Table of Contents

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

This is the first of 4 datasets associated with "Incubation Experiment (Liau) CB Sediments". There is additionally an amplicon dataset (16S rRNA gene, RNA and DNA) associated with this experiment: NCBI Sequence Read Archive, under BioProject PRJNA833464, accession numbers SAMN27993143 to SAMN27993196. The NCBI data will not be publicly available in May 2023.

The source material was collected from Chesapeake Bay (38.55505 N, 76.42794 W) (mesohaline), water column depth 26 meters, sediment horizon 0-10 centimeter below sea floor.

Incubation conditions: 16 degrees Celsius, S=15.5, dark, aerated.

Incubation Set-up: Sediments were homogenized and packed into polycarbonate core liners, sealed with a stopper at the bottom, and open to aerated aquarium water at the top. In a subset of cores, a polycarbonate filter (pore size 0.2 microns) was secured at 0.5 cm depth, to prevent downward growth of cable bacteria in these treatments. Sediments were incubated for up to 46 days. At 6 time points, microsensor profiles were measured, followed by destructive sampling.

Porewater extraction: Sediments were sectioned at 0.5 cm depth increments in an anaerobic glove bag under nitrogen atmosphere. Porewaters were separated by centrifugation (3500 rpm for 10 minutes), and filtered (0.2 micron) and aliquoted in the anaerobic glove bag. Samples for ferrous iron measurements were preserved with trace-metal grade nitric acid (final pH < 2).

Geochemical Measurements: Porewater sulfate and chloride were analyzed by suppressed ion chromatography following 100-fold dilutions (Dionex Integrion IC). Porewater ferrous iron (Fe2+) was measured colorimetrically using the Ferrozine assay. Ammonium was measured colorimetrically using the phenol hypochlorite method.

Cell Enumeration: Samples for microbial cell enumeration were preserved in 96% molecular grade ethanol (1:1 v/v), gently mixed with sterile autoclaved toothpicks, and stored at -20°C. Sediment-associated cells were separated from sediment grains using an acetate buffer to dissolve carbonates, followed by washing with NaCl solution, and then gentle vortexing with a detergent mixed with methanol (Lunau et al. 2005, Kallmeyer et al. 2008). Cells were recovered using density centrifugation with Nycodenz (50% w/v). Cable bacteria filaments were enumerated using fluorescence in situ hybridization (FISH) with the DSB706 oligoprobe (Malkin et al. 2022), and total cell counts were enumerated following staining with SyBR Green I.

Data Processing:
Standard curves with a minimum of 5 concentrations were analyzed to calibrate the geochemistry data. Microscopy counts were computed based on minimum cell counts. For cable bacteria, a minimum of 200 fields or 30 filaments were counted (whichever came first). For single total cell counts, a minimum of 400 cells were counted, appropriately diluted to be captured in 10-20 fields. The proportion of the slide examined was computed based on the field size, confirmed with a stage micrometer.

BCO-DMO Processing:
- replaced "NA" with "nd" (no data value);
- renamed fields to comply with BCO-DMO naming conventions.

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.