Technical note: Development of a gradient tube method for examining microbial population structures in floating sulphur biofilms

Abstract

Floating biofilms occur in thin layers of between 50 μm and 500 μm on the surface of certain organic, sulphidic aquatic environments and, at times, may only be several cells deep. While these structures may be important in terms of energy flow pathways, and possibly also in wastewater treatment operations, little is known about their structural/functional properties. This is due, in part, to their flimsy nature but also to methodological constraints related to their sampling and manipulation. We have investigated floating sulphur biofilms that appear as white layers on the surface of anoxic sulphidic organic wastewaters and describe here the development of a novel gradient tube method for investigating these systems. This approach enables testing of the hypothesis that these floating sulphur biofilms are complex well-differentiated structures rather than disordered dispersions of microbial biomass as has been previously thought. Furthermore, if the former is correct, they would seem to resemble the structure and functionality of comparable complex bioflms that are attached to solid substrates. The gradient tube method involves the establishment of apposing gradients of sulphide and oxygen that are expanded across a tube of agarose 10 cm in length; this simulates the oxic/anoxic interface that occurs over only several micrometres in the natural biofilm system. A plug of sulphide-enriched agarose is first placed in the base of the tube. Samples of the floating sulphur biofilm are then mixed into agarose growth medium and, before it sets, this is overlaid on top of the plug. The tubes are then open capped and incubated. A variety of different microbial populations may thus become established in the separate physiological niches that are set up in this way within the gradient tube. The populations may be quite robustly sampled by extruding and then sectioning the agarose plug. This expansion of the biofilm enables more detailed molecular phylogenetic studies of the populations found in the various niches within the biofilm and also measurement of physico-chemical parameters within the system.

Keywords: gradient tube method, floating biofilms, floating sulphur biofilms, microbial ecology, sulphur biotechnology, acid mine drainage wastewaters