Vol. 55, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1989, p. 219-2230099-2240/89/010219-05$02.00/0Copyright © 1989, American Society for Microbiology

Polyphosphate-Degrading Enzymes in Acinetobacter spp. andActivated Sludge

JOHAN W. VAN GROENESTIJN,t MICHAEL M. A. BENTVELSEN, MARIA H. DEINEMA,AND ALEXANDER J. B. ZEHNDER*

Department of Microbiology, Wageningen Agricultural University, Hesselink van Suchtelenweg 4,6703 CT Wageningen, The Netherlands

Received 20 June 1988/Accepted 17 October 1988

Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate:AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. Theactivity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a

temperature of 40°C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able todegrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity,adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities ofpolyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotrans-ferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well withthe ability of the sludge to remove phosphate biologically from wastewater.

Acinetobacter spp. are important in biological phosphateremoval from wastewater. Some strains of this strictlyaerobic bacterium are able to accumulate large amounts ofpolyphosphate in the form of granules (9). Activated sludgein wastewater treatment plants is enriched with Acinetobac-ter spp. if alternating aerobic and anaerobic conditions areapplied. Like activated sludge, pure cultures of an Acineto-bacter sp. degrade polyphosphate and release Pi anaerobi-cally, while Pi is taken up aerobically and converted intopolyphosphate. By the combined action of polyphosphate:AMP phosphotransferase, which catalyzes (polyphosphate),,+ AMP -> (polyphosphate)n-1 + ADP, and adenylate ki-nase, which catalyzes 2ADP t± ATP + AMP, ATP isproduced from polyphosphate in Acinetobacter strain 210A(26). This enzyme system allows the Acinetobacter strain touse polyphosphate as an energy reserve in times whenenergy generation becomes difficult or is not possible at all,e.g., in case the electron donor (organic carbon compound)or acceptor (oxygen) or both become limiting or are absent.

Besides polyphosphate:AMP phosphotransferase, otherpolyphosphate-hydrolyzing enzymes have been reported. Ina polyphosphate-accumulating Acinetobacter strain, poly-phosphatase was detected (20). This enzyme was able todegrade synthetic polyphosphate with a chain length of 15phospho-groups. In Escherichia coli polyphosphate kinasewas found (13), whereas Mycobacterium phlei containedpolyphosphate glucokinase (23), and polyphosphate-depen-dent NAD-kinase was detected in Acetobacter, Achromo-bacter, Brevibacterium, Corynebacterium, and Micrococcusspecies (18). Activities of the last three enzymes were absentin cell extracts of Acinetobacter strain 210A (26). Since mostAcinetobacter species do not possess a glycolytic pathway(4), degradation of polyphosphate by means of 1,3-phos-

* Corresponding author.t Present address: Department of Bioconversions and Bacterial

Fermentation, Gist-brocades N.V., Wateringseweg 1, 2600 MADelft, The Netherlands.

phoglycerate:polyphosphate phosphotransferase (16) is notexpected.

In the study presented here, the properties of the poly-phosphate-degrading enzymes polyphosphate:AMP phos-photransferase and polyphosphatase in Acinetobacter strain210A are reported. Six Acinetobacter strains have beenanalyzed for enzymes that are responsible for the productionof ATP from polyphosphate. The biochemical findings ob-tained with pure cultures were compared with findingsobtained in practice, i.e., with activated sludge from dif-ferent wastewater treatment plants in which biological phos-phate removal occurs.

MATERIALS AND METHODS

Organisms. Acinetobacter strains 210A, B8, P, 132, and124 were isolated from activated sludge by the methoddescribed by Deinema et al. (5). Acinetobacter calcoaceticus(NCIB 8250) was a kind gift of C. A. Fewson (3). Theseorganisms were maintained on yeast extract agar slants (5 gof glucose, 2.5 g of yeast extract, and 12 g of agar per liter oftap water, pH 7.0), subcultured every 2 months, and storedat 4°C.

Activated sludges. Samples of activated sludge were takenfrom the aerated zones or stages of the following wastewatertreatment plants: a labscale fill-and-draw reactor (2), pilotplant P1 (6), full-scale plant Renkum train 3 (with an anaer-obic and an aerobic zone) and train 2 (completely aerated)(17), full-scale plant Bunschoten, full-scale plant Bunnik(12), and a conventional pilot plant (completely aerated) (22).Growth conditions. Acinetobacter cells were cultivated in

shaken Erlenmeyer flasks on a butyrate medium (26), strains210A and P at 15°C and strains B8, 132, and 124 and A.calcoaceticus at 25°C, the optimal temperatures for theaccumulation of polyphosphate in these strains. Cells ofAcinetobacter strain 210A without polyphosphate, contain-ing only 13 mg of phosphorus per g of dry biomass, werecultivated in the same way under phosphorus limitation (26).In the experiments in which the polyphosphate:AMP phos-photransferase activity in Acinetobacter strain 210A was

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220 VAN GROENESTIJN ET AL.

determined as a function of growth rate, cells were contin-uously cultivated in a 2-liter bioreactor (Applikon, Schie-dam, The Netherlands) with automatically controlled pH(7.0) and temperature (25°C). The medium used in thisexperiment was modified by omitting Tris and replacing 2.29g of sodium butyrate per liter with 5.67 g of sodiumacetate 3H20 per liter.

Preparation of cell extracts. Cell extracts of Acinetobacterspp. were prepared by sonication and centrifugation asdescribed elsewhere (11). The preparation of cell extractsfrom activated sludge was the same but extended with thefollowing procedure: just before use, the frozen extractswere thawed and centrifuged for 10 min at 7,000 rpm in aHeraeus Biofuge A centrifuge (Federal Republic of Ger-many) to remove precipitated material.

Calculation of Km. Calculation of the K,M, for AMP as asubstrate for polyphosphate:AMP phosphotransferase wascarried out with a computer program with a nonlinearregression fitting procedure by the least squares method.Chemical and biochemical determination. Pi and total phos-

phorus (persulfate digestion method) were determined asdescribed by the American Public Health Association (1).The protein concentration was measured by the method ofLowry (16a) with bovine serum albumin as the standard. Theconcentrations of ATP, ADP, and AMP in the reactionmixtures were determined as described by Pradet (21) andVan Groenestijn et al. (26). By this method, samples wereboiled for 4 min in a Tris hydrochloride-EDTA solution, andATP, ADP, and AMP were determined with an adenylateenergy charge kit, on the basis of bioluminescence, by usingluciferine and luciferase.Enzyme assays. All assays were carried out at 30°C.(i) Polyphosphate:AMP phosphotransferase (assay 1, con-

tinuous method). The first assay was the continuous spectro-photometric method described by Van Groenestijn et al.(26). The reaction mixture in this assay contained 40 mM(NH4)2SO4 as a result of the addition of enzymes which weresuspended in 3.2 M (NH4)2SO4. In the pH experiments, theTris hydrochloride buffer was replaced by equimolaramounts of a MES (2-morpholinoethanesulfonic acid)-NaOH buffer at pHs lower than 7.0. In the experiment inwhich the activity in different Acinetobacter strains wasmeasured, the reaction mixture was supplied with 600 pg ofGraham salt per ml.

(ii) Polyphosphate:AMP phosphotransferase (assay 2, dis-continuous method). For assay 2, 0.18 ml of cell extract wasincubated in 1 ml of reaction mixture containing 3 mMMgCl2, 16 mM Tris hydrochloride (pH 7.0), and 0.3 mMAp5A [Pl,P5-di(adenosine-5')-pentaphosphate]. The reactionwas started by adding AMP, resulting in a concentration of 1mM. At intervals, samples of 25 ,ul were taken from thereaction mixture to determine ADP concentrations.

(iii) Polyphosphatase (EC 2.7.4.3). Cell extract (0.99 ml)was mixed with 0.01 ml of 50 mM MgCl2, which resulted ina final concentration of 0.5 mM MgCl, (pH 7.0). At intervals,samples of 100 .I1 were taken and immediately analyzed forPi.

(iv) Adenylate kinase. A volume of 0.16 ml of cell extractper ml of reaction mixture was added in a cuvette to 7 mMMgCl,2 90 mM Tris hydrochloride (pH 7.0), 200 mM D-glucose, 0.6 mM NADP, and 3.4 U of HK and 1.7 U ofglucose 6-phosphate dehydrogenase (G6P-DH) per ml. Thereaction was started by adding ADP, resulting in a concen-tration of 1 mM. The cuvette was placed in a spectropho-tometer (Beckman Instruments, Inc., Fullerton, Calif.), andthe production of NADPH, was measured at 340 nm. A

reaction mixture with additional 0.3 mM Ap5A, a specificinhibitor of adenylate kinase (8), was used as a control.

(v) Polyphosphate kinase (EC 2.7.4.1). A total of 0.18 ml ofcell extract and 600 p.g of Graham salt per ml of reactionmixture were added in a cuvette to 8 mM MgCl,, 100 mMTris hydrochloride (pH 7.0), 200 mM D-glucose, 0.65 mMNADP, 0.9 mM Ap5A, and 3.4 U of HK and 1.7 U ofG6P-DH per ml. The reaction was started by adding ADP,resulting in a final concentration 1 mM. The producedNADPH, was measured spectrophotometrically.

Chemicals. HK, G6P-DH, adenylate kinase. NADP, ATP,ADP, and AMP were purchased from Boehringer MannheimBiochemicals (Indianapolis, Ind.). Ap5A was obtained fromSigma Chemical Co. (St. Louis, Mo.), and the adenylateenergy charge kit was from LUMAC/3M, Schaesberg, TheNetherlands. MES was purchased from E. Merck AG,Darmstadt, Federal Republic of Germany. Synthetic poly-phosphate in the form of Graham salt, which represents amixture of linear sodium polyphosphates [(NaPO3),,; na102], was prepared by heating NaH2PO4 for 3 h at 750°C andthen cooling it in the air outside the furnace. Potassiumpolyphosphate (Kurrol salt; na 2 104) was prepared byheating KH,P04 for 2 h at 260°C (18). Sodium polyphosphatewas derived from this salt by cation exchange with DOWEX-50. When equivalent amounts of MgCl2 were added to thedissolved sodium polyphosphate, the insoluble magnesiumpolyphosphate precipitated.

RESULTS

Polyphosphate:AMP phosphotransferase activity as a func-tion of the growth rate of Acinetobacter strain 210A. Thepolyphosphate:AMP phosphotransferase activity and thephosphorus content of cells of Acinetobaccter strain 210Adepended on the growth rate. The enzyme activity decreasedfrom 36 to 25 nmol/min per mg of protein with increasinggrowth rates, measured at dilution rates between 0.05 and0.42/h, while the cellular phosphorus content was at anoptimum at a growth rate of 0.11/h.

Properties of polyphosphate:AMP phosphotransferase inAcinetobacter strain 210A. The effects of pH and temperatureon the activity of polyphosphate:AMP phosphotransferasein cell extracts of Acinetobacter strain 210A are presented inFig. 1. The optimal pH was 8.5, and the optimal temperaturewas 40°C. Two methods exist for the assay of this enzyme:(i) a spectrophotometric method, in which the reactionproduct is continuously converted into ATP, and (ii) adiscontinuous method, in which samples are taken at inter-vals from the reaction mixture (see Materials and Methods).The continuous method resulted in activities 30 times higherthan those obtained by the discontinuous method. One of thedifferences between the two assays is the presence of(NH4)2SO4 in the spectrophotometric method. The additionof 40 mM (NH4)2SO4 to the reaction mixture of assay 2stimulated the activity of polyphosphate:AMP phospho-transferase about threefold. The Pi concentration, tested inthe range of 0.1 to 10 nM, did not affect the activity. Theactivity was dependent on the AMP concentration, and thisdependency obeyed the law of Michaelis and Menten. A K,,,of 0.58 + 0.13 mM and a V,n,,x of 60 + 4 nmol/min per mg ofprotein were obtained after assay 1 at pH 7.0 and 30°C.

Properties of polyphosphatase in Acinetobacter strain 210A.Polyphosphatase in cell extracts of Acinetobacter strain210A was stimulated by the presence of NH4C1 or KCI. Thestimulation by NH4C1 was optimal at 300 to 400 mM. Theactivity increased from 1 to 24 nmol/min per mg of protein as

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POLYPHOSPHATE-DEGRADING ENZYMES 221

-15~~ ~ ~ ~ ~ ~ ~

>

6 7 8 9 20 30 40 50

E opH Ternperature(0C)

FIG. 1. Effect of pH (at 30°C) and temperature (at pH 7) on

polyphosphate:AMP phosphotransferase activity in cell extracts of

Acinetobacter strain 210A. At pHs lower than 7, the Tris hydro-

chloride buffer in the reaction mixture was replaced by a MES-

NaOH buffer.

a result of the addition of 300 to 400 mM NH4CI. A furtherincrease of the NH4Cl concentration, however, caused a

decrease of the activity. Only 1 nmol/min per mg of proteincould be measured at 1,000 mM NH4Cl. Stimulation upon

addition of KCI was less pronounced. An optimal activity of5 nmol/min per mg of protein was found at 300 mM KCI. Noactivity was found after heating cell extracts for 5 min at80°C. This is a strong indication that polyphosphate hydro-lysis is catalyzed by an enzyme. The substrate for thisreaction was native polyphosphate which was alreadypresent in the cell extracts. In an extract with 566 ,ug ofphosphorus per ml, of which 38 ,ug/ml consisted of Piphosphorus, the Pi phosphorus content per ml increased to287 ,ug after 9 h of incubation in the polyphosphatase assay.

In addition, the degradation of synthetic polyphosphates incell extracts without native polyphosphate was studied. In a

cell extract of cells cultivated under phosphorus limitationonly 83 ,g of phosphorus per ml was present, of which 21,g/ml was in the form of Pi phosphorus. The enzyme activitywas measured in a reaction mixture with 1 mg of polyphos-phate-phosphorus per ml but no NH4Cl. Magnesium poly-phosphate (prepared from Kurrol salt) was the only polymerhydrolyzed in this assay (3.2% within 7 h at a rate of 0.7nmol/per mg of protein). Hardly any Pi was produced in theabsence of polyphosphate or in the presence of sodiumpolyphosphate or potassium polyphosphate. In a controlexperiment, magnesium polyphosphate was incubated in a

reaction mixture without cell extract. Less than 0.1% was

degraded within 7 h.Enzymes involved in polyphosphate degradation in different

Acinetobacter strains. The amount of phosphorus and theactivities of polyphosphate:AMP phosphotransferase, ade-nylate kinase, and polyphosphate kinase in different Acine-tobacter strains are shown in Table 1. The activity ofpolyphosphate:AMP phosphotransferase of the strains cor-

related positively with phosphorus accumulation, and theadenylate kinase activity correlated best with polyphos-phate:AMP phosphotransferase activity. Significant activi-ties of polyphosphate kinase were detected only in strain P,which did not show any polyphosphate:AMP phosphotrans-ferase activity.

TABLE 1. Phosphorus accumulation and activities ofpolyphosphate:AMP phosphotransferase, adenylate kinase, and

polyphosphate kinase in six Acinetobacter strains

Enzyme activity (nmol/min per mgPhosphorus of protein) of:

Strain in cells(% of dry Polyphosphate:biomass) AMP phospho Adkenylate Polyphosphate

transferase

210A 10 43 89 0B8 6 10 80 1P 4 0 54 3NCIB 8250 2.5 2 60 0124 2.7 1 46 0132 1.6 0 39 0

Adenylate kinase in activated sludge. The activities ofadenylate kinase in cell extracts of activated sludge ofdifferent wastewater treatment plants are presented in Table2. The adenylate kinase activity in the sludge correlated wellwith the percentage of phosphorus removed from the waste-water.

DISCUSSION

Polyphosphate:AMP phosphotransferase activities werenot proportional to the cellular polyphosphate content atvarious growth rates of Acinetobacter strain 210A. Theinduction of higher enzyme activities at low growth rates isa well-known property of many catabolic enzymes, espe-cially those involved in early metabolism of substrates (10).Polyphosphate:AMP phosphotransferase can be regarded assuch an enzyme, because it catalyzes the first reaction in thecatabolic pathway of the degradation of polyphosphate andthe subsequent production of ATP.The properties of polyphosphate:AMP phosphotransfer-

ase in Acinetobacter strain 210A differed from those foundfor this enzyme in Corynebacterium xerosis. The optimal pHfor the enzyme in Acinetobacter strain 210A was 8.5, whilethe optimal pH found by Dirheimer and Ebel (7) for poly-phosphate:AMP phosphotransferase in C. xerosis was 6.5.The physiological function of the enzyme in C. xerosis is notyet fully understood, because of its high Km for AMP,namely 20 mM (14). The affinity of polyphosphate:AMPphosphotransferase in Acinetobacter strain 210A for AMP,however, is very high. The physiological significance of anenzyme with a Km of 0.6 mM can hardly be doubted. It hasalready been demonstrated that this enzyme plays a role in

TABLE 2. Removal of phosphorus from wastewater by differenttypes of activated sludge and their adenylate kinase activities

% of phosphorus AdenylateWastewater treatment plant removed from kinase activity

wastewater (nmol/min permg of protein)

Fill-and-draw system 100 680Pilot plant P1 (Sept. 1987) 100 146Pilot plant P1 (May 1987) 87 132Full-scale plant Bunschoten 81 87Renkum train 3 55 78Full-scale plant Bunnik 90 56Conventional pilot plant (Sept. 1987) ND" 64Renkum train 2 16 40Conventional pilot plant (May 1987) ND 13

"ND, Not determined.

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the use of polyphosphate as an energy reserve in Acineto-bacter strain 210A (26). The polyphosphate:AMP phospho-transferase activity in cell extracts was stimulated by(NH4)2SO4 just as in C. xerosis. This (NH4)2SO4 effect partlyexplains the different activities found with assays 1 and 2.

Polyphosphatase has been detected in cell extracts ofAcinetobacter strains 210A in significant amounts. Nativebacterial polyphosphate as well as highly polymeric syn-

thetic magnesium polyphosphate could be hydrolyzed bypolyphosphatase. Sodium polyphosphate and potassiumpolyphosphate could not act as substrates, despite the smallamounts of Mg2+ in the reaction mixture. Bacterial poly-phosphate in Acinetobacter strain 210A also contains highamounts of Mg2+ (27). Magnesium polyphosphate may bethe actual substrate that is accepted by the enzyme, as

magnesium PP1 is accepted by mouse duodenal pyrophos-phatase (19). The need for Mg2+ of polyphosphatase hasbeen found in other organisms (11, 15). The stimulation ofpolyphosphatase by NH4Cl is a specific NH4' effect and nota general salinity effect, because equal concentrations ofKCI caused weaker stimulation. NH4' may change theenzyme or the substrate (e.g., cation composition of poly-phosphate). An effect of NH4' on polyphosphatase hasnever been reported in biology, but the activating effect ofK+ on this enzyme from Aerobacter aerogenes has beenobserved (11). Kulaev and Konoshenko (15) also observedstimulation of polyphosphatase from Neurospora crassa

with a factor of 3 by 200 mM KCl. Polyphosphatase may

have a role in the degradation of polyphosphate in cells ofAcinetobacter strain 210A growing under phosphorus limi-tation. Under these conditions, polyphosphate can be usedas a phosphorus reserve (24). Since the cells do not containany detectable amounts ofAMP under phosphorus limitation(data not shown), degradation of polyphosphate by poly-phosphate:AMP phosphotransferase is unlikely.

Increased levels of adenylate kinase in different Acineto-bacter strains correlated with the polyphosphate:AMP phos-photransferase activity and phosphorus accumulation bythese strains. This correlation reflects the role of adenylatekinase in the degradation of polyphosphate in combinedaction with polyphosphate:AMP phosphotransferase. Bothenzymes play a role in the production of ATP from poly-phosphate in acinetobacters in vitro and in vivo and there-fore probably in biological phosphate removal by activatedsludge as well (26). This role was indicated by the positivecorrelation between the activity of adenylate kinase and theability of the sludge to remove phosphate biologically fromwastewater. Very high adenylate kinase activities were

found in the intermittently fed and aerated labscale fill-and-draw system, which contained a sludge (with more than 60%acinetobacters) that was able to release accumulated phos-phate very quickly under anaerobic conditions (2). Thehigher phosphate release rate in this sludge (71 mg ofphosphorus per h per g of dry biomass) compared with therates measured in pure cultures of Acinetobacter strain 210A(8 mg of phosphorus per h per g of biomass) (Van Groe-nestijn et al., submitted for publication) might be caused bythe higher adenylate kinase activity in the sludge. Furtherresearch is needed to explain the higher activities. Becauseof its high activities and its good correlation with biologicalphosphate removal, adenylate kinase might be used as an

indicator for monitoring this process. Earlier studies (25)demonstrated the correlation between the activity of poly-phosphate:AMP phosphotransferase in sludge and biologicalphosphate removal. Cell extracts of sludges that were able toremove phosphorus biologically produced more ADP from

AMP and Graham salt within 2 h than conventional sludgedid. The presence of Graham salt in the cell extracts of thesludge was a prerequisite for this reaction; without thissynthetic polyphosphate no ADP was produced.

In conclusion, it can be said that among the three enzymesthat can degrade polyphosphate in Acinetobacter strains(polyphosphate:AMP phosphotransferase, polyphospha-tase, and polyphosphate kinase), polyphosphate:AMP phos-photransferase plays, in combined action with adenylatekinase, the most important role in anaerobic ATP productionin Acinetobacter strains that accumulate aerobically largeamounts of polyphosphate. A link between the findingsobtained with pure cultures and those obtained in practicehas been made by showing the correlation between theactivities of polyphosphate:AMP phosphotransferase andadenylate kinase in activated sludge and the ability of thesludge to remove phosphate biologically from wastewater.The suitability of the adenylate kinase activity as an indica-tor for this process has been suggested. This study shows thevalidity of pure culture studies for the application.

ACKNOWLEDGMENTS

We thank A. J. van Milaan, D. E. M. Sijpkens, and H. M.Sleiderink for their practical assistance and the Department ofBiochemistry of Wageningen Agricultural University for use of theirequipment.

This research was supported by a special grant from WageningenAgricultural University.

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Uptake and release of phosphate by a pure culture of Acineto-bacter calcoacetic us. Water Res. 19:1587-1594.

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24. Van Groenestijn, J. W., and M. H. Deinema. 1985. Effects ofcultural conditions on phosphate accumulation and release byAcinetobacter strain 210A, p. 405-410. In J. N. Lester andP. W. W. Kirk (ed.), Proc. Int. Conf. Management StrategiesPhosphorus Environ.. 1985. Selper, London.

25. Van Groenestijn, J. W., and M. H. Deinema. 1987. The utiliza-tion of polyphosphate as an energy reserve in Acinetobacter sp.and activated sludge, p. 1-6. In R. Ramadori (ed.), Biologicalphosphate removal from wastewaters. Adv. Water Poll. Con-trol. Pergamon Press, Inc. Elsmford. N.Y.

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