FEMS Microbiology Ecology 74 (1990) 59-72
Published by Elsevier
59
FEMSEC 00277
Interactions between nitrogen fixation and oxegenic
photosynthesis in a marine cyanobacterial mat
Marlies Villbrandt
', Lucas J. Stal
and Wolfgang E. Krumbein
'
' Geomicrobiology Division, University of Oldenburg, Oldenburg. F.R. G.
and
Laboraroty for Microbiology, University of Amsterdam. Amsterdam, The Netherlands
Received 1 February 1990
Revision received 23 May 1990
Accepted 23 May 1990
Key words: Cyanobacterial mat; Nitrogen fixation; Oxygenic photosynthesis; Oxygen profiles;
Microelectrode measurements; Cyanobacteria; Diurnal cycle; Light; Oscillaroria
1. SUMMARY
Cyanobacterial mats developed on fine sandy
sediments of the upper littoral of the island of
Mellum (North Sea). Freshly colonized sediment
was dominated by the non-heterocystous, nitrogen-fixing cyanobacterium Oscillatoria limosa.
Well established mats in which the cosmopolitan
cyanobacterium Microcoleus chthonoplastes was
the dominant organism also usually contained 0.
1 h l O S A as a minor component. This mat was about
1 mm thick and contained high biomass. Photosynthesis was maximal at about 150 pm depth and
reached values of 280 pmol oxygen. 1-' . min-I.
O n the other hand, in the dark, high respiratory
activity turned the mat anaerobic within minutes.
Freshly colonized sediment consisted of low
cyanobacterial biomass loosely attached to the
sand grains and present up to a depth of 2.5 mm.
Respiratory activity was low and the sediment
remained aerobic to a depth of 2 mm throughout
Correspondence to: M. Villbrandt, Geomicrobiology Division,
University of Oldenburg, D-2900 Oldenburg, F.R.G.
0168-6496/90/$03.50
0
the night. Nitrogen fixation (acetylene reduction)
was measured during 24-h periods in both types of
mats in order to elucidate interactions with oxygenic photosynthesis and oxygen concentration.
Acetylene reduction in the mats showed very different diurnal patterns which depended on the
type of mat investigated and the time of year. The
results indicated that a temporary separation of
oxygenic photosynthesis and nitrogen fixation occurred in the mat. Established mats fixed nitrogen
predominantly during the transition from dark to
light and vice versa, when oxygenic photosynthesis
was reduced or absent. Freshly colonized sediment-fixed nitrogen throughout the night but often
a stimulation was seen at dawn. The latter showed
much higher specific activities than the established
type. Also in spring, specific activities were much
higher.
2. INTRODUCTION
In nature the process of nitrogen fixation is of
utmost importance because it counterbalances
losses of combined nitrogen from the environment
1990 Federation of European Microbiological Societies
60
by denitrification [l]. The reduction of dinitrogen
(N2) is catalyzed by the enzyme-complex nitrogenase that occurs exclusively in procaryotic
organisms [2]. Nitrogen fixation demands a high
amount of energy and a low potential electron
donor. It is assumed that at least 4-5 ATP are
required for the transfer of 2 electrons to
nitrogenase [31. Usually reduced ferredoxin is the
biological electron donor. Ni trogenase also requires an anaerobic environment because oxygen
is known to be a severe inhibitor of this enzyme
causing irreversible damage [4,5].
Many cyanobacteria are known as nitrogen
fixers. This seems in paradox to the fact that these
organisms are also oxygenic phototrophic organisms. Cyanobacteria are not only exposed to
atmospheric oxygen. They also produce it. A
variety of different mechanisms have been proposed by which cyanobacteria protect their
nitrogenase against atmospheric oxygen [6,7]. Two
principally different strategies are known by which
cyanobacteria bypass the problem of photosynthetically evolved oxygen: (i) Some filamentous
cyanobacteria form specialized cells - heterocysts.
The latter, in which nitrogen fixation takes place,
have lost the capacity of oxygenic photosynthesis
[8-lo]. Such organisms thus separate oxygenic
photosynthesis and nitrogen fixation spatially. (ii)
In non-heterocystous filamentous and nitrogenfixing unicellular cyanobacteria the temporary
separation of nitrogen fixation and oxygenic photosynthesis is of utmost importance [6,11]. The
most simple way to separate oxygenic photosynthesis from dinitrogen fixation in a diurnal
light/dark cycle is to carry out the latter process
during the dark period. All non-heterocystous,
nitrogen-fixing cyanobacteria investigated thusfar
show this type of adaptation [ll-131. However,
experiments with alternating light/dark periods
usually were done under aerobic conditions. Even
in continuous light most of the non-heterocystous
cyanobacteria seem to reduce oxygenic photosynthesis or even change to respiration when
nitrogenase is induced [ll].Although it is believed
that in the unicellular Gloeothece sp. nitrogen fixation and oxygenic photosynthesis take place
simultaneously in one single cell (71,the ultimate
proof for this still stands out.
Several investigations have been addressed to
the question of the diurnal behaviour of
nitrogenase activity in natural communities of
cyanobacteria but few payed attention to the interactions with oxygenic photosynthesis and
oxygen concentration [ 14-21]. The majority of
these investigations concentrated on populations
of heterocystous cyanobacteria. In such populations diurnal variations of nitrogenase activity
showed close correlation with light intensity. During the night no or only little activity is observed.
Intertidal sediments are often colonized by
cyanobacteria [22]. The microbial mats of the intertidal flats of the southern North Sea are dominantly formed by non-heterocystous cyanobacteria. The cyanobacterial mats that develop on the
intertidal sediments of the North Sea island of
Mellum were described by Stal et al. [22]. The
cyanobacteria form dense communities in which
the organisms are attached to the sand grains and
to neighbouring individuals. Eventually, a tough
microbial mat is established. The environment is
low in combined nitrogen 1151. Therefore nitrogen
fixation should play an important role during colonization of the barren sand. Stal and Krumbein
[23] isolated many of the cyanobacteria present in
these mats and discovered an aerobic nitrogen-fixing Oscillatoria. Heterocystous cyanobacteria were
never observed in the mats. The non-heterocystous
nitrogen-fixing Oscillatoria limosa was found as
pioneer organism to colonize the sand initially
[22]. Stal et al. [15] already pointed out the correlation between nitrogen fixation and the presence
of 0. limosa and the importance of the fixation of
dinitrogen for mat development. The mechanisms
by which 0. limosa protects nitrogenase against
oxygen have recently been elucidated in part
[ 11,24,25]. The cosmopolitan cyanobacterium Microcoleus chthonoplastes becomes the dominant
species in mature mats. The latter organism does
not fix nitrogen in laboratory culture. Often mature mats of M. chrhonopfastescontain significant
numbers of 0. limosa. Several other species of
cyanobacteria are present in the mats but very
rarely are they of quantitative importance. Mature
mats are also inhabited by many other groups of
microorganisms. Among them are anoxyphotobacteria, sulfate-reducing bacteria, colorless sul-
61
Table 1
Sampling site and time, level, chlorophyll content, dominant cyanobacteria species and application of the samples
Station I
Station I1
Main sea level
+ 1.50 m NN
+ 1.60 m N N
Sampling time
(month. year)
07.87
06.88
09.88
07.87
06.88
09.88
Chlorophyll content
(mg CM o.m-')
n.d.
22.8
86.1
186.4
129.8
355.6
Dominant cyan*
bacteria species
Oa
0
0
0
M
M
M
Application of
samples (Figs.)
1B
2B
4A
4B
-
Mb
4c
4D
1A
2A
3A-E
0 = Oscillatoria limosa.
M
Microcoleus chthonoplastes.
fur-oxidizing bacteria and methanogenic bacteria.
Some communities of microorganisms are vertically stratified: e.g. underneath the green layer of
cyanobacteria sometimes a pink layer of purple
sulfur bacteria is found. These organisms carry
out anoxygenic photosynthesis using sulfide as
electron donor. The sulfide is produced by sulfate
reducing bacteria that form a black layer beneath
the purple bacteria. Such laminated microbial
communities are characterized by steep and
fluctuating gradients of light, oxygen and sulfide
[26]. However, also established mats would require
nitrogen-fixing species since it is known that actively denitrifying bacteria inhabit such systems as
well (Stal, unpublished) causing a continuous loss
of combined nitrogen. An earlier investigation of
diurnal nitrogenase activity [15] in field samples
showed two maxima: a large peak at sunrise and a
smaller one at sunset. This pattern differed from
culture experiments with 0. limosa in which
fitrogenase activity was found exclusively during
the dark period [24]. However, when cultures of 0.
&imosawere grown under an alternating light/dark
cycle with aerobic conditions during the light and
anaerobic conditions during the dark period, a
similar pattern of nitrogenase activity as obtained
in the field, was found [27].These results confirmed the importance of oxygen for dark nitrogen
fixation.
The aim of the present investigation was to
look more carefully at the interactions between
oxygenic photosynthesis, oxygen concentration
and nitrogen fixation in 24-h periods to study the
mechanisms that are operative in natural rnicrobial mats. Two sites of different mat development
stages were choosen which differed in standing
crop biomass (Table 1). The site with low biomass
was essentially aerobic during the night, whereas
the other turned anaerobic very soon after oxygenic photosynthesis ceased.
3. MATERIALS AND METHODS
3.1. Area of investigation
The microbial mats studied were located on the
North Sea island of Mellum. The island is situated
in the southern North Sea, close to the coast of
Germany at a latitude of 59"55' North and a
longitude of 34"44' East. Mellum forms part of a
chain of islands that separates the Shallows (Waddensea) from the North Sea. Microbial mats are
found on the westbank of the island which is an
extended intertidal flat. This intertidal flat is especially characterized by the deposition of fine sandy
sediments which form an excellent substrate for
cyanobacteria to attach. Well established mats are
found as a small zone contiguous to the vegetation
62
border. In summer a very large part of the intertidal flat is colonized by cyanobacteria that do not
form the tough and leathery structure typical for
established mat systems. The cyanobacterial colonization at this site usually disappeared during
the winter months and reestablished afresh the
next year. In general, cyanobacterial mats are
found from 1.5 and 2.0 m above mean sea level.
The measurements were carried out in summer
1987 and in the year 1988. Two stations were
chosen for the present investigation. Station 1
represented a young cyanobacterial community
and was situated at 1.5 m above mean sea level.
Sampling station 2 was 1.6 m above mean sea
level and consisted of a well-developed microbial
mat. The mature mats passed the winter in a more
or less inactive state.
3.2. Assay of nitrogenase activity
Nitrogenase activity was determined using the
acetylene reduction test [28]. In situ measurements
were carried out with the bell-jar technique as
described by Stal [29]. Bottomless serum bottles
(50 ml) were pushed at random in the sediment of
an experimental area of 1 m2. A gas volume of
30-35 ml of air was enclosed by the bottles. The
bottles were then sealed using rubber stoppers and
subsequently, 5 ml of acetylene (15% v/v) were
injected with a gas-tight syringe. Every 15 min
during 24-h periods a new bottle was incubated.
The incubations were done at ambient light and
temperature. During some measurements the experimental area was flooded at high tide. In most
cases sampling of already incubated bottles was
still possible under such circumstances but the
experiment had to be interrupted for a short time
because the incubation of new bottles was not
possible until the water had run off. Each bottle
was incubated for 2 h. At the end of each incubation period the gas phase was sampled using
Vacutainers (Becton and Dickinson) and stored
for analysis of acetylene and ethylene in the
laboratory. The sediment that was enclosed by the
serum bottle was stored at -2OOC for later pigment analysis.
3.3. Analysis of acetylene and ethylene
Acetylene and ethylene were determined by
gas-chromatography (Varian model 3700). The gas
chromatograph was equipped with a Flame Ionization Detector. The 3-m glass column was packed
with Poropak R (50-80 mesh). The gas chromatograph was run at 35 O C. The injector and detector
temperatures were 70 and 90 O C, respectively.
Nitrogen was used as carrier gas at a flow rate of
20 ml/min. The flow rates of hydrogen and air
were 15 and 300 ml/min, respectively. The chromatograph was calibrated with 100 ppm ethylene
in helium (Scotty Gases) and 100%acetylene. The
total amount of ethylene produced per incubation
bottle was calculated with acetylene as an internal
standard [29].
3.4. Determination of oxygen and photosynthesis
Dissolved oxygen was measured with custommade microelectrodes constructed as described by
Revsbech et al. [26]. Microelectrode-tips were approximately 5 pm. The polarographic oxygen measurements were done with a voltage of 0.75 V
applied over the oxygen electrode and an external
reference electrode. The current was measured with
an autoranging picoamperemeter (Keithley, model
485). The linear response of the electrode was
checked in the laboratory. Prior to the measurements a 2-point calibration was carried out in the
field. Air-saturated sea water from the same location was used as the reference. The actual oxygen
concentration in the sea water was measured with
an Orbisphere model 2609 (Switzerland) oxygen
indicator. Corrections were made to account for
temperature and salinity effects. Zero oxygen was
read in the anoxic part of the sediment. Photosynthesis was measured according to Revsbech et
al. [26]. At steady state oxygen concentration the
mat was shaded during 1-2 s and the decrease in
oxygen concentration was recorded automatically
at a rate of 3 readings/s. The initial rate of
oxygen decrease was assumed to be equal to the
photosynthetic rate [26]. Oxygen and photosynthesis were measured in the same experimental area
where nitrogenase measurements were done or in
artificially illuminated (slide projector) cores in
the field laboratory. Measurements in sediment
cores were started within 1 h after collecting the
samples. Possible effects of heating through the
slide projector were avoided by the cooling system
63
into which the microbial mat cores were embedded.
4. RESULTS
3.5. Determination of chlorophyll a
Chlorophyll a and pheophytin a were determined by the method of Stal et al. [30]. Sediment samples were extracted twice with an adequate volume of methanol at ambient temperature
in the dark. The extracts were partitioned with
n-hexane and absorbance was read in the hexanephase at 660 nm, before and after acidification
with 5 N HCl.
4.1. Photosynthesis and oxygen profiles
The first two figures will deal with results from
laboratory experiments as opposed to the in situ
measurements that will follow.
In the mature mat of M. chthonoplastes high
rates of photosynthesis were observed in a core
illuminated with a slide projector (Fig. 1A). Under
the prevailing conditions during the measurements
oxygen solubility was 6.8 mg.1-I at air saturation. Nevertheless no oxygen supersaturation was
seen in the mat (Fig. 2A). This was probably a
result of a very high (phot0)respiratory activity of
the dense biomass at this station. All cores studied
were water-saturated but the surfaces of the mats
were exposed to air. This is the situation which
cyanobacterial mats on intertidal sediments experience most of the time [22]. Corrections were
made for temperature and salinity effects. Even
3.6. Other m e t h d
Temperature was measured with a mini-pt-100sensor. Light intensity was measured with a battery-operated luxmeter. Salinity was measured with
a refractometer. The cyanobacterial species composition was estimated by microscopic observations which were carried out in the field laboratory within 1 h after collection of the samples.
j
0
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20
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64
under these conditions the mat surface contained
less oxygen than that of a sterile sediment in
equilibrium with air. This was explained by photorespiration caused by the light intensity that occurred at the surface. The M. chthonoplastes mat
studied was approximately 1 mm thick. In the
light, oxygen was detectable to a depth of 1.2 mm
(Fig. 2A). Photosynthesis, however, was not detectable below a depth of 0.7 mm (Fig. 1A). Light
allowing oxygenic photosynthesis obviously did
not penetrate beyond that depth. When the light
was switched off, oxygen disappeared within 6
min (Fig. 2A). In the dark, oxygen penetrated only
to 0.3 mm depth as a result of respiration. Prolonged dark incubation did not alter the oxygen
profile (Fig. 2A). However, when the mats were
covered with a thin (1 mm) layer of water, diffu-
A
sion of air was limited and the mat turned
anaerobic up to the surface (results not shown).
The mat reacted instantaneously upon turning on
the light. Within 1 min the mat became supersaturated with photosynthetically produced
oxygen. A steady state in which photosynthesis,
respiration and diffusion were in equilibrium was
obtained after 30 min.
The young microbial community at station 1
was very different from the established system. At
t h s station we found cyanobactena (mainly 0.
limosa) loosely associated with the fine sandy
sediment. The sediment contained low cyanobacterial biomass. The average amount of chlorophyll a was only 45.8 mg. rn-’ which is low
compared to the biomass of station 2 (178.9 mg
m-2). At station 1 the cyanobactena were found
B
Fig. 2. Oxygen profiles measured by microelectrodes in cores taken at station 2 (Fig. A) and station 1 (Fig. B) and illuminated by a
slide projector with 60 klux. The cores were placed in seawater but the sediment surface was exposed to air. The cyanobacterial layers
in the freshly colonized sandy sediment (station 1, Fig. B) and the well established mat (station 2, Fig. A) were about 1 mm thick. The
cyanobacterial layer of station 1 was covered by a thin layer of sand. In station 2 the cyanobacterial layer was at the sediment
surface. The corres were illuminated ( 0 )or incubated in the dark ( 0 ) .
65
in the upper 3 mm of the sediment. Photosynthesis
and oxygen profiles were measured in a watersaturated core of which the surface was exposed to
air. The depth profile of photosynthesis reflected
the extreme vertical patchiness of this mat (Fig.
1B). Photosynthesis was detected between 0.2 mm
and 1.4 mm depth. The cyanobacteria in station 1
occurred under a thin layer of sand. This would
result in a sufficient light attenuation to prevent
photo-oxidative damage of the organisms. The
rates of photosynthesis at different depths varied
greatly. A few high peaks of photosynthesis probably coincided with local high concentrations of
cyanobacteria. Due to the low overall cyan@
bacterial biomass light attenuation was presumably much less than in the established mat system.
ms allowed photosynthesis at a much greater
depth than in station 2.
m e profile Of Oxygen at station 1 showed that
oxygen penetrated 3 mm in the sediment. Also the
oxygen profile was not as smooth as seen in station 2 indicating the patchiness of the system.
Because of the low microbial biomass present at
this station, respiratory activity was also low in
station 1. Therefore, after switching off the light,
the mat became more slowly depleted of oxygen
than in station 2 (Fig. 2B). Even after prolonged
dark incubation (6 h) the sediment still contained
considerable concentrations of oxygen up to a
depth of 1.5 mm (Fig. 2B). We observed a transient increase in oxygen concentration at 1s-2.0
mm depth after switching off the light, but we
were not abIe to explain this phenomenon. Switching on the light had a similar effect as in station 2.
The total photosynthesis integrated over the vertical was about the same in both cores. This indicated that light limited photosynthetic activity under the conditions applied and not biomass.
In situ measurements of photosynthesis were
done as well. However, at station 1 in situ photosynthetic activity was below the limit of detection.
The results of measurements at station 2 are show
in Fig. 3A. Total photosynthesis was determined
by integrating the depth profiles. The measurements were done every hour during a sunny,
cloudless day in July 1987 (Fig. 3B). Photosynthesis was first detected at 9.00 h. At that time light
intensity had already reached a value of more than
50 klux. During the following hours light intensity
gradually increased to about 100 klux at noon. At
the same time photosynthetic activity increased
linearly to reach the very high activity of 120 mg
0, 1-’ . min-’. Although the light intensity remained high after noon, the photosynthetic rate
dropped drastically to values of about 40 mg
02 1-’ min-’ and successively decreased as light
intensity started to decrease. At 20.00 h photosynthesis was nil. Concominantly with photosynthesis and light intensity, nitrogenase activity
(acetylene reduction), sediment temperature and
oxygen profiles were measured (Fig. 3C-E).
Acetylene reduction was high before sunrise. It
decreased to a low value before oxygenic photosynthesis was detected. The sediment temperature
followed essentially the light intensity. We measured a night value of 15OC and a maximum
value of 24’ C around noon.
-
4.2. Nitrogen fixation
Diurnal variations of nitrogenase activity were
measured at stations 1 and 2. The measurements
were performed in June and September 1988
jointly accompanied with measurements of light
intensity and temperature. The results are shown
in Fig. 4A-D.
The specific nitrogenase activities, expressed as
pmol C2H2 reduced per mg chlorophyll a and h
showed two obvious aspects. Differences occurred
between the two stations and between the two
sampling dates (June and September). Specific
nitrogenase activities were much higher in station
1 (Fig. 4C.D) compared with station 2 (Fig. 4A,B).
Maximum specific activities measured in June were
less than 1pmol C,H, mg-’ chl - h-’ in station 2
(Fig. 4A), whereas values of over 20 pmol C2H4*
mg-’ chl h-’ where detected in station 1 (Fig.
4C). The latter activity was in the range of the
highest nitrogenase activity recorded for the
cyanobacterium 0.limosa in culture [ll].This was
in agreement with the observation that the
cyanobacterial biomass in station 1 consisted Virtually exclusively of 0.limosa. In station 2 M.
chrhonoplastes is the dominant species. This
organism is not reported to fix nitrogen [23]. Maximum specific nitrogenase activities were highest
in June (Fig. 4A,C). In September, station 2 (Fig.
-
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67
4B) showed rates of acetylene reduction that were
an order of magnitude lower than in June (Fig.
4A). In station 1, the maximum September rate
(Fig. 4D) was 20% of the rate measured in June
(Fig. 4C).
Complex diurnal patterns of acetylene reduction were observed in both systems. In station 2,
in June nitrogenase activity occurred as two sharp
peaks at sunrise and sunset (Fig. 4A). Station 1 on
the contrary does not turn anaerobic during the
night. In this station a high night activity was
observed (Fig. 4c). This activity increased during
the night, reaching a maximum at sunrise. In
September a similar pattern was observed (Fig.
4 ~ ) However,
.
in this case the dark activities were
low and the peak at sunrise was much more distinct. Station 2 showed only very low nitrogenase
activities in September. However, activity was seen
exclusively during the day period and no peaks
were found at sunrise or sunset. This observation
combined with the fact that 0. limosa was virtually absent in September at station 2, hinted to the
presence of other nitrogen-fixing organisms.
For reasons of comparison the days during
which the measurements were done were very
similar with respect to light and temperature. Light
intensity at noon amounted to 90-100 klux. The
sediment temperature was 20-24' C during the
day and l l - 1 3 O C during the night. An important
factor might be the length of the dark period. The
dark period lasted about 7 h in June but increased
to 10 h in September.
5. DISCUSSION
After detailed studies of the temporary occurrence and separation of nitrogenase activity and
oxygenic photosynthesis in non-heterocystous
cyanobacteria in the laboratory under anaerobic
and aerobic conditions we have now verified these
effects under natural conditions in the field. The
results presented here confirmed that diurnal variations of nitrogenase activity occurred in situ.
Especially the nitrogenase activity during the early
morning seems to be important (Fig. 3C), when
oxygen reached only the first 200 pm of the
cyanobacterial mat (Fig. 3E). However, in June
1988 station 2 showed two very distinct and similar maxima at dusk and dawn (Fig. 4A). This
pattern was expected because of the incompatibility of oxygenic photosynthesis and nitrogen fixation. Thus during daytime nitrogenase would not
be active when the onset of light leads to photosynthetic evolution of oxygen, which, once a certain 0, concentration is reached, will inactivate
the nitrogenase complex. On the other hand, station 2 is characterized by anaerobic conditions
during the night (Fig. 2A). Such conditions will
not favour the energetically expensive nitrogenase.
At dusk and dawn all conditions are met for
nitrogenase to be active. Light is then present,
though at low intensity (Figs. 3B and 4). Photosynthetically oxygen evolution is absent or low
(Fig. 3A) and, consequently, the concentration of
oxygen in the sediment is low (Fig. 3E). Apparently light energy is harvested for nitrogen
fixation without causing a net production of
oxygen.
In combination with the proposed mechanism,
other regulating factors are the fixed nitrogen
species nitrate and ammonia. The concentrations
of ammonium, nitrate and nitrite in the interstitial
water of the Mellum microbial mats were measured by Stal et al. [22]. Their measurements
showed that only very low concentrations of nitrate
and nitrite were present. The concentrations of
ammonium, on the other hand, were considerable
(127-358 p g NH,-N 1-' in station 1 and 430-666
pg NH,-N. 1-' in station 2). Nevertheless, these
rather high levels of ammonium did not eliminate
nitrogenase activity [15]. The assessment of the in
situ role of ammonium in the regulation of
nitrogenase activity is hindered by the lack of
suitable methods to determine this compound at
the pm-scale. Sampling of a sufficient volume of
interstitial water gives at the best a resolution of a
few millimeters in the vertical profile. Nitrate- and
ammonium ion-selective microelectrodes interfere
with high salt concentrations and, consequently,
can not be used in marine systems [31]. One
possible mechanism that inhibits nitrogenase during the night could be: During the day the deep
penetration of oxygen allows nitrification to go on
in the deeper parts of the mat and ammonia does
therefore not reach the photosynthetic active
-
68
The situation in station 1 is different because
the cyanobacterial mat will not turn anaerobic
during the night (Fig. 2B). Aerobic dark conditions are preferred by non-heterocystous cyanobacteria to fix dinitrogen. Energy is available
through aerobic respiration. This was observed in
station 1. Apparently, energy is still limited because the mornings' dim light greatly stimulated
nitrogenase activity. This stimulation is even
stronger in September. Mat development might at
layers. During the night the lower penetration of
oxygen lowers nitrification and ammonia can diffuse all the way up to the active layers. This
explanation requires that ammonia is more effective as inhibitor of nitrogenase than nitrate. It is
also possible that not all of the produced nitrate
will reach the photosynthetic active layers because
some of it will get denitrified. This does, however,
only explain the absence of nitrogen fixation during the dark in station 2.
0.9
-
06-
03-
14
16
22
2
6
timefhl
10
Fig. 4. Diurnal pattern of acetylene reduction measured in situ with the beli-jar technique. Fig. A + B show the results of
measurements camed out in station 2 in June (A) and September (B) 1988. Fig. C + D show the results of measurements carried out
in station 1 in June (C) and September (D) 1988. The top and bottom graphs show light intensity and sediment temperature
respectively. Note different scaling of A + B and C + D.
69
this point have reached a state in which the sediment had accumulated considerable biomass and
consequently oxygen might have been more limiting in the dark than in an early state of development (June). The fact that nitrogen fixation is not
inhibited during the dark period at station 1 could
also be due to a lower mineralization, and therefore lower flux of ammonia from the deeper parts
of this newly established mat. In addition to this
the oxygen conditions at this station changes less
from light to dark situations. Therefore tKe nitrification must also be more constant.
The mat of M . chthonoplastes showed only very
low nitrogenase activity in September. Growth of
the cyanobacterial mat will decrease at the end of
the vegetation period [22] possibly resulting in a
lower demand of combined nitrogen. It is also
observed, that virtually no 0. limosa were present
in the mat at that time. There was absolutely no
nitrogenase activity during the night. Daytime activities were extremely low and fluctuated strongly.
It cannot be excluded that other organisms than
cyanobacteria contribute to total nitrogenase activity. The maximum nitrogenase activity of over
1
L
1L
time Ih)
Fig. 4 (continued).
I
18
.
22
I
2
I
6
time lhl
10
70
-
-
20 pmol C,H, mg-' chl a h-', measured in situ
is in agreement with the maximum measured in
cultures of 0.limosu [ll]. The patterns of acetylene
reduction in the field experiments, however, were
consistent with the physiology of nitrogen fixation
in 0. limosa in the laboratory [ll]. This leads to
the conclusion that 0. limosa was responsible for
the bulk of nitrogen fixation, at least in station 1.
The low nitrogenase activities measured in station
2 could be explained by other bacteria.
The results obtained, clearly showed that a
temporal separation of oxygenic photosynthesis
and nitrogen fixation is indeed operative in this
cyanobacterial mat. Photosynthetic oxygen evolution in the mat is not detectable below an incident
light intensity of about 10 klux. Light intensity
below this value, however, may be sufficient to
support nitrogenase activity through photosynthetic energy gains (e.g. cyclic photophosphorylation).
The in situ measurements of acetylene reduction with the gas-dome technique minimize disturbances of the microbial community. This results in more realistic numbers than earlier measurements where samples from the mat were incubated under rather artificial conditions [15].
However, the disadvantage of the gas-dome technique is that the vertical distribution of nitrogenase
activity in the mat is not known. The possibility of
a spatial separation of nitrogen fixation and oxygenic photosynthesis in a vertical zonation within
the mat was not investigated here, though it would
be imaginable. Stal et al. [15] showed that specific
nitrogenase activity in the mat increased considerably with depth. Only light of longer wavelength
penetrates deeper into the cyanobacterial mat [22].
This light could provide nitrogenase with energy
[25] without supporting oxygenic photosynthesis.
The light diffusion pattern in microbial mats is
known to vary largely with composition and mat
topology [32]. The vertical migration of the motile
organisms could be important as well.
From the results presented in the Figs. 3 and 4
it is evident that light triggers the diurnal pattern
of nitrogenase activity rather than temperature.
Culture experiments with 0. limosa have shown
that nitrogenase activity but not photosynthesis in
this organism was extremely susceptibleto elevated
temperatures (25-30 O C). From cultures of 0.
limosa grown at temperatures above 25OC it is
known that nitrogenase activity is strongly inhibited, whereas low temperatures (10-15 C) did
not show any effect (unpublished results). Because
the sediment cooled down to 11- 13 C during the
night it can be concluded that 0. limosa is remarkably well adapted.
The regulating mechanisms of controlling factors of nitrogen fixation and potential inhibitors
of nitrogenase are complex. Nitrogenase activity
in field communities without heterocysts can be
stimulated or blocked additionally by other changing parameters such as pH, salinity, water availability or flooding frequency. The extreme and to a
certain degree unpredictable diurnal variations of
nitrogenase activity in the microbial mats of the
North Sea show that it is not easy to extrapolate
from one or a few measurements to annual rates
of nitrogen fixation. Moreover, not only temporal
variations play a role but also the patchiness of
the sediment ecosystem makes such calculations
difficult. Continuous readings of nitrogenase activity in cultures or even under field conditions
would represent an important method towards a
better understanding of the complex relations discussed here.
ACKNOWLEDGEMENTS
We sincerely thank the Mellumrat for allowing
to carry out the fieldwork on the nature reserve
Mellum. We also thank U.Wollenzien for Skilled
technical assistance. This work was supported by
grant No. 333/23-1 of the Deutsche Forschungsgemeinschaft.
US
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