Multidisciplinary methodologies used in the study of cable bacteria

Abstract

Cable bacteria are a unique type of filamentous microorganism that can grow up to centimetres long and are capable of long-distance electron transport over their entire lengths. Due to their unique metabolism and conductive capacities, the study of cable bacteria has required technical innovations, both in adapting existing techniques and developing entirely new ones. This review discusses the existing methods used to study eight distinct aspects of cable bacteria research, including the challenges of culturing them in laboratory conditions, performing physical and biochemical extractions, and analysing the conductive mechanism. As cable bacteria research requires an interdisciplinary approach, methods from a range of fields are discussed, such as biogeochemistry, genomics, materials science, and electrochemistry. A critical analysis of the current state of each approach is presented, highlighting the advantages and drawbacks of both commonly used and emerging methods.

Keywords: Cable bacteria; enrichment cultures; genomics; microbial community; microprofiling; microscopy.

Figure 1.

Schematic of the position of CB in sediment and the half-redox reactions they perform in the oxic and anoxic regions of sediment.

Figure 2.

Diagram highlighting the eight major categories of CB research and the corresponding key methods discussed in this review. Culturing and filament collection are the starting points for most CB experimental designs. Potential workflows are depicted with arrows, and dashed lines link methods that are used for different categories. Advantages and disadvantages for each method are summarized in Table 1.

Figure 3.

The key steps involved in sediment collection, preparation, and incubation for creating CB enrichment cultures. There are multiple options for each step, which can be chosen based on field site and sediment characteristics and the type of enrichment culture needed for experiments.

Figure 4.

Schematic showing the steps to inoculate autoclaved sediment with a singular CB, creating a single-species culture. This involves taking sediment from an autoclaved core and an active core (A), picking a cable bacterium (B), and placing it into the autoclaved sediment (C), then removing the water between the two (D), following which autoclaved sediment is placed into a core (Thorup et al. 2021). Reproduced with permission from Elsevier Publishing under CC BY 4.0 licensing.

Figure 5.

(A) Profiles of pH, O₂, and H₂S in sediment with an active CB population (Hermans et al. 2020). Reproduced with permission from Copernicus Publications under CC BY 4.0 licensing. (B) Electrical potential profiles of a core with (right) and without (left) active CB, showing the increase in EP from the surface to greater depths (dotted profile) (Thorup et al. 2021). Reproduced with permission from Elsevier Publishing under CC BY 4.0 licensing.

Figure 6.

General protocol for performing FISH for CB filament quantification. The steps shown here are (i) slicing core into segments as required and homogenizing each sediment sample. (ii) Preserving sediment in ethanol. (iii) Preparing a pipette tip by slicing the end to minimize clogging when transferring sediment samples. (iv) Diluting sediment in appropriate solutions. (v) Filtering the diluted sample (sediment in water) onto a 0.2 µm polycarbonate filter with a membrane support filter (0.45 µm). (vi) Coat the filter in agar by briefly placing it sample-side-up on an agar drop and then letting it dry sample-side-down on an agar drop overnight; filters can be kept frozen at this stage for preservation. (vii) In the dark, the probe made up with hybridization buffer is added to the sample filter, with other sections kept for positive and negative controls. (viii) Filter-probe samples are incubated for 2 h at 46°C in a sealed tube with the excess buffer used to maintain humidity. (ix) Filter-probe samples are placed in a washing buffer for 15 min at 48°C. (x) Filters are placed onto a slide with a mounting medium. (xi) Microscopy images are then used to calculate the density of cable filaments in the initial sediment sample.

Figure 7.

Schematic of the steps involved in extracting and preparing CB filaments using glass hooks to move CB between drops of water and treatment solutions.

Figure 8.

Examples of the types of images that can be acquired using atomic force microscopy on CB. (A) 3D representation of the surface of a filament, (B) error signal image, and (C) the corresponding height measurement. (D) Topography of a larger part of the filament showing cell junctions with (E) cross-section measurements. (F) Image of a smaller area, showing greater detail, and (G) the ridges on the surface of the cell (Cornelissen et al. 2018). Reproduced with permission from Frontiers Publishing under CC BY 4.0 licensing.

Figure 9.

(A) Diagram of a trench slide setup with CB emerging from sediment, and dark-field microscopy images of the clear part of the slide showing the microaerophilic veil (B) with a CB filament crossing through the boundary (C). (D) shows the oxygen gradient using planar optodes and (E) the corresponding oxygen profile (Scilipoti et al. 2021). Reproduced with permissions from Science Journals under CC BY-NC 4.0 licensing.