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.
Settling problems caused by pin-point sludge constitute a serious problem in biological wastewater treatment, particularly in many industrial plants. Until now, most studies focused on the relationship between pin-point sludge formation and either shearing forces or the impact of toxicants. This study deals with the community structure in both the micro- and macrofloc fraction which was analyzed by fluorescent in situ hybridization (FISH) and BIOLOG substrate utilization patterns. It was shown that each fraction consisted of different microbial communities with unique metabolic profiles suggesting that pin-point sludge formation is not due to dispersal of intact flocs but to microcolonies growing separately. Alternatively, macroflocs may have an architecture leading to segregation of microbial communities after floc dispersal. Further it could be shown that the formation of microflocs was influenced by sludge age. The best sludge sedimentation was obtained for a sludge age of 5 and 10 days. Additional analysis of extracellular polymeric substances (EPS) suggested that the lower protein to carbohydrate ratio of 10-day-old sludge led to better flocculation compared to 20-day-old sludge containing similar total amounts of EPS. From a practical point of view, addition of potassium (0.1 g/l) effected a noticeable improvement of sludge settleability.
The influence of temperature on activated sludge in a bench-scale SBR fed with a synthetic medium simulating paper mill effluents was determined using biological methods based on the measurement of enzymatic activities. Redox activity inside cells and in the extracellular polymeric substances as well as the proportion of active cells in the sludge were measured by the reduction of the tetrazolium salts XTT and CTC, respectively. A significant shift in the size distribution of lignin at 30°C correlated with a maximum of extracellular redox activity. The percentage of active cells in the biocoenosis was independent of temperature. α-glucosidase and L-alanine-aminopeptidase activities were steady at 20, 30 and 40°C. After shifting the temperature to 50°C there was an enormous short-time increase of both enzyme activities because of the change from mesophilic to thermophilic conditions. Of the biological parameters tested, only extracellular redox activity correlated with degradative activity as detected by a change in molecular size distribution of lignin molecules.
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