Recent developments in the biochemistry and ecology of enhanced biological phosphorus removal.

Most of the genes encoding the enzymes involved in polyP synthesis and degradation and in phosphate transport have been studied in various Gram-negative bacteria. Progress has also been made in studying the biochemical mechanisms underlying the process of enhanced biological phosphorus removal (EBPR), in particular in lab-scale systems fed with acetate or acetate plus glucose as the sole carbon and energy sources. By applying 13C-NMR, previous models concerning anaerobic carbon metabolism have been advanced and the role of glycogen in providing reducing equivalents in EBPR is definitely demonstrated. The role of the citric acid cycle in supplying reducing equivalents for the conversion of acetyl-CoA into poly-beta-hydroxybutyrate and poly-beta-hydroxyvalerate has been discussed. An incomplete citric acid cycle has been proposed to provide a small part of the reducing equivalents. Polyphosphate:AMP phosphotransferase and polyphosphatase were readily detectable in EBPR sludge fed with acetate plus glucose, but polyphosphate kinase remained undetected. In a lab-scale EBPR system, fed for several months with only acetate as carbon source, a Rhodocyclus-like bacterium (R6) was highly enriched and is therefore probably responsible for EBPR in systems fed with acetate only. This R6-type bacterium was however also present in other EBPR sludges (but to a lesser extent), and may therefore play an important role in EBPR in general. This organism accumulates polyhydroxyalkanoates anaerobically and polyP under aerobic conditions. Unlike members of the genus Rhodocyclus, bacterium R6 cannot grow phototrophically. Therefore a provisional new genus Candidatus and species Accumulibacter phosphatis was proposed.

Purification and properties of pyrophosphatase of Acinetobacter johnsonii 210A and its involvement in the degradation of polyphosphate.

Inorganic pyrophosphatase (E.C. 3.6.1.1) of Acinetobacter johnsonii 210A was purified 200-fold to apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and triphosphate to orthophosphate. No activity was observed with other polyphosphates and a wide variety of organic phosphate esters. The molecular mass of the enzyme was estimated to be 141 kDa by gelfiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a subunit composition of six identical polypeptides with a molecular mass of 23 kDa. The cation Mg2+ was required for activity, the activity with Mn2+, Co2+ and Zn2+ was 48, 48 and 182% of the activity observed with Mg2+, respectively. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The analysis of the kinetic properties of the enzyme revealed an apparent Km for pyrophosphate of 0.26 mM. In A. johnsonii 210A, pyrophosphatase may be involved in the degradation of high-molecular polyphosphates under anaerobic conditions: (i) it catalyses the further hydrolysis of pyrophosphate and triphosphate formed from high-molecular weight polyphosphates by the action of exopolyphosphatase, and (ii) it abolishes the inhibition of polyphosphate: AMP phosphotransferase-mediated degradation by pyrophosphate and triphosphate.

Anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) by a denitrifying bacterium.

The anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) was studied in a denitrifying bacterium. Cells grown with 2-hydroxybenzoate were simultaneously adapted to degrade benzoate. Extract of these cells formed benzoate or benzoyl-CoA when incubated under reducing conditions with salicylate, MgATP, and coenzyme A, suggesting a degradation of 2-hydroxybenzoate via benzoate or benzoyl-CoA. This suggestion was supported by enzyme activity measurements. In extracts of 2-hydroxybenzoate-grown cells, the following enzyme activities were detected: two CoA ligases, one specific for 2-hydroxybenzoate, the other for benzoate, and two different enzyme activities catalyzing the reductive transformation of 2-hydroxybenzoyl-CoA. These findings suggest a degradation of salicylic acid by two new enzymes, 2-hydroxybenzoate-CoA ligase (AMP-forming) and 2-hydroxybenzoyl-CoA reductase (dehydroxylating), catalyzing (1) 2-hydroxybenzoate + MgATP + CoASH --> 2-hydroxybenzoyl-CoA + MgAMP + PPi (2) 2-hydroxybenzoyl-CoA + 2[H] --> benzoyl-CoA + H2O Benzoyl-CoA was dearomatized by reduction of the ring. This represents another case in which benzoyl-CoA is a central intermediate in anaerobic aromatic metabolism.

Biology of polyphosphate-accumulating bacteria involved in enhanced biological phosphorus removal.

Recent research on the process of biological phosphorus removal in lab-scale treatment systems has indicated that: (i) the development of an actively polyP-accumulating bacterial community after the introduction of an anaerobic period may take at least 4 months; (ii) up to 80% of all aerobic bacteria isolated from these communities are able to accumulate polyP; (iii) polyP synthesized by the bacterial communities of lab-scale treatment systems is probably mainly low polymeric, not exceeding 20 P-residues, and this polyP is rapidly degraded during the anaerobic period; (iv) the enzymatic hydrolysis of polyP under anaerobic conditions is accompanied by PHB formation from exogenous acetate, reducing equivalents are provided by the degradation of carbohydrates; and (v) nitric oxide inhibits the release of phosphate under anaerobic conditions in Renpho and F&D sludges. Bacteria belonging to the genus Acinetobacter occur in a wide variety of activated sludges in which enhanced biological phosphate removal is observed. A. johnsonii 210A was studied in detail with respect to the elemental composition of polyP granules, the enzymatic synthesis and degradation of polyP, the regulation of polyP metabolism, and the transport of phosphate. A. johnsonii 210A reflects activated sludge in a number of ways as far as polyP metabolism is concerned but its polyP is highly polymeric and the phosphate efflux rate under anaerobic conditions is relatively low and not increased by exogenous acetate. In addition to Acinetobacter, other polyP-accumulating microorganisms may be involved in biological phosphorus removal. The isolation of polyP-accumulating denitrifying bacteria may well have interesting implications for a new process design in wastewater treatment systems. Further studies on the enzymes involved in polyP biosynthesis and on the uptake and efflux systems of phosphate, potassium, magnesium and lower fatty acids in pure cultures will enlarge our insight in the energetics of the metabolism of polyP. In addition, the regulation of the metabolism of polyP-accumulating organisms needs to be studied in more detail.

Properties of polyphosphatase of Acinetobacter johnsonii 210A.

Polyphosphatase, an enzyme which hydrolyses highly polymeric polyphosphates to Pi, was purified 77-fold from Acinetobacter johnsonii 210A by Q-Sepharose, hydroxylapatite and Mono-Q column chromatography. The native molecular mass estimated by gel filtration and native gel electrophoresis was 55 kDa. SDS-polyacrylamide gel electrophoresis indicated that polyphosphatase of Acinetobacter johnsonii 210A is a monomer. The enzyme was specific for highly polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. The enzyme was inhibited by iodoacetamide and in the presence of 10 mM Mg2+ by pyro- and triphosphate. The apparent Km-value for polyphosphate with an average chain length of 64 residues was 5.9 microM and for tetraphosphate 1.2 mM. Polyphosphate chains were degraded to short chain polymers by a processive mechanism. Polyphosphatase activity was maximal in the presence of Mg2+ and K+.

Properties of polyphosphate: AMP phosphotransferase of Acinetobacter strain 210A.

Polyphosphate:AMP phosphotransferase, an enzyme which catalyzes the phosphorylation of AMP to ADP at the expense of polyphosphate, was purified more than 1,500-fold from Acinetobacter strain 210A by streptomycin sulfate precipitation and by Mono-Q, Phenyl Superose, and Superose column chromatography. Streptomycin sulfate precipitation appeared to be an effective step in the purification procedure. During the following chromatographic steps, there was a 29-fold increase in specific activity but the yield was low (0.3%). Kinetic studies showed apparent Km values of 0.26 mM for AMP and 0.8 microM for polyphosphate with an average chain length of 35 phosphate groups. The highest activities were found with polyphosphate molecules of 18 to 44 phosphate residues. The polyphosphate chain was degraded completely to ADP. The mechanism of degradation is processive. No activity was obtained with ortho-, pyro-, tri-, and tetraphosphate. The enzyme was inhibited by pyro-, tri-, and tetraphosphate. The inhibition by tri- and tetraphosphate was mixed with polyphosphate as a substrate. The inhibition constants for the dissociation of the enzyme-inhibitor complex and for the enzyme-inhibitor-substrate complex were 0.9 and 6.5 mM, respectively, for triphosphate and 0.7 and 1.5 mM, respectively, for tetraphosphate.