Accumulation and Degradation of Polyphosphate in Ac1netobacter Sp. 0000 0284 4245

Home , Aerobic organism, Polyphosphate kinase

ACCUMULATION AND DEGRADATION OF POLYPHOSPHATE IN AC1NETOBACTER SP.

CENTRALE LANDBOUWCATALOGUS

0000 0284 4245 Promotor: dr A.J.B. Zehnder, hoogleraar in de microbiologic

Co-promotor: dr M.H. Deinema, universitair docent ^uo$to\, il'ZT-

J.W. van Groenestijn

ACCUMULATION AND DEGRADATION OF

POLYPHOSPHATE IN ACINETOBACTER SP.

Proefschrift ter verkrijging van de graad van doctor in de landbouwwetenschappen, op gezag van de rector magnificus, dr C.C. Oosterlee, in net openbaar te verdedigen op vrijdag 17 juni 1988 des namiddags te vier uur in de aula van de Landbouwuniversiteit te Wageningen

BIBLIOTHEEK. LANDBOUWUNIVERSITEIT .WAGENINGEN JO/OO^O'I^^

STELLINGEN

1. Debiochemisch emodelle nva nhe tmechanism eva nd ebio - logischedefosfaterin g vanafvalwater ,zoal sgepresenteer d ophe tinternational econgre sove rdi tproces ,gehoude nva n 28t/ m 30Septembe r 1987t eRome ,kunne nnie tverklare n waaromhe tPhostri pproce sz ogoe dwerkt . -R .Ramador i(ed. )Biologica lphosphat eremova lfro mwast e waters. Proceedingso fa nIAWPR Cspecialize dconference , Rome,28-3 0Sept .1987 .Adv .Wat. Poll .Control .Pergamo n Press.

2. Integenstellin gto td ebewerin gva nKulae vhoef td epro - ductieva nAT Pui textracellulai radenosin ee nintracel lular:polyfosfaa tdoo rafstervend ecelle nva nCorynebac - teriumsp .nie tt eduide no pee npolyfosfaat:AM Pfosfo - transfersaseactiviteit . -V.D . Butukhanov, M.A. Bobyk, V.Zh. Tsyrenov and I.S. Kulaev (1979)Possibl e role of high-molecular-weight polyphosphate inAT Psynthesi sfro mexogenou sadenin eb y acultur eo fCorynebacteriu mspecies ,strai nVSTI-301 . Biokhimiya44:1321-1327 . - I.S.Kulae van dV.M .Vagabo v(1983 )Polyphosphat emeta bolism inmicroorganisms .Adv .Microbiol .Physiol .24:83 - 171.

3. Deconclusi eva nNeijsse le nTempes tda tglucos egelimi - teerdegroe iva nKlebsiell a aerogenesNCT C 418koolsto f gelimiteerdmoe tzij nspreek td ehypothes eva nLinto ne n Stephensontegen . -J.D . Linton and R.J. Stephenson (1978)A preliminary studyo ngrowt h yields in relation to the carbon and energyconten to fvariou sorgani cgrowt hsubstrates .FEM S microbiol.Lett .3:95-98 . -O.M .Neijsse lan dD.W .Tempes t (1976)Th erol eo fenergy - spillingreaction si nth egrowt ho fKlebsiell aaerogene s NCTC41 8i naerobi cchemosta tculture .Arch .Microbiol . 110:305-311.

4. Hethomogenisere nva naktie fsli bmet ee nvortex ,da ti n sommigepublicatie sword tbeschreve nal senig evoorbehan - deling ten behoeve van celtellingen op platen,ka n tot foutieveconclusie s leiden omtrentd e aantallen cellen in ditslib . 5. Zolanggrot ehoeveelhede nfosfaa taanwezi gblijve ni nd e effluentenva nafvalwate rzuiveringsinstallaties , isd e verwijdering vand e laatsteppm' sNH 4 enorganisch ekool - stofverbindingenui tafvalwate rvaa kee nzinloz ebezigheid . Deintroducti eva nfosfaa ti nd eheffingsformul evoo rd e lozingva neffluen ti she tgewenst epolitiek einstrumen t datdez evaa ktoegepast ehandelswijz eka nvoorkomen .

6. Doorhe t introduceren van fosfaatvrije wasmiddelen zal biologischedefosfaterin gva nafvalwate rsteed sgunstige r wordent.o.v .chemisch edefosfatering .

7. Dehoudin gva nregeringe ninzak ed eemissi eva nafgasse ni s eentypisch e'na-mij-de-zonvloed-politiek' .

8. Detijdelijk evolksverontwaardigin g tijdensd eintroducti e vand eeerst etreine ni nEuropa ,word tvaa kte nonrecht e aangehaaldal se rwee ree ncontroversiel etechnologisch e innovatiegeintroduceer dmoe tworden .

9. Natuurtalenten zijnvaa kslecht edocente nomdat zi jnie t inzienwaaro mee nleerlin gd esto fnie tbegrijpt .

10. Vooree nwetenschappe rme tee nachternaa mva nvie rletter - grepenwaari n 'oe'e n 'ij' voorkomen zal internationale bekendheidnie tsne lzij nweggelegd .

Stellingen behorende bij het proefschrift "Accumulation and degradationo fpolyphosphat ei nAcinetobacte rsp. "va nJ.W .va n Groenestijn.

Wageningen,1 7jun i198 8 VOORWOORD

Voordatu i ndi tproefschrif tgaa tleze nmoe tu beseffe nda t de verschillende activiteiten die hebben geleid tot de tot- standkomingva ndi tboe knie the twer ki sgewees tva nee nenke - ling,d eauteur ,maa ri ngrot emat eondersteun dzij ngewees tdoo r anderen.Dez emense nwi li khie rbedanken : Prof.Zehnde re nMi aDeinem avoo rhe tmogelij kmake nva ndi t onderzoek,voo rnu nwetenschappelijk ebegeleiding ,he tkritisc h doorlezenva nhe tmanuscrip te nhu nstimulerend einvloed . De studentenJacque sva nd eWorp ,Gerar dVlekke ,Martin aZuide - ma, Peter Okkerman, Michael Bentvelsen, DesireaAnink , Eric Boschker,Pir nVis ,The oDemmers ,Harri ePeters ,Roelo fRongen , Antoinetteva nMilaan ,Dorin eSijpken se nHedwi gSleiderink ,di e eendirect eo f indirectebijdrag ehebbe ngelever daa nd e inhoud vandi tproefschrift , enwaarme ei kmet vee lplezie rsame nhe b gewerkt. AnsBroersma-D eHaa nvoo rhe ttypwerk . NeesSlotboo mvoo rd etekeningen . FritsLa pvoo rd eelectro-technisch eondersteuning . WimRoelofse nvoo rd eanalytisch eassistentie . Chris Schouten voor de assistentie in de bacteriologische keuken. Hetpersonee lva nd eTFD Lvoo rd eEM-opnamen . Dr.A.J.W.G .Visse r enJ.S .Santem ava nd evakgroe p Biochemie voorhe tmogelij kmake nva nd ebioluminescenti emetingen . Jo Jacobs en Johannes van der Laan van de vakgroep Water- zuiveringvoo rhu nassistenti eme td eAAS-metingen . Envel eander emedewerker sva nd evakgroepe nMicrobiologi ee n Waterzuiveringdi eme traa de ndaa dhebbe nbijgedrage naa nd e totstandkoming van dit proefschrift. Hun gezelschap en be- langstellingvormde nee nhartverwarmend estimulan se nee nbe - langrijkesteunpilaa ronde rhe tsoirt sz ozwar epromotie-werk . Ook mijnouder s komtall edan ktoe :zi jhebbe nmi jgemotiveer d engestimuleer do mt egaa nstuderen . CONTENTS

page

Chapter1 : Introduction 1

Chapter2 : Regulationo fpolyphosphat eaccumulatio ni n Acinetobactersp . 27

Chapter3 : Therol eo fcation si nth eaccumulatio nan d releaseo fphosphat ei nAcinetobacte r strain210 A 45

Chapter4 : ATPproductio nfro mpolyphosphat ei nAcineto bacterstrai n210 A 63

Chapter5 : Polyphosphatea sa nenerg yreserv ei nviv o inAcinetobacte rstrai n21O A 77

Chapter6 : Polyphosphatedegradin genzyme si nAcineto bactersp .an dactivate dsludg e 93

Chapter7 : Summary+ Samenvattin g 107 CHAPTER 1

INTRODUCTION

PREAMBLE

A study on polyphosphate accumulation and degradation by Acinetobacter sp.i s important forth e following tworeasons : (1)Polyphosphat e accumulating strains of Acinetobacter play an important role in the biological phosphate removal from waste water, a process that is performed in a number of wastewater treatment plants and results in activated sludge enriched with polyphosphate accumulating bacteria. (2)Acinetobacte r sp. are able to accumulate exceptionally high amounts of phosphorus (up to 100 mg per g dry biomass), mainlyi nth efor m of polyphosphate, and this property makes this bacterium very suitable for research on the reserve functions of polyphosphate. The role of inorganic polyphosphate in biology has not yet been elucidatedver ywell ,especiall y itsrol ea sa n energy reserve invivo ,whic h stillremain s tob ea controversia l subject.I n this chapter the available information about both the appli cation of polyphosphate accumulating bacteria in wastewater treatment and the physiological role of polyphosphate in micro organisms are summarized.

THE ROLE OF PHOSPHATE IN EUTROPHICATION OF SURFACE WATERS

Inth e early ninetheenseventies the awareness for water pol lution grew exponentially and wastewater treatment plants were introduced on a large scale in the industrialized countries. Thesetreatmen t plants were designed toreduc eth e biological oxygen consuming potential of the waste waters, i.e. organic carbon and reduced nitrogen. Theeffluent s of these plants still may contain largeamount s ofphosphat e and nitrate,whic h are both the major compounds responsible for the eutrophication of surfacewaters .Eutrophication ,a noverloa d of surface waters with nutrients, has become a growing problem, and many negative effects have been reported. Examples are:alga l blooms, decrease of ecological quality and organism diversity in surface waters, and livestock deaths duet o ingestion of toxic algae. The treat ment of eutrophied water inconventiona lpotabl ewate r treatment plants also presents problems. They include penetration of fil- tersb y algae,filte rclogging ,taste ,odour ,colour ,turbidit y andth eformatio no ftrihalomethane s from organic compounds releasedb yalga ea sa resul to fchlorination .Deat ho falga e canlea dt oanaerobi ccondition si nth ewate rcausin gfis hkil l andodou rnuisance .Excretio no fprotein sb yalga ei nth ese a cancaus e foamwhic hma y interferwit h fishery.Th e flow of irrigationwate ri ncanal sca nb einterferre db yth eabundan t developmento ffilamentou salga e(Wiecher san dBes t 1985). Ina majorityo fsurfac ewaters ,phosphoru si sth elimitin gnutrient . Itsadditio n tothi swater s will cause eutrophication (Vollen- weider 1985).Accordin gt oVa nDiere ne tal .(1985 )th eeutro phication policy in The Netherlands should consist of four tracks:(1 )phosphat eba nfro mdetergents ,(2 )wate rtreatment (phosphateremova lfro mwast ewater ,dredgin go fsediment scon taining phosphate), (3)restrictio n ofphosphat eemissio nb y agriculture (e.g.runoff )an d (4)reductio no f foreigninflu xo f phosphate (byrivers) . Only an integral approachwil l reduce algalgrowth .Muc hattentio ni sgive nt oth eremova lo fphos phatefro m municipalwast ewater ,whic h iscurrentl y inth e Netherlandsth emos timportan t sourceo fphosphat ea sa pol lutant (Olsthoorn1986) .

CHEMICALPHOSPHAT EREMOVA LFRO MWASTEWATE R

Theconventiona lan dmos tapplie dmetho dfo rphosphat ere moval is its precipitation with chemicals, like alum (Al2(S04)3.18 H20), ferric chloride, lime and AVR (Swedish abbreviationfo rwastewate rtreatment )whic h isa mixtur eo f AlJ andF e compounds.I nadditio nt othes esalt sals opoly mersca nb euse dt oimprov eflocculatio no rchemical st ocorrec t thepH .Dependin g on thestage so fth ewastewate r treatment processi nwhic hth eprecipitatio nproces stake splace ,thre e categories canb edistinguished : (1)Pre-precipitation : thechemical sar eadde dt oth era wwast e wateran dth eprecipitat ei sremove di nth eprimar y settling tank. (2)Simultaneou s precipitation: thechemical sar eadde d toth e tank inwhic hbiodegradatio ntake splac ean dth eprecipitat ei s removedi nth esecundar ysettlin gtank . (3)Post-precipitation :th echemical sar eadde dt oth esecundar y effluentan dth eprecipitat ei sremove di na separat eplant . The application of the precipitation technique isno t wide spreadi nwastewate rtreatmen ti nTh eNetherlands ,becaus eo f thefollowin gdisadvantage so fth eprocess :hig hcosts ,pro - ductiono flarge rquantitie so fchemica lsludge ,formatio no f worthlessby-product s likehydroxyapatit e and similar highly insolublephosphates ,introductio no fextr aanion ssuc ha sCI " andSO 4 intoth eeffluent ,an dth enecessit yo fa nadvance d process control. New low-cost processes inwhic h phosphateca n berecovere d ina nusefu l formhav ebee nfoun di npost-precipi tation technology. Examplesare :th eprecipitatio no fphosphat e insecundar yeffluent sb ymean so flim eo ngrain so f sand ina n upflow reactor (VanDij kan dBraakensie k 1984), orth erecover y asMgNH^PO *b ymean so fa combine d anion exchange/precipitation process(Libert iet al .1985) .

BIOLOGICALPHOSPHAT EREMOVA LFRO MWASTEWATE R

An alternative for chemical phosphateremova l isbiologica l phosphateremova lfro mwast ewater ,whic hi sbase do nth een richmento factivate d sludgewit hpolyphosphat e accumulating bacteria.Thi ssludg ei sabl et oremov ephosphat ealmos t com pletely from the waste water.Th e process does not have the abovedescribe d drawback of chemical phosphate removal.Th e process of biological phosphate removal hasalread y proved its practicalapplicability .However ,th efailure swhic hstil loccu r occasionally,ar emostl ydifficul tt ounderstand ,du et oa lac k ofexperienc ean da lac ko funderstandin g of the fundamental principleso fth eprocess .

Theproces s

Treatment plants that spontaneously removed more phosphate than expected have been reported already in 1959 (Srinath et al.) and 1961 (Alarcon). The first investigations concerning this process started in the mid-sixties. From studies with full scale plants it appeared that biological phosphate removal took place in treatment plants with plug flow aeration reactors in which the return sludge and influent were added at the beginning of the reactor (Vacker et al. 1967, Milburg et al. 1971, Garber 1972, Yall et al. 1972). Barnard (1976) discovered that treat ment plants designed for nitrogen elimination, with nitrifica tion in aerobic zones and denitrification in anoxic zones, were able to remove large amounts of phosphate as well. A phosphate removal of 97% was possible. The presence of an anaerobic zone in the reactors was compulsory (Davelaar et al. 1978). Biologi cal phosphate removal appeared in reactors with two zones namely an anaerobic zone at the beginning of the reactor, to which influentan dretur nsludg ewer eadded ,an da naerobi c zonea t theend .I nth eanaerobi czon eth esludg erelease dlarg eamount s ofphosphate ,whil ei nth eaerobi czon ephosphat ewa stake nu p almostcompletel y(Fig . 1). secondary aeration tank darifier

effluent

Fig. 1. Flow sheet of an activated sludge plant with biological phos phate removal; and the ortho-phosphate concentration in the aeration tank.

The above described process has not always been recognized as a biological process. Twenty years ago a chemical and a biologi cal model were developped. According to the chemical model the improved phosphate removal was due to chemical precipitation reactions, and the temporary phosphate release was assigned to be due to a pH decrease (Menar and Jenkins 1970) . However, evidence for a biologically catalyzed reaction was growing as Levin and Shapiro (1965) indicated that the excess phosphate removal was a biological process: the addition of 2,4-dinitrophenol to the sludge stopped the uptake of phosphate completely. The pH optimum for phosphate removal appeared to be 7.8. At pH 9.0, in comparison, less phosphate was removed which could not be explained by the chemical precipitation model of Menar and Jenkins. Arvin (1983) proposed that phosphate removal in waste water treatment plants operating without chemicals was actually a combination of a biological and chemical process. He hypo thesized that phosphate release and uptake were of biological nature, but because of the high phosphate concentration occurring under anaerobic conditions precipitation of calcium phosphates could take place at a pH higher than 7.5. In 1975 Fuhs and Chen isolated several Acinetobacter strains from phos phate removing activated sludge able to store phosphate intra- cellularly as polyphosphate in form of granules. Pure cultures accumulated phosphorus up to 50 mg per gram dry biomass in continuousaerate dgrowt hmedia ,withou tadaptation .Acinetobac - terappeare dt ob eth edominan tgenu si nwastewate r treatment plantswher ephosphat ei sremove dbiologicall y (Nichollsan d Osborn 1979).Bucha n (1983)reporte dtha tAcinetobacte r could accountfo r66 %o fth enumbe ro fcell si nthi styp eo factivate d sludge.I na nacetat efe dpilo tplan twit halternatin gaerobi c andanaerobi ccondition sth emai npar to fth eorganism sculture d aerobically were acinetobacters (Wentzel et al. 1987). These microbiological findings were important indications thatth e observedphosphat eremova lwa sindee do fbiologica lnature .

Basedo nrecen tadvancement si nwastewate rengineerin gan d appliedresearc htw one wproceeding shav ebee ndeveloppe dfo r biological phosphate removal.The y are the Phostrip process (Levin and Sala 1987) and the Renpho process (Rensink, in press). Inth ePhostri pproces s (Fig.2 )th einfluen ti sadde d ina completel yan dcontinuou saerate dreactor .A par to fth e return sludgei smad eanaerobi ct ostri pth ephosphat efro mth e sludge.Th esludg e issubsequentl y separated from theortho - phosphateb ysettlin gan dreturne dt oth eaerobi creactor .

Influent

\ Phosphate-Rich Waste Activated Sludge

Phosphate-rich Supernatant KEY Sludge

Wastewater Primaiy Clarifier Aeration Tank Secondary Clarifier Stripper Tank Mixing Tank Lime Storage

Fig.2 .Flo wshee to fa standar dPhostri pprocess .

TheRenph oproces s(Fig .3 )i sabl et oremov et oa grea texten d both phosphorus and nitrogen from wastewater .Influen tan d returnsludg ear eadde dint oa nanaerobi c zonei nwhic hphos phatei sreleased , themixe d liquori ssubsequentl ytransporte d toa naerate dzon ei nwhic hphosphat ei stake nu pan dnitrifi cation takes place. Ina third, anoxic zonedenitrificatio n occurs.A par to fth einfluen ti sdirectl yadde dt oth edenitri

ll, <:pcpcp LJ

b" "c5 t> V 1. t» «^P 1. phosphate removal tank 2.secondar y clarifier 3.strippe r U. sludge thickener 5.influen t to comportment 3 6. effluent la and b) 11. 7 return sludge 8.concentrate d sludge 9. excess sludge 10. addition Na-acetate 11.strippe d sludge Fig.3 .Flo wshee to fth eRenph oprocess . ficationzone ,becaus eher eth eorigina l influent isalread y devoid of degradable organic carbon compounds and the fresh wastewate radd sth enecessar yelectro ndonor sfo rdenitrifi - cation.Phosphat ei sno trelease d inthi sstage ,sinc edenitri fyingcondition ssee mt oinhibi tphosphat erelease .At th een d ofth ereacto ra nadditiona laerobi czon ei sinserte dt oremov e the last traces of biodegradable organic pollutants. After settling, the sludge isdevide d into two parts: onepar t is returneddirectl yt oth ebeginnin go fth eplant ,an dfro mth e secondpar tphosphat ei sstrippe dbefor ei ti sreturne dt oth e first aerated zone. This stripping process is necessary if biologicalphosphat eremova lha st ob eapplie d inlo w loaded wastewater treatment processes; becausea tlo w sludgeloading s theamoun to fsludg eproduce di sto olo wt otak eu pal lphos phorusfro mth ewast ewater .

Biochemicalmodel s

Afterth ediscover yo fth ebiologica lnatur eo fbiologica l phosphateremoval ,researc ho nth ebiochemica lmechanism swa s startedt odevelo pa biochemica l model forthi sprocess .Th e necessityo fa nanaerobi czon ewa smos tintriguin gan dattracte d theattentio n of many scientists.Sinc emos t acinetobacters cannotus eglucos ea ssubstrat ebu tgro wwel lo nfatt yacid san d alcohols,Fuh san dChe n(1975 )state dtha tth eanaerobi cstag e playesa nimportan trol ei nth eproductio no fshor tchai nfatt y acidsb y facultativeanaerobi cbacteria .Rensin k(1981 )demon strateda lowe rfatt yaci dproductio nfro msewag ei nth eanaero - bicstage .Th efre efatt yaci dconcentratio ndecrease di nth e anaerobiczon ewithi na fe wweek ssimultaneousl ywit hth ede velopmento fenhance dbiologica lphosphat eremova li na pilo t plant.Fuh san dChe n(1975 )suggeste dtha tth elowe r fattyacid s were consumed anaerobically by polyphosphate accumulating bac teria,an dstore di nfor mo fPH B(poly-/3-hydroxybutyrate) .PH B couldb edetecte d inpur eculture so fAcinetobacte r (Fuhsan d Chen1975 ,Nicholl san dOsbor n1979) ,an di twa s hypothesized thatthi scompoun dwa suse da sa nenerg yan dcarbo nsourc efo r growthan dpolyphosphat eaccumulation ,a si nth eaerobi czon en o lowerfatt yacid swer edetectable .PH Baccumulate di nth esludg e in the anaerobic stage of thebiologica l phosphate removal process,an dwa sbroke ndow ni nth eaerobi cperio d (Fukaseet al. 1982,Comea ue tal . 1987,Min oe t al. 1987). Lower fatty acids were taken up in the anaerobic period and thisuptak e correlated with theaccumulatio no fPH Ban dpoly-/3-hydroxyvale - rate (Comeaue tal .1987) .Bordac san dChies a (1987)di dexperi mentswit h14 C labeledacetat ewhic hwa sadde dt oth esludg ea t thebeginnin go fth eanaerobi cperiod .Sevent ypercen to fth e Cwa sincorporate dint oPH Bafte ranaerobiosis .Th emas so f anaerobically released phosphateappeare d tob eproportiona lt o themas so facetat eadde d(Rensin k1981 ,Wentze le tal .1985) , ethanoladde d (DeVrie se tal .1985 )o rCO Dadde d (Hascoetan d Florentz 1985).

Concomitentlywit hth eabov edescribe dobservation sbiochemi calmodel swer edesigned . Ina nearl ymode lNicholl san dOsbor n (1979) suggested that both PHB and polyphosphate were accumulatedi nth esam eorganism ,probabl yAcinetobacter ,an d thatbot hwer eimportan ti nth esurviva lan dcompetitio no fthi s organismi nactivate dsludge .I nthei rmode lPH Bwa saccumulate d under anaerobic conditions as an electron sink of anaerobic fermentationproducts ,an dpolyphosphat eacte da sa phosphoru s reserve.A more sophisticated modelwa ssuggeste db yRensin k (1981)an dMarai se tal .(1983) . Inthei rmode l polyphosphate acts as an energy reserve, a hypothesis which has been put forward byman y microbiologists (Harold 1966, Dawes and Senior 1973, Kulaev 1979)an dwa sdemonstrate d invitr oi nEscherichi a coli(Kornber g 1957).Wit hth eenerg ygenerate db yth edegra dationo fpolyphosphat elowe rfatt yacid swer etake nu pwhe nth e systemwa sanaerobi can dwer etransforme d intoPH Bwhic h was storedi nth ecells ,jus ta sha sbee nsuggeste db yFuh san dChe n (1975). The model of Rensink (1981)an d Marais et al.(1983 ) explainsth ePH Baccumulation , thephosphat ereleas ean dit s responset olowe rfatt yaci dadditio ni nth eanaerobi cstag ean d thesubsequen tphosphat euptak ei nth eaerobi cperiod .Moreover , itexplain swh ypolyphosphat eaccumulatin gbacteri a likeAcine - tobacterbecom edominan t ina treatmen t plantwit halternatin g anaerobic and aerobic conditions.Accordin g to the model of Rensinkan dMarai se tal .Acinetobacte rwoul db eovergrow nb y morecompetitiv eaerobe slik ePseudomona san dFlavobacteriu mi n continuouslyaerate dplants .Th epresenc eo fa nanaerobi czon e givesa distinc tadvantag efo rAcinetobacte rsinc ei tca nthere , incontras tt omos tothe rstrictl yaerobi cbacteria ,tak eu p fermentation products.The y can immediately be utilized for energyproductio nwhe nth esludg eenter sth eaerobi czon ewher e freesubstrate sar escarce .I nthei rmode lComea ue tal .(1986 ) hypothesizetha tth euptak eo flowe rfatt yacid sdissipate sth e transmembraneprotongradien tan dtha ta sa consequenc eortho - phosphateha st ob etransporte dacros sth emembran et orestor e theprotongradient .Th emode lo fWentze le tal . (1987)combine s themodel so fComeau ,Rensin kan dMarais .However ,on eha st o keepi nmin dtha tal lmodel sar ehypothetica l andmainl ybase d onassumptions .I nmos tmodel sth eutilizatio no fpolyphosphat e asa nenerg yreserv eunde ranaerobi ccondition splay sa nimpor tant role. Many authors are of opinion that this energy is generatedb yth eactio no fpolyphosphatekinas ewhic hcatalyses : (poly-P)n + ADP—>-(poly-P)n_1 + ATP. This enzyme has been chosenfo rth emodel sbecaus ei twa sdetecte d inman ymicroor ganisms other thanAcinetobacte r (Romberg 1957,Kulae v and Vagabov 1983). Despite all these models and hypotheses ATP production frompolyphosphat eha sneve rbee ndemonstrate d in Acinetobactero ri nactivate d sludgean dth emicrobia lutiliza tiono fpolyphosphat ea sa nenerg yreserv ei nviv oha sals ono t yetbee nproved .

Asecon dno tye tprove dassumptio ni nal lthes emodel ssay s thatth ePH Baccumulatio n and"polyphosphat e degradation in the anaerobic stage takeplac e inth e same organisms,namel y in acinetobacters. Although polyphosphate degradation by pure cultureso fAcinetobacte rsp .ha sbee ndemonstrate dunde ran aerobiccondition s (Adamsee tal .1984 ,Ohtak ee tal .1985) ,th e accumulationo fPH Bha sonl ybee nshow nt ooccu runde raerobi c conditions.Acinetobacte r strainshav ebee nisolate d from acti vatedsludg ei nman yresearc hlaboratories ,bu tth eanaerobi c uptakeo f lower fattyacid s and accumulation of PHBb y pure cultures of this genus has never been reported. The causal relationbetwee nanaerobi cpolyphosphat edegradatio n andPH B synthesiswa sdoubte db yFukas ee tal .(1985) ,wh oincubate dtw o typeso factivate d sludgeanaerobically ,namel ysludg efro mth e biological phosphate removal process with adr y weight con taining10 %phosphorus ,mainl ypoly-phosphorus ,an dconventiona l activated sludgewit honl y1.9 %phosphorus .Bot hsludge stoo ku p thesam eamoun to ftota lorgani ccarbo n(TOC )unde ranaerobi c conditionsan d+ 34 %o fthi sTO Cwa saccumulate di nfor mo fPHB . Nevertheless theseauthor sobserve dth esam eeffec ta sreporte d byothers : thephosphat eric hsludg erelease dphosphat epropor tionalt oth eamoun to fTO Cincorporate dint oth ebiomass .Th e phosphatepoo rsludg eaccumulate dequa lquantitie so fTO Cbu t released only small amounts of phosphate.Mos t research and modeldevelopment swer ebase do nth eproces sillustrate di nFig . 1.However ,th e Phostripproces s works at the crucial steps completelydifferen tan di snevertheles sabl et oremov ephos phatesuccesfull yprimaril yb ybiologica lmean s(Fig .2 ,Levi n and Sala 1987). Biological phosphate removal inth ePhostri p processcanno tb ecouple dt oth eanaerobi caccumulatio no fPH B bypolyphosphat e containing bacteria, since theinfluen t is added first to the aerobic part, leaving so little organic substrates forfermentatio ni nth eanaerobi cstep . Itca nb econclude dtha tth efundamenta lprinciple so fth e processo fbiologica lphosphat eremova l from wastewate rar e stillunknow nan dtha tit sworkin gmechanis m ismainl ybase do n empericalexperience .

POLYPHOSPHATEI NMICROORGANISM S

Chemicalstructur eo finorgani ccondense dphosphate s

Inorganiccondense dphosphates ,th egenera lter mapplie dt o allpentavalen tphosphoru scompound si nwhic hvariou snumber so f tetrahedralPO 4group sar elinke dtogethe rb yoxyge nbridges , fall intothre eclasse s (Harold 1966): (1)Cycli ccondense dphosphates ,o rmetaphosphates :Onl ytri - and tetrametaphosphate are known.The y are present in some microorganisms(Kulae v 1979). (2)Linea r condensed phosphates:Unbranche d structureswit hth e M p M= cat;i on elementarycompositio n n+2 n°3n+l( - /generall yme tals)ar ecalle dpolyphosphates .Th enumbe ro fphosphat eresi dues inthes e compounds from living organisms,ma yvar yno ticeably: from two in pyrophosphate, the simplest type, to severalhundred san dthousand si nhig hmolecular-weigh tpoly phosphates (Kulaevan dVagabo v 1983). Linearpolyphosphate sare , basedo nthei rabilit yt odissolv ei ncol d5 %TCA ,divide d into two classes: acid solublepolyphosphate s (n<20)an daci dinsol - ublepolyphosphate s (witha highe rmolecula rweight )(Kulae v 1979). (3)Cross-linke dcondense dphosphates ,o rultraphosphates :Thei r characteristic feature is the presence of branching points, phosphategroup s inwhic h threeoxyge natom sar eshare dwit h neighboringphosphat egroups .Branchin gpoint sar ereadil yhy - drolyzed inwate r andtherefor eth eoccurrenc eo fultraphos phatesi nlivin gcell swoul dno tb eexpected .

Accumulationo fpolyphosphat eb ymicroorganism s

In188 8Lieberman ndiscovere dpolyphosphate si nyeast .U pt o now thisbiopolyme rha sbee nfoun di nman ytaxonomica lorders , fromprokaryote st ohighe rplant san danimals .Th eamoun to f polyphosphatepresen ti nth ecell so fmicroorganism sca nvar y considerably.I nyeast sth epolyphosphat econten tca naccoun t for20 %o fth ebiomas sdr yweight (Lissan dLange n 1960)an d strainso fAcinetobacte rar eabl et oaccumulat ethi scompoun du p to 24%o fthei rdr yweigh t (Deinemae tal . 1985). Insevera l fungi0. 2t o1 %polyphosphate-phosphoru s hasbee nfound .Mor e than14 0specie so fmicroorganism s containingpolyphosphat ear e currentlyknow n (Kulaev 1979).Th efor mi nwhic hhig hmolecula r weightpolyphosphate sar epresen ti nth ecel li sno tye tver y clear.Chai nlength so f50 0t o60 0phosphate-group shav ebee n found inAerobacte r aeroqenes (Harold 1963), andi nth eflagel lateCrithidi afasciculat ath epolyme rconsist so f 850monomer s (Janakidevie tal .1965) .Th eaci dsolubl efor mca nb epresen t ina fre estat e (MacFarlan e 1939), whileth eaci d insoluble formwil lb eprecipitated ,possibl yi ncombinatio nwit hRNA , linkedb yMg 2+ andCa 2+, orlinke d toproteins ,acidi cpoly saccharideso rphospholipid s(Kulae v1979) .Thes eprecipitate s formmetachromati cgranule so rvoluti ngranule s (Wiame1949) . Polyphosphate granules usually do not contain membrane structures,bu ti nmycobacteria ,cultivate do nolei cacid ,th e polyphosphategranule sar esurrounde db ya lipi dlaye r(Schaefe r etal .1965) .Polyphosphat eca nb elocalize da tsevera lplace s ino routsid eth ecytoplasm :o nth ecel lsurfac eoutsid eth e cytoplasmicmembran eo fyeast s (VanSteveninc k andBooi j 1964), inth enucle io fyeast san dfung i(Kulae v1979) ,i nvesicle so f theendoplasmi creticulu mi nyeast s(Kulae van dVagabo v1983 ) and in the chloroplasts of algae (Rubtsov et al. 1977). Two typeso fpolyphosphat egranule sar efoun di nyeasts ,a vacuola r and cytoplasmic form (Indge 1968). Inprokaryote s granules are foundi nth eregio no fth enucleoplas mo rnea rth ethylakoid so f cyanobacteria (Jensenan dSick o 1974),i nth eperiplasmi cregio n

10 ofMycobacteriu m smeqmatis(Ostrovsk ye tal .1980 )o ri nas sociationwit hglycoge ninclusion si nMyxococcu sxanthu s(Voel z etal .1966) .

The accumulation of polyphosphate bymicroorganism sdepend s onth eavailabilit y ofphosphorus ,oxidizabl esubstrates ,K or othermeta lcation san dth eactivit yo fenergy-providin gpro cessessuc ha srespiration ,fermentation ,an dphoto-phosphoryla - tion.Th especifi ccondition sfo rpolyphosphat eaccumulatio nca n bedivide dint othre ecategories : (1)Th eoverplu sphenomeno n(Harol d 1966),als ocalle dUeberkom - pensation(Lis san dLange n1962 )o rpolyphosphat esupersynthesi s (Kulaev1979 )whic hoccur safte rphosphat e isadde d tophos phorus starved cells.Unde rphosphoru s limitation the cells synthesizelarge ramount so fenzyme stha tpla ya rol ei nth e polyphosphatemetabolism , assoo na sphosphat ei sintroduce d into the medium unusual large amounts ofpolyphosphat e are temporarilyaccumulate dan dbroke ndow nagain .Thi seffec twa s firstobserve db yLis san dLange n(1962 )i nyeast ,an db yHarol d (1966)i nAerobacte raerogenes ,i nwhic ha nincrease dbiosyn thesiso fpolyphosphatekinas ean dpolyphosphatas ewa sdemon strated.Thi smechanis mha sals obee nfoun di nHydroqenomona s (Kaltwasser 1962), Micrococcusdenitrifican s (Kaltwasser etal . 1962)an di nvacuole so fSaccharomyce scarlsberqensi s(Lichk oe t al. 1982). (2)Luxur yuptak eo fphosphat eunde rcondition sunfavourabl efo r growth: This reaction is performed by microbial cells that accumulate largeamount s of polyphosphate in stationaryphase s orothe rcondition swhe ngrowt hdeclines ,provide dtha tsuf ficient energy,phosphoru s and cations are available.Thes e organismsdon' taccumulat epolyphosphat eunde rnorma l growth conditions.Luxur yuptak eha sbee ndemonstrate d inman ybac teria,mostl yunde rcondition so fnutrien timbalanc e (e.g.N o r S starvation). Examples are:Corynebacteriu m xerosis,mycobac teria (Kulaev 1979), Aerobacteraerogene s (Wilkinson andDugui d I960),Bacillu scereu san dB .meqaterium ,Aorobacte rradiobacte r (Szymonae tal .1962 )an dChlorobiu mthiosulfatophilliu m(Sha - poshnikov and Fedorov 1960). (3)Luxur yuptak eo fphosphat eunde rgrowt hconditions :i na fe w speciespolyphosphat ei saccumulate di nth egrowt hphase .Fo r examplei nAcinetobacte r sp.i nth elogarithmi cgrowt hphas e (Fuhsan d Chen 1975,Deinem ae tal .1980 )o r inActinomyce s aureofacienswit ha maximu maccumulatio ndurin gth esecon dhal f ofth elogarithmi can dth ebeginnin go fth estationar ygrowt h phases(Kulae ve tal .1976) .I nChlorell aa specifi cpolyphos -

11 phate fraction was accumulated onlyunde rcondition so fphoto synthesis. This fraction, called poly-P "C",wa s insoluble eitheri ncol dPC A(5% )o ri ncol dwea kalkal i(p H8-10 )bu ti t wasextractabl ewit h2 N KO Ha t37° Can dco-precipitabl ewit h KCIO4whic hi sforme dupo nneutralizatio no fth eKOH-extrac t withHC10 4(Miyach ie tal .1964) .

More examples of polyphosphate accumulationhav ebee nfoun d inothe rbacteria .Polyphosphat eca nb eaccumulate db yEsche richiacol ia tth einitia lstage so flogarithmi cgrowt h(Nes - meyanovae tal .1973 )o rdurin gth elaten tperio dwhic hprecede s thephas eo frapi dgrowt ha sfoun di nCorynebacteriu mxerosi s (Kulaev 1979).O nenterin g thelogarithmi cgrowt hphas ebot h organismsutiliz ethei rpolyphosphates . Insynchronou s cultures ofCorynebacteriu m diphteria (Saile tal .1958 )an dAzotobacte r sp. (Kulaev1979 )i twa sdemonstrate dtha tpolyphosphate spla ya specificrol edurin gcel ldivision .Polyphosphat eaccumulate da t thestag eprecedin gcel ldivisio nan dwa sconsume dver yrapidl y duringth edivisio nprocess .Polyphosphat eaccumulatio nca nals o bedependen t on the carbon substrate used.A formation ofa polyphosphatestorag eoccurre d inPropionibacteriu m shermanii grownwit hlactat ebu tno ti nth ecell sgrow nwit hglucose .Whe n lactate-growncell swer esuspende di na solutio no fglucose ,th e contento fpolyphosphat ei nth ecell sdecrease d rapidly. Itwa s proposedtha tcell sgrow no nglucos ea sth esubstrate ,d ono t accumulatepolyphosphat esinc ethi spolyme ri sutilize da srap idlya si ti sforme db yth ephosphorylatio no fth eglucos etha t isbein gmetabolize d (Wood 1985).Recentl ya Thiobacillu sstrai n hasbee nfoun dtha taccumulate spolyphosphat eu pt o50 %o fit s dryweight .Thi soccur si ncontinuou sgrowin g culturesunde r carbonan denerg y limitation, butonl ya t high ortho-phosphate concentrations inth e medium (6.8 mM). At low concentrations (0.68mM )n oluxur yuptak eca nb efoun d (Gommersan dKuenen ,i n press).

Therol eo fcation si npolyphosphat eaccumulatio n

Cations mayb enecessar y inpolyphosphat eaccumulatio n asa counterion of the polyanion or as a prerequisite for other processestha tar enecessar yfo rth esynthesi so fpolyphosphate . Accordingt oKulae v(1979 )th eimportanc eo fK ,Be ,Mg ,Mn ,Zn , Fean dC aha sbee nreporte db ysevera lauthors .M g asa poly phosphatecounterio nha sbee nfoun di nMicrococcu slysodeikticu s (Friedbergan dAviga d1968 )an di nDesulfovibri oqiqa s inwhic h largeamount so fMg-tripolyphosphat ewer epresen t (Jonesan d

12 Chambers 1975).I nvacuole so fNeurospor acrass a considerable quantities of polyphosphate, Mg and Ca have been found (Crameran dDavi s 1984).Th epresenc eo fK +a sa polyphosphat e counterioni nChlorell asp .ha sbee nreporte db yCembell ae tal . (1984).Accordin g toBaxte ran dJense n(1980 )K +i spresen ti n polyphosphategranule so fPlectonem aboryanum ,bu tdependen to n theirconcentration si nth eenvironmen tals odivalen tcation s could be found in the granules. The importance of K+ in the medium asa crucia l factorfo rpolyphosphat eaccumulatio ni n Corynebacteriumdiphteria ewa sfoun db ySai le tal . (1956). Both —+— 7+ — K and Mgz in the medium were essential for polyphosphate accumulation in Aerobacter aeroqenes (Wilkinson and Duguid 1960). Thefunction so fpolyphosphat ei nmicroorganism s

According to Kulaev (1979)th ephysiologica l functions of polyphosphatesremai nunknown .Becaus eo fthei rdifferen tmol ecularweights ,divers eresponse st oth ephysiologica l stateo f thecell ,an dvariou slocalizatio nwithi nth ecell ,i tappear s tob ever ylikel ytha tpolyphosphat e mayperfor m more thanon e functioni nth evita lprocesse so fthos eorganism si nwhic hthe y arepresent . Polyphosphates can act as aphosphoru s reserve which enable themicroorganism s toincreas e theirbiomas sman y folddurin ggrowt hi na phosphorus-fre emediu m (Harold 1966,Va n Groenestijn andDeinem a 1985). Moreover,the yrepresen ta valu able pool of activated phosphate which can be utilized in variousmetaboli cprocesses ,primaril yi nthos econnecte dt o differentstage so fcarbohydrat ean dnuclei c acid metabolism (Kulaevan dVagalo v 1983).I tha sbee nsuggeste dtha tth eear liest organisms inth eevolutio n of lifeuse d polyphosphate or pyrophosphate as the energy carrier instead of ATP (Harold 1966).Polyphosphate sar eregulator so f the intracellularcon centrationo fimportan tmetabolite sincludin gATP ,ADP ,othe r nucleosidepolyphosphates ,pyro -an d ortho-phosphate.Ther ei s substancial evidence forth eparticipatio n ofpolyphosphate si n the transport of glucosethroug h the plasmamembrane ofyeasts . Thepolyphosphate so nth eoutsid eo fth emembran ear eth eulti matephosphory ldonor si ntransport-associate d phosphorylation (Van Steveninck et al. 1986). The practical significance of polyphosphatea sa biologica l ion-exchanger isunknow nan donl y hypothesized (Baxteran dJense n1980 ,Kulae van dVagabo v 1983). Polyphosphatei sprobabl yimportan tfo rcel ldivisio no fCoryne bacterium diphtheria. Sail et al. (1958) hypothesized that during thisdivisio n polyphosphate is utilized as a phosphagen

13 asa reactio n toth e increased cellularenerg ydemand . Many authorshav especulate dabou ta functio no f inorganicpolyphos phatea sa nenerg ysourc eo rreserv ei nviv o(Dawe san dSenio r 1973),bu tther ear eonl ya fe wexperimenta lobservation stha t may supportthi shypothesis .Th egrowt h of someanaerobi cbac teriaha sbee nreporte dt ob estimulate db ypyro -an dpolyphos phatesi nth emediu m (Varmaan dPec k1983 ,Crude ne tal .1983) . Streptomyceslevori suse dinorgani cpolyphosphate san dnot AT P as an energy source during the synthesis of the antibiotic levorin(Zyuzin ae tal .1981) .However ,u pt ono wther ei sstil l a lacko fconvincin gevidenc efo ra rol eo fpolyphosphat ea sa n energyreserve .

Polyphosphatei nAcinetobacte rsp .

Thegenu sAcinetobacte ri sdefine da sa nonmotil eshor tro d whichi sunabl et odenitrify .Cell sar eGra m negative,cyto chromeoxydas enegative ,heterotrophic ,an dobligat eaerobic . Theygro wwel lo nman ysubstrate ssuc ha salcohols ,hydrocar bons, aliphatic acids,aromati c compounds,amin o acids and amines, but mostly not on glucose as the sole carbon source (Baumanne tal .1968 ,Jun i1978) .I n197 5Fuh san dChe nisolate d differentAcinetobacte r strainsfro m awastewate r treatment plantwit h biological phosphateremoval , anddemonstrate d that theseorganism swer eabl et oaccumulat e largeamount s ofpoly phosphate.Afte rtha trepor tpolyphosphat eaccumulatin gacineto - bactershav ebee nisolate di na numbe ro flaboratories .Thei r abilityt otak eu plarg eamount so fphosphat ei nth elogarithmi c phasewa sdemonstrate d byDeinem ae tal .(1980) .Th ebacteri a contained granules which were electrondense (Fig. 4),an d colouredviolet-blu ewit hNeisse rstainin g(Gur r1965) .B ymean s of energy dispersive microanalyses with X-rays (EDAX)th ehig h phosphorusconten to fthes egranule swa sdemonstrate d (Buchan 1983,Va nGroenestij nan dDeinem a 1985).Th epresenc eo fpoly phosphatei nAcinetobacte rsp .wa sprove dqualitativel yb yNM R analyses (Florentz 1983,Ohtak ee tal .1985 )an d quantitatively bychemica l fractionation (Fuhsan dChe n 1975,Ohtak e etal . 1985).Th epolyphosphat ewa smainl ypresen ti nth eaci dinsol ubleform .Th ephosphoru sconten to fdifferen t strainsdiverge d strongly. Fuhs and Chen (1975)di d not find more than 50rag phosphoruspe rg dr ybiomass ,Ohtak ee tal .(1985 )foun daroun d 30 mg phosphorus, Deinema et al. (1985)reporte d 54-120 mg phosphorusan dHa oan dChan g (1987)investigate da strai ntha t contained4 8m gphosphoru spe rg dr ybiomass .Unde ranaerobi c

14 Fig. 4. Electron micrograph of Acinetobacter strain 210A. The black polyphosphate granules are distributed throughout the cell. The bar indicates 0.1 um. The micrograph was made with the EM Philips-400T of the Technical and Physical Engineering Research Service in Wageningen.

15 conditionsortho-phosphat ei srelease db yth ecell s(Adams ee t al. 1984).Th ebiochemica lpathwa yo fth epolyphosphat edegrada tioni sstil lunknown .U pt ono wonl ya polyphosphatas eactivit y hasbee nfoun d(Ohtak ee tal .1985) .Fo rth esynthesi so fpoly phosphatepolyphosphatekinas eha sbee ndetecte di nsom eAcineto - bacter strains (T'Seyen et al. 1985), however, in very low activities of around 1nmol.min~.mg -1 protein.Th ebest in vestigated strain so far is Acinetobacter strain 210A (Van Groenestijnan dDeinem a 1985).Thi sstrai ngrow swel lo nfermen - tationproducts,lik eacetate ,lactat ean dethanol .NH^"*" ,NO3 - and ureum can serve asnitroge n sources,an d S04 ,S2O 3 , methioninean dcystin ea ssulphu rsources .Th estrai ndoe sno t requiregrowt hfactors .Th e Y^fo rdissolve doxyge nwa smeasure d tob e0.0 8 mg.l-1.

ENZYMESO FTH EPOLYPHOSPHAT EMETABOLIS M

Biosynthesiso fpolyphosphate s

Theenzym epolyphosphatekinas ecatalysin gth ereactio n

ATP+ (poly-P) n^i(poly-P)n+1 +ADP . wasfirs tdemonstrate db yYoshid ai n195 3wh owa sabl et osyn thesizepolyphosphate sfro mAT Pwit hth eai do fcell-fre eex tracts fromyeas t (Rulaev 1979).Romber g etal .(1956 )isolate d thesam eenzym efro mEscherichi acol ian dpurifie di thundre d fold.I twa sshow ntha tth eenzym ewa srequirin gMg ^ andwa s 94- stronglyinhibite db yADP .I n195 7Romber greporte dtha tth e reversereactio nwa sals opossible .Muhamme d (1961),however , wasno t able to demonstrate such a reverse reaction with a purifiedpolyphosphatekinas efro mCorynebacteriu mxerosis .B y 1979polyphosphatekinase shav ebee ndetecte di n1 6specie so f bacteriaan di nyeast san dfungi ,i nsom ecase sth ereactio nwa s reversible, in other cases only the synthesis reaction was catalyzed (Rulaev et al.1971 ,Rulae v 1979).Tw o isoforms of polyphosphatekinasehav ebee nfoun di nyeasts .Th emai nisofor m islocalize d inth ecytoplas m andappeare st operfor monl yth e degradationo fhig hmolecula rweigh tpolyphosphates .Th eisofor m inth evacuola rfraction ,however ,catalyze dbot hsynthesi san d therevers ereactio n(Shabali ne tal .1977) .Accordin gt oNes - meyanova (1973)th edirectio n inwhic hpolyphosphatekinas eope ratesma ychang ea tdifferen tphase so fgrowt hi nEscherichi a coli.

16 Besides polyphosphatekinase also 1,3-diphosphoglycerate:polyphosphate phosphotransferase is able to synthesize polyphosphate:

1,3-diphosphoglycerate + (poly-P)n^=±3-phosphoglycerate + (poly-P)n+1

This reaction was first detected by Kulaev et al. (1968) in Neurospora crassa. The enzyme associates polyphosphate synthesis with glycolytic phosphorylation reactions.

A number of microorganisms contain only polyphosphatekinase, others only l,3-diphosphoglycerate:polyphosphate phosphotrans ferase, and in some both enzymes are present (Kulaev 1979). There are at least some organisms in which polyphosphatekinase is wholy or largely responsible for polyphosphate biosynthesis, for example in Aerobacter aeroqenes. Polyphosphatekinase nega tive mutants of this bacterium were unable to synthesize any polyphosphate (Harold and Harold 1963).

Biodegradation of polyphosphates

In addition to polyphosphatekinase and 1,3-diphosphoglyce rate:polyphosphat e phosphotransferase, which have been described above, other enzymes are able to degrade polyphosphate: (1)Polyphosphate:AM Pphosphotransferase :

(poly-P)n+ AMP—*(poly-P) n_1 +AD P

The first indications for the existence of polyphosphate:AMP phosphotransferase in mycobacteria has been reported by Winder and Denneny (1957). Dirheimer and Ebel (1965) isolated the enzyme from Corynebacterium xerosis and purified it partly. The enzyme needed Mg2+ and was specific for AMP and high molecular weight polyphosphates. Because the 1^ for AMP was 2.10 M, the physiological significance was doubted. This enzyme has been found only in mycobacteria and corynebacteria. (2) Polyphosphatase:

(poly-P)n+ H 20~*(poly-P)n_1+ PL

Polyphosphataseha sbee nisolate d fromman yorganism s (Kulaev andVagabo v 1983).Polyphosphatase s witha hig haffinit yfo r highmolecula rweigh tpolyphosphate shav ebee nreporte d (Muham- med 1959)a swel l as seperate enzymes forth ehydrolyse so f

17 tetra-,tri -an dpyrophosphat e (Kulaev 1979).Th eactivit yi s mostlydependen t on theM g concentration,an d canb e sti mulatedb yK +(Kulae v1979) .Th eenzym eca nb elocalize dintra cellular^o ro nth eoute rsid eo fth eplasm amembran e (Kulaev andVagabo v 1983).I nE .col ithi senzym ei sderepresse dunde r phosphorusstarvation ,an d itssynthesi si sregulate d by the samegene swhic hals ocontro lth eformatio no falkalin ephospha tase (Kulaevan dVagabo v 1983). Otherpolyphosphat edegradin genzyme sdetecte di nmicroorganism s arepolyphosphat edepolymerase ,polyphosphat edependen tNAD - kinase,polyphosphat e glucokinase,polyphosphat efructokinase , polyphosphate mannokinase and polyphosphate gluconatokinase (Kulaev1979 ,Kulae van dVagabo v 1983).

OUTLINEO FTH EPRESEN TRESEARC H

Tofull ycontro lth ebiologica lphosphat eremova lfro mwast e water,th efundamenta lmicrobiologica lan dbiochemica lprin cipleso fthi sproces shav et ob eelucidated .Knowledg eo nth e microbiologicalfundamental so fphosphat eremova lwil lallo wt o taylorth etreatment proces si nsuc ha wa ytha tth especifi c needso fth emicroorganism sinvolve dca nbes tb esatisfied .Onl y thethoroug h understanding of themicrobiolog y ofphosphat e removal will enable theengineer s tooptimiz ethi sproces san d toincreas eth esafet yo foperatio nfo rth emos tdivergen twast e waters in large scale plants. Since organisms of the genus Acinetobactersee m tob eresponsibl efo rth eremova lo fphos phatei nactivate dsludge ,i twa sappropriat et oinvestigat eth e physiologyo fthes eorganism si nmor edetails . Theai mo fthi sthesi swa stherefor et ostud yth eenvironmen talan dregulator yfactor sresponsibl efo rth eaccumulatio nan d degradation of polyphosphate in Acinetobacter sp.. Special attention was givent o thepossibilit y ofpolyphosphat eactin g asa nenerg yreserve . Theaccumulatio no fpolyphosphat ei nAcinetobacte rsp .an d theinfluenc eo fenvironmenta l factorso nthi sproces s is des cribed inchapte r2 .Accumulatio nan dreleas eo fphosphat e are consideredt ob edependen to ncation sbecaus eo fth eexpecte d roleo fthes eion si nth eneutralizatio no fth enegativ echarge s ofpolyphosphat e andortho-phosphate . Chapter 3deal s with the rolean d behaviour of cations inphosphat eaccumulatio n and release.Emphasi si sgive nt oth eelucidatio no fth epolyphos phatedegradatio npathwa yt oobtai ninformatio no nth efunction s ofthi spolyme ri nth ecell .Th eproductio no fAT Pfro mpoly -

18 phosphate by the combined action of polyphosphate:AMPphospho transferasean dadenylat ekinas ei ncell-fre eextract so fAcine - tobacter is reported in chapter 4.Chapte r 5deal s with the conditionsunde rwhic h polyphosphatei sdegrade d inviv oan d ortho-phosphatei srelease dfro mth ecell .Th epropert yo fthi s compound to act as an energy reserve has been demonstrated. Other polyphosphate degrading enzymes that reflect other functionso fth epolyme rar ereporte di nchapte r6 .I naddition , the involvement of themos t important enzymesi npolyphosphat e breakdownhav ebee nmeasure d inactivate d sludget o show the validityo fpur ecultur estudie sfo rth eapplication .

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26 CHAPTER 2

REGULATION OF POLYPHOSPHATE ACCUMULATION IN ACINETOBACTER SP.

Johan W. van Groenestijn, Martina Zuidema, Jacques J.M. van de Worp, Maria H. Deinema & Alexander J.B. Zehnder.

ABSTRACT

The regulation of andth eoptimu m conditions forpolyphosphat e accumulationi nAcinetobacte rsp .wer edetermined .Acinetobacte r strain 210Aaccumulate d polyphosphate inth epresenc e of an intra-o rextracellula renerg ysource .Th eaccumulatio no fpoly phosphatedurin gendogenou srespiratio nwa sstimulate db ystrep tomycinan dinhibite db yKCN .Th ehighes tamoun to fpolyphos phatewa sfoun di ncell si nwhic henerg ysuppl ywa sno tlimited , namelya tlo wgrowt hrate sunde rsulphu rlimitation ,an di nth e stationary phase of growth when either the nitrogen or the sulphursourc ewa sdepleted .Th ephosphoru saccumulatio nwa sno t affected by thep Hbetwee n 6.5an d 9.Ther ewa s apronounce d effecto fth e temperature onphosphoru s accumulation but it varied from straint ostrain .Acinetobacte rstrai n210 Aaccumu latedmor ephosphat ea tlo wtemperatures ,strai nB 8showe da n optimumaccumulatio na t27.5°C ,whil estrai nP accumulate dphos phorusindependentl yo fth etemperature .Th eoptimu mtemperatur e forgrowt ho fAcinetobacte rstrain steste drange d from 25t o 33°C,an dth eoptimu mp Hwa sbetwee n6 an d9 .

accepted forpublicatio ni nAntoni eva nLeeuwenhoe k

27 INTRODUCTION

In197 5Fun san dChe ndiscovere dtha tsom eAcinetobacte rstrain s were able to accumulate large amounts of phosphate. These strainsha d been isolated from activated sludge-type sewage treatmentplant sdesigne dfo rbiologica lphosphat e removal.Es sentiali nthi sproces s isth ecirculatio no f sludgethroug h anaerobic and aerobic zones.Durin g aerobiosis phosphate is takenu pb yth esludg ean drelease dagai nunde ranaerobi ccondi tions(Barnar d1976 ,Rensin ke tal .1981) .Enhance dbiologica l phosphateremova ldepend so nth eenrichmen to fth esludg ewit h Acinetobacter(Bucha n1983) . Microorganisms mostlyaccumulat epolyphosphat eunde rcertai n growthcondition san da tcertai nstage so fthei rdevelopment .I n generalth eaccumulatio ndepend so nth eavailabilit yo fphos phorus,oxidizabl esubstrates ,K +o rothe rmeta lcations ,an d thefunctio no fenerg yproviding-processes ,suc ha srespiration , fermentationan dphotophosphorylation .Ther ei sa considerabl e variationo fspecifi ccondition sunde rwhic hmicroorganism ssyn thesizepolyphosphat e (Kulaev 1979).Th ephysiologica l functions ofinorgani cpolyphosphate sremai nobscur ean dman yspeculation s havebee nmad eabou tthei rrol ea sa nenerg ystorag eo rreserv e material (Dawes& Senio r 1983).Th emai ncriterio nfo ra com poundt oac ta sa nenerg ystorin gmateria li sth efac ttha ti t isaccumulate dunde rcondition swhe nth esuppl yo fenerg yi nth e celli si nexces so ftha trequire dfo rgrowt han drelate dpro cessesa ttha tparticula rmomen to ftim e(Wilkinso n1959) .Th e quantity of the excess energy will firstly be dependent on availabilityan ddepletio no fdifferen telements ,an dsecondl y ongrowt hrate ,an dphysical-chemica l limitations sucha stem peraturean dpH . Thecurren t investigationwa stherefor eundertake nt odeter minean dquantif yth eeffec to ftemperature ,pH ,an dvariou s nutrientlimitation sa tdifferen tgrowt hrate so nth esynthesi s ofpolyphosphat ei nthre estrain so fAcinetobacte risolate dfro m threedifferen tactivate d sludgeplants .

MATERIALSAN DMETHOD S

Organisms

ThreeAcinetobacte r strainswer e isolated fromdifferen tacti vatedsludg eplant saccordin gt oth emetho do fDeinem ae tal . (1980).Strai n210 Aoriginate dfro msludg ei nRenkum ,strai nP

28 from Bennekom and strain B8 from Bunnik (all three in The Netherlands). The strains were maintained on yeast extract agar slants (5 g glucose, 2.5 g yeast extract and 12 g agar per litre tapwater, pH = 7), subcultured every two months and stored at 4°C.

Media

The basic medium, used for the temperature experiments contained per 1000 ml demineralized water: 5,67 g Na-acetate.3H20, 1 g NH4C1, 0,6 g MgS04.7H20, 0,4 g KH2P04, 0.1 g Na2S203.5H20, 0,07 g CaCl2.2H20 and 2 ml of a trace mineral solution (containing 50 g EDTA, 5 g FeS04.7H20, 1,6 g CuS04.5H20, 5 g MnCl2.4H20, 1,1 g (NH4)6 Mo7024.4H20, 2,2 g ZnS04.7H20, 50 mg H3BO3, 10 mg KI and 50 mg CoCl2.6H20 per litre demineralized water). The pH was brought to 7.0 with 6 N HC1. In the experiments in which the effect of the pH was measured NH4C1 was replaced by 1.6 g.l NaN03 (to avoid the precipitation of NH4MgP04 at high pH values). Where indicated acetate has been replaced by lactate and ethanol both also at a concentration of 1 g carbon per litre. Continuous cultivation under different limitations was carried out with the following media: Substrate limitation: Basic medium. Sulphur limitation: Basic medium but MgS04.7H20 was replaced by equimolar amounts of MgCl2.6H20, and a sulphate poor trace ele ment solution was used as well (Van Groenestijn et al. 1987). 5 mg Na2S2C>3.5H20 per litre were added as sulphur source. To assure sufficient carbon source the acetate concentration was increased 1.7 times to guarantee an excess of energy source for 28 h in the experiments in which substrate addition was stopped after continuous cultivation. Nitrogen limitation: Basic medium with only 0.25 g NH4C1 per litre. Phosphorus limitation (for cultivation of phosphorus starved cells): Basic medium with only 35 mg KH2P04 and an additional 0.1 MTris-HC l per litre for the buffering of the medium. The phosphate uptake medium contained 66 mg KH2P04, 600 mg MgCl2.6H20 and 4 g Tris per litre demineralized water. The pH was adjusted to 7 with 6 N HC1.

Analytical methods

Dry weight of bacteria was measured after centrifugation of a 50 ml sample, washing it with demineralized water and drying at 100°C overnight. After treating the supernatant with amberlite

29 IR-120Hio nexchanger ,i twa sanalyse dfo racetat ewit ha gas - chromatograph (Varian 2400Aerograph ,Adams e 1980). NH^+ and ortho-phosphate (ascorbicaci dmethod )wer equantifie daccordin g toStandar dMethod s (1976),an dthiosulphat ewa smeasure d spectrophotometrically (Sorbo 1957). The phosphorus and nitrogen contento fth ebiomas swa scalculate dfro mth eincreas eo fth e biomassdr yweigh tan dth edecreas eo fth eortho-phosphat ean d NHU concentrationsi nth ebul kliquid .A quic kestimatio no f thebiomas sdr yweigh twa smad eb ymeasurin gth eoptica ldensit y at66 0n man dusin ga calibratio ncurv e (ODggna sfunctio no f dry weight). Polyphosphate granules were stained followingth e methodo fNeisse r(Gur r1965) .

Experimentalprocedure s

Forth ephosphat euptak eexperimen tcell swer eprecultivate di n aphosphoru slimite dmediu mi nErlenmeye rflask si na shake ra t 15°Cunti lgrowt h stopped.Th ecell swer eharveste d andwashe d withdemineralize dwater ,a par twa suse dfo rdr yweigh testima tion,th eothe rpar twa sresuspende d inth ephosphate-uptak e medium and incubated in Erlenmeyer flasks at 25°C.A t time intervalssample swer etake nt omeasur ephosphat econcentratio n anddr yweight .Paralle lexperiment swer edon eb yaddin g KCN (lmmol per litre phosphate uptake medium) or streptomycin sulphate(20 0mg.l" 1). Themaximu m specificgrowt hrat ewa sdetermine di na batc h culturei na 2 1 bioreacto r (Applikon,Schiedam ,Th eNether lands)wit hp Han dtemperatur econtrol .At tim einterval sth e dryweigh twa sestimate db ymeasurin gth eODggQ .Th emaximu m specific growth ratewa s calculated byplottin g the natural logarithmo fth edr yweigh tagains ttime .Th ephosphoru sconten t ofth ebiomas sfro mth egrowt hphas ewa scalculate dfro mth e ortho-phosphate concentration in the supernatant and in the initialmedium ,an dth ebiomas sdr yweight . Forcontinuou s cultivationa 2 1 bioreacto rwa sused .Al l measurementswer edon ewhe nstead ystat ewa sreached .

30 RESULTS

Growthan dphosphoru saccumulatio na sa functio no fphysical - chemicalparameter s

The effect of the temperature on growth and polyphosphate accumulation forthre e strainso fAcinetobacter .Th e maximum specificgrowt hrat eo nacetat ei nbatc hculture s forth ethre e strainsinvestigate dwa s 0.69 h-1 (Fig. la).Th eoptimu m growth temperaturerange dfro m2 5t o33°C .Temperatur eha da nimportan t effecto nth ephosphat eaccumulatio n (Fig.lb) ,bu tthi seffec t wasdifferen t forever ystrain .Strai n210 Aaccumulate d high amountso fphosphoru s (100mg.g -1dr yweight )a tlo wtempera tures and phosphorus accumulationdecrease d with increasing temperatures.Thi sbehaviou ro fstrai n210 Awa sno ta neffec to f

10 15 20 25 30 35 W temperature (°C)

Fig. 1. The effect of the temperature on the maximum specific growth rate (a) and phosphorus accumulation (b) during growth in acetate medium for three different Acinetobacter strains: 210A (# ), P (D) and B8 (o )•

31 the growth rate (Fig. 4) since at 16°C a higher phosphorus content was found as compared to 25°C at several fixed growth rates. In contrast to strain 210A, strain B8 accumulated in creasing amounts of phosphorus with increasing temperatures with an optimum at 27.5°C. For strain P temperature had hardly any effect on phosphate incorporation.

The effect of the pH on growth and polyphosphate accumulation for Acinetobacter strain 210A. Acinetobacter strain 210A was able to grow well between a pH of 6 and 9, some growth was still observed at a pH of 4.7 or at a pH of 9.5 (Fig. 2a). The optimum pH for growth was dependent on the nature of the carbon source. The carbon sources tested were acetate, ethanol and lactate. All three substrates yielded high growth rates of Acinetobacter strain 210A (Van Groenestijn &Deinem a 1985). If acetate was used as substrate, an optimum for growth was found at pH 9 but no growth occurred below a pH of 6.0, while with the other substrates, ethanol and lactate, the optimum pH range for growth was very broad. The phosphorus accumulation by Acinetobacter

Fig. 2. The effect of the pH on the maximum specific growth rate (a) and phosphorus accumulation (b) during growth of Acinetobacter strain 210A on different substrates at 25°C: acetate (•), ethanol (•), lactate (O ); every point in 2b represents the mean value of several measurements with an average standard deviation of + 3.5 mg phosphorus per g dry biomass.

32 strain210 Awa sp Hindependen tabov ea p Ho f6. 5 (Fig.2b) ,a t lowerpH-value sa clea rp Heffec tcoul db eobserved .Growt ho n acetatea ta p Ho f6. 3resulte di na significantl yhighe ramoun t ofphosphoru s inth ebiomas s compared to its content athighe r pH's. Inthes eexperiment sth eJi max of 0.69h was notreache d probablybecaus eNH^C lwa sreplace db yNaNC^ .Thermodynami ccon siderationsindicat etha tmor eenerg yi sneede d forcel lsyn thesiswhe nnitrat ei suse da snitroge n sourceinstea do fammo nium(MoCart y1965) .

Polyphosphateaccumulatio ndurin gunlimite dgrowt h

In batch at pH 7.0 and 25C Acinetobacter strain 210A was growing exponentially forsevera lhour s (Fig.3) .Durin gthi s experiment,th ephosphoru sconten to fth ebiomas swa sconstantl y 58m gpe rg biomas sdr yweigh tan ddi dno tincreas eafte rgrowt h _ hadstopped .Continuou scultivatio na t)i max (0.69h ),p H7. 0 and25° Cresulte dals oi na phosphoru sconten to f5 8m gpe rg biomassdr yweigh t (Fig. 4).

1200

1000

0 2 4 6 8 v 23 time (hours) Fig. 3.Growt han d accumulationo fphosphoru sb yAcinetobacte rstrai n 210Ai nbatc hcultur ei nbasi cmediu ma t25° Can dp H7.0 .Th epresen teddat aar efro mon eexperimen ttha trepresent s5 othe rexperiment s withgrowt hrat edeviation so f4Z .

Theeffec to fsubstrat elimitatio no npolyphosphat eaccumulatio n byAcinetobacte rstrai n210 A

Theeffec to fth egrowt hrate .Th eaccumulatio no fpolyphosphat e inAcinetobacte rstrai n210 Aa t25° Cdepende do nth egrowt hrat e (Fig. 4).A t lowgrowt hrate so faroun d0. 1h thesubstrat ei n the chemostat was no longer detectable and the phosphorus

33 accumulation increased, reaching amaximu m of 83m gphosphoru s perg biomas sdr yweigh ta ta growt hrat eo f0.1 1h .

100 1.0

90 X16°C -0.9 -O 80- -0.8 S rj\_• 70 yzst 0.7 / 0.6 / •~ -• 50 • -0.5

__ -m 40 • •0.4

• ^^ • u 30 / yield 0.3

20-_ • 0.2

10 -0.1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 growth rate (h_1) Fig. 4. Phosphorus accumulation by continuously growing Acinetobacter strain 210A at various dilution rates under substrate limitation at pH 7.0 and 25°C and 16°C. The biomass yield is given for growth at 25°C. Every phosphorus content represents the mean value of several measure ments with an average standard deviation of 4 mg per g dry biomass.

Polyphosphate accumulation in absence of an extracellular substrate. A batch of Acinetobacter strain 210A was precultivated under phosphorus limitation until growth stopped. The cells were washed and subsequently suspended in the phosphate uptake medium. One g of these cells containing only 13 mg phos phorus was able to take up an additional 27 mg phosphorus from the medium within 9 hours, the largest part during the first 35 minutes (Fig. 5). Initially no polyphosphate granules were de tectable with Neisser staining but after 1 hour granules were clearly visible. KCN almost entirely inhibited phosphate uptake indicating that phosphate accumulation is somehow depending on energy from the respiration process (endogenous respiration). As the medium did not contain any energy source, electrons for respiration had to be derived from intracellular storage products, e.g. lipids or other polymers. Interestingly strepto-

34 50

streptomycin

-£ 40

10-

01 23456789 time (hours) FiB- 1L Effect of streptoniycinsulphate (200 mg.l"1) and 1 mM KCN on the phosphorus accumulation in cells of Acinetobacter strain 210A in a mineral medium in absence of an external energy substrate (mean values of two independent experiments).

mycin stimulated phosphate uptake (Fig. 5). Since protein syn thesis is an energy consuming process its inhibition by strepto mycin might increase the energy available for phosphate uptake and polyphosphate synthesis. Both the KCN and the streptomycin experiment indicate that energy, probably from respiration pro cesses, is used for polyphosphate synthesis.

The effect of nutrient limitation on polyphosphate accumulation by Acinetobacter strain 21QA

The effect of the growth rate during nitrogen limitation. Acine tobacter strain 210A was continuously cultivated under nitrogen + limitation. During this cultivation no NH4 was detectable in the supernatant after harvesting the cells and acetate was still present in considerable amounts, while the biomass concentration was significantly lower than found under substrate limitation. The phosphorus accumulation was independent of the growth rate (Fig.6). The nitrogen content of the biomass of the nitrogen limited cultures remained constant at 120 + 10 mg nitrogen per g

35 90

80 S-limited

60-

£ 50 N-limited

0.1 0.2 0.3 0.4 0.5 0.6 0.7 growth rate (h~1)

Fig. 6. Phosphorus accumulation by continuous cultivated Acinetobacter strain 210A under sulphur limitation and nitrogen limitation, both at pH 7.0 and 25°C. dry biomass over the whole range of growth rates, indicating that no carbon reserve material was formed. The experiment was repeated 2 additional times at a growth rate of 0.11 h~ , the cells contained 56 and 54 mg phosphorus per g dry biomass, thus similar amounts as in the first experiment.

Polyphosphate accumulation during a nitrogen limited stationary phase. An extra phosphorus accumulation was found in stationary cultures of Acinetobacter strain 210A during nitrogen limitation (Table 1). This stationary situation was reached after stopping the substrate addition (time = 0 h) to nitrogen limited cultures growing at a dilution rate of 0.31 h -1

Theeffec to fth egrowt hrat edurin gsulphu rlimitation .Durin g thecontinuou scultivatio no fAcinetobacte rstrai n210 Aunde r sulphurlimitatio nn othiosulphat ewa sdetectabl ei nth esuper natantafte rharvestin gth ecells ,acetat ewa s stillpresen ti n highamount san dth ebiomas sconcentratio nwa slowe rtha nfoun d undersubstrat e limitedcontinuou scultivation .Th ephosphoru s accumulationi nth ebiomas sdepende do nth egrowt hrate .I twa s highesta tlo wgrowt hrate s (88m gphosphoru spe rg dr ybiomas s

36 at a growth rate of 0.04 h_1) decreasing to 58 mg phosphorus per g dry biomass around the maximum growth rate (Fig. 6). To check the reproducability 5 independent experiments were carried out at 0.12 h_1. The cells contained 96, 88, 88, 81 and 74 mg phosphorus per g dry biomass.

Table 1. Accumulation of phosphorus by stationary cultures of Acinetobacter strain 210A precultivated in a chemostat under nitrogen limitation at D = 0.31 h . Medium and substrate addition were stopped at time -Oh. The data presented are from one experiment which was representative of 3 similar experiments.

Time acetate Biomas s dryweigh t phos phorusconten t in themediu m biomass -1 (h) (mM) (m gl"1) (mg g drywt. )

0 17 480+ 1 0 53+ 2 0.75 15 490 65 1.75 11 500 65 2.75 6.3 507 74 4.50 0.5 525 71 5.75 0a 525 70 6.75 0 530 68

a: detection limit for acetate was 0.05 mM

Table 2. Accumulation of phosphorus by cultures of Acinetobacter strain 210A precultivated in a chemostat under sulphur limitation at D = 0.20 h . Medium and substrate addition were stopped at time = 0 h. The presented data are from one experiment which was representative of 3 similar experiments.

Time acetate Biomassdr y weight P--contentbiomas s in themediu m _1 (h) (mM) (mgl" 1) (mg g drywt. )

0 42 655+ 10 64+ 2 4 31 810 71 8 22 880 76 13 15 965 77 24 4.5 1060 79 28 1.5 1070 80 32 0a 1080 80 80 0 1010 80

a: detection limit for acetate was 0.05 mM

37 Polyphosphate accumulation during prolonged sulphurstarvation . Aftercontinuou scultivatio no fAcinetobacte r strain210 Aunde r sulphurlimitatio na ta growt hrat eo f0.2 0h thesubstrat e additionwa sstopped .A ttha ttim eacetat ewa sstil lpresen ti n themedium .Th ecultur ewa sstil labl et ogro wslowly ,an dth e cellularphosphoru sconten tincrease dt oa hig hleve l (Table 2). Growthwa smeasure db ymean so fODgg gan db ydeterminin gth edr y weight.

DISCUSSION

Growtho fAcinetobacte rsp .

Thetemperatur e optimafo rgrowt ho fth eAcinetobacte r strains investigatedrange dfro m2 5t o33° C(Fig .la) .Thi sagree swit h temperature optima forAcinetobacte r sp.strain sisolate d from soil, sedimentsan dactivate d sludgereporte db yothe r authors (Abbote tal . 1973,Fuh san dChe n1975 ,D uPree z1978 ,Ha o& Chang1987) .Growt hi spossibl ebetwee na p Ho f4. 8an d9.5 ,bu t growtho nacetat ei scompletel yinhibite da ta p Hlowe rtha n6 (Fig.2a) .Accordin gt oPir t(1975 )th etoxicit yo fwea kacid s sucha saceti caci di softe np Hdependent .Th efre eaci di smor e lipidsolubl etha nth eionize d form,henc ereductio ni np Hwil l favourfre ediffusio no fth emor ehydrophobi caci dint oth ecel l affectings oth ep Hgradien tacros sth emembrane .Thi seffec ti s strongerfo raceti caci dtha nfo rlacti cacid ,becaus eaceti c acidi sa weake raci dan dmor ehydrophobi ctha nlacti caci d(lo g Pocto flacti caci d= -0.6 2an dlo gP octo faceti caci d= -0.17 ; Pocti sth equotien to fdistributio no fa compoun dbetwee nocta - nolan dwater ;Verschuere n 1983).Highe rhydrophobicit yfacili tatesdiffusio nthroug hth ecellmembrane .

Polyphosphateaccumulatio nb yAcinetobacte rsp .

Theamoun to fpolyphosphat eaccumulate di nAcinetobacte rsp . dependso ntemperature ,pH ,natur eo fth ecarbo nan dnitroge n source,growt hrate ,growt hphase ,limitation so fdifferen tele ments,an do nth eselecte d Acinetobacter sp.strains .I ti s possiblet ounderstan dmos to fth eabov edescribe d observations ifth eassumptio ni smad etha tpolyphosphat eaccumulatio ni sth e resulto fth eproductio no fexces senerg yi nth ecell ,o rth e inhibitiono fth esynthesi so fothe rpolymers .A spolyphosphat e inAcinetobacte rsp .ca nac ta sa nenerg yreserv ei nviv o(Va n Groenestijne tal .1988 , submitted),it saccumulatio nma yobe y

38 the criterion stated byWilkinso n (1959)tha ta nenerg yreserv e is accumulated if the energy supply is in excess of that requiredb yth ecel lfo rgrowt han drelate dprocesses .A nenerg y storageproduc t like glycogen for example is accumulated if excess energy and carbon is available in the cell (Dawes & Senior 1973). Itca nb eexpecte dtha tth ecriteri a forpolyphos phatestorag ear esurplu so fenerg yan dphosphoru si nth ecell . Polyphosphate inAcinetobacte rsp .ca nb eaccumulate d onth e expenceo fa nextra -o rintracellula renerg ysourc e(Figs .3 an d 5). Inabsenc eo fa nextracellula r substrate intracellular com poundsar erespire dt osuppl yenerg yfo rmaintenanc ean dpoly phosphateaccumulation .I nth epresenc eo fstreptomyci nth enee d for energy formaintenanc e andbiosynthesi s willb elower ,an d thehighe ramount so faccumulate dpolyphosphat eunde rthes econ ditionsca nb eexplaine db ya navailabilit yo fa greate rsurplu s energy. Cultureso fAcinetobacte r strain210 Agrowin gwithou tnutri tionallimitatio na tthei rmaximu mrat eo nacetat ea tp H7. 0an d 25°Ccontai n5 8m gphosphoru spe rg biomas sdr yweigh t (Figs.3 , 4an d 6), butunde rcertai ncondition smor ephosphoru s canb e accumulated, at the same temperature and pH, using the same substrate.Dependen to nenvironmenta l conditions growing cells maygenerat emor eenerg ytha ni snecessar y forbiosynthesi san d maintenance.Th esurplu senerg yca nb ewaste d in the form of heatb ymean so f slip-reactions (energyspillin g reactions). This was found by Neijssel & Tempest (1976) in substrate (glucose)limite d continuous growing cultures of Klebsiella aeroqer.eswhic h were carbon limited butnot energy limited. Insteado fwastin gthi ssurplu senerg yb ymean so fslip-reac tions the formation of anenerg yreserv e such as polyphosphate mayb euse db yAcinetobacte rstrai n210 Aa sa nenerg ysink . Excessenerg yca nb eobtaine dwhe ncel lsynthesi si slimite d byth elac ko fcertai nessentia lnutrients ;e.g .sulphu rlimita tion(Fig .6 )o rnitroge nan dsulphu rdepletio n(Tabl e1 an d2) . Cultureso fAcinetobacte rstrai n210 Agrowin gcontinuousl yunde r sulphurlimitatio nwil lrestric tthei rbiosynthesi sa tlo wdilu - tionrates inth ebioreacto rdu et osulphu rstarvation .At lower growthrate sth eenerg ygenerate d fromth eextracellula r energy sourcewil lfor ma large rexcess ,givin gris et oth eformatio n oflarge ramount so fpolyphosphat ea sa nenerg ysin kproduct ,a s canb eobserve di nFig .6 .A simila robservatio nha sbee nrepor ted by Wilkinson & Munro (1967). Bacillus meqaterium only accumulated polyphosphate at low dilution rates (0.11 h ) duringsulphu rlimitation .Thi seffec tma yb ecomparabl ewit h the accumulation of increasing amounts ofglycoge nb ynitroge n

39 limited microorganisms atdecreasin g growth rates (Dawes & Senior 1973).Th eobservatio ntha tnitroge nlimite d cultureso f Acinetobacter strain 210A don't accumulate the same large amountso fpolyphosphat ea tlo wgrowt hrate sa ssulphu rlimite d culturesma yb edu et odifferen tresponse so fth eregulator y mechanismso fassimilatio n anddissimilatio n when nitrogenin steado fsulphu ri sdeficien t(Fig . 6). Ina stationar yphas eafte rnitroge no rsulphu rdepletion ,a largeexces so fcellula renerg yca nb eexpected ,whil ebiosyn thesiswil lb estrongl yreduce db yth ecomplet eabsenc eo fth e assimilablefor mo fth estructura lelement snitroge nan dsulphu r inth emedium .Unde rthes econdition sAcinetobacte r strain210 A isabl et otak eu pa nadditiona lamoun to fphosphoru sabov eth e amounttha ti salread ypresen ti nth ebiomas s (Tables1 an d2) . A similareffec tha sbee nfoun di nEnterobacte r (formerlyAero - bacteraeroqene swhic haccumulate dpolyphosphat eunde rnitroge n orsulphu rstarvatio ni nth epresenc eo fexces scarbo nsource . Thesecondition sreduce dnuclei caci d synthesis and favoured polyphosphateaccumulatio n (Harold1963) .Contrar yt oAcineto bactersp .thi sorganis mwa sno tabl et oaccumulat epolyphos phateunde rsubstrat elimitation . Interestingly,Acinetobacte r strain 210Ashow sa maximu m of polyphosphate accumulation under substrate limitation around a dilutionrate of 0.11 h (Fig- 4).Accordin g to Linton and Stephenson(1978 )growt ho nlimite d amountso facetat emean s energy limitationan dno tcarbo nlimitation ,whic hmake spoly phosphateaccumulatio na tthes eenerg yrestricte d conditions difficultt ounderstand . Specificregulator ymechanism sa spar t ofa surviva lstrateg yma ycaus ea shif ti nth ebalanc eo fsyn thesiso fdifferen tbiopolymer si nfavou ro fpolyphosphat epro duction. Innatura l environments energy limitation canb ea precursor of energydepletion . Inth eabsenc eo fa nexterna l energysourc epolyphosphat ewil lb eneede da sa nenerg yreserve . Asimila reffec tha sbee nfoun db yWilkinso nan dMunr o(1967 ) forth eaccumulatio no fpoly --hydroxybutyrat ei nBacillu smega - terium growingunde rsubstrat e (glucose)limitation .A maximum accumulationwa sfoun da ta growt hrat eo f0. 4h ~ ,a t0. 8h _1 twotime s lesspoly --hydroxybutyrat ewa spresen ti nth ecells . Thedecreas eo f thephosphoru s content inAcinetobacte r strain 210Aa tgrowt hrate slowe rtha n0. 1h _1 atwhic hals oa decreas e ofth eyiel doccurs ,ma yb ecause db yth eregulator yeffect so f alarmones.Th e concentrationo fthes eregulator y nucleotides increaseswhe nth eyiel dbecome slowe rtha n75 %o fit smaximu m (VanVersevel de tal . 1984).Thes ecompound smobilis ecarbo nan d energyreserve s(Rickenber g1974 )an dca ninhibi tsom eenerg y

40 consuming processes like RNA synthesis (Gallant 1979) and maybe also the production of polyphosphate. Polyphosphate accumulation by Acinetobacter sp. is strongly dependent on temperature and to a less extent on pH (Figs, lb and 2b). If it is assumed that the amount of accumulated poly phosphate reflects the imbalance between energy generation and biosynthesis, then the overall effect of temperature and pH can be based on effects on energy generation, synthesis of structural biopolymers and synthesis of polyphosphate. These effects can be different for every strain, e.g. the polyphos phate accumulation in Acinetobacter strains 210A, B8 and P is influenced by temperature in different ways (Figs, lb and 2b). Probably the effect of the growth rate on polyphosphate accumulation shows the same strain specificity, since Hao & Chang (1987) found an increase of polyphosphate accumulation with increasing growth rates of their Acinetobacter sp., which is contrary to the effect found in Acinetobacter strain 210A. From a fundamental point of view the differences between the strains are confusing, but from a practical point of view this means that biological phosphate removal from wastewater may be possible under divergent conditions, and is always based on Acinetobacter strains that are best adapted to the prevailing conditions. For example excellent biological phosphate removal from wastewater has been found at 5°C (Krichten et al. 1985) as well as at 20°C (Meganck et al. 1985).

ACKNOWLEDGEMENTS

We thank Prof. Dr. O.M. Neijssel for his helpful advice. This research was supported by a special grant from Wageningen Agri cultural University.

REFERENCES

Abbot, B.J., Laskin, A.I. & McCoy, C.J. (1973) Growth of Acine tobacter calcoaceticus on ethanol. Appl. Microbiol. 25:787- 792 Adamse, A.D. (1980) New isolation of Clostridium aceticum (Wieringa). Antonie van Leeuwenhoek 46:523-531 Barnard, J.L. (1976) A review of biological phosphorus removal in the activated sludge process. Water SA 2:136-144 Buchan, L. (1983) Possible biological mechanism of phosphorus removal. Wat. Sci. Techn. 15:87-103

41 Dawes, E.A. & Senior, P.J. (1973) The role and regulation of energy reserve polymers in micro-organisms. Adv. Microbiol. Physiol. 10:135-266 Deinema, M.H., Habets, L.H.A., Scholten, J., Turkstra, E. & Webers, H.A.A.M. (1980) The accumulation of polyphosphate in Acinetobacter spp. FEMS Microbiol. Lett. 9:275-279 Du Preez, J.C. Si Toerien, D.F. (1978) The effect of temperature on the growth of Acinetobacter calcoaceticus. Water SA 4:10- 13 Fuhs, G.W. &Chen , M. (1975) Microbiological basis of phosphate removal in the activated sludge process for the treatment of wastewater. Microb. Ecol. 2:119-138 Gallant, J.A. (1979) Stringent control in E coli. Ann. Rev. Genet. 13:393-415 Groenestijn, J.W. van & Deinema, M.H. (1985) Effects of cultural conditions on phosphate accumulation and release by Acineto bacter strain 210A. In: J.N. Lester & P.W.W. Kirk (Eds), Proceedings of the international conference on management strategies for phosphorus in the environment, Lisbon (p.405- 410). Selper, London Groenestijn, J.W. van, Deinema, M.H. & Zehnder, A.J.B. (1987) ATP production from polyphosphate by Acinetobacter strain 210A. Arch. Microbiol. 148:14-19 Gurr, E. (1965) The rational use of dyes in biology, Leonard Hill, London Hao, O.J. &Chang , d.H. (1987) Kinetics of growth and phosphate uptake in pure cultures studies of Acinetobacter species. Biotechnol. Bioengg. 29:819-831 Harold, F.M. (1963) Accumulation of inorganic polyphosphate in Aerobacter aerogenes. I. Relationship to growth and nucleic acid synthesis. J. Bact. 86:216-221 Krichten, D.J., Hong, S.N. & Tracy. K.D. (1985) Applied bio logical phosphorus removal technology for manicipal waste water treatment by the A/0 process. In: J.N. Lester &P.W.W . Kirk (Eds),Proceedings of the international conference on management strategies for phosphorus in the environment, Lis bon (p.405-410). Selper, London Kulaev, I. (1979) The biochemistry of inorganic polyphosphates. John Wiley and Sons, Chichester, New York, Brisbane, Toronto Linton, J.D. & Stephenson, R.J. (1978) A preliminary study on growth yields in relation to the carbon and energy content of various organic growth substrates. FEMS Microbiol. Lett. 3:95-98 McCarty, P.L. (1965) Thermodynamics of bacterial synthesis and growth. Proceedings second international conference on water

42 pollution research (p.169-199). Pergamon, New York Meganck, M., Malnou, D., Le Flohic, P., Faup, G.M. &Rovel , J.M. (1984) The importance of the acidogenic microflore in biolo gical phosphorus removal. Wat. Sci. Techn. 17:199-212 Neijssel, O.M. &Tempest , D.W. (1976) The role of energy-spil ling reactions in the growth of Klebsiella aeroqenes NCTC 418 in aerobic chemostat culture. Arch. Microbiol. 110:305-311 Pirt, S.J. (1975) Principles of microbe and cell cultivation. Blackwell Scientific Publications, Oxford London Edinburgh Melbourne Rensink, J.H., Donker, H.J. &D e Vries, H.P. (1981) Biological P-removal in domestic wastewater by the activated sludge process. Proceedings 5th European sewage and refuse sympo sium. EAS, Munich 487-502 Rickenberg, H.V. (1974) Cyclic AMP in prokaryotes. Ann. Rev. Microbiol. 28:353-369 Sorbo, B. (1957) A colorimetric method for the determination of thiosulfate. Biochim. Biophys. Acta 23:412-416 Standard methods for the examination of water and wastewater 14th edn, 1976. Am. Publ. Health Ass., Washington Verschueren, K. (1983) Handbook of environmental data on organic chemicals, 2nd ed.. Van Nostrand Reinhold Company, New York Verseveld, H.W. van, Arbridge, M. & Chesbro, W.R. (1984) Con tinuous culture of bacteria with biomass retention. Trends Biotechnol. 2:8-12 Wilkinson, J.F. (1959) The problem of energy-storage compounds in bacteria. Exptl. Cell. Res. Suppl. 7:111-130 Wilkinson, J.F. &Munro , A.L.S. (1967) The influence of growth- limiting conditions on the synthesis of possible carbon and energy storage polymers in Bacillus meqaterium. In: E.D. Powell, C.G.T. Evans, R.E. Strange & D.W. Tempest (eds) Microbial physiology and continuous culture. HMSO, London, 173-185

43 CHAPTER 3

THE ROLE OF CATIONS IN THE ACCUMULATION AND RELEASE OF PHOSPHATE BY ACINETOBACTER STRAIN 210A

Johan W. van Groenestijn, Gerard J.F.M. Vlekke, Desiree M.E. Anink, Maria H. Deinema and Alexander J.B. Zehnder

ABSTRACT

Cells of the strictly aerobic Acinetobacter strain 210A, con taining aerobically large amounts of polyphosphate (100 mg phos phorus per g dry biomass), released in absence of oxygen 1.49 mmol inorganic phosphate, 0.77 mEg Mg2+, 0.48 mEq K+, 0.02 mEg 2+ + Ca and 0.14 mEq NH4 per g dry biomass. The pH drop during this anaerobic phase was caused by the extrusion of 1.8 protons per PO4 ~ molecule. Cells of Acinetobacter strain 132 which did not accumulate polyphosphate aerobically, released per g dry O-i- biomass only 0.33 mmol ortho-phosphate and 0.13 mEq Mg , but amounts of K+ comparable to strain 210A. Stationary cultures of Acinetobacter strain 210A in which polyphosphate could not be detected with Neisser staining were taking up aerobically phos phate simultaneously with Mg , the most important counterion in polyphosphate. In absence of dissolved phosphate in the medium no Mg was taken up. Cells containing polyphosphate granules were able to grow in a Mg-free medium, while cells without these granules were not. Mg was not essential as counterion, because it could be replaced by Ca . The presence of small amounts of K+ was essential for polyphosphate formation in cells of strain 210A. In batch experiments with media without K , Acinetobacter strain 210A took up 59% less phosphate, in spite of a higher cellular ATP content during this K depletion. During continuous cultivation under K+-limitation, cells of Acinetobacter strain 210A contained only 14 mg phosphorus per g dry biomass, while this element was accumulated in amounts of 59 mg per g dry biomass under substrate limitation and 41 mg per g dry biomass under Mg -limitation. For phosphate uptake in acti vated sludge the presence of K seems to be crucial.

submitted for publication.

45 INTRODUCTION

Enhancedbiologica l phosphate removal from wastewater ,a biologicalalternativ et ochemica lphosphat eprecipitation ,i s based onth eenrichmen to factivate d sludgewit hpolyphosphat e accumulatingbacteri a (12).Acinetobacte r isth emos timportan t genusi nthi sproces s(4 )an dsom estudie so nit spolyphosphat e accumulationan ddegradatio nhav ebee ncarrie dou trecentl yi n pureculture s (8,12 ,14 ,21) .Jus tlik eactivate d sludgei n which biological phosphate removal was observed,Acinetobacte r takesu pphosphat eunde raerobi c conditions andrelease s it anaerobically (9).Polyphosphat e inAcinetobacte r ispresen t in granules(12 )an dth enegativ echarg eo fthi spolyanio ni sneu tralizedb ycation s (4,13) .A sa resul t simultaneously with phosphatea nuptak ean dreleas eo fcation sca nb eexpected .I n facti nwastewate rtreatmen tplant swit hbiologica l phosphate removal thiseffec tha salread ybee nobserved . Inth eanaerobi c zoneso fthes eplant sphosphat ei srelease d togetherwit h Mg2+ andK + (3,6 ,19 ,26) ,an ddurin gaerobi ccondition sth ephos - phatei stake nu pagai nsimultaneousl y with Mg and K and smallamount so fCa 2+ (6,26) .Kainrat he tal .(17 )analyse dth e drymatte ro fth esludg efro m thebiologica lphosphat eremova l processan dfoun da correlatio no fth etota lP-conten twit hM g andK +bu tno twit hC a ,Fe 3+an dA l .Fro mthes eobservation s itwa sconclude dtha tth eenhance dbiologica lphosphoru sremova l fromwast ewate rwa sindee dmainl ya biologica lprocess ,wit h chemicalprecipitatio nplayin ga mino rrole .However ,i tcoul d bepossibl etha twit hanothe rcompositio no fth einfluent ,e.g . water with highcalciu m concentrations, chemicalprecipitatio n mayb emor eimportant .A nexampl eo fa combine dchemica lan d biologicalphosphoru sremova lha sbee nreporte d byArvi nan d Kristensen (3). Theyfoun dtha tdurin ganaerobi ccondition sth e phosphatereleas efro mth esludg ewa salway saccompanie db ya releaseo fK + andM g ,an d adecreas eo f the Ca concen tration.I nth etw oplant sinvestigated ,50-60 %o fth ephosphat e inth esludg ewa sprecipitate dan dth edominatin gcounterion si n the precipitate were Ca^ (high amounts) and Mg (lower amounts).Fro m theseobservation s itmight b econclude d that onlyMg * andK areimportan tcounterion s inbiologica lphos phateremoval ,howeve rthi si sa tvarianc ewit hth efinding so f Buchan (4).B ymean so fenerg ydispersiv eanalyse swit hX-ray s (EDAX)o f theelectrondens e granules in sludge, this author proved thatcalciu m playsa predominan t rolei nth estabilisa tiono fpolyphosphate-ion san dtha ta calciu mrequiremen tfo r enhancedphosphoru suptak edoe sno tnecessaril ypoin tt ochemi -

46 calprecipitation . Todistinguis h betweenbiologica l andchemica l removal of phosphate and cations a study was undertaken with two pure cultureso fth eAcinetobacter ,namel y strain210 Aan d 132,bot h isolatedfro mactivate dsludg e(strai n210 Adi daccumulat ephos phatei nfor mo fpolyphosphat ewherea s 132di d not). Thispape r reportsth eresult so fth eexperiment so nth eeffec to fK ,Mg -' and Ca2+ onpolyphosphat e formation anddegradatio n aswel l as onth erol eo fpolyphosphat ea spossibl ecatio n (Mg )reserve .

MATERIALSAN DMETHOD S

Organisms.Acinetobacte r strain 210Aan d strain 132wer eiso lated fromactivate d sludgeb yth emetho d described by Deinema etal . (8).Strai n210 Ai sabl et oaccumulat elarg eamount so f polyphosphate,whil estrai n13 2i snot .Th eorganism swer emain tained onyeast extrac taga r slants (5g glucose ,2. 5g yeas t extractan d1 2g aga rpe rlitr etapwater ,p H= 7), subcultured everytw omonth san dstore da t4°C . Media.- Mediu mfo rbatc hcultivatio n containing butyratea s carbonan denerg ysourc ewa sprepare daccordin gt oVa nGroene - stijn et al. (14).Mediu m for batch cultivation containing acetatea scarbo nan denerg ysourc ewa sth esam ea sth emediu m withbutyrat eexcep ttha tNa-butyrat ewa sreplace db y5.6 7g Na - acetate.3H20.Fo roptima lgrowt han dpolyphosphat eaccumulation , theacetat ean dbutyrat emediu mwer eused ,respectivel y(13) . -Mediu m for cultivation under phosphorus limitation contained per litre demineralized water: 7 g Na-acetate.3H20, 0.1 g

Na2S203.2H20, 1g NH 4C1,0. 2g MgS0 4.7H20,4 0m gKH 2P04,0.0 6g CaCl2.2H20,2 m ltrac eelement s(14 )an d 12g Tris .Th ep Hwa s adjustedt o7. 0wit h6 N HC1 . -Phosphat ereleas emediu m containedpe rlitr edemineralize d water: 2.5g aceti caci dan d 4g Tris .Th ep Hwa s adjusted to 7.5wit h 6N HC1 . -Unbuffere d phosphate release medium (used inth eexperiment s inwhic hth ep Hdecreas ecause db yphosphat eefflu xwa sstudied ) containedpe rlitr edemineralize dwater :2 0mmo lbutano lan d6 g NaCl.Th ep Hwa sadjuste dt o7. 5wit h0. 1N NaOH .Butanol ,als o a substrateo fAcinetobacte r strain210A ,wa schose nbecaus ei t doesno tbuffe rth emediu ma sacetat eo rbutyrat ewoul ddo . -Phosphat euptak emediu m forAcinetobacte r contained perlitr e demineralized water: 136 mg KH2P04, 11 mg NH4C1, 44 mg CaCl2.2H20,6 1m gMgCl 2.6H20,1 2m gNaCl ,30 0m gaceti cacid ,6 g Trisan d 200m g streptomycinsulphate.Th ep Hwa s adjusted to

47 7.0wit h 6N HC1 .Experiment swit hvariou sconcentration so fK + were carried out replacing KH2P04 by equimolar amounts of + NaH2P04.2H 20.K wa sadde da sKC1 . -Phosphat euptak emediu m foractivate d sludgecontaine dpe r litre demineralized water: 0.6 g MgS04.7H20, 0.25 g NaH2P04.2H20, 1g NH4C1, 0.07 gCaCl 2.2H20, 1.5 g Tris and variousamount so fKC1 .Th ep Hwa sadjuste dt o7. 0wit h6 NHC1 . -Mediu mwithou tMg 2+ (usedi nth eMg-reserv ebatc hexperiments ) containedpe rlitr edemineralize dwater :5.6 7g Na-acetate.3H 20, 1g NH 4C1,0.4 4g KH 2P04,0. 1g Na 2S203.5H20,0.0 7g CaCl 2an d2 mlo fa trac eminera lsolutio n (14).Th ep Hwa sbrough tt o7. 0 with6 N HC1 . Continuouscultivatio nunde r substratean dcatio nlimitatio n wascarrie dou twit hth efollowin gmedia : -Substrat elimitation :Basi cmediu mcontaine dpe rlitr edemine ralized water: 5.67 g Na-acetate.3H20, 1 g NH4CI, 0.6 g MgS04.7H20,0.4 4g KH 2P04,0. 1g Na 2S203.5H20,0.0 7g CaCl 2an d 2m lo fa trac eminera l solution (containing 50g EDTA , 5g FeS04.7H20, 1.6 g CuS04.5H20, 5 g MnCl2.4H20, 1.1 g (NH4)6Mo7024.4H20,2. 2g ZnS0 4.7H20,5 0m gH3BO3 ,1 0m gK Ian d 50m g CoCl2.6H20pe r litredemineralize d water). Thep Hwa s broughtt o7. 0wit h6 N HC1 .Th etrac eminera lsolutio nuse di n thisexperimen tdiffere dfro mtha tuse di nth ebatc hexperi ments,becaus eo fpractica lreasons :n oprecipitatio nwa sforme d afterextende d sterilization of thecomplet emediu m in1 01 carboys.

- M.g* -limitation:Basi cmediu mwit honl y8 0m gMgS0 4.7H20pe r litre. -K -limitation :Basi cmedium ,howeve rKH 2P04wa sreplace db y equimolaramount so fNaH 2P04.2H20.1 5m gKC 1wa sadded . Phosphatereleas e experiments.Acinetobacte rstrai n210 Aan d strain13 2wer egrow na t15° Ci nshake nErlenmeye rflask si na mediumcontainin gbutyrat ea scarbo nan denerg ysource .Culture s from the log-phaseo f strain 210Acontainin g 100m g phosphorus perg biomas sdr yweigh tan do fstrai n13 2containin gonl y2 6m g phosphoruspe rg biomas sdr yweigh twer ecentrifuge dan dwashe d withdemineralize dwater .Th epellet swer eresuspende di nse - rumvials containing aphosphat ereleas emedium . Thevial swer e made free of oxygen by flushing them with agas-mixtur e of N2:C02 (99%:1%,v/v) .Befor eincubatio nsample swer etake nwit h a syringefo rth edeterminatio no fth ebiomas scontent .Th ese - rumvialswer eplace di na shake r(t okee pth ebiomas si nsus pension)a t 30°C.At time intervals aliquots were takenan d centrifuged.Th esupernatan twa sanalyse dfo rK +,C a ,M g , + + NH4 , Na andortho-phosphate .

48 Phosphateuptak eexperiments .Cell so fAcinetobacte r strain 210A were precultured under P-limitation at 25°C in shaken Erlenmeyerflask sunti lgrowt hstopped .A ttha tmomen tn opoly phosphategranule scoul db esee ni nstrai n210 Aafte rstainin g followingth emetho do fNeisse r (15).Grow nculture swer ecen - trifugedan dwashe dtwic ewit ha Tris/HC lbuffer ,an dth epel letswer eresuspende d ina phosphat euptak emedium ,t oobtai na biomassconcentratio no f0. 5g dr yweigh tpe rlitre .Th eacetat e inthi smediu mserve da sth eenerg ysourc efo rth euptak epro cesses,whil estreptomyci nwa sadde dt opreven tgrowt ho fth e cells. During the experiments thedr yweigh t content of the biomassincrease db yabou t8% ,whic hwa sth eresul to fphosphat e andcation suptake .Afte rth ephosphat ewa stake nu ppolyphos phategranule s werevisibl ewit h Neisser staining. Experiments wereals ocarrie dou ti nphosphat euptak emedi afro mwhic hK , 9+ 9+ Mg ,C a orortho-phosphat ewer eomitted .I naddition ,th eAT P contento fth ecells ,expose dt ovariou sK + concentrations,wa s measured after 0.5hour s ofincubation . The uptake of phosphate by activated sludge in a mineral mediumwit hvaryin gK concentrationswa sinvestigate da swell . Sludge was obtained from anintermittentl y aerated labscale pilotplan twhic hbiologicall yremove dphosphat efro m synthetic wastewate r (2).Whe nth esludg esampl ewa stake na tth een do f theanaerobi cperio dphosphat eha dbee nrelease dfro m thecells . Toremov eadsorbe dK fromth esludge ,i twa scentrifuge dan d washed twice at 4°C with a solution of 9 g NaCl per litre demineralized water.Th epelle twa sresuspende d ina minera l phosphate uptake medium. All experiments with sludge were carriedou ti nErlenmeye r flasksa t25° Co na rotar yshake r(20 0 rpm).A ttim einterval ssample swer etake nt omeasur eth eortho - phosphatean ddr yweight . Continuous cultivation.Acinetobacte r strain 210Awa scon - +~~ 9+ tinuouslycultivate dunde rsubstrate ,K - o rM g -limitationi n a 21 bioreacto r (Applikon, Schiedam, TheNetherlands )wit h automaticallycontrolle dp H(7.0 )an dtemperatur e (25°C).Whe n cultures had reached a steady state,sample s were taken and + 9+ centrifuged.Th esupernatan twa sanalyse dfo rK ,M g ,ortho - phosphatean dacetate ,th epelle twa swashe dwit hdemineralize d wateran duse dfo rth edeterminatio no fth edr yweigh tcontent . Mg-reserveexperiments .Cell so fAcinetobacte r strain 210A, containing7 5m gP pe rg biomas sdr yweight ,wer ecultivate di n batch in a medium containing acetate as carbon and energy source, harvested,washe d withdemineralize d wateran dcentri fuged as described in thephosphat ereleas eexperiments . Phos phoruspoo rcell swit honl y2 0m g Ppe rg biomas s dry weight 49 wereobtaine da sdescribe dabov ei nth ephosphat euptak eexperi ments.Al lpreculture s were grown at 15°C.Th epellet s were resuspended ina mediu mwithou tMg 2+wit hacetat ea scarbo nan d energysource .Th ecel lsuspension swer eshake ni nErlenmeye r flasksa t25°C .A ttim einterval ssample swer etake nt oestimat e thebiomas sdr yweight . Reproduceability.Figs .1 ,3 an d4 an dTabl e1 sho wdat afro m oneexperimen ttha trepresent sothe rexperiment s with thesam e procedure.Althoug hth erate san dquantitie svarie dconsiderabl y between the different experiments (the most extreme cases differedb y 30%),th estoichiometr ybetwee nphosphat ean dth e various cationsan damon g thecation sthemselve s remainedcon stanti neac hexperiment .Th edat apresente di nFig .4 dar emea n valuesfro mexperiment si nfivefol di nwhic hthre eseperatel y grownculture swer euse dtha ttoo ku p0.8 5+ 0.0 7mmo lphosphat e perg dr ybiomass . Chemicals.Streptomycinsulphat ewa sobtaine dfro mSigm aChemi calCo. ,St.Louis ,MO .Th eAT Pbioluminescenc eCL Stest-combina tion including luciferase/luciferin reagent and Na2ATPwa sob tainedfro mBoehringer ,Mannheim ,W-Germany . Analyses.Ortho-phosphat ewa sdetermine dspectrophotometrical - lyaccordin gt oStandar dMethod s (24)usin gth eascorbi caci d method.K +,M g , Car,+an dNa +wer equantifie dusin ga natomi c + absorptionspectrophotometer .NH 4 wa sanalyse dwit hth eNessle r reagentaccordin gt oStandar dMethod s (24).Acetat ewa smeasure d afteradditio no famberlit eIR-120 Hionexchange rusin gGL Cb y meanso fa Varia n240 0Aerograp h (1).Bacteria ldr yweight wa s determined after centrifugation of a 50 ml sample of the culture,washin gth epelle tonc ewit hdemineralize dwate ran d dryinga t100° Covernight .Dr yweight swer eals oestimate dfro m opticaldensit ya t66 0n mb yusin ga calibrationcurv e (ODggQa s functiono fdr y weight). TheAT P content of the culture was quantifiedaccordin gt oPrade t (22).Sample so f0. 5m lcultur e wereboile dfo r4 minute si n5 m lo fa solutio ncontainin g5 0m M Tris/HClan d4 m M EDTAa tp H7.7 5i norde rt oextrac tAT Pfro m thecell san dsto pal lenzym eactivity .Luciferase/luciferi n reagentwa sadde dt oa subsampl eo fthi ssolutio nan dth ere sultinglight-emissio nwa smeasure dwit h a luminescence-meter (madeb yth eElectroni cDesig nan dMaintenanc eSho pDivisio no f theGenera lResearc hDepartment ,Universit yo fGeorgia ,Athens , USA).Polyphosphat e was stained using themetho d of Neisser (15).

50 RESULTS

Release of phosphate and cations. The anaerobic efflux of + + phosphate, Mg , K , Ca and NH4 by cultures of Acinetobacter strain 210A containing 100 mg phosphorus per g dry biomass is 9+ + presented inFig .1 .M g andK wereth emos timportan tcation s released under anaerobic conditions. For comparison theex - trusiono fphosphate ,M g andK byAcinetobacte rstrai n132 , which isunabl et oaccumulat epolyphosphate ,wa sals odeter mined.Strai n13 2release danaerobicall yth esam eamoun to fK asstrai n210 Abu tmuc h lessphosphat ean dMg 2+ (Fig. 1).Th e releaseo fK + in strain 210A seems tob e independent of the phosphaterelease ,becaus eK +wa sextrude dwit ha highe rinitia l ratetha nphosphat ean dM g (Fig.l)an da tlo wp Hth eefflu xo f K+ was lessinhibite d thanth ereleas eo fphosphat e(dat ano t shown).A tp H5. 9an d30° Con eg biomas srelease danaerobicall y within threehour s 0.13mmo lK +bu tonl y0.0 5mmo lphosphate . 1.5 • b a a E strain 210A strain 132

oc t5

o a. C a

Ik 0 time (hours)

2+ + + 2+ FIG. 1. Release of ?i (1 mEq = 1 mmol), Mg , K , NH4 and Ca by Acinetobacter strain 210A and strain 132 under anaerobic conditions at 30°C and pH 7.5. The presented data were determined in one experiment and are representative for five (strain 210A) or three (strain 132) other experiments.

In a phosphate release medium without a Tris-HCl buffer the pH decreased from 7.4 to 6.4 within 6 hours while 10.5 mg ortho- phosphate-phosphorus per litre (0.34 mM) was released by cells

51 ofstrai n210 A(Fig .2) .I na contro lexperimen twit hth esam e mediumbu twithou tbiomas sth ep Hdecrease dfro m7. 4t o6. 4b y theadditio no f0.2 8m M KH2P04 plus0.0 6m M KNaHP04.Fro mthes e results canb e calculated thatth ep Hdro p is caused by the 3 extrusiono f 1.8proton spe rP0 4 ~molecule .

time (hours)

FIG. 2. Decrease of the pH during the release of phosphate by 625 mg.l-1 biomass of Acinetobacter strain 210A at 30°C in an unbuffered anaerobic phosphate release medium.

i/i i/) c° a

s cr LU e

time(hours )

FIG. 3. The uptake of phosphate (1 mEq = lmmol), Mg 2+ K+ and Ca2+ by stationary cultures of Acinetobacter strain 210A in a medium with acetate and streptomycin (pH = 7, 25°C).

52 Uptake of phosphate and cations. Concomintently with ortho- phosphate cations were taken up by a culture of Acinetobacter strain 210A devoid of polyphosphate (Fig. 3). This experiment has been carried out in a complete phosphate uptake medium. To prevent growth and thus interference between phosphate uptake for polyphosphate formation and phosphate incorporation in newly formed cells (biomass) streptomycin was added to the medium.

FIG. A. The uptake of phosphate (1 mEq - lmmol), Mg2+ , K+ and Ca2+ by stationary cultures of Acinetobacter strain 210A in a medium with acetate and streptomycin (pH - 7, 25°C) under different limitations a: without phosphate, b: without Ca 2+ c: without K , and d: without Mg2+. To measure the mutualistic relationship between phosphate and the different cations, individual cations were omitted from the complete uptake medium (Figs. 4a-d). Simultaneously with the uptake of ortho-phosphate, K+ and Mg2+ were also incorporated into the cells. All three ions, if present, were absorbed fast in the first hour (Figs. 3 and 4b-d). Later, the rate diminished + + steadily. The concentration of Na and NH4 did not change much and there was no correlation with phosphate uptake. The largest uptake or release of NH..+ and Na+ mostly occurred in the first 15 minutes (results not presented). Mgz correlated best with the amount of phosphate that was accumulated, if no phosphate was taken up by the cells, Mg2+ uptake was reduced strongly

53 9+ (Fig.4a) .Th eequivalent so fMg' ' was6 0- 100 %o fth eequiva lentso fphosphat etake nup .Th euptak eo fK +wa sindependen to f thephosphat eabsorption .I nabsenc eo fphosphat ei nth emediu m K+uptak eshowe dth esam ebehaviou ra si nth ecomplet emediu m (Figs.3 an d 4a).Th eabsenc eo fC a didno taffec tth euptak e ofphosphate ,Mg 2+an dK + (Fig.4b) ,whil ei nabsenc eo fK +les s phosphatean dMg 2+wer eabsorbe d(Fig .4c) .Th eomissio no f Mg2+ fromth emediu mdi dno treduc eth einflu xo fphosphate ,bu tCa 2 wastake nu pi nlarg eamount sinstea do fM g (Fig. 4d)2+. Ina separateexperiment , cellso fAcinetobacte r strain210 A containingn opolyphosphat ewer eresuspende di nth ephosphat e uptakemediu m atvariou s initial concentrations ofK .Thi s cationstimulate dth euptak erat eo fphosphat ean dincrease dth e finalconcentratio no fphosphat ei nth ecell s(Tabl e 1).Fo ra maximumphosphat euptake ,K +concentration shighe rtha n1 0mg.l - 1 werenecessary .Concentration so f1.0 ,1. 5an d3. 5m gK per litrewer eteste da swell ,bu tth eamount so fphosphat etake nu p atthes econcentratio ndi dno tdiffe rmuc h (±10%)fro mthos ea t 0.5 mg K+ per litre.Afte r 0.5hour s of incubation, theAT P contento fth ecell swa smeasured . Itdecrease dwit hincreasin g K+ concentrations,namel yfro m 7.2t o2. 6an d3. 0umo lAT Ppe rg drybiomas sa ta K + concentrationo f 0.5, 10an d 100 mg.l-1, respectively. TABLE1 . Theeffec to fth eextracellula rK concentrationo n theuptak eo fphosphat efro ma mediu mwit hacetat ean dstrepto mycina t25° Can dp H7b ystationar yculture so fAcinetobacte r strain210A .Th enumber sar egive ni nm gPO.- Ppe rg dr ybio mass.

K-concentration s(mg. l ) Time (h) 0.5 10 100

0.1 0.16 0.21 0.13 0.5 0.40 0.55 0.63 1 0.51 0.72 0.82 2 0.65 0.96 1.11 4 0.74 1.04 1.12

Thephosphat euptak eb yactivate dsludg eshowe da simila rdepen dencyo nth eadditio no fK + (Fig.5) ,bu ti nthes eexperiment sa lowerK + (=5 mg. l )concentratio nwa salread ysufficien tfo ra maximumphosphat euptake .Concentration so f12 ,25 ,4 0an d7 0m g K perlitr ewer eals oteste dwit hth esam esludg esample .Th e

54 phosphateuptak edi dno tdiffe rmor etha n30 %fro mth euptak e measured at 5 and 100 mg K+ per litre. A different sludge sample,take non ewee kearlie rfro mth epilo tplant ,accumulate d 30%mor ephosphat ei nth epresenc eo f10 0m gK +pe rlitre ,whil e thesam euptak eperformanc ea sb yth efirs tsludg esampl ewa s foundi nabsenc eo fK .

40-

100m g K\f1

o E o

en e

O CL

time(hours ) FIG. 5. The uptake of phosphate by activated sludge in an aerobic mineral medium with different initial K concentrations.

Polyphosphate accumulation in continuous cultures under various limitations. Acinetobacter strain 210A was continuously cultivated at different dilutionrates under substrate, K+- or Mg^ -limitation. The effect of these limitations on the accumulation of phosphate by the cells is presented in Table 2. During cultivation with limited amounts of cations always a steady state was reached in which acetate was still present in considerable amounts while the biomass concentration was not more than half of that found in substrate limited cultures. Cells cultivated under Mg -limitation were still able to accumulate considerable amounts of phosphate, while K+-limita-

55 TABLE2 .Growt h and P-content ofAcinetobacte rstrai n210 Ai na continuou scultur e 1 9j . witha nacetat emediu m under substrate,K orM g limitation.Th edat apresente d wereobtaine dunde rstead ystat econdition sa ttw odilutionrate sa t25° Can dp H7.0 . Limitingnutrien t Dilution- Biomass Acetate K+ Mg*+ P-content rate in biomass (h-1) (mgdr ywt .1 " h (mM) (mg.I' 1) (mg.g "1dr ywt. ) acetate(carbo n 0.09 624 0.2 12 59 59 andenerg ysource ) 0.18 844 0.3 14 53 66

Mg2+ 0.06 314 21 85 0.2 41 0.11 290 27 89 0.3 55

K+ 0.08 362 4.8 1.4 80 14 0.20 310 20 2.6 77 26

10 15 time(hours )

FIG.6 . Growtho fcell so fAcinetobacte r strain210 Awit h polyphos phate(positiv ewit hNeisse r staining;containin g 75m gP pe rg dr y biomass), suspended ina nacetat emediu m withoutM g (•), andcell s without polyphosphate (negative with Neisser staining; containing 15 mgP pe rg dr ybiomass) , suspended ina mediu mwithou t (O) o rwit h (•) Mg2+ (25°C,p H= 7) .

56 tionha da strongl ynegativ eeffec to nthi saccumulation ,an da t lowdilutio nrates ,wher eth estead ystat econcentratio nwa s lowest,n opolyphosphat ewa saccumulated :th ecell scontaine d onlysmal lamount so fphosphoru san ddi dno tcolou rwit hNeisse r staining.

Polyphosphategranule sa sa Mg-reserve .Tw odifferen tculture s ofAcinetobacte rstrai n210 Awer eresuspende d ina Mg 2 -free growthmedium .On ecultur econtainin g7 5m gphosphoru spe rg dr y biomasswa sabl et ogrow .Th esecon dcultur ewit honl y2 0m g phosphoruspe rg dr ybiomas sgre wconsiderabl yless .Cell sfro m thiscultur ewer eonl yabl et ogro wa thig hrate si na mediu mt o whichmagnesiu m wasadde d (Fig. 6).

DISCUSSION

9+ Theexperiment sreporte dher eindicat etha tMg' ' isth emos t importantcounterio no fpolyphosphat ei nAcinetobacte rstrai n 210A.I nthi sstrai nMg 2+wa salway srelease dan dtake nu psi multaneouslywit hphosphat e(Fig .1 an d4a-c) ,whil estrai n132 , whichdoesn' taccumulat e polyphosphate, released only small amountso fM g under the sameconditions . Inth e phosphate uptakeexperiment sever ymmo lphosphat ewa stake nu ptogethe r with0. 6t o1 mE gM g .Thi si senoug ht ocontrabalanc ea majo r parto fth enegativ echarg eo fpolyphosphate ,sinc eeac hphos phate group in this molecule carries one negative charge. Releaseo fphosphat eb ystrai n210 Awa sals ocouple d toa p H decreasei nth emediu m (Fig.2),whic hwa scause db yth eefflu x + of1. 8mmo lH permmo lP0 4 .Thi sapproache sth eamount o f protonspe rphosphat eexpecte ddurin gth enett ohydrolysi so f Mg-polyphosphate. Theuptak ean d release ofK +,however ,wa s independento f phosphate uptake and release, since (i)strai n 132 extruded underanaerobi ccondition sth esam eamoun to fK +a sstrai n210 A (Fig.1) ,(ii )K wasrelease dfaste rtha nphosphat eb ystrai n 210Aan dreache dit smaximu mconcentratio nmuc hearlie r(Fig.l) , (iii)a ta lo wp Hphosphat ereleas ewa sslowe rtha nth ereleas e ofK ,an d(iv )independen to fth epresenc eo rabsenc eo fphos phatestrai n210 Atoo ku pa smalle ramoun to fK + ascompare dt o phosphate(Figs .3 ,4a,4 ban d4d) .Th euptak ean dreleas eo fK + mightno tdirectl yb ecouple d toth eaccumulatio n anddegrada tiono fpolyphosphat ebu trathe rt oa reestablishmen to rdeclin e ofa specifi cenergeti cstat eo fth ecell .A swa sindicate db y Muldere tal . (20)th emaintenanc eo fa stee pK +gradien tacros s

57 thecell-membran ecost senergy .Sinc eAclnetobacte r isa stric t aerobe, energy supply under anaerobic conditions is restricted anda leakag eo fK + canb eexpected .Althoug hK +doe sno tpla ya quantitatively important rolea spolyphosphat e counterion in Acinetobacter strain 210A, the presence of K in the medium appearedt ob ea crucia lfacto rfo rphosphoru saccumulatio nb y thisstrai n (Table1 an d 2)a swel la si nactivate dsludg e(Fig . 5).Ther ecoul db eman yreason s forthi sdependency :K + could eitherpla y a role inphosphat etranspor t (18),o ri nenerg y generation (20),o ri nstimulatio no fenzyme-acitivitie s (10)o r mayb eo f importance forth estructur eo fpolyphosphat egra nules. The suboptimal polyphosphate accumulation during growth under K-limitatio n points in thedirectio n of energy shortage whichi si naccordanc et oth efinding so fMulde re tal . (20)an d Saile tal .(23) .Mulde re tal .(20 )reporte dtha t ^-limita tioncause senerg ylimitation ,an d Saile tal .(23 )foun dtha t K+-limited cellso fCorynebacteriu mdiphteria econsume d less oxygenan daccumulate d lessphosphate .However ,th ebatc hex perimentspresente di nTabl e1 i nwhic hphosphat euptak ean dAT P contenti nAcinetobacte r strain210 Awer emeasure d show that energyshortag ema yno tb eth eonl yreaso nfo rth einhibitio no f polyphosphate accumulation byK -limitation . At low K concen trationsles sphosphat e wastake nu pan d theuptak erat ewa s slowerbut th ecellula rAT Pconten twa shigher .A sAT Pi smos t probablyth eenerg yan dphosphat edono ri npolyphosphat esyn thesisb yAcinetobacte r sp.(25) ,a neffec to fK -limitatio n on phosphatetranspor tan denerg ygeneratio ncanno tb eth emajo r causeo f adecrease d polyphosphate accumulation under these conditions.Therefore ,K +mus thav ea tleas ton eothe rrol ei n polyphosphateaccumulatio ne.g .th estimulatio no fth eactivi ties of enzymes involved in polyphosphate synthesis or a structural role in the formation of polyphosphate granules. Furtherresearc h isneede d toelucidat eth erelatio n between polyphosphatean dK + inAcinetobacter . AlthoughMg ^ waspreferre da sa counterion ,thi scatio nwa s notessentia lfo rpolyphosphat eaccumulatio n byAcinetobacte r strain210A .I fM g wasno tavailabl ea sa counterio ni twa s partlyreplace db yCa 2+ (Fig.4d) .Phosphat eaccumulatio n was still possible when Mg supply was limited in continuous cultureso fAcinetobacte r strain210 A (Table2) . Fromth eresult so fth eexperiment so ngrowt ho fcell swit ho r without polyphosphate ina M g -freemedium , itca nb econ - eludedtha tth epolyphosphat egranule sca nals oac ta sa Mg' '- 2+ reserve.A function ofpolyphosphat e asa cation reserve has alwaysbee nhypothesize d butwa sdifficul tt oprove ,becaus e

58 mostmicroorganism saccumulatin gpolyphosphat econtai nrelative lysmal lamount so fthi scompound .A sAcinetobacte rstrai n210 A accumulatesexceptionall y largeamount so fpolyphosphat eth e role of this compound as a cation reserve could clearly be demonstrated. Many cations can serve as counterions inpolyphosphat e in differentmicroorganisms .I nMicrococcu s lysodeikticus (11)an d inDesulfovibri ogiqa s(16 )Mg 2+wa sth emost importan tpoly phosphatecounterion , while Mg and Ca2+ had been found in vacuolescontainin gpolyphosphat ei nNeurospor acrass a(7 )an d K+ was apolyphosphat e counterion inChlorell a sp. (5).Th e findingso fBucha n(4 )tha tCa 2+ isth epredominan tpolyphos phate counterion inacinetobacter s inactivate d sludge wasno t confirmedb you rexperiments .Sinc eBucha nuse dEDAX ,studie s havebee n carried outi nou r laboratory on the quantitative composition of polyphosphate granules using also the same technique. The first results indicate the presence of high amountso fMg 2+ andCa 2+ andminima lamount so fK + (unpublished results).On epossibl eexplanatio no fth edifference sbetwee n EDAXstudie san dreleas eexperiment si stha tCa 2+ isa polyphos phatecounterio ni nAcinetobacte rsp .whic hi sno trelease dwhe n thepolyphosphat ei sbroke ndow n(Ca 2+might sta yi nth ecel l whileortho-phosphat e isreleased) . Inconclusio ni tca nb esai dtha tth euptak ean dreleas eo f cationsb ypur eculture so fAcinetobacte r strain 210A is in accordancewit h theresult sobtaine d inexperiment s withacti vatedsludg efro m wastewate rtreatmen tplant s showing bio logicalphosphat eremoval .M g hasbee nreporte da sth emos t important counterion during phosphate uptakean d release, fol lowedb yK +,whil eCa 2+ wasfoun dt opla ya ninsignifican trol e inthi sproces s (3,6 ,17 ,19 ,26) .Arvi ne tal . (3)conclude d thatMg 2+ isa typica lcounterio ni nth epolyphosphat eformatio n insludge , while Ca isaccumulate d in sludge by means of chemicalprecipitation .Ou rresult swit hAcinetobacte rstrai n 210Aconfir m theimportanc eo fMg 2+ asa counterio no fpoly phosphate.Th euptak ean dreleas eo fK +b yactivate dsludg ema y nothav ean yrelatio nwit hphosphat euptak ean drelease .Thi s cationi sconcentrate daerobicall yinsid eth ecells ,bu tno ti n polyphosphategranules ,an dma yb erelease dunde renerg yunfa vourablecondition ssuc ha sanaerobiosis .A shortag eo fK +i n thewast ewate rma yhav ea negativ eeffec to nbiologica lphos phateremova l(Fig . 5).

59 ACKNOWLEDGEMENTS

We thank Mr. R.M.L. Rongen for his practical assistance and the Department of Water Pollution Control and the Department of Biochemistry of Wageningen Agricultural University for using their equipment. This research was supported by a special grant from the Agricultural University Wageningen.

REFERENCES

1. Adamse, A.D., 1980. New isolation of Clostridium aceticum (Wieringa). Antonie van Leeuwenhoek 46:523-531. 2. Appeldoorn, K.J., and M.H. Deinema. 1987. Biological phos phate removal under defined conditions in a fill-and-draw system, p. 309-311. In R. Ramadori (ed.), Biological phos phate removal from waste waters. Proceedings of an IAWPRC specialized conference, Rome, Sept., Adv. Wat. Poll. Con trol. Pergamon Press. 3. Arvin, E., and G.H. Kristensen. 1985. Exchange of organics, phosphate, and cations between sludge and water in biologi cal phosphorus and nitrogen removal processes. Wat. Sci. Technol. 17:147-162. 4. Buchan, L., 1983. Possible biological mechanism of phos phorus removal. Wat. Sci. Technol. 15:87-103. 5. Cembella, A.D., N.J. Antia, and P.J. Harrison. 1984. The utilization of inorganic and organic phosphorus compounds as nutrients by eukaryotic microalgae: a multidisciplinary per spective: part 2. Crit. Reviews Microbiol. 11:13-81. 6. Comeau, Y., K.J. Hall, R.E.W. Hancock, and W.K. Oldham. 1986. Biochemical model for enhanced biological phosphorus removal. Wat. Res. 20:1511-1521. 7. Cramer, C.L., and R.H. Davis. 1984. Polyphosphate-cation interaction in the amino acid-containing vacuole of Neuro- spora crassa. J. Biol. Chem. 259:5152-515. 8. Deinema, M.H., L.H.A. Habets, J. Scholten, E. Turkstra, and H.A.A.M. Webers. 1980. The accumulation of polyphosphate in Acinetobacter spp. FEMS Microbiol. Lett. 9:275-279. 9. Deinema, M.H., M. Van Loosdrecht, and A. Scholten. 1985. Some physiological characteristics of Acinetobacter spp. accumulating large amounts of phosphate. Wat. Sci. Technol. 17:119-125. 10. Epstein, W., and M. Davies. 1970. Potassium-dependant mutants of Escherichia coli K-12. J. Bacteriol. 101:836-843. 11. Friedberg, I., and G. Avigad. 1968. Structures containing

60 polyphosphatei nMicrococcu s lysodeikticus.J .Bacteriol . 96:544-553. 12.Funs ,G.W. ,an d M. Chen.1975 .Microbiologica l basiso f phosphateremova li nth eactivate d sludgeproces sfo rth e treatmento fwastewater .Microb .Ecol .2:119-138 . 13.Groenestijn ,J.W .van ,an dM.H .Deinema .1985 .Effect so f culturalcondition so nphosphat eaccumulatio nan dreleas eb y Acinetobacter strain 210A,p .405-410 .I n J.N. Lester and P.W.W.Kir k (ed.), Proceedings of theinternationa l con ferenceo nmanagemen t strategies forphosphoru s in theen vironment,Lisbon . 14.Groenestijn , J.W. van, H.H. Deinema, and A.J.B. Zehnder. 1987. ATPproductio n frompolyphosphat e inAcinetobacte r strain210A .Arch .Microbiol .148:14-19 . 15.Gurr ,E. ,1965 .Th erationa lus eo fdye si nbiology .Leonar d Hill,London . 16.Jones ,H.E. ,an dL.A .Chambers .1975 .Localize d intracel lularpolyphosphat e formationb yDesulfovibri ogigas .J . Gen. Microbiol.89:67-72 . 17.Kainrath , P., W. Maier, R.Wagner , and Kh.Krauth .1985 . Investigationso nth ecompositio no factivate dsludg efro m biologicalphosphoru sremova lprocesses ,p .411-416 .I nJ.N . Lester and P.W.W. Kirk (ed.), Proceedings of the inter nationalconferenc eo nmanagemen tstrategie sfo rphosphoru s inth eenvironment ,Lisbon .Selper ,London . 18.Medveczky ,N. ,an dH .Rosenberg .1971 .Phosphat etranspor t inEscherichi a coli.Biochim .Biophys .Acta .241:494-506 . 19.Miyamoto-Mills ,J. , J.Larson , D.Jenkins , and W. Owen. 1983. Designan doperatio no fa pilot-scal ebiologica lphos phateremova lplan ta tth eCentra lContr aCost aSanitar y District.Wat. Sci .Technol .15:153-179 . 20.Mulder ,M.M. ,M.J .Teixeir ade Mattos ,P.W .Postma ,an dK . van Dam.1986 .Energeti cconsequence so fmultipl eK +uptak e systems in Escherichia coli. Biochim. Biophys. Acta. 851:223-228. 21.Ohtake , H., K. Takahashi, Y.Tsuzuki , and K.Toda .1985 . Uptakean dreleas eo fphosphat eb ya pur ecultur eo fAcine tobactercalcoaceticus .Wat .Res .19:1587-1594 . 22.Pradet ,A. ,1967 .Etud ede sadenosines-5'-mono ,di -e ttri phosphatesdan sle stissue svegetaux .I Dosag eenzymatique . Physiol.Veg .5:209-221 . 23. Sail, T., S. Mudd, and J.C. Davis.1956 .Factor s con ditioning theaccumulatio n anddissappearanc e of metaphos- phatei ncell so fCorynebacteriu mdiphtheriae .Arch .Bio - chem.Biophys .60:130-146 .

61 24. Standard methods for the examination of water and wastewa ter, 14th edn. 1976. Am. Publ. Health Ass. Washington. 25. T'Seyen, J., 0. Malnou, J.C. Block, and G. Faup. 1985. Poly phosphate kinase activity during phosphate uptake by bac teria. Wat. Sci. Technol. 17:43-56. 26. Vries, H.P. de, M. van Loosdrecht, and J.H. Rensink. 1985. Nieuwste ontwikkelingen met betrekking tot biologische defosfatering. H20 18:358-362.

62 CHAPTER 4

ATP PRODUCTION FROM POLYPHOSPHATE IN ACINETOBACTER STRAIN 210A

Johan W. van Groenestijn, Maria H. Deinema and Alexander J.B. Zehnder

ABSTRACT

Incell-fre e extractso fAcinetobacte r strain 210A polyphosphate:AMPphosphotransferas ean dadenylat ekinas eactivi tywa s measured. Polyphoshate glucokinasean dpolyphosphat ede pendentNA Dkinas ewer eno tdetected .Th especifi cactivit yo f poly-phosphate:AMP phosphotransferase was found to be 43 nmolmin -1mg -1protei ni npresenc eo f1 nuno l1 AMP.Th eadeny late kinase reaction had an equilibrium constant ([ATP][AMP][ADP]~2)o f 0.7,a nactivit y of 54nmo l rain-1 mg-1 protein,an dwa salmos tcompletel y inhibitedb y0. 3m M P ,P- di(adenosine-5')-pentaphosphate .AT Pwa sforme dthroug hth ecom binedactio no fpolyphosphate:AM Pphosphotransferas ean dadeny latekinas ei ncell-fre eextract sfro mbacteria lpolyphosphat e andfro m chemicallyprepare dpolyphosphat e (Graham'ssalt) .A spectrophotometricmetho d forth econtinuou smonitorin go fpoly phosphate:AM Pphosphotransferas ei sals opresented .

Arch.Microbiol .(1987 )148:14-1 9

63 INTRODUCTION

Somestrain so fth egenu sAcinetobacte rma ypla ya nimportant roledurin genhance dbiologica lphosphat eremova l from waste water (Fuhsan dChe n 1975). Theseorganism sar eabl et oaccumu latephosphat eintra-cellularl yi nfor mo fpolyphosphat e(Ohtak e et al. 1985). This polymer can, under certain conditions, accountfo rove r30 %o fth ebiomas sdr yweigh t(Deinem ae tal . 1985). Invariou sbacteri apolyphosphat eac ta sreserv emateria l forphosphorus .However ,mor einterestingly ,i tmigh tals ob e useda sa nenerg yreserv ei nviv o(Kulae v1979) .I nth ecas eo f Acinetobacter,a strictlyaerobi cbacterium , phosphatei stake n upi nexces st ofor mpolyphosphat edurin gaerobi cgrowt han di s released whenth eenvironmen tbecome sanaerobi c (Deinemae tal . 1985). It ispossible , by alternating aerobic and anaerobic conditions,t oforc esom eAcinetobacte r strainsint oa cycli c process during which phosphate is accumulated and released again.Whe nthes ealternatin gcondition sar eintroduce di nwast e watertreatmen tplants ,th enumbe ro fAcinetobacte rincrease s andlarg eamount so fphosphat ear eremove d (Buchan1983 ,Rensin k etal .1981) . Therol eo fpolyphosphat ei nAcinetobacte rha sno tye tbee n elucidated,but man yhypothese sexist .Th emos tobviou son ei s thatAcinetobacte r mayus eth eenerg ystore di nfor mo fpoly phosphatedurin gperiod so fstarvatio no rothe radvers econdi tions.I nsom eothe rbacteri apolyphosphat eha sbee nshow nt ob e able tophosphorylat e adenylates, pyridine nucleotides or some otherorgani ccompound s (amoder nan dcomprehensiv erevie wo n thissubjec tha sbee nwritte nb yKulae van dVagabo vi n 1983). Examplesare : polyphosphatekinas e (Romberg 1957)whic hcatalyses :

(polyphosphate)n+ AD P* *(polyphosphate) n_^ +ATP , polyphosphate:AMPphosphotransferas e (Dirheimeran dEbe l1965 ) catalysing:

(polyphosphate)n+ AM P >-(polyphosphate)^ ^+ ADP , polyphosphateglucokinas e(Szymon aan dOstrowsk i 1964)cataly sing:

(polyphosphate) n +glucos e *•(polyphosphat e) n_1 +glucose-6- P and polyphosphate dependent NAD-kinase (Murata et al. 1980) catalysing:

(polyphosphate) n +NA D *•(polyphosphat e) n-1 +NADP . Especiallypolyphosphatekinas e and glucokinase hasbee n studied thoroughly.Ne w insightsint oth ereactio nmechanis man dkin eticso fpolyphosphatekinas e (Robinsonan dWoo d 1986)an dgluco kinase (Pepinan dWoo d 1986)ha sbee ndevelope drecently .

64 Inth epresen tstud yi ti sshow ntha ti ncell-fre eextract s ofAcinetobacte r strain 210AAT P is formed from native and syntheticalpolyphosphat evi ath ecombine dactio no ftw oen zymes,namel ypolyphosphate:AM Pphosphotransferas ean dadenylat e kinase.

MATERIALSAN DMETHOD S

Organism.Acinetobacte rstrai n210 Awa sisolate d fromactivate d sludgewit hth emetho ddescribe db yDeinem ae tal .(1980) .Thi s organism was maintained on yeast extract agar slants, subculturedever ytw omonth san dstore da t4 °C .Th estrai nca n beobtaine d fromth eauthors .

Growthconditions .Polyphosphat eric hAcinetobacte rcell scon taining10 0m g phosphoruspe rg biomas sdr yweigh twer ecul tivatedi nErlenmeye rflask si na shake ra t15°C .Th emediu m containedpe r100 0m ldemineralise dwate r2.2 9 gNa-butyrate , 0.1g Na2S203-5H20, 1.0g NH4C1, 0.6 g MgS04-7H20, 0.44g KH2PO4,0.0 6g CaCl2 ,2H20,6 g Tris(hydroxymethyl)aminomethane and 2m l of a trace mineral solution (containing 1500 mg FeCl3-6H20,5 0m g H3BO3, 10m g CuS04-5H20, 10m g Nal, 40 mg MnCl2-4H20, 20m g Na2Mo04-2H20, 40m g ZnS04•7H 20 and 50m g CoCl2-6H20pe rlitr edemineralise dwater) .Th ep Hwa sadjuste d to7 wit h6 N HC1 . Cellswithou tpolyphosphat e werecultivate dunde rP-limitatio n ina mediu ma sdescribe dabov eexcep ttha ti tcontaine d only 34m gKH 2P04pe rlitre .Polyphosphat efre ecell sdi dno tstai n followingth emetho do fNeisse r (Gurr1965 )an dcontaine d the minimumamoun to f1 3m gphosphoru spe rg dr yweight .

Preparationo fcell-fre eextracts .Th eculture swer eharveste d inth elat elog-phas ean dcentrifuged ,washe dwit hdemineralise d wateran dcentrifuge dagain .Th epellet swer eresuspende di na 30m MTris-HC lbuffe rp H7.0 ,an dthe nsonifie dwit ha nultraso nicdisintegrato r (modelB12 ,Branso nSoni cPowe rCompany) . The membranesan dstil lintac tcell swer eremove db ycentrifugatio n at 35,000 x g for 30minutes . Under these conditions poly phosphateremaine d inth esupernatant .Th epolyphosphat eric h extract contained 1.94m g protein per ml, and 360u g total phosphoruso fwhic h3 3u g (asP )wa sortho-phosphate .Th eex tractwithou tpolyphosphat econtaine d1.3 1 mgprotei npe rml , and 59u g totalphosphoru s ofwhic h 10u g (asP )wa sortho -

65 phosphate.

Chemicalan dbiochemica ldeterminations .Ortho-phosphat ean d totalphosphoru s (persulphatedigestio nmethod )wer e determined spectrophotometricallyaccordin g toth eStandar dMethod s (1976). Theprotei nconcentratio nwa smeasure dwit hth emetho do fLowr y usingbovin eseru malbumi na sstandard .ATP ,AD Pan dAM Pconcen trations incell-fre e reaction mixtures were determined ac cordingt oPrad^t (1 967 )an dth einstruction sbelongin gt oth e AdenylateEnerg yCharg e (AEC)ki to fLUMAC/3M .Th esample swer e boiled for 4minute s in 5m l ofa solutio n containing 50m M Tris-HClan d4 m M EDTAa tp H7.75 ,i norde rt osto pal lenzym e activity.Thes esample swer ethe ndivide dint othre esubsamples . Thefirs tobtaine dluciferin ean dfirefl yluciferase .Th ere sultinglight-emissio nwhic hwa sdependen to nth eAT Pconcen trationwa smeasure dwit ha luminescentionmete r (madeb yth e Electronics Designan dMaintenanc eSho pDivisio no fth eGenera l ResearchDepartment ,Universit yo fGeorgia, Athens ,USA) .Th e secondsubsampl ewa sfirs tpretreate dwit hP-enolpyruvat ean d pyruvatekinaset oconver tAD Pint oAT P and further treated as the first aliquot, while athir d subsamplewa s pretreated with adenylatekinas et oconver tAM Pint oAD Pfollowe d byth e same treatmenta sapplie dt oth esecon dsubsample .

Enzymeassays .Al lassay swer ecarrie dou ta t3 0C an dp H7.0 . Thereactio nmixtur econtaine d 0.18m lcell-fre eextrac tpe rm l whichresulte di n 238u gprotei npe rm l inth ecas eo fextract s withoutpolyphosphat e and 353u gprotei nan d6 5u gbacteria l totalphosphoru spe rm lwhe nextract swit hpolyphosphat ewer e used.

Adenylatekinase,E C2.7.4.3 :3 m MMgCl 2an d2 0m MTris-HCl , thereactio nwa sstarte db yaddin gADP,resultin gi na concentra tiono f 0.6 mM.A ttim einterval ssample so f5 0u lwer etake n fromth ereactio nmixtur et odetermin eAMP ,AO Pan dAT Pconcen trations. Polyphosphate:AMP phosphotransferase (Dirheimer and Ebel 1965):

3m M MgCl2 and 16 mM Tris-HCl, thereactio n was started by addingAMP ,resultin gi na concentratio no f1 mM .A ttim einter vals2 5u lwer etake nfro mth ereactio nmixtur et odetermin eAM P andAD Pconcentrations . Thefollowin gassay swer ecarrie dou ti na cuvett econtainin g

areactio nmixtur ewit h0. 5 mMKH 2P04 and 100m M Tris-HClp H 7.0.Th ecuvett ewa splace d ina Beckman nspectrophotomete ran d theproductio no fNADPH 2wa smeasure dcontinuousl ya t34 0nm . Polyphosphate:AMP phosphotransferase: 8 mM MgCl2, 220 mM

66 glucose, 0.67 mM NADP, 3.4 U (units) hexokinase (HK)/ml, 1.7 U D-glucose-6-P-dehydrogenase (G6P-DH)/ml and 1 U adenylate kinase/ml, if not sufficient suitable polyphosphate was present Graham's salt was added, the reaction was started by adding AMP, resulting in a concentration of 1 mM. The reaction scheme is given in Fig.l. From this scheme it is evident that the conver sion of one AMP to one ADP will result in the synthesis of one NADPH2. Polyphosphate glucokinase,EC 2.7.1.a: 8 mM MgCl2/ 0.67 mM NADP 1.7 U/ml G6P-DH, the reaction was started by adding glucose, resulting in a concentration of 220 mM. Polyphosphate dependent NAD kinase: 5.0 mM MgCl2, 1-7 UG6P-DH/m l and 1.2 mM D-glucose-6-P, the reaction was started by adding NAD,resulting in a concentration of 5.0 mM. ATP production in cell-free ex tracts: 8 mM MgCl2, 220 mM glucose, 0.67 mM NADP, 3.4 U HK/ml and 1.7 U G6P-DH/ml, the reaction was started by adding AMP or ADP.

D-glucos e ATP- (D ADP-

(poly-P),,., D-glucose-6 NA0P-^| 1© NADPH2 • - 6- P- glucono- i - lactone

Fig. 1. A method to measure polyphosphate:AMP phosphotransferase (1) continuously. The formed ADP conducts the reactions of the added enzymes which form a part of the detection system: adenylate kinase (2), hexokinase (3) and glucose-6-P-dehydrogenase (4). The produced NADPH? is measured spectrophotometrically and recorded.

Chemicals. HK (EC 2.7.1.1), G6P-DH (EC 1.1.1.49), adenylate ki nase (EC 2.7.4.3), NADP, NAD, AMP, ADP, ATP and D-glucose-6-P 1 5 were purchased from Boehringer, Mannheim. Ap5A (P ,P -di(adenosine-5')-pentaphosphate) was obtained from Sigma Chemical Co., St.Louis,MO. The AEC-kit was from LUMAC/3M bv. Synthetic poly phosphate in form of Graham's salt, which represents a mixture 2 of linear sodium polyphosphates [(NaPC>3)n/ n=10 ], was prepared by heating NaH2P04 3 hours at 750 °C followed by cooling in the air outside the furnace (Kulaev 1979).

67 RESULTS

Measuremento fpolyphosphat e qlucokinase,polyphosphat edepen dentNA Dkinas ean dadenylat ekinas e

Incell-fre eextract sn opolyphosphat eglucokinas ean dpolyphos phatedependen tNA Dkinas eactivitie s were found. Incontro l experiments D-glucose-6-P andNAD Pwer eadde drespectively . In bothcase sNADPH 2wa sforme drapidly .Adenylat ekinas ewa smea suredi ncell-fre eextract s containing nopolyphosphate .It s activityamounte dt o5 4nmo lmin -1 mg-1 proteinan dit sequili brium constant ([ATP][AMP][ADP]-2)wa s0. 7 (Fig.2).Thi scon stanti ssomewha thighe rtha nth econstant sreporte db yBowe n andKerwi n (1954)fo radenylat ekinas e (0.3- 0. 4 at ap Ho f 6.0).

6001

80 120 time (min)

Fig. 2. The adenylate kinase reaction (2 ADP2=AM P + ATP), measured in a cell-free extract of Acinetobacter strain 210A without polyphos phate. At time intervals samples were taken in which the concen trations of AMP (•), ADP (O) and ATP (D) were determined.

68 Measuremento fpolyphosphate :AM Pphosphotransferas e

Cell-freeextract sfro mAcinetobacte r strain210 Acontainin g native polyphosphate phosphorylated AMP (Fig.3b). Cell-freeex tractswithou tbacteria lpolyphosphat ewer eno tabl et ocatalys e thisreactio n(Fig.3a) .Durin gphosphorylatio no fAM Pth econ centrationo fAD Pwa salway shighe rtha ntha to fATP .Thi smigh t bedu et oth eactio no fadenylat ekinas ewhic hha sa nequilib riumconstan t([ATP ][AMP ][ADP] -2)o f0.7 .Th ehig hamount so f AMP added (1mM) , caused therefore a change inrelativ eAD P concentration.Additio no fsyntheti cpolyphosphat ei nth efor m of6 m M (asP )Graham' ssal tplu s8 m MMgCl 2t ocell-fre eex tractcontainin gn obiologica lpolyphosphat eresulte dwithi n44 0

2i a 1- -" 0- ,/:— 1 D B-1 1 B 1

3 Time (h) Fig. 3. ADP (O) and ATP (D) formation during incubation of cell-free extracts of Acinetobacter strain 210A with 1 mM AMP (a: without poly phosphate, b: with bacterial polyphosphate).

69 minutesi na nAD Pan dAT Pproductio no f13 0u Man d1 9pM ,res pectively(result snot shown) .Thi sexperimen t clearlydemon strated thatpolyphosphat e was thephosphoru s donor and agrees withth efinding so fDirheime ran dEbe l(1965 )tha tfo rpartl y purified polyphosphate:AMPphosphotransferas e from Corynebacte- riumxerosi sGraham' ssal tcoul d servea sa substrate . To establish whether AMP was first converted into ADP and whetherAT Pwa sforme dsubsequentl yfro mAD Pb yadenylat ekinas e (firstAT Pan dthe nAD Pcoul dals ob epossible )th efollowin g experimentswer ecarrie dout: I na firs texperimen tAMP ,ADP ,

ATPan dNADPH 2wer emeasure dsimultaneousl yi ncell-fre e ex tractswit hbacteria lpolyphosphate .Th eexperimen twa s started byaddin gAMP .Th eresult ssummarize di nTabl e1 sho wclearl y thatAM Pwa srecycle di nthi sproces san dth eequilibriu m ofth e adenylatekinas ereactio nwa sno treache d sinceAT Pwa suse du p immediatelyfo rNADPH 2formation .Tha tmean ttha tAD Pwa scon sumedb yth eadenylat ekinas ereactio ni norde rt oproduc eAT P andAMP .Th edecreasin gAM Pconcentratio nan dth eincreasin gAD P concentrationi nth efirs thou rindicate da conversio no fAM P intoADP .Durin gth efirs thou rAD Paccumulated ,probabl ybe causeth epolyphosphate:AM Pphosphotransferas e activity was highertha nth eadenylat ekinas eactivity .

Table1 .Th eproductio no fAD Pan dAT P(measure dvi aNADPH 2wit h theHK/G6P-D Hmethod )afte radditio no fAM Pt oa polyphosphat e containingcel lfre eextrac t ofAcinetobacte rstrai n210A .

ratio

Time NADPH2 ATP ADP AMP TAMPl TATP1 2 (min) (uM) (uM) (uM) ()iM) [ADP]

0 0 0.3 12 3304 6.9 10 4 0.2 151 3382 0.03 20 35 0.1 301 3210 0.004 40 118 2.1 578 2883 0.02 60 245 2.8 658 2455 0.02 100 335 2.4 534 2933 0.02 140 395 1.8 428 2953 0.03

In a second experiment adenylatekinas ewa s specifically inhi bitedwit hApcj A(Feldhau set al . 1975).A control experiment revealedtha tth emeasuremen to fAT Pb yth eHK/G6P-D Hmetho dwa s notaffecte db y0. 3 mMAp 5A.I nthi sassa ycell-fre e extract containing bacterialpolyphosphat e was incubated with 5m M AMP and0. 3m MAp^A ,an dAT Pwa squantifie daccordin gt oth eHK/G6P -

70 DH method. Very low amounts of NADPH2 were formed (Fig.4) indi cating a very low ATP production, while high amounts of ADP accumulated. The results from both experiments showed clearly that in cell-free extracts of Acinetobacter strain 210A ATP are formed from polyphosphate by the combined action of polyphos phate:AM P phosphotransferase and adenylate kinase.

3000-1

En . 2000 Q.

Q- 1000 O<

80 120 160 Time (min) Fig. 4. The accumulation of ADP (o) and the production of ATP (measured as NADPH, (•)) during the incubation of a polyphosphate con taining cell-free extract of Acinetobacter strain 210A with AMP and ApcA (inhibitor of adenylate kinase).

In case polyphosphate kinase would also be present in the cell- free extract it could be expected that ATP production would not specifically be inhibited by Ap5A. Ap5A has never been reported to inhibit polyphosphate kinase (Feldhaus et al. 1975, Kulaev 1979). The very low ATP production during incubation (Fig.4) suggests that polyphospate kinase plays (if any) merely an infe rior role in the conversion of ADP to ATP as compared to the polyphosphate:AMP phosphotransferase/adenylate kinase system.

Continuous monitoring of the polyphosphate:AMP phosphotrans ferase activity

A method for the continuous measurement of polyphosphate:AMP phosphotransferase activity was developed. The incubation mix-

71 ture usually used to determine the ATP production in cell-free extracts, was supplied with additional adenylate kinase (commer cially available as myokinase). This was necessary because the native activity of adenylate kinase in cell-free extracts of Acinetobacter strain 210A was too low to convert all the ADP produced fast enough to ATP. NADPH2 formation depended strictly on the addition of AMP and became linear after 8 minutes (Fig.5). At that moment the increase of ADP concentration leveled off and reached an equilibrium concentration of 100 uM. Subsequently all the ADP produced was turned over immediately by the adenylate kinase.

350

10 15 20 25 Time (min) Fig. 5. NADPH2 (- -) and ADP (O) production during the polyphos phate:AM P phosphotransferase assay (HK/G6P-DH method). At 18 minutes 100 nmol ADP was added per ml reaction mixture to demonstrate that the activities of the enzymes of the detection system were not saturated.

Polyphosphate:AMP phosphotransferase activity can only be measured if the reaction catalysed by this enzyme is the rate- limiting step; i.e. adenylate kinase, HK and G6P-DH have to be present in excess. The free capacity of these three enzymes can be tested by adding extra ADP at the end of the experiment (in Fig.5 100 uM at 18 minutes). As depicted by Fig. 5 a higher rate of NADPH2 production was indeed measured. During the assay the phosphate ion concentration in the reac-

72 tionmixtur eremaine dconstan tan dn ofre ephosphat ewa spro duced.Thi smean stha tphosphorylatio no fon eAM Pdiminishe sth e polyphosphatemolecul eb yon ephosphat egrou ponly . Thehighes tpolyphosphate:AM Pphosphotransferas e activities were found during these HK/G6P-DH assay's, namely 43nmo lmin -1 mg-1 proteina scalculate dfro mFig .5 a swel la s fromTabl e1 . Cell-free extractswithou tpolyphosphat eproduce dn oNADPH2 . Uponadditio no f6 m M (asP )Graham' ssal tNADPH 2wa s formeda t a rate corresponding toabou thal fo fth epolyphosphate:AM P phosphotransferaseactivit ymeasure di ncell-fre eextract swit h bacterialpolyphosphate .

DISCUSSION

Theenzyme spolyphosphat edependen tNA Dkinas ean dpolyphosphat e glucokinasewer eno tfoun di nAcinetobacte rstrai n210A ,thoug h these enzymes have been found in many other polyphosphate accumulatingbacteri a (Murata etal . 1980,Kulae v 1979). The absenceo fpolyphosphat eglucokinas ewa sno tsurprisin gsinc e Acinetobacter strain 210A is not able to assimilate glucose (Deinemae tal .1980) . Theenzyme spolyphosphate:AM Pphosphotransferas ean dade nylate kinase enableAcinetobacte r cells to form ATP under energy-starvation, as long aspolyphosphat e ispresent . This systemi si na wa y superiort opolyphosphat ekinas esinc ei ti s ablet ophosphorylat eAMP .Th econsiderabl epolyphosphat ere servei nAcinetobacte rstrai n210 Aca ntherefor eb ever yvalu able under conditions when this strict aerobe has no other possibility toobtai n energy, e.g. inabsenc e of oxygen. The abilityt oaccumulat epolyphosphat ean dt ous ei ta sa nenerg y sourceunde radvers econdition sma ygiv eAcinetobacte ra dis tinct competitive advantagei na nactivate d sludge systemsub jectedt orepeate d anaerobic/aerobic cycles.Kulae v andVagabo v (1986)conclude dtha tth egenera lfunctio no fhig hmolecular - weightpolyphosphate si nmicroorganism s isth eregulatio no f intracellular concentrations ofimportan tmetabolite sincludin g ATP, ADP,othe rnucleosid epolyphosphates ,an dfinall ypyro -an d particularlyortho-phosphate . Incertai naerobi cmicroorganism s polyphosphatei sno tbroke ndow ni fenerg ygeneratio ni slimite d orblocke d (Harold1966) .I nthes eorganism spolyphosphat emigh t onlyb ea phosphoru s storagematerial ,whic h isvaluabl e in biotopeswher ephosphat ei sgrowt hlimiting .A sAcinetobacte r strain210 Ai sisolate dfro ma phosphate-ric henvironmen ti tca n

73 beexpecte dtha tth epolyphosphat eaccumulate db ythi sstrai n hasa nothe rfunctio ntha nphosphoru sstorage . Themeasure d activities ofpolyphosphate:AM Pphosphotrans ferasean dadenylat ekinas ear ei naccordanc et oth erate sb y whichphosphat ei srelease db ypur eculture so fAcinetobacte r sp.( 1nmo lPO4- P min mg-1dr yweigh tfoun db yAdams ee tal . -1 -1 1984)o r activated sludge (0.6nmo lP0 4-Pmin mg dryweigh t foundb yRensin ket al .1981) . 2 Dirheimer and Ebel (1965)foun d aK m of 2 10" M AMP for polyphosphate:AMPphosphotransferas e inCorynebacteriu m xerosis and needed in their assay 50m MAM P toreac ha nactivit yo f about1 7nmo l min" mg proteina t3 7C .Becaus eo fit s low activitiesa tlo wAM Pconcentrations ,th ephysiologica lsignifi canceo fthi senzym ei sstil lunclea r (Kulaev 1979).However , from ourexperiment s itappeare dtha tAcinetobacte r strain210 A disposeso fa polyphosphate:AM Pphosphotransferas eactivit ytha t isalread ysufficientl yhig ha tlo wAM Pconcentrations .Assumin g a cellular protein content of 500m g per g dry weight, the specific activity of polyphosphate:AMP phosphotransferase of4 3 nmolmin -1m g proteincoul db eresponsibl e forth eproductio n of1. 5mmo lAT P h g-1 dryweight .Whe ngrow nunde roptima l conditions onegra m ofAcinetobacte r strain 210Acontain sabou t 100m g phosphorus (Deinemae tal .1985 )o fwhic habou t8 0m go r 2.6mmo l areincorporate d intopolyphosphate .A ta nAT P pro ductionrat eo f1. 5mmo lh -1 g-1dr yweight ,polyphosphat ecoul d servea sa nenerg ysourc efo r1. 7hours . From chemostat experiments with strain 210A (Deinema etal . 1985)0. 6mmo lacetat eh g-1dr yweigh tar eneede dfo rmainte nance,whic hi nit sbes tcas etranslate st o6. 6mmo lAT Ph g-1 dryweight .Thi si sabou tfou rtime smor etha nth epolyphos - phate:AMPphosphotransferas esyste m cansuppl y invitro .Ac cording toPir t (1975)microorganism s need during growth under carbon limitation 0.5 to 14mmo l ATP h g_1 dryweigh t for maintenance.On eha st orealize ,however ,tha tthes eenergie s necessary formaintenanc ewer eextrapolate dfro mwel lgrowin g cultures.Thes evalue smigh tb esignificantl ydifferen tfro mth e maintenanceenerg yrequire dunde rnon-growt hcondition s(Maso n etal .1986 )an dunde rcircumstance swher en oexterna lenerg y source is available.A s amatte r of factmicroorganism s are actuallycapabl et osurviv efo ra ver ylon gperio do ftim ei n theirvegetativ eform si nseverel yenergy-limite d systemsi n nature(e.g .ope nocean ,dee pgroundwate rsystems ,etc.) .Thi s indicatestha tth emaintenanc eenerg ymigh tno tb econstan tbu t might reach a value close to zero for some organisms under certainadvers ecircumstances .

74 Whether Acinetobacter strain 210A can really benefit in vivo from polyphosphate as an energy reserve and whether this reserve is able to cover the maintenance needs for a longer period of time, remains to be answered. Research is in progress in our laboratory concerning these problems.

Acknowledgements. We thank Mr. G.J.F.M. Vlekke for his prelimi nary researchwork, while Dr. G.J.J. Kortstee and Dr. W.J. Mid- delhoven are gratefully acknowledged for their helpfull advices and the Department of Biochemistry of the Agricultural Universi ty Wageningen for using its equipment. This research was suppor ted by a special grant from the Agricultural University Wa geningen .

REFERENCES

Adamse AD, Deinema MH, Zehnder AJB (1984) Studies on bacterial activities in aerobic and anaerobic waste water purification. Antonie van Leeuwenhoek J Microbiol 50:665-682 Bowen WJ, Kerwin TD (1954) The kinetics of myokinase. I. Studies of the effects of salts and pH and of the state of equilib rium. Arch Biochem Biophys 49:149-159 Buchan L (1983) Possible biological mechanism of phosphorus removal. Wat Sci Techn 15:87-103 Deinema MH, Habets LHA, Scholten J, Turkstra E, Webers HAAM (1980) The accumulation of polyphosphate in Acinetobacter spp. FEMS Microbiol Lett 9:275-279 Deinema MH, Van Loosdrecht M, Scholten A (1985) Some physio logical characteristics of Acinetobacter spp. accumulating large amounts of phosphate. Wat Sci Techn 17:119-125 Dirheimer G, Ebel JP (1965) Caracterisation d'une polyphosphate- AMP-phosphotransfSrase dans Corynebacterium xerosis. C R Acad Sc Paris 260:3787-3790 Feldhaus P, Frohlich T, Goody RS, Isakov M, Schirmer RH (1975) Synthetic inhibitors of adenylate kinases in the assays for ATP-ase and phosphokinases. Eur J Biochem 57:197-204 Fuhs GW, Chen M (1975) Microbiological basis of phosphate re moval in the activated sludge process for the treatment of waste water. Microb Ecol 2:119-138 Gurr E (1965) The rational use of dyes in biology. Leonard Hill, London Harold FM (1966) Inorganic polyphosphates in biology: structure, metabolism, and function. Bac Rev 30:772-794

75 Romberg SR (1957)Adenosin e triphosphatesynthesi sfro mpoly phosphateb ya nenzym efro mEscherichi acoli .Biochi mBiophy s Acta 26:294-300 KulaevI (1979 )Th ebiochemistr yo finorgani cpolyphosphates . JohnWile yan dSons ,Chicheste rNe wYor kBrisban eToront o Kulaev I, VagabovV M (1983)Polyphosphat e metabolism inmicro organisms.Ad vMicro bPhy s24:83-17 1 Mason CA, Hamer G, Bryers JD (1986)Th e death and lysis of microorganismsi nenvironmenta lprocesses .FEM SMicrobio lRe v 39:373-401 MurataK ,Uchid aT ,Tan iK ,Kat oJ ,Chibat aI (1980 )Metaphos - phate: ane w phosphoryldono rfo rNA Dphosphorylation .Agri c Biol Chem (Tokyo)44:61-6 8 Ohtake H, Takahashi K, Tsuzuki Y, Toda K (1985)Uptak e and release of phosphate by a pure culture of Acinetobacter calcoaceticus.Wate rRe s19:1587-159 4 PepinCA ,Woo dH G (1986)Polyphosphat eglucokinas e fromPropio - nibacteriumshermanii .Kinetic san ddemonstratio nthat th e mechanism involvesbot hprocessiv e andnonprocessiv e type reactions.J Bio lChe m 261:4476-4480 Pirt SJ (1975) Principles of microbe and cell cultivation. BlackwellScientifi cPublications ,Oxfor dLondo nEdinburg h Melbourne PradetA (1967 )Etud ede sadenosine-5'-mono ,di -e ttri -phos phates dans les tissus ve'ge'taux. I. Dosage enzymatique. PhysiolVS g 5:209-221 RensinkJH ,Donke rHJGW ,Vrie sH Pd e(1981 )Biologica lP-remova l indomesti cwastewate rb yth eactivate d sludgeprocess .Pro ceedings 5th European Sewage and Refuse Symposium. EAS, Munich487-50 2 Robinson NA, WoodH G (1986)Polyphosphat ekinas efro mPropioni - bacteriumshermanii .Demonstratio n thatth e synthesis and utilization of polyphosphate isa processiv e mechanism.J BiolChe m 261:4481-4485 Standardmethod s forth eexaminatio no fwate ran dwastewate r 14theditio n (1976)A mPub lHealt hAss ,Washingto n Szymona M, Ostrowski W (1964)Inorgani c polyphosphateglucoki naseo fMycobacteriu mphlei .Biochi mBiophy sAct a85:283-29 5

76 CHAPTER5

POLYPHOSPHATE ASA N ENERGYRESERV E INVIV O IN ACINETOBACTER STRAIN 210A

JohanW .va nGroenestijn ,Mari aH .Deinema ,an dAlexande rJ.B . Zehnder

ABSTRACT

The regulation of the degradation of inorganic polyphosphate and the use of this compound as an energy reserve in vivo in Acine- tobacter strain 210A has been studied. The response of this organism to various energetic conditions was determined. The ortho-phosphate release and ATP content of strain 210A under anaerobic conditions was compared to another polyphosphate con taining Acinetobacter sp. and to two acinetobacters and a Pseu- domonas sp. which are unable to accumulate polyphosphate. It could be concluded that the prerequisites for polyphosphate degradation were a low energy state of the cell and the presence of AMP. The degradation and synthesis of polyphosphate coincided with low and high cellular ATP contents, respectively. This degradation was affected by the pH and was stimulated by alco hols. Under anaerobic conditions cells with polyphosphate released more ortho-phosphate and contained more ATP than cells without polyphosphate. The transmembrane protongradient played an important role in the anaerobic energy metabolism of this strictly aerobic organism. The addition of DNP decreased the cellular ATP concentration and stimulated the polyphosphate degradation.

submitted for publication

77 INTRODUCTION

Accumulationo fpolyphosphat ei sa wel lknow nphysiologica lpro pertyo fman ymicroorganisms ,bu tth efunctio no fthi sbiopoly - meri sno tye ttotall yclear .Fro mth egrea tnumbe ro fstudie s onpolyphosphat emetabolis m thegenera lconclusio nca nb edraw n thatthi sinorgani cpolyme rca npla ya nessentia lrol ei nth e metabolismo forganism sa sa nhigh-energ yphosphoru scompoun d functionallyalternativ et oATP ,an dtha ti tca nb eimportan ti n theregulatio no fnumerou sbiochemica lprocesse s (Kulaevan d Vagabov1983) .Man yauthor shav especulate dabou tth efunctio n ofinorgani cpolyphosphat ea sa nenerg yreserv ei nvivo .I n197 3 Dawesan dSenio rconclude dtha tth estatu so fpolyphosphat ea s anenerg yreserv ewa sb yn omean sclearl y established. Their majorargumen tagains tpolyphosphat efunctionin ga sa nenerg y reservewa sbase d on theobservatio n that inman y organisms polyphosphatepresent i nth ecel lma yonl yaccoun tfo ra smal l proportion of the totalAT P generated. In order to show the energy-storagefunctio no fa compound ,i ti snecessar yt odemon strate three points (Wilkinson 1959): (1) The compound is accumulated under conditions when the supply of energy from exogenoussource si si nexces so ftha trequire db yth ecel lfo r growthan drelate dprocesse sa ttha tparticula rmomen to ftime ; (2)th e compound isutilise d when the supply of energy from exogenoussource si sinsufficien tfo rth eoptima lmaintenanc eo f thecell ,eithe rfo rgrowt han ddivisio no rfo rmaintenanc eo f viability and other processes; and (3)tha t the compound is brokendow nt oproduc eenerg yi na formutilisabl eb yth ecell , andtha ti tis ,i nfact ,utilise dfo rsom epurpos ewhic hgive s thecel la biologica ladvantag ei nth estruggl efo rexistenc e overthos ecell swhic hd ono thav ea comparabl ecompound .Ther e area number of polyphosphate accumulating bacteria thatdon' t obeythes econdition s (Dawesan dSenio r1973 ,Harol d 1966).A fewauthor srepor tth eus eo fpyro -an dpolyphosphate s asa n energy reserve invivo .Pyrophosphat eincrease d thegrowt hrat e andyiel d ofClostridiu m sp.(Crude ne t al. 1983). Pyro-an d polyphosphates stimulatedgrowt h ofanaerobi cbacteri ao fseve ralunidentifie d speciesi na minima lmediu m inwhic hortho - phosphatewa s produced.Ortho-phosphat ealon edi dno thav ethi s stimulatoryeffec t(Varm aan dPec k 1983).Zyuzin ae tal .(1981 ) reportedtha tStreptomvce slevori suse dinorgani cpolyphosphate s andnot ATP as an energy sourcedurin g the synthesis of the antibioticlevorin . Somestrain so fth estrictl yaerobi cAcinetobacte raccumulat e aerobicallylarg eamount so fpolyphosphat e (upt o10 0m gP pe rg

78 drybiomass ,Deinem ae tal .1980 )an dwer eabl et odegrad ethi s compound in the absence of oxygen (Adamse et al. 1984). Re cently,i tha sbee ndemonstrate dtha tAT Pca nb eproduce dfro m polyphosphatei ncell-fre eextract so fAcinetobacte rstrai n210 A byth ecombine d actiono fpolyphosphate:AM Pphosphotransferas e andadenylat ekinas e (VanGroenestij ne tal . 1987). This report presents evidence thatpolyphosphat e canac ta s sourceo fenerg yi nwhol ecell so fAcinetobacte rstrai n210 Aan d thatth ephosphorylatio n of adenylatesb ypolyphosphat edepend s onth epresenc eo fAM Pan da lo wenerg ystat eo fth ecel l(lo w protonmotiv e forcean dlo wAT P level).

MATERIALSAN DMETHOD S

Organisms.Acinetobacte r strains 210A,B8 ,13 2 and 124 were isolated from activated sludgewit h themetho d described by Deinemaet al .(1980) .Thes estrain swer emaintaine do nyeast extractaga rslant s( 5g glucose ,2. 5g yeas textrac tan d 12g agarpe rlitr etapwater ,p H= 7) ,subculture dever ytw omonth s andstore da t4°C .Strain s210 Aan dB 8wer eabl et oaccumulat e largeamount s ofpolyphosphate ,whil e strains 132an d 124wer e unable to store this compound.Pseudomona s strain 10,whic h doesn'tcontai ndetectabl eamount so fpolyphosphate ,wa sob tainedfro mth ecultur ecollectio no fou rlaboratory . Itwa s maintainedo nnutrien tbrot haga r( 8g nutrien tbrot han d1 2g agar per litre tapwater), subcultured every two months and storeda t4°C .

Media.Th ecell swer eprecultivate di na standar dphosphat eric h medium containingbutyrat ea sonl ycarbo nan denerg ysourc ean d in one experiment ina phosphat epoo r (phosphorus limited)me dium.Bot hmedi awer edescribe delsewher e(Va nGroenestij ne t al. 1987).Butyrat ei sa noptima lsubstrat efo rpolyphosphat e accumulation inAcinetobacte rstrai n210A . Experimentswer ecarrie dou twit hdifferen tphosphat ereleas e mediawhic hwer eal lbase do ndemineralize dwater ,namel yth ep H experimenti na mediu mwit h5 g Na-acetate.SH^O ,6 g NaC lan d4 gtri spe rlitre .Th edesire dp Hwa sobtaine db yadjustin gth e mediumwit h6 N HCl .Th eexperiment swit hdifferen talcohol san d theexperiment si nwhic hth eeffec to fdifferen ttype so fenerg y starvationwa sdetermined ,wer ecarrie dou twit ha mediu mcon taining6 g.l -1NaC lan d3 0m M Tris-HCl.Th ep Ho fth emediu m used foranaerobi c incubationwa sadjuste dt o8. 0an dth eaero bic medium had a pH of 7.5. The anaerobic medium used for

79 measuringth ecellula rAT Pconten tcontaine d3 0i HTris-HC lp H 8.0.

Experimentalprocedures .Cell swer e precultivated inErlenmeye r flasksi na shaker .Acinetobacte rstrai nB 8wa sgrow na t25°C , itsoptimu m temperature forpolyphosphat eaccumulation .Al l other strains were grown at 15°C, the temperature at which Acinetobacter strain210 Aaccumulate dhig hamount so fpolyphos phate.I nth elat elog-phas eon egra mbiomas sdr yweigh tcon tained 100 mg P (strain 210A), 54m g P (strain B8), 30m g P (strain 124),1 7m g P (strain 132)an d 20 mg P (Pseudomonas strain10) .Thes elat elog-phas eculture swer eharvested ,cen - trifuged,washe dwit ha 8 m M Tris-HClbuffe rp H7. 5an dcentri - fugedagain .Th epellet swer eresuspende d inth eappropriat e releasemediu mresultin gi nbiomas sconcentration so f0. 5t o1. 0 g dryweigh tpe rlitre .Medi awer emad eanaerobi ci nserumvial s of3 0o r10 0m lb yflushin gthe mwit ha gas-mixtur eo f ^tCC^ (99%:l%,v/v).Th ep Ho fth emedi awhic hwa sinitiall yadjuste d to8. 0decrease d to7. 5a sa resul to f additiono f carbondi oxide.I nexperiment swher ealcohol swer euse dthe ywer edi rectlyinjecte dint oth evials ,containin galread yth eanaerobi c cellsuspension .Serumvial san dErlenmeye rflask swer eincubate d ona rotar yshake r (200rpm )a t30°C .A ttim einterval ssample s weretake nwit ha syringe .Car ewa stake nt okee pth econten to f thevial sanaerobic . In a seperate set of experiments the effect of acetate additiont oaerobicall ystarve dculture swa sinvestigated .Cell s (0.74g dr yweigh tpe rlitre )wer eincubate di nshake nErlen meyerflask si na releas emediu m containingNaC lan dTris-HC l butn oacetate .A ttim einterval sth emediu mwa sanalyse dfo r ortho-phosphate,whic hwa sinitiall yrelease db yth ecell sbut takenu pafte rth eadditio no facetate .Thes eexperiment swer e carriedou ta t25°C ,becaus ea tthi stemperatur emor epolyphos phatei saccumulate dtha na t30°C . Chemicalan dbiochemica ldeterminations . Ortho-phosphate and totalphosphoru s (persulphatedigestio nmethod )wer edetermine d spectrophotometricallyaccordin gt oth eStandar dMethod s (1976). Theprotei nconcentratio nwa smeasure dwit hth emetho do fLowr y usingbovin eseru malbumi na sstandard .Bacteria ldr yweigh twa s determined after centrifugation of a 50 ml sample of the culture,washin gwit hdemineralize dwate ran ddryin gi ta t100° C overnight. The concentrations of ATP, ADP and AMP in the cultureswer e determined according to Pradet (1976)an d Van Groenestijne tal .(1987) .Sample so f0. 5m lwer etake n(wit ha gastight glass syringe inth e case of anaerobic cultures to

80 prevent oxygen contamination) and boiled for 4 minutes in 5 ml of a Tris-HCl/EDTA solution. ATP, ADPan d AMPwer e determined by means of an AEC (adenylate energy charge) kit, based on bioluminescence, using luciferine and luciferase.

Chemicals. The AEC-kit was from LUMAC/3M bv. (Schaesberg, The Netherlands), «-dinitrophenol (DNP) and N,N'-dicyclohexylcarbo- diimide (DCCD) were purchased from Merck, Darmstadt, Federal Republic of Germany.

RESULTS

ATP levels in cells of Acinetobacter strain 210A

The ATP content of anaerobically incubated cells of Acinetobac ter strain 210A varied with the presence of different salts in the medium besides Tris-HCl (Table 1). All nine salts tested (concentration 100 mEq per litre) decreased markedly the ATP- level of the cells as compared to a medium containing Tris-HCl buffer only. K+- and NH^-salts had a stronger effect than Na - salts. Therefore in all experiments the anaerobic cellular ATP- levels were determined in a medium made of demineralized water and Tris-HCl (30 mM). Table 1. Effect of 100mEq' l of different salts on the ATP content (expressed as pmol ATP per g dry bicmass) of Acinetobacter strain 210A two hours after oxygen depletion.

cation

Na NH.

control (onlyTris-HCl ) 1.76 anion:CI " 0.71 0.40 0.41 NO," 0.41 0.37 0.29 SO,/ 0.38 0.24 0.28

Regulation of the polyphosphate degradation in Acinetobacter strain 210A

The degradation of polyphosphate and subsequent release of ortho-phosphate by intact cells of Acinetobacter strain 210A took place only when energy generation in the cell was not possible, inhibited or uncoupled (Fig. 1). These conditions were: lack of substrate under aerobic conditions, inhibition of

81 cr Q.

12 16 Time(h) Fig, 1. Release of ortho-phosphate by Acinetobacter strain 210A incubated at pH 7.5 and 30°C under anaerobiosis and different aerobic conditions: with 4 mM DNP, with 1 mM KCN, with 4 mM DCCDan d without additions. The presented data are from one experiment which was repre sentative of 4 similar experiments.

respiration by KCN, inhibition of membrane bound ATPase by DCCD, dissipation of the protongradient with the uncoupler DNP or lack of oxygen as electron acceptor (anaerobic conditions). Under aerobic conditions in the absence of an extracellular energy source phosphate release started as soon as the cellular ATP content was low (Fig. 2). Addition of acetate (arrow in Fig. 2 after 12.7 hours) increased rapidly the ATP-level and turned on

82 again phosphate uptake.

2.6

% <1J a. F 12 "O <1u0 r) 10 0) i— a. 8 rf a.

6 8 Time (h)

Fig. 2. Release of ortho-phosphate, ATP content and 0Dg60 of an aerobically incubated culture of Acinetobacter strain 210A without an extracellular carbon source (pH 7.5, 25°C). At 12.7 h 4 mmol Na- acetate was added per litre. The presented data are from one experi ment which was representative of 3 similar experiments.

The rate of phosphate release by the cells depended on the pH, which was optimal at 7.5, while lower pH's had a strong negative effect on this process. The efflux rate was positively- affected by the presence of various alcohols. The stimulatory effect of alcohols increased with their molecular weight and with their concentration in the medium (the tested alcohols were methanol, ethanol, propanol-1, butanol-1 and pentanol-1 at a

83 concentration of 40 mM). The presence of 40 mM pentanol-1 doubled the rate of phosphate release. Pentanol-1 stimulated the phosphate release of Acinetobacter strain 210A up to a concen tration of 60 mM. At that concentration the release stopped abruptly after one hour, though the cells still contained poly phosphate. The cause of this inactivation was investigated in seperate experiments. Acinetobacter strain 210A was anaerobically incubated with 60 mM and 80 mM pentanol-1. Cells and medium were analysed for intra- and extracellular adenylates at the moment the phosphate release stopped (between 1 and 2 hours at 60 mMpentanol- 1 and at 0.67 hours at 80 mM). When the polyphos phate degradation ceased most of the adenylates had leaked out of the cells. Since AMP is needed for the enzymatic degradation of polyphosphate (Van Groenestijn et al. 1987), its lacking in the cell might have stopped the hydrolysis of the polymer. In fact, after 0.67 hours in the experiment with 80 mM pentanol-1 phosphate release ceased at a time when AMP had completely disappeared from the cells. Besides adenylates and 7.5 mg P04-P also 6 mg P from complex compounds and 29 mg protein were detec ted in 1 1 supernatant. As these amounts were low compared with 1 gram biomass dry weight initially containing 0.5 g protein and 100 mg P, it can be concluded that lysis of cells or leakage of

Table 2. Concentration of adenylates (umol.g dry biomass) in the total culture and in the waterphase during the anaerobic incubation of Acinetobacter strain 210Awit hpentanol- 1o r theaerobi cincubatio nwit hDCCD .

i Additionst o themediu m

DCCD pentanoL- 1

(4mM ) (4mM ) (0mM ) (60mM ) (60mM ) (80mM )

Timeelapse d (hours)8 2 8 24 1 2 0.67

Adenylates

ATP total 0.5 2.7 0.1 3.0 1.2 1.2 waterphase 0.04 2.7 0.01 0.3 1.2 0.3

ADP total 2.8 11.7 4.6 7.3 7.1 4.6 waterphase 0.6 8.7 0.05 1.1 5.5 2.9

AMP total N.D.b N.D. 4.8 0 11.5 3.1 waterphase N.D. N.D. 0.05 0.6 12.8 3.3 a: after addition of DCCD or pentanol-1 b: N.D. not determined

84 highmolecula rcompound sfro mth ecell swer equit enegligibl e after0.6 7hour so fanaerobi cincubatio nwit h 80m M pentanol-1. Anaerobicincubatio no fAcinetobacte rstrai n210 Awit hDCC Dals o causeda slo wreleas eo fadenylates ,whil e2 4hour sincubatio n without pentanoldi dno tresul ti na detectabl e leakageo fATP , ADPo rAM P (Table2) .

Polyphosphatea sa nenerg yreserv ei nwhol ecell so fAcineto bactersp .

Phosphate release and cellularAT P content were measured in anaerobically incubated cultureso fAcinetobacte r strains 210A, B8,12 4an d13 2an dPseudomona sstrai n10 .Strain s210 Aan dB 8 were used because they are able to accumulate the highest amounts of phosphorus compared to the other Acinetobacter strains in our collection. Strains 124 and 132 are typical representatives ofacinetobacter swhic hcanno taccumulat epoly phosphate.Pseudomona sstrai n1 0serve da sa contro lt odemon stratetha tth ebehaviou ro fAcinetobacte rstrai n12 4an d13 2 was comparable toothe r fastgrowin gaerobi cbacteri ano tabl e toaccumulat epolyphosphate . Thestrain s 124an d 132showe d no oronl ya ver ysmal lreleas eo fphosphat ean dth eAT Pconten to f the cells immediately dropped to low levels when incubated anaerobically(Fig .3) .Pseudomona sstrai n1 0reacte dth esam e asAcinetobacte rstrai n13 2(result sno tshown) .Th epolyphos phate accumulating strains 210Aan dB8 ,however , releasedhig h amountso fphosphat ean dwer eabl et omaintai na rathe rhig hAT P levelfo rsevera lhours .T oinvestigat ewhethe rth ehighe rAT P contentso fstrai n210 Aan dB 8durin ganaerobiosi swer estrai n specifico ra resul to fth epresenc eo fpolyphosphate ,strai n 210A was cultivated under P-limitation or at 35°C. At 35°C strain210 Adoe shardl yaccumulat epolyphosphat e (VanGroene - stijn et al. submitted). Inbot h cases, 35°C and P-limited, culturescontaine donl y1 5m gP pe rg dr ybiomass .I nabsenc eo f oxygenphosphat ereleas ewa snegligibl ean dth eAT Pconten to f thesecell swa smuc hlowe rtha ncompare d tocell scontainin g highamount so fpolyphosphat e(Fig .3) .Th eadditio no fDN Pt o anaerobicculture s ofAcinetobacte rstrai n210 Aresulte di na higherphosphat erelease ,bu tlowe rcellula rAT Plevel s(Fig . 4), indicating thatphosphat ereleas e isstimulate d by lowAT P concentrationsi nth ecell .

85 16 20 Time (h)

Fig. 3. Release of ortho-phosphate and cellular ATP content in anaerobically incubated cultures of polyphosphate accumulating Acinetobacter strains 210A (•) and B8 (A), Acinetobacter strains without poly phosphate 132 (o) and 124 (•), and of cultures of Acinetobacter strain 210A which were precultivated at 35°C (no polyphosphate accumulation) (V) and under P-limitation (no polyphosphate accumula tion) (A). The anaerobic incubation was at pH 7.5 and 30°C. The curves are representative of curves determined in sevenfold (210A, 15°C), quadruplicate (132), duplicate (B8) and singular (124; 210A, 35°C; 210A, P-limited).

86 E o jA IS no addition "5! £• 3 -a — Ol £: <- \—

0 30

Fig. 4. Effect of 4 mM and 10 mM DNP on the release of ortho-phosphate and cellular ATP content of anaerobically incubated cultures of Acine- tobacter strain 210A at pH 7.5 and 30°C. The presented data are mean values from an experiment in duplicate, which was representative of other experiments showing the same effects.

DISCUSSION

Polyphosphate degradation and release of ortho-phosphate by Aci- netobacter strain 210A was observed when energy became scarce regardless of the cause of the energy shortage (Fig. 1). How ever, when sufficient oxygen and energy source were available polyphosphate was not broken down (Fig. 2). As phosphate was

87 releaseda ta lo wcellula rAT Pconcentratio nan dtake nu pa ta highAT Pconcentratio n (Fig.2) , itca nb econclude d thatth e polyphosphate synthesis anddegradatio n iscontrolle d byth e energystat eo fth ecells .Simila robservation shav ebee nmad e in Micrococcus lysodeikticus in which the polyphosphate accumulated during theexponentia l phase of growth and dis appeared graduallydurin g carbonstarvatio n (Friedbergan dAvi - gad1968) .Rhodopseudomona sspheroide san dAnabaen avariabili s accumulated polyphosphate under anaerobic conditions inth e presenceo flight ,whil ei nth edark ,i nabsenc eo fphotophos - phorylation, the amount of polyphosphate declined (Carran d Sandhu 1966). Besidesth eAT Pleve lals oth eavailabilit yo fAM Pseem st o becrucia lfo rpolyphosphat edegradation .Polyphosphat ehydro lysisstoppe dwhe nth eadenylate sha dleake dou to fpermeabi - lizedcell s(Tabl e2) .Interestingly ,phosphat ereleas ecease d completelyafte r 0.67 ho fincubatio nwit h 80m M pentanol-1; thoughonl yAM Pha dleake dou tentirel yint oth emedium ,AT Pan d ADPwer e stillpresen ti nth ecells .Thi sindicate stha tth e presenceo fAM Pi sa nimportan tfacto rfo rpolyphosphat edegra dationi nviv oan dtha tpolyphosphat ei nanaerobicall yincubate d Acinetobacterstrai n210 Ai smainl ydegrade db yth eenzym epoly phosphate:AM Pphosphotransferas e (asmeasure di ncell-fre eex tractso fthi sstrai nb yVa nGroenestij ne tal . 1987),an dno t by polyphosphatase, whichwa s found inAcinetobacte rcalcoace - ticus(Ohtak ee tal .1985) . Cultureso fAcinetobacte rstrain sabl et oaccumulat epoly phosphate(210 Aan dB8 )release danaerobicall yhig hamount so f phosphatean dwer eabl et omaintai na nexceptionall yhig hAT P concentration for several hours, while the ATP content of cultureswithou tan ydetectabl epolyphosphat eimmediatel ydrop pedt oalmos tzero .A decreas eo fth eAT Pconten twit h 90o r morepercen twithi na fe wminute safte rth eenerg ygeneratin g system stoppes,seem s tob e common for many other bacteria (Chapmanan dAtkinso n1977 ,Ott oe tal .1984) .Th ecausa lrela tionbetwee nhig hanaerobi cATP-level san dpolyphosphat edegra dation (andsubsequen treleas eo fortho-phosphate) ,coul db e explainedb yproductio no fAT Pfro mpolyphosphat e invivo ,viz . polyphosphatea sa nenerg yreserve .A possibl eproductio no fAT P bya K + efflux (Mulder etal .1986 )durin g anaerobiosis was considereda snegligible ,sinc eAcinetobacte rstrai n13 2wa sno t ablet omaintai nhig hAT Plevel sdurin gthes econdition sdespit e ofth ereleas eo fth esam eamount so fK +a scompare dt ostrai n 210A(Va nGroenestij ne tal .submitted) . Besidesth eproductio n ofAT Pvi apolyphosphate:AM Pphospho -

88 transferase and adenylate kinase (Van Groenestijn et al. 1987), additional ATP could be produced via a protongradient generated by the efflux of ortho-phosphate. Acinetobacter strain 210A releases H2P04~ plus a metal ion as the result of polyphosphate degradation (Van Groenestijn et al. submitted). Anaerobically incubated Acinetobacter strain 210A appeared to have a transmem brane protongradient that plays an important role in the anaero bic energy metabolism of this strain, since the addition of DNP, which decreases this gradient, had a clear effect on the cellular ATP-level and the phosphate release rate (Fig. 4). The experiments with DNP also showed that lower cellular ATP con tents correlated with faster phosphate releases. An additional indication of the role of the protongradient in ATP generation by anaerobically incubated Acinetobacter strain 210A, is the observation that phosphate is released faster in a medium with DCCD, in the first two hours, as compared to an anaerobic medium (Fig. 1). DCCD inhibits ATPase and therefore the pro duction of ATP by a protongradient (Abrams and Baron 1970), thus resulting in lower cellular ATP concentrations and a faster polyphosphate degradation. The observation that the phosphate release rate decreased after 5 hours of incubation with DCCD can be explained by the leakage of adenylates from the cells (Table 2). Alcohols have been shown to augment membrane fluidity and reduce membrane order. Membrane fluidity correlates positively with increasing chain length (increasing hydrophobicity) of the alcohols added (Ingram and Buttke 1986). The higher freedom of motion within the membrane can stimulate phosphate transport (Molitoris et al. 1985) or facilitate the leakage of small mole cules such as protons. In both cases a higher rate of phosphate release would be expected which actually occurred. Polyphosphate as an energy reserve may be useful for Acine tobacter sp. under conditions of temporary anaerobiosis by supplying enough energy for the maintenance of a well func tioning cell metabolism. This might give these species a com petitive advantage over other strictly aerobic bacteria in a habitat where oxygen lacks occasionally. In the process of biological phosphate removal from waste water, in which acti vated sludge is subjected to alternating cycles of anaerobiosis and aerobiosis, conditions are created where the capacity of polyphosphate accumulation can be beneficial. In fact this kind of sludge is enriched with polyphosphate accumulating Acineto bacter sp. (Fuhs and Chen 1975, Buchan 1983). The role of poly phosphate as an energy reserve under anaerobic conditions in this specific wastewater treatment process has been hypothesized

89 (Rensink 1981,Marai se tal .1983 ,Comea ue tal . 1986)an di s nowsupporte db you rfindings .

Acknowledgements.W e thank theDepartment ofBiochemistr y of WageningenAgricultura lUniversit yfo rusin git sequipment .Thi s researchwa s supported bya specia lgran tfro mWageninge nAgri culturalUniversity .

REFERENCES

AbramsA ,Baro nC (1970 )Inhibitor yactio no fcarbodiimide so n bacterial membraneATPase . Biochem BiophysRe s Comm 41:858- 863 AdamseAD ,Deinem aMH ,Zehnde rAJ B(1984 )Studie so nbacteria l activitiesi naerobi can danaerobi cwast ewate rpurification . Antonieva nLeeuwenhoe k50:665-68 2 BuchanL (1983)Possibl e biological mechanism of phosphorus removal.Wat Sc iTech n15:87-10 3 CarrNG ,Sandh uG R (1966)Endogenou s metabolism ofpolyphos phates in two photosynthetic microorganisms. Biochem J 99:P29-P30 ComeauY ,Hal lKJ ,Hancoc kREW ,Oldha mW R (1986)Biochemica l model forenhance d biological phosphorus removal. Water Res 20:1511-1521 Cruden DL,Durbi nWE ,Markovet z (1983)Utilizatio no fPP. ^a sa n energysourc eb ya Clostridiu m sp..App lEnviro n Microbiol 46:1403-1408 ChapmanAG ,Atkinso nD E(1977 )Adenin enucleotid econcentration s andturnove rrates .Thei rcorrelatio nwit hbiologica lactivi tyi nbacteri aan dyeast .Ad vMicrobio lPhysio l15:253-30 6 DawesEA ,Senio rP J (1973)Th erol ean d regulation of energy reservepolymer si nmicro-organisms .Ad vMicrobio lPhysio l 10:135-266 Deinema MH,Habet s LHA,Scholte n J,Turkstr a E,Weber s HAAM (1980)Th eaccumulatio n ofpolyphosphat e inAcinetobacte r spp..FEM SMicrobio lLet t9:275-27 9 Friedberg I,Aviga dG (1968)Structure scontainin g polyphosphate inMicrococcu slysodeikticus .J Bacteri d 96:544-553 FunsGW ,Che nM (1975)Microbiologica lbasi so fphosphat ere movali nth eactivate d sludgeproces sfo rth etreatmen to f wastewater.Micro bEco l 2:119-138 Harold FM (1966)Inorgani c polyphosphates inbiology : structure metabolism andfunction .Bac tRe v 30:772-794

90 Ingram LO, Buttke TM (1986)Effect s of alcohols on micro organisms.Ad vMicrobio lPhysio l25:253-30 0 Kulaev I, VagabovV M (1983)Polyphosphat emetabolis m inmicro organisms.Ad vMicrobio lPhysio l24:83-7 1 MaraisGvR ,Loewentha lRE ,Siebrit zI P(1983 )Review :observa tions supporting phosphate removal by biological excess uptake.Wa tSc iTech n15:15-4 1 MulderMM ,Teixeir ad eMatto sMJ ,Postm aPW ,Da mK va n (1986) Energetic consequences of multiple K uptake systems in Escherichiacoli .Biochi mBiophy sAct a851:223-22 8 MolitorisBA ,Alfre yAC ,Harri sRA ,Simo nF R(1985 )Rena lapica l membranecholestero lan dfluidit y inregulatio n ofphosphat e transport.Ame rJ Physio l249:F12-1 9 Ohtake H, Takahashi K, Tsuzuki Y, Toda K (1985)Uptak e and release of phosphate by a pure culture of Acinetobacter calcoaceticus.Wate rRe s19:1587-159 4 OttoR ,Klon tB ,Brin kB ten ,Koning sW N (1984)Th ephosphat e potential,adenylat eenerg ycharg ean dproto nmotiv eforc ei n growing cells of Streptocuccus cremoris. Arch Microbiol 139:338-343 PradetA (1967)Etud ede sadenosine-5'-mono ,di -e ttriphos phates dans les tissues vegetaux. I. Dosage enzymatique. PhysiolVe g 5:209-221 RensinkJ H (1981)Biologisch edefosfaterin ge nprocesbepalend e factoren.In :Symposiu m Boek- Defosfatering ;nieuw eontwik - kelingene npraktijkervaringe ni nNederlan de nZweden .NV A Symposium (indutch ) Standardmethod sfo rth eexaminatio no fwate ran dwastewater , 14thed n (1976).A mPub lHealt hAss ,Washingto n VanGroenestij n JW, Deinema MH, ZehnderAJ B (1987)AT P pro ductionfro m polyphosphatei nAcinetobacte r strain210A . ArchMicrobio l148:14-1 9 VarmaAK , PeckJ rH D (1983)Utilizatio no fshor tan dlong-chai n polyphosphatesa senerg ysource sfo rth eanaerobi cgrowt ho f bacteria.FEM SMicrobio lLet t16:281-28 5 WilkinsonJ F(1959 )Th eproble mo fenergy-storag ecompound si n bacteria.Ex pCel lRe sSupp l7:111-13 0 ZyuzinaML ,Kulae v IS,Boby kME ,Efimov aTP ,Tereshi n (1981) Interrelationshipo fpolyphosphat emetabolis mwit hlevori n biosynthesisi nStreptomyce slevoris .Biokhimiy a46:782-78 8

91 CHAPTER 6

POLYPHOSPHATE DEGRADING ENZYMES IN ACINETOBACTER SP. AND ACTIVATED SLUDGE

Johan W. van Groenestijn, Michael M.A. Bentvelsen, Maria H. Deinema and Alexander J.B. Zehnder

ABSTRACT

Polyphosphatedegradin genzyme swer estudie di nAcinetobacte r andactivate dsludge .Polyphosphate:AM Pphosphotransferas eac tivityi nAcinetobacte rstrai n210 Adecrease dwit hincreasin g growthrates .Th eactivit yo fthi senzym ei ncell-fre eextract s ofAcinetobacte rstrai n210 Awa smaxima la ta p Ho f8. 5an da temperatureo f40°C ,an dwa sstimulate db y(NH^^SO ^ TheKJJ ,fo r AMPwa s0. 6m Man dV maxwa s6 0nmol.mi n .mg protein. Cell- freeextract so fthi s strainals ocontaine dpolyphosphatase , whichwa sabl et odegrad enativ epolyphosphat ean dsyntheti cMg - polyphosphate,an dwa sstrongl ystimulate db y300-40 0m M NH4CI. Apositiv ecorrelatio nwa sfoun dbetwee npolyphosphate:AM Pphos photransferaseactivity ,adenylat ekinas e activity and phos phorusaccumulatio n insi xdifferen tstrain so fAcinetobacter . Significantactivitie so fpolyphosphatekinas ewer eonl ydetecte d instrai nP ,whic hcontaine dn opolyphosphate:AM Pphosphotrans ferase.I nsample so factivate dsludg efro mdifferen tplant sth e activitieso fpolyphosphate:AM Pphosphotransferas ean dadenylat e kinasecorrelate dwel lwit hth eabilit yo fth esludg et oremov e phosphatebiologicall yfro mwast ewater .

93 INTRODUCTION

Acinetobacteri simportan ti nbiologica lphosphat eremova lfro m wastewater .Som estrain so fthi sstrictl yaerobi cbacteriu mar e ablet oaccumulat e largeamount so fpolyphosphat ei nfro mo f granules (Fuhsan dChe n 1975).Activate d sludgei nwastewate r treatment plants is enriched withAcinetobacte r ifalternatin g aerobican d anaerobic conditions areapplied .Lik e activated sludgepur e cultureso fAcinetobacte r sp.degrad epolyphosphat e andreleas eortho-phosphat eanaerobically ,whil eaerobicall y ortho-phosphatei stake nu pan dconverte dint opolyphosphate .B y thecombine d action of polyphosphate:AMP phospotransferase, whichcatalyse s

(polyphosphate)n + AMP—*(polyphosphate)n_1 + ADP, and ade nylate kinase, which catalyses: 2AD P5= ^AT P + AMP, ATP is produced frompolyphosphat ei nAcinetobacte rstrai n210 A(Va n Groenestijne tal . 1987). Thisenzym esyste mallow sthu sAcine tobactert ous epolyphosphat ea sa nenerg yreserv ei ntime swhe n energygeneratio nbecome sdifficul to ri snot possibl eat all ; e.g. incas eth eelectro ndono r(organi ccarbo ncompound )and/o r acceptor (oxygen)becom e limiting orar eabsent . Besidespolyphosphate:AM Pphosphotransferas eals oothe rpoly phosphatehydrolyzin genzyme shav ebee nreported .I na polyphos phateaccumulatin g Acinetobacter strainpolyphosphatas e was detected (Ohtakeet al .1985) .Thi senzym ewa sabl et odegrad e synthetic polyphosphate with a chain length of 15phospho - groups. In Escherichia coli polyphosphatekinase was found (Komberg 1957),wherea sMycobacteriu mphle icontaine dpolyphos phateglucokinas e (Szymonaan dOstrowsk i1964 )an dpolyphosphat e dependentNAD-kinas ewa sdetecte di nAcetobacter ,Achromobacter , Brevibacter,Corynebacteriu man dMicrococcu s species (Muratae t al.1980) .Activitie so fth elas tthre eenzyme swer eabsen ti n cell-freeextract so fAcinetobacte rstrai n210 A (VanGroenestij n etal .1987) .Sinc emos tAcinetobacte rspecie sdon' tposses sa glycolyticpathwa y (Baumanne tal .1968 )a degradatio n ofpoly phosphateb ymean s of l,3-phosphoglycerate:polyphosphate phos photransferase(Kulae ve tal .1968 )i sno texpected . In thepresent study thepropertie s of the polyphosphate degradingenzyme spolyphosphate:AM Pphosphotransferas ean dpoly phosphatase inAcinetobacte r strain 210Aar ereported . Sixdif ferentAcinetobacte rstrain shav ebee nanalyze dfo renzyme stha t areresponsibl efo rth eproductio no fAT Pfro mpolyphosphate . Thebiochemica l findings obtained with pure cultureswer e com paredt opractice ,i.e .activate dsludg efro mdifferen twaste watertreatmen tplant si nwhic hbiologica lphosphat e removal

94 occurs.

MATERIALSAN DMETHOD S

Organisms.Acinetobacte rstrai n210A ,B8 ,P ,13 2an d 124wer e isolated from activated sludgewit h themetho d described by Deinemae tal . (1980).Acinetobacte rcalcoaceticu s (NCIB8250 ) wasa kin dgif to fProf .Fewso n (Barrowmanan dFewso n 1985). Theseorganism swer emaintaine do nyeas textrac taga rslant s( 5 gglucose ,2. 5g yeas textrac tan d1 2g aga rpe rlitr etapwater , pH= 7.0),subculture dever ytw omonth san dstore da t4°C .

Activatedsludges .Sample so factivate dsludg ewer etake nfro m theaerate dzone so rstage sof :labscal efil lan ddra wreacto r (Appeldoorn and Deinema 1987), pilot plant PI (DeVrie s and Rensink 1985), fullscal eplan tRenkum :trai n3 (wit ha nan aerobican da naerobi czone )an dtrai n2 (completel y aerated) (Mulderan dRensin k 1987),pilo tplan tBunschoten ,ful lscal e plantBunschoten ,ful lscal eplan tBunni k(Jansse nan dRensin k 1987),conventiona l pilotplan t (completelyaerated )(Spanjer s andKlapwij k 1986).

Growthconditions .Acinetobacte rcell swer ecultivate di nshake n Erlenmeyerflask so na butyrat emediu m (VanGroenestij ne tal . 1987).Strain s 210Aan dP a t15° Can dstrain sB8 ,132 ,12 4an d Acinetobactercalcoaceticu sa t25°C ,th eoptima l temperatures forth eaccumulatio no fpolyphosphat ei nthes estrains .Cell so f Acinetobacterstrai n210 Awithou tpolyphosphate ,containin gonl y 13m gphosphoru spe rg dr ybiomass ,wer ecultivate di nth esam e wayunde rphosphoru slimitatio n(Va nGroenestij ne tal .1987) . In the experiments in which the polyphosphate:AMPphospho transferaseactivit yi nAcinetobacte rstrai n210 Awa sdetermine d as functiono fgrowt hrate , cellswer e continuously cultivated ina 2 1 bioreacto r(Applikon ,Schiedam ,Th eNetherlands )wit h automatically controlled pH (7.0)an dtemperatur e (25°C). The mediumuse di nthi sexperimen twa smodifie db yomittin gTri san d replacing 2.29 gNa-butyrat epe rlitr eb y5.6 7g Na-acetate.3H 20 perlitre .

Preparationo fcell-fre eextracts .Cell-fre eextract so fAcine tobacterwer e prepared by sonication and centrifugation accor dingt oVa nGroenestij ne tal .(1987) .Th epreparatio no fcell - freeextract sfro mactivate d sludgewa s thesame ,bu textende d withth efollowin g procedure: justbefor eus eth efroze nex -

95 tractswer ethawe d andcentrifuge d 10minute sa t7,00 0rp m ina Heraeus BiofugeA centrifuge (West-Germany)t o removeprecipi tatedmaterial .

Calculation1^ .Calculatio no fth e K^ forAM Pa sa substrat efo r polyphosphate:AMP phosphotransferase was carried out with a computerprogramm ewit ha non-linea rregressio n fittingpro cedureusin gth eleas tsquare smethod .

Chemicalan dbiochemica ldetermination . Ortho-phosphate and totalphosphoru s (persulphatedigestio n method)wer edetermine d according toStandar d Methods (1976). Theprotei n concentration wasmeasure dwit hth emetho do fLowr yusin gbovin eseru malbumi n as standard. The concentrations of ATP,AD P and AMP in the reactionmixture swer edetermine daccordin gt oPrade t (1976)an d Van Groenestijn et al. (1987). In this method samples were boiledfo r4 minute si na Tris-HCl/EDT Asolution ,an dATP ,AD P andAM Pwer edetermine d bymean so fa nAE C (adenylateenerg y charge)kit , based on bioluminescence, using luciferine and luciferase.

Enzymeassays .Al lassay swer ecarrie dou ta t30°C . Polyphosphate:AMP phosphotransferase (assay 1, continuous method): continuousspectrophotometrica lmetho daccordin gt oVa n Groenestijne tal .(1987) .Th ereactio nmixtur e inthi sassa y contains4 0m M (NH^^SO^du et oth eadditio no fenzyme swhic h were suspended in 3.2M (NH4)2S04. Inth e pH experiments the Tris-HClbuffe rwa sreplace db yequimola ramount so fa ME S(2 - morpholinoethanesulfonicacid )- NaO Hbuffe rat pH' slowe rtha n 7.0.I nth eexperimen ti nwhic hth eactivit yi ndifferen tAcine - tobacterstrain swa smeasure dth ereactio nmixtur ewa s supplied with 600u gGraham' ssal tpe rml . Polyphosphate:AMP phosphotransferase (assay 2, discontinuous method):0.1 8m lcell-fre eextrac twa sincubate di n1 m lreac

tionmixtur econtainin g3 m M MgCl2, 16m M Tris-HCl (pH=7.0)an d 1 0.3 mM Ap5A (P ,P^-di(adenosine-5')-pentaphosphate),th ereac tionwa sstarte db yaddin gAMP ,resultin gi na concentratio no f 1mM .I nth eextract so fAcinetobacte rstrai n210 Aenoug hnativ e polyphosphatewa spresent ,bu tcell-fre eextract so fsludg eha d tob esupplemente d with 600u gGraham' ssal tpe rml .A si ti s expected thatGraham' s saltbind sM g anadditiona l 1200u g

MgCl2.H20wa sadde dpe rm lt oassur ea nexces so ffre eM g in thereactio nmixture .A ttim einterval ssample so f2 5u lwer e takenfro mth ereactio nmixtur et odetermin eAD Pconcentrations . Polyphosphatase (EC 2.7.4.3): 0.99 ml cell-free extract was

96 mixedwit h0.0 1m l5 0m MMgCl 2/whic hresulte di na fina lcon centrationo f0. 5m MMgCl 2 (pH=7.0).A ttim einterval s samples of10 0p iwer etake nan dimmediatel yanalyse dfo rortho-phos phate. Adenylatekinase :0.1 6m lcell-fre eextract spe rm lreactio n mixturei na cuvette ,7 m MMgCl 2,9 0m MTris-HC l (pH=7.0),20 0 mMD-glucose ,0. 6m MNADP ,3. 4U (Units )H Kan d1. 7UG6P-D Hpe r ml, thereactio n wasstarte d byaddin g ADP, resulting ina concentrationo f1 mM . Thecuvett ewa splace d ina Beckman n

spectrophotometeran dth eproductio no fNADPH 2wa smeasure da t 340run .A reactio nmixtur ewit hadditiona l0. 3m MAp 5A,a speci ficinhibitio no fadenylat ekinas e(Feldhau se tal .1975) ,wa s useda scontrol . Polyphosphatekinase(E C2.7.4.1) :0.1 8m lcell-fre eextrac tan d 600u gGraham' ssal tpe rm lreactio nmixtur ei na cuvette ,8 m M MgCl2,10 0m MTris-HC l (pH=7.0),20 0m MD-glucose ,0.6 5m MNADP , 0.9m MAp 5A,3. 4UH Kan d1. 7U G6P-D Hpe rml ,th ereactio nwa s startedb yaddin gADP ,fina lconcentratio n1 mM .Th eproduce d NADPH2wa smeasure d spectrophotometrically.

Chemicals.HK ,G6P-DH ,adenylat ekinase ,NADP ,ATP ,AD Pan dAM P werepurchase dfro mBoehringer ,Mannheim .Apc Awa sobtaine dfro m SigmaChemica l Co.,St.Louis ,th eAEC-ki twa sfro m LUMAC/3M, Schaesberg, TheNetherlands . MESwa spurchase d from Merck, Darmstadt.Syntheti c polyphosphate infor m ofGraham' ssalt , which represents amixtur e oflinea r sodium polyphosphates [(NaP03)n,n = 1 0 ],wa sprepare db yheatin gNaH 2P043 hour sa t 750°Cfollowe db ycoolin gi nth eai routsid eth efurnace .K - polyphosphate (Kurrol'ssalt ,n = 2.1 0)wa sprepare db yheatin g KH2P04 2hour sa t260° C (Kulaev 1979). Na-polyphosphatewa s derived from this saltb ycatio n exchangewit h DOWEX-50.B y addingequivalen tamount so fMgCl 2 theNa-polyphosphat ewa s precipitateda sth einsolubl eMg-polyphosphate .

RESULTS

Polyphosphate:AMP phosphotransferase as a function of the growth rate of Acinetobacter strain 210A

The polyphosphate:AMP phosphotransferase activity and the phosphorus content of cells of Acinetobacter strain 210A de pended on the growth rate. The enzyme activity decreased from 36 to 25 nmol.min~.mg-1 protein with increasing growth rates, measured at dilutionrates between 0.05 and 0.42 h-1, while the

97 cellularphosphoru sconten tha da noptimu ma ta growt hrat eo f 0.11 h-1.

Propertieso fpolyphosphate;AM Pphosphotransferas ei nAcineto - bacterstrai n210 A

Theeffec to fp Han dtemperatur eo nth eactivit yo fpolyphos - phate:AMPphosphotransferas ei ncell-fre eextract so fAcineto - bacterstrai n210 Ai spresente di nFig .1 .Th ep Hoptimu m was

(2U l/> c 2 o SIa. (/> 100 .8 Q. £• Q. z: > < S 80 F A- A -S a XL E~> 60 a. X C3 a. E o • f 8. 40 y / / •6

rfc 01 20 =5 1— a. 01 7 0 I > 6 7 8 9 10 10 20 1 30 1 VO 1 50 »— ^o • pH Temperature(°C ) Fig. 1. Effect of pH (at 30°C) and temperature (at pH=7) on polyphos phate:AM P phosphotransferase in cell-free extracts of Acinetobacter strain 210A. At pH's lower than 7 the Tris-HCl buffer in the reaction mixture was replaced by a MES-NaOH buffer.

8.5 and the temperature optimum was 40°C. Two methods exist for the assay of this enzyme: (1) A spectrophotometrical method in which the reactionproduct is continuously converted into ATP, and (2) a discontinuous method in which at time intervals sam ples are taken from the reaction mixture (see Materials and Methods section). The continuous method resulted in 30 times higher activities than the discontinuous method. One of the differences between the two assay's is the presence of (NH4)2S04 in the spectrophotometrical method. The addition of 40 mM (NH4)2S04 to the reaction mixture of assay 2 stimulated the activity of polyphosphate:AMP phosphotransferase about 3 times The ortho-phosphate concentration, tested in the range of 0.1 to 10 mM did not affect the activity. The activity was dependent on

98 the AMPconcentratio n andthi s dependency obeyed thela wo f

Michaelis andMenten .A K m of0.5 8+ 0.1 3m M anda V max of6 0+ 4 nmol.min-*.mg~1 protein wasmeasure d following assay1 a ta p H of 7.0an d30°C .

Properties ofpolyphosphatas e inAcinetobacte r strain 210A

Polyphosphatase incell-fre e extracts ofAcinetobacte r strain

210A wasstimulate d byth epresenc e ofNH 4C1 orKC 1 (Fig. 2). 25-

1000 NH;an dK *(mM ) Fig. 2. Polyphosphatase activity in cell-free extracts of Acinetobac ter strain 210A as function of the concentration of K and NH^ (added as KC1 and NH,C1) in the reaction mixture.

The stimulation by NH4CI was optimal at 300-400 mM. No activity was found after heating cell-free extracts 5 minutes at 80°C. This is a strong indication that polyphosphate hydrolysis is catalyzed by an enzyme. The substrate for this reaction was native polyphosphate which was already present in the cell extracts. In an extract with a total of 566 ug phosphorus per ml of which 38 ug.ml""1 consisted of ortho-phosphate phosphorus, the ortho-phosphate phosphorus content per ml increased to 287 ug after 9 hours of incubation during the polyphosphatase assay. The degradation of synthetic polyphosphates was also studied in cell-free extracts without native polyphosphate. In a cell-free extract of cells cultivated under phosphorus limitation only 83 ug phosphorus per ml was present of which 21 ug.ml-''- was in the form of ortho-phosphate phosphorus. The enzyme activity was measured in a reaction mixture with 1 mg polyphosphate-phos-

99 phoruspe r ml but no NH4C1. Mg-polyphosphate (prepared from Kurrol'ssalt )wa s theonl ypolyme rhydrolize d inthi sassa y (3.2%withi n 7hour sa ta nrat eo f 0.7nmol.min -1.mg-1protein) . Hardlyan yortho-phosphat ewa sproduce d inabsenc eo fpolyphos phateo r inpresenc e ofNa -o rK-polyphosphate . Ina contro l experimentMg-polyphosphat ewa sincubate d ina reactio nmixtur e butwithou t cell-freeextract .Les stha n 0.1percen twa sde gradedwithi n 7hours .

Enzymesinvolve di npolyphosphat edegradatio ni ndifferen t strainso fAcinetobacte rsp .

Theamoun t of phosphorus and theactivitie s ofpolyphos - phate:AMPphosphotransferase ,adenylat ekinas ean dpolyphos - phatekinasei ndifferen tstrain so fAcinetobacte rar eshow ni n Table1 .Th eactivit yo fpolyphosphate:AM Pphosphotransferas eo f the strains correlated positivelywit hphosphoru saccumulation , andth eadenylat ekinas eactivit ycorrelate dbes twit hpolyphos phate:AM Pphosphotransferas eactivity .Significan tactivitie so f polyphosphatekinasewer eonl ydetecte d instrai nP , whichdi d notsho wan ypolyphosphat e:AM Pphosphotransferas eactivity .

Table1 .Phosphoru s accumulation andactivitie so fpolyphosphate:AM P phos photransferase, adenylate kinasean d polyphosphatekinase in sixdifferen t strains ofAcinetobacter .

Strain phosphorus incell s enzyme activity (nmol.min .mg'narotein) (percentage ofdr ybiomass ) polyphosphate:AMP adenylate polyphosphate- phosphotransferase kinase kinase

210A 10 43 89 B8 6 10 80 P 4 0 54 NCIB 8250 2.5 2 60 124 2.7 1 46 132 1.6 0 39

Polyphosphate:AMPphosphotransferas ean dadenylat ekinas ei n activatedsludg e

Theactivitie s ofpolyphosphate:AM Pphosphotransferas ean d adenylatekinas ei ncell-fre eextract so factivate d sludgeo f differentwastewate r treatmentplant sar epresente di nTable s2 and 3.Sludge stha twer eabl et oremov ephosphoru sbiologicall y alsoproduce dmor eAD Pfro mAM Pan dGraham' ssal twithi n2 hour s thanconventiona l sludge,althoug hth eactivitie swer e low(as -

100 Table 2.Biologica l removalo fphosphoru sfro mdomesti cwastewate rb ydifferen ttype so f activated sludge and theproductio no fAD Pfro mGraham' s salt and AMP by cell-free extractso fthes esludges .

Typeo factivate dsludg e percentagephosphoru s ADPproductio n removedfro mwastewate r (nmolpe rm gprotei ni n2 hours )

Phosphorusaccumulatin g sludge pilotplan tP I(Sep t 1986) 99 64 pilotplan tP I(Feb . 1986) 99 25 pilotplan t Bunschoten 70 16 Renkumtrai n3 55 13

Conventional sludge Renkumtrai n2 16 conventionalpilo tp Lant notdetecte d

Table3 .Th eremova lo fphosphoru sfro mwastewate rb ydifferen ttype so factivate dsludg e andthei ractivit yo fadenylat ekinase .

wastewatertreatmen tplan t percentagephosphoru s adenylatekinas e removedfro mwastewate r (mnol.ain.mg~1 protein)

fillan ddra wsyste m 100 680 pilotplan tP I(Sept .1987 ) 100 146 pilotplan tP I(Ma y1987 ) 87 132 fullscal eplan tBunschote n 81 87 Renkumtrai n3 55 78 fullscal eplan tBunni k 90 56 conventionalpilo tplan t (Sept.1987 ) N.D. 64 Renkumtrai n2 16 40 conventionalpilo tplan t (May1987 ) N.D. 13

N.D. notdetecte d

say2 ,Tabl e 2).Assa y2 wa suse dbecaus eo fit shig hsensitivi tyt olo wactivitie so fpolyphosphate:AM Pphosphotransferase . Thepresenc eo fGraham' ssal ti nth ecell-fre eextract so fth e sludgewa sa prerequisit efo rthi sreaction ,withou tthi ssyn theticpolyphosphat en oAD Pwa sproduced .Th eadenylat ekinas e activityi nth esludg ecorrelate dwel lwit hth epercentag eo f phosphorusremove dfro mth ewast ewate r (Table 3).

101 DISCUSSION

Polyphosphate:AMPphosphotransferas e activities were notparal lelledb ycellula rpolyphosphat econten ta tvariou sgrowt hrate s ofAcinetobacte r strain210A .Th einductio no fhighe renzym e activitiesa tlo wgrowt hrate si sa wel lknow npropert yo fman y catabolicenzymes ,especiall ythos einvolve di nearl ymetabolis m ofsubstrate s (Harderan dDijkhuize n 1983).Polyphosphate:AM P phosphotransferaseca nb eregarde da ssuc ha nenzyme ,becaus ei t catalysesth e firstreactio n inth ecataboli cpathwa y of the degradationo fpolyphosphat ean dth esubsequen tproductio no f ATP. Thepropertie s ofpolyphosphate:AM P phosphotransferase in Acinetobacter strain 210Adiffere d from those found for this enzymei nCorynebacteriu m xerosis.Th ep Hoptimu mfo rth eenzym e inAcinetobacte rstrai n210 Awa s8.5 ,whil eth ep Hoptimu mfoun d byDirheime r and Ebel (1965)fo rpolyphosphate:AM P phospho transferasei nCorynebacteriu m xerosiswa s 6.5.Th ephysiologi calfunctio no fth eenzym ei nCorynebacteriu mxerosi si sno tye t fullyunderstood ,becaus eo fit shig hK _fo rAMP ,namel y2 0m M (Kulaev 1979).Th eaffinit yo fpolyphosphate:AM Pphosphotrans ferasei nAcinetobacte rstrai n210 Afo rAMP ,however ,i sver y

high.Th ephysiologica lsignificanc eo fa nenzym ewit ha K mo f 0.6m Mca nhardl yb edoubted .I tha salread ybee ndemonstrate d thatthi senzym eplay sa rol ei nth eus eo fpolyphosphat ea sa n energyreserv ei nAcinetobacte r strain 210A (VanGroenestij ne t al.submitted) .Th epolyphosphate:AM Pphosphotransferas eacti vityi ncell-fre eextract swa sstimulate db y(NH^^SC ^jus ta s inCorynebacteriu m xerosis.Thi s (NH^^SO^effec tpartl yex plainsth edifferen tactivitie sfoun dwit hassa y1 an dassa y2 .

Polyphosphataseha sbee ndetecte d incell-fre eextract so f Acinetobacterstrain s210 Ai nsignifican tamounts .Nativ ebac terialpolyphosphat ea swel la shig hpolymeri csyntheti cMg - polyphosphatecoul db ehydrolize db ypolyphosphatase .Na -an dK - polyphosphate couldno tac ta ssubstrates ,despit eo fth esmal l amountso fMg 2+ inth ereactio nmixture .Bacteria lpolyphosphat e inAcinetobacte r strain210 Aals ocontain shig hamount so f Mg2+ (VanGroenestij ne tal .submitted) .Mg-polyphosphat ema yb eth e actualsubstrat etha ti saccepte db yth eenzyme ,lik eMg-pyro - phosphateb ymous eduodena lpyrophosphatas e (Nayaduan dMile s 1969).Th enee d forM g bypolyphosphatas eha sbee nfoun di n other organisms (Harold andHarol d 1965, Kulaev and Konoshenko

1971).Th estimulatio no fpolyphosphatas eb yNH 4C1i sa specifi c NH4"1"effec tan dno ta genera l salinity effect,becaus e equal

102 concentrationso fKC 1cause da les sstron gstimulation .NH 4 may changeth eenzym eo rth esubstrat e(e.g .catio ncompositio no f + polyphosphate).A n effecto fNH 4 onpolyphosphatas e hasneve r beenreporte d inbiology ,bu tth eactivatin g effecto fK on thisenzym efro mAerobacte raeroqene sha sbee nobserve dearlie r byHarol d andHarol d (1965). Kulaevan dKonoshenk o (1971)foun d alsoa stimulatio no fpolyphosphatas e fromNeurospor acrass a witha facto r3 b y20 0m MKC1 .Polyphosphatas ema yhav ea rol e inth edegradatio no fpolyphosphat ei ncell so fAcinetobacte r strain210 Agrowin gunde rphosphoru slimitation .Unde r these conditionspolyphosphat eca nb euse da sa nphosphoru sreserv e (VanGroenestij nan dDeinem a 1985).A sth ecell sd ono tcontai n any detectable amounts of AMP under phosphorus limitation (unpublishedresults) ,a degradatio no fpolyphosphat eb ypoly phosphate:AM Pphosphotransferas e isunlikely . Increased levelso fadenylat ekinas ei ndifferen tstrain so f Acinetobactercorrelate d withth epolyphosphate:AM Pphospho transferase activity and phosphorus accumulation by these strains. This correlationreflect sth erol eo fadenylat ekinas e inth edegradatio n ofpolyphosphat e incombine d action with polyphosphate:AMPphosphotransferase .Bot henzyme spla ya rol e inth eproductio no fAT Pfro mpolyphosphat ei nacinetobacter si n vitro and inviv o and therefore probably also in biological phosphateremova lb yactivate d sludge (VanGroenestij ne tal . 1987,Va nGroenestij ne tal .submitted) .Thi srol ewa sindicate d byth epositiv ecorrelatio nbetwee nth eactivitie so fpolyphos phate:AM Pphosphotransferas ean dadenylat ekinas ean dth eabili tyo fth esludg et oremov ephosphat ebiologicall y fromwast e water.Ver yhig hadenylat ekinas eactivitie swer efoun di nth e intermittentlyfe dan daerate d labscalefil lan ddra w system, whichcontaine d asludg ewit hmor etha n60 %acinetobacter stha t wasabl et oreleas eaccumulate dphosphat ever yfas tunde ran aerobic conditions (Appeldoornan dDeinem a 1987).Th ehighe r phosphatereleas erat ei nthi ssludg e (71m g phosphorus.h-1.g-1 drybiomass )a scompare dt oth erate smeasure di npur eculture s ofAcinetobacte rstrai n210 A (8m gphosphorus.h -1.g-1biomass , VanGroenestij ne tal .submitted )migh tb ecause db yth ehighe r adenylatekinas eactivit y inth esludge .Furthe r research is needed toexplai nth ehighe ractivities .Becaus eo f itshig h activitiesan dit sgoo dcorrelatio nwit hbiologica lphosphat e removal adenylate kinase might be used as an indicator for monitoring thisprocess . Inconclusio n itca nb esai dtha tamon gth ethre eenzyme s that can degrade polyphosphate in Acinetobacter (polyphos phate:AM Pphosphotransferase ,polyphosphatas ean dpolyphosphate -

103 kinase),polyphosphate:AM Pphosphotransferas eplays ,i ncombine d actionwit hadenylat ekinase ,th emos timportan trol ei nanaero bicAT P production inAcinetobacte r strains that accumulate aerobicallylarg eamount so fpolyphosphate .A lin kbetwee nth e findingsobtaine d withpur eculture san dpractic eha sbee nmad e by showing the correlation betweenth eactivitie s ofpolyphos - phate:AMPphosphotransferas ean dadenylat ekinas ei nactivate d sludgean dth eabilit yo fth esludg et oremov ephosphat ebio logically from wastewater .Th esuitabilit yo fth eadenylat e kinaseactivit ya sa nindicato rfo rthi sproces sha sbee nsug gested.Th estud ypresente d inthi spape rshow sth evalidit yo f purecultur estudie s forth eapplication .

Acknowledgements.W ethan kMis sA .va nMilaan ,Mis sD .Sijpken s andMis sH .Sleiderin kfo rthei rpractica lassistance ,whil eth e Departmento fBiochemistr yo fWageninge nAgricultura lUniversit y isgratel yacknowledge d forusin gthei requipment .Thi sresearc h wassupporte db ya specia lgran tfro mWageninge nAgricultura l University.

REFERENCES

Appeldoorn, K.J., Deinema, M.H. 1987. Biological phosphorus removalunde rdefine dcondition si na fill-and-dra w system. In: R. Ramadori (ed.)Biologica l phosphate removal from wastewaters.Proceeding so fa nIAWPR Cspecialize dconference , Rome, 28-30 Sept. (p. 309-312). Adv. Wat. Poll. Control. PergamonPress . Barrowman, M.M., Fewson, C.A. 1985. Phenylglyoxylate decar boxylasean dphenylpyruvat edecarboxylas e from Acinetobacter calcoaceticus.Curr .Microbiol .12:235-24 0 Baumann,P. ,Doudoroff ,M. ,Stanier ,R.Y .1968 .A stud yo f the Moraxella group. IIOxidase-negativ e species (genusAcineto bacter) . J. Bacteriol. 95:1520-1541 Deinema,M.H. ,Habets ,L.H.A. ,Scholten ,J. , Turkstra,E. ,We - bers, H.A.A.M. 1980.Th e accumulation of polyphosphate in Acinetobacter spp..FEM SMicrobiol .Lett .9:275-27 9 Dirheimer,G. ,Ebel ,J.P .1965 .Caracterisatio nd'un epolyphos - phate-AMP-phosphotransferase dans Corynebacterium xerosis. C.R.Acad .Sc .Pari s260:3787-379 0 Feldhaus, P., Frohlich, T.,Goody ,R.S. ,Isakov , M.,Chirmer , R.H.1975 .Syntheti cinhibitor so fadenylat ekinase si nth e assays for ATP-ase and phosphokinases. Eur. J. Biochem.

104 57:197-204 Fuhs, G.W., Chen, M. 1975. Microbiological basis of phosphate removal in the activated sludge process for the treatment of wastewater. Microb. Ecol. 2:119-138 Groenestijn, J.W. van, Deinema, M.H. 1985. Effects of cultural conditions on phosphate accumulation and release by Acineto- bacter strain 210A. In: J.N. Lester and P.W.W. Kirk (eds). Proceedings of the international conference on management strategies for phosphorus in the environment, Lisbon (p. 405- 410). Selper, London Groenestijn, J.W. van, Deinema, M.H., Zehnder A.J.B. 1987. ATP production from polyphosphate by Acinetobacter strain 210A. Arch. Microbiol. 148:14-19 Harder, W., Dijkhuizen, L. 1983. Physiological responses to nutrient limitation. Ann. Rev. Microbiol. 37:1-23 Harold, F.M., Harold, R.L. 1965. Degradation of inorganic poly phosphates in mutants of Aerobacter aeroqenes. J. Bacteriol. 89:1262-1270 Janssen, P.M.J., Rensink, J.H. 1987. High biological P- and N- removal on a full-scale activated sludge plant with alterna ting aeration. In: R. Ramadori (ed.) Biological phosphate removal from wastewaters. Proceedings of an IAWPRC specia lized conference, Rome, 28-30 Sept. (p. 365-368). Adv. Wat. Poll. Control. Pergamon Press. Romberg, S.R. 1957. Adenosine triphosphate synthesis from poly phosphate by an enzyme from Escherichia coli. Biochim. Bio- phys. Acta 26:294-300 Kulaev, I.S., Szymona, O.V., Bobyk, M.A. 1968. The biosynthesis of inorganic polyphosphates in Neurospora crassa. Biokhimiya 33:419-434 Kulaev, I.S., Konoshenko, G.I. 1971. The detection and proper ties of polyphosphatases of Neurospora crassa which hydrolyse inorganic polyphosphates to orthophosphate. Biokhimiya 36:1175-1182 Kulaev, I.S. 1979. The biochemistry of inorganic polyphosphates. John Wiley and Sons, Chichester, New York, Brisbane, Toronto Mulder, J.W., Rensink, J.H. 1987. Introduction of biological phosphorus removal to an activated sludge plant with practi cal limitations. In: R. Ramadori (ed.) Biological phosphate removal from wastewaters. Proceedings of an IAWPRC specia lized conference, Rome 28-30 Sept. (p. 213-224). Adv. Wat. Poll. Control. Pergamon Press Murata, K., Uchida, T., Tani, K., Kato, J., Chibata, I. 1980. Metaphosphate: a new phosphoryl donor for NAD phosphoryla tion. Agric. Biol. Chem. (Tokyo) 44:61-68

105 Nayadu,P.R.V. ,Miles ,P.L .1969 .Inhibitio no fpyrophosphatas e activityo fmous eduodena lalkalin ephosphatas eb ymagnesiu m ions.Biochem .J .115:29-3 5 Ohtake,H. ,Takahashi ,K. ,Tsuzuki ,Y. ,Toda ,K . 1985.Uptak e andrelas eo fphosphat eb ya pur ecultur eo fAcinetobacte r calcoaceticus.Wate rRes .19:1587-159 4 Pradet,A .1967 .Etud ede sadenosine-5'-mono ,di -e ttri-phos - phates dans les tissues vegetaux. I. Dosage enzymatique. Physiol.Veg .5:209-22 1 Spanjers,H. ,Klapwijk ,A .1986 .Ee ncontin urespiratiemete r- nieuweperspectieve nvoo rhe tbeheerse nva nhe tactief-slib -

proces.H 2019:432-43 4 Standardmethod sfo rth eexaminatio no fwate ran dwastewater , 14thedn ,1976 .Am .Publ .Healt hAss. ,Washingto n Szymona, M.,Ostrowski ,W . 1964.Inorgani cpolyphosphat egluco - kinaseo fMycobacteriu mphlei .Biochim .Biophys .Act a85:283 - 295 Vries,H.P .de ,Rensink ,J.H . 1985.Biologica l phosphorusre movala tlo wsludg eloading sb ypartia lstripping .In :J.N . Lester and P.W.W.Kir k (eds.)Proceeding s of the interna tionalconferenc eo nmanagemen t strategiesfo rphosphoru si n theenvironment , Lisbon, (p.54-65) . Selper,London .

106 CHAPTER 7

SUMMARY + SAMBMVATTING

SUMMARY

Biologicalphosphat eremova lfro mwast ewate ri sa biotechno - logicalalternativ et ochemica lphosphoru sprecipitation .Thi s processi sobtaine db yrecyclin gth esludg ethroug hanaerobi c andaerobi czones .I nth eanaerobi cpart sphosphat e isrelease d byth e sludgean ddurin g anaerobiosisphosphat e istake nup . Biological phosphateremova l isdependen t onth e enrichmento f activated sludgewit hpolyphosphat eaccumulatin gAcinetobacter . Likeactivate d sludge,pur eculture so fstrictl yaerobi cAcine tobactersp .absorb ephosphat e (upt o10 0m gphosphoru spe rg drybiomass )durin gaerobi ccondition san dreleas ei tanaero - bically.Th eai mo fthi sstud ywa st ogathe rknowledg eo nth e uptakean dreleas eo fphosphat eb yAcinetobacte ran dth emetabo lic functionso fpolyphosphate . Theaccumulatio no fpolyphosphat eb ypur eculture so fAcine tobacterstrai n210 Adepende do nth epresenc eo fa nintra -o r extracellularenerg ysourc e(chapte r 2).Th ehighes tamoun to f polyphosphatewa sfoun di ncell si nwhic henerg ysuppl ywa sno t limited,namel ya tlo wgrowt hrate sunde rsulphu rlimitation , andi nth estationar yphas eo fgrowt h wheneithe r thenitroge n orth esulphu rsourc ewa sdepleted .Accumulatio no fpolyphos phatewa sals opossibl edurin gendogenou srespiration .Whe nthi s respirationwa sblocke dwit hKC Nth ephosphat euptak estopped , whileth einhibitio no fth eprotei nsynthesi swit h streptomycin enhanced the accumulation ofphosphate ,whic h indicated the competitionbetwee nprotei n synthesisan dpolyphosphat esyn thesis forenergy . Therewa sa pronounce deffec to fth etempe ratureo nphosphoru saccumulatio nbu tthi seffec tvarie dfro m straint ostrain . Therol ean dbehaviou ro fcation si nth eaccumulatio nan d releaseo fphosphat ewa sstudie d (chapter 3). PO4~ wa srelease d togetherwit h1. 8protons .Mg 2+appeare dt ob eth emos timpor tantcounterio no fpolyphosphat ei nAcinetobacte r strain210A . Itwa srelease dan dtake nu psimultaneousl ywit hphosphate .Mg 2+ was not an essential polyphosphate counterion. If Mg2+ was depleted, stationary cultureso fAcinetobacte r strain 210Atoo k upth esam eamoun to fphosphat ewit hCa 2+ asth emos timportan t counterion. Inth epresenc eo fMg 2+ stationaryculture sdi dno t needCa 2+ forthei rphosphat eabsorption ,bu tth epresenc eo fK +

107 seemed tob ecrucia l forthi sprocess ,althoug hthi scatio ndi d notpla y aquantitativel y important role asa polyphosphate counterion.I naddition ,th einflu xan defflu xo fK +wa sinde pendento fphosphat euptak ean drelease.Continuou s cultivation atlo wgrowt hrate sunde rK +-limitationdi dno tresul ti npoly phosphateaccumulation ,whil eunde rsubstrat eo rM g -limitation largeamount so fpolyphosphat ewer epresen ti nth ecells .Th e sameeffec twa sfoun di nactivate dsludge .5 m gK +pe rlitr ewa s neededfo ra satisfactor ybiologica lphosphat eremova li nth e aerobiczon eo fa wastewate rtreatment plant .Granule so fMg - polyphosphate in Acinetobacter strain 210A could serve as a 2+ Mq -reserve.Cell swit hthes egranule swer eabl et ogro wi na mediumfre eo fM g ,wherea scell swithou tgranule swer enot , theyonl ygre wi nth epresenc eo fextracellula rM g . Polyphosphatei ncell-fre eextract so fAcinetobacte rstrai n 210Acoul db edegrade db yth eenzyme spolyphosphatas e or poly- phosphaterAMPphosphotransferas e(chapter s4 an d 6).Polyphos phateglucokinase ,polyphosphat edependen tNAD-kinas ean dpoly - phosphatekinase were notdetectable .PolyphosphateiAM Pphospho transferasewa sals ofoun di nAcinetobacte rstrai nB8 ,but not inAcinetobacte r strain P, which contained onlypolyphosphate - kinase.Bot hstrain swer eabl et oaccumulat elarg eamount so f polyphosphate.I nstrain stha tcanno taccumulat ethi sbiopoly - mer,n oo rver y small activities ofpolyphosphatekinas e and polyphosphate:AMPphosphotransferas e were found. All strains showed activities ofadenylat ekinase .I twa sdemonstrate d that byth ecombine d actiono fpolyphosphate:AM Pphosphotransferas e and adenylate kinase a continuous regeneration ofAT P from AMP orAD Pwa s possiblea slon ga spolyphosphat ewa spresent .Poly phosphate:AM Pphosphotransferas e could usenativ e and synthetic polyphosphatea ssubstrat ean dshowe da maximu mactivit ya ta p H of8.5 .It sactivit ywa sstimulate db y (NH^j^SC^,th eKJJ ,fo rAM P -1 appearedt ob e0. 6mM ,an dV maxwa s6 0nmol.min~.mg protein . Polyphosphatasei ncell-fre eextract so fstrai n210 Awa sabl et o hydrolysenativ epolyphosphat ean d syntheticMg-polyphosphate . TheK -an dNa-form ,however ,wer eno tdegraded . The activities ofpolyphosphate:AM Pphosphotransferas e and adenylatekinas ei n activatedsludg ecorrelate dwel lwit hth eabilit yo fth esludg e toremov ephosphat ebiologicall yfro mwast ewater . Degradation ofpolyphosphat ei nviv o inAcinetobacte r strain 210Aoccurre d ifth eenerg ysuppl yi nth ecel lwa sstopped ,fo r exampleunde ranaerobiosi so ri nth epresenc eo fKCN ,«.-dinitro - phenolo rN-N'-dicyclohexylcarbodiimi d (chapter 5). Thedegrada tionan d synthesiso fpolyphosphat ewa s dependent on theAT P concentrationi nth ecells .Lowe rAT Pconcentration scause da

108 faster phosphate release.Thi sreleas ewa s stimulated byalco hols.Th etransmembran eprotongradien tseeme dt opla ya nimpor tantrol ei nth eanaerobi cenerg ymetabolis mo fthi sstrictl y aerobicbacterium .Additio no fo<-dinitrophenol ,a protoniono - phore, decreased the cellularAT P concentration and stimulated thepolyphosphat e degradation.Th e roleo f polyphosphate as an energyreserv e inviv oha sbee ndemonstrate d by experiments in whichfiv estrain swer eincubate danaerobically .Cell so fAcine - tobacter strains 210Aan d B8,whic h were ablet oaccumulat e polyphosphate,release dlarg eamount so fortho-phosphat eanaero bically and contained highlevel so fATP .Cell so ftw o other strainso fAcinetobacte ran don estrai no fPseudomona swhic h didn't accumulatepolyphosphate ,showe d amuc h smallerreleas e ofphosphat ean dcontaine donl ylo wAT Pconcentrations .Cell so f strain210 Acultivate dunde rphosphoru slimitatio no ra t35° C didno tcontai ndetectabl eamount so fpolyphosphate .A sa resul t theirAT Pleve lwa slo wan dthe yrelease donl ysmal lo rnegli gibleamount so fphosphat eunde ranaerobi ccondition s Mg-polyphosphatei nAcinetobacte r sp.i sa multifunctional compound.I tca n serveas :(1 )a nenerg yreserv ei fi ti sde gradedb ya reactio nwit hAMP ,catalyze db ypolyphosphate:AM P phosphotransferase, (2)a phosphoru sreserv ei fi ti shydrolyze d bypolyphosphatase ,an d(3 )a Mg 2+reserv ewhereb yM g canb e replacedb yCa 2+ asa counterion .Th emos timportant roleo f polyphosphatei nwastewate rtreatmen tplant swit h biological phosphateremoval ,an dprobabl yals oi nnatura lenvironments ,i s itsus ea sa nenerg yreserv et osustai ntemporar yanaerobiosis . This propertymigh t explainth eenrichmen to f activated sludge subjected to alternating anaerobic and aerobic conditionswit h polyphosphateaccumulatin gAcinetobacte rsp. .

109 SAMENVATTING

Biologischedefosfaterin gva nafvalwate ri see nbiotechnolo - gischaltematie fvoo rd echemisch efosfaatverwijdering .Di t procesword tverkrege ndoo rhe tsli bt erecirculere ndoo ran aerobee naerob ezones .I nd eanaerob egedeelte ngeef the tsli b fosfaata fe ntijden saerobi eword te rfosfaa topgenomen ,Biolo gischedefosfaterin gi sgebaseer do pd everrijkin gva naktie f slibme tpolyfosfaa tophopend eacinetobacters .Reinculture nva n strictaerob eacinetobacter sneme nfosfaa to ptijden saerobi ee n onderanaerob eomstandighede nword the tfosfaa twee rafgegeven , zoalsda too kbi jaktie fsli bword twaargenomen .Het doe lva n hethie rbeschreve nonderzoe ki she tverzamele nva nkenni sove r de fosfaatopnam ee nafgift edoo rAcinetobacte re n de functies vanpolyfosfaa t indez ebacterie .Di tonderwer pi sva ngroo t belangdoo rd ezee rgrot ehoeveelhede n fosfordi eAcinetobacte r sp.ka nopneme n(to t10 0m gfosfo rpe rg drog ebiomassa )e nd e roldi edi tgeslach tspeel tbi jd ebiologisch edefosfatering . Ditproefschrif t beschrijft deregulati eva nd e ophopinge n afbraakva npolyfosfaa ti nAcinetobacte rsp. ,alsmed ed eenzyme n diebi jd eafbraa kbetrokke nzij ne nd emogelijk efunctie sva n ditbiopolymee ri ndez ebacterien . Deophopin gva npolyfosfaa tdoo rreinculture nva nAcinetobac tersta m 210Ahin ga fva nd eaanwezighei d vanee n intra-o f extracellulaire energiebron (hoofdstuk 2). Degrootst ehoeveel hedenpolyfosfaa twerde ngevonde ni ncelle nwaari nd eaanvoe r vanenergi enie tbeperken dwas ,namelij kbi jlag egroeisnelhede n tijdenszwavellimitati eo fi nd estationair efas en auitputtin g vand estikstof -o fzwavelbron .Ophopin gva npolyfosfaa twa soo k mogelijktijden s endogene respiratie.D e opname van fosfaat stoptewannee rdez erespirati egeblokkeer dwer dme tKCN ,terwij l dezeopnam ewer dbevorder ddoo rd eeiwitsynthes et eremme nme t streptomycine.Di teffec tduidd eo pee nenergi e concurrentie tusseneiwit -e npolyfosfaatsynthese .D etemperatuu rha dee n duidelijkeffec to pd efosfo rophoping ,maa rdi teffec tvarieer - deva nsta mto tstam . Hoofdstuk 3 beschrijft het onderzoek naar de rol en het gedragva nkatione n bijd e ophoping en afgifte van fosfaat. P04 werd samenme t 1.8protone nafgegeven .M g bleekhe t belangrijkstetegenio nt ezij nva npolyfosfaa ti nAcinetobacte r stam210A .Di twer dsteed sgelijktijdi gme tfosfaa tafgegeve ne n opgenomen. Mg2+ wasgee nessentiee l tegenion, want stationaire culturenva nAcinetobacte rsta m210 Akonde nwannee rMg 2+uit - geputwas ,dezelfd ehoeveelhede nfosfaa topneme nme tCa 2+al s belangrijkste tegenion. De fosfaat opname door stationaire

110 culturenwa snie tafhankelij kva nd eaanwezighei dva nC a ,mit s 2+ erMg 2+ aanwezigwas ,maa rd eaanwezighei dva nK leekcruciaa l voordi tproces ,hoewe ldi tkatio nkwantitatie fgee nbelangrijk e rolspeeld eal spolyfosfaa ttegenion .D einflu xe nefflu xva nK + wasonafhankelij kva nd efosfaa topnam ee nafgifte .Continu e cultiveringbi jlag egroeisnelhede nonde rK -limiterend eomstan - digheden resulteerdenie ti npolyfosfaa tophoping ,terwij ltij - denssubstraa to fMg 2+-limitatiewe lgrot ehoeveelhede npolyfos faataanwezi gware ni nd eeel .I naktie fsli bwer dee nzelfd e effectwaargenomen .Voo ree ngoed ebiologisch edefosfaterin g in deaerob e zoneva nee nzuiveringsinstallati e was5 m gK per liternodig .Korrel sme tMg-polyfosfaa ti nAcinetobacte rsta m 210Akonde nal see nMg 2+ reservedienen .Celle nmet dez ekorrel s wareni nstaa to mi nee nmediu mzonde rMg ~ tegroeien ,terwij l cellenzonde rkorrel s alleenkonde ngroeie nal se rextracel - lulairMg ~ aanwezigwas . Hetpolyfosfaa t incelvrij eextracte nva nAcinetobacte rsta m 210Ako nworde nafgebroke nme td eenzyme npolyfosfatas ee npoly fosfaat:AM P fosfotransferase (hoofdstukken 4e n 6). Polyfosfaat glucokinase, polyfosfaat-afhankelijk NAD-kinasee npolyfosfaat - kinasekonde nnie tworde naangetoond .Polyfosfaat:AM Pfosfo transferas ewer doo kgevonde n inAcinetobacte r stam B8, maar niet inAcinetobacte r stam P,dez elaatst esta m bevattewe l polyfosfaatkinase.Beid estamme nkunne ngrot ehoeveelhede npoly fosfaatophopen .I nstamme ndi eda tnie tkunne nwerde ngee no f zeerlag epolyfosfaatkinas ee npolyfosfaat:AM P fosfotransferase activiteitengevonden .All estamme nbezate nee nadenylaa tkinas e activiteit.Doo rmidde lva nd egecombineerd ewerkin gva npoly fosfaat:AM P fosfotransferase enadenylaa tkinas eko ne rcontin u ATPworde ngeregenereer dui tAM Po fAD Pzolan ge rpolyfosfaa t aanwezig was. Zowel synthetisch alsbacteriee lpolyfosfaa tko n als substraatdiene nvoo rpolyfosfaat:AM P fosfotransferase. Dit enzymvertoond eee nmaximal eactivitei tbi jee np Hva n8. 5e n werdgestimuleer ddoo r(NH 4)2S04.D e1 ^voo rAM Pblee k0. 6m Mt e 1 zijn,e nd eV max was 60nmol.min.mg~ eiwit.Polyfosfatas ei n celvrijeextracte nva n stam 210Ako nbacteriee l en synthetisch Mg-polyfosfaat hydrolyseren,maa rgee nNa -e nK-polyfosfaat .D e + activiteitva ndit enzy mwer dster kgestimuleer ddoo rNH 4 .D e polyfosfaat:AMP fosfotransferase enadenylaa tkinas eactivitei teni naktie fsli bcorreleerd egoe dme thet vermoge nva ndi t slibo m fosfaatbiologisc hui tafvalwate rt everwijderen . Deafbraa kva npolyfosfaa ti nviv oi nAcinetobacte rsta m210 A trado pwannee rd eenergi evoorzienin gi nd eee lstopte ,bij - voorbeeldtijden sanaerobi eo fi nd eaanwezighei dva nKCN ,

111 afbraake nopbou wva npolyfosfaa tblee kafhankelij k tezij nva n deAT P concentratie ind e eel. Een lagere ATP concentratie veroorzaakteee nsneller efosfaa tafgifte .D eafgift ewer dge - stijmuleerddoo ralcoholen .D etransmembraa n protonengradient leek een belangrijke rol te spelen in het anaerobe energie metabolismeva ndez estric taerob ebacterie .D etoevoegin gva n -dinitrofenol, eenprotonionofoor ,verlaagd ed eAT Pconcentra tiei nd eee le nstimuleerd ed epolyfosfaa tafbraak .D ero lva n polyfosfaatal see nenergi ereserv ei nviv ower dgedemonstreer d doorvij f stammenanaeroo bt eincuberen .Celle nva nAcinetobac - terstamme n210 Ae nB8 ,di epolyfosfaa tkunne nophopen ,gave n tijdensanaerobi egrot ehoeveelhede n ortho-fosfaat af enbevat - ten hoge ATP niveau's. Cellen van twee andere Acinetobacter stammene nee nPseudomona sstam ,di egee npolyfosfaa t kunnen ophopen,liete nee nvee llager efosfaa tafgift ezie ne nbevatte n slechts lageAT P concentraties.Oo k cellenva n stam 210Adi e onder fosforlimitatieo f bij35° Cware ngekweekt ,waardoo r zij geenaantoonbar ehoeveelhede npolyfosfaa tbevatten ,gave nklein e of verwaarloosbare hoeveelheden fosfaat af enbezate n lageAT P niveau's. Mg-polyfosfaati nAcinetobacte rsp .i see nmultifunctionel e verbinding.He tka ndiene nal s(1 )ee nenergi ereserve ,waarbi j hetword tafgebroke ndoo ree nreacti eme tAMP ,gekatalyseer d doorpolyfosfaatrAM Pfosfotransferase ,(2 )ee nfosfo rreserv e waarbij het wordt gehydrolyseerd met behulp vanpolyfosfatase , en(3 )ee nM g reservewaarbi jMg 2+ doorCa 2+ alstegenio nka n worden vervangen. De belangrijkste functiedi e dit biopolymeer vervulti nbiologisc hdefosfaterend e zuiveringsinstallaties en waarschijnlijk ooki nnatuurlijk emilieus ,i see nro lal sener giereserv eo m tijdelijkeanaerob esituatie st edoorstaan .Dez e eigenschap zoud everklarin gkunne nzij nvoo rd everrijkin gva n aktief slibdat onderworpen isaa n afwisselend anaerobie en aerobiemet polyfosfaa tophopend eAcinetobacte rsp. .

112 CURRICULUM VITAE

Deauteu rwer do p2 8me i195 8gebore nt eTiel ,alwaa rhi ji n 1976 aan deRijksscholengemeenscha phe tAtheneum- Bdiplom abe - haalde.I nda tjaa rwer dbegonne nme tee nstudi eMilieuhygien e aand eLandbouwhogeschoo l teWageningen .Het doctoraalpakke t bestondui tWaterzuivering ,Microbiologi ee nProceskunde .Prak - tijkervaringwer dopgedaa nbi jhe tZuiveringsscha pWest-Over - ijsselt e Zwollee nbi jhe tNationa l Environmental Engineering Research Institute teNagpu r inIndia .I n198 4wer d hetdoc - toraalexamenme tlo fbehaald .Va nfebruar i198 4to tjanuar i198 8 isd eauteu rwerkzaa mgewees tbi jd evakgroe pMicrobiologi eva n deLandbouwuniversitei tt eWageningen .He tonderzoe kda ti ndez e periodei sverrich theef tgelei dto tdi tproefschrift .