Marine sediments microbes capable of electrode oxidation as a surrogate for lithotrophic insoluble substrate metabolism

Abstract

Little is known about the importance and/or mechanisms of biological mineral oxidation in sediments, partially due to the difficulties associated with culturing mineral-oxidizing microbes. We demonstrate that electrochemical enrichment is a feasible approach for isolation of microbes capable of gaining electrons from insoluble minerals. To this end we constructed sediment microcosms and incubated electrodes at various controlled redox potentials. Negative current production was observed in incubations and increased as redox potential decreased (tested -50 to -400 mV vs. Ag/AgCl). Electrode-associated biomass responded to the addition of nitrate and ferric iron as terminal electron acceptors in secondary sediment-free enrichments. Elemental sulfur, elemental iron and amorphous iron sulfide enrichments derived from electrode biomass demonstrated products indicative of sulfur or iron oxidation. The microbes isolated from these enrichments belong to the genera Halomonas, Idiomarina, Marinobacter, and Pseudomonas of the Gammaproteobacteria, and Thalassospira and Thioclava from the Alphaproteobacteria. Chronoamperometry data demonstrates sustained electrode oxidation from these isolates in the absence of alternate electron sources. Cyclic voltammetry demonstrated the variability in dominant electron transfer modes or interactions with electrodes (i.e., biofilm, planktonic or mediator facilitated) and the wide range of midpoint potentials observed for each microbe (from 8 to -295 mV vs. Ag/AgCl). The diversity of extracellular electron transfer mechanisms observed in one sediment and one redox condition, illustrates the potential importance and abundance of these interactions. This approach has promise for increasing our understanding the extent and diversity of microbe mineral interactions, as well as increasing the repository of microbes available for electrochemical applications.

Keywords: Halomonas; Marinobacter; Pseudomonas; electromicrobiology; geobiology; iron oxidation; lithotrophy; sulfur oxidation.

Figure 1

Schematic diagram of sediment microcosms with window illustrating three electrode system incubated in 10 gallon aquaria. Working electrodes placed between 2–4 cm in the sediment water column, while reference and counter electrodes remained in surface waters. A flow of UV treated and aerated water was added at a continuous rate to facilitate oxygen and nutrient replenishment and waste removal. P designates a potentiostat. TEA stands for terminal electron acceptor and the redox state—reduced (red) or oxidized (ox)—is indicated.

Figure 2

Sediment free bioreactor schematic including three electrode system (A). Inserted elements diagramed left to right include a gas diffuser, a working electrode, a counter electrode and a reference electrode. Example negative current production for a sediment incubated working electrode vs. a sterile or non-inoculated control both batch fed of nitrate shown in (B).

Figure 3

Phyla observed in solid substrate enrichments using nitrate as a terminal electron acceptor. The phyla recovered from the original biomass used for inoculation of cultures is designated original electrode enrichment/nitrate. Phyla were determined from 16S rRNA tagged pyrosequencing analysis of DNA extracts from enrichments, analyzed using Qiime (see Materials and Methods).

Figure 4

Maximum likelihood estimation phylogenetic tree based on 900 bp of the 16 rRNA gene. sequences aligned using the SINA aligner. Nearest neighbors obtained from the Green Genes database (accession numbers indicated). The scale bar indicates nucleotide substitutions computed using RaxML. The Thalassospira sp. (HM057728) was designated as the tree root.

Figure 5

Representative chronoamperometry profiles for oxygen fed electrochemical cells with various electrode oxidizing isolates compared to a control (non-electrochemically active) Streptococcus mutans strain. Electrodes were poised at −400 mV vs. Ag/AgCl. Graphs illustrate the first 30 h post inoculation with biomass.

Figure 6

Representative CV plots for: (A) Marinobacter sp. FeSN3, (B) Idiomarina sp. SN11 and (C) Thallassospira sp. SN3. Plots are displayed for control (SWB media), planktonic cells (suspended biomass removed from electrochemical cell), and biofilm (electrode attached biomass) samples for each organism. Each CV was run with a scan rate of 5 mV/s over a voltage range from −600 to +500 mV with the exception of the biofilm sample from Idiomarina sp. SN11 (B) which was run from −800 to +600 mV.