Long-Distance Electron Transport in Multicellular Freshwater Cable Bacteria

Reviewer #1 (Public Review):

Summary:

This work provides significant insight into freshwater cable bacteria (CB) and is an important contribution to the emerging CB literature. In this manuscript, Yang et al. describe current-voltage measurements on CB collected from two freshwater sources in Southern California. The studies use electrostatic and conductive atomic force microscopies, as well as four-probe measurements. These measurements are consistent with back-of-the-envelope calculations on conductivities needed to sustain CB function. The data shows that freshwater CB have a similar structure and function to the more studied marine cable bacteria.

Strengths:

Excellent measurements on a new class of cable bacteria.

Weaknesses:

The paper would benefit from additional analysis of the data.

Reviewer #2 (Public Review):

Summary:

In this work, Mohamed Y. El-Naggar and co-workers present a detailed electronic characterization of cable bacteria from Southern California freshwater sediments. The cable bacteria could be reliably enriched in laboratory incubations, and subsequent TEM characterization and 16S rRNA gene phylogeny demonstrated their belonging to the genus Candidatus Electronema. Atomic force microscopy and two-point probe resistance measurements were then used to map out the characteristics of the conductive nature, followed by microelectrode four-probe measurements to quantify the conductivity.

Interestingly, the authors observe that some freshwater cable bacteria filaments displayed a higher degree of robustness upon oxygen exposure than what was previously reported for marine cable bacteria. Finally, a single nanofiber conductivity on the order of 0.1 S/cm is calculated, which matches the expected electron current densities linking electrogenic sulphur oxidation to oxygen reduction in sediment. This is consistent with hopping transport.

Strengths and weaknesses:

A comprehensive study is applied to characterise the conductive properties of the sampled freshwater cable bacteria. Electrostatic force microscopy and conductive atomic force microscopy provide direct evidence of the location of conductive structures. Four-probe microelectrode devices are used to quantify the filament resistance, which presents a significant advantage over commonly used two-probe measurements that include contributions from contact resistances. While the methodology is convincing, I find that some of the conclusions seem to be drawn on very limited sample sizes, which display widely different behaviour. In particular:

The authors observe that the conductivity of freshwater filaments may be less sensitive to oxygen exposure than previously observed for marine filaments. This is indeed the case for an interdigitated array microelectrode experiment (presented in Figure 5) and for a conductive atomic force microscopy experiment (described in line 391), but the opposite is observed in another experiment (Figure S1). It is therefore difficult to assess the validity of the conclusion until sufficient experimental replications are presented.

The calculation of a single nanofiber conductivity is based on experiment and calculation with significant uncertainty. E.g. for the number of nanofibres in a single filament that varies depending on the filament size (Frontiers in microbiology, 2018, 9: 3044.), and the measured CB resistance, which does not scale well with inner probe separation (Figure 5). A more rigorous consideration of these uncertainties is required.