Vol. 69, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL

ACETYLENE INHIBITION

OF NITROUS OXIDE REDUCTION

BY DENITRIFYING Tadashi Department

Received

RESEARCH COMMUNICATIONS

Yoshinari

BACTERIA

and Roger

Knowles

of Microbiology, Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec HOA lC0, Canada.

February

lo,1976

Summary reduction

Acetylene (0.1 atm) caused complete or almost complete inhibition of of N20 by whole cell suspensions of Pseudomonasperfectomarinus, P. aermginosa and Micrococcus denitrificans. Acetylene did not inhibit reduction of N03- or NO*- by these organisms. In the presence of acetylene there was stoichiometric conversion of N03- or NOp- to N20 with negligible subsequent reduction of the latter. In the absence of acetylene there was no or only transient accumulation of N20. The data are consistent with the view that N20 is an obligatory intermediate in the reduction of NOz- to N2 in all of the three organisms studied. The pathway by denitrifying cation

by which bacteria

of enzyme

of inhibition

nitrogenous appears

fractions

of nitrous

to depend

are reduced on the

(N20)

reductase

(DNP) in Tseudomonasdenitrificans

was an obligatory

intermediate

studies

of the

in

effects

the reduction

denitrificans

and k'~crococcus

Partial

of inhibitors

(11)

suggested

that

purifi-

(lo),

and studies cyanide

(8) showed

that

(N02-)

such as N3-, (1))

(N2)

(N3-),

of nitrite

of N02- and N20 in Pseudomonasstutzeri

reduction

species.

by azide

and dinitrophenol

However,

to dinitrogen

Pseudomonasperfectomarinus

from oxide

oxides

(CN-) N20

to N2.

CN- and DNP on

P. denitrificar,s

N20 was not

(12)

an obligatory

intermediate. Fedorova reported

et al.

that

(4),

acetylene

in a study (C2Hx)

of extra-terrestrial

inhibited

life

the reduction

detection,

of N20 during

denitri-

fication. We confirm ing

bacteria,

report

also

here

C2H2 inhibits

P. perfectomarinus, that

during

of NzOwith

astoichiometry

mediate

the reduction

in

that

reduction which

reduction

P. aeruginosa

suggests

that

705

denitrify-

and M. denitrificans.

of N03- or N02-,

of NOz- to N2 in all

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

of N20 by three

N20 is three

C2H2 causes an obligatory species

We

accumulation inter-

of denitrifier.

Vol. 69, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL

MATERIALS

RESEARCH COMMUNICATIONS

and METHODS

Organisms Pseudomonasperfectomarinus was obtained from Dr. W.J. Payne, University of Georgia. Micrococcus denitrificans (NRC 814029) was obtained from the National Research Council of Canada, Ottawa. Pseudomonasaeruginosa was from the Macdonald College culture collection (Mac W67). Media and Incubation P. perfectomarinus was grown aerobically on a rotary shaker for 24 hr at 25" C in the medium of Best and Payne (2) modified as follows: 0.2 M NaCl, 0.05 M MgS@h, 0.01 M KCl, 0.01 M CaC12, 0.01 M NaN03, 0.5% Tryptone and 0.15% Yeast Extract (Difco). Late log phase cells were harvested from 60 ml of growth medium and washed three times by centrifugation (3300 g at 4" C for 15 min) and resuspension in nitrate-free artificial sea water (ASW: NaCl, MgS04, KC1 and CaCl as above). Aliquots (0.5 ml) of the cell suspension containing (1.6 x 10 1I cells) were transferred to 50-ml sterile Erlenmeyer flasks each containing 10 ml of the above medium without nitrate. After closing with sterile serum stoppers (Suba-Seal, England), the flasks were evacuated and back-filled with sterile helium to one atmosphere three times. When required, 5.0 ml of gas phase was replaced with the same volume of acetylene (C2H2) to give a final concentration of 0.1 atm. Also, when desired 0.5 ml N20, or 0.4 ml of 190 I+ NaN02 or NaN03 was added to give a final concentration of 560 ug N per flask. Flasks were incubated at 24" C with occasional shaking. P. aeruginosa and M. denitrificans were grown in Vernon's medium (14) under initially aerobic conditions on a rotary shaker for 24 hr at 32" C. Late log phase cells were harvested and treated similarly to those of P. perfectomarinus except that they were washed with 0.85% sodium chloride (NaCl) solution. Aliquots (0.5 ml) of the cell suspension (containing 2.6 and 3.4 x 1Ol1 cells for P. aeruginosa and M. denitrificans, respectively) were transferred to 50-ml Erlenmeyer flasks each of which contained 0.5% peptone and 0.15% yeast extract in 10 ml of 10 mM sodium phosphate (pH 6.8). Subsequent procedures were as described above. Analytical Bacteria were counted in a Petroff-Hausser chamber Protein was estimated (7) with priate dilution of the samples. as standard after washing the cells with ASW or 0.85% NaCl.

after approbovine albumin

Nitrate was determined by the Griess reaction (13) and nitrate by the brucine method (5). Samples were kept frozen prior to analysis. Samples for nitrite and nitrate analysis were withdrawn from flasks at various times by and diluted 20 times with distilled water before they hypodermic syringe, were frozen. Gas chromatographic analysis of N20 wasaspreviously Corrections for N20 solubility and normalization of initial N20 were made.

described (9). concentration

of

RESULTS Fig.

1 shows data in

P. perfectomarinus, of C2H2 (Fig.

lB),

N20 proceeded

rapidly

C2H2. Reduction trol,

while

in

N02- to N20 with N02- was complete

were

from an experiment the absence supplemented in the

control

of N02- occurred the presence

with

cell

reduction

suspensions

1A) and in the presence N20, N02- and N03-.

more slowly.

8 hr whether

dense

but was completely

of CzH2 there

no subsequent within

(Fig.

in which

of 0.1 Reduction

inhibited

No N20 accumulated

was stoichiometric of the

latter.

C2H2 was present

by 0.1

or not.

atm of atm

in the con-

conversion Reduction

of

of

of N03- to Subsequent

Vol. 69, No. 3, 1976

BIOCHEMICAL

+ N,O

AND BIOPHYSICAL

T-vO2-

RESEARCH COMMUNICATIONS

1 --+ I

600

Time

NO,’

(h)

Fig. 1. Reduction of added N20, N02- and N03- by dense suspensions of P. perfectomarinus incubated in a He atmosphere at 24" C (A) in the absence and (B) in the presence of 0.1 atm pCzH2. Symbols are . X20, o N02- and o N03-.

reduction

of the accumulated

C2H2 no N20 accumulated, conversion Thus caused

of the

the

effect

Figs.

added

delay

in

the reduction

were

very

in P. aeruginosa

fzkrn.s.

organisms

These

showed

In the absence

of CzH2 there

no further was very

of the

pronounced. did

of

was stoichiometric

reduction

of N02- but

results

not

latter.

Acetylene affect

the

of reduction

complete

of C2H2. Compared than

707

could

complete

inhibition

from P. perfectomarinus

greater

using

rate

P. aeruginosa

of the nitrogenous

and C2H2 caused

and almost

differed

experiments

of reduction

similar

of N20 in the absence rates

of similar

The patterns

hT20 reduction

they

the presence

N to N20 with

2 and 3 show the

two organisms

accumulation

more slowly.

of NOj-.

and M. denitrificans. these

in

of C2H2 on N20 reduction

a slight

of reduction

but

NOp- occurred

oxides inhibition

in M. denitriin

showing

transient

to P. perfectomarinus

be accounted

for

by the

in of

Vol. 69, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

+ NO;

z

+

NO,-

600-

200

-

0

2

4

6240

2

4

Time

6240

2

4

6

24

(h)

Fig. 2. Reduction of added N20, NOg- and NOg- by dense P. aeruginosa. Otherwise, the same as for Fig. 1.

suspensions

of

suspensions

of

+ NO,-

Z

600-

0

I’=

0.

--

’

n

024660246602468

Time Fig.

3. Reduction

M. denitrificans.

of added Otherwise,

(h)

N20, N02- and N03- by dense the same as for Fig. 1. 708

Vol. 69, No. 3, 1976

TABLE 1.

BIOCHEMICAL

Initial

and Final

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Bacterial

Time of incubation

Initial protein

U-1

(mg/flask)

40

0.45

P. perfectomarinus

Protein

Concentrations

Acetylene (0.1

Final

atm)

+

P. aeruginosa

0.70

24

+

12

M. denitrificms

0.73 +

small

differences There

in cell

and protein

was no evidence

protein

(mg/flask)

M20

N02-

N03-

0.63

0.69

0.93

0.51

0.55

0.67

0.77

0.99

1.14

0.74

0.80

1.11

0.87

1.00

1.30

0.81

0.89

1.11

concentrations.

of reduction

of C2H2 to ethylene

(C2H4)

in these

experiments. Table final

1 shows that

protein

experiments. were

only

small

concentrations) Growth

slightly

occurred

increments

greater

amounts in

increased

in the

absence

of growth

(measured

the dense

suspensions

in the

order

of C2H2 than

in

by initial used in

N20 < N02its

and the

< N03- and

presence.

DISCUSSION The present complete

data

or almost

affecting

other

complete

inhibition

experimental < N03- were

denitrificans site

in

complete

stages

consistent

inhibition

is also

of N20 reduction

with

of 0.1 atm, without

Our unpublished

shown by 0.01

Such growth

(6) and with

oxide

C2H2, at a concentration

of denitrification.

conditions.

the presence

Nitrous

show that

increments

the growth

in

and energy

the possible

loss

of at

the order yields least

significantly

data

atm C2H2 depending

causes

show that

on organisms

and

N20 < N02-

reported

for

P.

one phosphorylation

of C2H2. and C2H2 are

substrates

709

for

and competitive

inhibitors

of

Vol. 69, No. 3, 1976

nitrogenase

BIOCHEMICAL

(3).

It

of N20 reductase.

is

therefore

Unpublished

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

interesting data

that

suggest

that

C2H2 should

be an inhibitor

the inhibition

is

non-compe-

titive. The reported

effects

are explainable there

are

in

chiometric that,

that

organisms

of steps

inhibits

systems.

but

N20 reduction

reduction

studied,

between

(10)

cytochrome

(1,8,11,12) However,

of CzH2 on such systems.

of N20 during

of P. perfectomarinus denitrificms

on specific

C2H2 specifically

in the

sequence

CN- and DNP on denitrification

of effects

of effects

accumulation

at least

in the

terms

no reports

The fact

of Ng-,

stoi-

of N02- and NOs- suggests

N20 is

an obligatory

N03- and N2. This not with

and causes

studies

is

intermediate

consistent

with

of P. stutzeri

(1)

studies

and M.

(11).

These

results

N20 reduction

suggest

in further

trification

in complex

elsewhere)

show that

lation

in

soils

N2/N20

formed

Acknowledgment Canada.

the potential biochemical

systems the presence

or sediments during This

(4).

utilization studies

as well

Preliminary of CzH2 permits

and may facilitate

of C2H2 inhibition as in

the assay

experiments

(to

measurements measurement

of of deni-

be published of N20 accumu-

of the ratio

of

denitrification. study

was supported

by the National

Research

Council

of

REFERENCES 1. Allen, M.B., and Van Niel, C.B. (1952) J. Bacterial. 64, 397-412. 2. Best, A.N., and Payne, W.J. (1965) J. Bacterial. 89, 1051-1054. 3. Burns, R.C., and Hardy, R.W.F. (1975) Nitrogen fixation in bacteria and higher plants. Springer-Verlag, New York. 4. Fedorova, R.I., Milekhina, E.I., and Il'yukhina, N.I. (1973) Izv. Akad. Nauk. SSSR, Ser. Biol. 1973(6), 797-806, 5. Jenkins, D., and Medsker, L.L. (1964) Anal. Chem. 36, 610-612. 6. Koike, I., and Hattori, A. (1975) J. Gen. Microbial. 88, 11-19. 7. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 8. Matsubara, T., and Mori, T. (1968) J. Biochem. (Tokyo) 64, 863-871. 9. Patriquin, D., and Knowles, R. (1974) Can. J. Microbial. 20, 1037-1041. 10. Payne, W.J., Riley, P.S., and Cox, C.D. (1971) J. Bacterial. 106, 356-361. 11. Pichinoty, F., and d'ornano, L. (1961) Ann. Inst. Pasteur (Paris) 101, 418-426. 12. Sacks, L.E., and Barker, H.A. (1952) J. Bacterial. 64, 247-252. 13. Strickland, J.D.H., and Parsons, T.R. (1968) Bull. Fish. Res. Board, Canada, 167, 77-80. 14. Vernon, L.P. (1956) J. Biol. Chem. 222, 1035-1044. 710