Stephen C. Nold

Molecular Diversity of Denitrifying Genes in Continental Margin Sediments within the Oxygen-Deficient Zone off the Pacific Coast of Mexico

ABSTRACT To understand the composition and structure of denitrifying communities in the oxygen-deficient zone off the Pacific coast of Mexico, the molecular diversity of nir genes from sediments obtained at four stations was examined by using a PCR-based cloning approach. A total of 50 operational taxonomic units (OTUs) for nirK and 82 OTUs for nirS were obtained from all samples. Forty-four of the nirS clones and 31 of the nirK clones were sequenced; the levels of similarity of the nirS clones were 52 to 92%, and the levels of similarity of the nirS clones were 50 to 99%. The percentages of overlapping OTUs between stations were 18 to 30% for nirS and 5 to 8% for nirK. Sequence analysis revealed that 26% of the nirS clones were related to the nirS genes of Alcaligenes faecalis (80 to 94% similar) and Pseudomonas stutzeri (80 to 99%), whereas 3 to 31% of the nirK clones were closely related to the nirK genes of Pseudomonas sp. strain G-179 (98 to 99%), Bradyrhizobium japonicum (91%), Blastobacter denitrificans (83%), and Alcaligenes xylosoxidans (96%). The rest of the clones, however, were less than 80% similar to nirS and nirK sequences available in sequence databases. The results of a principal-component analysis (PCA) based on the percentage of OTUs and biogeochemical data indicated that the nitrate concentration and oxygen have an effect on the denitrifying communities. The communities at the stations in oxygen-deficient zones were more similar than the communities at the stations in the oxygenated zone. The denitrifying communities were more similar at the stations that were closer together and had similar nitrate levels. Also, the results of PCA based on biogeochemical properties suggest that geographic location and biogeochemical conditions, especially the nitrate and oxygen levels, appear to be the key factors that control the structure of denitrifying communities.

Pseudomonas stutzeri nitrite reductase gene abundance in environmental samples measured by real-time PCR.

ABSTRACT We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific fornirS sequences that corresponded approximately to thePseudomonas stutzeri species. The assay was linear from 1 to 106 gene copies (r2 = 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. AlthoughP. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier.

Pacific Northwest marine sediments contain ammonia-oxidizing bacteria in the beta subdivision of the Proteobacteria.

ABSTRACT The diversity of ammonia-oxidizing bacteria in aquatic sediments was studied by retrieving ammonia monooxygenase and methane monooxygenase gene sequences. Methanotrophs dominated freshwater sediments, while β-proteobacterial ammonia oxidizers dominated marine sediments. These results suggest that γ-proteobacteria such asNitrosococcus oceani are minor members of marine sediment ammonia-oxidizing communities.

Patterns and governing forces in aquatic microbial communities

In this review we survey recent publications employing molecular techniques to investigate the distribution of microbial species in aquatic environments. We analyzed the occurrence of microbial phyla in freshwater and marine habitats and observed patterns of distribution that could be explained by the adaptation of microorganisms to physical and biological parameters that vary in aquatic habitats. The gram-positive bacteria, the Verrucomicrobiales and the α- and γ-subdivisions of the Proteobacteria are distributed throughout a range of aquatic habitats, while other phylogenetic groups appear to be adapted to more narrowly defined environmental niches such as anoxic water and sediments (δ-Proteobacteria) or floating aggregates (Cytophaga-Flexibacter-Bacteroides phylum). β-proteobacterial sequence types have been detected throughout freshwater habitats, but these organisms are largely absent from open ocean environments. Within several of these divisions, clusters of closely related small sub unit ribosomal RNA sequence types have been detected in geographically disparate environments, suggesting that some microbial species are globally distributed. In addition to physical variables such as salinity and pH, biological variables also influence microbial community composition. This was illustrated by changes that occurred in the eukaryotic and bacterial species composition in laboratory mesocosms after a viral outburst. We conclude that physical and biological forces govern the composition of aquatic microbial communities and result in divergent evolutionary histories of the indigenous microbial species.

Benthic Bacterial Diversity in Submerged Sinkhole Ecosystems

ABSTRACT Physicochemical characterization, automated ribosomal intergenic spacer analysis (ARISA) community profiling, and 16S rRNA gene sequencing approaches were used to study bacterial communities inhabiting submerged Lake Huron sinkholes inundated with hypoxic, sulfate-rich groundwater. Photosynthetic cyanobacterial mats on the sediment surface were dominated by Phormidium autumnale, while deeper, organically rich sediments contained diverse and active bacterial communities.

Eukaryal and archaeal diversity in a submerged sinkhole ecosystem influenced by sulfur-rich, hypoxic groundwater

ABSTRACT Middle Island sinkhole (23 m depth) is an underwater karst feature in Lake Huron (MI, USA) inundated with hypoxic, high-conductivity groundwater. Here, microbial mats composed of purple-pigmented filamentous cyanobacteria cover carbon-rich sediments. To study the species diversity of Archaea and Eukarya in this habitat, we constructed clone libraries and sequenced the small subunit ribosomal RNA genes from sediment cores sectioned into five visually distinct layers: the surface cyanobacterial mat (0–0.2 cm), an underlying white crystalline layer (0.2–0.5 cm), and three sub-sections of black organic-rich sediment chosen from distinct layers in the cores (0.5–2.0 cm, 7.5–9.5 cm, and 24.5–28.5 cm). Clone libraries from the cyanobacterial mat were dominated by eukaryal 18S rRNA gene sequences such as nematodes (Tobrilus gracilis), ciliates (Frontonia vernalis), and tardigrades (Isohypsibius granulifer). Shallow organic-rich sediments shared clones with the overlying mat but also included seed shrimp (Cyprididae sp.) and copepods (Leptodiaptomus spp.). Clone libraries from the deepest sediments were dominated by archaeal sequences similar to known methanogens (Methanosphaerula and Methanosaeta) and uncultivated Archaea, including non-thermophilic Crenarchaeota. Phylogenetic trees revealed representation in diverse eukaryotic and archaeal lineages. By chronicling the species composition of freshwater sinkholes, this study expands our knowledge of microbial communities in habitats influenced by hypoxic, sulfur-rich groundwater.

Observations of the Middle Island Sinkhole in Lake Huron: A Unique Hydrologic and Glacial Creation of 400 Million Years

In the northern Great Lakes region, limestone sediments deposited some 400 million ybp during the Devonian era have experienced erosion, creating karst features such as caves and sinkholes. The groundwater chemical constituents of the shallow seas that produced these rock formations now contribute to the formation of a unique physical (sharp density gradients), chemical (dissolved oxygen-depleted, sulfate-rich) and biological (microbe-dominated) environment in a submerged sinkhole near Middle Island in freshwater Lake Huron. A variety of methods including aerial photography, physico-chemical mapping, time series measurements, remotely operated vehicle (ROV) survey, diver observations and bathymetric mapping were employed to obtain a preliminary understanding of sinkhole features and to observe physical interactions of the system’s groundwater with Lake Huron. High conductivity ground water of relatively constant temperature hugs the sinkhole floor creating a distinct sub-ecosystem within this Great Lakes ecosystem. Extensive photosynthetic purple cyanobacterial benthic mats that characterize the benthos of this shallow sinkhole were strictly limited to the zone of ground water influence. tions of chloride, and 100-fold higher concentrations of sulfate (Ruberg et al., 2005). A variety of non-photosynthetic benthic microbial mats were observed in this deepwater aphotic sinkhole system. Water samples collected from the sinkhole plume contained bacterial concentrations (~9x109 cells l-1) an order of magnitude higher than ambient lake concentrations (~1x109 cells l-1), and showed evidence for the occurrence of significant chemosynthesis in this lightless deep water environment (Biddanda et al., 2006). These rates of chemosynthesis occurring in the Lake Huron Isolated sinkhole were comparable to those measured in thermal vents in Yellowstone Lake (Cuhel et al., 2002). Understanding the nature of the groundwater emerging in Lake Huron’s sinkholes requires an introduction P A P E R

A Call to Develop Course-Based Undergraduate Research Experiences (CUREs) for Nonmajors Courses

A discussion of course-based undergraduate research experiences (CUREs) for non–science majors (nonmajors) that summarizes the state of knowledge of best practices for nonmajors CUREs, identifies future research priorities, and recommends tools to align research questions with student outcomes.