Dark anaerobic nitrogen fixation (acetylene reduction) in the cyanobacterium Oscillatoria sp.

FEMS Microbiology Ecology 45 (1987) 227-232 Published by Elsevier

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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

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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-

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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

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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

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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

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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).

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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 .

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