APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1988, p. 2220-2225 Vol. 54, No. 90099-2240/88/092220-06$02.00/0Copyright © 1988, American Society for Microbiology

Heat Production by the Denitrifying Bacterium Pseudomonasfluorescens and the Dissimilatory Ammonium-Producing BacteriumPseudomonas putrefaciens during Anaerobic Growth with Nitrate as

the Electron AcceptorM.-O. SAMUELSSON,l* P. CADEZ,2 AND L. GUSTAFSSON'

Department of Marine Microbiology, University of Goteborg, Carl Skottsbergs Gata 22, S413 19 Goteborg, Sweden,'and E. Kardelj, University of Ljubljana, Biotechnical Faculty, 61000 Ljubljana, Yugoslavia2

Received 22 March 1988/Accepted 16 June 1988

The heat production rate and the simultaneous nitrate consumption and production and consumption ofnitrite and nitrous oxide were monitored during the anaerobic growth of two types of dissimilatory nitratereducers. Pseudomonasfluorescens, a denitrifier, consumed nitrate and accumulated small amounts of nitriteor nitrous oxide. The heat production rate increased steadily during the course of nitrate consumption anddecreased rapidly concomitant with the depletion of the electron acceptors. A mean experimental enthalpychange value of -800 kJ/mol of nitrate and a mean growth yield value of 33 g (dry weight)/mol of nitrateconsumed were obtained for different concentrations of nitrate. For Pseudomonas putrefaciens, a dissimilatoryammonium producer, the nitrate consumption resulted in an accumulation of nitrite and nitrous oxide. Nitriteconsumption commenced after depletion of the nitrate; consequently, two phases were noted in the heatproduction rate curve during growth. A mean experimental enthalpy change value of -810 kJ/mol of nitratewas obtained for different concentrations of nitrate.

Dissimilatory nitrate reduction, which utilizes nitrate asthe electron acceptor, operates via different pathways. Thebest known of these is denitrification; during this processdinitrogen gas is produced and the reduction of nitrateoccurs via nitrite and nitrous oxide through a respiratorychain. Earlier reports have pointed out that oxidative phos-phorylation in the case of a denitrifying Pseudomonas per-fectomarinus (6, 7) is only coupled to the reduction of nitrateto nitrite and nitrous oxide to dinitrogen gas. Koike andHattori (15), however, have shown that three different cellyields were obtained when a denitrifying Pseudomonasdenitrificans was grown on nitrate, nitrite, and nitrous oxide,suggesting that an oxidative phosphorylation is also coupledto the reduction of nitrite to nitrous oxide.Another pathway encompasses dissimilatory ammonium

producers (23), for which the reduction of nitrate to nitrite iscoupled to a respiratory chain and the further reduction toammonium is regarded as a fermentative process in somebacteria (5, 24). In other studies, bacteria such as Vibriosuccinogenes have been shown to have the capacity toproduce ammonium by utilizing a respiratory pathway (3).

Denitrification is characterized by a reduction of nitrate todinitrogen gas during totally anaerobic conditions. In thepresence of oxygen, nitrite can accumulate because of thecompetition between oxygen and nitrite for the electrons (8,13). During dissimilatory ammonium production, however,nitrite accumulation occurs despite anaerobic conditions.After depletion of nitrate, the accumulated nitrite is reducedto ammonium (21, 23). Nitrous oxide has been reported to beproduced through a side reaction (23, 26). Other investiga-tors have shown that some dissimilatory ammonium-pro-ducing bacteria, V. succinogenes and Pseudomonas putre-faciens, are able to reduce nitrous oxide to dinitrogen gas (3,21, 28).

* Corresponding author.

Microcalorimetry is a suitable tool to monitor microbialmetabolism and gives a measure of the total cellular activity.The technique allows measurements of the heat productionrate (dQldt, where Q is evolved heat quantity and t is time)of the culture, which is related to the amount of biomass, thekind of metabolism used, the rate of growth, and the energyspent on reactions not directly coupled to biosynthesis (2,11). Reiling and Zuber (19) have reported that there are twodistinctly different periods in the heat production rate curvefor nitrate-limited growth of a denitrifying strain of Bacillusstearothermophilus. The first period was attributed to areduction of nitrate to nitrite and the second to the reductionof nitrite, probably to yield nitrous oxide.The aim of the present study was to compare the growth of

a denitrifier, Pseudomonas fluorescens, and a dissimilatoryammonium producer, P. putrefaciens, during anaerobicgrowth in nitrate- or nitrite-limited medium. The heat pro-duction rate, consumption of nitrate, and production andconsumption of nitrite and nitrous oxide were monitored.

MATERIALS AND METHODS

Organisms, media, and growth conditions. P. fluorescens2799, a denitrifying bacterium that reduces NO3 to N2 byrespiration (17), and Pseudomonas putrefaciens, which wasisolated from the Baltic Sea and which produces NH4+ fromN03 through a dissimilatory pathway (21, 22), were used inthis study.

P. fluorescens was cultured in a defined liquid mediumthat contained glucose (3 g/liter) as the carbon source andNH4Cl (1 g/liter) as the nitrogen source (10). For the exper-iment for which the data are shown in Fig. 4A to C, acomplex medium consisting of peptone (6 g/liter) and yeastextract (3 g/liter) was used. The initial concentrations ofN03 were approximately 2, 3, 6, or 12 mM; and those ofNO2 were approximately 1, 2, or 4 mM.

P. putrefaciens, for which we have not been successful in

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Stainlesssteel tubes

loutletFIG. 1. Experimental design used to monitor the anaerobic in-

cubation of the various strains. The water bath was heated to 28°Cand placed on a magnetic stirrer; it provided the simultaneousstirring of several incubation flasks.

designing a suitable defined medium, was cultured in trypticsoy broth (TSB; Difco Laboratories, Detroit, Mich.). Thismedium was prepared by using synthetic seawater supple-mented with glucose (3 g/liter) (21). The initial concentra-tions of NO3- or NO2- were approximately 1 or 3 mM.The autoclaved media were dispensed aseptically (70 ml)

into sterilized 100-ml flasks. The flasks were sealed, andanaerobic conditions were created by evacuating and flush-ing the flask with He gas while the contents were stirredvigorously. This gas phase replacement procedure was re-

peated 3 times. Inocula (5 ml) were obtained from 24-hcultures of P. fluorescens and 48-h cultures of P. putrefa-ciens, which were grown anaerobically in the same mediathat were used in the experiments described above. The ageof the inocula was chosen to minimize the addition to theexperimental flasks ofN03- and NO2- from the precultures.All liquid cultures were incubated in a water bath at 28°C andstirred mechanically (Fig. 1).

Microcalorimetry. The experimental design used in thisstudy is shown in Fig. 1, unless stated otherwise. The heatproduction rate (dQldt) was measured by using a heatconduction multichannel microcalorimeter (thermal activitymonitor 2277; Thermometric AB, Tartalla, Sweden) (25).The microcalorimeter was fitted with two channels, each ofwhich was equipped with a flowthrough cell to permit thesimultaneous recording of two cultures. Each channel con-tained a measuring and a reference cell. The latter containedatmospheric air. The voltage signal was monitored by usinga two-channel potentiometric recorder (1,000-mV range;2210-022; LKB). Peristaltic pumps (Micro Perpex pump2132; LKB) were used to circulate the cultures from theincubation flasks to the microcalorimeter and then back tothe flasks at a constant flow rate of 50 ml/h. Before thecommencement of each experiment, the flowthrough celland the connected tubes were sterilized with 70% (wt/vol)ethanol and then rinsed with sterile water until base-linestability was obtained. The base line fluctuated less than 1,uW over 24 h in the 300- and 1,000-,uW ranges used. Forevery experiment the external calibration was performed byusing an external current supplied to produce 100 or 300 ,uW.Internal calibration was performed with a chemical reaction(4); this gave an effective volume of 0.53 ml of the flow-through cell. The microcalorimeter was operated at 28°C.The evolved heat quantity (Q) is proportional to the area

under the heat production rate curve (Q = constant x area).Thus, by integration of the heat production rate curve

obtained, the enthalpy change (AH) of a process can becalculated, if no external work is performed.

Growth determinations. Growth was monitored turbidimet-rically at a wavelength of 610 nm by using a spectrophotom-eter (DU; Beckman Instruments, Inc., Fullerton, Calif.).Standard curves of turbidity values plotted against dryweight were obtained by measuring various dilutions ofstationary-phase cultures. Dry weight determinations wereperformed in duplicate on samples that were washed twice indistilled water. Duplicates differed by +2%.

Chemical analyses. Samples of 4 ml were withdrawn asep-tically from the culture and immediately filtered through aNO3-- and N02 -free filter (GF/F; Whatman, Inc., Clifton,N.J.). The subsequent reduction in volume was compen-sated for by injection of 4 ml of He gas into the flasks. Theconcentrations of N03 and N02 were analyzed directlyby using an Auto-Analyzer (Technicon Instruments Corp.,Tarrytown, N.Y.) (1). By using a gas-tight syringe, a 100-,ulsample was withdrawn from the gas phase and immediatelyanalyzed for N20 content by using a gas chromatograph(model 428; Packard Instrument Co., Inc., Rockville, Md.)equipped with an electron capture detector (model 902;Packard) (duplicates differed by +2%). Because the cultureswere stirred continually during the course of the experiment,it was assumed that the N20 was in a state of equilibriumbetween the water and gas phases. The concentration ofglucose was measured by using a commercial enzyme com-bination kit (Boehringer GmbH, Mannheim, Federal Repub-lic of Germany) to ensure that the medium was not glucoselimiting.The results presented are typical for experiments that

were performed at least twice for each nitrate or nitriteconcentration.

RESULTS

Anaerobic growth of P. fluorescens. When P. fluorescenswas grown anaerobically on glucose with NO3- as theelectron acceptor, only a small amount of NO2 was accu-mulated (0.5% of the added NO3) during the early log phase(Fig. 2A and B). The NO2- that was produced was thereafterconsumed simultaneously with NO3-. The N20 concentra-tion remained low during the whole growth period (Fig. 2A),but more N20 was produced when the electron acceptor wasN02- than when NO3- was the electron acceptor. An N20value as high as 130 ,uM was obtained when an initialconcentration of 4 mM NO2 was used (Table 1).During the period of logarithmic growth, the heat produc-

tion rate increased steadily (Fig. 2B and C). Only one phasethat resulted in a maximum was obtained in the heat produc-tion rate curve (Fig. 2C and 3) when either NO3- or N02was used as the electron acceptor. The maximum in the heatproduction rate was followed by a sharp decrease to zero,and this was concomitant with commencement of the sta-tionary phase. At that time, only trace amounts of theelectron acceptors remained (Fig. 2A). The absence of bothgrowth and heat production during the stationary phase is inagreement with the fact that no other electron acceptor wasavailable.The biomass and total heat production increased in rela-

tion to an increased N03 concentration, resulting in a meanvalue of the experimental enthalpy change of -800 + -100(standard deviation [SD]) kJ/mol ofN03 and a growth yieldof 33 + 4 (SD) g/mol of N03 consumed (Table 2).

In our preliminary experiments, oxygen leaked into thesystem (322 ppm [,ul/liter] at a flow rate of 50 ml/h), and threeseparate phases, each with one maximum, occurred in theheat production rate curve, both in complex media (Fig. 4A

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OM

i

0

o

'o:0'Iz__E-o06

J _0

24r

3

z

201

16

12

8

4

01

E

z

-

z

Time (h)FIG. 2. (A) Concentrations of N03- (0), N02- (l), and N20 (A) (B) Log dry weight (0). (C) Heat production rate for P. fluorescens

grown anaerobically on the defined medium containing 3 mM N03- as the electron acceptor.

to C) and in defined media (data not shown). The first phasecorresponded to growth in which oxygen and some nitratewere used, the second phase corresponded to growth inwhich nitrate was used, and the third phase corresponded togrowth on the nitrite that was accumulated when the nitratereduction occurred. At an initial concentration of 2 mMnitrate, 60% of this added nitrate accumulated as nitrite.

Anaerobic growth of P. putrefaciens. When NO3 was usedas the electron acceptor during anaerobic growth of P.putrefaciens, NO2 accumulated (Fig. 5A). At the time ofNO3- depletion, the maximum concentrations of NO2 andN20 corresponded to 40 and 0.04%, respectively, of theinitial N03 concentration (1 mM). When the NO3 concen-tration used was 3 mM, an even greater accumulation ofNO2- occurred, together with an increase in N20, amount-ing to 70 and 0.2% of added NO3-, respectively.When N03 was used as the electron acceptor, two

phases were observed in the heat production rate curves

TABLE 1. Maximum concentrations of N20 measured duringanaerobic growth of P. fluorescens in defined medium with

different initial concentrations of N03- or N02-Electron acceptor Maximum concnand concn (mM) (pLM) of N20

N03-3.2 ........................................................6.1 ........................................................12.1.......................................................

0.71.70.4

N02-2.2 .................................... 17.74.4 ................................... 130

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Nio;110-

100

C

090 N

40 / m m

0

0

to

60

50

40

3mM 6mM

30

20

10

0 2 4 6 8 10 12 14

Time (h)FIG. 3. Heat production rate of P. fluorescens grown anaerobi-

cally on the defined medium supplemented with various initialconcentrations of NO3- or N02 . No samples were withdrawnduring the course of the incubation.

(Fig. 5C and 6). The highest rate of biomass productionoccurred during the first phase, when N03 was reduced toN02- (Fig. SB). At the end of the second phase, the heatproduction rate dropped sharply when the N02 was de-pleted. In addition, the concentration of N20 was reducedduring this latter phase, presumably by production of N2(21). In accordance with these results, only one phase wasdetected in the power-time curve when N02 was added asthe electron acceptor (Fig. 6). Independent of the electronacceptor used, a slow increase in the heat production rate

TABLE 2. Growth yields and experimental enthalpy changevalues for P. fluorescens grown anaerobically in defined medium

with different concentrations of N03-

NO3- concn YNO3- concnAHc.p(mM) (g [dry wt]/mol)a (kJ/mol)b

2.8 31 -8803.3 32 -8903.4 40 -7306.0 36 -7706.1 28 -6506.3 33 -850

a YNO3 , Growth yield. The mean for all six N03- concentrations was 33+ 4 (SD).

b AkXp, Enthalpy change. The mean for all six N03- concentrations was-800 ± -100 (SD).

C0

Z._

0.

i

E

03zn o

o s

0o

z

L

4w

Time (h)FIG. 4. (A) Concentrations of N03- (0) and N02- (O). (B) Log

dry weight (0). (C) Heat production rate for P. fluorescens grown oncomplex medium with 2 mM N03- under mixed aerobic andanaerobic conditions. During this experiment the precautions shownin Fig. 1 for the avoidance of oxygen leakage into the experimentalsystem were not undertaken. No pump hood was used, and thetubes consisted of Teflon (E. I. du Pont de Nemours & Co., Inc.,Wilmington, Del.) instead of stainless steel.

and biomass was detected, however, during the stationaryphase, despite the fact that N037, N027I or both wereexhausted.The total heat production increased in relation to in-

creased concentrations of N03 or N027, and mean exper-imental enthalpy change values of -810 + -130 (SD) and-920 + -120 (SD) kJ/mol of N03 or N02 consumed,respectively, were obtained (Table 3).

DISCUSSION

When P. fluorescens was grown anaerobically, only asmall accumulation of nitrite and nitrous oxide occurred.According to Downey et al. (8) and Fenchel and Blackburn(9), the induction of nitrate reductase and nitrite reductaseoccurs in sequence when nitrate is in excess. The smallaccumulation of nitrite therefore seemed to be sufficient toinduce the production of nitrite reductase. This probablyresulted in both the simultaneous consumption of nitrate andnitrite (Fig. 2A) and in a continuous reduction of nitrate todinitrogen gas. This was confirmed by the detection of just

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c

0

320 '

CO % 1 L

X \0E. 0, J\

'Cl

E O' NO%oC

C4

0 2 4 6 8 10

Time (h)FIG. 5. (A) Concentrations of N03- (0), N07- (OI), and N20

(A\). (B) Log dry weight (0). (C) heat production rate for P.putrefaciens grown anaerobically on TSB medium supplementedwith glucose as the energy source and with 1 mM N03- as theelectron acceptor.

one experimental growth rate (Fig. 2B) and a steady increasein the heat production rate that resulted in one maximum(Fig. 2C and 3). These results reveal that the rate of nitratereduction to nitrite is lower than or equal to the nitritereduction to dinitrogen gas, as found by Kaspar (14).

It is interesting from an ecological point of view that nonitrite accumulation occurred during anaerobic conditions,but when our strain of P. fluorescens had access to bothoxygen and nitrate as electron acceptors at the same time,nitrite accumulated (Fig. 4). Denitrification in the presenceof oxygen has been reported by some investigators (16, 20;J. C. G. Ottow and W. Fabig, Abstr. Proc. 6th Int. Symp.Environ. Biogeochem., 1983, p. 61), and other investigators(8, 13) have suggested that nitrite then can be accumulatedbecause of a competition between oxygen and nitrite for theelectrons.The mean growth yield value obtained for P. fluorescens

cultured anaerobically on a defined medium with glucose asthe energy source and with nitrate as the electron acceptorwas 33 ± 4 (SD) g (dry weight)/mol of nitrate consumed(Table 2). This value is in agreement with the reportedgrowth yield value of 28.6 glmol of nitrate for P. denitrificansgrown in a nitrate-limited continuous culture system (15).The growth of P. fluorescens yielded a mean experimentalenthalpy change value of -800 ± -100 (SD) kJ/mol ofnitrate consumed (Table 2), and this resulted in a heat

30

0 2 4 6 8 10 12 14 16 18 20

Tilm (h)FIG. 6. Heat production rate of P. putrefaciens grown anaerobi-

cally on TSB medium supplemented with glucose as the energysource and with different initial concentrations of N03- or N07- asthe electron acceptors. No samples were withdrawn during thecourse of the incubation.

evolution of -24 kJ/g of biomass produced. This latter valueis somewhat lower than the -30 to -35 kJ/g (dry weight)reported for the anaerobic, denitrifying thermophilic B.stearothermophilus by Reiling and Zuber (19).For growth on nitrite compared with that on nitrate, P.

fluorescens largely increased its nitrous oxide accumulation(Table 1). This may be due to a toxicity of nitrite ascompared with that of nitrate (12, 14). Kaspar (14) hassuggested that nitrite reduction to nitrous oxide could be adetoxication process.

In contrast to the denitrifying P. fluorescens, nitrite wasaccumulated under anaerobic conditions because of nitratereduction by the dissimilatory ammonium producer P. pu-trefaciens (Fig. 5A). This fact is in agreement with theprevious findings of Samuelsson (21). The first phase of theheat production rate curve was mainly due to nitrate reduc-

TABLE 3. Experimental enthalpy change values forP. putrefaciens grown anaerobically in TSB medium andsupplemented with glucose and different concentrations ofNN3 or N07

Electron acceptor AH..Pand concn (mM) (kJ/mol)a

N03-1.1 ........ ............................... -1,0101.3 ........... ................................. -8503.0 ........... ................................. -7103.1 ........... ................................. -680

N02-1.0 ............ ................................ -7801.0 ............ ................................ -9602.5 ............ ................................ -1,0902.5 ........... ................................. -830

a For N03- as the electron acceptor, the mean value was -810 ± -150(SD). For N02- as the electron acceptor, the mean value was -920 ± -140(SD).

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tion. Because less than 100% of the nitrate consumed couldbe detected as nitrite, some of the nitrite produced duringthis period must have been reduced simultaneously with thenitrate.The total heat production was approximately proportional

to the initial concentrations of either nitrate or nitrite (Table3). This indicates that the nitrate and nitrite were thegrowth-limiting factors during anaerobic growth in TSBmedium that was supplemented with an excess of glucose asthe energy source. A slow increase in the heat productionrate and biomass was detected, however, after the sharpdrop in the heat production rate curves that coincided withnitrite depletion (Fig. 5 and 6). When nitrate or nitritesupplements were not added to the TSB medium, steady butslow biomass production was obtained during incubation.Consequently, either some other component(s) within themedium must be able to serve as an electron acceptor orsome other fermentative pathway could be used. Fumarate,for example, has been shown to be used as an electronacceptor for a dissimilatory nitrate reducer (V. succino-genes) (18, 27). The use of an unidentified electron acceptoror a fermentative pathway does not seem to be in functionuntil nitrate or nitrite is exhausted, as the heat productionrate curve dropped to a very low value simultaneously withthe depletion of these electron acceptors.

P. putrefaciens grew more slowly in nitrite concentrationsof 1 to 3 mM than it did in the same concentrations of nitrate(Fig. 6). This shows that it utilizes the two electron acceptorswith different efficiencies, which is consistent with the heatevolution of the same order (or slightly increased) whennitrite was used as the electron acceptor compared withwhen nitrate was used as the electron acceptor (Table 3).The different phases of the heat production rate curves,

the patterns of nitrate and nitrite reduction during growth,and previous findings (21) indicate that the dissimilatoryammonium producer P. putrefaciens, during anaerobicgrowth on nitrate, subsequently produces energy duringreduction of nitrate to nitrite, during nitrite reduction toammonium, and by some unidentified energy-producing me-tabolism (fermentation or respiration). Our denitrifyingstrain, P. fluorescens, produced energy by reduction ofnitrate and nitrite. In contrast to P. putrefaciens, it reducednitrate during anaerobic growth with only a minor accumu-lation of nitrite.

ACKNOWLEDGMENTS

We thank P. Conway, B. Norkrans, U. Ronner, and P. Ronnow,Department of Marine Microbiology, University of Goteborg, forvaluable discussions.

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21. Samuelsson, M.-O. 1985. Dissimilatory nitrate reduction tonitrite, nitrous oxide, and ammonium by Pseudomonas putre-faciens. Appl. Environ. Microbiol. 50:812-815.

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26. Tiedje, J. M. 1981. Use of nitrogen-13 and nitrogen-15 in studieson the dissimilatory fate of nitrate, p. 481-497. In J. M. Lyon, R.C. Valentine, D. A. Phillips, D. W. Rains, and R. C. Huffaker(ed.), Genetic engineering of symbiotic nitrogen fixation andconservation of fixed nitrogen. Plenum Publishing Corp., NewYork.

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