A CURE for Meat: Comparing Bacterial Contaminants on Different Ground Beef Sources Emphasizes Process of Science and Quantitative Reasoning.

To broaden and emphasize the educational benefits of research to more biology majors in a course setting, we developed and assessed a microbiology-focused course-based undergraduate research experience that utilizes culture-based bacterial enumeration to compare contamination present on different ground beef sources (conventional vs. organic). During the final 3 weeks of the quarter, students learned and practiced common microbiology techniques like dilution math, selective and differential media-based identification, and statistical analysis to evaluate data and test hypotheses. Students were assessed primarily via a formal lab report and a lab practical focused on evaluating process of science and quantitative reasoning skills. The majority of students could write hypotheses and describe variables but were challenged when asked to describe the limitations of the experiments that were conducted as part of this research project. Most students could perform Excel-based graphing and a t test, but many could not solve the complex dilution math required for this project. The greatest barriers to skills mastery represented microbiology-focused concepts, like understanding selective media biases and the nuances of multistep viable counting procedures and outcomes.

Community structure and function of high-temperature chlorophototrophic microbial mats inhabiting diverse geothermal environments.

Six phototrophic microbial mat communities from different geothermal springs (YNP) were studied using metagenome sequencing and geochemical analyses. The primary goals of this work were to determine differences in community composition of high-temperature phototrophic mats distributed across the Yellowstone geothermal ecosystem, and to identify metabolic attributes of predominant organisms present in these communities that may correlate with environmental attributes important in niche differentiation. Random shotgun metagenome sequences from six phototrophic communities (average ∼53 Mbp/site) were subjected to multiple taxonomic, phylogenetic, and functional analyses. All methods, including G + C content distribution, MEGAN analyses, and oligonucleotide frequency-based clustering, provided strong support for the dominant community members present in each site. Cyanobacteria were only observed in non-sulfidic sites; de novo assemblies were obtained for Synechococcus-like populations at Chocolate Pots (CP_7) and Fischerella-like populations at White Creek (WC_6). Chloroflexi-like sequences (esp. Roseiflexus and/or Chloroflexus spp.) were observed in all six samples and contained genes involved in bacteriochlorophyll biosynthesis and the 3-hydroxypropionate carbon fixation pathway. Other major sequence assemblies were obtained for a Chlorobiales population from CP_7 (proposed family Thermochlorobacteriaceae), and an anoxygenic, sulfur-oxidizing Thermochromatium-like (Gamma-proteobacteria) population from Bath Lake Vista Annex (BLVA_20). Additional sequence coverage is necessary to establish more complete assemblies of other novel bacteria in these sites (e.g., Bacteroidetes and Firmicutes); however, current assemblies suggested that several of these organisms play important roles in heterotrophic and fermentative metabolisms. Definitive linkages were established between several of the dominant phylotypes present in these habitats and important functional processes such as photosynthesis, carbon fixation, sulfur oxidation, and fermentation.

Manipulatives-based laboratory for majors biology - a hands-on approach to understanding respiration and photosynthesis.

The first course in our year-long introductory series for Biology majors encompasses four learning units: biological molecules and cells, metabolism, genetics, and evolution. Of these, the metabolism unit, which includes respiration and photosynthesis, has shown the lowest student exam scores, least interest, and lowest laboratory ratings. Consequently, we hypothesized that modeling metabolic processes in the laboratory would improve student content learning during this course unit. Specifically, we developed manipulatives-based laboratory exercises that combined paper cutouts, movable blocks, and large diagrams of the cell. In particular, our novel use of connecting LEGO blocks allowed students to move model electrons and phosphates between molecules and within defined spaces of the cell. We assessed student learning using both formal (content indicators and attitude surveys) and informal (the identification of misconceptions or discussions with students) approaches. On the metabolism unit content exam, student performance improved by 46% over pretest scores and by the end of the course, the majority of students rated metabolism as their most-improved (43%) and favorite (33%) subject as compared with other unit topics. The majority of students rated manipulatives-based labs as very helpful, as compared to non-manipulatives-based labs. In this report, we will demonstrate that students made learning gains across all content areas, but most notably in the unit that covered respiration and photosynthesis.

Formation of multilayered photosynthetic biofilms in an alkaline thermal spring in Yellowstone National Park, Wyoming.

In this study, glass rods suspended at the air-water interface in the runoff channel of Fairy Geyser, Yellowstone National Park, WY, were used as a substratum to promote the development of biofilms that resembled multilayered mat communities in the splash zone at the geyser's source. This approach enabled the establishment of the temporal relationship between the appearance of Cyanobacteria, which ultimately formed the outer green layer, and the development of a red underlayer containing Roseiflexus-like Chloroflexi. This is the first study to define time-dependent successional events involved in the development of differently colored layers within microbial mats associated with many thermal features in Yellowstone National Park. Initial (1-month) biofilms were localized below the air-water interface (60 to 70 degrees C), and the majority of retrieved bacterial sequence types were similar to Synechococcus and Thermus isolates. Biofilms then shifted, becoming established at and above the air-water interface after 3 months. During winter sampling (6 to 8 months), distinct reddish orange microcolonies were observed, consistent with the appearance of Roseiflexus-like sequences and bacteriochlorophyll a pigment signatures. Additionally, populations of Cyanobacteria diversified to include both unicellular and filamentous cell and sequence types. Distinct green and red layers were observed at 13 months. Planctomycetes-like sequences were also retrieved in high abundance from final biofilm layers and winter samples. Finally, biomass associated with geyser vent water contained Roseiflexus-like sequence types, in addition to other high-abundance sequence types retrieved from biofilm samples, supporting the idea that geothermal water serves as an inoculum for these habitats.

Molecular characterization of novel red green nonsulfur bacteria from five distinct hot spring communities in Yellowstone National Park.

We characterized and compared five geographically isolated hot springs with distinct red-layer communities in Yellowstone National Park. Individual red-layer communities were observed to thrive in temperatures ranging from 35 to 60 degrees C and at pH 7 to 9. All communities were dominated by red filamentous bacteria and contained bacteriochlorophyll a (Bchl a), suggesting that they represented novel green nonsulfur (GNS) bacteria. The in vivo absorption spectra of individual sites were different, with two sites showing unusual Bchl a protein absorption bands beyond 900 nm. We prepared and analyzed 16S rRNA libraries from all of these sites by using a combination of general bacterial primers and new GNS-specific primers described here. These studies confirmed the presence of novel GNS-like bacteria in all five communities. All GNS-like clones were most similar to Roseiflexus castenholzii, a red filamentous bacterium from Japan that also contains only Bchl a. Phylogenies constructed by using GNS-like clones from Yellowstone red-layer communities suggest the presence of a moderately diverse new "red" cluster within the GNS lineage. Within this cluster, at least two well-supported subclusters emerged: YRL-A was most similar to Roseiflexus and YRL-B appeared to be novel, containing no known isolates. While these patterns showed some site specificity, they did not correlate with observed Bchl a spectrum differences or obvious features of the habitat.

Bacterial Diversity Studies Using the 16S rRNA Gene Provide a Powerful Research-Based Curriculum for Molecular Biology Laboratory.

We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods.