Protocol for using autoclaved intertidal sediment as a medium to enrich marine cable bacteria.

Cable bacteria (CB) are non-isolated filamentous bacteria in the family of Desulfobulbaceae, known for fostering centimeter-long electron transfer in sediments with pronounced redox zonation. This protocol details steps to extract CB filaments from cultured natural sediment, inoculate autoclaved sediment with extracted filaments, and subsequently evaluate the growth and enrichment of CB. We also describe the approaches for collecting suitable sediment, preparing autoclaved sediment, and manufacturing glass needles and hooks for the extraction of CB.

Aquatic Eddy Covariance: The Method and Its Contributions to Defining Oxygen and Carbon Fluxes in Marine Environments.

Aquatic eddy covariance (AEC) is increasingly being used to study benthic oxygen (O2) flux dynamics, organic carbon cycling, and ecosystem health in marine and freshwater environments. Because it is a noninvasive technique, has a high temporal resolution (∼15 min), and integrates over a large area of the seafloor (typically 10-100 m2), it has provided new insights on the functioning of aquatic ecosystems under naturally varying in situ conditions and has given us more accurate assessments of their metabolism. In this review, we summarize biogeochemical, ecological, and biological insightsgained from AEC studies of marine ecosystems. A general finding for all substrates is that benthic O2 exchange is far more dynamic than earlier recognized, and thus accurate mean values can only be obtained from measurements that integrate over all timescales that affect the local O2 exchange. Finally, we highlight new developments of the technique, including measurements of air-water gas exchange and long-term deployments.

Benthic microbial fuel cell as direct power source for an acoustic modem and seawater oxygen/temperature sensor system.

Supported by the natural potential difference between anoxic sediment and oxic seawater, benthic microbial fuel cells (BMFCs) promise to be ideal power sources for certain low-power marine sensors and communication devices. In this study a chambered BMFC with a 0.25 m(2) footprint was used to power an acoustic modem interfaced with an oceanographic sensor that measures dissolved oxygen and temperature. The experiment was conducted in Yaquina Bay, Oregon over 50 days. Several improvements were made in the BMFC design and power management system based on lessons learned from earlier prototypes. The energy was harvested by a dynamic gain charge pump circuit that maintains a desired point on the BMFC's power curve and stores the energy in a 200 F supercapacitor. The system also used an ultralow power microcontroller and quartz clock to read the oxygen/temperature sensor hourly, store data with a time stamp, and perform daily polarizations. Data records were transmitted to the surface by the acoustic modem every 1-5 days after receiving an acoustic prompt from a surface hydrophone. After jump-starting energy production with supplemental macroalgae placed in the BMFC's anode chamber, the average power density of the BMFC adjusted to 44 mW/m(2) of seafloor area which is better than past demonstrations at this site. The highest power density was 158 mW/m(2), and the useful energy produced and stored was ≥ 1.7 times the energy required to operate the system.

Using Oxidative Electrodes to Enrich Novel Members in the Desulfobulbaceae Family from Intertidal Sediments.

Members in the family of Desulfobulbaceae may be influential in various anaerobic microbial communities, including those in anoxic aquatic sediments and water columns, and within wastewater treatment facilities and bioelectrochemical systems (BESs) such as microbial fuel cells (MFCs). However, the diversity and roles of the Desulfobulbaceae in these communities have received little attention, and large portions of this family remain uncultured. Here we expand on findings from an earlier study (Li, Reimers, and Alleau, 2020) to more fully characterize Desulfobulbaceae that became prevalent in biofilms on oxidative electrodes of bioelectrochemical reactors. After incubations, DNA extraction, microbial community analyses, and microscopic examination, we found that a group of uncultured Desulfobulbaceae were greatly enriched on electrode surfaces. These Desulfobulbaceae appeared to form filaments with morphological features ascribed to cable bacteria, but the majority were taxonomically distinct from recognized cable bacteria genera. Thus, the present study provides new information about a group of Desulfobulbaceae that can exhibit filamentous morphologies and respire on the oxidative electrodes. While the phylogeny of cable bacteria is still being defined and updated, further enriching these members can contribute to the overall understanding of cable bacteria and may also lead to identification of successful isolation strategies.

Temporal patterns of microbial community structure in the Mid-Atlantic Bight.

Although open ocean time-series sites have been areas of microbial research for years, relatively little is known about the population dynamics of bacterioplankton communities in the coastal ocean on kilometer spatial and seasonal temporal scales. To gain a better understanding of microbial community variability, monthly samples of bacterial biomass were collected in 1995-1996 along a 34-km transect near the Long-Term Ecosystem Observatory (LEO-15) off the New Jersey coast. Surface and bottom sampling was performed at seven stations along a transect line with depths ranging from 1 to 35 m (n=178). Microbial populations were fingerprinted using ribosomal 16S rRNA genes and terminal restriction fragment length polymorphism analysis. Results from cluster analysis revealed distinct temporal patterns among the bacterioplankton communities in the Mid-Atlantic Bight rather than grouping by sample location or depth. Principal components analysis models supported the temporal patterns. In addition, partial least squares regression modeling could not discern a significant correlation from traditional oceanographic physical and phytoplankton nutrient parameters on overall bacterial community variability patterns at LEO-15. These results suggest factors not traditionally measured during oceanographic studies are structuring coastal microbial communities.

Harnessing microbially generated power on the seafloor.

In many marine environments, a voltage gradient exists across the water sediment interface resulting from sedimentary microbial activity. Here we show that a fuel cell consisting of an anode embedded in marine sediment and a cathode in overlying seawater can use this voltage gradient to generate electrical power in situ. Fuel cells of this design generated sustained power in a boat basin carved into a salt marsh near Tuckerton, New Jersey, and in the Yaquina Bay Estuary near Newport, Oregon. Retrieval and analysis of the Tuckerton fuel cell indicates that power generation results from at least two anode reactions: oxidation of sediment sulfide (a by-product of microbial oxidation of sedimentary organic carbon) and oxidation of sedimentary organic carbon catalyzed by microorganisms colonizing the anode. These results demonstrate in real marine environments a new form of power generation that uses an immense, renewable energy reservoir (sedimentary organic carbon) and has near-immediate application.

The Identification of Cable Bacteria Attached to the Anode of a Benthic Microbial Fuel Cell: Evidence of Long Distance Extracellular Electron Transport to Electrodes.

Multicellular, filamentous, sulfur-oxidizing bacteria, known as cable bacteria, were discovered attached to fibers of a carbon brush electrode serving as an anode of a benthic microbial fuel cell (BMFC). The BMFC had been operated in a temperate estuarine environment for over a year before collecting anode samples for scanning electron microscopy and phylogenetic analyses. Individual filaments were attached by single terminus cells with networks of pilus-like nano-filaments radiating out from these cells, across the anode fiber surface, and between adjacent attachment locations. Current harvesting by the BMFC poised the anode at potentials of ~170-250 mV vs. SHE, and these surface potentials appear to have allowed the cable bacteria to use the anode as an electron acceptor in a completely anaerobic environment. A combination of catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH) and 16S rRNA gene sequence analysis confirmed the phylogeny of the cable bacteria and showed that filaments often occurred in bundles and in close association with members of the genera Desulfuromonas. However, the Desulfobulbaceae Operational Taxonomic Units (OTUs) from the 16S sequencing did not cluster closely with other putative cable bacteria sequences suggesting that the taxonomic delineation of cable bacteria is far from complete.

Influence of substrate on electron transfer mechanisms in chambered benthic microbial fuel cells.

This research investigated whether the addition of an exogenous electron donor would affect power production in laboratory-scale benthic microbial fuel cells (BMFC) by differentially influencing microbially mediated electron transfer processes. Six BMFCs were operated for over one year in a temperature-controlled laboratory. Three BMFCs relied on endogenous electron donors, and three were supplemented with lactate. The supplemented BMFCs generated more cumulative charge, but did not generate higher average current between periods of lactate enrichment. Coulombic efficiencies during the lactate treatments ranged from 25 to 65% suggesting that lactate utilization was variably coupled to power production. Cumulative electron flux resulting from lactate additions and chemical changes within the anode chamber, as well as a difference in the anode-hosted microbial communities indicated that lactate supplementation promoted sulfate reduction. After the addition of molybdate to suppress sulfate reduction and sulfur disproportionation, all BMFCs continued to produce current, but no longer responded to lactate additions. Chemical data support a two-step cycle in which endogenous organic carbon and/or supplemented lactate fuel sulfate reduction resulting in sulfide and simple organic molecules (such as acetate) that can act as the electron donors for the BMFC.

Quantitative population dynamics of microbial communities in plankton-fed microbial fuel cells.

This study examines changes in diversity and abundance of bacteria recovered from the anodes of microbial fuel cells (MFCs) in relation to anode potential, power production and geochemistry. MFCs were batch-fed with plankton, and two systems were maintained at different potentials whereas one was at open circuit for 56.8 days. Bacterial phylogenetic diversity during peak power was assessed from 16S rDNA clone libraries. Throughout the experiment, microbial community structure was examined using terminal restriction fragment length polymorphism. Changes in cell density of key phylotypes, including representatives of delta-, epsilon-, gamma-proteobacteria and Flavobacterium-Cytophaga-Bacteroides, were enumerated by quantitative PCR. Marked differences in phylogenetic diversity were observed during peak power versus the final time point, and changes in microbial community structure were strongly correlated to dissolved organic carbon and ammonium concentrations within the anode chambers. Community structure was notably different between the MFCs at different anode potentials during the onset of peak power. At the final time point, however, the anode-hosted communities in all MFCs were similar. These data demonstrate that differences in growth, succession and population dynamics of key phylotypes were due to anode potential, which may relate to their ability to exploit the anode as an electron acceptor. The geochemical milieu, however, governs overall community diversity and structure. These differences reflect the physiological capacity of specific phylotypes to catabolize plankton-derived organic matter and exploit the anode of an MFC for their metabolism directly or indirectly through syntrophy.

Enhanced power from chambered benthic microbial fuel cells.

We describe a new chamber-based benthic microbial fuel cell (BMFC) that incorporates a suspended, high surface area and semi-enclosed anode to improve performance. In Yaquina Bay, OR, two chambered BMFC prototypes generated current continuously for over 200 days. One BMFC was pumped intermittently, which produced power densities more than an order of magnitude greater than those achieved by previous BMFCs with single buried graphite-plate anodes. On average, the continuous power densities with pumping were 233 mW/m2 (2.3 W/m3); peak values were 380 mW/m2 (3.8 W/m3), and performance improved over the time of the deployments. Without pumping, high power densities could similarly be achieved after either BMFC was allowed to rest at open circuit. A third chambered BMFC with a 0.4 m2 footprint was deployed at a cold seep in Monterey Canyon, CA to test the new design in an environment with natural advection. The power density increased 5-fold (140 mW/m2 vs 28 mW/m2) when low-pressure check valves allowed unidirectional flow through the chamber.

Substrate degradation kinetics, microbial diversity, and current efficiency of microbial fuel cells supplied with marine plankton.

The decomposition of marine plankton in two-chamber, seawater-filled microbial fuel cells (MFCs) has been investigated and related to resulting chemical changes, electrode potentials, current efficiencies, and microbial diversity. Six experiments were run at various discharge potentials, and a seventh served as an open-circuit control. The plankton consisted of a mixture of freshly captured phytoplankton and zooplankton (0.21 to 1 mm) added at an initial batch concentration of 27.5 mmol liter(-1) particulate organic carbon (OC). After 56.7 days, between 19.6 and 22.2% of the initial OC remained, sulfate reduction coupled to OC oxidation accounted for the majority of the OC that was degraded, and current efficiencies (of the active MFCs) were between 11.3 and 15.5%. In the open-circuit control cell, anaerobic plankton decomposition (as quantified by the decrease in total OC) could be modeled by three terms: two first-order reaction rate expressions (0.79 day(-1) and 0.037 day(-1), at 15 degrees C) and one constant, no-reaction term (representing 10.6% of the initial OC). However, in each active MFC, decomposition rates increased during the third week, lagging just behind periods of peak electricity generation. We interpret these decomposition rate changes to have been due primarily to the metabolic activity of sulfur-reducing microorganisms at the anode, a finding consistent with the electrochemical oxidization of sulfide to elemental sulfur and the elimination of inhibitory effects of dissolved sulfide. Representative phylotypes, found to be associated with anodes, were allied with Delta-, Epsilon-, and Gammaproteobacteria as well as the Flavobacterium-Cytophaga-Bacteroides and Fusobacteria. Based upon these results, we posit that higher current efficiencies can be achieved by optimizing plankton-fed MFCs for direct electron transfer from organic matter to electrodes, including microbial precolonization of high-surface-area electrodes and pulsed flowthrough additions of biomass.

Activity and distribution of bacterial populations in Middle Atlantic Bight shelf sands.

Abstract Spatiotemporal variation and metabolic activity of the microbial community were studied in coarse-grained Middle Atlantic Bight shelf sediments in relation to pools of dissolved and particulate carbon. Algal cells were present 8->70 mum) fraction of the sediment held the major share (61-98%) of benthic bacteria. Bacterial and algal cell abundances, exoenzymatic activity, and [DOC] generally showed higher values in May/July 2001 than in August/December 2000. Carbohydrates and proteins were hydrolyzed at potential rates of 1-12 nmol cm(-3) h(-1) (beta-glucosidase) and 3-70 nmol cm(-3) h(-1) (aminopeptidase), respectively. Fluorescence in situ hybridization analyses of the benthic microbes assigned 45-56% of DAPI-stained cells to Eubacteria and less than 2% to Eukarya. The prokaryotic community was dominated by planctomycetes and members of the Cytophaga/Flavobacterium cluster. Near the sediment surface, iodonitrotetrazolium violet reducing cells, that are considered actively respiring, amounted to 15-29% of total bacteria. Despite a low organic content (particulate organic carbon <0.03%) and relatively low bacterial abundances (<10(9) cm(-3)), the Middle Atlantic Bight shelf sediments showed organic matter turnover rates that are comparable to those found in organic-rich finer-grained deposits. Our findings suggest a high biocatalytic filtration activity in these coarse permeable sediments.