Enrichment of Root Endophytic Bacteria from Populus deltoides and Single-Cell-Genomics Analysis.

UNLABELLED: Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. Here, we describe a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria. IMPORTANCE: Plant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses of genomes of uncultured bacteria to provide genomics information that will facilitate future cultivation attempts.

Quantification of Rare Earth Elements in the Parts Per Million Range: A Novel Approach in the Application of Laser-Induced Breakdown Spectroscopy.

This work extends a previous percentage level concentration study of the optical emission spectra for six rare earth elements, europium (Eu), gadolinium (Gd), lanthanum (La), praseodymium (Pr), neodymium (Nd), and samarium (Sm), along with the transition metal, yttrium (Y) using laser-induced breakdown spectroscopy (LIBS). The concentration of these six rare earth elements and yttrium has been attempted for the first time systematically down to parts per million (ppm) concentration levels ranging from 30 to 300 ppm. The authors have developed multivariate models for each element capable of predicting concentration with acceptable to excellent levels of accuracy. Additionally, partial least squares regression coefficients were used to identify key spectral features able to be used in this lower concentration regime. This study has demonstrated that it is conceivable to quantify the six rare earth elements along with yttrium at low concentrations in the parts per million levels.

Caldicellulosiruptor obsidiansis sp. nov., an anaerobic, extremely thermophilic, cellulolytic bacterium isolated from Obsidian Pool, Yellowstone National Park.

A novel, obligately anaerobic, extremely thermophilic, cellulolytic bacterium, designated OB47(T), was isolated from Obsidian Pool, Yellowstone National Park, WY. The isolate was a nonmotile, non-spore-forming, Gram-positive rod approximately 2 microm long by 0.2 microm wide and grew at temperatures between 55 and 85 degrees C, with the optimum at 78 degrees C. The pH range for growth was 6.0 to 8.0, with values of near 7.0 being optimal. Growth on cellobiose produced the fastest specific growth rate at 0.75 h(-1). The organism also displayed fermentative growth on glucose, maltose, arabinose, fructose, starch, lactose, mannose, sucrose, galactose, xylose, arabinogalactan, Avicel, xylan, filter paper, processed cardboard, pectin, dilute acid-pretreated switchgrass, and Populus. OB47(T) was unable to grow on mannitol, fucose, lignin, Gelrite, acetate, glycerol, ribose, sorbitol, carboxymethylcellulose, and casein. Yeast extract stimulated growth, and thiosulfate, sulfate, nitrate, and sulfur were not reduced. Fermentation end products were mainly acetate, H2, and CO2, although lactate and ethanol were produced in 5-liter batch fermentations. The G+C content of the DNA was 35 mol%, and sequence analysis of the small subunit rRNA gene placed OB47(T) within the genus Caldicellulosiruptor. Based on its phylogenetic and phenotypic properties, the isolate is proposed to be designated Caldicellulosiruptor obsidiansis sp. nov. and OB47 is the type strain (ATCC BAA-2073).

Miniature sensor suitable for electronic nose applications.

A major research effort has been devoted over the years for the development of chemical sensors for the detection of chemical and explosive vapors. However, the deployment of such chemical sensors will require the use of multiple sensors (probably tens of sensors) in a sensor package to achieve selective detection. In order to keep the overall detector unit small, miniature sensors with sufficient sensitivity of detection will be needed. We report sensitive detection of dimethyl methylphosphonate (DMMP), a stimulant for the nerve agents, using a miniature sensor unit based on piezoresistive microcantilevers. The sensor can detect parts-per-trillion concentrations of DMMP within 10 s exposure times. The small size of the sensor makes it ideally suited for electronic nose applications.

Multiple single-cell genomes provide insight into functions of uncultured Deltaproteobacteria in the human oral cavity.

Despite a long history of investigation, many bacteria associated with the human oral cavity have yet to be cultured. Studies that correlate the presence or abundance of uncultured species with oral health or disease highlight the importance of these community members. Thus, we sequenced several single-cell genomic amplicons from Desulfobulbus and Desulfovibrio (class Deltaproteobacteria) to better understand their function within the human oral community and their association with periodontitis, as well as other systemic diseases. Genomic data from oral Desulfobulbus and Desulfovibrio species were compared to other available deltaproteobacterial genomes, including from a subset of host-associated species. While both groups share a large number of genes with other environmental Deltaproteobacteria genomes, they encode a wide array of unique genes that appear to function in survival in a host environment. Many of these genes are similar to virulence and host adaptation factors of known human pathogens, suggesting that the oral Deltaproteobacteria have the potential to play a role in the etiology of periodontal disease.

Anaerobic high-throughput cultivation method for isolation of thermophiles using biomass-derived substrates.

Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium thermocellum (T(opt) = 55 °C), Caldicellulosiruptor obsidiansis (T(opt) = 78 °C) and the fermentative hyperthermophiles, Pyrococcus furiosus (T(opt) = 100 °C) and Thermotoga maritima (T(opt) = 80 °C). Multi-well plates were incubated at various temperatures for approximately 72-120 h and then tested for growth. Positive growth resulting from single cells sorted into individual wells containing an anaerobic medium was verified by OD(600). Depending on the growth substrate, up to 80 % of the wells contained viable cultures, which could be transferred to fresh media. This method was used to isolate thermophilic microbes from Rabbit Creek, Yellowstone National Park (YNP), Wyoming. Substrates for enrichment cultures including crystalline cellulose (Avicel), xylan (from Birchwood), pretreated switchgrass and Populus were used to cultivate organisms that may be of interest to lignocellulosic biofuel production.

Improved measurement for volatile particles: vapor-particle separator design and laboratory tests.

Sampling and measurement of volatile particles is a challenging task. It has been hampered by lack of a reliable technique capable of accurately capturing the phase-partition process of the pollutants without generating bias and artifacts in the data. The objective of this research is to design a new vapor-particle separation technique for performing the phase separation on-line (the sampling aspect), which, simultaneously, enables characterization of the vapors and particles. The new vapor-particle separator (VPS) consists of a thin metallic microporous membrane for (1) extraction of vapor molecules that are thermally desorbed from the condensed particulate phases and (2) collection of the vapors for subsequent chemical analysis. We evaluated this new separator using synthetic particles made of nonvolatile and or semi-volatile chemicals, and reported the laboratory test results in this paper. The laboratory particle test results showed reasonably high particle transmission efficiency across all particle sizes. The thermal dynamics of nanoparticles was succinctly observed on-line. The results successfully demonstrated the ability of VPS to separate particles and vapors thus enabling a faithful observation of the thermal behavior. We believe the new technology will make a great contribution to the measurement of volatile particles.