Journal of General Microbiology (1989), 135, 2445-245 1. Printed in Great Britain
2445
The Effect of Oxygen on Denitrification in Paracoccus denitrzfians and
Pseudomonas aeruginosa
By K A T H R Y N J . P . DAVIES,? D A V I D LLOYD* A N D L Y N N E B O D D Y
Department of Microbiology, University College, Newport Road, Card18 CF2 I TA, UK
(Received 23 March 1989; revised 9 May 1989; accepted 6 June 1989)
Denitrification by Paracoccus denitrijicans and Pseudomonas aeruginosa was studied using
quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously
dissolved gases. Evidence was provided for aerobic denitrification by both species: in the
presence of 0 2N
, 2 0production increased in Pa. denitrijicans, while that of N2 decreased; with
Ps. aeruginosa, the concentrations of both N2 and N 2 0 increased on introducing O2 into the gas
phase. Disappearance of NO; was monitored in anaerobically and aerobically grown cells
which were maintained either anaerobically or aerobically: the rate and extent of NO5
utilization by both species depended on growth and maintenance conditions. The initial rate of
disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred
when cells were grown anaerobically and maintained aerobically. In nitrogen balance
experiments both species converted over 87% of the added NO? to N2 and N 2 0 under both
anaerobic and aerobic maintenance conditions.
INTRODUCTION
Denitrification, the dissimilatory reduction of nitrate to nitrogen-containing gases such as
dinitrogen (N2)and nitrous oxide (N20), is believed by many workers to be a strictly anaerobic
process (Kaspar, 1982; Tiedje et al., 1982; Payne, 1983). This view probably stems largely from
the fact that O2 has been shown to suppress the synthesis of denitrifying enzymes and to inhibit
their activity (John, 1977; Payne, 1981;Tiedje et al., 1982) and that theoretically, denitrification
produces less energy than O2 respiration under aerobic conditions (Thauer et al., 1977). Such
considerations, however, provide no proof that aerobic denitrification does not occur.
Whilst many biochemical studies have been done on bacterial denitrification, few have
monitored dissolved O2 concentrations or even gaseous O2 (e.g. Nakajima et al., 1984); hence
they are able to provide little evidence for or against the idea (Koike & Hattori, 1975; Oren &
Blackburn, 1979). However, in those studies where dissolved O2 concentration was measured,
there have been several reports of nitrate utilization and the presence of nitrate reductase in the
presence of dissolved 02.For example, Krul(l976) and Krul & Veeningen (1977) reported that,
in a study of denitrifying bacteria isolated from activated sludge and drinking water under
anaerobic conditions, there was a range between total O2repression of synthesis of dissimilatory
nitrate reductase to almost non-repressed synthesis, e.g. in Alcaligenes strains. Also, nitrate
reductase was induced in chemostat cultures of Hyphomicrobium species at dissolved O2
concentrations below 28 % air saturation (Meiburg et al., 1980).
Presence of nitrate reductase provides indirect evidence of denitrification, but production of
nitrogen-containing gases would provide direct evidence, and the latter has been achieved under
aerobic conditions in several recent studies. For example, Aquaspirillum magnetotacticum - an
organism which has an obligate requirement for oxygen - was shown to reduce NO: to N 2 0and
t Present address : Gesellschaft fur Biotechnologische Forschung mbH, Mascheroder Weg 1, D-3300
Braunschweig, FRG.
0001-5491 0 1989 SGM
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2446
K . J . P . DAVIES, D . L L O Y D A N D L . B O D D Y
N2 in the presence of 02,albeit at low levels
(0.2-1%) (Bazylinski & Blakemore, 1983).
Robertson & Kuenen (1983; 1984a, b) demonstrated the presence of nitrate reductase and the
production of nitrogen-containing gases from nitrate by Thiosphaerapantotropha at dissolved O2
concentrations of up to 90% air saturation. These observations find application in the treatment
of highly nitrogenous waste waters (Voets et al., 1975). Lloyd et al. (1987) demonstrated the
persistence of denitrification by cultures of Paracoccus denitrijcans, Pseudomonas aeruginosa,
Pseudomonas stutzeri and Propionibacterium thoenii which were grown anaerobically and then
maintained aerobically.
This paper reports on denitrification by Pa. denitrijcans and Ps. aeruginosa under four
combinations of growth and maintenance conditions : the bacteria were grown under both
anaerobic or aerobic conditions and sets of each of these then maintained under anaerobic and
aerobic conditions. Denitrification was observed in all cases; it occurred most slowly when
anaerobically grown cells were tested under aerobic conditions (e.g. 25% of the rate observed
anaerobically in Ps. aeruginosa).
METHODS
Maintenance andgrowth of organisms. Paracoccus denitrijcans NCIB 8944 and Pseudomonas aeruginosa P A 0 129
(from Dr W.A. Venables of this Department) were maintained aerobically on slopes of nitrate agar (Difco). Both
organisms were grown in batch cultures on defined medium (Burnell et al., 1975) containing 100 mM-KNO, and
50 mM-sodium succinate as sole carbon source or on nitrate broth (Difco) which contains 10 mM-KN03; Pa.
denitrijicans was grown at 30 "C and Ps. aeruginosa at 37 "C. Aerobic cultures (250 ml) were grown with forced
aeration and the presence of dissolved 0,was checked using an O2 electrode (Uniprobe, type G) just before
harvesting : 0,concentrations in excess of 200 PM indicated adequate maintenance of aerobiosis. Anaerobic
cultures in screw-capped bottles were sparged aseptically with argon before inoculation.
When the cultures attained an OD400of 1.0 (Pye SP 1800 spectrophotometer)cells were harvested at 4 "C in a
6 x 250 ml rotor of a Sorvall RC5B centrifuge at loo00 r.p.m. for 20 min (3 x lo5g,, min). Pellets were washed
twice by resuspension in 20 m-potassium phosphate buffer (pH 7.2). For aerobic preparations air-saturated
buffer was used and suspensions were force aerated ; anaerobic suspensions were prepared in argon-saturated
buffer and bubbled with argon until injection into the reaction vessel. Washed organisms were used immediately
(within about 2 min).
Dissolved gas anafysis by mass spectrometry. A quadrupole mass spectrometer type SX 200 and associated
DPP 16 digital peak programmer (VGGas Analysis) fitted with a turbomolecular pump (Pfeiffer) was used. The
mass spectrometer inlet was fitted with a PTFE membrane (thickness 50 pm) in a vessel (8 ml total volume; 5 ml
working volume) open to gases as described previously(Lloyd &Scott, 1983,1985;Lloyd, 1985;Degn et al., 1985).
The temperature of the vessel was maintained at 30 "C and the stirring speed was 1200 r.p.m.
Where samples were required for estimations of NO; and NO?, a reaction volume of 70 ml stirred at 100 r.p.m.
was used. Continuous mass spectrometric monitoring of dissolved gases was done with a quartz probe
( 3 4 mm 0.d.) sealed at the apex distal to the mass spectrometer, and with an entry port for gases 5 mm from the
tip. The entry port was a 0-25mm diameter hole drilled through the tube wall and covered with a sleeve of silicone
rubber (0.19 mm o.d., 0.15 mm id.).
In both systems levels of dissolved N2,NO, and 0,were followed simultaneously and continuously (at mjr
values 28 for N2,30 for NO,, and 32 for 0,) under either anaerobic conditions, maintained by passing argon at
50 ml min-l over the surface of the reaction mixture, or at fixed 0,concentrations established by introducing
known proportions of 0,into the mobile gas phase. Calibrations were with air- and N20-saturated buffers in the
absence of organisms and used solubility data from tables (Wilhelm et al., 1977).
Other analytical methods. Protein was determined by the Lowry method. Nitrate was measured by the brucine
sulphate method and nitrite by diazotization using Griess llosvays reagents (Department of Environment, 1969).
Discrimination between various NO, species was by gas chromatography on a CTR,5 mm x 2 m, glass column
(Alltech, type 8700) with helium as carrier gas and a Katharometer detector. N20was the only species detectable
in these experiments.
RESULTS
Eflects of O2 on denitrijcation in anaerobically grown bacteria
Anaerobically grown Ps. aeruginosa incubated anaerobically with 5 mM-KN03 and 5 m M sodium succinate produced only N2 (Fig. !). In the presence of 1 0 0 dissolved
~ ~
02,N 2 0
was also produced. Increasing O2 to 1 5 0 increased
~ ~
N 2 0 production, whereas the N 2
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Aerobic bacterial denitr$cation
PO2 &Pa)
16
12 n
3
8
4
i
20
I
40
Time (min)
I
60
2
8
z"
I
80
Fig. 1. Effects of O2on gas production from NO, in a washed cell suspension of anaerobically grown
Ps.aeruginosa. Mass spectrometric monitoring of dissolved gases was done in a stirred vessel open for
gas flow. Initially, the reaction mixture contained 5 mM-KNO, and 5 mM-sodium succinate and the gas
phase was argon. O2was added as indicated into the gas stream. The cell suspension contained 0-2mg
protein ml-l; the temperature was 37 "C. Dissolved O2 (---.-.), N2 (-)
and N 2 0 (------) were
monitored at m/z 32, 28 and 30 respectively.
20 kPa 0,
2
120
n
2
E
W
n
E 80
5
2
W
1 u0
0
z
5
0
10
15 20
25 30
Time (min)
Fig. 2. Effects of O2on gas production from NO, in a washed cell suspension of aerobically grown Pa.
denirrificans.Conditions and symbols are as in Fig. 1 except that concentrations of nitrogen and carbon
sources were both 2 mM initially, the protein concentration was 0-8 mg ml-l, and the temperature
30 "C. A, NO, concn.
0
concentration remained at the anaerobic level; these changes are further evidence for the O2
sensitivity of nitrous oxide reductase to inhibition by O2 (Hochstein et al., 1984). Further
increases in dissolved O2 increased the rates of evolution of both the gaseous products of
denitrification ; this observation cannot be explained on the basis of present understanding of
possible control mechanisms.
Efects of O2 on denitr8cation in aerobically grown bacteria
Incubation of aerobically grown Pa. denitr$cans under anaerobic conditions in the presence
of 2 mM-KNO, and 2 mM-sodium succinate gave N2 as the sole detectable gaseous product of
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K . I . P. DAVIES, D . LLOYD A N D L. BODDY
1250
c
n
z
3.
W
0
E:
u
0
Time (min)
Fig. 3. Production of gases from NO; by aerobically grown Pa. denitrijicans. Conditions and symbols
as in Fig. 1 except that 2 mM-sodium succinate was added with NO, as shown (arrowed) to a suspension
containing 0432 mg protein ml-l under a gas phase of 20 kPa O2 in argon at 30 "C.
Table 1. Disappearance of NO? and appearance of NO: in Pa. denitriJicansand Ps. aeruginosa
Organisms were incubated as non-proliferating cell suspensions with 0.5 mM-KNO,. Values are means for
experiments done in triplicate.
Rates of:
r
Organism
Pa. denitrijicans
Ps. aeruginosa
Growth
conditions
Maintenance
conditions
Anaerobic
Anaerobic
Aerobic
Aerobic
Anaerobic
Anaerobic
Aerobic
Aerobic
Anaerobic
Aerobic
Aerobic
Anaerobic
Anaerobic
Aerobic
Aerobic
Anaerobic
I
NO?
NO,
disappearance
production
[nmol min-' (mg protein-')]
130
40
95
80
120
30
75
35
2
7
4
2
7
7
4
2
Final NO,- concn
01M)
50
150
60
60
40
185
70
50
denitrification (Fig. 2). Introduction of 20 kPa O2 into the gas phase gave an overshoot in
dissolved O2 to 195 PM and eventually to a steady-state level of 100 PM accompanied by
increased production of N2 and the appearance of N 2 0as a second product. The concentration
of K N 0 3 decreased to 0.2 mM during the experiment and the concentrations of gaseous products
eventually declined (not shown). A more detailed analysis of NO: disappearance would be
necessary to reveal a possible discontinuity resulting from the introduction of 02.Similar
changes were seen when non-proliferating suspensions of aerobically grown Ps. aeruginosa
were subjected to anaerobic-aerobic transitions except that both products were produced
anaerobically in this case.
Comparisons of denitr9cation rates, residual nitrate and nitrogen balances under anaerobic and
aerobic conditions
Table 1 shows the rates of denitrification measured in washed cell suspensions under
anaerobic and aerobic conditions after growth in the absence or presence of 02.For both
organisms the highest rate of nitrate disappearance was observed when anaerobically grown
cells were tested under anaerobic conditions. In the presence of O2 this rate was diminished by a
factor of 3- to 4-fold.
Initial rates of accumulation of NO? were increased when anaerobically grown organisms
were maintained aerobically for Pa. denitrijicans but remained similar for Ps. aeruginosa.
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Aerobic bacterial denitrijication
Table 2. Nitrogen balances in aerobically grown Pa. denitrijicans and Ps. aeruginosa maintained
under aerobic and anaerobic conditions
Experimental details are as described in the text; results are mean values of experiments with two different
suspensions.
Organism
Pa. denitrificans
Ps. aeruginosa
Maintenance
conditions
NOT-N added
(pg-atoms)
N recovered
(N2 + N2O)
(pg-atoms)
Conversion of NOT-N
into N 2 0 and N 2
Anaerobic (1)
(2)
Aerobic (1)
(2)
Anaerobic (1)
(2)
Aerobic (1)
(2)
139
277
139
277
139
277
277
554
125
256
123
243
123
252
250
5 20
90
94
92
88
88
91
90
94
(%)
Aerobically grown organisms also showed rapid utilization of NO, when incubated under
aerobic conditions : these values were 73 % and 62.5 % of the maximum rates for Pa. denitrijicans
and Ps. aeruginosa respectively. Lowest rates for both organisms were obtained for aerobically
grown cells under anaerobic conditions. NO? levels remaining after 24 h incubation gave an
indication of the effectiveness of scavenging at low concentrations (not shown). Anaerobically
grown organisms (both species) exposed to O2 were noticeably less efficient in this respect. A
) found here for Pa. denitrijicans was indicated
higher affinity for NO, (apparent K,,, < 5 p ~ than
in experiments in which small pulses of NO? were provided and rates of disappearance
monitored with a nitrate electrode (Parsonage et al., 1985).
Mass spectrometric measurements of denitrification were used to assess the extent of NO,-N
conversion to gaseous end products. Fig. 3 shows a typical experiment in which a washed
suspension of aerobically grown Pa. denitrijicans was provided with a known amount of KN03
(139g-atom N) and total gas production was monitored in the presence of 0 2 .Addition of
KN03 2 mM-sodium succinate stimulated respiration as indicated by a decrease of dissolved
O2 from 210 to 50 p ~ Both
. N2 and N 2 0 were immediately evolved. Steady-state levels were
briefly attained before exhaustion of the nitrogen source led to cessation of denitrification and
disappearance of both products.
Total gas production was calculated from the integrated areas under the curves for each gas by
using the appropriate gas-exchange coefficients (measured from the t l l Zvalues for the system in
the absence of organisms). Table 2 summarizes the results for both organisms after aerobic
growth and under anaerobic and aerobic test conditions. In aerobically grown bacteria
denitrification proceeded effectively (more than 88% NOT-Nwas recovered as N2 N 2 0in all
cases) and rapidly, irrespective of the absence or presence of 0 2 .
+
+
DISCUSSION
The utilization of NO: and production of nitrogen-containing gases, in the presence of
relatively high concentrations of dissolved O2 (sometimes greater than air saturation), by the
facultative anaerobes Pa. denitrijicans and Ps. aeruginosa, provides strong additional evidence
that denitrification is not a strictly anaerobic process. Indeed, with anaerobically grown Ps.
aeruginosa, not only were N2 and N 2 0produced in the presence of 0 2but
, also their production
was increased.
Similar experiments with anaerobically grown Pa. denitrijicans indicated some differences
,
(Lloyd et al., 1987). Both N2 and N 2 0were produced under argon, and on introducing 0 2N2
production was decreased whereas that of N 2 0 increased. In order to suppress completely N2
production hyperbaric O2 concentrations (e.g. 360 PM) were required, and even under these
conditions denitrification to N 2 0 continued.
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K . J . P . DAVIES, D . LLOYD A N D L. BODDY
These data imply that, although the sensitivity of nitrous oxide reductase to inhibition by O2
varies between species, the enzymes responsible for denitrification were produced and remained
stable under a wide range of aerobic-anaerobic growth and maintenance conditions. This
contrasts with what might have been expected, since dissimilatory nitrate reductase - a key
enzyme in the NO? reduction process during denitrification - has previously been reported as
being induced in the presence of NO:, under anaerobic conditions (Pichinoty, 1973).
Mechanisms whereby existing nitrate reductase is reversibly inactivated are not well
understood; rather than a specific and direct O2effect, a control mediated via the redox state of
ubiquinone has been experimentally implicated (Alefounder et al., 1983). Evidence for
simultaneous utilization of O2 and NO? as terminal electron acceptors by a wide variety of
bacterial species (Ottow & Fabig, 1985) confirms that further studies are needed to elucidate
control mechanisms modulating the synthesis and activities of electron transport components
involved in denitrification.
This work was carried out during tenure of a NERC postgraduate research studentship (K. J. P. D.)
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