FEMS Microbiology
Ecology
Published by Elsevier
365
45 (1987) 365-376
FEC 00141
Pigments, light penetration, and photosynthetic activity
in the multi-layered microbial mats of Great Sippewissett
Salt Marsh, Massachusetts
Beverly Pierson a, Adair Oesterle
u Biology
Department,
L. Murphy
b
University of Puget Sound, Tacoma, WA, and ’ Department of Microbiology and Immunology,
Carolina, School of Medicine, Chapel Hill, NC, U.S.A.
Received
Accepted
Key words: Bacteriochlorophyll;
University of North
17 July 1987
22 July 1987
Anoxygenic photosynthesis; Green sulphur bacterium; Purple sulphur
bacterium: Cyanobacterium
chlorophyll
1. SUMMARY
The multi-layered microbial mats in the sand
flats of Great Sippewissett Salt Marsh were found
to have five distinct layers of phototrophic
organisms. The top 1-3 mm contained oxygenic
phototrophs. The lower 3-4 mm contained anoxygenie phototrophic bacteria. The uppermost gold
layer contained diatoms and cyanobacteria, and
chlorophyll a was the major chlorophyll. The next
layer down was green and was composed of primarily filamentous cyanobacteria containing chlorophyll a. This was followed by a bright pink layer
of bacteriochlorophyll
u-containing purple sulfur
bacteria. The next layer was a peach-colored layer
of bacteriochlorophyll
b-containing purple sulfur
bacteria. The lowest layer was a thin dull green
layer of green sulfur bacteria containing bacteriochlorophyll c. The distribution of the chlorophylls
with depth revealed that two-thirds of the total
Correspondence to: B. Pierson, Biology Department,
of Puget Sound, Tacoma, WA 98416, U.S.A.
0168-6496/87/$03.50
.a, George
0 1987 Federation
University
of European
Microbiological
in the mat
was composed
of
bacteriochlorophylls
present
in the anoxygenic
phototrophs.
The cyanobacterial
layers and both
purple
sulfur bacterial
layers had photoautotrophic activity. Light was attenuated
in the uppermost layers so that less than 5% of the total
radiation at the surface penetrated
to the layers of
anoxygenic phototrophs.
2. INTRODUCTION
Multi-layered
(laminated)
microbial mats composed primarily
of phototrophic
prokaryotes
develop in a variety of environments
exposed to
light. The development
of such mats occurs as
long as some aspect of the environment
suppresses
the activity of invertebrate
grazers that otherwise
would consume
the mat-forming
organisms
[l].
Some of the environmental
parameters that can be
sufficiently
extreme
to restrict
the activity
of
grazers while permitting the growth of prokaryotes
include
temperature,
pH, salinity and the concentration of sulfide. The laminated microbial mats
Societies
366
that have been most extensively studied include
those found in hot springs [2] and in hypersaline
and intertidal environments [3,4]. Such mats are
commonly composed of a surface layer of oxygenic cyanobacteria which may cover one or more
layers of anoxygenic phototrophic bacteria, although exceptions to this mat structure do exist
151. In the intertidal environment, a green surface
layer of filamentous and/or unicellular cyanobacteria is usually found covering a pink to red
layer of purple sulfur bacteria [6]. If adequate
organic matter is present in the substrate, biogenic
sulfide produced by sulfate-reducing bacteria diffuses up through the lower layers and may be used
to support anoxygenic photosynthesis [6]. Phototrophic bacteria may be found at considerable
depths in these mats and several different types
may be present. The presence of light and its
quality and intensity are likely to be very significant in determining the activity of these bacteria.
Recently published data by Jorgensen and Des
Marais [7] on the penetration of light in mats and
its relationship to the activity of purple sulfur
bacteria support this idea.
In Great Sippewissett Salt Marsh on the southern part of Cape Cod, MA, U.S.A., we found an
intertidal zone in which some sand flats were
covered by mats of considerable thickness and
complexity. These mats consisted of a surface
layer of cyanobacteria that frequently occurred in
two recognizable layers, an upper golden layer of
cyanobacteria mixed with diatoms and a lower
dark green layer of primarily cyanobacteria. Under the layer(s) of cyanobacteria there were three
distinct layers of anoxygenic phototrophic bacteria: a pink colored layer of purple sulfur bacteria
which covered an orange or peach-colored layer of
purple sulfur bacteria which in turn covered a dull
green layer of green sulfur bacteria. The individual
layers ranged in thickness from 1 to 2 mm and the
mat as a whole varied from 4 to 9 mm in thickness.
We were not aware of any other intertidal mats
having so many different layers of phototrophs.
Most mats that have been studied contain only
two phototrophic layers and the photosynthetic
activity is confined to only the top one or two
millimeters. Many phototrophic bacteria are also
capable of chemotrophic growth and the mere
presence of phototrophic organisms does not mean
that they are necessarily photosynthesizing [8].
Because of the unusually large number of layers in
this mat, its overall thickness, and its complexity
we wanted to determine how much light penetrated
into the mat, what pigment complexes were present in the mat layers that could absorb this light
and if the organisms in the mat layers were photosynthetically active.
This paper describes the results of our observations. An accompanying paper [9] describes the
results of an ultrastructural examination of the
mat community to determine the types and relative numbers of the different phototrophs present.
3. MATERIALS
AND METHODS
3.1. Light penetration in the mats
The penetration of total photosynthetically significant radiation (visible and near infrared)
through the mat layers was measured with an
LI-COR LI-185B radiometer equipped with an
LI-200SB pyranometer sensor. This sensor has
maximum sensitivity at 950 nm and ranges from
20% maximum at 450 nm to 40% maximum at
1050 nm. Cores 1.7 cm in diameter were taken
with a brass cork borer and placed over the sensor.
A tight-fitting plunger could be used to adjust the
position of the core within the borer. Total radiation penetrating to the bottom of the core was
measured. Slices 1 mm in thickness were then cut
sequentially from the bottom of the core, and the
total radiation penetrating through the remaining
core was measured. Since the core was in a brass
sleeve, only the radiation impinging directly upon
the surface of the core contributed to the radiation
measured within the core. Alternatively, penetration of radiation in the mat was measured by
taking a large core with a glass petri dish cover.
The core was then sandwiched between the petri
dish cover and the inverted bottom of the dish.
The sensor was placed in the hole in the mat left
by the core. The large core sandwiched in glass
was then re-inserted in the mat on top of the
sensor. Mat layers were then sequentially removed
by scraping from the bottom of the core to the
361
top, and total penetration of radiation was measured after the removal of each layer. In this way,
radiation coming to each mat layer from a much
larger angle was measured. No attempt was made
to measure radiation reflected back from lower
layers.
3.2. Pigmentation in the mat layers
The pigments in the colored layers were determined by slicing l-mm thick disks from the
1.7-cm diameter cores described in Section 3.1 and
extracting the pigments from each disc with absolute methanol. Alternatively, the pigment-protein complexes were prepared in cell-free extracts.
All absorbance spectra were recorded with a Cary
14 spectrophotometer.
3.2.1. Methanol extracts. Discs (1 mm in thickness) cut from cores (1.7 cm in diameter) contained 227 mm3 sand mat and were extracted in
3-5 ml of absolute methanol for 5 min in the
dark. Alternatively, specific colored layers of mat
were selected (approx. 200 mm3 sample volumes)
for extraction. Nearly all pigments were removed
by a one-step extraction with methanol. Subsequent extractions with fresh methanol were done
as needed until the remaining sand-mat debris was
colorless. Extracts were clarified by pressure filtration through 2.4 cm Whatman glass fiber (GF/C)
filters. The specific absorption coefficient of 75.0
lg-i. cm-’ in methanol at 665 nm [lo] was used
to determine the concentration of chorophyll a.
The molar extinction coefficient of 60 mM-’ .
cm-’ at 773 nm in methanol was used to determine the concentration of bacteriochlorophyll
a [ll]. The specific absorption coefficient of 86
lgg’ . cm-’ in methanol at 670 nm was used to
determine the concentration
of bacteriochlorophyll c [12]. The molar extinction coefficient of
bacteriochlorophyll
b in methanol at 790 nm was
estimated to be 66 mM_’ . cm-’ (10% greater
than that of bacteriochlorophyll a) since the coefficient in ether is thought to be approximately 10%
greater than that of bacteriochlorophyll a in ether
[ll]. No other values of molar extinction coefficients or specific absorption
coefficients for
bacteriochlorophyll
b were available.
3.2.2. Pigment/protein complexes in cell-free extracts. The in vivo absorption spectra were de-
termined on cell-free extracts of microorganisms
from the slices of cores or selected colored layers
obtained as in Section 3.2.1. In this case the sand
mat was mixed with Tris-sodium-magnesium
(TSM) buffer [13] instead of methanol and agitated
to dislodge all organisms from the sand. Cell
suspensions in buffer were then decanted from the
colorless sand grains. The cells were disrupted
ultrasonically for 2 min in 15-30 s intervals with
an Ultrasonic Processor (Heat Systems-Ultrasonics Inc. Model W220) using a standard tip.
Debris was removed by low-speed centrifugation
and the absorption spectra were recorded using
the clarified extracts.
3.3. Photosynthetic activity
Photosynthetic activity was determined by measuring the uptake of [‘4C]bicarbonate. Mat layers
were carefully divided on the basis of color. Cells
were removed from sand grains by gentle agitation
with filtered seawater. No attempt was made to
maintain anoxic conditions during these manipulations. Appropriate volumes were dispensed into
2-ml glass vials. All cells were incubated in a total
volume of 1.0 ml seawater. Cells from the cyanobacterial layer were incubated with 0.1 pCi/ml
NaH14C0, (New England Nuclear, specific activity 8.4 mCi/mmol). Cells from the purple sulfur
bacterial layers were incubated in 0.5 pCi/ml
NaHi4C0,, 0.1 mM Na, S. 9H,O, and 5. lop6
M 3-(3,4-dichlorophenyl)-l,l-dimethylurea
(DCMU). The DCMU was added to these samples to
inhibit oxygenic
photosynthetic
activity by
cyanobacteria present in these layers. All incubations reported here were done under 150 W incandescent reflector flood lamps. Light intensity
was regulated by distance from the lamp or by
using layers of cheesecloth as a neutral density
filter.
All incubations were at ambient temperatures
(20-25” C) and for 30 min except for the time
course experiments in the dark. At the end of
incubations, activity was stopped with the addition
of 0.1 ml of 37% formaldehyde. Samples were
stored in the dark at 4°C until processing. Samples were filtered through membrane filters with
0.45 pm pore size (Gelman). The cells on the filter
were washed with 3.0 ml 1 M NaHCO, followed
368
by 1.0 ml 2% HCl and 1.0 ml distilled water.
Filters were suspended in 10 ml Aquasol- (New
England Nuclear) and counted in a Beckman liquid
scintillation counter (LS 7800).
4. RESULTS
4.1. Description of the mat layers
The different colors of the mat layers were due
to the different pigments present in the microorganisms making up the layers. A detailed
description of the cellular composition of the layers
is given in Nicholson et al. [9]. Five different
colored layers were numbered sequentially beginning with the surface layer for purposes of discussion. The surface layer (layer 1) was a golden color
and consisted microscopically
of diatoms and
filamentous cyanobacteria. It was a fairly loosely
packed layer and was not always present. The next
layer down (layer 2) was green and consisted of
densely packed filamentous cyanobacteria (species
of Oscillatoria) and some unicellular organisms. It
was very cohesive. Layer 3 was pink and consisted
primarily of unicellular purple sulfur bacteria
(Thiocapsa roseopersicina and species of Thiocystis
and Chromatium)
in clusters with some filamentous cyanobacteria. Layer 4 was a peach color
and consisted primarily of unicellular purple sulfur
bacteria (Thiocapsa pfennigii), most of which were
in clusters. Layers 3 and 4 were dense and cohesive. The deepest layer in the mat (layer 5) was a
dull green color and consisted of primarily unicellular green sulfur bacteria (Prosthecochloris
aestuarii). It was a less cohesive layer. The boundaries
between the layers were very distinct. Many different cores were taken from this mat system for the
analyses reported below. Figs. 2, 4 and 5 illustrate
the correlation between the l-mm segments of
each core and the actual positions of the colored
layers within that core. In all figures in which data
are presented from l-mm segments of such cores,
the depth profile for each core under analysis is
presented on the left side of the figure using the
same symbols as in Fig. 2 to show the position of
the colored layers in that particular core. Each of
the colored layers, except for layer 5, ranged from
1 to 2 mm in thickness in all of the cores. Layer 5
was usually less than 1 mm thick.
4.2. Analysis of chlorophyll pigments in the mat
layers
The colored layers were carefully separated
from each other and the pigments were extracted
in methanol. The absorption spectra of all five
layers had intense and overlapping broad maxima
in the region from 400 to 500 nm (data not
shown). However, the spectra were distinctly different in the red and near infrared part of the
spectrum (Fig. 1). The absorption spectra for layers
1 and 2 were qualitatively identical in this region
with only two absorption maxima at 665 and 614
nm due to chlorophyll a. The spectrum from layer
3 had the same two maxima as well as a much
larger peak at 773 nm due to bacteriochlorophyll
a. The spectrum from layer 4 had two prominent
maxima, one at 790 nm due to bacteriochlorophyll
Absorption
Spectra
af
0.5.
0.5
04.
A
0.4
R
0.3
03.
614
0.z
mat Lagers
Layers 1 + 2
665
Layer 3
614
0.1
!u!!A
SOD
700
800
600
700
800
Lc!!L!
nm
0.4
773
665
0.2
0.1.
0.5
(methand)
nm
Layer 4
I
Layer 5
0.5
669
I
OR3
669
790
Oq3
0.2
0.2
0.1
0.1
790
600
7,0,0
800
0.4 lil,
600
700
nm
800
Fig. 1. The red and near infrared absorption spectra of
methanolic extracts of the different colored layers. Layers 1
and 2 had identical spectra and only one is shown. The layers
had been carefully separated from each other to avoid crosscontamination. Peaks at 614 and 665 nm are due to chlorophyll 0. The peak at 773 nm is due to bacteriochlorophyll a.
The peak at 790 nm is due to bacteriochlorophyll b. The peak
at 669 nm is due to bacteriochlorophyll c.
369
b and one at 669 nm due to bacteriochlorophyll
c.
The spectrum from layer 5 had a small peak at
790 nm and a prominent
peak at 669 nm due to
bacteriochlorophyll
c.
When the mat was sectioned in 1 mm increments from top to bottom and the distribution
of
the chlorophylls
was determined
quantitatively
throughout
the mat (Fig. 2 and Table l), the
maximum
concentration
of chlorophyll
u (0.159
pg. mmm3 sediment) was found at a depth of 2-3
mm in the green cyanobacterial
layer, although
significant
amounts
of chlorophyll
a were also
found in the next mm section in the pink layer.
Bacteriochlorophyll
a was found to have a more
narrow distribution
and the maximum concentration (0.242 I-18* mm-j
sediment)
occurred
at a
depth of 3-4 mm in the middle of the pink layer.
Significant amounts of bacteriochlorophyll
b were
found in the lower green layer but the maximum
concentration
(0.052
pg . mmm3
sediment)
occurred at a depth of 4-6 mm at the bottom of
the pink and in the peach-colored
layer. Significant amounts of bacteriochlorophyll
c occurred in
the peach layer, but the maximum concentration
(0.117 pg. mme3 sediment) was found at a depth
of 6-7 mm in the lower green layer (layer 5). The
only pigments detected in the gray sand below 7
mm were traces of bacteriochlorophyll
b and c.
Neither of these pigments was detected at depths
of less than 4 mm. Neither chlorophyll
a nor
bacteriochlorophyll
a were detected below 5 mm
in the mat, and although both were found in the
top mm of the mat, only very low levels of the
bacteriochlorophyll
a were detected in the top 2
mm.
To determine
the absorption
characteristics
of
each of the colored layers in vivo, cell-free extracts
were prepared
for each layer. The absorption
properties
in the region of 400-500 nm were intense and showed extensive
overlap in all the
layers (data not shown). The distinctive
absorption characteristics
of each layer were in the red
and near infrared parts of the spectrum.
Fig. 3
includes
the absorption
spectra of cell-free extracts of all the layers. Layers 1 and 2 were
identical with a single large maximum at 676 nm
due to chlorophyll
a and a smaller peak at 620 nm
due to phycocyanin.
Layer 3 had two prominent
Pigment Distribution with Depth
Extracts in Illethanol
core
’ i
A Chl a (665 nm)
0 BChl a (773 nm)
0 BChl b (790 nm)
0:04 0:08 0:12 0:16 0:20 0124 I 28
Pigment Concentration
(ug mrnm3sediment)
Fig. 2. Pigment distribution
with depth (extracts in methanol).
A 1.7 cm diameter core was sectioned in l-mm increments. The
sections were extracted in methanol and the concentrations
of
the various chlorophylls
(in pg.rnrn-’
sediment)
were determined for each section. The concentrations
of the pigments
were plotted as a function of depth in the mat. The five distinct
and differently
colored mat layers are designated
by different
symbols in the core diagram on the left side of the figure. The
horizontal dashes from 0 to 1.5 mm represent layer 1; the solid
lines from 1.5 to 3 mm represent layer 2: the widely spaced
dots from 3 to 4.5 mm represent layer 3; the closely spaced
dots from 4.5 to 6 mm represent layer 4; the diagonal dashes
from 6 to 7 mm represent layer 5. The same symbols used in
this diagram to represent the different colored layers are used
in all other core diagrams. The diagonal pattern from 7 to 8
mm represents
a zone of gray sediments,
and the diagonal
pattern from 8 to 9 mm represents the zone of black sediments.
Chl. chlorophyll;
Bchl, bacteriochlorophyll.
maxima
at
844
and
794
nm
due
to
bacteriochlorophyll
a. Layer 4 had a prominent
maximum at 1015 nm due to bacteriochlorophyll
b and a significant
peak at 748 nm due to
bacteriochlorophyll
c. The absorption
at 844 nm is
due mostly to bacteriochlorophyll
u and some
bacteriochlorophyll
b (which also absorbs near
840 nm). The bacteriochlorophyll
a also caused
370
I
Table
Chlorophyll
distribution
Chl, chlorophyll;
with depth
Bchl, bacteriochlorophyll.
Concentration
of each chlorophyll
(pg.mmm3
(fraction of total))
Depth
(mm)
Chl (I
Total
chlorophylls
Bchl a
Bchl b
Bchl c
(pg.mmm3)
0.005
0.009
0.087
0.242
0.059
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0.074
0.090
0.246
0.399
0.234
0.166
0.157
0.032
O-l
1-2
2-3
3-4
4-5
0.069
0.081
0.159
0.157
0.066
5-6
6-7
7-8
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0.052
0.052
0.040
0.008
Total pigment (pg)
(in column 1 X 1 X 8 mm deep)
0.532
0.402
0.152
(0.93)
(0.90)
(0.65)
(0.39)
(0.28)
the band at 794 nm. Layer 5 had only one absorption maximum
(748 nm) due to bacteriochlorophyll c.
A core of the mat was sectioned
in 1 mm
intervals and the depth distribution
of the in vivo
04
I
i!
i!
i !
-.
n 1
-,
600
700
800
Wavelength
900
(nm)
1000
(0.07)
(0.10)
(0.35)
(0.61)
(0.26)
(0.22)
(0.31)
(0.26)
(0.26)
0.057
0.114
0.117
0.024
0.312
(0.24)
(0.69)
(0.74)
(0.74)
1.398
absorption
maxima was determined
(Fig. 4). A
pattern
similar to that found in the methanol
extracts was observed. The maximum
ACT6(chlorophyll a) was found at a depth of l-2 mm in the
green layer (layer 2). Significant amounts of chlorophyll a were also found throughout the mat to a
depth of 5 mm. The maximum A,, (bacteriochlorophyll a) was found at a depth of 3-4 mm in
layer 3 (the pink layer). Significant amounts were
also found in the green cyanobacterial
layer although very little was found in the top 2 mm of
the mat. The maximum A,,,, (bacteriochlorophyll
b) was found at a depth of 4-5 mm in layer 4 (the
peach layer). None was found in the top 3 mm.
c) was
The maximum
A,,, (bacteriochlorophyll
found at a depth of 4-6 mm, in layers 4 and 5.
Traces were found in the 3rd mm of the mat, and
none was found above this.
_ .. .
II00
Fig. 3. Red and near infrared absorption
spectra of cell-free
extracts in buffer. Absorption
spectra of the cell-free extracts
in buffer were prepared
for each of the colored layers. The
layers had been carefully separated
from each other to avoid
cross-contamination.
The spectra
for layers 1 and 2 were
identical. The peak at 676 nm is due to chlorophyll
a. The
peaks at 844 nm and 794 nm are due to bacteriochlorophyll
n.
The peak at 748 nm is due to bacteriochlorophyll
c. The peak
at 1015 nm is due to bacteriochlorophyll
b. Bacteriochlorophyll
b also contributes
to absorption
at 844 nm but
probably not at 794 nm.
4.3. Light penetration in the mat
The penetration
of visible and near infrared
radiation through the various mat layers was measured on clear days during periods of high incident solar radiation. All measurements
were made
using natural sunlight. The total incident radiation
reaching
the surface of each colored layer was
measured by burying the sensor in the mat so that
it was covered only by the appropriate
layer(s).
With a total incident
radiation
(TIR) of 970
Wmd2, 66% of this (640 WmP2) penetrated
the
371
Pigment
_
Distribution
In Vivo
with Depth
Light Penetration and
Relative Rbsorbance of Illat Layers
core
core
Penetration
0
1
-1
A
0
0
8
Chl a
BChl a
BChl b
BChl c
(676nm)
(844 nm)
(1015 nm)
(748 nm)
II
Cl
0.2
0.4
0.6
0.8
Rbsorbance
1.0
1.2
Fig. 4. Pigment distribution
with depth (in viva). A 1.7 cm
diameter
core was sectioned
in l-mm increments.
The microbial cells were dislodged
from the sand grains and were
disrupted ultrasonically
in buffer. The absorption
spectra were
recorded
for each of the cleared extracts. The relative absorbances for each pigment at its far-red absorption
maximum
(see Fig. 3) were plotted as a function of depth in the mat.
top gold layer of the mat reaching the surface of
the green layer. The surface of the pink layer,
however, received only 10.4 WmP2 (1.1% TIR).
The surface of the peach layer received 0.18 Wmm2
(0.02% TIR). The surface of the lower green layer
received 0.10 Wm-* (0.01% TIR). The total radiation penetrating
through all of the colored layers
of the mat was 0.04 Wme2 (0.004% TIR).
The penetration
of radiation
through a core of
the mat was measured in l-mm increments
(Fig.
5). The incident radiation at each depth in Wmm2
is listed in the figure and the log of the incident
radiation
is plotted with depth. Again it can be
seen that while only about 20% of the TIR was
attenuated
in the top mm (the gold layer), 97% of
3
s
I
Legend
(log I)
2
Surface
lmm
2mm
3mm
4mm
5mm
6mm
7mm
8mm
9mm
Rbsorbance
= 1000 W/m*
= 850
= 28.0
= 23.0
=
2.8
=
2.4
q
1.4
q
1.0
=
0.65
q
0.45
log (IO / I)
Fig. 5. Penetration of total radiation through the mat and
relative absorbance of the mat in l-mm increments. Total
radiation (visible and near infrared) was measured with a
LI-COR pyranometer (Wmm2) through a 1.7 cm diameter core
held within a brass sleeve. 1 mm sections were sequentially cut
from the bottom of the core and total radiation penetrating the
remaining core was measured after each cut. The total incident
radiation at the surface of the mat was 1000 Wmm2.The log of
incident radiation (log I) at each depth is plotted and the bars
indicate the absorbance of each millimeter segment calculated
from the log of the ratio of the total incident radiation at the
surface of each segment to the total radiation transmitted
through each segment, log ( I,/1 ).
the TIR at the surface was attenuated
at a depth
of 2 mm. These particular
core measurements
were made in the presence of a very high total
surface radiation
of 1000 Wme2. Under
these
conditions,
0.65 Wme2 (0.065% TIR) penetrated
the entire mat.
The absorbance
(A) of each millimeter of mat
in the core was calculated
by determining
the
logarithm
of the ratio of the incident
radiation
(I,) at that depth in the core to the total radiation
312
reaching the next mm in the core (Z). The relative
of l-mm sections of the core in situ
absorbances
are presented
in Fig. 5. In this particular
core,
layer 1 was exactly 1 mm thick and layers 2, 3 and
4, were all nearly 2 mm thick. Since the core was
sectioned
in l-mm increments,
the sections fortuitously
coincided
nearly
perfectly
with the
boundaries
between the colored layers, and an
interesting
and predictable
pattern of absorbance
was revealed (Fig. 5). The first layer had a low
absorbance
factor, but in layers 2, 3 and 4, the top
millimeter
of each colored layer had a very high
absorbance
factor, while the 2nd mm of each
colored layer had a much lower absorbance
factor.
The absorbance
factors for the 1st millimeter segments of these three different colored layers decreased linearly, while the absorbance
factors for
the 2nd mm in each of these layers remained fairly
constant.
UPTAKE
vs LIGHT INTENSITY
LAYERS 1 + 2
( Cyanobacteria and Diatoms - Chl a)
2500 -,
OJ
IdO
I
I
I
I
500
I
I
I
I
f
1000
Wm -2
4.4. Photosynthetic activity in the mat layers
bicarbonate
as a
The uptake of 14C-labeled
function of light intensity was determined
in three
of the colored layers: layers 1 and 2 combined,
layer 3 and layer 4. Activity in layer 5 was not
determined
in this study. The chlorophyll
a-containing cyanobacterial
layers found in the top 2-3
mm of the mat, showed maximal uptake at 1000
Wm-‘, the highest light exposure measured at the
surface under natural conditions
and used in this
study (Fig. 6). At lower intensities,
a second peak
of activity was observed in the range of 400-500
Wm-*. In cells from layer 3 found between 3 and
5 mm deep in the mat, maximum
activity was
found between 300 and 400 Wm-*
(Fig. 7). A
second although somewhat lower peak of activity
was found at 800 WmP2. In cells from layer 4
found between 5 and 7 mm deep in the mat,
maximum activity was found at 50 Wme2 (Fig. 8).
A second although
lower peak of activity was
found at 200 Wrn-*.
All of the layers showed substantial
uptake of
14C-labeled bicarbonate
in the dark. The activity
in the dark was determined
as a function of time
of incubation
from 0 to 60 min. The uptake in
layers 1 and 2 was constant over this time interval.
The uptake in layer 3 increased slightly with time
to reach a maximum at 30 min and then remained
Fig. 6. Uptake of [‘4C]bicarbonate
as a function
of total
incident radiation (layers 1 and 2). A suspension of cells from
the cyanobacterial
layers was divided into equal aliquots. Each
sample was incubated with [‘4C]bicarbonate
at a final radioactive concentration
of 0.1 pCi/ml
for 30 min at eight different
light intensities obtained from a 150 W reflector flood lamp.
Samples contained
no added sulfide or DCMU. The dashed
line represents uptake in the dark. Chl, chlorophyll.
constant.
The uptake in layer 4 increased slowly
with time for the entire 60 min. The dark uptake
in layers 1 and 2 was 21% of the light uptake at 30
min under optimum
conditions
of 1000 WmP2.
The dark uptake in layer 3 was 26% of the light
uptake under optimum conditions
of 300 Wme2
for 30 min. The dark uptake in layer 4 after 30
min was 30% of the light uptake under optimum
conditions
of 50 WmP2.
5. DISCUSSION
The distribution
pattern of the layers of phototrophic organisms in the Sippewissett
Salt Marsh
mat was similar to that reported for other mats in
similar habitats [6,14] and to the plates of phototrophic
microorganisms
that form in aquatic
habitats
such as freshwater
lakes and marine
373
UPTAKE
vs LIGHT
LAYER
INTENSITY
3
( Purple sulfur bacteria
OJ
I
IdO
I 500
I
- Bchl a)
I
1
1
1 10008
Wm -*
Fig. 7. Uptake of [r4C]bicarbonate
as a function
of total
incident radiation
(layer 3). A suspension
of cells from the
purple sulfur bacterial layer (layer 3) was divided into equal
aliquots which were incubated with [‘4C]bicarbonate
at a final
radioactive
concentration
of 0.5 pCi/ml
for 30 mm at eight
different light intensities obtained from a 150 W reflector flood
lamp. Samples contained
Na,S,9H,O
(0.1 mM) and DCMU
(5.10m3 mM). The dashed line represents uptake in the dark.
Bchl, bacteriochlorophyll.
UPTAKE
vs LIGHT
LAYER
(Purple
6000
DPM
INTENSITY
4
- Bchl b)
sulfur bacteria
1
4000 -
160
260
360
Wm -*
Fig. 8. Uptake of [t4C]bicarbonate
as a function
of total
incident radiation
(layer 4). A suspension
of cells from the
purple sulfur bacterial layer (layer 4) was divided into equal
aliquots which were incubated with [t4C]bicarbonate
at a final
radioactive
concentration
of 0.5 pCi/ml
for 30 min at nine
different light intensities obtained from a 150 W reflector flood
lamp. Samples contained
Na,S.9H,O
(0.1 mM) and DCMU
(5.10m3 mM). The dashed line represents uptake in the dark.
Bchl, bacteriochlorophyll.
lagoons [15-171. The major difference between the
Sippewissett
mat and other previously
described
mat systems was the presence of the distinct layer
of purple sulfur bacteria containing
bacteriochlorophyll b in addition to the layer containing
bacteriochlorophyll
(I and a distinct layer of green
sulfur bacteria containing
bacteriochlorophyll
c.
Such layers of green sulfur bacteria occurring beneath layers of purple sulfur bacteria have been
frequently
described
from planktonic
systems
[15-171, but we are unaware of any other mats or
lacustrine systems that contain two distinct layers
of purple sulfur bacteria, one with bacteriochlorophyll a and one with bacteriochlorophyll
b, as
well as a layer of green sulfur bacteria. The presence of Thiocapsa pfennigii in mats has been observed in electron micrographs
of other ecosystems, but not as a distinct layer [18]. A similar
peach colored layer beneath
the bacteriochlorophyll u-containing
layer has been observed
in
some of the gelatinous
mats at Laguna Figueroa,
Baja, Mexico (Pierson, B., and Stolz, J., unpublished observations).
While the in vivo absorption
spectra of the
carefully isolated layers (Fig. 3) showed remarkable homogeneity
of the chlorophyll
pigments present, it is clear from the depth profiles (Fig. 2 and
4) that there was considerable
overlap in the distribution
of different
chlorophylls
across
the
boundaries
of the layers. Since action spectra of
photosynthetic
activity
have not yet been determined,
we do not know which pigments
are
responsible
for sustaining
most of the photosynthetic activity in each layer. Quantitative
analysis
of the total chlorophylls
present in each millimeter
of mat (expressed in micrograms
of chlorophyll
per cubic mm of wet sediment)
revealed some
interesting
characteristics
of the distribution
(Table 1). In the top 2 mm, 90-93s
of the total
chlorophyll
was chlorophyll
a, only 7-10s
being
bacteriochlorophyll
a. The chlorophyll
a decreased to 65% of the total in the 3rd mm and to
39% in the 4th mm. The 4th mm of the mat (found
in the dense pink layer) was distinctive
in having
the highest total chlorophyll
content of any depth
(an impressive 0.399 pg. mmm3). The majority of
the pigment at this depth (61%) was bacteriochlorophyll a. The 5th mm of the mat (also in the
314
dense pink layer) was of interest, since it contained nearly identical amounts of all four chlorophylls for a total chlorophyll
content
of 0.234
pg. mme3 of sediment. Below the 5th mm in the
mat only bacteriochlorophylls
b and c were present. Bacteriochlorophyll
c was present in higher
amounts (approximately
70% of the total chlorophyll) than bacteriochlorophyll
b at all depths.
When the total amount of each chlorophyll
was
determined
for the entire core (Table l), chlorophyll a was the most abundant
chlorophyll
with
0.532 pg in the upper 5 mm of a one square
millimeter
column
followed
by bacteriochlorophyll
a with 0.402 pg in the same 5 mm.
Bacteriochlorophyll
c was third with a total of
0.312 pg in the lower 4 mm of the same column,
and bacteriochlorophyll
b was present in the least
amount with 0.152 pg in the lower 4 mm of the
same column.
For comparison
with other ecosystems the total
pigment content of the g-mm deep mat can be
expressed
in mg * m-2. The total pigment
was
1398 mg . mm2 with 532 mg . m 2 due to chlorophyll a, 402 mg 1m-2 due to bacteriochlorophyll
a, 152 mg . mP2 due to bacteriochlorophyll
b and
312 mg . me2 due to bacteriochlorophyll
c. This
value (1398 mg * rne2) is within the range of total
chlorophyll content of highly productive terrestrial
systems and is much higher than that reported for
most other aquatic
and mat systems [3,14,19].
However, if one considers the chlorophyll
a only
(523 mg . mP2) then this mat is comparable
in
pigment
content
to other
highly
productive
cyanobacterial
mats [3,19]. It is the presence of the
bacteriochlorophylls
a, b, and c in the anoxygenic
phototrophs
comprising
the lower layers of the
mat that drives the total chlorophyll
content to
such high levels. These bacteriochlorophylls
together account for almost 2/3 of the total chlorophyll in the mat. These chlorophylls
and the
potential
contribution
of the anoxygenic
phototrophs to total productivity
in mats have only
occasionally
been considered [3,7,14,19].
Although actual productivity
was not measured
in this study, it appears likely that the bacteriochlorophyll-containing
anoxygenic
phototrophs
make a significant
contribution
to total mat productivity
in this particular
mat system. We are
currently
determining
their role in primary productivity.
The data on light penetration
(Fig. 5) also
support this hypothesis, since it was evident from
these data that only certain wavelengths
of light
were attenuated
by each colored layer. While examination
of the in vivo absorption
spectra (Fig. 3
make this statement
seem obvious, it is not clear
from the absorption
spectra alone (Fig. 3) that all
of the radiation at the wavelengths absorbed by a
particular
layer of phototrophs
is actually completely attenuated
by that layer. For example, one
might imagine that the cyanobacteria
in the top 3
mm of the mat might not absorb all of the radiation in the range absorbed
by phycobilins
and
chlorophyll
a, leaving significant amounts of radiation at these wavelengths
available
to be absorbed by cyanobacteria
present as a minority
population
in the 4th mm of the mat. Recent data
obtained by Jorgensen and Des Marais [7] on the
penetration
of radiation in hypersaline mats argue
against this possibility.
Examination
of the total
radiation absorbance
factors (A) for each millimeter of mat plotted in Fig. 5 also argues against this
possibility. While the concentration
of chlorophyll
a throughout
the green layer is fairly constant
(Fig. 2) the attenuation
absorptivity
of the layer is
not (Fig. 5). It appears that most of the radiation
that can be absorbed by the cyanobacterial
pigments is absorbed
in the 1st mm of the 2-mm
thick layer, leaving very little for absorption in the
2nd mm of this layer. This suggests that cells in
the lower part of any given layer receive far fewer
quanta at the wavelengths
they can absorb than
cells in the upper portion
of that layer. Since
wavelengths
not absorbed by the pigments in the
green layer pass right through this layer, the cells
in the pink layer below having pigments that can
absorb these transmitted
wavelengths contribute
a
high absorbance
factor in the top millimeter of the
pink layer.
In any given layer, then, it is clear that the
population
of cells in the upper portion of that
layer receive much more light than the cells in the
lower portion. It is possible that this might explain
the two different light intensity maxima observed
in the light-dependent
uptake of [‘4C]bicarbonate
(Figs. 6-8). The cell preparations
for each of the
375
uptake experiments
included cells from both the
upper and lower portions of each colored layer.
The cells in the upper portion of each layer may
be adapted to higher incident radiation than those
in the lower part of each layer. It will be necessary
to separate each colored layer into at least two
sub-layers
and to determine
the light intensity
optimum for each sub-layer to verify this suggestion.
While a light-stimulated
uptake of NAHCO,
was observed in all layers studied, the dark uptake
was also high, suggesting the presence of significant chemoautotrophic
activity in these mat layers.
Chemolithoautotrophic
growth in isolates of Chromatium gracile and Thiocapsa roseopersicina can
be sustained by hydrogen and reduced sulfur compounds [20]. Since no attempt was made to exclude oxygen during the preparation
and incubation of cells in the uptake studies and since 0.1
mM sulfide was included in the incubations,
conditions were satisfactory
to sustain chemolithoautotrophic activity. We do not yet know how significant chemolithotrophy
is to the overall activity
of this mat.
Comparison
of the uptake in each layer as a
function of light intensity (Figs. 6-8) revealed that
the maximum
uptake in the deeper layers in the
mat (layers 3 and 4) occurred at lower light intensities than in the upper layers, suggesting
that
indeed populations
of cells located deeper in the
mat are adapted to lower levels of total incident
radiation.
White incandescent
light was used for
all the uptake experiments
reported here. Since
cells in all but the topmost layer are receiving light
that is highly filtered by the colored layers above,
it would be most desirable to determine the action
spectra for photosynthetic
activity in each of these
layers. Indeed, preliminary
experiments
on light
penetration
using interference
filters and spectroradiometry
(Pierson, B., unpublished
results) have
shown that only near infra-red
radiation
reaches
the deeper layers of the mat, and one would
expect phototrophs
here to be using exclusively
these wavelengths
to sustain photosynthesis.
We
are currently attempting
to determine the spectral
distribution
as well as intensity
of all radiation
reaching
lower layers and to determine
which
wavelengths
are being used to sustain
photo-
synthesis in each layer.
The Sippewissett Salt Marsh and sediments as a
whole have been found to be highly productive
ecosystems
[21]. Our study has shown that the
multi-layered
phototrophic
microbial
sand mat
communities
within the salt marsh are extremely
high in chlorophyll
content, suggesting that they
too are highly productive ecosystems. We are currently attempting
to determine the productivity
in
situ of each of these mat layers.
ACKNOWLEDGEMENTS
We thank the Marine Biological Laboratory
at
Woods Hole, MA, for providing space and facilities to George Murphy and Adair Oesterle (students) and Beverly Pierson (instructor)
at the MBL
during the summer of 1984 when this research was
conducted
as part of the course, Microbiology:
Molecular Aspects of Cellular Diversity. We also
thank the Division of Biological Energy Research
(DOE) and the Foundation
for Microbiology
for
support of this work.
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