20
Chemical Technology • September 2013
water treatment
T
he examination of the population structure of
biofilms, and particularly attempts to correlate
structure and function in a way that rigorously
represents the actual situation within the biofilm,
remains an exacting methodological task. The require-
ments are particularly acute when studying floating
biofilms that occur as layers of around 50 μm to
Technical note
Development of a gradient
tube method for examining
microbial population structures
in floating sulphur biofilms
by ML Bowker, Environmental Biotechnology Research Institute, Rhodes University, Grahamstown, South Africa,
JB Molwantwa, Digby Wells Associates, Johannesburg, South Africa, J Gilfillan and R Dorrington Department of
Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown, South Africa, R Kirby, Department
of Life Sciences and Institute of Genome Science, National Yang-Ming University, Taiwan and PD Rose, Department
of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown, South Africa
500 μm on water surfaces in some aquatic environ-
ments and are, during the early stages of formation,
possibly only several cells deep (Gilfillan, 2000).
We have examined floating sulphur biofilms that
form at the oxic/anoxic interface on the surface of tan-
nery evaporation ponds (Figure 1A), which receive high
organic and sulphide loads (Gilfillan, 2000; Bowker,
This article, by
ML Bowker, JB
Molwantwa,
J Gilfillan, R
Dorrington,
R Kirby and
PD Rose was
first published
in ‘Water SA’
Vol 39 No. 4
July 201, pp
523-528 and
is republished
here with kind
permission.
Available on
website http://
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 dis-
ordered 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 float-
ing sulphur biofilm are then mixed into agarose growth medium and, before it sets, this is over-
laid 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.
