Instrument Type: Push Corer

Description from NSF award abstract:
Current estimates suggest that the volume of ocean crust capable of sustaining life is comparable in magnitude to that of the oceans. To date, there is little understanding of the composition or functional capacity of microbial communities in the sub-seafloor, or their influence on the chemistry of the oceans and subsequent consequences for global biogeochemical cycles. This project focuses on understanding the relationship between microbial communities and fluid chemistry in young crustal fluids that are responsible for the transport of energy, nutrients, and organisms in the crust. Specifically, the PIs will couple microbial activity measurements, including autotrophic carbon, nitrogen and sulfur metabolisms as well as mineral oxide reduction, with quantitative assessments of functional gene expression and geochemical transformations in basement fluids. Through a comprehensive suite of in situ and shipboard analyses, this research will yield cross-disciplinary advances in our understanding of the microbial ecology and geochemistry of the sub-seafloor biosphere. The focus of the effort is at North Pond, an isolated sediment pond located on ridge flank oceanic crust 7-8 million years old on the western side of the Mid-Atlantic Ridge. North Pond is currently the target for drilling on IODP expedition 336, during which it will be instrumented with three sub-seafloor basement observatories.

The project will leverage this opportunity for targeted and distinct sampling at North Pond on two German-US research cruises to accomplish three main objectives:

1. to determine if different basement fluid horizons across North Pond host distinct microbial communities and chemical milieus and the degree to which they change over a two-year post-drilling period.

2. to quantify the extent of autotrophic metabolism via microbially-mediated transformations in carbon, nitrogen, and sulfur species in basement fluids at North Pond.

3. to determine the extent of suspended particulate mineral oxides in basement fluids at North Pond and to characterize their role as oxidants for fluid-hosted microbial communities.

Specific outcomes include quantitative assessments of microbial activity and gene expression as well as geochemical transformations. The program builds on the integrative research goals for North Pond and will provide important data for guiding the development of that and future deep biosphere research programs. Results will increase understanding of microbial life and chemistry in young oceanic crust as well as provide new insights into controls on the distribution and activity of marine microbial communities throughout the worlds oceans.

There are no data about microbial communities in ubiquitous cold, oceanic crust, the emphasis of the proposed work. This is an interdisciplinary project at the interface of microbial ecology, chemistry, and deep-sea oceanography with direct links to international and national research and educational organizations.

Iron is a critical element for life that serves as an essential trace element for eukaryotic organisms. It is also able to support the growth of a cohort of microbes that can either gain energy for growth via oxidation of ferrous (Fe(II)) to ferric (Fe(III)) iron, or by utilizing Fe(III) for anaerobic respiration coupled to oxidation of simple organic matter or H2. This coupled process is referred to as the microbial iron cycle. One of the primary sources of iron to the ocean comes from dissolved iron (dFe) that is produced through oxidation and reduction processes in the sediment where iron is abundant. The dFe is transported into the overlaying water where it is an essential nutrient for phytoplankton responsible for primary production in the world’s oceans. In fact, iron limitation significantly impacts production in as much as a third of the world’s open oceans. The basic geochemistry of this process is understood; however important gaps exist in our knowledge about the details of how the iron cycle works, and how critical a role bacteria play in it.

Intellectual Merit. Conventional wisdom holds that most of the iron oxidation in sediments is abiological, as a result of the rapid kinetics of chemical iron oxidation in the presence of oxygen. This proposal aims to question this conventional view and enhance our understanding of the microbes involved in the sedimentary iron cycle, with an emphasis on the bacteria that catalyze the oxidation of iron. These Fe-oxidizing bacteria (FeOB) utilize iron as a sole energy source for growth, and are autotrophic.  They were only discovered in the ocean about forty-five years ago, and are now known to be abundant at hydrothermal vents that emanate ferrous-rich fluids. More recently, the first evidence was published that they could inhabit coastal sediments, albeit at reduced numbers, and even be abundant in some continental shelf sediments. These habitats are far removed from hydrothermal vents, and reveal the sediments may be an important habitat for FeOB that live on ferrous iron generated in the sediment. This begs the question: are FeOB playing an important role in the oxidative part of the sedimentary Fe-cycle? One important attribute of FeOB is their ability to grow at very low levels of O2, an essential strategy for them to outcompete chemical iron oxidation. How low a level of O2 can sustain them, and how this might affect their distribution in sediments is unknown. In part, this is due to the technical challenges of measuring O2 concentrations and dynamics at very low levels; yet these concentrations could be where FeOB flourish. The central hypothesis of this proposal is that FeOB are more common in marine sedimentary environments than previously recognized, and play a substantive role in governing the iron flux from the sediments into the water column by constraining the release of dFe from sediments. A set of experimental objectives are proposed to test this. A survey of near shore regions in the Gulf of Maine, and a transect along the Monterey Canyon off the coast of California will obtain cores of sedimentary muds and look at the vertical distribution of FeOB and putative Fe-reducing bacteria using sensitive techniques to detect their presence and relative abundance. Some of these same sediments will be used in a novel reactor system that will allow for precise control of O2 levels and iron concentration to measure the dynamics of the iron cycle under different oxygen regimens. Finally pure cultures of FeOB with different O2 affinities will be tested in a bioreactor coupled to a highly sensitive mass spectrometer to determine the lower limits of O2 utilization for different FeOB growing on iron, thus providing mechanistic insight into their activity and distribution in low oxygen environments.

Broader Impacts. An important impact of climate change on marine environments is a predicted increase in low O2 or hypoxic zones in the ocean. Hypoxia in association with marine sediments will have a profound influence on the sedimentary iron cycle, and is likely to lead to greater inputs of dFe into the ocean. In the longer term, this increase in dFe flux could alleviate iron-limitation in some regions of the ocean, thereby enhancing the rate of CO2-fixation and draw down of CO2 from the atmosphere. This is one important reason for developing a better understanding of microbial control of sedimentary iron cycle. This project will also provide training to a postdoctoral scientist, graduate students and undergraduates. This project will contribute to a student initiated exhibit, entitled ‘Iron and the evolution of life on Earth’ at the Harvard Museum of Natural History providing a unique opportunity for undergraduate training and outreach.

Project description from C-DEBI:
Heterotrophic organisms are central to subsurface microbial communities and play an important role in carbon cycling. Most approaches to measuring enzymatic activities rely on the addition of a fluorescently labeled substrate to a sediment incubation. However, quantifying rates of extracellular enzymatic hydrolysis of organic matter is often problematic due to the tendency for a fluorescently labeled organic substrate to sorb to the sediment matrix. This results in lower fluorescence intensities and distorted, inaccurate hydrolysis rate calculations. In this project, a desorption treatment was developed to counteract the adverse effects of sorption on enzymatic activity measurements. Upon subsampling a sediment incubation amended with a fluorescently labeled substrate, the subsample is treated with a concentrated solution of unlabeled substrate, along with 0.2% sodium dodecyl sulfate (SDS), in order to competitively desorb the adsorbed, fluorescent substrate target. This treatment improves measured fluorescence intensities by a median of 62.5%, and is particularly effective at desorbing high molecular weight substrate products, resulting in debiased hydrolysis rates that are 14.75 nM/hr lower on average. Competitive desorption treatment was demonstrated to be effective for multiple substrates and in a broad range of sediments from diverse geological and geochemical contexts. Future applications of this method will result in more quantitative and comparable hydrolysis rates in subsurface sediments, will enable enzymatic activity measurements in problematic sediments that were previously infeasible, and will facilitate physiological characterization of microbial communities and model organisms in order to better understand heterotrophic carbon cycling in the subsurface environment.

This project was funded by a C-DEBI Graduate Fellowship.

Description from C-DEBI:
The goal of this project was to explore the mechanisms of subsurface organoheterotrophy by identifying the range of extracellular peptidases present in sediments of the White Oak River, NC, consistent with C-DEBI Research Theme 1, Activity in the Deep Subseafloor Biosphere: function & rates of global biogeochemical processes. This grant funded two sampling expeditions to the White Oak River as well as extensive laboratory work with a purified peptidase that was supplied by collaborators Andrzej Joachimiak and Karolina Michalska of Argonne National Laboratory and preparatory work on peptidases of the Tennessee River. So far this dataset has led to the submission of two manuscripts, with one more manuscript in preparation. The White Oak River work showed that a wide range of peptidases are present in depths up to 80 cm in the White Oak River, which is deeper than the zone of methanogenesis. Although absolute peptidase activities declined with depth, activities normalized to cell abundance were roughly constant, and activities normalized to organic carbon oxidation rates increased nearly two orders of magnitude relative to the surface, indicating that extracellular peptidases were important to the subsurface ecosystem. Biochemical analysis of a purified peptidase that was expressed by the Argonne group showed it to be a novel aminopeptidase with specificity for N-terminal cysteine, a function not previously observed in peptidses. In summary, in situ and in vitro studies of subsurface peptidases revealed that they are ecologically important and may contain novel properties.

Description from C-DEBI:
The goal of this project was to explore the mechanisms of subsurface organoheterotrophy by identifying the range of extracellular peptidases present in sediments of the White Oak River, NC, consistent with C-DEBI Research Theme 1, Activity in the Deep Subseafloor Biosphere: function & rates of global biogeochemical processes. This grant funded two sampling expeditions to the White Oak River as well as extensive laboratory work with a purified peptidase that was supplied by collaborators Andrzej Joachimiak and Karolina Michalska of Argonne National Laboratory and preparatory work on peptidases of the Tennessee River. So far this dataset has led to the submission of two manuscripts, with one more manuscript in preparation. The White Oak River work showed that a wide range of peptidases are present in depths up to 80 cm in the White Oak River, which is deeper than the zone of methanogenesis. Although absolute peptidase activities declined with depth, activities normalized to cell abundance were roughly constant, and activities normalized to organic carbon oxidation rates increased nearly two orders of magnitude relative to the surface, indicating that extracellular peptidases were important to the subsurface ecosystem. Biochemical analysis of a purified peptidase that was expressed by the Argonne group showed it to be a novel aminopeptidase with specificity for N-terminal cysteine, a function not previously observed in peptidses. In summary, in situ and in vitro studies of subsurface peptidases revealed that they are ecologically important and may contain novel properties.

Marine sediments harbor a vast amount of Earth’s microbial biomass, yet little is understood regarding how cells subsist in these low-energy environments without forming endospores. DNA methylation, a reversible process that involves the addition of a methyl group to a nucleotide base via a methyltransferase, exists as a possible epigenetic mechanism for non-sporulated cells in low-energy sediment environments to regulate gene expression and potentially lower cellular activity. To investigate the presence and scope of this phenomenon in estuarine sediment microbial communities, we sequenced three metagenomic and 16S rRNA gene amplicon libraries extracted from a sediment core collected from the banks of the Oyster Rocks site of the Broadkill River, Milton, Delaware, USA. We targeted 5-methylcytosine at CpG sites by digesting metagenomic libraries with the methylation-sensitive restriction endonuclease HpaII. By quantitatively distinguishing “mixed” methylation states for populations of CpG site copies, we identified dynamic, non-binary shifts in CpG methylation for community taxa and function.

Description from NSF award abstract:
Pristine fluids from a typical ridge-flank hydrothermal system have never been sampled, mainly because it has not been possible to locate a site of focused discharge where representative samples could be collected. The PIs have located a small basement feature, Dorado outcrop, on 23 m.y.-old seafloor on the eastern flank of the East Pacific Rise that they plan to sample to determine the fluid composition, and to assess the rate of discharge from the outcrop, so that they can quantify the chemical impact of this hydrothermal system. They plan an 18-day expedition that combines the surveying capabilities of the AUV Sentry (bathymetric, sub-bottom sonar, photo mosaics, water column anomalies) and an ocean-class vessel capable of collecting high-quality multi-beam data and CTD samples, and supporting the survey and sampling capabilities of the ROV Jason II for collection of spring and plume fluids, heat flow data, sediment push cores, and still and video photography. These data and samples will be combined hopefully to generate the first well-constrained estimates of hydrothermal flows from Dorado outcrop. This expedition will result in the collection of samples and data from a “fire hose” of ridge-flank, hydrothermal system, challenging the commonly held view that discharge from ridge flank hydrothermal systems occurs primarily from diffuse seeps.

Description from NSF award abstract:
Pristine fluids from a typical ridge-flank hydrothermal system have never been sampled, mainly because it has not been possible to locate a site of focused discharge where representative samples could be collected. The PIs have located a small basement feature, Dorado outcrop, on 23 m.y.-old seafloor on the eastern flank of the East Pacific Rise that they plan to sample to determine the fluid composition, and to assess the rate of discharge from the outcrop, so that they can quantify the chemical impact of this hydrothermal system. They plan an 18-day expedition that combines the surveying capabilities of the AUV Sentry (bathymetric, sub-bottom sonar, photo mosaics, water column anomalies) and an ocean-class vessel capable of collecting high-quality multi-beam data and CTD samples, and supporting the survey and sampling capabilities of the ROV Jason II for collection of spring and plume fluids, heat flow data, sediment push cores, and still and video photography. These data and samples will be combined hopefully to generate the first well-constrained estimates of hydrothermal flows from Dorado outcrop. This expedition will result in the collection of samples and data from a “fire hose” of ridge-flank, hydrothermal system, challenging the commonly held view that discharge from ridge flank hydrothermal systems occurs primarily from diffuse seeps.

Description from NSF award abstract:
Pristine fluids from a typical ridge-flank hydrothermal system have never been sampled, mainly because it has not been possible to locate a site of focused discharge where representative samples could be collected. The PIs have located a small basement feature, Dorado outcrop, on 23 m.y.-old seafloor on the eastern flank of the East Pacific Rise that they plan to sample to determine the fluid composition, and to assess the rate of discharge from the outcrop, so that they can quantify the chemical impact of this hydrothermal system. They plan an 18-day expedition that combines the surveying capabilities of the AUV Sentry (bathymetric, sub-bottom sonar, photo mosaics, water column anomalies) and an ocean-class vessel capable of collecting high-quality multi-beam data and CTD samples, and supporting the survey and sampling capabilities of the ROV Jason II for collection of spring and plume fluids, heat flow data, sediment push cores, and still and video photography. These data and samples will be combined hopefully to generate the first well-constrained estimates of hydrothermal flows from Dorado outcrop. This expedition will result in the collection of samples and data from a “fire hose” of ridge-flank, hydrothermal system, challenging the commonly held view that discharge from ridge flank hydrothermal systems occurs primarily from diffuse seeps.