Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Processing Description
• Data Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

A time series incubation experiment was performed to investigate potential interactions between marine cable bacteria and their associated microbial community. Sediment was collected from the main channel of Chesapeake Bay from a mesohaline region that experiences severe and prolonged seasonal oxygen depletion. The upper 10 cm of sediments were homogenized under a gentle N2 gas stream to minimize oxidation, and re-packed into polycarbonate core liners (Ø = 2.5 cm, height = 8 cm), sealed with a silicone stopper at the bottom, and left open to aquarium water at the top. The sediment cores were constructed so that the sediment surface was flush with the core liner. Sediment cores were incubated in continuously aerated aquaria maintained at bottom water temperature (16oC), in artificial seawater (Red Sea Salts TM), in the dark, at bottom water salinity (S=15.5), and this enabled a rapid proliferation of cable bacteria. Water level and salinity in the aquaria was monitored daily and topped with deionized water as necessary to maintain water level and salinity (approximately twice weekly). At six time points (Days 3, 6, 10, 14, 20, 46), triplicate cores were selected at random for microsensor profiling, followed by destructive sampling. One sediment core was sampled for microscopy and nucleic acid preservation, and a separate sediment core was sampled under anaerobic conditions for porewater geochemistry. Sediment cores were each sectioned at 0.5 cm increments to 3 cm. In a subset of sediment cores, downward growth of cable bacteria was blocked by embedding a 25 mm filter (0.2 μm polycarbonate; Millipore) at 0.5 cm depth. The filter was held in place with a specially constructed filter-holder equipped with a rubber Oring to ensure a water-tight seal. Sediment cores with barrier filters were similarly sampled on Days 3, 20, 46 for microsensor profiling, microscopy, nucleic acid analyses, and porewater geochemistry.

For nucleic acid sequencing analysis, sediments were collected in cryogenic tubes, flash frozen in liquid nitrogen, and stored at -80oC. DNA and RNA were extracted separately. Extracted RNA was purified with Norgen CleanAll DNA/RNA Clean-Up and Concentration Kit (Norgen Biotek). Residual DNA in the RNA extracts was digested with two rounds of TURBO DNAse treatment (Invitrogen) and cDNA synthesis was accomplished by reverse transcription-polymerase chain reaction using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen), according to manufacturer’s protocol. DNA and cDNA concentrations were quantitated with Qubit dsDNA Broad Range assay kits using Qubit 2.0 fluorometer (Invitrogen). The V4-V5 region of the microbial 16S rRNA gene (DNA and cDNA) was amplified using the modified primer pair of the Earth Microbiome Project (515F-Y/926R; Parada et al., 2016). For DNA samples, amplicons were amplified, barcoded, and sequenced at Bioanalytical Services Laboratory (Baslab) at the Institute of Marine and Environmental Technology (IMET; Baltimore, MD, USA) using an Illumina Miseq platform (2 x 300 bp). For RNA samples, cDNA amplicons were sequenced using an Illumina Miseq platform (2 x 250 bp) by Genewiz (NJ, USA).

- Imported "SRA_samples_ChesapeakeBaySediments_IncubationExpt(Liau).xlsx" into BCO-DMO system
- Replace spaces in parameter names with underscores to comploy with BCO-DMO requirements and style
- Export file as "963432_v1_sra_cb_incubation.csv"

NSF Award Abstract:
Marine sediments represent the world's largest repository of stored organic carbon, and understanding how microorganisms break down this carbon is an imperative for understanding global carbon cycling. Yet long-standing questions remain regarding how networks of microorganisms work together to accomplish the complete breakdown of organic carbon in marine sediments. Sediment microbes interact in a myriad of ways that couple their metabolism to the break down of organic carbon, including by sharing products of metabolism. Accumulating evidence further suggests that some microorganisms can interact by transferring electrons directly to other unrelated microorganisms. This ability occurs across diverse microorganisms and appears to be widespread in the biosphere, particularly in anaerobic environments such as marine sediments. This project addresses emerging questions about the identity and metabolic linkages between microorganisms that work together in natural anaerobic marine and estuarine sediments to break down organic carbon. The investigators approach these questions by focusing on the influence of a keystone bacterium on its surrounding microbial community. "Cable bacteria" are a recently discovered group of long filamentous bacteria that act as electrical conductors in aquatic sediments providing a conduit for electrons to commute from deeper sulfidic sediments up to the surface oxygen layer by the process of centimeter-scale electron transport. Since their discovery about 6 years ago, these bacteria have been observed in a wide range of depositional sedimentary environments, often at extremely high cell densities. Where these bacteria are abundant, such as in coastal marine muds, they drive intense localized changes in pH and strongly influence the mineral cycling. This research explores the direct and indirect influence of cable bacteria on the metabolic activity of associated microorganisms. This project also advance the education and training of two early-career investigators, two PhD students, and undergraduate students. The skills and expertise gained from these PhD research projects will enable the students to be competitive in academic pursuits and in bioinformatics and technology applications relevant to private industry. The scientific discoveries emerging from this work is being incorporated into undergraduate and graduate level courses in marine microbial ecology. The research team will reach out to the broader community by hosting public lectures promoting a better understanding of environmental microbial ecology.

The proposed work is to investigate the role of cable bacteria in structuring sediment microbial communities. Due to their growth strategy and morphology, cable bacteria are particularly amenable to experimental manipulation, providing an outstanding opportunity to better understand community interactions among microorganisms in a natural and complex anaerobic environment. The investigators will explore the interactions and relationships between cable bacteria and their associated microbial community by manipulating the growth and activity of cable bacteria and quantifying the resultant microbial community response. Specifically, this project aims to (1) identify microorganisms whose growth is enhanced by cable bacteria, (2) identify metabolic processes linked with cable bacteria activity using metatranscriptomics, (3) test specific metabolic links between sediment microorganisms and cable bacteria activity using a DNA-stable isotope probing (SIP) approach, and (4) visually confirm the identity and quantify key microorganisms associated with cable bacteria using microscopy. As more is learned about the identity and the mechanisms by which microorganisms are metabolically linked in anoxic sediments, we will be better able to understand and make predictions about how microorganisms function in their environment and how they can be utilized in bioengineered systems.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.