Microbial reduction of indigo (fermentation vat)

States explicitly that reducing bacteria, grown from spores, produce a reducing enzyme, and it is this enzyme that reduces indigo — not the bacteria themselves.

States that in the chemical vat the chemical itself reduces the indigo, while the fermentation vat requires arranging the nutrients and environment needed by the reducing bacteria and enzyme, which is why fermentation building is said to be difficult.

States that the temperature best suited to activating the reducing enzyme is 45–50°C, which differs from the liquid temperature (30°C) suited to bacterial growth and enzyme production, and that briefly raising the liquid temperature can help reduction proceed when it is low.

States that the alkalinity most favourable for producing the reducing enzyme is pH 11.0, falling off sharply above that; enzyme activity starts at pH 9.6 or higher with an optimum of pH 10.5, dropping sharply below pH 9.6, and adds that this range is slightly narrower than the bacteria's own growth range (pH 10.0–11.5).

States that the reducing bacteria develop if oxygen is present but develop poorly with little oxygen and not at all with none, whereas activating the reducing enzyme and reducing indigo call for limiting contact with oxygen; stirring should only be enough to neutralise the acid produced, since over-stirring oxidises the reduced indigo and prevents dyeing.

Traces bacterial community change with PCR-based methods and shows that the dominant genus changes at different stages of fermentation; states that more than 10 species of indigo-reducing bacteria have been identified, but that the reduction mechanism of the indigo particle is a "riddle."

Introduces the alkaliphile Bacillus pseudofirmus MC02, which can transfer electrons to solid electron acceptors such as Fe(III) oxides, and suggests that reduction of the indigo particle may involve an analogous mechanism of discarding metabolically generated electrons to an extracellular insoluble substance.

Reports that by 16S rRNA gene clone library analysis, the major genus-level constituents on day 3 were Halomonas (54%) and Tissierella (14%), while by day 4 they were Tissierella (35%), Amphibacillus (19%) and Paenibacillus (11%), with Halomonas dropping to 8%; concludes that this decrease in Halomonas together with the rise of Amphibacillus led to the large change in dyeing intensity. Also states that between day 1 and day 2, ORP changed from −440 to −640 mV and pH from 11.3 to 10.2.

States that an even lower redox potential (−600 mV) is considered necessary for indigo reduction in industrial practice, citing Bechtold et al. (1993).

States that bacteria present in the snail's flesh must have served as the reducing agent in antiquity, drawing an analogy to Clostridium isatidis, the bacterium known to reduce plant indigo.

States that the only successful reconstruction relies on bacteria naturally present in the snail's own flesh, which over a few days of moderate warming turned the dye bath yellow (its soluble leuco form) before the fibre was dipped in and left to oxidise purple on exposure to air.