Architecture of a nascent Sphingomonas sp. biofilm under varied hydrodynamic conditions

Abstract

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 10(5) microm(2)) or microcolony size (1 x 10(6) microm(2)) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 mum above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-a-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.

FIG. 1.

Simplified schematic of a multichannel flow cell used for experiments on biofilm formation by Sphingomonas sp. strain L138 under different hydrodynamic conditions. For details of the experimental setup, see reference .

FIG. 2.

Analysis of minimum representative area of Sphingomonas sp. strain L138 biofilm to be sampled when cell coverage is used as the quantifying parameter.

FIG. 3.

Analysis of minimum representative area of Sphingomonas sp. strain L138 biofilm to be sampled when mean size of microcolonies is used as the biofilm parameter.

FIG. 4.

Sagittal (x-z) section of a Sphingomonas sp. strain L138 biofilm showing a mushroom-shaped microcolony. Bar, 8 μm.

FIG. 5.

Changes in Sphingomonas sp. strain L138 biofilm thickness as a function of biofilm age at low Reynolds number (Re = 0.07). Error bars indicate standard deviations.

FIG. 6.

Distribution of cell area coverage in the confocal image slices of a Sphingomonas sp. strain L138 biofilm (grown at Re = 0.07) plotted as a function of biofilm depth for different biofilm ages. Symbols: ♦, 1 day; ▴, 2 days; ○, 3 days; ▵, 4 days.

FIG. 7.

Changes in EPS area coverage in Sphingomonas sp. L138 biofilm (grown at Re = 0.07) plotted as a function of biofilm age (in days). Symbols: ▴, 1 day; ▪, 2 days; ○, 3 days; ▵, 4 days.

FIG. 8.

Changes in diffusion length and void (water channel) width as a function of biofilm age. Data are presented based on analysis of images of cells alone (without EPS [▴]) and images of cells and EPS digitally combined (with EPS [○]) to delineate the effect of EPS production. Error bars are standard errors of the means, with n = 160; that is, the values plotted are the means of 160 measurements per image. For details of the image analysis protocol, see the text.

FIG. 9.

Changes in biovolume (calculated per confocal stack) during early stages of biofilm development in Sphingomonas sp. strain L138 (grown at Re = 0.07). Confocal image stacks were collected from the same biofilm location, with a stationary (x-y) microscope stage.

FIG. 10.

Digitally superimposed images of Sphingomonas sp. strain L138 biofilm taken 18 and 19 h after flow cell initiation (grown at Re = 0.07). Microcolonies are outlined in red (18 h) and black (19 h). An increase in the size of the microcolony at the upper right-hand corner is clearly seen, while some other colonies did not exhibit any observable growth during the same period. Arrow indicates flow. Bar, 12 μm.

FIG. 11.

Digitally superimposed images of Sphingomonas sp. strain L138 biofilm taken 23 and 25 h after flow cell initiation (grown at Re = 0.07). Microcolonies are outlined in red (23 h) and black (25 h). The microcolony on the upper right-hand side has migrated in a direction opposite to that of the flow (arrow). Coalescence of two microcolonies in the center can also be seen. Bar, 12 μm.