FEMS Microbiology Ecology 45 (1987) 227-232
Published by Elsevier
227
FEC 00125
Dark anaerobic nitrogen fixation (acetylene reduction)
in the cyanobacterium Oscillatoria sp.
Lucas J. Stal and Heike Heyer
Geomicrobiology Division, University of Oldenburg, Oldenburg, F.R.G.
Received 25 February 1987
Revision received 14 April 1987
Accepted 21 April 1987
Key words: Oscillatoria sp.; Nitrogen fixation; Light-dark cycle
1. SUMMARY
The filamentous, non-heterocystous, nitrogenfixing cyanobacterium Oscillatoria sp. strain 23
(Oldenburg) showed cycling of acetylene reduction in light-dark cycles. Under aerobic conditions
nitrogenase activity is exclusively present during
the dark period. However, if anaerobic conditions
were applied during the dark period, two activity
maxima were observed. A relatively small activity
peak occurred during the first few hours of the
dark period and a high peak as soon as the light
was switched on. A low activity remained during
the second half of the dark period. This pattern of
acetylene reduction in Oscillatoria agrees well with
the field data on nitrogen fixation [Stal, L.J. and
Krumbein, W.E. (1984), Mar. Biol. 82, 217-224].
2. INTRODUCTION
Cyanobacterial mats develop on marine intertidal sediments [1-3]. Because the marine environ-
Correspondence to: L.J. Stal, Geomicrobiology Division, University of Oldenburg, P.O. Box 2503, D-2900 Oldenburg, F.R.G.
ment generally is low in combined nitrogen [4],
colonization of the sediments coincides with the
appearance of nitrogen-fixing cyanobacteria [5].
Cyanobacteria which form heterocysts are considered to be best adapted to nitrogen fixation under
aerobic and oxygenic phototrophic conditions [6].
Such organisms, however, generally are not found
in microbial mats.
Oscillatoria sp., a filamentous, non-heterocystous, aerobic nitrogen-fixing cyanobacterium [7]
was found to initially colonize North Sea intertidal sediments [1]. Nitrogenase activity in these
mats corresponded very well with the presence of
Oscillatoria.
Nitrogenase, the enzyme responsible for nitrogen fixation, is extremely sensitive to molecular
oxygen [8]. Thus far, the mechanisms by which the
oxygenic phototrophic cyanobacteria protect
nitrogenase from inactivation by oxygen are not
completely understood. Non-heterocystous nitrogen-fixing cyanobacteria, when grown in the
laboratory under light-dark cycles, show nitrogenase activity only during the dark period [9-12].
This was explained as a temporal separation of the
principally incompatible processes of nitrogen
fixation and oxygenic photosynthesis. Also in continuous light, nitrogenase showed a cyclic pattern
0168-6496/87/$03.50 © 1987 Federation of European Microbiological Societies
228
if the cultures were previously adapted to lightdark cycles [9,12,13] or in synchronized cultures
[9,14]. Very recently, it has been shown, that even
in continuous light a temporal separation of oxygenic photosynthesis and nitrogen fixation occurs
in Oscillatoria sp. (unpublished results).
Diurnal variation of nitrogenase activity in natural populations of heterocystous cyanobacteria
and the non-heterocystous Trichodesmium (Oscillatoria) thiebautii showed a typical light dependency [15,16]. The latter organism probably has a
spatial separation of photosynthesis and nitrogen
fixation, similar to that in heterocystous species.
In microbial mats with Oscillatoria sp., diurnal
cycling of nitrogenase activity showed a pattern
which can neither be explained by the typical
light-dependence of heterocystous species nor by
the cycling found in the laboratory in non-heterocystous cyanobacteria, including Oscillatoria sp.
[5]. Nitrogenase activity in microbial mats was
characterized by a relatively small activity peak at
sunset and a very high activity at sunrise. Because
microbial mats may turn anaerobic 15-60 min
after photosynthesis has ceased [1,18], it is suggested that not only a shift from light to dark and
vice versa, but also the concomitant shift to
anaerobic conditions in the dark regulated synthesis and activity of nitrogenase. By growing Oscillatoria sp. in the laboratory in light-dark cycles
under anaerobic conditions during the dark phase
it was possible to demonstrate that such a biphasic
diurnal cycle can really be induced.
2. MATERIALS A N D M E T H O D S
2.1. Organism and growth conditions
Oscillatoria sp. was isolated from North Sea
microbial mats [7]. The structure and population
dynamics of these mats were extensively described
in [1]. Oscillatoria sp. was grown in ASN~'H medium
[19]. This medium did not contain any source of
combined nitrogen. The medium contained 10 mM
TES [N-Tris (hydroxymethyl) methyl 2-aminoethane-sulfonic acid] buffer, adjusted to pH 7.9 with
1 N NaOH. Cultivation was done in cottonplugged 50-ml erlenmeyer flasks containing 30 ml
of medium. The erlenmeyer flasks were incubated
in a Gallenkamp orbital shaking illurmnated incubator. The temperature was 20 °C, fluorescent
light intensity was 1.3 klux and the shaking rate
was 100 rpm. A light-dark cycle of 16-8 h was
used. One hour after the light was switched off,
the cotton plug was exchanged with a sterile rubber
stopper and the culture liquid was gassed for 10
min with oxygen-free nitrogen until no measurable
oxygen was present (_+ 100 volume changes). During gassing the cultures were kept in the dark. One
hour after the light was switched on again, the
rubber stopper was simply changed back to a
sterile cotton plug. The cultures were thus grown
during 2 weeks.
The test for long-term nitrogenase activity under anaerobic conditions in the dark, was done
with cultures grown under aerobic light-dark
cycles. The cultures were taken from the incubator
1 h after the light was switched off and nitrogenase
was induced [9]. Cultures grown under aerobic
conditions did not contain the TES buffer.
2.2. Acetylene reduction
Nitrogenase activity was determined by the
acetylene reduction technique [20]. Every 30 min a
culture was taken from the incubator. The whole
hairy clump of one culture was used for one assay.
The cultures were incubated according to the
respective growth conditions in the incubator. The
7-ml assay bottle contained 1 ml of a 2.5% NaC1
solution with 10 mM TES buffer adjusted to pH
7.9 with 1 N NaOH.
Anaerobic conditions were obtained by gassing
with nitrogen. Aerobic incubations were done under air. The transition from aerobic to anaerobic
conditions was delayed 1 h with respect to the
transition from light to dark. The assay was started
immediately after the culture was taken from the
incubator by adding 15% acetylene.
The test for anaerobic dark nitrogenase activity
was done under an atmosphere of helium and 15%
acetylene. In this experiment acetylene reduction
was followed over 48 h.
2.3. Determination of chlorophyll a
Chlorophyll a was extracted with methanol
and absorption was read at 665 nm. An absorp-
229
tion coefficient of 74.5 m l . m g -a was used to
calculate the amount of chlorophyll a [21].
50
3. RESULTS A N D DISCUSSION
Oscillatoria sp. strain 23 has been shown to
reduce acetylene to ethylene (nitrogenase activity)
anaerobically in the dark [7]. The nitrogenase activity observed under these conditions, was not
due to some contamination with oxygen. Cyanobacteria have been shown to possess a very high
affinity for oxygen [22]. Previously reported
anaerobic dark nitrogenase activity in the unicellular cyanobacterium Gloeothece sp. apparently was
due to incomplete removal of oxygen from the
assay system. Maryan et al. [22] did not observe
any nitrogenase activity in Gloeothece sp. PCC
6909 under dark anaerobic conditions. This was
confirmed in our laboratory (unpublished results).
Also none of the 7 heterocystous strains, isolated
from microbial mats and Anabaena PCC 7120 did
reduce acetylene to ethylene under such conditions [7,23]. Therefore, we conclude that dark
anaerobic nitrogenase activity in Oscillatoria is
independent of oxygen.
Acetylene reduction under dark anaerobic conditions under an atmosphere of helium or argon
was linear and proceeded for 12-24 h (Fig. 1).
Specific nitrogenase activity was typically 2 /~mol
C z H 2 reduced per hour and mg Chl. a. Nitrogen
fixation in terms of the energy demand, is an
expensive process. Oscillatoria sp. apparently is
able to generate energy anaerobically in the dark
from endogenous reserve material by a heterofermentative lactic acid fermentation [24,25]. Such
metabolic pathways were not found in Gloeothece
PCC 6909 and several heterocystous cyanobacteria (unpublished results). Doubtlessly, the
energy yield by fermentation is low and it might
be questioned whether under natural conditions,
fermentation provides only the energy for purposes of maintenance rather than allowing
processes with very high energy demand such as
nitrogen fixation.
Microelectrode measurements of photosynthesis and oxygen concentrations in cyanobacterial
mats have shown that at sunset photosynthesis
La
~]o
10
12
24
36
8
hours
Fig. 1. Acetylene reduction in Oscillatoria sp. strain 23 incubated anaerobically in the dark.
ceased and a high rate of respiration provided for
anaerobic conditions within 15-60 min ([18]; unpublished observations). The cyanobacterial mat
persists in anaerobiosis throughout the night and
turns aerobic as soon as light intensity allows net
photosynthesis. During diurnal measurements of
nitrogenase activity in cyanobacterial mats of the
North Sea, Stal et al. [5] observed a pattern with,
surprisingly, two activity maxima. A small peak
was observed at sunset and a large one at sunrise.
Between the maxima, nitrogenase activity decreased to virtually zero at midnight and noon.
Diurnal measurements of nitrogenase activity in
natural populations of heterocystous planktic
Nodularia sp. in the Baltic [15] and of non-heterocystous planktic Trichodesmium in the ocean [16],
showed typical light-dependency. This was also
shown for heterocystous cyanobacteria grown in
the laboratory under light-dark cycles [9,11]. In
studying nitrogenase activity in Oscillatoria in
light-dark cycles Stal and Krumbein [9] found
acetylene reduction only to occur during the dark
period. Only one maximum was observed at the
onset of the dark period. It was suggested, that
under natural conditions not only the transition
from light to dark regulates synthesis and activity
230
of nitrogenase, but also the concomitant change to
anaerobic conditions in the dark. Therefore, we
cultivated Oscillatoria in a 1 6 / 8 h light-dark cycle
and changed the culture atmosphere to oxygen-free
nitrogen 1 h after the light was switched off. The
culture was changed back to air, 1 h after the light
was switched on again. Oscillatoria was grown
under such conditions during two weeks and the
organism grew remarkably well. Growth was only
slightly inhibited as compared to cultures grown
under aerobic light-dark cycles. Nitrogenase activity measured in cultures of Oscillatoria grown
under such conditions now showed a pattern which
was very similar to the natural situation (Figs. 2
and 3). Nitrogenase was induced as soon as the
light was switched off. Activity reached a small
peak of 4.5 ffmol acetylene reduced per hour and
mg Chl. a., similar to the value observed in aerobic
'5rl3
i
i
L
I
t
i
t
tL
q
/
I l l r l l l l l J
~
air
11
~
20
t
15
19
23
03
07
11
lime
15
kJ
Fig. 3. Pattern of acetylene reduction in light-dark, aerobicanaerobic grown cultures of Oscillatoria sp. (closed circles,
solid line) compared to diurnal variation of nitrogenase activity
in microbial mats of the North Sea (open circles, dotted line)
[Stal, L.J. and Krumbein, W.E. (1984) Mar. Biol. 82, 217-224].
Closed arrows indicate the beginning and the end of the dark
period in the Oscillatoria sp. culture at time 19.00 h and 03.00
h, respectively. The open arrows indicate the approximate
times of sunset (time 21,00 h) and sunrise (time 05.00 h).
--r
~
15
o
N2
E
10
~ 2,,^ J,-^/I
I h*l
~
I
5
I
10
15
lime (h)
Fig. 2. Rates of acetylene reduction in cultures of Oscillatoria
sp. strain 23, grown under a 1 6 / 8 h light-dark regime with
anaerobic conditions during the dark period. The arrows show
the periods of time when the cultures were incubated in the
dark or in the light and under an anaerobic nitrogen atmosphere or air. The transition from aerobic to anaerobic conditions was delayed 1 h compared to the light-dark cycle.
light-dark cycle [9]. Nitrogenase activity decreased
to about 2.5 ~mol C 2 H 4 . m g tChl. a . h -~1 after
changing to anaerobic conditions and remained
constant for 3.5 h, then suddenly falling to a low
rate of about 0.7 /~mol C z H 4 • mg-~Chl, a - h -1
for the rest of the dark period. As soon as the light
was turned on, a large peak was observed.
Nitrogenase activity eventually reached a maxim u m of almost 20/~mol C 2 H 4 - mg-aChl, a - h -]
and decreased rapidly to zero after changing back
to aerobic conditions. This high activity was also
observed if aerobic light-dark grown Oscillatoria
was transferred to continuous light [9].
231
C o m p a r i n g this p a t t e r n with the n a t u r a l situation, the s i m i l a r i t y of b o t h curves is evident (Fig.
3). T h e shifts in the m a x i m a can be e x p l a i n e d b y
the fact that the t r a n s i t i o n f r o m light to d a r k a n d
vice versa in n a t u r e is n o t as a b r u p t as it was in
the l a b o r a t o r y . P h o t o s y n t h e s i s m a y d r o p to very
low levels b e f o r e sunset a n d is then limited to the
u p p e r p a r t of the m a t [18]. It has p r e v i o u s l y been
shown that highest specific n i t r o g e n a s e activity
occurs in the lower p a r t of the m a t [5].
T h e a b r u p t decrease in activity after 3.5 h
u n d e r a n a e r o b i c c o n d i t i o n s in the d a r k is n o t
c o m p l e t e l y u n d e r s t o o d . This o b s e r v a t i o n seems in
c o n t r a d i c t i o n with the e x p e r i m e n t shown in Fig. 1.
H e r e we o b s e r v e d a c o n s t a n t activity d u r i n g 1 2 - 2 4
h. However, this e x p e r i m e n t was d o n e using an
a t m o s p h e r e of an inert gas (helium or argon).
Hence, no n i t r o g e n can be fixed a n d therefore,
n i t r o g e n a s e activity can n o t be r e g u l a t e d b y the
i n t r a c e l l u l a r p o o l of fixed nitrogen. Because
n i t r o g e n was p r e s e n t in the l i g h t - d a r k e x p e r i m e n t ,
the i n t r a c e l l u l a r p o o l of fixed nitrogen will increase. However, the high activity in the light
suggested that this was not yet sufficient to i n h i b i t
nitrogenase. M o s t p r o b a b l y , the decrease in activity can be e x p l a i n e d b y a s s u m i n g l i m i t a t i o n in
energy a n d electrons. As this a p p a r e n t l y was not
the case in the s a m e p e r i o d of time u n d e r an
a t m o s p h e r e of inert gas a n d acetylene, it seems
likely, that the r e d u c t i o n of m o l e c u l a r nitrogen
has a c o n s i d e r a b l y higher energy d e m a n d t h a n the
r e d u c t i o n of acetylene. If this is the case, it can be
a s s u m e d that the o r g a n i s m c o n t a i n s virtually no
e n d o g e n o u s reserve m a t e r i a l b y the end of the
d a r k period. Nevertheless, the high activity
o b s e r v e d in the light showed that n i t r o g e n a s e is
p r o v i d e d with energy a n d electrons b y p h o t o synthesis. This is in c o n t r a s t with the o b s e r v a t i o n s
of Ernst et al. [26]. T h e y showed that n i t r o g e n a s e
activity in the h e t e r o c y s t o u s A n a b a e n a d e p e n d e d
on the presence of glycogen. I n v e s t i g a t i o n s on the
role of glycogen in n i t r o g e n fixation in Oscillatoria
are in progress.
T o our knowledge, this is the first r e p o r t of
n i t r o g e n fixation in a c y a n o b a c t e r i u m u n d e r d a r k
a n a e r o b i c conditions. O s c i l l a t o r i a sp. strain 23
grew well u n d e r a l i g h t - d a r k regime with a n a e r o b i c
c o n d i t i o n s d u r i n g the d a r k period.
ACKNOWLEDGEMENTS
T h e a u t h o r s are very m u c h i n d e b t e d to Dr.
W . E . K r u m b e i n for his c o n t i n u o u s interest a n d
s u p p o r t in this w o r k a n d for his critical c o m m e n t s
on the m a n u s c r i p t .
REFERENCES
[1] Stal, L.J., Van Gemerden, H. and Krumbein, W.E. (1985)
Structure and development of a benthic marine microbial
mat. FEMS Microbiol. Ecol. 31,111-125.
[2] Bauld, J. (1984) Microbial mats in marginal marine environments: Shark Bay, Western Australia, and Spencer
Gulf, South Australia. In: Microbial Mats - Stromatolites
(Cohen, Y., Castenholz, R.W. and Halvorson, H.O., Eds.),
pp. 39-58. Alan R. Liss Inc., New York.
[3] Javor, B. and Castenholz, R.W. (1981) Laminated microbial mats, Laguna Guerrero Negro, Mexico. Geomicrobiol. J. 2, 237-273.
[4] Thomas, W.H. (1969) Phytoplankton nutrient enrichment
experiments off Baja California and in the eastern equatorial Pacific Ocean. J. Fish. Res. Bd. Can. 26, 1133-1145.
[5] Stal, L.J., Grossberger, S. and Krumbein, W.E. (1984)
Nitrogen fixation associated with the cyanobacterial mat
of a marine laminated microbial ecosystem. Mar. Biol. 82,
217-224.
[6] Haselkorn, R. (1978) Heterocysts. Ann. Rev. Plant Physiol. 29, 319-344.
[7] Stal, L.J. and Krumbein, W.E. (1981) Aerobic nitrogen
fixation in pure cultures of a benthic marine Oscillatoria
(cyanobacteria). FEMS Microbiol. Lett. 11, 295-298.
[8] Robson, R.L. and Postgate, J.R. (1980) Oxygen and hydrogen in biological nitrogen fixation. Ann. Rev. Microbiol. 34, 183-207.
[9] Stal, L.J. and Krumbein, W.E. (1985) Nitrogenase activity
in the non-heterocystous cyanobacterium Oscillatoria sp.
grown under alternating light-dark cycles. Arch. Microbiol. 143, 67-71.
[10] Huang, T.-C. and Chow, T.-J. (1986) New types of N2-fixing unicellular cyanobacterium (blue-green alga). FEMS
Microbiol. Lett. 36, 109-110.
[11] Mullineaux, P.M., Gallon, J.R. and Chaplin, A.E. (1981)
Acetylene reduction (nitrogen fixation) by cyanobacteria
grown under alternating light-dark cycles. FEMS Microbiol. Lett. 10, 245-247.
[12] Mitsui, A., Kumazawa, S., Takahashi, A., Ikemoto, H.,
Cao, S. and Arai, T. (1986) Strategy by which nitrogen-fixing unicellular cyanobacteria grow photoautrophically.
Nature 323, 720-722.
[13] Grobbelaar, N., Huang, T.-C., Lin, H.-Y. and Chow, T.-J.
(1986) Dinitrogen-fixing endogenous rhythm in Synechococcus RF-1. FEMS Microbiol. Lett. 37, 173-177.
[14] Leon, C., Kumazawa, S. and Mitsui, A. (1986) Cyclic
appearance of aerobic nitrogenase activity during syn-
232
[15]
[16]
[17]
[18]
[19]
[20]
chronous growth of unicellular cyanobacteria. Curr. Microbiol. 13, 149-153.
Hiibel, H. and Hiibel, M. (1974) In situ determinations of
the circadian N2-fixation of microbenthos on the Baltic
coast. Arch. Hydrobiol. Suppl. 46, 39-54.
Saino, T. and Hattori, A. (1978) Diel variation in nitrogen
fixation by a marine blue-green alga, Trichodesmium
thiebautii. Deep-Sea Res. 25, 1259-1263.
Carpenter, E.J. and Price, C.C. (1976) Marine Oscillatoria
(Trichodesmium): explanation for aerobic nitrogen fixation
without heterocysts. Science 191, 1278-1280.
Revsbech, N.P., Jergensen, B.B., Blackburn, T.H. and
Cohen, Y. (1983) Microelectrode studies of the photosynthesis and 02, H2S, and pH profiles of a microbial
mat. Limnol. Oceanogr. 28, 1062-1074.
Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M.
and Stanier, R.Y. (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J.
Gen. Microbiol. 111, 1-61.
Stewart, W.D.P., Fitzgerald, G.P. and Burris, R.H. (1967)
In situ studies on N2-fixation using the acetylene reduction technique. Proc. Natl. Acad. Sci. U.S.A. 58,
2071-2078.
[21] McKinney, G. (1941) Absorption of light by chlorophyll
solutions. J. Biol. Chem. 140, 315-322.
[22] Maryan, P.S., Eady, R.R., Chaplin, A.E. and Gallon, J.R.
(1986) Nitrogen fixation by Gloeothece sp. PCC 6909:
Respiration and not photosynthesis supports nitrogenase
activity in the light. J. Gen. Microbiol. 132, 789-796.
[23] Stal, L.J. and Krumbein, W.E. (1985) Isolation and characterization of cyanobacteria from a marine microbial
mat. Bot. Mar. 28, 351-365.
[24] Stal, L.J. and Krumbein, W.E. (1986) Metabolism of
cyanobacteria in anaerobic marine sediments. In:
GERBAM
Deuxi~me Colloque International de
Bact6riologie marine (1FREMER Actes de Colloques, No.
3), pp. 301-309.
[25] Heyer, H. and Stal, L.J. (1986) Heterofermentative lactate
fermentation, sulfur reduction and acetylene reduction
(N2-fixation) in the cyanobacterium Oscillatoria sp. incubated anaerobically in the dark. Abstracts of the 4th
Int. Symp. Microbial Ecol., Ljubljana, Yugoslavia.
[26] Ernst, A., Kirschenlohr, H., Diez, J. and B~ger, P. (1984)
Glycogen content and nitrogenase activity in Anabaena
t,ariabilis. Arch. Microbiol. 140, 120-125.
-