Selective enrichment and characterization of a phosphorus-removing bacterial consortium from activated sludge.

Under alternating aerobic/anaerobic conditions and without additional carbon sources, a bacterial consortium consisting initially of 18 bacterial strains was obtained in a sequence batch reactor. The phosphorus removal capability could only be maintained using sterile filtrate of activated sludge as medium. The addition of calcium and magnesium salts, as well as vitamins and trace elements, to autoclaved sterile filtrate of activated sludge was not sufficient to achieve stable phosphorus removal. A further enrichment by subcultivation on solid, agar, freezing, and shortening of the aerobic and anaerobic phases led to a defined bacterial consortium consisting of four strains. On the basis of physiological and chemotaxonomic characterization, and partial 16S rRNA sequencing, one of the organisms was identified as Delftia acidovorans. A further isolate belonged to the Bacillus cereus group, and the third isolate was identified as Microbacterium sp.. The remaining strain seems to represent a new genus within the Flavobacteriaceae. Under continuous chemostat conditions, this consortium was able to remove up to 9.6 mg P/l phosphate in the aerobic phase and released up to 8.5 mg/l in the anaerobic phase. Up to 25 mg P-polyphosphate/g dry mass was stored under aerobic conditions.

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids.

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins.

A key feature of the Perutz stereochemical model for cooperativity in hemoglobin is a strong coupling between quaternary structure and the spin state of the heme iron [Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386]. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin in equilibrium low-spin equilibrium. To test this hypothesis, we have measured the changes in spin equilibria upon conversion of six mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20 degrees C in 20 mM maleate buffer, pH 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz stereochemical model [Cho, K. C., & Hopfield, J. J. (1979) Biochemistry 18, 5826-5833]. That is, these data do not support a view that the low affinity of the T state is due to restraints acting through the iron-proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.

Diamagnetism of human apo-, oxy-, and (carbonmonoxy)hemoglobin.

In recent years, a controversy has arisen over the magnetic properties of oxyhemoglobin (HbO2) and (carbonmonoxy)hemoglobin (HbCO). At present, it is unclear which, if any, conditions give a completely diamagnetic state for the heme-ligand complex which can be used as a diamagnetic reference state. In order to establish a diamagnetic reference independent of assignments of electronic configurations, we have measured the magnetic susceptibilities of apohemoglobin solutions and powdered iron-free protoporphyrin IX. We have also reexamined the magnetism of HbO2 and HbCO solutions at 20 degrees C and at several ionic strengths. We find no difference in magnetism between HbO2 and HbCO and no changes in their magnetism with solution conditions. Furthermore, relative to the new (apohemoglobin + porphyrin) diamagnetic reference, our data are consistent with complete diamagnetism for both HbO2 and HbCO under all conditions we have studied. Our data imply that any low-lying triplet state must lie at least 900 cm-1 above the diamagnetic ground states. These results disagree strongly with reports of substantial room temperature paramagnetism for HbO2 and a smaller paramagnetism for HbCO which disappears at high ionic strength [see Cerdonio, M., Morante, S., Vitale, S., Giacometti, G., & Brunori, M. (1982) in Hemoglobin and Oxygen Binding (Ho, C., Ed.) pp 63-68, Elsevier/North-Holland, Amsterdam, and references cited therein].