were harvested by centrifugation (10 000/g for 20
min) in the exponential phase and concentrated in
potassium phosphate buffer (100 mM, pH 7) to a
final cell density of 5 mg cell wet weight per ml.
Bacterial decolourisation was achieved by incubating
the dyes (0.1 ml, 100 mg l1) with bacteria (0.3
ml) and glucose (0.1 ml, 1% w/v) in potassium
phosphate buffer (4.5 ml, 100 mM, pH 7) at
37 8C. The incubation period was 10 min for
non-reactive dyes and 60 min for Reactive Black 5.
The absorption spectra of decolourised samples
were measured over the range 200/700 nm (scan
speed 4 nm s1, 1 cm path length) using a Unicam
SP1800 Ultraviolet Spectrophotometer against a
reference blank of sample matrix. All samples (1
ml) were centrifuged in a microfuge at 11 600/g
for 5 min before analysis. The requirement for
reducing equivalents for decolourisation activity
was demonstrated by the replacement of glucose
from the assay with additional potassium phosphate
buffer. Where necessary bacterial cultures
and reactions were incubated in a Compact
anaerobic cabinet (Don Whitley Scientific Ltd.,
UK). Chemical decolourisation was achieved by
the addition of 6 or 8 mg sodium dithionite to 10
ml hydrolysed and parent Reactive Black (500 mg
l1 in phosphate buffer), respectively.