Use of metabolic inhibitors and gas chromatography/mass spectrometry to study poly-β-hydroxyalkanoates metabolism involving cryptic nutrients in enhanced biological phosphorus removal systems

The role of the tricarboxylic (TCA) cycle and the glyoxylate pathway in anaerobic accumulation of poly-β-hydroxyalkanoates by enhanced biological phosphorus removal (EBPR) activated sludge was investigated. Accumulation of both poly-β-hydroxybutyrate (PHB) and poly-β-hydroxyvalerate (PHV) was observed, after feeding acetate to fresh activated sludge collected from a pilot-scale EBPR facility. Based on the supplied amount of carbon nutrient the level of PHA synthesis exceeded the level of expected synthesis. Feeding of pyruvate combined with acetate resulted in accumulation of PHB and PHV, in approximately a 1:1 ratio. Citrate addition did not result in PHA accumulation but both PHB and PHV were formed when citrate combined with acetate was fed to the sludge. Succinate or succinate combined with acetate stimulated 2-fold higher accumulation of PHV than PHB. In most cases of synthesis, total PHA accumulation exceeded total carbon input. This excess accumulation suggests the presence of nutrients that are normally unusable or cryptic in the absence of additional complementary nutrients such as short chain fatty acids. This hypothesis is consistent with the fragmentation patterns produced during gas chromatography-mass spectrometric (GC/MS) analyses of PHA extracted from sludge fed with 13C-labeled nutrients. Treating the EBPR sludge by adding different nutrients combined with malonate, α-ketoglutarate and monofluoroacetate, three common inhibitors of steps in the TCA cycle significantly impaired PHA accumulation and implied that these steps can be important for PHA synthesis. A biochemical model that incorporates elements of the TCA cycle and the glyoxylate pathway can account for the observed patterns of PHA accumulation.