The tetrazolium salts triphenyltetrazolium chloride (TTC) and 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) have been used extensively for the measurement of respiratory activity in natural waters, drinking water, sediments and activated sludge. Usually this is done spectrophotometrically after extracting the reduced formazan salt. Other tetrazolium salts like 5-cyano-2,3-di-4-tolyl-tetrazolium chloride (CTC) are reduced to formazan salts which are fluorescent. This property has been used to microscopically determine the proportion of metabolically active cells in a sample. The observation that CTC is also reduced extracellularly prompted an investigation of the role of this activity in activated sludge. In order to test the assumption that extracellular reduction is stimulated by recalcitrant molecules a bench-scale sequencing batch reactor (SBR) was fed with synthetic wastewater containing either lignin or amylose as well as hexanoic acid, glycerol and sucrose as organic constituents. About 4 times as much tetrazolium salt 3′-{1-[(phenylamino-)carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) was reduced by extracts containing extracellular polymeric substances (EPS) produced in the presence of lignin than by extracts derived from amylose-fed cells. These differences are statistically significant at P > 0.05. Extracellular redox activity in lignin-containing activated sludge accounted for approximately 2% of total redox activity. In addition to experiments involving reactors fed with different substrates, the extracellular redox activity was measured microscopically using a Zeiss confocal laser microscope. Planktonically grown cells of Escherichia coli or activated sludge flocs were treated with CTC and the nucleic acid stain PicoGreen which fluoresces in the red and green visible light regions, respectively. In Escherichia coli preparations, CTC-formazan crystals were found exclusively inside cells. By contrast, activated sludge flocs revealed crystals both inside and outside the cells. Formaldehyde-treated flocs did not show any reduction of CTC. The extracellular crystals accounted for up to 50% of the total CTC reduction. It follows that extracellular redox activity is associated with the production of EPS. Its role may be to help microbial systems to deal with organic materials which are difficult to utilize.

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