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Smallwood, Chuck R.

Publications and source records attributed to Smallwood, Chuck R..

8 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Biotic countermeasures that rescue Nannochloropsis gaditana from a Bacillus safensis infection

The natural assemblage of a symbiotic bacterial microbiome (bacteriome) with microalgae in marine ecosystems is now being investigated as a means to increase algal productivity for industry. When algae are grown in open pond settings, biological contamination causes an estimated 30% loss of the algal crop. Therefore, new crop protection strategies that do not disrupt the native algal bacteriome are needed to produce reliable, high-yield algal biomass. Bacteriophages offer an unexplored solution to treat bacterial pathogenicity in algal cultures because they can eliminate a single species without affecting the bacteriome. To address this, we identified a highly virulent pathogen of the microalga Nannochloropsis gaditana, the bacterium Bacillus safensis, and demonstrated rescue of the microalgae from the pathogen using phage. 16S rRNA amplicon sequencing showed that phage treatment did not alter the composition of the bacteriome. It is widely suspected that the algal bacteriome could play a protective role against bacterial pathogens. To test this, we compared the susceptibility of a bacteriome-attenuated N. gaditana culture challenged with B. safensis to a N. gaditana culture carrying a growth-promoting bacteriome. We showed that the loss of the bacteriome increased the susceptibility of N. gaditana to the pathogen. Transplanting the microalgal bacteriome to the bacteriome-attenuated culture reconstituted the protective effect of the bacteriome. Finally, the success of phage treatment was dependent on the presence of beneficial bacteriome. This study introduces two synergistic countermeasures against bacterial pathogenicity in algal cultures and a tractable model for studying interactions between microalgae, phages, pathogens, and the algae microbiome.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Singleplex, multiplex and pooled sample real-time RT-PCR assays for detection of SARS-CoV-2 in an occupational medicine setting

Abstract For workplaces which cannot operate as telework or remotely, there is a critical need for routine occupational SARS-CoV-2 diagnostic testing. Although diagnostic tests including the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (CDC Diagnostic Panel) (EUA200001) were made available early in the pandemic, resource scarcity and high demand for reagents and equipment necessitated priority of symptomatic patients. There is a clearly defined need for flexible testing methodologies and strategies with rapid turnaround of results for (1) symptomatic, (2) asymptomatic with high-risk exposures and (3) asymptomatic populations without preexisting conditions for routine screening to address the needs of an on-site work force. We developed a distinct SARS-CoV-2 diagnostic assay based on the original CDC Diagnostic Panel (EUA200001), yet, with minimum overlap for currently employed reagents to eliminate direct competition for limited resources. As the pandemic progressed with testing loads increasing, we modified the assay to include 5-sample pooling and amplicon target multiplexing. Analytical sensitivity of the pooled and multiplexed assays was rigorously tested with contrived positive samples in realistic patient backgrounds. Assay performance was determined with clinical samples previously assessed with an FDA authorized assay. Throughout the pandemic we successfully tested symptomatic, known contact and travelers within our occupational population with a ~ 24–48-h turnaround time to limit the spread of COVID-19 in the workplace. Our singleplex assay had a detection limit of 31.25 copies per reaction. The three-color multiplexed assay maintained similar sensitivity to the singleplex assay, while tripling the throughput. The pooling assay further increased the throughput to five-fold the singleplex assay, albeit with a subtle loss of sensitivity. We subsequently developed a hybrid ‘multiplex-pooled’ strategy to testing to address the need for both rapid analysis of samples from personnel at high risk of COVID infection and routine screening. Herein, our SARS-CoV-2 assays specifically address the needs of occupational healthcare for both rapid analysis of personnel at high-risk of infection and routine screening that is essential for controlling COVID-19 disease transmission. In addition to SARS-CoV-2 and COVID-19, this work demonstrates successful flexible assays developments and deployments with implications for emerging highly transmissible diseases and future pandemics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Cellulosome Localization Patterns Vary across Life Stages of Anaerobic Fungi

Anaerobic fungi ( Neocallimastigomycota ) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Calcareous organic matter coatings sequester siderophores in alkaline soils

Although most studies on organic matter stabilization in soils have focused on adsorption to aluminosilicate and iron oxide minerals due to their strong interactions with organic nucleophiles, stabilization within alkaline soils has been empirically correlated with exchangeable Ca concentrations. Yet the extent of competing processes within natural soil remain unclear. We employed sub-micron scale techniques to investigate the minerology and organic carbon on individual fine particles within an alkaline soil from Eastern Washington. XRD, Mössbauer spectroscopy, TEM-EDX-SAED, and XPS analysis demonstrated a mineral assemblage dominated by quartz, feldspars, iron oxides, and calcite. Although exposed Si, Al, and Fe accounted for ~30% of the surface elemental composition, most of the organic carbon and nitrogen was associated with Ca-rich coatings. Adsorption isotherms of two siderophores with the same soil showed greater adsorption of pyoverdine compared with less polar enterobactin. Using NanoSIMS to map the distribution of isotopically labeled siderophores across the surfaces of fine grains, we observed a preference for binding to surfaces heavily coated with organic matter. We propose a mechanism of adsorption by which organic molecules aggregate within calcareous soils via calcium bridging, favoring the stabilization of larger molecules with a greater number of nucleophilic functional groups.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose

The conversion of lignocellulose-rich biomass to bio-based chemicals and higher order fuels remains a grand challenge, as single-microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydrate active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar-rich hydrolysate readily supports growth of Saccharomyces cerevisiae, which can be engineered to produce a range of value-added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2017

Multimodal microfluidic platform for controlled culture and analysis of unicellular organisms

Modern live-cell imaging approaches permit real-time visualization of biological processes, yet limitations exist for unicellular organism isolation, culturing and long-term imaging that preclude fully understanding how cells sense and respond to environmental perturbations and the link between single-cell variability and whole-population dynamics. Here we present a microfluidic platform that provides fine control over the local environment with the capacity to replace media components at any experimental time point, and provides both perfused and compartmentalized cultivation conditions depending on the valve configuration. In this work, the functionality and flexibility of the platform were validated using both bacteria and yeast having different sizes, motility and growth media. The demonstrated ability to track the growth and dynamics of both motile and non-motile prokaryotic and eukaryotic organisms emphasizes the versatility of the devices, which with further scale-up should enable studies in bioenergy and environmental research.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Multimodal hyperspectral optical microscopy

We describe a unique and convenient approach to multimodal hyperspectral optical microscopy, herein achieved by coupling a portable and transferable hyperspectral imager to various optical microscopes. The experimental and data analysis schemes involved in recording spectrally and spatially resolved fluorescence, dark field, and optical absorption micrographs are illustrated through prototypical measurements targeting selected model systems. Namely, hyperspectral fluorescence micrographs of isolated fluorescent beads are employed to ensure spectral calibration of our detector and to gauge the attainable spatial resolution of our measurements; the recorded images are diffraction-limited. Moreover, spatially over-sampled absorption spectroscopy of a single lipid (18:1 Liss Rhod PE) layer reveals that optical densities on the order of 10-3 may be resolved by spatially averaging the recorded optical signatures. We also briefly illustrate two applications of our setup in the general areas of plasmonics and cell biology. Most notably, we deploy hyperspectral optical absorption microscopy to identify and image algal pigments within a single live Tisochrysis lutea cell. Overall, this work paves the way for multimodal multidimensional spectral imaging measurements spanning the realms of several scientific disciples.

47 OTHER INSTRUMENTATION↗