FEMS Microbiology Ecology 74 (1990) 325-336
Published by Elsevier
325
FEMSEC 00303
Anoxygenic microbial mats of hot springs:
thermophilic C ~ ~ ~ r osp.
biu~
R.W. Castenholz ', J. Bauld
2,*
and B.B. Jmgenson
'
I Department of Biology, University of Oregon, Eugene, OR U.S.A.; Baas Becking Geobiological Laboratory
and CSIRO Diu. of Mineral Physics and Mineralogy, Canberra, Australia; and Insritute of Ecology and Genetics, University of Arhus,
Arhus, Denmark
Received 25 July 1990
Accepted 30 August 1990
Key words: Bacteria, green; Photosynthesis; Microelectrode
1. SUMMARY
Non-laminated, green to yellow-green microbial mats, with Chlorobium sp. as the only phototroph, occurred from 55 to about 40°C in hot
springs in and near Rotorua, New Zealand. The
pH ranged from 4.3 to 6.2 and sulfide from 0.2 to
1.8 mM. This Chlorobium sp. is unique in its
ability to form populations at temperatures as
high as 55 O C. Spectroradiometric measurements
with a fiber-optic microprobe in the intact Chlorobium mass showed great opacity with less than
0.1% of the incident radiation (at photosynthetically usable wavelengths) available at 0.7 mm depth within the mat, although the concentrated
Chlorobium population sometimes extended to 3
mm depth. Sulfide-dependent, anoxygenic photosynthesis was demonstrated by [14C]bicarbonate
Correspondence to: R.W.Castenholz, Department of Biology,
University of Oregon, Eugene, OR 97403. U.S.A.
* Present address: Division of Continental Geology, Bureau
of Mineral Resources, P.O. Box 378, Canberra, ACT 2601,
Australia.
assimilation in mat suspensions and in intact mats
by a sulfide-specific microelectrode. No oxygen
evolution occurred and no 0, was present within
the mat. A light-enhanced uptake of [I4C]acetate
also occurred in cell suspensions. This rate was
not enhanced by sulfide.
2. INTRODUCTION
Past studies of a large diversity of hot springs
in the pH range of 6.0-10.0 have established that
the predominant phototrophic mat-forming
bacteria in hot springs are cyanobacteria (up to
74" C) or communities of cyanobacteria and Chloroflexus (below 70 O C) or cyanobacteria and Chromatiaceae (below 57 C) [l-31. When free sulfide
levels exceed ca. 10 pM in waters above 55OC,
cyanobacteria that otherwise live at this temperature and above are excluded and Chlorofexus
(Chloroflexaceae) mats form up to higher temperatures ( < 66 O C) using a sulfide-dependent,
anoxygenic mode of photosynthesis [I,431. Below
about 55 O C, several species of cyanobacteria,
Chlorofexus aurantiacus, purple sulfur bacteria
0168-6496/90/$03.50 0 1990 Federation of European Microbiological Societies
326
(Chromatiaceae), and sometimes Heliothrix predominate in hot springs, depending on sulfide
concentration, pH, and other chemical factors
[2,6,71.
Recently, thermal outflows were discovered in
New Zealand that had microbial mats dominated
by the green sulfur bacterium Chlorobiurn sp. as
the sole phototroph in the temperature range of 40
to 5 5 O C [2]. These waters had pH levels from
4.3-6.2 (sometimes rising to ca. 7) and sulfide
(H,S, HS-, and S2-) concentrations above 50
pM. This rare combination of temperature, pH,
and sulfide occurs in a few natural and artificial
hot springs (Rotorua area of New Zealand), possibly not elsewhere. These springs and some of the
characteristics of the green bacterial mat are described, using methods which include sulfidespecific microelectrodes, micro-spectroradiometry,
and [’4C]bicarbonateand [I4C]acetatephotoincorporation.
3. MATERIALS AND METHODS
3.1. Preparation of mat and cells
Microelectrode and spectroradiometric measurements were made with undisturbed cores collected in 26 mm (i.d.) open-ended Plexiglas tubes
with beveled lower rim for easy penetration of the
mats and the silty clay sediment. The corers were
gently, but firmly, pushed through the mat and
sediment with a turning movement. Retrieval was
accomplished by inserting a rubber stopper in the
top to provide suction, allowing a core to be
brought up without loss. Cores from the ‘Travelodge Stream’ (TLS) mat were covered with
sulfide-rich spring water and topped with paraffin
oil to reduce oxidation and gas exchange with air.
A concentrated sulfide solution (pH 6.5) was added to the water periodically to maintain a concentration of about 250 pM, but little addition
was necessary since sulfide was also generated
from the mat below. The core was placed in a
thermostat-controlled water bath at 43-45 O C and
kept in semi-darkness for 3-6 h before use.
Cells to be used for photoincorporation experiments were collected from the hot spring by syringe
(without needle) and maintained anaerobically in
semi-darkness until use 1-2 h later. Since the mat
was a dense green ooze of essentially pure unicellular Chlorobiurn, the only care in collection was
to “blow away” the light coating of elemental S (if
present) with the syringe and then remove only
the remaining top 1 mm of green cells and not the
deeper material. Microscopic inspection showed
only Chlorobiurn-like cells. The collected material
was diluted with anoxic spring water to an O.D.
that corresponded to about 1-4 pg bacteriochlorophyll (BChl c ) ml-’. This was dispensed in
equal amounts (after thorough mixing) into 7 ml
capacity glass scintiIlation vials (Wheaton, Glass,
Millville, NJ). After standing for 30 min the suspensions were measurably micro-oxic; however,
0,was further reduced by the addition of fresh
sodium ascorbate. The subsequent addition of
other reagents to some of the vials was done
rapidly so that incubation at the appropriate ternperature could begin within 0.5-1.0 h. Reagent
grade chemicals were used in all cases and added
as solutions only after pH adjustment to about
6.5. DCMU (3-(3,4-dichlorophenyl)-l,l-dimethy~urea) was a gift from E.I. DuPont de Nemours
(Inc.). A specific absorption coefficient of 86 liter
g-’ cm-’ in methanol at 670 nm was used to
calculate the concentration of BChl c. Incubations
were done in the shallow hot springs. Cultured
Chlorobiurn sp. from TLS were used in some experiments. These were isolated by shake culture in
agar solidified medium after initial enrichment in
liquid medium of the same type (see ref. 8). CUItures used for incubations were grown at about
100 W mP2 under combined coolwhite fluorescent
and incandescent flood lamps. Incubations Were
in a water bath at 43-45”C.
Incubations were stopped in 50-75 min by a
final concentration of 4% formalin, and usually 1
ml was filtered on 0.45 pm pore size “Millipore
HA” filters, washed with 2% w/v HCI to remove
extracellular carbonates, and counted in a LKB
Wallac 1217 Rackbeta liquid scintillation counter
after dissolution of the filter in a cocktail composed of toluene, 4 g 1-’ PPO, and 0.1 g 1-1
POPOP. Counting efficiency was determined by a
channels-ratio method of counting a set of
quenched standards with automatic conversion of
sample cpm to dpm. In the laboratory experi-
321
ments with cultures in Oregon, cpm were not
converted.
3.2. Light
A halogen lamp with dual fiber optic light
guides and collimating lenses provided a rough
approximation of daylight irradiance on mat core
surfaces in the laboratory. An irradiance of up to
300 W m-* was used. This is about 0.3 of full
solar irradiance in summer. For spectroradiometric measurements, monochromatic illumination of
the mat surface was provided as described by
J~rgensenand Des Marais [7,9]. The white light
from the halogen lamp was passed through a set
of apertures and a continuous interference filter
ranging from 400 to 1025 nm with a half-bandwidth of 25-32 nm.
Gradients of spectral irradiance were measured
in the mats using a fiber optic microprobe [7,9].
The probe was constructed from a single optical
fiber of 80 pm diameter. The fiber was tapered
and rounded at the sensing tip which had a diameter of 25 pm and a light-collecting half angle of
20". The detector was an ultra low-noise, hybrid
photodiode-amplifier (EG & G, TCN-1000). The
probe was mounted on a micromanipulator and
measurements of spectral light gradients were
taken at 0.05-0.1 mm depth intervals.
The mats were prepared for light measurements
by transferring the uppermost 5 mm of a core to a
short tube with a plug at the bottom of 2% agar
made up in spring water. This core with about 1
cm of overlying spring water was placed on a
plastic plate over a hole through which the optical
fiber could penetrate up into the mat. In this way,
measurements of the downwelling irradiance were
made at O o light-fiber angle.
The spectral light gradients were calculated for
each wavelength relative to the irradiance of the
collimated light beam at the mat surface. Measurements of total artificial or natural irradiance
were made with a Lambda Li-Cor LI 185 instrument with radiometer probe.
3.3, Microelectrode measurements
Measurements of oxygen, sulfide and pH were
done simultaneously in the mats by the use of
microelectrodes (e.g. ref. 10). Oxygen microelec-
trodes were of the Clark-type with built-in reference and a sensing tip of about 5 pm diameter
[ll]. Sulfide microelectrodes were made from a
silver/ silver sulfide-coated platinum electrode
with a sensing tip of about 50 pm diameter [ l l ] .
pH microelectrodes were glass electrodes with a
sensing tip of about 40 pm [Ill.
Each electrode was attached to a separate micromanipulator, and the electrode tips penetrated
into the mat surface within 1 mm horizontal distance of each other. The position of the tips as
they touched the mat surface was determined under a dissecting scope mounted on a boom. Other
positions were then read on the micromanipulators.
The oxygen microelectrodes were calibrated
during the experiments by taking, (a) the reading
deep within a mat as the zero value, and (b) the
reading at the air-water interface or in the water
during bubbling with air as the air saturation
value. The linearity of the electrode response was
checked before experiments were started. The response of the 0, microelectrode was measured
with a Kiethly Model 485 picoammeter with a
separate voltage source negatively polarizing the
cathode at 0.75 V (see ref. 11). Recordings were
with a Cole-Parmer Model 8377-15 portable recorder.
The sulfide microelectrodes were calibrated
during the experiments by taking simultaneous
measurements in the stirred water above the mat
of sulfide electrode potential, pH, and total sulfide
concentration. Sulfide and pH values were read in
mV with digital display Extech Model 611 pH and
mV meters with calomel reference electrodes. In
order to apply the reference to a small (26 mm
diameter) container, the end was narrowed by a
series of telescoping Tygon tubes of different sizes
filled with electrolyte (1 M KCI) solidified with
agar. Thus, a tube of only a few mm diameter was
submerged in the test container with mat. For
each measurement, the sulfide potential was corrected to total sulfide for each pH value [12].
Samples and standards for total sulfide were taken
with a 1-ml syringe and injected into 2% Zn-acetate
for preservation. Sulfide concentration was determined after appropriate dilutions by the Methylene blue technique [ 131. The pH microelectrodes
328
were calibrated with standard buffers of pH 4, 7,
and 10.
4. DESCRIPTION OF SPRINGS WITH
I
1
LAKES
CHLO-
ROBIUM MATS
Several unnamed hot springs in the Rotorua
and Lake Rotoiti area possess the unusual characteristics that result in the predominance of Chlorobium sp. (Table 1). In these springs Chlorobium
formed a slimy, dull greenish to greenish-yellow
dense ooze over the sedimentary substrate from
about 55 to about 40°C. The thin mat was unlaminated and contained no other recognizable
phototrophs.
The characteristics and location of New Zealand springs in which Chlorobium-Like mats were
identified are shown in Table 1 and Fig. 1. Although others surely exist, those described here
probably represent the majority of such habitats
in New Zealand. Similar Chforobium-dominated
hot springs have not been reported elsewhere.
Most discharges in the Rotorua area are slightly
alkaline, chloride-rich waters. However, Chlorobium grows in a small number of associated acidsulfate discharges. These springs or wells possess a
combination of high sulfide and pH (about 4.36.2) at the surface in a range tolerated by thermophilic Chlorobium but not by other photosynthetic
prokaryotes [14.15 1.
“Travelodge Stream” (TLS), adjacent to the
Travelodge Hotel on Sulphur Bay, Rotorua was
r‘
Fig. 1. Map of ‘Chlorobium’ springs in Rotorua area. New
Zealand. e in the inset map indicates the area of enlargement
of the city of Rotorua. c is the rim of the Rotorua caldera. A.
Amohau St.; F. Fenton St.; G. “Government Vent Pool”; P,
Polynesian pools; SB, Sulphur Bay; SF, Sulphur Flats; SP,
Sulphur Point; T, Travelodge Hotel; Tt, Tudor Towers; 1.
“Sulphur Point-1” Spring; 2, “Travelodge” stream; 3, Sulphur
Flats Springs 1 and 2; 4, Parengarenga Springs; 5. Manupairua
Springs.
studied most extensively (Table 1, Fig. 1). The
source of this flow is the by-pass from the geothermal well of the hotel. Nearby wells contained the
following major constituents (in mg/l): Na’,
170-245; K + , 15-21; Ca2+/Mg2+. 21-27; C1-,
141-191; SO:-, 490-625; H3BO3, 7-8; S O 2 ,
155-195; HCO;, 63-93; and H,S, 52-58 (1.5-1.7
mM) [MI.
A few additional small springs with hospitable
Table 1
New Zealand hot springs possessing mats of Chlorobium in 1986
Name/location
Source temp.
Water over Chlorobium
(“C)
Temp. range
PH
Sulfide
conc. (mM)
(OC)
Travelodge Stream
Sulphur Point-1 Spring
Sulphur Bay Flats Spring 1
Spring 2
Whakarnvarewa “Cirque-2” Spring b
Parengarenga Spring “7”
Manupairua Spring
70
ca. 52-40
62
35
34
47
50
45
55-43
35-33
34-33
47-35
50-42
45-ca. 40
5.3-7.1
5.6-5.9
4.7
4.3
6.1
5.7
4.8
p H values at 45OC were between 5.3 and 6.2, but rose to 7.1 at one sampling time in March.
a
0.28-0.85
0.68-1.75
( H 2 Sodor)
0.60
(H2S odor)
0.35
0.38
329
chemical and thermal properties occurred along
the shore of Lake Rotorua to the north of TLS
(Fig. 1). A few springs on the southern shore of
western Lake Rotoiti (Fig. 1) also sustained a
Chlorobium mat (Table 1 and ref. 2). Manupairua,
on Rotoiti, is an acid-sulfate hillside spring with a
relatively low pH (4.8). The single retaining cistern
at 45-43°C was covered by an apparently pure
Chlorohium mat (Table 1, Fig. 1).
5. RESULTS
5.1. The Chlorobium mats
The Chlorobium mats in “Travelodge Stream”
(TLS) were sustained at temperatures of about
50 “ C (Fig. 2). In the natural ‘Sulphur Point-1’ (S.
Pte.-1) spring a constant 55-56 ” C was the upper
limit for the Chlorobium mat. The water velocity
over the main area of Chlorobium mat in TLS was
about 0.4-0.6 m s-’. The downstream limit for
the Chlorobium mat here and in the other springs
was not clearly defined, but in TLS and S . Pte.-1
Chlorobium was at least partly replaced by the
dark brown cyanobacterium Oscillatoria botyana,
below about 42-43°C at which point sulfide had
decreased and pH had increased.
The Chlorobium mat in TLS was about 0.5-3.0
mm thick, unlaminated, highly pigmented, compact ooze, and quite slimy (Figs. 3, 4). The top
0.1-0.2 mm was usually a yellowish-green color,
the yellow color contributed by elemental sulfur.
Below that the color was a deep green (Fig. 3).
The transition between the Chlorobium and compact clay-like substrate was a black layer 1- > 2
mm thick (Fig. 4).
Freshly collected Chlorobium from all of the
springs appeared similar when examined microscopically (Fig. 5a). The cells were rods, 0.5-0.7
pm in diameter and about 1.0-1.5 pm long, often
adhering end to end to form chains (Fig. 5a). The
individuality of the cells was apparent and no
trichomes of the Chloroflexus type were seen. Pure
cultures, obtained from three of the springs all
seem to be of the “Chlorobium” type morphologically (Fig. 5b).
‘In vivo’ absorption spectra of Chlorobium collected from TLS and S. Pte.-1 springs were very
Fig. 2. “Travelodge Stream’’ (TLS) (9 March. 1986). looking
upstream from a position with about 45 O C water. The clay-like
channel floor is completely covered with a green mat of Chlorohrurn (C) up to about 50-52OC. Black “streamers” with
metal sulfide precipitates abound along the edge (arrows) at
the surface water temperatures above 52OC (B). Liter bottle in
foreground indicates scale.
similar to the spectrum of a culture isolated from
TLS (Fig. 6)
5.2. Light penetration in the mat
For downwelling light of 400-1000 nm, there
was an extremely high attenuation rate, especially
between 0.1 and 0.2 mm (Fig. 7). T h s increased
attenuation at the surface may have been an
artifact due to the failure of the probe tip to
cleanly penetrate the surface and the frequent
adherence of the slimy mat to the tip. The results,
however, clearly show preferential absorption in
the blue/blue-green region (BChl c and carote-
330
Fig. 3. Close-up of TLS Chlorohium mat a t edge of stream (1 1 April. 1986). The lighter color is a milky ycllow-green :ind contains
much elemental sulfur, The darker areas (arrows) are a very deep green and are composed of Chhrohruni with the lighter 0.1 -0.2 mm
top layer removed by syringe. The 8-ml-capacity vials are ahout 1.7 cm in diameter.
Fig. 4. A core taken from TLS in September. 1986. The Chlorohrum mat resta on lop of a clay-like haae. The lighter surface cover
(yellow-green) is rich in elemental sulfur. The dark mat (dark green mixed with black) (arrow) composed o f (7ilorohruni and metal
sulfide precipitates.
noids) and a maximum in the 740-750 nrn region
(BChl c ) , essentially an inverse image of the in
vivo absorption spectrum of collected Chlorohium
mat (Fig. 6). In the spectral regions of maximum
absorption, less than 0.1% of the incident light
remained at 0.7 mm depth. This represents an
average absorption coefficient (log,, mm- ' ) of
10.95 at 740 nrn, or 0.002% transmittance per mm.
The total irradiance from 380 to 850 nm at 1
mm depth (<0.1 W r n 2 - ~ )was approximately
equivalent to the lowest intensity (5 lux) capable
of sustaining growth in Prosthecochloris strain
5071 [16]. Therefore, there is some possibility of
photosynthesis t o the 1 -1.2 mm depth. However,
the cells below this depth are also fully pigmented
and are probably viable.
5.3. Microelecmxle dutu
Sulfide, 02.and pH microelectrodes were inserted into the mat core simultaneously and within
a horizontal distance of 1 mm. There was n o
detectable O2 within the Chlorohium mat at any
time during measurements.
After 3 h of darkness. the range o f sulfide
concentrations was between about 150 and 200
pM from 1 rnm above the mat to 3 mm depth
331
.
Fig. 5. (a) Photomicrograph o f ChkJrohium (arrows) removed from TLS. Detritus includes diatom frustules, since sediment of stream
is limnic. Bar = 50 pm. (b) Photomicrograph of Chlorobium sp. (Strain NZ-TLS-2-C) isolated from TLS. Bar 100 pm.
0600
'
500
nk 600
'
700
*
800
-'
'
Fig. 6. Absorption spectra of TLS Chlorohium () prepared as "sonicate" in p H 7.8 "TSM" buffer (NaCI. 80 mM;
MgSO,, 5 mM; Tris. 20 mM); of a cell suspension of the
Chlorohium mat o f "Sulphur Point - 1" (S. he.-1) (- - - - - -);
and of a suspension of Chhrohium culture (NZ-TLSZ-C)
(- .-. -). The absorption spectra of Chlorohiurn sp. cultures
isolated from S. Pte.-I and "Government Vent Pool" were
similar. Absorption maximum in far red = 750 nm.
within the mat. The pH gradient was only from
6.7 to 6.6 (Fig. 8a).
The light was turned on about 15 min after the
sulfide depth profile was made in darkness. There
was a rapid loss of sulfide in the mat surface
(electrode tip at 0.2 mm depth in mat) over the
first 2 min (Fig. 8b), but this rate slowed considerably over the next 3 min, indicating a probable
limitation by low sulfide concentration on the rate
of sulfide oxidation. Although not obvious from
Fig. 8b, there was a substantially lower rate of
sulfide utilization during the first minute of illumination than during the second minute (data
not shown). The cause of this lag is unknown.
After 8 min of light there was a severe depletion
of sulfide throughout the upper 1.2 mm of mat
(Fig. 8c).
332
6.2
The depth of the depletion corresponds to the
probable maximum depth of photosynthesis with
an incident irradiance of 300 W m-2, although, at
best, the photosynthetic rate must be minimal at
1.0-1.2 mm (see SECTION 5.2 and Fig. 7). Photosynthesis had little effect on pH, at any depth,
presumably as a result of effective buffering.
The sulfide levels below 2 mm in the mat were
consistently above those of the water phase above
the mat, and the increase of sulfide in the surface
layer of mat when lights were turned off (Fig. 8b,
d) could be accounted for only by sulfide generaWavelength (nm)
6.6
.05
I
.I5
7.0
.2
p~
6.6
6.2
.25 rnM9.S
.05
7.0
.2
.25
4 0 9 5 0
!&
.l
.15
i
5
E
j:b\
1
0
05
10
15
20
25
:Ju 35
min
Fig. 8. Microelectrode data from TLS mat, 7 September, 1986.
(a) Profile of p H (open circles) and sulfide (closed circles) in
darkness at approximately 5 min before the time course in b.
(b) Time course of sulfide concentration at 0.1-0.2 mm below
the mat surface from darkness to light (L) (270 W m - 2 ) a t
time 0 to 20 min when the light was again turned off (D). (c)
Profile of p H (open circles) and sulfide (closed circles) after 10
min of light during the time course shown in b. (d) Second
time course of sulfide concentration at 0.1-0.2 mm below mat
surface begun with light-on after about 20 min of darkness..
The core used was collected approximately 4 h before use. and
maintained at 43-45 C in semi-darkness.
Fig. 7. The attenuation of downwelling light within TLS C h b
mat (7 September, 1986). from about 400 to 1000 nm.
and at depths in the mat of 0.0 (surface), 0.1, 0.2, 0.4, 0.7, and
1.0 mm.
robrum
tion within the mat and/or sediment (Fig. 8, and
data not shown). The biogenic source of the sulfide
is unknown, although Chlorobiurn itself could be
the producer [17].
When the lamp was shut off (after 20 min)
there was a fairly rapid increase in sulfide for 2
min, although the original concentration of 20 min
earlier was not attained (Fig. 8b). Another trial,
after 20 min in darkness, was run at a different
point in the same mat core with more frequent
measurements during the first minute (Fig. 8d).
The depletion of sulfide in the light was again
rapid, and the lag occurred primarily during the
first 0.25 min. For the remainder of the first
minute the rate of sulfide depletion was maximal.
Again, the rate slowed considerably after the
sulfide concentration had dropped to 30-40 F M
333
(Fig. 8d). When the lamp was shut off after 9 min,
the increase in sulfide was rapid and returned to
dark levels within 2.5 min. In both this and the
earlier trial (Fig. 8d, b) there was a dower rate of
sulfide increase in the first 0.25 min of darkness
than in the subsequent 0.5-0.75 min (data not
shown).
Table 3
Effects of sodium ascorbate and/or sodium sulfide or sodium
thiosulfate on ['4C]bicarbonate photoincorporation in Chlorobium sp. (Culture NZ-TLS-2-C (R.W.C.)) isolated from TLS,
Rotorua, N.Z.
Light (cpm)
Dark (cpm)
Control (no ascorbate, no S2- )
41
Ascorbate (9 mM)
68
Ascorbate (30 mM)
281
Ascorbate (90 mM)
1301
Ascorbate (9 mM)
+sulfide (0.7 mM)
5 184
Ascorbate (9 mM)
+ thiosulfate (4.3 mM)
4451
5.4. Relative photosynthetic rates measured by [I4C]
bicarbonate and ["C]acetate assimilation
Suspensions of feral Chlorobium were prepared
from collections of TLS mat. These cells were
treated as described in MATERIALS AND METHODS
and incubated in filled 7-ml vials under natural
light, using a shallow hot spring as the incubation
bath. Cultures isolated from TLS were also used
in some experiments.
When NaHI4CO, was used as the radio-label
the results show that sulfide in the range of 0.7-1.0
m M consistently stimulated photoassimilation of
CO, above that of the light controls (Tables 2, 3
and data from other experiments not shown). As
expected, photoincorporation was insensitive to 7
pM DCMU (Table 2). Sodium thiosulfate was
initially ineffective in enhancing photoincorporation (data not shown) but, after a 40 min lag
period, was almost as effective as sulfide (Table
35
18
34
32
47
35
Values are in cpm pg-' BChl c (-background) after 70 min
at 43°C and an irradiance of 400 W m-' (coolwhite fluorescent + incandescent (GE QF-500A, 500 W floodlight)); 0.143
pCi m1-I; BChl c: 1.02 pg ml-'; Initial O2 values were less
than 0.1 mg I - ' (as measured by O2 microelectrodes in replicate samples) except in controls without ascorbate or sulfide
(about 1 mg I-').
dent acetate assimilation. No attempt was made to
exclude CO, from the preparations. Sulfide additions stimulated the dark uptake rates slightly
but had little or no effect on the photoassimilation
of acetate (Table 2). In comparing the actual
uptake rates of C-1 labelled and C-2 labelled
acetate there was little difference (cf. 35 809 dpm
pg-' BChl c h-' for C-1 and 37515 dpm pg-'
BChl c h-' for C-2 light controls).
3).
The assimilation rates of acetate in the light
were similar irrespective of treatments (Table 2).
All treatments showed pronounced light-depen-
Table 2
Effects ofsulfide on photoincorporation of['4C]bicarbonatc (Expts. a and b), [l-'4C]acetate (Expt. c), and [2-14C)acetate (Expt. d) by
c h-I.
a Chlorobium population from Travelodge Spring, Rotorua. as percentage of light control dpm pg-' BChl
[I4C]bicarbonate
-
Control (no S2-)
Formalin control
DCMU (7 pm)
Sulfide (0.7 mM)
(1.1 mM)
Light
a 100
b 100
a 0.01
b 1.5
b 106
a 173
b 186
[1-I4C]acetate
[2-'4C]acetate
Dark
Light
Dark
Light
Dark
0.9
1.1
1.2
0.9
1.2
c 100
4.8
d 100
1.4
c
-
d 0.1
-
5.5
7.9
d 100
d 102
5.2
8.8
0.2
c 120
c 108
Incubations at 44-45°C for 50 min (Expt. b), 75 min (Expts. a, c, d); cells collected at 46°C; irradiance, 60-90 W m-*; sodium
ascorbate, 90 mM. ["CINaHCO,, 0.140 pCi m1-I; 11 or 2-14C]Na acetate, 3 pM. 0.106 pCi ml-'. BChl c: 3.25 pg ml-' (Expt. a),
3.28 pg ml-' (Expts. b, c. d). Light control counts (dpm pg-' BChl c h-'): Expt. a, 1094; b, 5164; c. 35809; d, 37515.
334
A high concentration of sodium ascorbate (90
mM) was used as an 0, scavenger in all incubations shown in Table 2. Since it was known from
previous experiments (data not shown) that
ascorbate promoted a light-enhanced bicarbonate
uptake even without sulfide, a series of experiments that tested the effect of various ascorbate
concentrations on bicarbonate uptake were carried
out. Table 3 is an experiment that demonstrates
the dependency of bicarbonate photoincorporation on the concentration of ascorbate. The range
was from less than a 2-fold enhancement at 9 mM
to over a 30-fold enhancement at 90 mM. Sulfide
or thiosulfate, however, each enhanced photoincorporation to a much greater extent than ascorbate alone (Table 3).
6. DISCUSSION
The existence of Chlorobiaceae at temperatures
up to 55 O C had not been reported prior to these
discoveries in New Zealand [2]. The combination
of relatively low pH (4.3-6.2), high primary sulfide
(0.2-1.7 mM) and temperatures of 40-55 O C, although rare, appears to be an environment that
enriches for Chlorobium. Chlorobium mats similar
in appearance to those of New Zealand have been
found in a tepid spring of saline 'oil field brine'
water in northern California (Wilbur Hot Springs).
The spring had a neutral pH, but with over 4 mM
sulfide (Castenholz, unpublished data). The temperature was merely 42OC. However, in 1990
Chlorobium from these springs was enriched for at
45 O C (Castenholz, unpublished results).
At non-thermal temperatures Chiorobium is a
common underlayer component of complex microbial mats, particularly in marine habitats (e.g.
ref. 18), but not as a semi-pure benthic top mat or
ooze-except in some sulfide-rich ponds in marine
marshes (Castenholz, unpublished observations).
The results of all experiments indicate that the
mats studied were those of typical Chiorobiurn, i.e.
with expected cell morphology, pigmentation, and
general physiology. The unique or previously unknown characteristic that this Chlorobiurn possesses is the ability to grow at temperatures above
40 C (up to 55 C). In addition, Chlorobium had
not been previously studied in essentially intact,
pure mat communities.
The responses to sulfide and light were as expected, The ['4C]bicarbonate uptake experiments
and those with microelectrodes showed that the
feral population of Chlorobium was photoautotrophic and dependent on sulfide. Laboratory experiments confirmed that thiosulfate could also be
used as an electron source after a short lag period
(Table 3). The surprising results are those that
show that the addition of sodium axorbate
accounted for the photo-enhanced incorporation
of bicarbonate even when no reduced sulfur compounds were added. The poising of the redox
potential to an appropriately negative level should
not in itself enhance photosynthesis when no photosynthetically usable electron donor is added.
Redox reagents such as thioglycollate or dithionite
were ineffective or inhibitory (data not shown).
The known photosynthetic electron donors for
Chlorobium are forms of reduced sulfur (e.g
sulfide, %O:-, polysulfides, and S2-) or H,.
Ascorbate is not known for this capacity. Ascorbate concentration used was high in one set of
experiments (Table 2). A separate experiment with
culture NZ-TLS-2-C showed that 90 mM sodium
ascorbate enhanced photoincorporation of bicarbonate 20-fold over the rates using 9 mM,
which also increased the light rate 2-fold over that
of the dark or light rate without any additions
(Table 3). It is unlikely that the more rapid or
efficient removal of 0, with increased ascorbate
concentration would account for these results.
Thus, an adequate explanation will await further
experiments with pure cultures.
Acetate can only be used as a source of carbon
together with CO, in all Chlorobiaceae examined
[19,20]. Its importance relative to CO, increases
with decreasing light intensities [21]. CO, was never
excluded in the present experiments. It has been
reported that the enzymes required for oxidizing
acetate are lacking in Chlorobium and therefore it
cannot be used as a source of reducing power or
as a source of CO, [22]. Thus, acetate is thought
to be assimilated whole and no difference should
be expected between the uptake rates of C-1 and
C-2 labelled acetate. Our results support this view.
Also, as may be the case with CO,, it seems
335
possible that in these experiments, ascorbate was
acting as electron donor in the assimilation of
acetate. Although the Chlorobium mats were not
analyzed for the presence of acetate it is apparently a common product of fermentative acetogenesis in other microbial mats of hot springs [23].
The studies of intact mat with microprobes
revealed the extreme optical opacity of the population, the probable depth of photosynthesis ( < 1.2
mm) and an approximation of the kinetics of
photosynthetic sulfide use (Figs. 7 and 8).
The kinetics of change in sulfide associated
with shifts from light to dark or vice versa might
be used as a fairly exact measure of anoxygenic
photosynthetic rate, much as 0, kinetics can be
used in oxygenic portions of microbial mats
[10,24]. However, the calculation of photosynthetic rates from these data require that several
assumptions be made. These calculations will not
be presented here. The light-dependent sulfide depletion data and dark repletion data (Fig. 8),
nevertheless, demonstrate that the natural density
of Chlorobium in the upper 1 mrn of mat is
sufficient to rapidly deplete a relatively high concentration of sulfide (0.2-0.3 mM). It is also apparent that the rapid sulfide repletion originated
from within the mat and not from the water
above.
In theory, the rate of sulfide increase immediately after the shift from a steady state light
value to darkness could give the rate of anoxygenic photosynthesis required to maintain the
sulfide at the low steady state value (i.e. normal
daytime rates). The total dynamics of H,S in the
mat could be calculated by combining the rates of
sulfide disappearance in the light, of reappearance
in the dark, and from the diffusion gradients in
the light steady state [25]. This would give a rough
estimate of the sulfide consumption and production rates in the mat.
Although such calculations would contain
several uncertainties, it is probable, from present
data, that Chlorohium surface populations under
high light rapidly became sulfide-concentration
limited but that high rates of photosynthesis may
continue, depending on the rate at which sulfide
becomes available. With the extremely high attenuation rate of photosynthetically useful radia-
tion within the mat, it would be interesting to
more accurately estimate the relative rates of photosynthesis by sulfide depeletion and repletion
rates at 0.1 mm depth increments in the photic
zone. In addition, a “time course” of sulfide depth
profiles after the dark/light shift should enhance
the accuracy of the estimates.
ACKNOWLEDGEMENTS
We thank the New Zealand Department of
Scientific and Industrial Research (Taupo Research Laboratory); the Forest Research Institute
(N.Z. Forest Service), Rotorua; the Precambrian
Paleobiology Research Group -Proterozoic
(PPRG-P) and the Center for the Study of Evolution and the Origin of Life (U.C.L.A.)for support
at various times during this research. R.W.C. also
thanks the U.S. National Science Foundation
(Grant BSR-8408179) for support and Dr. E. White
and Dr. W. Vincent for providing space, support,
and facilities during the stay in New Zealand. The
Baas Becking Geobiological Laboratory was supported by CSIRO, the Bureau of Mineral Resources, and the Australian Mineral Industries
Research Association, Ltd.
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