The Porous Surface Model, a Novel Experimental System for Online Quantitative Observation of Microbial Processes under Unsaturated Conditions

Abstract

Water is arguably the most important constituent of microbial microhabitats due to its control of physical and physiological processes critical to microbial activity. In natural environments, bacteria often live on unsaturated surfaces, in thin (micrometric) liquid films. Nevertheless, no experimental systems are available that allow real-time observation of bacterial processes in liquid films of controlled thickness. We propose a novel, inexpensive, easily operated experimental platform, termed the porous surface model (PSM) that enables quantitative real-time microscopic observations of bacterial growth and activity under controlled unsaturated conditions. Bacteria are inoculated on a porous ceramic plate, wetted by a liquid medium. The thickness of the liquid film at the surface of the plate is set by imposing suction, corresponding to soil matric potential, to the liquid medium. The utility of the PSM was demonstrated using Pseudomonas putida KT2440 tagged with gfp as a model bacterium. Single cells were inoculated at the surface of the PSM, and the rate at which colonies expanded laterally was measured for three matric potentials (؊0.5, ؊1.2, and ؊3.6 kPa). The matric potential exerted significant influence on colony expansion rates, with a faster rate of spreading at ؊0.5 than at ؊1.2 or ؊3.6 kPa (diameter increase rate, ca. 1,000, 200, and 17 m h ؊1 , respectively). These differences can be attributed to cell motility, strongly limited under the most negative matric potential. The PSM constitutes a tool uniquely adapted to study the influence of liquid film geometry on microbial processes. It should therefore contribute to uncovering mechanisms of microbial adaptation to unsaturated environments.

Hydration-controlled bacterial motility and dispersal on surfaces

“…The mild matric potential that we prescribed does not per se restrict bacterial motion (12), but it acts through its control of the effective liquid-film thickness on the ceramic-plate surface. The relationship between matric potential and liquid-film thickness depends solely on surface wettability and roughness (15).…”

“…The PSM allows growing and observing fluorescent cells at the surface of a porous ceramic plate (diameter = 4.2 cm; maximum pore size = 1.7 μm) under prescribed suction, which controls surface liquidfilm thickness (12). Liquid FAB medium with 5 mM benzoate as the sole carbon source was used to wet the plate and sustain microbial growth.…”

“…Individual trajectories were detected and analyzed with Image Pro Plus (MediaCybernetics). Population-scale dispersal was quantified as previously described (12).…”

“…Here, to avoid the complexity inherent to natural partially saturated microbial habitats such as soil matrixes and benefit from direct observation of bacterial dispersal at both individual and population scales under conditions of controlled hydration, we used the porous-surface model (PSM) (12). In this experimental system, bacteria are grown on a porous ceramic surface in thin aqueous films, whose effective thickness is controlled by applying a prescribed suction similar to how matric potential controls hydration in soils.…”

Microbial dispersal in unsaturated porous media: Characteristics of motile bacterial cell motions in unsaturated angular pore networks

“…Self-propulsion by rotating flagella is the common form of motion among many motile prokaryotes in aqueous environments [Grossart et al, 2001]. Other forms of motility such as swarming, gliding, twitching, and sliding are limited to a few species [Harshey, 2003;Dechesne et al, 2008]. Microbial life in natural soils under most climatic conditions is characterized by motion in small pores and in thin liquid films governed by low Reynolds numbers that also limit the role of passive (advective) transport to a few rare events where the soil becomes very wet [Purcell, 1977;Bitton and Harvey, 1992;Fenchel, 2002;Berg, 2005;Or et al, 2007].…”