No abstract

Coastal sediments are rich in conductive particles, possibly affecting microbial processes for which acetate is a central intermediate. In the methanogenic zone, acetate is consumed by methanogens and/or syntrophic acetate-oxidizing (SAO) consortia. SAO consortia live under extreme thermodynamic pressure, and their survival depends on successful partnership. Here, we demonstrate that conductive particles enable the partnership between SAO bacteria (i.e., Geobacter spp.) and methanogens (Methanosarcina spp.) from the coastal sediments of the Bothnian Bay of the Baltic Sea. Baltic methanogenic sediments were rich in conductive minerals, had an apparent isotopic fractionation characteristic of CO2-reductive methanogenesis, and were inhabited by Geobacter and Methanosarcina. As long as conductive particles were delivered, Geobacter and Methanosarcina persisted, whereas exclusion of conductive particles led to the extinction of Geobacter. Baltic Geobacter did not establish a direct electric contact with Methanosarcina, necessitating conductive particles as electrical conduits. Within SAO consortia, Geobacter was an efficient [13C]acetate utilizer, accounting for 82% of the assimilation and 27% of the breakdown of acetate. Geobacter benefits from the association with the methanogen, because in the absence of an electron acceptor it can use Methanosarcina as a terminal electron sink. Consequently, inhibition of methanogenesis constrained the SAO activity of Geobacter as well. A potential benefit for Methanosarcina partnering with Geobacter is that together they competitively exclude acetoclastic methanogens like Methanothrix from an environment rich in conductive particles. Conductive particle-mediated SAO could explain the abundance of acetate oxidizers like Geobacter in the methanogenic zone of sediments where no electron acceptors other than CO2 are available.

The nanoSIMS-based chemical microscopy has been introduced in biology over a decade ago. The spatial distribution of elements and isotopes analyzed by nanoSIMS can be used to reconstruct images of biological samples with a resolution down to tens of nanometers, and can be also interpreted quantitatively. Currently, a unified approach for calculation of single cell assimilation rates from nanoSIMS-derived changes in isotope ratios is missing. Here we present a comprehensive concept of assimilation rate calculation with a rigorous mathematical model based on quantitative evaluation of nanoSIMS-derived isotope ratios. We provide a detailed description of data acquisition and treatment, including the selection and accumulation of nanoSIMS scans, defining regions of interest and extraction of isotope ratios. Next, we present alternative methods to determine the cellular volume and the density of the element under scrutiny. Finally, to compensate for alterations of original isotopic ratios, our model considers corrections for sample preparation methods (e.g., air dry, chemical fixation, permeabilization, hybridization), and when known, for the stable isotope fractionation associated with utilization of defined growth substrates. As proof of concept we implemented this protocol to quantify the assimilation of 13C-labeled glucose by single cells of Pseudomonas putida. In addition, we provide a calculation template where all protocol-derived formulas are directly available to facilitate routine assimilation rate calculations by nanoSIMS users.

Coastal sediments are rich in conductive minerals, which could impact microbial processes for which acetate is a central intermediate.

Phenotypic heterogeneity within microbial populations arises even when the cells are exposed to putatively constant and homogeneous conditions. The outcome of this phenomenon can affect the whole function of the population, resulting in, for example, new "adapted" metabolic strategies and impacting its fitness at given environmental conditions. Accounting for phenotypic heterogeneity becomes thus necessary, due to its relevance in medical and applied microbiology as well as in environmental processes. Still, a comprehensive evaluation of this phenomenon requires a common and unique method of quantitation, which allows for the comparison between different studies carried out with different approaches. Consequently, in this study, two widely applicable indices for quantitation of heterogeneity were developed. The heterogeneity coefficient (HC) is valid when the population follows unimodal activity, while the differentiation tendency index (DTI) accounts for heterogeneity implying outbreak of subpopulations and multimodal activity. We demonstrated the applicability of HC and DTI for heterogeneity quantitation on stable isotope probing with nanoscale secondary ion mass spectrometry (SIP-nanoSIMS), flow cytometry, and optical microscopy datasets. The HC was found to provide a more accurate and precise measure of heterogeneity, being at the same time consistent with the coefficient of variation (CV) applied so far. The DTI is able to describe the differentiation in single-cell activity within monoclonal populations resolving subpopulations with low cell abundance, individual cells with similar phenotypic features (e.g., isotopic content close to natural abundance, as detected with nanoSIMS). The developed quantitation approach allows for a better understanding on the impact and the implications of phenotypic heterogeneity in environmental, medical and applied microbiology, microbial ecology, cell biology, and biotechnology.