Bioenergetic models for acetate and phosphate transport in bacteria important in enhanced biological phosphorus removal

Abstract

Most of our understanding of the physiology of microorganisms is the result of investigations in pure culture. However, in order to understand complex environmental processes, there is a need to investigate mixed microbial communities. This is true for enhanced biological phosphorus removal (EBPR), an environmental process that results in the enrichment of the polyphosphate-accumulating organism Accumulibacter spp. and the glycogen non-polyphosphate accumulating organism Defluviicoccus spp. We investigated acetate and inorganic phosphate (P(i)) uptake in enrichments of Accumulibacter spp. and acetate uptake in enrichments of Defluviicoccus spp. For both enrichments, anaerobic acetate uptake assays in the presence of the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the membrane potential (Delta psi) uncoupler valinomycin, indicated that acetate is likely to be taken up by a permease-mediated process driven by the Delta psi. Further investigation with the sodium ionophore monensin suggested that anaerobic acetate uptake by Defluviicoccus spp. may in part be dependent on a sodium potential. Results of this study also suggest that Accumulibacter spp. generate a proton motive force (pmf or Delta p) for anaerobic acetate uptake by efflux of protons in symport with P(i) through an inorganic phosphate transport (Pit) system. In contrast, we suggest that the anaerobic Delta p in Defluviicoccus spp. is generated by an efflux of protons across the cell membrane by the fumarate respiratory system, or by extrusion of sodium ions via decarboxylation of methylmalonyl-CoA. Aerobic P(i) uptake by the Accumulibacter spp. enrichment was strongly inhibited in the presence of an ATPase inhibitor, suggesting that the phosphate-specific transport (Pst) system is important even under relatively high concentrations of P(i). Acetate permease activity in these microorganisms may play an important role in the competition for acetate in the often acetate-limited EBPR process. Activity of a high-velocity Pst system in Accumulibacter spp. may further explain its ability to compete strongly in EBPR.

Anaerobic glyoxylate cycle activity during simultaneous utilization of glycogen and acetate in uncultured Accumulibacter enriched in enhanced biological phosphorus removal communities

“…The microbial community structure in subsamples taken from the parent bioreactor were similar (although not identical) to those reported by Burow et al (2008), as biomass was sampled from the same Table S4). The range is statistically insignificant (P ¼ 0.12), providing evidence that the community structure was stable across each biomass sample used in acetate uptake assays.…”

“…Protein concentration was determined using the BCA protein assay kit using bovine serum albumin as a standard (Pierce Biotechnology, Rockford, IL, USA). The HEPES-buffered salts medium and acetate uptake assays have been previously described (Burow et al, 2008). Briefly, anaerobic uptake assays were carried out in triplicate in 100 ml batch tests in conical flasks that used oxygen-free nitrogen or helium sparging (15 min) to obtain anaerobic conditions.…”

“…Determination of anaerobic acetate uptake rates and P i release rates in the presence of oxantel was previously reported (Burow et al, 2008). All assays performed with oxantel were carried out at pH 8.…”

“…In one PAO, known as Accumulibacter, acetate uptake is driven by active transport processes utilizing the electrochemical gradient of the proton-motive force (PMF). Generation of the PMF is hypothesized to occur via efflux of phosphate (P i ) (derived from polyphosphate (polyP) breakdown) in symport with protons across the cell membrane (Saunders et al, 2007;Burow et al, 2008). Reducing equivalents for PHA formation are generated by simultaneously catabolizing intracellular glycogen.…”

Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge, a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions

“…Accumulibacter" (11,36) The capacity for storage of acetate as PHA by "Ca. Accumulibacter" is controlled by the intracellular pools of poly(P) and glycogen (2,17,37,38). Glycine uptake by Tetrasphaera is likely also controlled by the amount of intracellular poly(P) available to provide energy for substrate uptake by the action of the low-affinity Pit transporter that is present in all known PAOs, including Tetrasphaera (12,35).…”