FEMS MicrobiologyEcology 73 (1990) 193-202
Published by Elsevier
193
FEMSEC 00250
Microbial sulfate reduction in acidic (pH 3) strip-mine lakes
R u t h A. G y u r e t A l l a n K o n o p k a t, Austin Brooks z a n d William D o e m e l 2
Department of Biological Sctences, Purdue University, 14,est Lalayetre,IN, U.S.A., and z Biology Department,
Wabash College Crawfordsmlle, IN, U.S.A.
Received 21 August 1989
Revision receivedand accepted ll October 1989
Key words: Acid mine drainage; p H optimum; Sediment
1. S U M M A R Y
~SSO4 reduction was detected in slurries of sediments obtained from Reservoir 29 (pH 3.8) and
Lake B (pH 6.2), two acid strip-mine lakes in
Indiana. The rates varied seasonally and were
higher in summer and fall than in the spring. The
optimal p H for sulfate reduction in Reservoir 29
sediments was 5, but samples had increased activity at p H 7 within 24 h after adjusting the p H to
this value. In Lake B, the optimal p H for sulfate
reduction was the in situ pH (6.2). Sulfate reduction in both lakes was stimulated 2-3-fold by
increasing Pn~- High concentrations (5 raM) of
organic acids inhibited sulfate reduction at p H
3.8, but stimulation was observed at concentrations of 0.1 raM. Acid-volatile sulfides accounted
for about 70% of the products of 35SO4 reduction.
2. I N T R O D U C T I O N
Acidification can have substantial effects on
the biological productivity of ecosystems. Acidic
environments occur naturally in peatlands [1] or in
Correspondence to: A. Konopka, Department of Biological
Sciences, Purdu~ University,West Lafayette, IN 47907, U.S.A.
areas where sulfides are exposed to oxygen, as in
geothermal areas [2]. H u m a n activities also may
acidify ecosystems. Acid precipitation has affected
aquatic ecosystems in several broad geographic
regions. In some of these, p H decreases to 4.5
have been observed [3]. More severe effects are
observed near surface or subsurface coal mines.
Acid mine drainage may reduce the p H of aquatic
systems below 3 [4] due to large inputs of sulfuric
acid.
Aquatic ecosystems that have been severely
acidified due to inputs of mine drainage have been
shown to have reduced species diversity of photo~
trophic and heterotrophic microorganisms [51. A
variety of microbial activities take place under
highly acidic conditions [41. If acidic inputs to the
ecosystems are stopped, the lakes return to a
neutral p H over a period of time [6]. This recovery
is due, in part, to biological processes. Thus, biological processes which consume hydrogen ions
are of interest in these environments because they
buffer the systems against p H decreases, and may
be useful in increasing the p H of acid-impacted
systems. Anaerobic bacterial processes such as
denitrification and sulfate reduction can fulfill this
function [7,8]. In sulfate-rich habitats sulfate reduction is the predominant terminal reaction ~n
anaerobic mineralization [9,10]. Sulfate reduction
has been shown to neutralize acidity in experimen-
0168-6496/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
194
tally acidified lakes [11,12], atmospherically
acidified lakes [3], impoundments receiving acid
mine drainage [13,14], wetlands receiving acid mine
drainage [15], and in peat [16].
Our interest is in the activity of sulfate-reducing bacteria at pH < 4 found in some coal stripmine lakes [4]. This contrasts with the studies
cited above, where sulfate reduction was measured
in sediments whose pH was greater than 5.5 in
most cases. Some investigators have increased the
pH of highly acidic mine drainage by stimulating
the activity of sulfate-reducing bacteria [17-20].
However, they were unable to isolate cultures of
acidophilic sulfate-reducing bacteria and postulated that the organisms were active in microzones of higher pH. In this study we have examined
).he pH optimura for sulfate reduction in a lake
sediment with a bulk pH < 4.
Primary productio, was found to be low in the
acid mine lakes examined in this study [4]. Therefore, the effect of electron donor concentration
upon sulfate reduction was of interest. Many compounds stimulate sulfate-reduction rates in pHneutral sediments [21,22]. However, at low pH
some of these substrates (organic weak acids) are
toxic to microorganisms [23-25]. la this study we
determined how the addition of electron donors
and variation in other rdevant environmental factors affected sulfate reduction in these acidic environments.
3. METHODS
3.1. Sampling site description
These studies were conducted at the GreeneSullivan State Forest (39°00'N, 87°15'W) near
Dugger, IN. This area had been actively stripmined for coal prior to 1967, resulting in extensive
acid contamination of surface waters. The pH of
Reservoir 29 was 2.7 due to acid leaching from
several abandoned coal refuse piles at the northern end. This 225-ha lake has an average depth of
5 m and maximum depth of 7-8 m. The source of
water for Lake B is seepage from Reservoir 29
through a connecting dam. Lake B has a surface
area of 20 ha and a highly irregular bottom that
includes areas of 8-9 m deep where the bottom
remained anaerobic throughout the study period.
Although the pH of the epilimnion and metalimnion of Lake B was 3.2, the hypolimnion and
sediments in the deep areas had pH > 6 [4].
3.2. Sampling procedure and fieM measurements
Sampling stations were located over the deepest
portions of the lakes. Water samples were collected from discrete depths by lowering Tygon
tubing, and drawing water with a peristaltic pump
(Cole-Parmer Instrument, Chicago, IL.). Reservoir
29 contained a 5-cm thick layer of black sediment
(13~ organic carbon by weight) overlying a dense
clay. Thus, it was difficult to collect sediment
cores, and therefore an Eckman dredge was used
to collect profundal surface sediments. These were
stored in 100-ml polypropylene jars filled to the
top to exclude air. All water and sediment samples
were stored on ice in the dark during transport
and were analysed within 8 h of collection.
3.3. Sulfate.reduction measurement~
Surface sediments were gently homogenized
with a stainless steel paddlp at 30 rpm in a sealed
polypropylene vessel that was kept on ice and
continuously flushed with N 2 gas. For anaerobic
experiments, all gases were passed through a
heated (340°C) copper colunm to remove traces
of 02. Sediment samples (5 cm~) were removed
through a port in the bottom of the po|ypropylen¢
vessel via a syringe aud dispensed into N2-flushed
30-ml serum vials by using the Hungate technique
[26]. During all manipulations prior to incubation
sediment was kept on ice. Vials were closed with
butyl rubber septa (Supelco, Belle[onte, PA) and
crimped with aluminum seals. Samples were
warmed to incubation temperature before injection of 5/ICi carrier-free H~SSO4, (43 Ci/mg S;
ICN Radiochemicals, irvine, CA) in 0.1 rnl deoxygenated distilled water.
Unless otherwise specified, samples were incubated for 6 h at 37 ° C, because rates of sulfate
reduction were highest at this temperature. Incubation was terminated by adding 0.5 mi of 10%
Zn.acetate. The samples were then frozen until
they were processed. Control samples were killed
in this manner immediately after addition of isotope.
195
For measurement of seasonal changes in s -'Irate
reduction, 5-cm~ sediment samples were incubated
at the in situ temperature, For experiments in
which amendments were made, slurries were used
to allow better mixing. To form slurries, 5-cm3
sediment samples were mixed with 5-ml deoxygenated distilled water. For pH adjustment,
deoxygenated 1 N NaOH or 2 N H2SO~ was
added to slurries in amounts determined by titration of sediment samples. During pH adjustment,
the slurries were gassed with N2. During the 6-h
incubation period with ~sSO4, pH never varied by
more than 0.2 units. Na-resazurin (10/xl of 0.2%
solution per lO-ml sample) was used as a redox
indicator to m o n i t o r reducing conditions
throughout the experiment.
The frozen samples from 35SO4 incorporation
experiments were processed within 1 week. These
samples were thawed 30 min at room temperature
and then attached to a stripping train [27]. While
continuously flushing vials with N z, liberated HzS
was trapped in a series of two scintillation vials,
each containing 5 ml of 10% Zn-acetate and 1
drop antifoam agent B. Over a period of 2 h, 8 ml
deoxygenated I N HCI was added gradually to the
sediment samples in 1-ml increments, This procedure liberates FeS and H2S ('acid-volatile sulfur"
or AVS). After stripping, 12 ml ACS liquid scintillation fluid (Amersham, Arlington Heights, IL)
was added to each trap for counting by liquid
scintillation. Quench correction was made by the
¢hannds ratio method.
The amount of nonradioactive sulfate in the
samples was determined by precipitation with
BaC! z [28] to permit calculation of AVS production from the data on t~e fraction of 3SSO4 that
was reduced. Sulfate was removed in a series of
aqueous washes. Samples were centrifuged at 8600
rpm for 10 rnin, and 0.1 ml supematant was
counted. The sediment pellet was resuspended in
30 ml distilled water and gently shaken at room
temperature for 10 rain. The centrifugation step
was then repeated, and supernatant was counted.
Washes were repeated and pooled until counts in
the 0.l-ml afiquot dropped below 100 cpm. Sulfate
concentrations in sediments ranged from 30-100
mM.
Sulfate reduction was measured in tr/plicate.
Values are reported as sample means + standard
deviation, T e determine treatment significance,
pair-wise comparisons between a treatment mean
and the appropriate eontrol value were performed
usin3 a Student's t-test.
In most experiments, only 3~SO4 reduction to
AVS was measured. However, a few measurements of ~SSO4 incorporation into other forms of
reduced, inorganic sulfur were made. Samples that
had been stripped of AVS were boiled with reduced CrC! 2 for 2 h in a distilling apparatus
[29,30], and evolved H2S was trapped with Znacetate as described above. Prior to this analysis,
35SO4 was removed by washing sediment with
distilled water. Sediment was recovered by eentrifugation at 8000 × g for 10 rain, and washing was
repeated until the supernatant contained < 100
cpm ml- ~.
3.4. Effect of electron donors on sulfate reduction
Sediment slurries from Reservoir 29 or Lake B
were pre-incubated for 12 h at 37°C. Samples
were then amended with deoxygenated stock solutions of one of the following substrates at 0.1, 1,
or 5 mM concentrations: acetate, alanine, casamino acids, ethanol, formate, fumarate, glutamate,
lactate, malate, propanol, propionate, pyrophosphate, pyruvate, or succinate. 5 ~Ci 3aSO4 were
added, and incubation was continued for 6 h at
37* C. The reactions were terminated by adding
zinc acetate and freezing the samples.
Hydrogen gas was tested as an electron donor
by injecting 10 ml H 2 into sealed vials containing
10 ml sediment slurry and 20 ml headspace.
4. RESULTS
Biological reduction of sulfate to sulfide occurred in sediments of Reservoir 29 (pH < 4) and
Lake B (pH 6.2). Activity was higher at 37°C
than at lower temperatures or at 55 *C (Table 1).
Although H2S concentrations as high as 1 mM
were measured in the hypoliranion of Reservoir 29
[4], no sulfate reduction was detected in hypolimnetic samples. The sulfate-reducing activity in
Reservoir 29 was restricted to the sediments.
196
Table 1
Sulfate reduction measured in Reservoir 29 and Lake B sediment slurdes incubated at four temporatures. Samples were
incubated with 5/~Ci 3sSO4 for 6 h
Temperature
{°C)
10
25
37
55
AVS production a
(nmol H~Scm -3 h -t)
Reservoir 29
Lake B
2.8±0.2
~5±0.9
19,3±2.4
0.2±0.1
0.7±0.2
3.1±0.3
33±1,3
0.8±0.2
= Values are means of three replicate samples± standard deviation.
The high concentrations ( > 1 m M ) of free H2S
that occurred in Reservoir 29 sediments at p H < 4
did not inhibit sulfate reduction. N o decrease in
sulfate reducing activity was observed in Reservoir
29 samples after a d d i t i o n of H2S up to 4 raM.
AVS (acid-volatile sulfide) production at in situ
temperatures ranged from 1 2 - 1 5 2 n m o l H2S cm -~
day -1 in Reservoir 29 a n d Lake B sediments
(Table 2). I n Reservoir 29, the rates were lower in
spring than in s u m m e r or fall, T h e rates also
varied d u r i n g the season; for example, rates were
lower in August, 1985, than on preceding or succeeding dates.
A d d i t i o n a l experiments on the effects of env i r o n m e n t a l factors u p o n sulfate reduction were
incubated at 37 ° C to optimize activity. F u r t h e r more, these experiments were d o n e in sediment
diluted 1 : 1 with water to facilitate mixing of
chemical a m e n d m e n t s .
W h e n Reservoir 29 sediment slurries were adjusted to a series of p H values (from in situ p H of
3.8), AVS production, measured d u r i n g the next 6
h, was highest at p H 5 (Fig. 1), Lake B slurries,
adjusted to these same values, had m a x i m u m activity at the in situ p H of 6.2. However, when
sulfide production of Reservoir 29 samples was
monitored for a longer period, increases in activity
were observed within 24 h at p H 5 a n d 7 (Fig. 2).
If samples were pre-incubated at the adjusted p H
for 12 h before the addition of ~SSO4, sulfate
reduction was greatest at p H 7 in sediments from
both lakes (Table 3).
A n u m b e r of electron donors were tested as
stimulators of sulfate reduction. Of these, hydro-
Table 2
AVS production measured in sediment samples from Reservoir
29 and Lake B during 1984-85, Samples were incubated 6 h
with ~/xCi ~SSO4at in situ temperatures. In both lakes, in situ
sediment temperature was I0-12°C throughout the sampling
sc/~son
Date
AVS produced ~ (nmol cm- 3day- 1)
30 VIII 84
2 X 84
23 Ill 85
Reservoir 29
Lake B
16
152
20
n.d. t,
12
9 IV 85
34
n.d.
n.d.
23 IV 85
26
|8
4 V 85
84
84
29 V 85
8 VII 85
12 VII 85
19 VII 85
30 ell 85
19 VIII 85
27 VIII 85
13 X 85
28 X 85
36
n.d.
44
n.d.
120
112
132
140
40
80
108
112
n.d.
n,d.
n.d.
100
136
n.d.
VMues are the means of three replicate subsamplcs.
h n.d. = not determined,
gen gas w a s m o s t effective. Increases in H z p a r t i a l
pressures e n h a n c e d sulfate-reducing activity in
sediments of both lakes at all times d u r i n g the
sampling season. Rates were as m u c h as 3-fold
higher after H 2 addition (Table 4). H 2 also
stimulated activity in Reservoir 29 sediments after
the p H was adjusted to 7.
f
I - - t ~aservoir 2 9
E~'.ake 8
I
2
2
•5
6
7
g
~H
Fig. t. Acid-volatile sulfide production measured over 6 h
immediately after pH adjustment. The pH of 10.ml sediment
slurries was adjusted with NaOH or H2SO4, 5/~Ci ~SSO4was
added, and the samples wcrc incubated at 37 ° C. Open bars,
Reservoir 29; hatched bars, Lake lB.
197
200
Table 5
/
pHi//0
160
Effect of 5.0 mM organic acid additions on sulfate re.ducdon
rales in Re~rvoir 29 and Lake B sediment slurries at in sitar
and adjusted pH values. Samples were incubated 6 h at 37 ° C
with 5 ixCi ~>SO,t
///
o
E 12~
nH3.8
0
~o---76~- -V~o
Treatment
AVS production,
( n m o l H 2 S c m -~ h z)
In situ p H
Adjusted pH
Resetwoir 20 b
No additions
5 mM acetate
5 mM citrate
5 mM lat;tatc
20.84-1,7
l.l ± 0.2
0.4:t 0.1
0.3.t:0.1
5.:] + 1,2
7.2 4- 0.6
22.3 :t 6,3
14.3+O.5
Table 3
Lake B c
No additions
5 m i acetate
5 mM citrate
5 mM lactate
3,3 _+0.6
5.1 + 1.0
3.74-t.0
1.84-0.9
0.5 + 0.1
t'Ll ±O.1
0.t +0.1
0.2::1:0.1
Sulfate reduction in Reservoir 29 and Lake B sediment slurries
measured after 12-h pre-inenbation at in situ or adjusted pH.
Samples were intmbated with 5/tCi 3SSO,= for 6 h at 37°C. In
situ p H values were 3.8 in Reservoir 29 and 6.2 in Lake B.
a Values are means of three replicate sample.s+_star~datd deviation.
h [n situ pH ~ 3.8: adjusted pH = 7 .
c In situ pH = 6.2; adjusted pH = 4.
0 20t~2
4
6
g
10
17
14
~B
18
20
T~AE (h)
Fig. 2. Acid-volatile sulfide production in sediment slurries
from Reservoir 29. 10-mI slurries were incubated at 37 • C after
adjustment to ( o ) pH 3.8, (12) pH 5, or (a) pH 7.
pH
3
4
5
6
7
A V S production a
(nmoi H2S c m - 3 h - I )
Reservoir 29
Lake B
n.d. ~
t2.9±0.3
20.64-0.7
n.d.
21.74-0.6
1.44-0.2
4.2~1.2
3.1~0.1
6.7~0.2
8.0~5
u Values are means of three replicate samples_+ standard deviation.
b n,d. = not determined.
Table 4
Effect of H 2 t!pon sulfate reduction. H 2 was added by injecting 10 ml H;~ overpressurc into vials eontaimng 10 ml sediment
slurry and 20 ml Nz headspaee. H23SSO4 was added immediately, and samples were incubated for 6 h
Source
pH
Reservoir 29
Reservoir 29
Lake B
3.8
7.0
6.2
AVS production a
(nmol H2S ¢m -3 h - i)
No addition
+ H2
18.74-0.8
5.0 + ].2
3.8:1:0.1
30.4:1: 5D
13.3 4- 0.8
11.3+0.8
= Values are means of '.hree replicate samples+standard deviation.
In the initial experiments with organic substrates, 5-mM concentrations of three organic acids
inhibited sulfate reduction m sediments from Reservoir 29 (pH 3.8). Only lactate caused a reduction in activity of sediments from Lake B (pH 6.2)
(Table 5), However, if the pH of Reservoir 29
samples was increased, the organic acids stimulated sulfate reduction. In contrast, when the pH
of Lake B samples was lowered, the addition of
organic acids inhibited sttlfa'~c reduction. A
detailed experiment using acetic acid additions t.o
Reservoir 29 sediments (pH 3.8) showed that the
highest rates of sulfate redaction occurred when
0.1-0.5 mM acetic acid was added. At concentrations above 2 raM, sulfate reduction was inhibited
relative to an unamended sample (Fig. 3).
A wide array of organic compounds (see Methods) were added to Reservoir 29 slumes at either
l-mM or 0.l-mM concentrations. None of them
stimulated sulfate reduction during the first 6 h
after addition. However. if samples were pre-incubated at 37°C for 12 h before addition of
substrate and 3sSOa, some additions were stimulatory. At l-raM concentrations, only glutamate and
alanine caused a statistically significant ( P < 0.05)
198
8o
s"
--...
tO
6~
\
O
E
°\
•~E~_~L=..o. . . . . . . . . .
r-
v
These sediments were greenish black in color and
smelled strongly of sulfide when brought to the
surface. The sediments of Lake B, in contrast, had
a p H > 6 and were inky black; free H2S was never
detected in the porewater. Chemical analysis (Table 6) showed that the amount of reduced sulfur
was greater in Lake B than in Reservoir 29. Acidvolatile S ( H ~ S + F e S ) comprised less of the
reduced S in Reservoir 29 than in Lake B. In
Reservoir 29 sediments (pH 3.8), tiffs fraction
consisted solely of H2S, because it could be purged
with N2 before acid stripping. In contrast, all the
AVS in Lake B (pH 6.2) consisted of FeS.
A set of samples to which 3sSO4 had been
added was chemically fractionated to measure both
AVS and chromium-reducible S (CRS) after 24 h
incubation at 37 ° C. In sediments from both Reservoir 29 and Lake B, AVS was the predominant
product of sulfate reduction (87% and 76% of the
total, respectively). In Reservoir 29 sediments 135[
of reduced ~5S was converted to CRS, whereas in
Lake B samples 24% of reduced S was in the CRS
fraction.
-~- ........
20
°---..o
D
0+I
1.0
10.0
ACETATE ADDED (mM)
Fig, 3. Effect of acetic acid upon sulfate reduction by Reservoir 29 sediment slurries. Slurries were pre-incuhated [or 12
h, then acetic acid and 3~SO4 were added, and the slurries were
incubat~ for 6 h at 370C. The dashed line indicates the
activity in samples to which no acetic acid was added.
increase in sulfate reduction. At concentrations of
0.I mM, all of the tested compounds except malate
were able to stimulate sulfate reduction in Reservoir 29 at pH 3.g. Sulfate reduction in sediment
from Lake B was significantly stimulated ( P <
0.05) by 5-mM additions of acetate~ casamino
acids, ethanol, formate, lactate, propanol, pyrurate, and succinate, but only after samples were
incubated 12 h before the addition of substrate.
The other tested compounds did not stimulate
sulfate reduction in Lake B.
Chemical fractionation of some reduced sulfur
compounds in the sediments of Reservoir 29 and
Lake B was conducted. At the porewater pH of
3.8 in Reservoir 29 sediment, volat;le H2S concentrations reached very high levels; a concentration of 2.86 mM was measured in October, 1985.
5. DISCUSSION
Reservoir 29 is an extreme environment with
pH values 3-4 units below neutrality and high
concentrations of sulfate and iron. While most
macroorganisms are absent, the habitat contains
simple microbial communities capable of primary
production and aerobic heterotrophic mineraliza-
Table 6
Sulfur contents of Reservoir29 and Lake B surface sediments
Fraction
AVS ~
CRS b
Total amount of reduced sulfur
Sulfate
Total amount of sulfur
Reservoir 29
Sulfur
contents
(pmol cm-3)
3.6
6.2
L~eB
Amount of
reduced sulfur
(%)
37
63
Sulfur
contents
(/zmol cm-3)
80
73
9,8
153
38,2
48.0
122
275
Acid-volatile sulfur, b Ozromium-rcducible sulfur.
Amount of
reduced sulfur
(%)
52
48
t99
lion [4]. The data presented here document that
anaerobic sulfate reduction ,also occurs. In Reservoir 29, substantial sulfate-reducing activity occurred at a bulk pH of 3.8 or tess. In general, the
pH minimum for growth of sulfate-reducing
bacteria has been thought to be about 5 [31].
However, there are several other reports of sulfate
reductioa at acidic pH. Rudd et at. [3] demonstrated sulfate reduction in sediments of pH 4.5
[3]. Satake [32] found that 3~SO4 was reduced in
anoxic water samples from a volcanic acid lake of
pH 2.0.
The rates of sulfate reduction found in Reservoir 29 surface sediments at in situ temperature
were 25-5095 of the rates measured in eutrophic
Lake Mendota [33]. The strip-mine lakes are
oligotrophic systems [4], and it may be the flux of
or~#nic matter to the sediments which limits the
rate of sulfate reduction in Reservoir 29. Planktonic algae ate the primary input of organic carbon
to Reservoir 29 sediments. Seasonal variations in
rates of sulfate reduction were correlated with
changes found in algal biomass (Brooks et aL, in
preparation). For e~anple, the lowest rates of
sulfate reduction during the summer of 1985 were
measured in August, in a period that lagged 1-2
weeks behind a decline in the size of the phytoplankton populations. The temperature optimum
for sulfate reduction in the two strip-mine lakes
~-ras much higher than the environmental temperature of 1 0 - I 2 ° C. This result is not unusual; other
studies of microbial sediment processe~ in tem~erate regions have reported similar findings
[33,34].
The effect of low pH on sulfate reduction in the
natural environment is not well understood.
A¢idophilic or acid-tolerant sulfate-reducing
bacteria have not b¢¢~ isolated in pure culture. If
these strains do not exist, the organisms must exist
in higher pH microzoncs. We made a number of
attempts to isolate cultures of sulfate-reducing
bacteria at pH 3-5, Primary enrichments could be
maintained and transferred at pH 3.8, but pure
culture~ that grew at acidic pH were not obtained.
With similar anaerobic techniques, sulfate-reducing bacteria with neutral pH optima for growth
were easily isolated from Reservoir 29 [35].
When the pH of Reservoir 29 sediment samples
was adjusted, sulfate reduction occurred at significant rates only when the bulk pH was low. Activity was 4-5-fold higher at pH 4 and 5 than at pH
7 during the first 6 h after p H alteration. If the
sulfate-reducing bacteria in this system were active
in microzones of higher pH, it is difficult to understand why neutralizing the pH caused a decrease in activ,ty, unless indirect effects such as
changes in chemical speciation were responsible.
"the results from Reservoir 29 were similar to
those in an acidic bog sediment, where the pH
optima for several anaerobic processes ranged from
4.2-5.6 [36]. The optimal pH for sulfate-reduction
•n
Reservoir 29 was lower than in pH-neutral
systems, or in Lake B and similar acid mine
drainage-impacted systems [13], where the water
column is acid but the sediments are circumneutral. The Lake B microbial community
which reduced sulfate functioned optimally at the
in situ pH (6.2), and acidification of samples
always resulted in significant inhibition of activity.
However, the effects of shifting pH in Reservoir 29 sediment samples were complex. For
example, ,,vithin 12 h of pH adjustment, the activity at pH 7 was as high as at the optimal pH of 5.
The observations may reflect physiological adaptations to pH by the existing population of sulfa:c.
reducers or changes in the metabolic pathways of
fermentative bacteria which provide energy substratus for sulfate reduction. Ciostridium acetobutylicum [37] and Sarcina uentriculi [24] provide
examples of this last phenomenon. Organic acids
are major fermentation products at neutral pH.
whereas neutral produc*.s such as acetone and
butanol predominate at pH < ~.
Sulfate reduction was stimulated by organic
acids at pH 3.8 but only if the concentrations were
less than 1 raM. The experiments with acelic acid
(Fig. 3) illustrated that organic acids inhibited
sulfate reduction at high concentrations in acidic
environments. Similar phenomena have been
observed in Candida utilis [25] and Clostridium
thermoaceticum [23]. In these cases, the effect was
~ttributed to the passage of undissociated a~ds
through the cell membrane; these molecules act as
uncouplers of proton motive force [23].
In sediments of neutral pH, a wide variety of
added organic compounds can stimulate sulfate-
200
reduction rates. These include organic acids, amino
acids, hydrogen gas, alcohols, and carbohydrates
[21,22,38]. In studies with natural samples, it is
unclear whether compounds serve directly as substrates for sulfate reduction or other microbial
processes which are coupled to sulfate reduction.
Despite this uncertainty, a variety of electron
donors were added to Reservoir 29 sediments to
determine what conditions might enhance sulfate
reduction,
Note that in these expel, aents organic compounds stimulated sulfate reduction relative to
unamended sediment samples only if samples were
pre-incubated for 12 h prior to substrate addition,
The absence of immediate stimulation may have
several bases; one is that adequate supplies of
energy substrates were available in the sediment.
If this were true, the pre-incubation may have
exhausted these supplies, and stimulation merely
indicates the potential of the system to use these
substrates to drive sulfate reduction. The level of
sulfate reduction in pre-incubated samples to
which stimulatory organic compounds were added
was 1 - I . 5 times the initial rate measnred in unamended sediments thai were not pre-incubated.
Hydrogen gas was the: one substrate whose
addition immediately stiraulated sulfate reduction
and resulted in rates me,re than twice as high as
those found during the first 6 h in unamended
sediments.
The sediments of guservoir 29 and Lake B
contained significant quantities of chromium-reducible sulfur (CRS). We determined what proportion of reduced sulfate accumulated as CRS
during sulfate reduction in these sediments. AVS
was the major product found in both sediments.
Moderate amounts of CRS were formed during
sulfate reduction in Reservoir 29. This result was
similar to those of R u d d e t ai. [3] in lake sediments at p H < 4.8. However, we found a similar
result in Lake B sediments (pH 6.2); in Lake B
samples the AVS : CRS ratio was approximately 3.
In the study of R u d d e t al. [3], lake sediments of
p H 5 - 6 had an AVS : CRS ratio of < 1. The CRS
analyses would detect S in the forms of pyrite or
elemental sulfur [30]. We did no analyses of organic
S formation; this fraction was very significant in
the study of Rudd et al. [3] at p H 5-6, but a
minor component in two lake sediments of p H <
4.8.
In this study, we showed that sulfate reduction
could occur in sediments that have a bulk p H less
than 4 and that the optimal p H for sulfate reduction in freshly collected sediments was 5. However, further work is necessary to explain why
increased rates of sulfate reduction occur at p H 7
within 24 h of a p H adjustment.
ACKNOWLEDGEMENTS
This research was supported in part by the
Purdue Water Resources Center (project G905-06).
REFERENCES
Ill Kivinen, E. (1977) Survey, classification, ecology, and
conservation of peatlands. Bull. Int. Peat Soc. 8, 24-25.
[2] Brock, T.D. (1978) Thermophilic Microorganisms and Life
at High Temperatures. SpringeT-Verlag, New York, NY.
[3] Rudd, J.W.M., Kelly, C.A. and Purntani, A. (1986) The
role of sulfate reduction in long-temx accumulation of
organic and inorganic sulfur in lake sediments. Lirrmol.
Oceanogr. 31, 12gl-1291.
141 Oyure, R.A., Konopka, A., Brooks, A. and Doemel, W.
(1987) Algal and bacterial activities in auidic (pH 3) strip
mine lakes. Appl. Environ. MicrobioL 53, 2069-2076.
[5l Joseph, J.M. (1953) Migrobio|ogical study of acid mlne
waters: preliminary report. Ohio J: Sci. 53, 123-137.
[6] Campbell, R.S. and Lind, O.1". 0969) Water quality and
aging of strip-mine lakes. J. Water Pollut. Control Fed.
41, 1943-1955.
[7] Kelly, C.A., Rudd, J.W.M., Cook, R.B. and Schindler,
D.W. (1982) The potential importance of bacterial
processes in regulating rate of lake acidification. Limnol.
Oceanogr. 27, 868-882.
[8] Kilham, P. (1982) Acid precipitation: Its role in the
alkalization of a lake in Michigan: Linmol. Oceanogr, 27,
856-867.
[9} Mountfovt, D.O. Asher~;R.A., Mayes, E.L. and Tiedje,
J.M. (1980) Carbon and electron flow in mud and sandflat
intertidal sediments at Delaware inlet, Nelson, New Zealand, AppL Environ. Micro~ioL 39, 686-694,
[10] Senior, E, IAndstrom, E.B., Banal, LM. and Nedwe[|,
D.B. (1982) Sulfate reduction and methanoganesis in the
sediment of a sahmarsh on the east coast of the United
Kingdom. Appl. Environ. Microbiol. 43, 987-996.
[1t1 Cook, R.B., Kelly, C.A., Sehindler, D.W, and Turner,
M.A. (1986) Mechanisms of hydrogen ion neutralization
in an experimentally acidified lake~ Linmol. Oceanogr. 31,
134-148.
201
[12] Schindlcr, D.W., Turner, M.A., Stainton, M.P. and Linsey, G.A. (1986) Natural sources of acid neutralizing
capacity in low alkalinity lakes of the Precambrian Shield.
Science 232* 844-847.
[13] Hevlihy, A.T. and Mills, A.L. (1985) Sulfate reduction in
freshwater sediments receiving acid mine drainage. Appl.
Environ. Microbiol. 49, 179-186.
[14] Hurley, AT., Mills, A.L, Homb~rger. G.M. and
Bruckner, A.F- (198"/) The importance of sediment sulfate
reduction to the sulfate budget of an impoundment receiving acid mine drainage. Water Resoun~,s Res. 23, 287-292.
[15] Wieder, R.K. and Lang, G.E. (1984) Influence of wetlands
and coal mining on streamwater chemistry. Water Air Soil
Pollut. 23. 381-396.
[161 Brown, K.A. (1986) Formation of organic sulphur in
anaerobic peat. Soil Biol. Biochem. 18, 131-140.
[171 Decker, C.S. and King, D.L. (1971) Accelerated recovery
of acid strip-mine lakes. Proc. 26th lndust. Wasle Conf.,
Purdue University, Lafayette, IN. pp. 208-216.
[18] King, D.L., Simnder, J.J., Decker, C.S. and Ogg, C.W.
(19"/4) Acid strip-mine lake recovery. J. Water Pollut.
Control Fed. 46, 2301-2315.
{19] Turtle, J.H., Dugan, P.R. and Randles, C.L (1969) Microbial sulfate reduction and its potential utility as an acid
mine water pollution abatement procedure. Appl. Microbiol. 17, 297-302.
[20] Turtle, ,LH,, Dugan, P.R., MacMillan, C.B. and Randles,
C.L (1969) Microbial dissimilatory sulfur cycle in acid
mine water. J. Bactcriol. 97, 594-602.
[21] Dicker, HJ. and Smith, D. (1985) Effects of organic
amendments on sulfate-reduction activity, H2 consumption, and H 2 production in salt marsh sediments. Microb.
~..oi. 11, 299-315,
[22] Smith, R.L. and Klug,, M.J. (1981) Electron donors utilized
by sulfate-reducing bacteria in eutrophic lake sediments.
App[. Environ. Mi~obioL 42, 116-121.
[231 Baronofsky, JJ., Schreurs, W.£A. and Kashket, E.R.
(1984) Uncoupling by acetic acid limits growth of and
acidogenesis by ClosLridium thermoaceticum, App1. Environ. Mics'obiol. 48, 1134-1139.
[24] Goodwin, S. and Zeikus. J.G. (1987) Physiological adaptations of anaexobi¢ bacteria to low pH: Metabolic control
of proton motive force in Sarciaa ventriculi. J. Bacleriol.
169, 2150-215"/.
[25] HuetinB, S. and Tempest, D.W. (1977) Influence of acetate
on the growth of Candida utilis in conl;auous culture.
Arch. Mierobio]. 115, 73-78.
[26] Hungate. R.E (1979) A roll tube method for cultivation
of strict anaerobes. In Metbods in Microbiology, VoL 3B
(Norris, J.IL and Ribbons, D.W., Eds.), pp. 117-132.
Academic Press, New York, NY.
1271 Smith. R.L and ~ u g MJ. (1981) Reduction of sulfur
compounds in the sediments of a eutrophic lake basin.
Appl. Environ. Mierobiol. 41, 1230-1237.
[28] TabatabaL M.A. (1974) Determination of sulphate in water
samples. Sulphur Inst. J. 10, 11-13.
[29] ghabina, N.N. and Volkov. I.L (1978) A method of
determination of various sulfur compounds in sea sediments and rocks. In Environmental Biogeochemistry and
O¢omicrobiology, Vol. 5, Methods, Metals, and Assessmeut (Krumbein, W.E., Ed.), pp. 735-746. Ann Arbor
Science,, Ann Arbor, MI.
[30] Wieder, R.K., Lang, G.E, and Granus, V.A. 0955) An
evaluation of wet chemical methods for quantifying sulfur
fractions in freshwater wcl.land peat. Limnol. Oceanogr.
30, 1109-1115.
[311 Poslgate, J.R. (1984) The Sulphate-reducing Bacteria. 2nd
edition, 208 pp. Carabridge University Press. Cambridge.
[32} Satake, K. (1977) Microbial sulphate reduction in a
volcanic acid lake having pH 1.8 to 2.0. Jpn. J. Limnol,
38, 33-35.
[33] lngvorseo, K., Zeikus, J.G. and Brock, T.D. (19gl) Dynamics of bacterial sulfate red~lction in a eutrophic lake.
AppL Environ. Microbiol. 42, 1029-1036.
[341 Zeikus, J.G. and Winfrey, M.IL (3976) Temperature limitation of methanogenesis in aquatic sediments. Appl. Environ. Mictobiol. 31, 99-107.
[35] Gyure, R.A, (1986) Microbial ecology of acid strip mine
lakes in southern Indiana, PhD. the_sis,Purdue University.
|36] Goodwin, S. and Zeikus, J.G. (1987) Ecophysio[ogical
adaptations of anaerobic bacteria to low pH: Analysis of
anaerobic digestion in acidic bog sediments. Appl. Environ. Microbiol. 53, 57-64.
[37] Ross, D. (1961) The acetone-butanol fcm'gmtafion. I~rc~.
Ind. Microbiol. 3, 73-90.
[38] Stamms, A.J.M., Hanson, T.A. and Skyrmg, G.W. (1985)
Utilization of amino acids as energy substrates by two
marine Desul/ouihrio strains. FEMS MicrobioL F_coL 31,
11-15.