BIOCHEMISTRY OF THE ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL SYSTEMS: INVESTIGATION OF THE ENZYME SYSTEMS

The biochemistry of enhanced biological phosphorus removal (EBPR) has been extensively studied by numerous researchers. However, these studies did not make use of some of the biochemistry and chemistry tools, such as enzyme assays and NMR techniques, to explore the metabolism of phosphorus removal under defined conditions. In this study EBPR sludges cultivated in two separate UCT systems operated at 5 and 20°C were used for examination of the anabolic and catabolic reactions taking place during the anaerobic and aerobic stages of phosphorus removal. At both temperatures, glycogen metabolism was shown to be essential for EBPR, and that the reducing equivalents for PHA synthesis were obtained through the EMP pathway. Enzyme assays performed on biomass samples indicated that the branched TCA pathway was operative during anaerobic metabolism at 20°C because an excess of NADH was produced during glycolysis. Low temperatures were shown to slow down glycogen metabolism significantly, thereby providing a competitive advantage to poly-P metabolism. At 5°C, the slower glycogen metabolism did not produce an excess of NADH, the branched TCA pathway was not operative, and the pyruvate generated by glycolysis was sent through the glyoxylate cycle under anaerobic conditions. Aerobic metabolism was also investigated, and the glyoxylate pathway was shown to be dominant because it is a good means of conserving carbon by minimizing the CO2 loss from the system at 20°C. On the other hand, at 5°C, the TCA cycle enzymes were more strongly induced under aerobic conditions. Inhibitor studies were found to be inconclusive because activated sludge has such a complex extracellular matrix, which reduces the efficiency of inhibitor compounds. Interpretation of such studies must be made with caution. To isolate poly-P metabolism from glycogen, techniques other than enzyme inhibition must be developed.