FEMS Microbiology Ecology 86 (1992) 195-203
0 1992 Federation of European Microbiological Societies 0168-6496/92/%05.00
Published by Elsevier
195
FEMSEC 00364
Microprofiles of photosynthesis and oxygen concentration
in Microcystis sp. scums
Bas W. Ibelings and Luuc R. Mur
Universiry of Amsterdam, Laboratory for Microbiology, The Netherlands
Received 25 March 1991
Revision received 18 September 1991
Accepted 20 September 1991
Key words: Cyanobacteria; Artificial scum conditions; Oxygen microelectrodes; Downwelling photon
irradiance; Colony size
SUMMARY
Oxygen microelectrodes were used to monitor
oxygen concentration and rates of gross photosynthetic activity in Microcystis sp. scums which
were formed and incubated under laboratory
conditions. The depth of the photic layer, rate of
photosynthesis, oxygen concentration and the location of the transition to anoxia in the scum
depended on irradiance levels and colony size.
Gross photosynthetic activity never extended below 2.5 mm depth in the scum. At high irradiance
levels oxygen concentration in the upper 1.5 m m
of the scum decreased and the oxygen concentration peak shifted to greater depth. Oxygen concentrations in scums composed of small colonies
( < 55 pm) were higher than concentrations in
large colonies scums (> 300 pm) but small
colonies showed stronger indications of photoin-
Correspondence to: B.W. Ibelings, University of Amsterdam,
Laboratory for Microbiology, Nieuwe Achtergracht 127, 1018
WS Amsterdam, The Netherlands.
hibition. In a natural scum small colonies are
presumably shielded from inhibitory intensities by
larger colonies which will dominate the upper
layers. Accumulation of low-light adapted, smaller
colonies in deeper layers likely yielded a second
peak in photosynthetic activity. In order to systematically discuss scums and scum formation a
distinction is made in three different scum types.
2. INTRODUCTION
The occurrence of cyanobacterial surface
blooms has been documented under different environmental conditions. During periods of calm
weather populations of buoyant, gas vacuolated
cyanobacteria float to the surface of a lake to
create nocturnal blooms [ 1,2]. During day time
mechanisms which cause loss of buoyancy operate through turgor-mediated collapse of gas vesicles [3,4] and ballast accumulation of photosynthetically fixed carbohydrate 151. Dynamic exchange of colonies at the lake surface as demonstrated in a computer model [61 may give the
196
impression of a static surface bloom (type I
blooms). In contrast a period of intensive mixing
below the euphotic zone, which makes the
colonies overbuoyant [7] induces surface blooms
at any time of the day. A surface bloom may
remain during the entire day even in the case of
optimal photosynthetic activity [8]. This type of
bloom is more likely to accumulate in a thicker
scum over longer periods at wind protected sites
(Type I1 blooms). Scum formation has also been
described as the result of failure of the regulating
mechanisms to correct overbuoyancy in senescent
populations [9] (Type I11 blooms).
It is still being questioned whether bloom formation is generally advantageous. In any type of
surface bloom colonies are exposed to potentially
lethal conditions such as photooxidation [lo] and
dehydration [ll] which would impair overall
growth of the population. This view was amended
in that cyanobacteria at or near the lake surface
could enhance their growth through interception
of atmospheric N, [12], CO, [13] or deposited N
compounds [14]. The negative effects of potential
photooxidation would be overcome by increased
carotenoid levels [15,16].
In this study oxygen microelectrodes were used
to monitor oxygen concentration and gross rates
of photosynthesis in Microcystis scums. Our expectation was that photosynthetic activity would
be restricted due to photoinhibition at the surface combined with a rapid light attenuation,
limiting photosynthesis in deeper layers and gradually developing carbon limitation. The aspect of
variation in photosynthetic activity with colony
size as demonstrated by Paerl [17] was included
in the study.
3. MATERIALS
AND METHODS
Description of the lake
Microcystis sp. was sampled from Lake Nieuwe
Meer near Amsterdam, The Netherlands. The
lake has a surface area of 1.5 km2,a mean depth
of 18 m and is surrounded by trees. Each year a
dense Microcystis sp. bloom develops, with extensive scum formation of type 11 (cf. INTRODUCTION).
Aged scums show crust formation trough desiccation and lysis of colonies in the top layer. The
scums that were used for microelectrode s t u d i e s
were formed in the laboratory and were m a d e up
of colonies collected in Lake Nieuwe Meer.
Oxygen microelectrode studies
Microcystis was sampled with a bucket in t h e
early morning from a mixed population near t h e
lake surface, i.e. no samples were taken f r o m
scums in the lake. Scums for microelectrode studies were formed subsequently in the laboratory by
allowing the buoyant colonies to float up i n a
small beaker. The laboratory formed scums simulated conditions in the early stages of type 11
scum formation in the lake. These scums had t h e
advantage of a absence of turbulence and a better control of external conditions. Scum formation took place during a l-h dark period prior to
the onset of illumination. Scums were between 5
and 8 mm thick. Each measurement was preceded by an illumination period of 30 min d u r i n g
which the oxygen profiles stabilized. The s c u m s
were illuminated from the top only. Light (cf.
RESULTS) was provided by glass fibre optics connected to a halogen lamp (Schott, KL 1500). An
infrared filter was placed between the light source
and the scum. Oxygen concentrations in the s c u m
were measured with Clark type oxygen microelectrodes [MI. The tip of the electrodes,,hada diameter around 7 p m and a 10-90% response time of
< 0.5 s. The electrodes were calibrated with a
saturated Na,SO, solution (0%) and air saturated water (100%). The tip of the electrode was
placed on the scum surface using a binocular
dissection microscope and moved downwards i n
steps of 100 km, using a micromanipulator
(Merzhauser). The signal was recorded and stored
with a Keithly 485 picoammeter. In principle t h e
experimental setup was the same as that shown in
[ 191. Light (downwelling photon irradiance; E d )
was measured as PhAR using a Licor LI185B
quantum photometer equipped with a 2 T ,cosine-corrected sensor. The sensor was held at the
position of the scum surface to measure the irradiance level.
Rates of photosynthetic activity
Gross photosynthesis was calculated as the initial rate of decrease in oxygen concentration after
197
Rate of gross photosynthesis (mM 0 2 / h)
Oxygen concentration ( m M )
000
020
040
060
080
100
0
10
I\
1
20
40
30
,
b
c
t
k
P
.
t
t
Fig. 1. Oxygen Concentration (a) and rate of gross photosynthetic activity (b) profiles in Microcysris scums at three different
irradiance levels: Ed = 50 (*k 200 ( W ) and 1200 ( A ) pmol photons m-’ s-I.
darkening of the scum [20]. In our study, rate of
photosynthesis was calculated via linear regression of 12 points during the first 4 s after the
scum was shielded from the light source. Samples
for chlorophyll analysis were taken after homogenizing the scum by inverting the beaker. Chlorophyll was extracted in hot ethanol and estimated
at 665 nm using an extinction coefficient of 8.16 1
(g.mrn1-I [211. Protein was measured by the Folin
method of Lowry with bovine serum albumin as
standard [22].
Colony size fractions
Samples from Lake Nieuwe Meer were separated into two different size fractions. A scum
consisting solely of larger, irregular shaped
colonies resulted from filtering a lake sample
over a 300-pm mesh size. Filtering with a plankton net of 55-pm mesh size yielded a fraction
with small, spherical colonies.
4. RESULTS
Reproducibility
Reproducibility in oxygen concentration profiles at random spots in the scum was acceptable
with 60% of the triplicate measurements having a
SE of less than 6% of the mean. The remaining
measurements had a SE between 6 and 12% of
the mean. Reproducibility in photosynthesis profiles was less, with over 50% of the triplicates
having a SE of more than 15% of the mean. This
was probably due to the heterogenic nature of
198
scums. Each of the three figures presents results
generated from observations on scums sampled at
different days within a month. True comparison
of graphs can only be made within a figure, since
colonies in different scums differed in conditions
they had encountered in the lake.
Oxygen concentration and production profiles at
different Ed
Fig. l a shows oxygen concentration profiles in
Microcystis scums at different irradiance levels.
Five different values of Ed were used: 5, 50, 200,
400, and 1200 pmol photons m-' s - ' . For reasons of clarity Fig. l a shows only three of the
profiles. A summary of all results is presented in
Table 1. At Ed = 5 pmol photons m-' s - ' anoxic
conditions (which had developed in the dark period during which the scums were formed) remained with exeption of a shallow top layer. Step
by step increases of Ed led to increases in depth
integrated oxygen concentrations and a shift of
anoxia to deeper layers (Table 1). The maximal
observed oxygen concentration was 0.81 mM, almost three times that in air-saturated water. In
the upper 400 p m of the scum, oxygen concentration at E d = 1200 pmol photons m-* s - ' was
lower than at Ed = 50 or 200 pmol photons m-'
s-I. The peak at Ed = 1200 pmol photons m-'
s- shifted to greater depth. These observations
are likely to be the result of photoinhibition
and/or photorespiration.
No gross photosynthetic activity was found a t
s-'. When Ed was
raised to 50 pmol m-* s-' gross photosynthetic
activity was restricted to the upper mm of the
scum (Fig. lb). Integrated over depth, gross photosynthesis resembled usual P-I measurements
and saturated at E d = 2 0 0 pmol photons rn-'
s-I (Table 1). The layer with gross photosynthetic
activity deepened with increases in Ed,but never
extended below the upper 2 mm of the scum. A
maximal rate of gross photosynthesis of 27.4 mM
0, h - ' was found at Ed= 1200 pmol photons
m - 2 s - I at a depth of 200 pm.
E d = 5 pmol photons
Oxygen concentration and production in rime
Type I1 scums are often exposed to high light
intensities over longer periods. TO test if scums
could maintain their initial photosynthetic activity
at high Ed scums were incubated for 12 h a t
Ed = 2000 pmol photons m-' s - I . Observations
were made at regular intervals after the onset of
illumination. Figs. 2a and 2b show the profiles
taken after 2.5, 6.5 and 12.5 h. Between 2.5- a n d
12.5-h incubation no significant changes in oxygen concentration or gross photosynthesis were
found. The invariability of the oxygen concentration profiles indicate a stable balance between
oxygen evolution and respiratory processes. The
maximal rate of gross photosynthesis was 38 rnM
0, h-' which was found directly below the scum
surface. The maximum depth at which gross pho-
Table 1
Depth where anoxia was reached, depth of photic layer, integrated oxygen concentration and integrated rates of gross photosynthetic activity in Microcystis scum at different irradiance levels
Ed
(pmol photons m-' s - ' )
Anoxia
(mm)
Euphotic
layer
2 [0,1
(rnmol o2m - 2 )
I Photosynthectic activity
-
( m m o l 0 , m-' h - ' )
(mm
0
5
50
200
400
1200
0. I
0.9
2.1
4
4.3
5
0
0
1.1
1.3
1.7
0.006
0
0.07
0.6
1.51
1.96
0
0.14
0.33
0.35
2
2.03
0.3
z
199
tosynthesis was still measurable was 2400 pm.
Fig. 2b shows a second peak in gross photosynthetic activity at 800-1300 p m depth. At lower
Ed a plateau in photosynthetic activity was often
found at the depth of this second peak (Fig. lb).
The effect of colony size on scum conditions
Colony sizes influenced oxygen concentrations
in the scum. Oxygen profiles in Figs. 3a and 3b
have been normalized on basis of chlorophyll
concentration (mg 1-’1 to enable comparison. It
was assumed that chlorophyll in the vertical direction was distributed homogenously. With Ed
= 250 pmol photons K 2s - ’ (Fig. 3a) highest
oxygen concentrations were measured in a ‘small
size fraction’ scum (‘SF scum’ composed of spherical colonies < 5 5 pm). Deeper than 1600 p m
Oxygen concentration
000
020
040
060
(
mM
)
080
the oxygen concentration in the ‘large size fraction’ scum (‘LF scum’ composed of irregular
shaped colonies > 300 pm) exceeded the concentration in the S F scum. Oxygen concentration in a
scum containing colonies of all sizes (‘field’) was
comparable to the LF in the upper 1500 p m of
the scum, but started to deviate at greater depths.
Anoxic conditions were reached at 2000 p m in
the SF scum and at 2200 p m in the ‘field’ scum,
but were absent in the large fraction scum.
At Ed=2000 pmol photons m-’ s - I oxygen
concentration in the SF scum was lower than at
Ed=250 pmol photons m-2 s - I but still exceeded concentrations in LF and ‘field’ scums. In
LF and ‘field’ scums oxygen concentration increased when the irradiance level was raised to
its maximum. LF scum did not become anaerobic
Rate of gross photosynthesis (mM 0 2 / h)
100
10
20
30
40
i
/*‘
Fig. 2. Oxygen concentration (a) and rate of gross photosynthetic activity (b) profiles in Microcystis scums. Time-course experiment:
2.5- (a), 6.5- (.)and 12.5- ( A ) h incubation time. Ed = 2000 wmol photons m - 2 s-’.
200
Oxygen concentration ( mM / mg chl
Oxygen cmcentration ( mM / mg chi )
)
5
000
005
010
015
020
025
I \
:t
me
m
I/
E,
1000
I
5
51
G
Fig. 3. Oxygen concentration profiles in Microcysfis scums composed of different colony sizes: field (untreated, 0); LF (cooln=,is.
larger than 300 p m , m ) and SF (colonies smaller than 55 p m , A). TWOdifferent surface light intensities: 250 (a) and 2000 pmol
photons m-‘ s - I (b). The profiles have been normalized on chlorophyll concentration (mg I - ’ ) to enable comparison.
even at greater depths. The chlorophyll/ protein
ratio of small colonies was higher than the ratio
of large colonies, 0.024 and 0.019 respectively.
5. DISCUSSION
Limitation of oxygen production by photosynthesis in scums is to be expected when a combination of inhibitory processes would be administered. It has been put forward that enhanced
carotenoid levels would protect Microcystis
against high light at the lake surface, where it
would have a better access to the preferred source
of carbon, CO, [13,15,16]. The microelectrode
studies on hyperscums in Hartbeespoort Dam
[23] did not support this view in that a gradual
decrease in oxygen concentration during prolonged incubation was ascribed to restricted diffusion of CO,. Our studies did not show decrease
in oxygen concentration or loss of gross photosynthetic activity during prolonged incubation (Fig.
2a and b). Nevertheless the graphs in Fig. l a
support the idea that net photosynthesis was restricted in scums. It is most likely that a combination of photoinhibition and photorespiration led
to decreased oxygen concentrations near the surface at high Ed. An imbalance between elevated
oxygen concentrations and restricted CO, availability would force RuBisCO to work as an oxygenase, thereby reducing net photosynthesis. Confirmation could not be obtained from production
measurements in Fig. l b since these graphs give
gross photosynthesis. In a study on Aphani-
20 1
zomenon blooms [ 131 a peak in photosynthesis
was found in the early morning, before high oxygen concentrations builded up. In our study oxygen concentrations reached a Ievel of 400 p M
(which is the I(, of the oxygenasel within 15 min
at Ed= 150 pmoI photons m-2 s-I. Photorespiration limits the ecological relevance of data on
gross photosynthetic activity as measured with
microelectrodes [24]. The increase in integrated
oxygen concentration with an increase in Ed (Table 1) indicated that net photosynthesis increased, despite a likely increase in photorespiration. At a change from E d = 2 0 0 to 400 pmol
photons m-’ s- oxygen concentration increased
while gross photosynthesis remained at the same
level. This would even suggest a decrease in oxygen consuming processes with this increase in
irradiance level. TO elucidate the role of photorespiration in Microcystis scums the effect of
variation in anorganic carbon to oxygen concentration ratios will be studied as was done for
diatom biofilms [241.
Low photosynthetic activity at a depth of 0
pm, even at low Ed (Fig. lb), was probably the
result of interference of a high-oxygen diffusion
rate with the production measurement. This
Seems to be a limitation of measurement of photosynthesis with microelectrodes [251.
The photic layer never extended below 2.5 mm
in a scum. In thicker scums of type I1 or 111 most
colonies therefore will be in the dark and nonproductive. The colonies in deeper layers work
like an air cushion, keeping the top layer at the
surface and prohibiting dynamic exchange of
colonies through variations in buoyancy [6].
In type I1 or I11 scums which exist over long
periods, desiccation of the top layer, leading to
crust formation, would further hamper photosynthetic activity [23]. In our study desiccation did
not influence the results. The incubations simulated only the initial stages of scum formation.
Desiccation is a slow process compared to photooxidation [23] and a time span of 12 h (Fig. 2) is
too short for desiccation to operate.
The question of whether photosynthetic activity and growth are enhanced in surface blooms
[131 would need a more detailed comparison between a population present in surface blooms and
’
a circulating population, including changes with
depth and time. The proposal to discriminate
between different types of scums as put forward
in the introduction could facilitate discussion and
perhaps solve some of the paradoxes cited at the
start of this discussion. Furthermore Reynolds [9]
concluded that there is evidence that only some
strains of Microcystis are adapted to withstand
prolonged surface exposure. Paerl [ 151 studied
Microcystis surface blooms where euphotic zone
in the lake was still 0.6 m (i.e. no real scum
formation, type I bloom) whereas in the hyperscum studies of Zohary [23] the photic layer was
only 0.01 m. Maximum photosynthetic rates in
scums in Hartbeespoort Dam reached 38.8 mM
0, h-I, which is almost identical to what was
found in Lake Nieuwe Meer (Fig. lb). Chlorophyll concentration in Hartbeespoort Dam was
1-3 g I - ’ at the scum surface, but less than 0.1 g
I-’ in Lake Nieuwe Meer. Thus specific rates of
photosynthesis (rates expressed per mg of chlorophyll) were much lower in Hartbeespoort Dam.
The difference emphasizes that hyperscums in
Hartbeespoort were of type 111, formed by a
senescent population at water temperatures below 12°C.
Higher oxygen concentrations in SF scum may
be the result of a more efficient light absorption
by small particles as predicted by the ‘sieve effect’:
absorbance in a suspension will be less when the
partition of pigments, perpendicular to the photon flux, is non-homogeneous. The partition of
large irregular shaped Microcystis colonies in a
scum, as well as the inhomogeneous partition of
cells in larger colonies [17], caused lower light
absorbtion. This resulted also in higher oxygen
concentrations in deeper layers of LF and ‘field’
scums and even absence of anoxia in LF scums.
For Microcystis in suspension a relationship between colony size and light attenuation was established [26]. Small colonies showed indications of
photoinhibition and/or photorespiration, the
photosynthetic activity decreased when Ed was
raised (Fig. 3). Changes in colony size could be a
protecting mechanism against high light. Aphanizomenon increased colony sizes at high light intensities [271.
The occurrence of a second, subsurface peak
202
in photosynthetic activity (Fig. 2b) could be explained using the following elements: i) according
to Stokes law [9] larger colonies will accumulate
faster at the lake surface, resulting in a spatial
separation of larger and smaller colonies in a
scum; ii) larger Microcystis colonies respond faster
to an increase in water-column stability. They
accumulate in the surface mixed layer, where
large colonies are relatively more abundant [28].
In this way, larger colonies increase the average
light dose received during the day; iii) Microcysris
adapts its photosynthetic activity to different light
intensities [29]. The small and large colonies that
made up the SF and LF scum were adapted to
the different light doses they received in the lake.
This was supported by the difference in chlorophyll/ protein ratios between the large and small
colonies fractions. Generally algae that are
adapted to growth at low-light conditions will
show higher chlorophyll/ protein ratios than
high-light adapted cells, e.g. [30]. The near surface peak in photosynthetic activity resulted from
photosynthesis of larger colonies, which were
adapted to high light. Low-light adapted, smaller
colonies deeper in the scum were responsible for
the second peak. Adaptation to lower light conditions might also have determined the susceptibility of small colonies to high light. Similarity between ‘field’ and LF-scums oxygen profiles near
the scum surface supports this hypothesis. Absence of photoinhibition in ‘field’ scums is in
accordance with dominance of larger colonies at
the scum surface. The large colonies shield
smaller colonies in deeper layers from inhibitory
intensities. Studies to confirm this hypothesis
would need a better understanding of the microstructure of scums. Studies of this type are in
preparation.
stricted by inhibitory processes near the surface
and a rapid attenuation of light, which prohibited
photosynthetic activity deeper in the scums. Even
if scum formation is judged advantageous by mere
notice of apparent adaptations to enhance photosynthesis, the risk of persistent scum formation a t
wind protected sites has to be taken into account.
Survival of scum conditions may be more important than the potential of growth enhancement in
surface blooms [9]. Incubation of Microcystis
colonies under conditions simulating bloom conditions (anoxia in the dark at 20°C) led to a rapid,
irreversible loss of photosynthetic capacity (in
preparation). Different strains of Microcystis may
differ in their abilities to enhance photosynthesis
in scums or to withstand scum conditions for
longer periods. Specific lake characteristics like
geographical latitude, morphometry and intensity
of wind stress determine the frequency and duration of scum formation, the type of scum (I, I1 or
111) and may influence adaptations in Microcystis
to the conditions encountered in a scum.
ACKNOWLEDGEMENTS
The authors would like to thank Dr. Ben de
Winder for originally suggesting the use of microelectrodes for assistance with the experimental
set-up and for discussions. Mr. Bert Groen is
acknowledged for skilfully constructing of the
electrodes. Dr. Hans C.P. Matthijs, Dr. Tineke
Burger-Wiersma and Dr. Lucas Stal are acknowledged for discussions. This work was supported
by a grant from the Dutch Ministry of Transport
and Public Affairs (DBW/RIZA).
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