Aerobic Denitrifying Bacteria That Produce Low Levels of Nitrous Oxide

A PPLIED AND E NVIRONMENTAL M ICROBIOLOGY ,June 2003,p.3152–3157Vol.69,No.6

0099-2240/03/$08.00?0DOI:10.1128/AEM.69.6.3152–3157.2003

Copyright ?2003,American Society for Microbiology.All Rights Reserved.

Aerobic Denitrifying Bacteria That Produce Low Levels of

Nitrous Oxide

Naoki Takaya,1Maria Antonina B.Catalan-Sakairi,1Yasushi Sakaguchi,1

Isao Kato,1Zhemin Zhou,1and Hirofumi Shoun 2*

Institute of Applied Biochemistry,University of Tsukuba,Tsukuba,Ibaraki 305-8572,1and Department of Biotechnology,

Graduate School of Agricultural and Life Sciences,University of Tokyo,Bunkyo-Ku,Tokyo 113-8657,2Japan

Received 25November 2002/Accepted 17March 2003

Most denitri?ers produce nitrous oxide (N 2O)instead of dinitrogen (N 2)under aerobic conditions.We isolated and characterized novel aerobic denitri?ers that produce low levels of N 2O under aerobic conditions.We monitored the denitri?cation activities of two of the isolates,strains TR2and K50,in batch and continuous cultures.Both strains reduced nitrate (NO 3?)to N 2at rates of 0.9and 0.03?mol min ?1unit of optical density at 540nm ?1at dissolved oxygen (O 2)(DO)concentrations of 39and 38?mol liter ?1,respectively.At the same DO level,the typical denitri?er Pseudomonas stutzeri and the previously described aerobic denitri?er Paracoccus denitri?cans did not produce N 2but evolved more than 10-fold more N 2O than strains TR2and K50evolved.The isolates denitri?ed NO 3?with concomitant consumption of O 2.These results indicated that strains TR2and K50are aerobic denitri?ers.These two isolates were taxonomically placed in the ?subclass of the class Proteobacteria and were identi?ed as P.stutzeri TR2and Pseudomonas sp.strain K50.These strains should be useful for future investigations of the mechanisms of denitrifying bacteria that regulate N 2O emission,the single-stage process for nitrogen removal,and microbial N 2O emission into the ecosystem.

Nitrous oxide (N 2O)is a gaseous nitrogen oxide that is present at a concentration of about 350ppb in the atmosphere.The concentration of this compound was maintained below 300ppb in the global nitrogen cycle before the 20th century.However,recent reports suggest that the atmospheric concen-tration of N 2O is now increasing at a rate as high as 0.3%per year (1).N 2O has a 200-to 300-fold-stronger greenhouse effect than carbon dioxide (CO 2)and has the potential to destroy the ozone layer (17).Therefore,the N 2O balance is critical to the natural environment.The proposed sources of N 2O are chem-ical industries,burning fossil fuels,and biomass,as well as soil denitri?cation of nitrogenous compounds resulting from excess agricultural fertilizer (3,6,25).Another critical source of N 2O is wastewater treatment plants,in which considerable amounts of nitrogen pollutants removed from treated water are released into the atmosphere as N 2O,as well as dinitrogen (N 2).

Currently,nitrogen removal in wastewater treatment plants is essentially based on the activity of nitrifying and denitrifying microorganisms,both of which are inhabitants of activated sludge.Nitrifying bacteria aerobically oxidize ammonium con-taminants to nitrite (NO 2?)and nitrate (NO 3?),which are then reduced by denitrifying bacteria to gaseous nitrogen forms such as N 2O and N 2.Ef?cient wastewater treatment relies on successively exposing water to aerobic and anaerobic conditions,since nitri?cation and denitri?cation are aerobic and anaerobic processes,respectively (4,18).These properties represent a shortcoming of current systems since both denitri-?cation and nitri?cation produce N 2O as a by-product in the absence of correctly regulated oxygen (O 2)concentrations (2,

20).For example,the denitrifying activity of most denitrifying bacteria is suppressed when anaerobiosis is insuf?cient and they cannot catalyze the ?nal step of denitri?cation (reduction of N 2O to N 2)and produce N 2O (2,8,13,19).Because com-plete removal of dissolved O 2is dif?cult before the anaerobic denitri?cation that follows aerobic nitri?cation,current sys-tems release considerable amounts of N 2O during denitri?ca-tion.

To overcome this problem,novel aerobic denitrifying bac-teria are required that could be used for constructing aerobic denitrifying processes.Paracoccus denitri?cans (formerly Thio-sphaera pantotropha )(2,19,20)is an aerobic denitri?er that has been isolated from activated sludge and it is the best-characterized such organism.This species reduces NO 3?even in the presence of a saturating concentration of O 2.More recent surveys of aerobic denitri?ers have revealed some novel species,such as Microvirgula aerodenitri?cans (16)and Thaurea mechernichensis (22);the former organism denitri?es as ef?-ciently as P.denitri?cans under aerobic conditions (15).Previ-ously published results show that these denitri?ers remove NO 3?quite ef?ciently from treated water or from culture medium.However,the previous reports did not address the effect of O 2on the reduction of N 2O to N 2.

In the present paper we describe a novel method for screen-ing and characterizing natural aerobic denitri?ers that produce N 2gas by reducing NO 3?under oxic conditions.The strains described here produce less N 2O under aerobic conditions than the previously described aerobic denitri?ers produce,in-dicating that they potentially could be used to construct an aerobic denitrifying system that emits low levels of N 2O.

MATERIALS AND METHODS

Strains and media.P.denitri?cans (T.pantotropha )ATCC 35512originated from the American Type Culture Collection.Pseudomonas stutzeri ZoBell (?

*Corresponding author.Mailing address:Department of Biotech-nology,Graduate School of Agricultural and Life Sciences,University of Tokyo,Bunkyo-Ku,Tokyo 113-8657,Japan.Phone and fax:81-35841-5148.E-mail:ahshoun@mail.ecc.u-tokyo.ac.jp.

3152

ATCC14405)was provided by W.G.Zumft.Other strains were isolated in this study.The following media were used:bromothymol blue(BTB)medium(0.1% L-asparagine,0.1%KNO3,0.1%KH2PO4,0.005%FeCl2?6H2O,0.02%CaCl2?2H2O,0.1%MgSO4?7H2O,1ml of BTB liter?1[1%in ethanol],2%agar;pH

7.0to7.3),screening medium(SM)[0.284%sodium succinate,10mM NaNO3, 0.136%KH2PO4,0.027%(NH4)2SO4,0.1%yeast extract(Difco),0.019% MgSO4?7H2O,1ml of a trace element solution(14)liter?1;pH7.2],denitri-?cation medium(DM)(0.472%sodium succinate,10mM Na15NO3,0.15% KH2PO4,0.042%Na2HPO4,0.06%NH4Cl,0.5%Casamino Acids[Difco],0.1% MgSO4?7H2O,2ml of a trace element solution[14]liter?1;pH7.2),arti?cial wastewater(AWW)(0.085%NaNO3,0.06%peptone,0.04%bouillon extract, 0.01%urea,0.003%NaCl,0.01%KH2PO4,0.0014%KCl,0.002%MgSO4?7H2O,0.00185%CaCl2?2H2O),and Luria-Bertani medium(1%tryptone,0.5% yeast extract,0.5%NaCl).

Screening of denitri?ers.Samples from rice ponds,domestic wastewater,and soil were transferred to200ml of SM in500-ml Erlenmeyer?asks with cotton plugs and incubated at30°C for3days.Fresh SM was inoculated with5ml of culture and incubated under the same conditions.These procedures were re-peated three times.The resultant bacterial suspension was streaked on BTB medium plates with8.5g of sodium succinate liter?1and incubated at30°C for 1to3days.Soil samples were sometimes suspended in0.9%NaCl and plated directly on BTB medium plates.Regardless,soil samples were incubated in DM or AWW instead of SM.Resulting blue colonies were isolated and screened as follows(second screening).The bacteria were transferred to200ml of Luria-Bertani medium with10mM NaNO3in a500-ml Erlenmeyer?ask.The?ask was sealed with a butyl rubber stopper and rotary shaken at120rpm at30°C(pre-culture).The atmospheric air in the headspace was not replaced,so the initial conditions were aerobic.A portion(50ml)of the preculture was collected by centrifugation,washed twice with0.9%NaCl,transferred to100ml of DM containing15N(Na15NO3)in a500-ml Erlenmeyer?ask,and incubated as described above.Aerobic denitri?cation by the bacteria was measured by deter-mining time-dependent production of15N2and the amount of residual O2in the headspace gas.

Batch culture.Batch cultures in?asks were incubated essentially under the conditions described above for the second screening.To investigate their effects on denitri?cation,various carbon sources were added to DM instead of succinate at a C/N ratio of24.

Continuous culture.Precultures(100ml)were transferred to1-liter fermen-tation jars(BMJ-01;Able,Tokyo,Japan)containing500ml of DM or AWW with formate in which Na15NO3was substituted for Na14NO3.Each culture was magnetically stirred at300rpm and30°C(pH7.4).Initially,20%O2balanced with argon was added to the cultures at a constant?ow rate of20ml/min. Exhaust gas that?owed into gas sampling tubes was collected after passing through a vapor condenser.The culture broth was aseptically withdrawn through sampling tubes,and then NO3?,NO2?,and biomass were analyzed.A contin-uous nutrient?ow was started when cultures reached the logarithmic growth phase and was continued at a dilution rate of0.14h?1.After48h,exhaust gas was analyzed in duplicate to con?rm the steady state.The O2supply conditions were manipulated by changing the feed gas composition and/or the stirring speed.The cultures were constantly supplied with nutrients for over48h,which was equivalent to6.7replacements of the working volume to ensure a steady state.

Determination of the16S rRNA gene sequences.Total bacterial DNA was puri?ed as described previously(14).The genes encoding16S rRNA were am-pli?ed by PCR by using total DNA(0.1?g)as the template and primers AGA GTTTGATCCTGGCTCAG and GGTTACCTTGTTACGACTT(26).Genes were ampli?ed by30cycles of94°C for20s,50°C for1min,and72°C for1.5min, followed by extension at72°C for10min.

Analytical methods.We analyzed gaseous O2and N2O by gas chromatography (GC)(23).Isotope-labeled N2gas was measured by isotope mass spectroscopy (Delta plus,Finnigan MAT)as described previously(24),and NO3?and NO2?were measured colorimetrically(12).

Enzyme assays.Nitrate reductase(Nar)activity was assayed as described previously(7)by using methylviologen-dithionite as the electron donor.Nitrite reductase(Nir)activity was assayed by using NADH-phenazine methosulfate as the electron donor by determining the amount of NO produced by the P450nor-trap method(7).Nitric oxide reductase(Nor)activity was determined by deter-mining NADH-dependent N2O formation by GC as described previously(11). Nitrous oxide reductase activity was assayed as described by Kshirsagar et al.(9). Nucleotide sequence accession numbers.The nucleotide sequence data have been deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession numbers AB096261(strain TR2)and AB096260(strain K50).

RESULTS

Isolation of novel aerobic denitri?ers.We developed plate assays to isolate bacterial strains that denitrify under aerobic conditions,.The method is based on the changes in the pH of a medium due to NO3?depletion by denitri?cation.The plates contained KNO3and the pH indicator BTB.The pH of the medium was initially adjusted to between7.0and7.3.Plates inoculated with a bacterial suspension were incubated for sev-eral days at the appropriate temperature,and then bacterial NO3?consumption was monitored by examining blue colonies and/or halos due to the increasing pH of the medium.Positive strains obtained from this initial screening analysis were screened further by culturing them in?asks containing DM under initially aerobic conditions.Denitri?cation was periodi-cally monitored by measuring N2O and15N2in the gas phase by GC and GC-mass spectrometry,respectively.Most strains completely consumed O2before they produced N2.However, some strains produced signi?cant amounts of N2even in the presence of3%O2in the gas phase of the?asks.Among these, strains that produced the least N2O(aerobic denitri?ers emit-ting low N2O levels)were selected.

In a typical experiment we screened97bacterial colonies, and we found29strains which were positive in BTB plate assays and9strains that produced N2even in the presence of 3%O2.Six of the nine strains evolved less than1?mol of N2O ?ask?1under the same culture conditions.The most promi-nent aerobic(O2-tolerant)N2producers(strains TR2and K50)from several screening analyses were analyzed as follows. Properties of denitri?ers in batch culture.The time-depen-dent denitri?cation by strain TR2or K50was compared with that of the typical denitri?ers P.stutzeri ZoBell and P.denitri-?cans ATCC35512during batch culture in?asks with a head-space that was initially?lled with air to create aerobic condi-tions.After the incubation was started,the O2concentration in

the gas phase was gradually decreased,and N2evolved(Fig.1). The rates of N2production were signi?cantly different for the different cultures.Initiation of N2production by P.stutzeri ZoBell required a lag period of2.5h,whereas the other three strains evolved N2without a lag phase,indicating that N2 production by P.stutzeri ZoBell is more sensitive to O2than N2 production by the other strains is(Fig.1A).The aerobic deni-tri?er P.denitri?cans produced N2without a lag but concom-itantly produced a considerable amount of N2O(Fig.1B). Strains TR2and K50produced N2without a lag period,and little N2O was detected during the entire incubation period. These results demonstrated that strains TR2and K50should be aerobic denitri?ers that produce low levels of N2O even under aerobic conditions.The NO3?initially added(1mmol) was completely consumed by these cultures(data not shown). The amount of consumed N atoms recovered in N2was great-est with strain K50,in which the0.27mmol of N2obtained corresponded to54%recovery(Fig.1C).The?nal level of recovery with strain TR2was28%(Fig.1D).

Denitri?cation in continuous culture.We further investi-gated the effects of aeration on N2and N2O production by the isolated bacteria.Table1shows the steady-state characteristics observed with continuous cultures of the strains isolated and of the typical denitri?ers.Changing the composition of the aer-ating gas and agitation speed allowed us to establish anoxic

V OL.69,2003AEROBIC DENITRIFYING BACTERIA3153

(dissolved oxygen [DO]concentration,0?mol liter ?1),hypoxic (DO concentration,5.3to 9.4?mol liter ?1),and oxic (DO concentration,28to 39?mol liter ?1)aeration conditions.Most of the cultures produced N 2under anoxic and hypoxic conditions;the only exception was a P.denitri ?cans culture in which there was too little growth to detect N 2produced under anoxic conditions.All of the strains produced N 2at a higher rate under hypoxic conditions than under anoxic conditions (data not shown).However,under anoxic conditions the rate of N 2production was higher than that under hypoxic condi-tions,as for other bacterial denitri ?ers (2,4),when the rates were expressed on the basis of cell mass (optical density at 540nm [OD 540]).This is because the cell mass was signi ?cantly larger under hypoxic conditions than under anoxic

conditions.

FIG.1.Denitri ?cation by new isolates and control denitri ?ers in initially aerobic batch cultures.The strains were cultured in ?asks containing DM,and the gas phase was monitored periodically as described in Materials and Methods.(A)P.stutzeri ZoBell;(B)P.denitri ?cans ATCC 35512;(C)strain K50;(D)strain TR2.Symbols:F ,N 2;?,N 2O;s ,O 2.Typical results from more than three independent experiments are shown.

TABLE 1.Steady-state characteristics of continuous cultures of bacterial strains a

Strain

Conditions

b

DO concn (?mol liter ?1)

OD 540

NO 3?consumption (?mol min ?1unit of OD 540?1)N 2O production (?mol min ?1unit of OD 540?1)

N 2production (?mol min ?1

unit of OD 540?1)

TR2A 00.5249.9?10?5 6.8H 5.3 1.37.39.7?10?4 2.8O 39 2.4 3.5 1.6?10?40.9K50

A 00.4309.5?10?3 6.5H 6.3 1.7 6.8 1.6?10?3 1.8O 38 3.70 6.2?10?40.03P.stutzeri ZoBell

A 00.524 1.5?10?2 2.9H 7.8 1.38.8 6.7?10?3 1.5O 38 1.08.4 6.2?10?30.0P.denitri ?cans ATCC 35512

A 00.2609.5?10?30.0H 9.4 3.2 2.7 4.1?10?3 1.4O 28 6.70.4 2.8?10?20.0

a DM was used for the cultures.The dilution rate was 0.14h ?1.The data are averages of two independent experiments.

b

A,anoxic (the in ?uent gas was 100%Ar,and the agitation rate was 300rpm);H,hypoxic (the in ?uent gas was 20%O 2–80%Ar,and the agitation rate was 300rpm);O,oxic (the in ?uent gas was 20%O 2–80%Ar,and the agitation rate was 430rpm).

3154TAKAYA ET AL.A PPL .E NVIRON .M ICROBIOL .

The value per cell for strain TR2(2.8?mol of N2min?1unit of OD540?1)was double that for P.denitri?cans(1.4?mol of N2min?1unit of OD540?1)under hypoxic conditions.When challenged with oxic conditions,P.stutzeri ZoBell and P.deni-tri?cans produced no N2and considerable amounts of N2O, indicating that O2inhibited the reduction of N2O to N2under these conditions.By contrast,strains TR2and K50could still produce N2under oxic conditions(Table1).The activity of strain TR2(0.9?mol of N2min?1unit of OD540?1)was as much as32%of the activity under hypoxic conditions.Both TR2and K50produced less N2O than the other strains pro-duced under the corresponding aeration conditions.TR2pro-duced less N2O than K50produced,indicating that strain TR2 reduces N2O to N2more ef?ciently than K50reduces N2O to N2.Our results showed that P.denitri?cans,a typical aerobic denitri?er,produced N2O at a much higher rate than the other strains produced N2O,indicating that this organism produces N2O instead of N2under oxic conditions.These?ndings indi-cate that the strains isolated in the present study are aerobic denitri?ers that produce low levels of N2O.

Electron donor speci?city.We investigated the electron do-nor speci?city of denitri?cation by strains TR2and K50in?ask cultures under initially aerobic conditions(Fig.1).Table2 shows that both strains consumed O2and produced N2and/or N2O with all carbon sources tested,indicating that they can use a variety of carbon sources as electron donors for O2respira-tion and denitri?cation.Of the carbon sources tested,succi-nate supported O2respiration and denitri?cation most ef?-ciently in TR2.Strain K50utilized O2ef?ciently in the presence of ethanol and acetate in addition to succinate.Al-though the N2production rates were lower and more N2O evolved than the N2O that evolved with the other carbon sources,C1compounds,such as methanol and formate,could support denitri?cation by both bacteria.Strain K50evolved little N2O irrespective of the electron donor.

Identi?cation of the strains.Strains TR2and K50were both gram-negative rods with catalase and oxidase activities.They also metabolized glucose to organic acid,indicating that they are pseudomonads.The nucleotide sequences of their16S rRNA genes and a phylogenetic analysis revealed that these strains are members of the genus Pseudomonas in the?sub-class the class Proteobacteria.The levels of identity of the sequences of strains TR2and K50were greatest with the se-quences of P.stutzeri(99%)and Pseudomonas mendocina NCIB10541(99%),respectively.The phenotype of strain TR2 was characteristic of P.stutzeri;namely,there were no?uores-

cent pigments,organic growth factors were not required,and the organism was gelatinase negative and amylase positive.The ability to grow at41°C is also a phenotypic criterion that dis-tinguishes P.stutzeri strains from other Pseudomonas spp. Thus,we identi?ed strain TR2as P.stutzeri and designated it P.stutzeri TR2.Strain K50accumulated poly?-hydroxybutyric acid,which P.mendocina does not do.Therefore,we could not identify this strain and designated it Pseudomonas sp.strain K50.

Performance in AWW system.We estimated the denitri?ca-tion by continuous cultures of P.stutzeri TR2in organic waste-water.We added formate to conventional wastewater as an additional electron donor assuming that it should provide se-lective pressure for survival that should allow P.stutzeri TR2to become the dominant species in the culture.Table3shows that neither P.stutzeri TR2nor the control strain P.stutzeri ZoBell evolved N2O at any DO level.TR2more ef?ciently reduced NO3?and produced more N2than P.stutzeri ZoBell.When the DO concentration was increased to113?mol liter?1,P. stutzeri ZoBell produced neither N2nor N2O.This is in con-trast to P.stutzeri TR2,which produced a signi?cant amount of N2even under highly aerobic conditions(DO concentration, 141?mol liter?1).The level of recovery of N atoms in N2in an aerobic culture of P.stutzeri TR2was14%of the consumed NO3?.Other N atoms of the consumed NO3?should have been incorporated into the biomass.These results indicate that the denitrifying system of P.stutzeri TR2is more resistant to

TABLE2.Carbon source utilization of strains TR2and K50a

Carbon source

Reaction rate(?mol h?1)

Strain TR2Strain K50

N2N2O O2N2N2O O2

Glycerol8.8 1.3230 4.80.05140

Glucose8.8 6.523015.0ND b390

Succinate36.3ND38042.5ND410

Citrate17.5 2.131016.30.23310

Acetate13.8 2.625028.8ND410

Ethanol 2.58.326328.8ND450

Methanol0.50 1.62300.040.73150

Formate0.1612.0200ND 1.0380

a The values are the initial reaction rates for N2and N2O production and O2

consumption during the?rst8h of?ask culture.N2,N2O,and O2concentrations

in the gas phase were determined by GC.The initial amounts of NO3?and O2

were1and4mmol,respectively.The data are averages of two independent

experiments.

b ND,not detected.

TABLE3.Steady-state characteristics of strains in AWW supplemented with formate a

Strain DO concn

(?mol

liter?1)

OD540

NO3?consumption

(?mol min?1unit

of OD540?1)

NO2?production

(?mol min?1unit

of OD540?1)

N2production

(?mol min?1

unit of

OD540?1)

P.stutzeri TR230.2251.47.7 5.5

720.5023.00.18 5.6

1410.6218.20.10 1.3 P.stutzeri ZoBell30.4523.10.89 1.3

1130.5022.40.14ND

1600.4922.70.08ND

a The dilution rate was0.14h?1.The data are typical results of three independent experiments.No N2O production was detected for any strain.

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O2than the denitrifying system of P.stutzeri ZoBell is and that the TR2strain produced N2under aerobic conditions in AWW.

Enzyme activities.The activities of respiratory NO3reduc-tase,NO2?reductase,NO reductase,and N2O reductase in crude extracts prepared from denitrifying cells of P.stutzeri TR2were determined,and the results obtained with3-day cultures are shown in Fig.1.The speci?c activities of these enzymes were4,400,63,6.8,and64nmol of product min?1mg of protein?1,respectively,indicating that the activity observed should have been responsible for denitri?cation by P.stutzeri TR2.

DISCUSSION

Although N2O production by denitrifying bacteria under insuf?cient anaerobic conditions is an established phenomenon that causes serious global warming,in most studies of aerobic denitri?ers the workers have described only NO3?and NO2?consumption under aerobic conditions,and studies in which the workers focused on denitri?ed gas(N2O and N2)have been limited.This is probably due to dif?culties in detecting deni-tri?ed N2under aerobic conditions;under such conditions contamination with atmospheric N2interferes with precise quantitation of denitri?ed N2.Here,we used heavy-isotope-labeled NO3?(15NO3?)and highly sensitive mass spectrome-try to screen and isolate denitrifying bacteria emitting low levels of N2O under aerobic conditions.To our knowledge,we are the?rst researchers to isolate and characterize denitri?ers that produce less N2O than other bacteria produce.Our screening method should be applicable to isolation of aerobic denitrifying bacteria that exhibit such properties.

Our results indicated that both of the denitri?ers isolated,P. stutzeri TR2and Pseudomonas sp.strain K50,produced N2 even under oxic conditions(DO concentration,38to39?mol liter?1)(Table1),conditions under which other typical aerobic denitri?ers could not produce N2,indicating that the novel strains are O2resistant,aerobic denitri?ers.Physiologically,O2 is the best electron acceptor for supporting growth,and it receives electrons through the respiratory chain.Most denitri-fying microorganisms perform O2respiration and repress the denitri?cation mechanism when O2is available.Therefore,the physiological signi?cance of denitri?cation under oxic condi-tions(aerobic denitri?cation)is quite intriguing.Aerobic deni-tri?cation has also been identi?ed in P.denitri?cans(19,20),as well as in the novel species M.aerodenitri?cans(15)and T. mechernichensis(22).The strains identi?ed here,together with these organisms,should be useful for future investigations of the mechanisms of aerobic denitri?cation by bacteria and the role of aerobic denitri?cation in the ecosystem.

We found that all of the bacteria tested emitted considerably more N2O under continuously aerobic conditions than under anaerobic conditions(Table1).This is consistent with the observation that in most denitri?ers,the activity of N2O re-ductase is inactivated by O2,which also represses expression of the encoding gene and N2production(4,8).We also found that both P.stutzeri TR2and Pseudomonas sp.strain K50 produced less N2O and more N2under our conditions than the other denitri?ers produced(Fig.1and Table1).Furthermore, our results demonstrated that the N2-and N2O-producing activities of the strains were dependent on the culture.For example,carbon sources seemed to affect denitri?cation to various degrees depending on the strain(Table2).Factors other than carbon sources can also affect aerobic denitri?ca-tion.In the presence of formate as an electron donor,P.stutzeri TR2produced N2O in batch culture(Table2),whereas little N2O was detected in continuous culture(Table3).This may have been due to differences in conditions between the batch and continuous cultures or to the compositions of the media. The mechanism with which the isolates suppressed N2O pro-duction remains to be studied.However,the results suggest that aerobic denitri?cation that produces less N2O should be a complicated system that is regulated by various factors,includ-ing the O2supply and the composition of the medium.It is interesting that P.stutzeri TR2and ZoBell have different deni-trifying properties,especially for N2O production under oxic conditions(Table1)and for N2production in response to sudden exposure to O2(Fig.1),despite their close phyloge-netic relationship.A comparison of the genes of these strains should reveal the critical gene(s)required for aerobic denitri-?cation and for N2O suppression.

The novel bacteria isolated in the present study denitri?ed under conditions that mimicked those in wastewater treatment plants(Table3).This?nding suggests that the isolates could be maintained in the mixed population of microorganisms found in sludge as is observed with P.denitri?cans,which reduces NO3?more ef?ciently than a control reactor without the strain when it is mixed with activated sludge(9).Sometimes,a C1 carbon source,such as methanol or formate,has been used to manipulate the dominant bacterial species in sludge(5,21). Our novel strains should be useful for constructing new aerobic denitri?cation processes that do not produce N2O,since these strains could be selected as the dominant organisms by using C1compounds as the electron donors.

ACKNOWLEDGMENTS

We thank Kota Hatayama(University of Tsukuba)for determining the nucleotide sequence of the16S rRNA gene.We also thank Norma Foster for critical reading of the manuscript.

This study was supported by PROBRAIN(Program for Promotion of Basic Research Activities for Innovative Biosciences)and by a grant-in-aid for scienti?c research from the Ministry of Education, Science,Culture and Sports of Japan.

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14.Ozeki,S.,I.Baba,N.Takaya,and H.Shoun.2001.A novel C1-utilizing

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