ASU Scholarship Showcase

Gas seeps emanating from Yanartaş (Chimera), Turkey, have been documented for thousands of years. Active serpentinization produces hydrogen and a range of carbon gases that may provide fuel for life. Here we report a newly discovered, ephemeral fluid seep emanating from a small gas vent at Yanartaş. Fluids and biofilms were sampled at the source and points downstream. We describe site conditions, and provide microbiological data in the form of enrichment cultures, Scanning electron microscopy (SEM), carbon and nitrogen isotopic composition of solids, and PCR screens of nitrogen cycle genes. Source fluids are pH 11.95, with a Ca:Mg of ~200, and sediments under the ignited gas seep measure 60°C. Collectively, these data suggest the fluid is the product of active serpentinization at depth. Source sediments are primarily calcite and alteration products (chlorite and montmorillonite). Downstream, biofilms are mixed with montmorillonite. SEM shows biofilms distributed homogeneously with carbonates. Organic carbon accounts for 60% of the total carbon at the source, decreasing downstream to <15% as inorganic carbon precipitates. δ¹³C ratios of the organic carbon fraction of solids are depleted (−25 to −28‰) relative to the carbonates (−11 to −20‰). We conclude that heterotrophic processes are dominant throughout the surface ecosystem, and carbon fixation may be key down channel. δ¹⁵N ratios ~3‰, and absence of nifH in extracted DNA suggest that nitrogen fixation is not occurring in sediments. However, the presence of narG and nirS at most locations and in enrichments indicates genomic potential for nitrate and nitrite reduction. This small seep with shallow run-off is likely ephemeral, but abundant preserved microterracettes in the outflow and the surrounding area suggest it has been present for some time. This site and others like it present an opportunity for investigations of preserved deep biosphere signatures, and subsurface-surface interactions.

Precise spatial positioning and isolation of mammalian cells is a critical component of many single cell experimental methods and biological engineering applications. Although a variety of cell patterning methods have been demonstrated, many of these methods subject cells to high stress environments, discriminate against certain phenotypes, or are a challenge to implement. Here, we demonstrate a rapid, simple, indiscriminate, and minimally perturbing cell patterning method using a laser fabricated polymer stencil. The stencil fabrication process requires no stencil-substrate alignment, and is readily adaptable to various substrate geometries and experiments.

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.

Over 100 hot spring sediment samples were collected from 28 sites in 12 areas/regions, while recording as many coincident geochemical properties as feasible (>60 analytes). PCR was used to screen samples for Korarchaeota 16S rRNA genes. Over 500 Korarchaeota 16S rRNA genes were screened by RFLP analysis and 90 were sequenced, resulting in identification of novel Korarchaeota phylotypes and exclusive geographical variants. Korarchaeota diversity was low, as in other terrestrial geothermal systems, suggesting a marine origin for Korarchaeota with subsequent niche-invasion into terrestrial systems. Korarchaeota endemism is consistent with endemism of other terrestrial thermophiles and supports the existence of dispersal barriers. Korarchaeota were found predominantly in >55°C springs at pH 4.7–8.5 at concentrations up to 6.6×10⁶ 16S rRNA gene copies g^-1 wet sediment. In Yellowstone National Park (YNP), Korarchaeota were most abundant in springs with a pH range of 5.7 to 7.0. High sulfate concentrations suggest these fluids are influenced by contributions from hydrothermal vapors that may be neutralized to some extent by mixing with water from deep geothermal sources or meteoric water. In the Great Basin (GB), Korarchaeota were most abundant at spring sources of pH<7.2 with high particulate C content and high alkalinity, which are likely to be buffered by the carbonic acid system. It is therefore likely that at least two different geological mechanisms in YNP and GB springs create the neutral to mildly acidic pH that is optimal for Korarchaeota. A classification support vector machine (C-SVM) trained on single analytes, two analyte combinations, or vectors from non-metric multidimensional scaling models was able to predict springs as Korarchaeota-optimal or sub-optimal habitats with accuracies up to 95%. To our knowledge, this is the most extensive analysis of the geochemical habitat of any high-level microbial taxon and the first application of a C-SVM to microbial ecology.

Many studies link the compositions of microbial communities to their environments, but the energetics of organism-specific biomass synthesis as a function of geochemical variables have rarely been assessed. We describe a thermodynamic model that integrates geochemical and metagenomic data for biofilms sampled at five sites along a thermal and chemical gradient in the outflow channel of the hot spring known as “Bison Pool” in Yellowstone National Park. The relative abundances of major phyla in individual communities sampled along the outflow channel are modeled by computing metastable equilibrium among model proteins with amino acid compositions derived from metagenomic sequences. Geochemical conditions are represented by temperature and activities of basis species, including pH and oxidation-reduction potential quantified as the activity of dissolved hydrogen. By adjusting the activity of hydrogen, the model can be tuned to closely approximate the relative abundances of the phyla observed in the community profiles generated from BLAST assignments. The findings reveal an inverse relationship between the energy demand to form the proteins at equal thermodynamic activities and the abundance of phyla in the community. The distance from metastable equilibrium of the communities, assessed using an equation derived from energetic considerations that is also consistent with the information-theoretic entropy change, decreases along the outflow channel. Specific divergences from metastable equilibrium, such as an underprediction of the relative abundances of phototrophic organisms at lower temperatures, can be explained by considering additional sources of energy and/or differences in growth efficiency. Although the metabolisms used by many members of these communities are driven by chemical disequilibria, the results support the possibility that higher-level patterns of chemotrophic microbial ecosystems are shaped by metastable equilibrium states that depend on both the composition of biomass and the environmental conditions.

With the successful development of organic/polymeric light emitting diodes, many organic and polymeric fluorophores with high quantum efficiencies and optical stability were synthesized. However, most of these materials which have excellent optical properties are insoluble in water, limiting their applications in biological fields. Herein, we used micelles formed from an amino-group-containing poly(ε-caprolactone)-block-poly(ethylene glycol) (PCL-b-PEG-NH₂) to incorporate a hydrophobic blue emitter oligofluorene (OF) to enable its application in biological conditions. Although OF is completely insoluble in water, it was successfully transferred into aqueous solutions with a good retention of its photophysical properties. OF exhibited a high quantum efficiency of 0.84 in a typical organic solvent of tetrahydrofuran (THF). In addition, OF also showed a good quantum efficiency of 0.46 after being encapsulated into micelles. Two cells lines, human glioblastoma (U87MG) and esophagus premalignant (CP-A), were used to study the cellular internalization of the OF incorporated micelles. Results showed that the hydrophobic OF was located in the cytoplasm, which was confirmed by co-staining the cells with nucleic acid specific SYTO 9, lysosome specific LysoTracker Red®, and mitochondria specific MitoTracker Red. MTT assay indicated non-toxicity of the OF-incorporated micelles. This study will broaden the application of hydrophobic functional organic compounds, oligomers, and polymers with good optical properties to enable their applications in biological research fields.

Hydrophobic platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) was physically incorporated into micelles formed from poly(ε-caprolactone)-block-poly(ethylene glycol) to enable the application of PtTFPP in aqueous solution. Micelles were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM) to show an average diameter of about 140 nm. PtTFPP showed higher quantum efficiency in micellar solution than in tetrahydrofuran (THF) and dichloromethane (CH₂Cl₂). PtTFPP in micelles also exhibited higher photostability than that of PtTFPP suspended in water. PtTFPP in micelles exhibited good oxygen sensitivity and response time. This study provided an efficient approach to enable the application of hydrophobic oxygen sensors in a biological environment.

Background: The use of culture-independent nucleic acid techniques, such as ribosomal RNA gene cloning library analysis, has unveiled the tremendous microbial diversity that exists in natural environments. In sharp contrast to this great achievement is the current difficulty in cultivating the majority of bacterial species or phylotypes revealed by molecular approaches. Although recent new technologies such as metagenomics and metatranscriptomics can provide more functionality information about the microbial communities, it is still important to develop the capacity to isolate and cultivate individual microbial species or strains in order to gain a better understanding of microbial physiology and to apply isolates for various biotechnological applications.

Results: We have developed a new system to cultivate bacteria in an array of droplets. The key component of the system is the microbe observation and cultivation array (MOCA), which consists of a Petri dish that contains an array of droplets as cultivation chambers. MOCA exploits the dominance of surface tension in small amounts of liquid to spontaneously trap cells in well-defined droplets on hydrophilic patterns. During cultivation, the growth of the bacterial cells across the droplet array can be monitored using an automated microscope, which can produce a real-time record of the growth. When bacterial cells grow to a visible microcolony level in the system, they can be transferred using a micropipette for further cultivation or analysis.

Conclusions: MOCA is a flexible system that is easy to set up, and provides the sensitivity to monitor growth of single bacterial cells. It is a cost-efficient technical platform for bioassay screening and for cultivation and isolation of bacteria from natural environments.

We have constructed a conceptual model of biogeochemical cycles and metabolic and microbial community shifts within a hot spring ecosystem via coordinated analysis of the “Bison Pool” (BP) Environmental Genome and a complementary contextual geochemical dataset of ∼75 geochemical parameters. 2,321 16S rRNA clones and 470 megabases of environmental sequence data were produced from biofilms at five sites along the outflow of BP, an alkaline hot spring in Sentinel Meadow (Lower Geyser Basin) of Yellowstone National Park. This channel acts as a >22 m gradient of decreasing temperature, increasing dissolved oxygen, and changing availability of biologically important chemical species, such as those containing nitrogen and sulfur. Microbial life at BP transitions from a 92°C chemotrophic streamer biofilm community in the BP source pool to a 56°C phototrophic mat community. We improved automated annotation of the BP environmental genomes using BLAST-based Markov clustering. We have also assigned environmental genome sequences to individual microbial community members by complementing traditional homology-based assignment with nucleotide word-usage algorithms, allowing more than 70% of all reads to be assigned to source organisms. This assignment yields high genome coverage in dominant community members, facilitating reconstruction of nearly complete metabolic profiles and in-depth analysis of the relation between geochemical and metabolic changes along the outflow. We show that changes in environmental conditions and energy availability are associated with dramatic shifts in microbial communities and metabolic function. We have also identified an organism constituting a novel phylum in a metabolic “transition” community, located physically between the chemotroph- and phototroph-dominated sites. The complementary analysis of biogeochemical and environmental genomic data from BP has allowed us to build ecosystem-based conceptual models for this hot spring, reconstructing whole metabolic networks in order to illuminate community roles in shaping and responding to geochemical variability.