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Leaves from the woad plant (Isatis tinctoria ) were prepared for the woad vat according to the mediaeval practice1 described3. Couched woad1,2 provided the substrate for setting up the woad vat, which has been successfully used to dye wool and cotton. Spontaneous fermentation in the woad vat resulted in the evolution of gases (identified as CO2and H2), acidification of the medium (neutralized by addition of alkali to pH 9), and solubilization of the indigo, causing the vat to turn greenish brown with a surface layer of oxidized blue indigo. The redox potential of the fermenting woad was −560 to−595 mV relative to the standard calomel electrode. The woad vat evolved a noxious odour that was notorious even in mediaeval times1. Analysis by gas chromatography mass spectrometry of the organic headspace volatiles showed they were predominantly dimethyl sulphide (45%), dimethyldisulphide (24%) and methanethiol (18%) (C. Duckham and J. Ames, unpublished data), which are characteristic volatile products of the anaerobic breakdown of cruciferous plant material4 as well as of microbial metabolism5.

We enriched five isolates of indigo-reducing Gram-positive anaerobic bacteria from the fermenting woad vat by using indigo-supplemented, reinforced clostridial agar (pH 9) incubated at 47 °C. Reduction of indigo was indicated by the medium turning colourless under anaerobic conditions, and becoming blue on exposure to air (Fig. 1). All of the isolates were rod-shaped, produced oval terminal endospores, and were negative for the enzyme catalase. Phylogenetic analysis and 16S ribosomal RNA gene sequence analysis6 of a representative strain (Wv6) showed that the unknown bacterium was a member of rRNA cluster I Clostridium, related to C. carnis. Clostridium strain Wv6 is a phylogenetically distinct species for which the name C. isatidis will be formally proposed elsewhere7.

Figure 1: Indigo reduction by Clostridium isolated from a woad vat.
figure 1

The plates were initially brown because, even though they contained indigo, the dye was not divided finely enough to colour the agar blue. Clostridium reduced the indigo anaerobically to its colourless form, then, when exposed to air, the indigo was reoxidized and the agar turned blue.

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We conclude that it was the reduction of indigo by Clostridium that enabled indigo to dissolve and so allowed its use as a dye for textiles in mediaeval times. This finding constitutes another important biotechnological application of anaerobic microbes8.

The mediaeval vat operators were aware of several requirements for the system to work: they had to keep the vat alkaline, adding wood ash1,2 to counter acid produced by fermentation; they provided the Clostridium with polysaccharide substrate, adding bran to maintain the fermentation for several months1,2; and they had to maintain the temperature at around 50 °C (1,2) as the Clostridium is thermophilic. The Clostridium also reduced indigo when inoculated as a pure culture in reinforced clostridial medium (pH 9), its growth being accompanied by the production of acetic, formic and lactic acids.

In current industrial practice, the dyeing of cotton yarn for denim production consumes about 2 kg of sodium dithionite for every 3 kg of indigo reduced9. Disposal of the by-products of dithionite oxidation is an environmental burden, which could be eliminated by replacing modern chemical methods of indigo reduction with one based on the fermentative biochemistry of indigo-reducing Clostridium.