoautrophic, phototrophic-independent metabolism in a high Arctic winter environment characterized by air temperatures commonly below -40 degrees C and with an annual average air temperature of -15 degrees C. These results broaden our knowledge of the physical and chemical boundaries that define life on Earth and have astrobiological implications for the possibility of life existing under similar Martian conditions.

Toxicity of CdTe quantum dots in bacterial strains.

Contradictory results on quantum dot cytotoxicity exist for many types of biological systems, especially microorganisms. In this study, we compare the cytotoxicity of CdTe quantum dots (QDs) to four very different environmental bacterial strains, giving quantitative models of the growth curves for exposed organisms. The mechanisms of toxicity are explored by measuring reactive oxygen species generation by the QDs alone and investigating the oxidative damage to mutant bacteria especially sensitive to ROS. Electron microscopic examination also reveals factors that may contribute to resistance to nanoparticles in some strains.

Method for simultaneous oxygen and hydrogen isotope analysis of water of crystallization in hydrated minerals.

The isotopic composition of water in hydrated minerals, such as gypsum and jarosite, has numerous applications in studies of recent climate change, ore formation, and soil development. However, oxygen and hydrogen isotope analysis of water of crystallization is currently a complex procedure. Commonly used techniques involve offline extraction of water from hydrated minerals and subsequent isotope analysis. Such methods are time-consuming, require relatively large sample sizes, and the stepwise procedure has to be carried out with extreme caution to avoid erroneous results. We present a novel online method for the oxygen and hydrogen isotope analysis of water of crystallization in hydrous minerals. Gypsum (CaSO 4.2H 2O) samples, 2 mg in size, are reacted in a simply modified carbon reducing furnace connected to a continuous-flow mass spectrometer system. Analysis time is less than 10 min/sample. The precision (2 std dev mean) of our method for 2-mg gypsum (30 mumol of H 2O) samples is 0.3 per thousand for oxygen and less than 1.4 per thousand for hydrogen isotope measurements. For oxygen isotope analysis alone, samples as small as 0.2 mg of gypsum can be analyzed with a precision of 0.3 per thousand.

Measurement of sulfur isotope compositions by tunable laser spectroscopy of SO2.

Sulfur isotope measurements offer comprehensive information on the origin and history of natural materials. Tunable laser spectroscopy is a powerful analytical technique for isotope analysis that has proven itself readily adaptable for in situ terrestrial and planetary measurements. Measurements of delta(34)S in SO2 were made using tunable laser spectroscopy of combusted gas samples from six sulfur-bearing solids with delta(34)S ranging from -34 to +22 per thousand (also measured with mass spectrometry). Standard deviation between laser and mass spectrometer measurements was 3.7 per thousand for sample sizes of 200 +/- 75 nmol SO(2). Although SO(2)(g) decreased 9% over 15 min upon entrainment in the analysis cell from wall uptake, observed fractionation was insignificant (+0.2 +/- 0.6 per thousand). We also describe a strong, distinct (33)SO(2) rovibrational transition in the same spectral region, which may enable simultaneous delta(34)S and Delta(33)S measurements.

Shewanella oneidensis MR-1 uses overlapping pathways for iron reduction at a distance and by direct contact under conditions relevant for Biofilms.

We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this "Fe-bead" system, Shewanella oneidensis reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by S. oneidensis, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in omcB or mtrB show about 50% of the wild-type levels of reduction, while a cymA mutant shows less than 20% of the wild-type levels of reduction and a menF mutant shows insignificant reduction. The Fe-bead reduction defect of the menF mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the menF gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance.