Stimulation of Sulfate-Reducing Bacteria in
Lake Water from a Former Open-Pit Mine
Through Addition of Organic Wastes
James M. Castro, Bruce W. Wielinga, James E. Gannon, Johnnie N. Moore
ABSTRACT: A method to improve water quality in a lake occupying
a former open-pit mine was evaluated in a laboratory-scale study.
Untreated pit lake water contained high levels of sulfate, iron, and
arsenic (I 200, 100, and 5 mg/L, respectively) and was mildly acidic
(-pH 6). Varying amounts of two locally available organic waste
products (waste from a potato-processing plant and composted steer
manure) were added to pit water and maintained in microcosms under
anoxic conditions. In selected microcosms, populations of sulfate-
reducing bacteria increased with time; sulfide was generated by sulfate
reduction; sulfate, iron, and arsenic concentrations approached zero; and
pH approached neutrality. Best results were obtained with intermediate
amounts of waste potato skin. Water Environ. Res., 71,218 (1999).
KEYWORDS: open-pit, mine, lake, acid, sulfate, reduction, remedia-
tion, bacteria.
Abandoned or discontinued open-pit, metal-mining operations
have resulted in the creation of pit lakes. Depending on the mineral
composition of rocks surrounding a pit, particularly the presence or
absence of carbonates, water in the resulting lake may be mildly to
severely acidic, near-neutral, or even alkaline (Plumlee et aI.,
1992). Depending on amounts and types of associated minerals,
the lake may contain varying dissolved concentrations of metals,
metalloids, and sulfate (Macdonald et aI., 1994, and Miller et aI.,
1996). Under current economic conditions, mining of relatively
low-grade gold deposits is profitable, and more than 100 such
deposits are mined throughout the world (Mining Journal Group,
1997). These mining activities will result in the creation of nu-
merous new pit lakes during the next 50 years (Macdonald et aI.,
1994). In the state of Nevada alone, more than 30 new pit lakes are
expected to be formed in the next 20 years that, when filled, will
contain more than 1.2 X 109 m3 (I X 106 ac-ft) of water (Miller
et aI., 1996). Thus, an increased need for pit lake remediation is
foreseen.
Although water in pit lakes may vary greatly in pH and metal-
metalloid content, they typically have high to very high sulfate
concentrations (up to several thousand parts per million) produced
by oxidation of sulfides. Mechanisms of sulfate formation arc well
known. Using pyrite (FeS2) as an example, oxidation produces
sulfuric acid (H2S04) by the following reactions (Stevenson,
1986):
12 FeS04 + 3 O2 + 6 H20 ~ 4 Fe2(S04)3 + 4 Fe(OH)3
Fez(S04h + FeS2 ~ 3FeS04 + 2 SO
218
Furthermore, ferric Iron (Fe3+) generates acid by hydrolysis
(Mills, 1985)
(5)
Sulfides of elements other than iron are also subject to oxidation,
which can release toxic levels of those elements. In the presence of
carbonate host rock, some or all of the acid may be neutralized, but
most of the sulfate and significant amounts of the dissolved metals
and metalloids typically remain in solution. Moreover, because
most pit lakes connect with aquifers and can be attractive to
animals in search of drinking water and migratory birds, they
present potential hazards for both public health and wildlife con-
servation. Sulfate concentrations alone can violate state and federal
water-quality regulations. When commonly high metal-metalloid
concentrations and acidity are considered, it is evident that cor-
rective action is necessary to improve such lakes' water quality.
No practical remediation method for water quality of large pit
lakes has yet been developed. Neutralization of acidic lake water
by addition of limestone or other bases has been proposed, but
such treatment is not enough. Neutralizing acidic, high-sulfate
water with high concentrations of dissolved metals typically pro-
duces neutral, high-sulfate water with high dissolved metal con-
centrations, which still does not meet environmental standards for
domestic or agricultural use.
However, past studies of acidic drainage from coal mines (Tuttle
et aI., 1968; Tuttle, Dugan, Macmillan, and Randles, 1969; and
Tuttle, Dugan, and Randles, 1969) and pit lakes at abandoned coal
mines (Campbell and Lind, 1969) have suggested an approach that
may remediate conditions in metal-mine pit lakes. Stimulation of
dissimilatory sulfate-reducing bacteria (SRB) by addition of or-
ganic nutrients would be a cost-effective means of dealing with
these contaminated waters.
Sulfate-reducing bacteria gain energy for growth by coupling
oxidation of organic compounds or hydrogen with the reduction of
sulfate to sulfide (Barton and Tomei, 1995; Perry, 1995; and
Widdell, 1988). Sulfate reduction has the added benefit of precip-
itating metals and metalloids by reactions of the typc
SO~- + M2+ +2C (org) ~ MS! + 2 CO2 (6)
(I)
where M is a dissolved metal and C (org) is organic carbon.
(2) Moreover, gradual conversion of amorphous iron sulfides to pyrite
(Berner, 1970) consumes acid
(3)
(4) (7)
Water Environment Research, Volume 71, Number 2
 Castro et al.

The result of these processes is water of near-neutral pH with low 0

levels of dissolved metals, metalloids, and sulfate, which may ''\1
approach or meet environmental standards.
2 <;7
Added organics have two effects: (I) reducing oxygen and other
"I
highly oxidizing species (NOx, Fe(III), etc.) in water and sedi-
4 '9
-
ments, thus establishing suitable redox conditions for dissimilatory
sulfate reduction, and (2) providing, either directly or indirectly, <;7

the low-molecular-weight organic compounds required by SRB. •...

lJ)
6
.•...
Q)

-
Examples of organic amendments that stimulate SRB are decom- Q)

posed sawdust (Tuttle, Dugan, Macmillan, and Randle, 1969),
substances present in drainage from cultivated fields (Campbell
and Lind, 1969), spent mushroom compost (Dvorak et aI., 1992),
hay and straw (Bechard et aI., 1993), and slurried cattle waste
(Ueki et aI., 199]).
This paper reports a laboratory-scale investigation of the feasi-
bility of remediating water from a pit lake at a gold mine in
northern Nevada. Two organic amendments are readily available
to the mine: waste from a local potato-processing plant and com-
posted cattle manure from a feed lot.
Materials and Methods
Site Description. The Summer Camp Pit is located at
the Getchell Mine in Humboldt County, Nevada (41 °13' N,
117 ° 16' W). A former open-pit mine, the Summer Camp Pit is
now filled with water to a depth of approximately 18 m and
receives precipitation, surface drainage, and, primarily, water
pumped from an underground gold mine. Water in this lake is high
in sulfates and mildly acidic; the acidity is partly moderated by
carbonate rock in contact with the lake (Silberman et aI., 1974).
The ore body is high in orpiment (As2S3) and realgar (AS4S4) but
low in metal sulfides other than pyrite. As a result, lake water is
low in metals other than iron, sodium, calcium, and magnesium but
uncommonly high in arsenic, typically containing several milli-
grams per liter. The drainage water pumped from the underground
mine contains small amounts of nitrate and nitrite, residue from
explosives used in the mine. Water temperature measured in June
1996 ranged from 9 °C at the bottom to 20°C at the surface;
measurements in November 1996 ranged from 10°C at the bottom
to 7 °C at the surface. Dissolved oxygen levels measured in
November ]996 decreased sharply from 7 to 9 mglL near the
surface to < 1 mg/L below 8 m (Figure ]).
Sample Collection. Sediment samples and depth-integrated wa-
ter samples were collected from the anoxic zone of the lake,
between depths of 9 and] 6 m, on November 5, ] 996. Three 20-L
carboys (Nalgene) were purged with N2 gas for 5 minutes before
being filled with a sample. Carboys were fitted with caps contain-
ing three ports that allowed continuous N2 purging during sample
collection. Water and sediment were retrieved through sterile
silicone tubing (6 mm i.d.) connected to a peristaltic pump. The
tubing was taped to the cable of an oxygen-temperature probe
(YSI Corporation. Yellow Springs, Ohio) that had been marked at
0.5-m intervals and connected to a YSI model 58 Dissolved
Oxygen Meter. The probe provided sufficient weight to get the
tubing to the lake bottom and allowed simultaneous collection of
Iimnological data. Carboy no. 1 contained bottom sediments and
water, whereas carboys nos. 2 and 3 contained depth-integrated
water samples. Once the carboys were filled, the pump was shut
off and the inlet port capped. Each sample was purged for an
additional 5 minutes with N2 and the vessels were sealed with
slight positive nitrogen pressure for transportation back to the
E 8 -.. ' -...... '\1
g ...
..•...
.c:. '\1
a.
Q) 10 !v
0
12
--- Temperature
14 .... 'V ... Dissolved oxygen
16
o 2 4 6 8 10 12
Temperature (OC) and D.O. (mg/L)
Figure 1-Dissolved oxygen concentration and temper-
ature profile as a function of depth in Summer Camp Pit
Lake, Humboldt County, Nevada (November 1996).
laboratory. Samples were kept on ice during transportation (22
hours).
Microcosm Set Up. Microcosms were contained in 5-L Nal-
gene polyethylene bottles. To reduce variation between micro-
cosms, each microcosm was established with water and sediment
from each of the three carboys. Initially, 750 mL of sediment
slurry from carboy no. ] was added to each microcosm, followed
by addition of I 000 mL of water from carboy no. 2. The appro-
priate quantity of each amendment was added and each microcosm
was then brought to a final volume of 3 250 mL with water from
carboy no. 3. Each of the 20-L carboys was continuously mixed
while water was being transferred. All transfers were made under
a constant stream of N2 gas to maintain anoxic conditions. Micro-
cosms were numbered I through II, with microcosm I I (MCll)
having no added amendments. Each of the other microcosms
received 0.5 gIL sodium nitrate (NaN03), 0.025 giL monobasic
potassium phosphate (KH2P04), and 0.025 giL dibasic potassium
phosphate (K2HP04) as bacterial nutrients. Microcosms 2 through
6 received, respectively, 365, I 825, 3 650, 7 300, and 3 650 mglL
organic carbon as waste potato peel. Microcosms 7 through 10
received 500,2500,5000, and 10 000 mglL, respectively, organic
carbon as composted manure. To study bacterial activity in the
absence of sulfate reduction, excess sodium molybdate (4.7 gIL)
was added to MC6 as a competitive inhibitor of sulfate metabo-
lism.
Microcosms were passed into an anaerobic glove box (Coy
Laboratories, Grass Lake, Michigan) containing an atmosphere of
88% N2, 5% carbon dioxide, and 7% hydrogen. The glove box was
equipped with an alarm to indicate oxygen contamination. All
sampling and microbiological processing were conducted under
this atmosphere, with the exceptions of the sampling at 150 and
2] 0 days. A methylene blue anaerobic indicator strip was placed in
the cap of each microcosm to indicate redox state. Between sam-

March/April 1999 219

Castro et al.
piing intervals the microcosms were sealed, removed from the
anaerobe chamber, and stored in an incubator at 15°C; this
temperature was used because it approximated the average tem-
perature in the lake.
Bacterial Enumeration. Sulfate-reducing bacteria were enu-
merated using a five-tube most probable number (MPN) method. A
l-mL aliquot from each thoroughly mixed microcosm was serially
diluted 10-fold in prereduced phosphate buffered saline solution
(pH 6.8). Diluted sample was used to inoculate the quantitative
enrichments. The Desulfomonile tiedjei medium used, ATCC me-
dium 1690 (American Type Culture Collection, Manassas, Virginia),
was modified as follows: the concentration of sodium bicarbonate
(NaHCO) was increased from 3 to 4.2 gIL (-50 mM); pyruvate
(40 mM) was replaced as the sole carbon source with a combina-
tion of pyruvate, acetate, and lactate (13.3 mM each); iron ammo-
nium sulfate (Fe(NH4MS04h . 6H20) (0.087 mM) was added to
enhance detection of sulfate reduction; and a reductant solution
consisting of 42.6 mM ascorbic acid and 65.7 mM sodium thio-
glycolate replaced 50 mM sodium dithionite. Enrichments were
incubated at 20°C in an atmosphere of N2:C02 (80:20) and
counted after 30 days. Sulfate reduction was indicated by black-
ening of the media as a result of precipitation of ferrous sulfide.
Analytical Methods. All reagents used were American Chem-
ical Society analytical reagent grade except as noted. Hydrochloric
and nitric acids were trace metal grade. All water samples were
filtered through O.20-J,1m membrane filters. Samples for metal and
metalloid determination were acidified to <pH 2 with nitric acid.
After sulfide was found to affect arsenic solubility, dissolved
arsenic was determined in unacidified samples. Solution pH was
measured using a glass combination electrode. Metals and metal-
loids were determined by inductively coupled argon-plasma emis-
sion spectroscopy (ICAPES) using a Thermo-Jarrell-Ash (Frank-
lin, Massachusetts) IRIS system according to U.S. Environmental
Protection Agency (U.S. EPA) method 200.7. Arsenic (UI) was
determined by hydride generation atomic absorption spectroscopy
(HG-AAS) using a Varian Model 875 spectrophotometer (Varian
Techtron Pty., Ltd., Mulgrave, Victoria, Australia) equipped with
a VGA-76 hydride generator. Anions were determined by ion
chromatography according to U.S. EPA method 300.0, using a
Dionex system (Dionex Corp., Sunnyvale, California) equipped
with an AS4A-SC column and an ASRS-I cation suppressor.
Nonpurgeable organic carbon was determined using a Shimadzu
TOC-5000A analyzer (Shimadzu Scientific Instruments, Inc., Co-
lumbia, Maryland) calibrated against a potassium hydrogen
phthalate primary standard. Soluble sulfides and ammonia nitrogen
were determined immediately upon sampling. Sulfides were deter-
mined by the methylene blue method using a Hach DREL/2000
field kit (Hach Co., Loveland. Colorado) according to U.S. EPA
method 376.2. Ammonia nitrogen was determined by the Nessler
method using a Hach DREL/2000 kit according to U.S. EPA
method 350.2. Total organic carbon in the composted manure was
detennined using a Coulometrics CO2 Coulometer system (UIC,
Inc., Joliet, Jllinois), and in the potato waste it was determined
using a Fisons Carlo Erba EA I L 10 (CE InstrumentslElantech,
Beverly, Massachusetts) calibrated against a 2,5-bis-(5-tert-butyl-
benzoxazol-2-yl)-thiophene standard.
For ICAPES analysis, solid organic amendments were digested
as follows: samples (0.5 g) of dried, ground material were sealed
in polytetraftuoroethylene vessels with 5-mL aliquots of aqua regia
and heated in a microwave oven. Digests were then cooled, diluted
to 50-mL volumes, centrifuged, filtered through 0.2-I.Lnl membrane
220
filters, acidified with 200 fLL nitric acid each, and stored in clean
polyethylene bottles for later analysis.
Organic Amendments. Potato waste and composted cattle ma-
nure were obtained locally by Getchell Mining Corporation. The
amendments contained 50.4 and 24.0% organic carbon (dry basis),
respectively.
Results and Discussion
In microcosms with added organic amendments, nitrate concen-
trations dropped from initial values near 90 mg NIL to near 0
within 31 days. At the same time, nitrite concentrations increased
from 0 to maxima near 30 mg NIL and then decreased to near 0
within 60 days. This pattern is consistent with either denitrification
or dissimilatory nitrate reduction by bacteria (Tate, 1995).
The concentration of ammonia-nitrogen gradually increased in
MC5 and MCl 0 from approximately 20 mglL to approximately 50
mglL. In MC2, MC4, MC6, MC8, and MC I I it remained approx-
imately constant at 5 to 10 mglL. In MC I it dropped within 5 days
from 7 mglL to O. In MC7, it remained constant at approximately
5 mglL for the first 2 months, then decreased to I mg!L thereafter.
Ammonium-nitrogen concentrations in MC9 fluctuated throughout
the study between 15 and 40 mglL. There were at least three
sources and one sink of ammonia-nitrogen in the systems. Likely
sources included dissolution of soluble ammonia from organic
amendments (especially the composted manure), ammonification
of organic nitrogen, and dissimilatory nitrate reduction; the most
likely sink was nitrogen immobilization by bacteria (Tate, 1995).
These multiple ammonia pathways probably explain the lack of
any readily apparent trend in ammonia-nitrogen concentrations in
the microcosms.
Lake water initially had low dissolved oxygen concentrations
(Figure I), and care was taken to avoid oxygen uptake during
transfer to the microcosms. Based on the appearance of methylene
blue indicator strips, all microcosms except 4, 5, and 6 remained
anoxic during processing. Addition of higher amounts of potato
wastes had the effect of slightly oxidizing these microcosms and
retarding the onset of sulfate reduction. Addition of nitrate as a
bacterial nutrient had the effect of elevating the redox potentials of
the microcosms and preventing sulfate reduction for several weeks
until the nitrate and nitrite were reduced. Many heterotrophic
bacteria can use nitrate and/or nitrite as a terminal electron accep-
tor when oxygen becomes limited. In environments where both
nitrate and sulfate are available, nitrate reduction is the predomi-
nant terminal electron-accepting process (Lovley, 1991). In pit
lake remediation, where promotion of bacterial sulfate reduction is
the objective, a better choice of nitrogen source would be an
ammonium salt, which would provide reduced nitrogen rather than
nitrate.
The water obtained from the lake was at pH 6.1. In most cases,
pH values increased to approximately 7, but in MC4 and MC5 the
pH decreased to values below 6. Microcosm 4 eventually recov-
ered to approximately pH 6, but the pH in MC5 remained low (5.2
at 210 days). The decrease in pH may have been caused by the
release of organic acids from the potato waste in these micro-
cosms. In MC] I, the control, after 150 days the pH fell sharply and
was at pH 4.95 after 210 days. This seems to have been caused by
oxygen diffusing into the microcosm, with resultant iron oxidation
and acid generation (see discussion below).
Concentrations of SRB, sulfate, and sulfide in all microcosms at
the start of the experiment were approximately equal (I X 102
bacterialmL, MPN estimated; I 200 mglL; and < I IJ.g!L, respec-
Water Environment Research, Volume 71, Number 2
 Castro at al.

!~:r
was oxidized by nitrate to Fe(III), which would precipitate as
oxyhydroxides at the prevailing pH. Iron then increased again at 30
to 60 days, as nitrates and nitrites became depleted and the Fe(Ill)

io~j .~.~ -. - tto

was reduced again to Fe(II). Finally, as sulfides appeared in
solution, Fe(II) precipitated as sulfides and the dissolved iron
concentration dropped again. Microcosms 5 and I I were notable
exceptions to this general rule.
o.~C3 1 en
As in most other microcosms, the iron in MC5 was resolubilized
between 31 and 60 days, apparently by reduction to Fe(Il). How-

6~t4
c::

....J
-.a::
E
-
iir
(I)
(')
o
ever, at 210 days the iron concentration was still more than 100
mglL, although it appeared to be dropping at this point. The deLay
in precipitation of iron sulfides in MC5 was likely caused by
a:l 5 C4 :::::J slower sulfate reduction in this microcosm compared to several
4 ~
(/) others (see Figure 2). Iron and sulfide concentrations reflect a slow

6~-t4
z 23 _ _ . .0 2
approach to equilibrium that may be related to the low pH and high
a.
::2
-.
1 ~.- 3 level of organic matter in the microcosm.
C) o 0 to Between 60 and 150 days, the dissolved iron concentration in
..Q
5 MC5
.c MC]] dropped from 156 to less than 1 mg/L (data not shown).
III
:::l During the same period, an orange-brown precipitate appeared on
4
a.
3 .0 2 (j)
the sides of the bottle containing the microcosm. A nitrogen-

l r; _~__
2 ••.. ----- II-:-':':':':"~. c:: carbon dioxide-hydrogen atmosphere was maintained in the mi-
3i

-
1 ···0·
o .... • 0 a. crocosm, and nothing had been added to the water and sediment.
(I)
The position of the precipitate suggests that oxygen had diffused
...•.
o

:MC'C .c-.
~~
J
1 ~....
.. ~
~ ~
, ~~
3
to
160r:
120

60

40
e-
•.••.••_----
-----J. MC1 6
4

5
4 0·····
.............,
'.
MC9 6
160o -----0 , 0

3 .... .... 4
..••... ....-----_ MC3

•• r~--------o_-_-O
2 c;' '. 120
1 .' _--- - .'W 2
80
o 0
I
o 50 100 150 200 250
40

-l
Time (days) o
![
Figure 2-Changes in SRB,sulfate, and sulfide concentra- »
Cil
tions over time in selected microcosms. Symbols: • = (1)
::I
sulfate-reducing bacteria, • = sulfate, and 0 = sulfide. c o'
e III
::I
a.
tively), In MCI, to which no organic nutrient was added, these
"0
Q) »
> ~
concentrations remained essentially constant over the 210 days of "0
III
III
the experiment (Figure 2). In MC6, which was amended with both
i:5 3'
a carbon source and molybdate to inhibit SRB, there was no
change in sulfate or sulfide concentrations, and SRB numbers 160 Mca
6 --
co
.!:
120 riA
dropped to <2/mL (data not shown). Following a variable lag
period, densities of SRB in microcosms 3, 4, 8, and 9 increased to
/?~ __-----
80
concentrations that varied between 2 X 104 and 3 X 105/mL 40 I ~ -0 _
(Figure 2). The rise in SRB concentrations was followed by a
decrease in sulfate and an increase in sulfide (Figure 2). 160o -
MC9
Iron (Figure 3) behaved as expected in an anoxic system sub- 120
jected to an initial addition of nitrate followed by a gradual drop in 80
redox potential. The redox potential for the Fe(III)-Fe(II) couple is
40
higher than that for the nitrate-nitrite couple under standard con-
o
ditions, that is, 1 m of all reactants. However, at pH 6 the nitrate 50 100 150 200 250

couple's potential (0.408 V) is considerably higher than that of the Time (days)
iron couple (0.173 V for L mM Fe2+).
The dissolved iron concentration in lake water was initially Figure 3-Concentrations of dissolved iron, total ar-
greater than 100 mglL because of Fe(II) in solution. Within 3 senic, and As(lII) as a function of time. Symbols: • =
weeks the dissolved iron concentration dropped to near 0 as Fe(II) dissolved iron, • = total arsenic, and 0 = As(III).

March/April 1999 221

Castro et al.
through the walls of the polyethylene bottle. Polyethylene is
known to be permeable to gases, and the time interval between 60
and ISO days was the longest time that the microcosms were stored
without being purged with fresh reducing gases. It is likely that
oxygen diffused into MCII, oxidizing the iron. This is consistent
with both the position of the precipitate on the walls of the
container and with the decrease in pH from 6.4 at 60 days to 5.6
at 150 days and 4.95 at 210 days because oxidation of Fe(II) with
precipitation of oxyhydroxides generates acid (equation 5).
Total dissolved arsenic concentrations were initially 1 to 3
mg/L. They increased somewhat during the first 5 days and in-
creased more slowly through the first 21 days (Figure 3). This was
unexpected because, at the time, iron was precipitating from so-
lution as oxyhydroxides in MCI through MClO, and arsenates
typically co-precipitate with iron oxyhydroxides. It is possible that
addition of 25 mM phosphate as a bacterial nutrient caused the
displacement of adsorbed arsenic from mineral or organic surfaces
because phosphate and arsenate ions would tend to compete for the
same sites.
Total arsenic and As(III) in MCll dropped from 5.0 and 1.35
mglL, respectively, at 60 days to near 0 at 150 days (data not
shown). This behavior is also consistent with inward diffusion of
oxygen because As(III) would be oxidized to arsenates, and ar-
senates tend to co-precipitate with iron oxyhydroxides (Dyck and
Lieser, 1981, and Moore, 1994).
In microcosms where anoxic conditions were established and
measurable sulfide appeared in solution, arsenic concentrations
fell. Arsenic concentrations then rose with further increases in
dissolved sulfide. This is consistent with the precipitation of ar-
senic sulfides such as As2S3, followed by re-dissolution of these
sulfides to form thioarsenite complexes. In water containing 60
mglL sulfide at pH 6, the solubility of arsenic is approximately 5
mg/L (Webster, 1990).
Determinations of total arsenic in water samples from micro-
cosms were performed by ICAPES, using solutions acidified to
<pH 2 with nitric acid, according to U.S. EPA method 200.7.
Arsenic(III) was determined in unacidified samples using HG-
AAS. At 31 days, for some of the more sulfidic microcosms,
measured As(III) concentrations were larger than total arsenic
measurements. This anomaly seems to have been the result of
sample handling. In highly sulfidic microcosms, As(III) solubility
was increased by sulfide through the formation of thioarsenite
complexes (Webster, 1990). In samples analyzed by ICAPES,
added nitric acid oxidized some of the sulfide and thus destabilized
the thioarsenite complexes, precipitating dissolved arsenic. By
contrast, samples analyzed for As(III) by HG-AAS were not acid-
ified. Sulfide concentrations in those samples were undisturbed,
and dissolved arsenic levels remained higher. Once this potential
nitric acid interference was noted, further ICAPES determinations
of arsenic in sulfidic solutions were performed on unacidified
samples (ICAPES determinations of iron and other metals were
still performed on acidified samples according to the U.S. EPA
method).
Between day 60 and 150, arsenic levels in MC4 and MC5 dropped
to near 0, although conditions in these microcosms (i.e., increasing
sulfide concentration and moderate pH) were still conducive to the
formation of thioarsenite complexes. This drop in dissolved arsenic is
possibly a result of conversion of amorphous ferrous sulfides in the
sediments (with the approximate composition FeS) to pyrite (FeS~.
Pyrite typically forms on a time scale of weeks to years (Berner, 1970)
but can form more quickly under favorable conditions (Howarth,
222
1979). Such favorable conditions require the introduction of small
amounts of oxygen to generate elemental sulfur by partial oxidation of
sulfide (Lord and Church, 1983)
S2- + 1/2 O2 + H20 ~ SO + 2 OH- (8)
followed by the reaction
FeS + SO~ FeS2 (9)
Oxygen can be introduced to sulfidic sediments by many mech·
anisms, including lake overturn, storm events, and bioturbation. In
this case, slow diffusion of oxygen through the walls of polyeth-
ylene containers might have brought about pyritization of sulfides
in the microcosms over a period of a few months. Authigenic
pyrite is known to be a strong arsenic scavenger that can accom-
modate significant amounts of arsenic in its crystal lattice. Thus,
formation of pyrite is commonly accompanied by a decrease in
dissolved arsenic (Belzile and Lebel, 1986, and Morse, 1995).
Subsequent small increases in arsenic concentrations in MC4
and MC5 after 210 days are not as easily explained. Sulfide levels
in these microcosms increased steeply during this interval and
approximately quadrupled between days 150 and 210. Any arsenic
released to solution would be kept in solution as thioarsenites. It is
possible that arsenic could have been released by slow-reacting
solid phases in the sediment, such as refractory oxides. Pyrite's
high thermodynamic stability (Berner, 1970) makes it unlikely that
arsenic would be released from that mineral by reaction with
sulfide.
Dissolved organic carbon concentrations were approximately
proportional to the amount of organic matter added. There was
approximately twice as much readily soluble organic carbon in the
compost as in the potato waste. (During the first 21 days of
incubation, an average 12.5% of organic carbon added to compost-
treated microcosms dissolved, while dissolution rates for carbon in
microcosms treated with potato waste averaged 6.3%.) Water
samples taken later than 12 days after the start of the experiment
from MC8, MC9, and MClO, which contained large amounts of
compost, showed an intense dark brown color; these samples also
produced a precipitate upon acidification with hydrochloric acid.
The only metals besides iron that were present at significant
concentrations in the pit water were calcium, sodium, and magne-
sium. Concentrations of these metals remained fairly constant,
except for increases in magnesium and sodium in samples from
microcosms containing large amounts of potato waste or compost
(data not shown).
This experiment has demonstrated the possibility, in principle,
of remediating water quality in a mildly acidic pit lake containing
high levels of sulfate, iron, and arsenic by addition of inexpensive
organic matter. In some of the microcosms, bacterial reduction of
sulfate produced sulfide and, after a period of months, the bulk of
the sulfate, iron, and arsenic in the microcosms was removed from
solution as precipitated sulfides. However, it is also evident that
there are multiple poorly understood variables that affect the rates
of sulfate reduction, sulfide precipitation, and the eventual forma-
tion of stable mineral phases. The amount and type of organic
additive had unexpected effects on the above processes and on
solution pH. Removal of sulfate, iron, and arsenic and neutraliza-
tion of acids did not improve uniformly with increased organic
matter; rather, there appears to be an optimum amount of organic
matter that affords the fastest remediation. Moderate amounts of
waste potato peel seemed to deliver the best combination of
sulfate, iron, and arsenic removal.
Water Environment Research, Volume 71, Number 2
 Castro et al.

Judging by these laboratory results, the best results in the field Dyck, W., and Lieser, K.H. (1981) Coprecipitation of Copper, Zinc,
might be anticipated using approximately 0.2 gIL (200 glm3) Arsenic, Silver, Cadmium, and Lead with Iron Hydroxide and Iron
organic carbon in the form of potato waste. This waste, which is Phosphate. Vom Wasser (Ger.), 56, 183.
Herlihy, A.T.; Mills, A.I.; Hornberger, G.M.; and Bruckner, A.E. (1987)
approximately 92% water, contains approximately 50% carbon on
The Importance of Sediment Sulfate Reduction to the Sulfate Budget
a dry basis, or approximately 4% carbon as received from the
of an Impoundment Receiving Acid Mine Drainage. Water Resour.
plant. As the lake volume is approximately 2 X 105 m3, it would
Res., 23, 287.
require 4 X 104 kg carbon, or approximately 900 Mg (I 000 ton) Howarth, R.W. (1979) Pyrite: Its Rapid Fonnation in a Salt Marsh and Its
of wet potato waste. This amount of material, delivered over a Importance in Ecosystem Metabolism. Science, 203, 49.
period of months, is well within the capacity of the potato-pro- Lord, C.l.I., and Church, T.M. (1983) The Geochemistry of Salt Marshes:
cessing plant. It would require approximately 50 trips by dump Sedimentary Ion Diffusion, Sulfate Reduction, and Pyritization.
trucks with a standard 18-Mg (20-ton) capacity, or correspond- Ceochim. Cosmochim. Acta (G.B.), 47, 1381.
ingly fewer trips by larger trucks. Lovley, D.R. (1991) Dissimilatory Fe(llI) and Mn(IY) Reduction. Micro-
After the initial treatment, it might be necessary to continue bioI. Rev., 55, 259.
Macdonald, M.S.; Miller, G.C.; and Lyons, W.B. (1994) Water Quality in
adding small amounts of potato waste to the lake as long as
Open Pit Precious Metal Mines, Reno, NY. Dep. Environ. Resour.
oxidizing waters from the underground mine are discharged to the
Sci., Univ. Nev., Reno.
lake. However, with establishment of eutrophic conditions, a
Miller, G.C.; Lyons, W.B.; and Davis, A (1996) Understanding the Water
healthy aquatic algal population might produce enough organic
Quality of Pit Lakes. Environ. Sci. Tee/mol., 30, 118A.
matter on an ongoing basis to consume the added nitrate from the Mills, A.L. (1985) Acid Mine Waste Drainage: Microbial Impact on the
mine drainage water and obviate further waste additions. The Recovery of Soil and Water Ecosystems, In Soil Reclamation Processes.
tendency of the lake to stratify at times during the year would also R.L.l. Tate and D.A. Klein (Eds.), Marcel Dekker, New York, N.Y., 35.
tend to maintain a reservoir of organic matter in an anoxic or Mining Journal Group (1997) Mining Activity Survey. Mill. Mag., 176, 39.
suboxic bottom layer. Even in much shallower lakes than the Moore, J.N. (1994) Contaminant Mobilization Resulting from Redox
Summer Camp Pit, sulfidic bottom sediments rich in organic Pumping in a Metal-Contaminated River-Reservoir System. In End-
ronmental Chemistry of Lakes and Reservoirs. Advances in Chemistry
carbon have been found to act as sinks for sulfates and dissolved
Series 237, 451.
metals (Herlihy et aI., 1987, and Pedersen et aI., 1993).
Morse, l.W. (1995) Dynamics of Trace Metal Interactions with Authigenic

Acknowledgments Sulfide Minerals in Anoxic Sediments. In Metal COIIWIIl;lIat;OI1;n

Aquatic Sediments. H.E. Allen (Ed.), Ann Arbor Press, Ann Arhor,
Credits. The authors gratefully acknowledge the M.J. Murdock
Mich., 187.
Charitable Trust for funding equipment used in this work and
Pedersen, T.F.; Mueller, B.; and McNee, J.J. (1993) The Early Diagenesis
thank John Barta (Getchell Mining Corporation, Golconda, Ne-
of Submerged Sulfide-Rich Mine Tailings in Anderson Lake, Mani-
vada), Tim Meikle and Paul Policastro (Bitterroot Restoration, toba. Can. J. Earth Sci., 30, 1099.
Inc., Corvallis, Montana), and Thomas DeLuca (University of Perry, K.A (1995) Sulfate-Reducing Bacteria and Immobilization of Met-
Montana) for assistance with field work. Funding was provided by als. Marine Ceoresour. Ceoteclmol., 13, 33.
Getchell Mining Corporation and the University of Montana. Plumlee, G.S.; Smith, K.S.; Ficklin, W.H.; and Briggs, P.H. (1992) Geo-
Authors. James Castro and Johnnie Moore are in the Depart- logical and Geochemical Controls on the Composition of Mine Drain-
ment of Geology, University of Montana, Missoula, Montana. ages and Natural Drainages in Mineralized Areas. In WlIIer-Rock
Bruce Wielinga and James Gannon are in the Division of Biolog- Interaction. Y.K. Kharaka and AS. Maest (Eds.), A.A. Balkema,
Rotterdam, Neth., 1,419.
ical Sciences, University of Montana, Missoula, Montana.
Silberrnan, M.L.; Berger, B.R.; and Koski, R.A. (1974) K-Ar Age Rela-
Correspondence should be addressed to James M. Castro, De-
tions of Granodiorite Emplacement and Tungsten and Gold Mineral-
partment of Geology, The University of Montana, Missoula, MT
ization Near the Getchell Mine, Humboldt County, Nevada. £Con.
59812-1019. Geol., 69, 646.
Submitted for publication November 4, ]997; revised manu- Stevenson, FJ. (1986) Cycles of Soil: Carbon, Nitrogen. P/lOsphorus.
script submitted May 27, 1998; accepted for publication October Sulfur, Micronutrients. Wiley & Sons, New York, N.Y.
]5, ]998. Tate, R.L.I. (1995) Soil Microbiology. Wiley & Sons, New York, N.Y.
The deadline to submit Discussions of this paper is June ]5, ]999. Tuttle, J.H.; Dugan, P.R.; Macmillan, C.B.; and Randles, c.1. (1969)
Microbial Dissimilatory Sulfur Cycle in Acid Mine Water. J. Bacte-
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