ELSEVIER
FEMS Microbiology
Sulfide-induced
Ecology 21 (1996) 131-138
dissimilatory nitrate reduction to ammonia in
anaerobic freshwater sediments
R.C. Brunet
*,
L. J. Garcia-Gil
Institute of Aquatic Ecology, Umioersity of Girona, Hospital 6, E-l 7071 Girona, Spain
Received 20 April 1994; revised 27 June 1996; accepted 28 June 1996
Abstract
Different reduced sulfur compounds (H .,S, FeS, S,O:- > were tested as electron donors for dissimilatory nitrate reduction
in nitrate-amended sediment slurries. Only-in the free sulfide-enriched
slurries was nitrate appreciably reduced to ammonia
(0.30 pmol NHl/ymol
NO;), with concomitant oxidation of sulfide to So (0.55 pmol S’/pmol
S*-). The initial
concentration of free sulfide appears as a factor determining the type of nitrate reduction. At extremely low concentrations of
free S’- (metal sulfides) nitrate was reduced via denitrification whereas at higher S*- concentrations, dissimilatory nitrate
reduction to ammonia (DNRA) and incomplete denitrification to gaseous nitrogen oxides took place. Sulfide inhibition of
NO- and N,O- reductases is proposed as being responsible for the driving part of the electron flow from S’- to NH:.
Keywords:
Sediment;
Denitrification;
Dissimilatory
nitrate reduction
1. Introduction
In anoxic,
pathways
identified:
nitrate-containing
of dissimilatory
denitrification,
environments,
two
nitrate reduction have been
by which nitrogen
oxides
(mainly NO; and NO;) are reduced to dinitrogen
gases (N,O and N,), and dissimilatory nitrate reduction to ammonia (DNRA), producing ammonia by
reduction of nitrate and/or
nitrite. The ability to
respire with nitrate under anaerobic conditions
is
widespread among several genera of heterotrophic
bacteria [l-3].
In addition, inorganic compounds
such as H,S and S,Ofcan also be used as electron
donors
by facultative
anaerobic
chemolithoau-
* Corresponding
[email protected]
author.
Fax:
+34
(72) 41 81 50; E-mail:
0168.6496/96/$15.00
Copyright 0 1996 Federation
PII SO168-6496(96)0005
1-7
of European
to ammonia;
Sulfide; Ferrous sulfide; Thiosulfate
totrophs such as Thiobacillus denitrzjkans in pure
and mixed culture [4-71.
The occurrence of denitrification
coupled to the
oxidation of reduced sulfur compounds
in natural
environments
has been previously
reported. FeSamended sediments from rice-fields in the Camargue
exhibited increased denitrification
rates [8] and FeSdependent saturation kinetics of nitrate reduction have
also been demonstrated [9]. Pyrite (FeS,) oxidation
in groundwater systems has been reported to result in
nitrate removal from such environments
[lo- 123.
Brettar and Rheinheimer [ 13,141 reported the occurrence of sulfide-dependent
in situ denitrification
at
the oxic-anoxic interface of the Gotland Deep in the
Baltic Sea, as well as the potential for oxidation of
S,Ogand H,S by nitrate in water samples collected above the interface.
Denitrification
to gaseous products is coupled to
Microbiological
Societies. Published
by Elsevier Science B.V
132
R.C. Brunet. L.J. Garcia-Gil/
FEMS Microbiology
electron transport phosphorylation
[ 151 whereas most
DNRA activity is postulated to act as an electron
sink [ 1,16,17]. Nevertheless,
membrane-bound
nitrate and nitrite reductases have been reported in
several genera as, for example,
Campylobacter
sputorum [ 181, Wolinella succinogenes [ 191, Desulfovibrio gigas [20] and D. desulfuricans [21]. All of
them couple H, or formate oxidation to ATP production via DNRA. In addition, D. desulfuricans and
D. propionicus are able to couple nitrate and nitrite
ammonification
to the oxidation of inorganic sulfur
compounds [22].
The removal of eight electrons during the reduction of NO; to NH: makes this process potentially
advantageous in reduced environments
[23]. In fact,
DNRA has been proposed to be quantitatively important in the nitrogen cycling of marine sediments [24].
The work reported in this paper was initiated when it
was observed that NH: was produced following
NO, addition to the sulfide-rich monimolimnion
of
an enclosure placed in the stratified Lake Vilar (N.E.
Spain). The objective was to reduce the organic
carbon pool by stimulating
denitrification
as described by Rip1 [25] as part of the restoration program for this eutrophic lake 1261. Similar results
were later obtained under laboratory
conditions,
where the capacity of several sulfur-reduced
compounds (H,S, FeS and S,O:- ) to act as electron
donors for dissimilatory nitrate reduction was tested.
2. Materials
and methods
The field experiment was initiated by the addition
of 320 g mm2 of Ca(NO,), .4H,O to the monimolimnion of an enclosure (approx. 590 m3, 5.75 m
in depth and 10 m diameter) made of butyl rubber.
Samples at different depths were collected daily over
five summer days by means of an electric pumpdriven water sampler [27]. The same device working
in reverse was used to inject the calcium nitrate
solution to the bottom of the enclosure. A second set
of experiments was performed in the laboratory with
slurries of anaerobic sediment from Lake Vilar. Slurries were prepared by mixing 10% (v/v)
anoxic
sediment with membrane
filtered (0.45 pm pore
size) deoxygenated tap water. The mixture was mechanically homogenized
and sieved through a 0.5
Ecology 21 (1996) 131-138
mm mesh, distributed in l-l sterile bottles and incubated at 25°C for 31 h. Four triplicate experimental
sets were prepared as follows: (1) NO, (referred to
as untreated); (2) NO; + H,S; (3) NO; + FeS; and
(4) NO; + S,O:-. The concentrations
of nitrate and
reduced sulfur compounds used were 2 mmol 1-l
and 1 mmol I-’ respectively. Two bottles of sterilized slurry were supplemented for each treatment as
described above and two more were left as blanks
without any addition. Samples were taken from each
slurry at 5 h intervals and centrifuged at 1000 X g
for 15 min (blank bottles were sampled at the beginning and at the end of the experiment). Pellets were
frozen and stored for later analysis of elemental
sulfur and metal-bound
sulfide. Free sulfide in the
supematant
was immediately
determined
by the
methylene blue method [28]. Sulfide was then removed from the remaining volume by precipitation
with zinc acetate. Aliquots of this sulfide-free supernatant were then collected to measure nitrogen
species and S,Og- concentration. Nitrate was determined after total ammonification
following TiCl 3
reduction [29]. Ammonia and nitrite were determined
using Nessler’s reagent, sulfanilamide,
and N-( Inaphtyl) ethylendiamine,
respectively
according to
the methods as outlined elsewhere [30]. The S,Osand So concentrations
were measured after cyanolysis [3 1,321. Sulfate was determined calorimetrically
following Golterman
and De Graaf BierbrauwerWiirtz [33]. Metal-bound
sulfide was extracted as
H,S by addition of 0.5 M H,SO, and trapped by
N,-flushing in 1 M Zn(CH,COO),
(De Groot, personal communication).
Gas samples (10 ml) were
extracted from each reaction bottle with a syringe
containing 5 ml of 2% w/v Zn(CH,COO),
to avoid
free-sulfide in the gas sample and stored at 4°C in
5-ml evacuated glass vials. Nitrous oxide was determined by gas chromatography
using a Carbomax
102 column and an E.C.D.
3. Results and discussion
3. I. Field experiment
In order to test the oxidant effect of NO, on the
sulfide-containing,
anoxic monimolimnion
of Lake
Vilar, calcium nitrate was added to the bottom of an
R.C. Brunet, L.J. Garcia-Gil/
FEMS Microbiology
Ecology 21 (1996) 131-138
3.2. Laboratory
transformations
1
2
3
4
5
Time (days)
Fig. 1. Time-course of concentration
values of nitrogen species
and H,S in the monimolimnion
(5.5 m) of the enclosure placed in
Lake Vilar after addition of 320 g m-’ of Ca(N0,),.4H20.N0,;
(0); NH: (0); NO; (A) and H2S (0).
enclosure at a depth of 5.75 m. The chemocline was
found at 5.25 m, confining a monimolimnion
of 0.5
m depth (approx. 40 m3). Conductivity values in the
monimolimnion
ranged from 1200 to 1400 &S cm-’
during the period of the experiment.
Nitrate and sulfide were completely
depleted
within 3 days. During sulfide oxidation ammonia
was simultaneously
produced, while nitrite was only
present in trace quantities. Nitrite started to increase,
once sulfide was almost completely
depleted and
ammonia production tended to decrease (Fig. 1). In
the absence of H,S, nitrate was reduced with a
transient accumulation of nitrite. However, no nitrite
accumulation was observed in the presence of H,S,
as nitrate was reduced to ammonia.
Both nitrate and sulfide were simultaneously
depleted as NH: accumulated.
However, since both
H,S and NO; were exhausted at the same time, it
cannot be deduced whether H,S oxidation and NH:
production were mediated by metabolically
related
processes.
Table 1
Transformation
Experimental
of nitrogen
condition
Untreated
+H,S
+ S-FeS
+s&
* Measured
species ( Fmol g-
in nmol g-
’ ) during S-compounds
ANO;
19.29
13.09
21.48
20.46
1.91
1.74
2.85
1.04
9.47
3.75
2.64
8.20
and sulfur
oxidation
ANO,
+
f
k
&
nitrogen
Slurries of anoxic sediment of Lake Vilar were
treated with different sulfur compounds (FeS, S,O:and H,S) and amended with nitrate, in order to
assess the effect of reduced sulfur compounds on
denitrification.
Nitrogen species evolution after addition of S,O:to the experimental slurries was fairly similar to the
untreated conditions. These were different from the
sulfide-containing
slurries, either in particulate (FeS)
or soluble (Hz S) form, which showed lower nitrite
accumulation.
Among the different S-compounds
tested, only
H,S caused the ammonia concentration
to increase.
The highest N,O concentrations
were found under
these conditions,
reaching transient
accumulation
values of 400 nmol gg’ (Table 1). This may be a
consequence of the inhibition of NO- and N,O-reductase activities [34-371. The rest of the sulfur
compounds did not result in the accumulation
of
N,O, which supports the concept of a better balanced steady state between production and consumption of this compound, pointing to N, as the major
final product. This happens with thiosulfate and particularly with FeS, which continually supplies S2- at
extremely low concentrations,
apparently enhancing
denitrification
to N, [8,9]. This situation can be
compared to sulfide-limited continuous cultures. Under these conditions,
steady state cultures
of
Thiobacillus denitrijkans
did not accumulate NO,
while low levels of N,O were detected in the gas
outlet [5]. In fact, the respiratory chain of T. denitrificans transfers electrons from sulfide and SOi- to
NO, and NO;, respectively [38]. This would partially explain the low accumulation
of nitrite in the
sulfide-amended
experimental systems.
0.0
0
experiments:
133
AN,0
+
f
+
+
0.04
0.43
0.22
0.06
’ . Maximum value reached during transient accumulation.
*
95 f 5
400521
0.47 * 0.05
3 + 0.53
ANH:
0
3.79 + 0.13
0
0
R.C. Brunet.L.J. Garcia-Gii/FEMS MicrobiologyEcology21 (1996) 131-138
134
Table 2
Transformation
of sulfur species ( pmol
g-’ ) during S-compounds
oxidation
ASO:-
AS0 *
Untreated
6.10 f 0.69
n.d.
_
- 6.24 f 0.65
+H,S
+ S-FeS
+ s,o:-
0.60 f 0.13
8.99 f 0.68
22.65 f 1.24
11.79 * 0.30
0.82 + 0.11
9.23 + 0.21
_
_
- 3.08 i 0.33
- 13.14 f 1.33
- 4.64 f 0.467
Experimental
condition
* Maximum value reached during transient
M.S.: Metallic sulfide (mainly FeS).
n.d.: Not determined.
AS&
- 5.48 i_ 0.31
ratios of redox species (N and S) in the experimental
Experimental
Untreated
+H,S
+ S-FeS
+s,o?
condition
AH?.?
_
- 23.08 + 0.78
_
accumulation
During the experiment the percentage of reduced
sulfur that was oxidized was variable, dependent on
the sulfur species involved. Thus, H,S was almost
totally depleted (99.2%); FeS in the untreated and
amended slurries was oxidized at 89.6% and 70.0%,
respectively,
and thiosulfate
the least, with only
57.7%.
Sulfate was the major product in all the sediments
except those containing H,S. This compound was
incompletely oxidized to elemental sulfur (Table 2).
This product was also produced in appreciable
amounts during thiosulfate oxidation. It has been
reported that some denitrifiers carry out this process
through the transient accumulation of intracellular So
[4,6,39]. In contrast, So was hardly found during FeS
oxidation.
The relationship between the oxidation of sulfur
compounds
and nitrate reduction can be demonstrated after the calculation
of some molar ratios
(Table 3). Moreover, additional information on the
main metabolic pathways can be obtained. Ammonia, for instance, is only produced during the oxidation of H, S. In this process, 30% of the reduced
nitrate was converted into ammonia.
Table 3
Calculated
AMS.
The ratio between the nitrate consumed and the
total reduced sulfur oxidized slightly exceeded the
theoretical values for complete oxidation of FeS and
S20iby nitrate, that is, 1.6. These differences are
attributable to heterotrophic activity. For the oxidation of H,S, this ratio was found to be closer to the
theoretical one for incomplete oxidation of H,S to
elemental sulfur (0.25). Actually, So was the main
oxidation product in HZ S-amended slurries (see Table
2). Finally, FeS, both basal and added, and S,Ozwere oxidized to SOiin stoichiometric amounts,
fairly close to the theoretical ratios of 1 and 2 for
sulfide and thiosulfate, respectively. As pointed out
above, sulfate production in H,S-amended
slurries
was small.
3.3. Effect of H,S on denitrijkation
Our results suggest that, in the conditions tested,
nitrate reduction to ammonia was related to the
presence
of sulfide.
Ammonia
production
was
recorded while sulfide was being oxidized and ceased
after the exhaustion of H,S (Fig. 2). Nitrite accumulation after HZ S depletion was faster than during
slurries during S-compounds
oxidation
ANHi/ - ANO,
ANO,/ - ASre.m
ANH:/
0
0.30 + 0.05
0
0
3.78
0.51
1.69
2.02
0
0.15 * 0.001
0
0
S r_,Tot: Added reduced S-species + basal metal bound S’-.
* Measured along all the experiment time-course (O-3 I h).
a Measured during H2 S oxidation (O-2 1 h).
&-0.31
f 0.06
* 0.30
* 0.07
- AS,,,
(O-21 h)
ASO:-/ - AS,,, *
1.21
0.03
1.28
2.66
f
+
+
k
0.07
0.006 a
0.09
0.09
R.C. Brunet. LJ. Garcia-Gil/
0
4
8
12
16 20
24
28
FEM.7 Microbiology
32
in H,S
amended
time-course oxidation. To a lesser extent, this observation is also applicable to the oxidation of metallic
sulfide such as FeS. These rates were, however,
substantially
lower than those registered for both
untreated and thiosulfate-amended
slurries (see Table
1).
The addition of sulfide to the nitrate-amended
slurries resulted in NO; and NH: dynamics compa-
Table 4
Rates of N and S changes
Experimental
condition
in the experimental
sediment (pm01
g-’
135
rable to those obtained in the enclosure experiment.
Nitrate depletion rates in the experimental
slurries
followed a zero order kinetics in agreement with
King and Nedwell [40], which is characteristic
at
high NO; concentration.
Rate values are presented
in Table 4. Addition of either S,O:and FeS did
not cause any apparent effect on nitrate depletion
rates with respect to the untreated slurries. In turn,
H,S caused the nitrate depletion rate to decrease by
21.6% with respect to the rest of sulfur compounds
tested. H,S was oxidized faster than FeS and S,O:-.
In addition, when both basal FeS and added H,S
were simultaneously present (H,S-amended
slurries),
H,S was preferentially
oxidized. This fact is attributable to the higher availability of free-S’- compared to insoluble (low KS) metal-bound sulfide.
The effect of reduced sulfur compounds on nitrate
reduction kinetics in natural environments
depends
on both the microbial process involved and the experimental
conditions.
Golterman
and Garcia-Gil
[8,9] reported increased denitrification
rates when
FeS was added to Rhone delta sediments. These
sediments were originally
oxidized and contained
low organic matter (< 2%), whereas the sediment of
Lake Vilar was highly reduced and had appreciable
amounts of organic matter (> 5%). Therefore, the
high basal heterotrophic
denitrification
activity in
our sediments was probably too high to detect any
possible effect of FeS or S,O:on nitrate reduction
rates. In addition, Brettar and Rheinheimer [ 131 reported increased denitrification rates both in situ and
in low H,S or S,O:(50 pmol l-‘1 and nitrateamended water samples of oxic-anoxic interface of
Gotland Deep (Baltic Sea). In these conditions, no
increase in NH,f concentration was detected. On the
Time (hours)
Fig. 2. Time-course of N and S transformations
slurries. Bars represent standard errors.
Ecology 21 (1996) 131-138
h-‘1
ANO,/At
ANH:/At
A%,
*/At
AM.S./At
Untreated
+H2S
+ S-FeS
- 0.84 + 0.08
- 0.66 + 0.04
- 0.84 + 0.09
0
0.17 + 0.02
0
_a
-0.84
-0.52
+ 0.1
* 0.1
-0.21 f 0.05
- 0.08 * 0.03
_b
+s,o:-
- 0.83 + 0.19
0
- 0.34 + 0.03
* Added reduced S-species: H,S, FeS and S,Of
M.S.: Metallic sulfide (mainly F&S).
a No S-addition.
b Not discernible from the FeS added.
-0.18
f 0.02
136
R.C. Brunet, L.J. Garcia-Gil/
FEMS Microbiology
Fig. 3. Proposed general reductive pathway for reduced sulfurmediated nitrate reduction to ammonia in freshwater sediments.
Black arrow heads represent nitrogen transformations
and white
ones the electron flow. Dashed lines indicate the particular H,S
derived electron flow and related nitrogen products. X: NO- and
N,O-reductase
inhibitions.
other hand, sulfide was seen to exert an inhibitory
effect on denitrification
[34-371 and yet stimulate
DNRA in waterlogged soils [41].
Some of the features pointed out above can be
explained after considering the differential free-S’initial concentration
supplied to the nitrate-amended
system. Because of its low solubility, ferrous sulfide
provides extremely low concentrations
of free-S*which could extensively reduce NO, to N,, enhancing the denitrification
process [8,9]. Accordingly,
among the different S-compounds tested, FeS evolved
the lowest N,O concentration.
Our data show that 1
mmol lP ’ H ZS inhibited NO- and N,O-reductase
activities [34-371, as supported by the accumulation
of N,O while H,S was present.
The partial inhibition by sulfide of the denitrification pathway to NZ probably causes part of the
electron pool coming from sulfide to be channelled
to NH: (Fig. 3). Nevertheless, the low calculated
ammonia:sulfide
ratio, as well as the lack of sulfate
production during H,S oxidation, suggest some nitric oxide accumulation
under NO- and N,O-reductase blockage. Whether ammonia comes from
NO; or NO reduction cannot be deduced from our
data. If NO was the precursor [42,43], the NO-reductase in sulfide-oxidizing
DNRA organisms should
be different from that described so far.
Sterile controls did not exhibit any N and S
transformation,
indicating that all the processes described are mediated by bacterial activity. Nevertheless, very little is known about the bacterial groups
involved in these processes, in particular those coupling H,S oxidation and DNRA.
Ecology 21 (1996) 131-138
Some authors [44-461 have proposed that aerobic
denitrifiers of the Pseudomonas group can actually
survive in nitrate-free
anaerobic environments
by
means of a low level fermentation activity. However,
since this group is strictly heterotrophic, it is very
unlikely to perform H,S-mediated
DNRA.
In turn, denitrifiers of the T. derzi@kwzs
group
would be good candidates because of their ability to
denitrify using either H 2S or FeS as electron donors
[4-71. However, no reports about ammonia production have been found in the literature. Furthermore,
many species of DesuJfovibrio are also able to use
NO;, and a restricted number of them NO,, as
electron acceptors 121,471. Dannenberg
et al. [22]
showed that D. desuljiiricans was able to completely
oxidize sulfide to sulfate either with NO; or NO;
yielding ammonia. As a general feature of sulfate-reducing bacteria, however, it is extensively
documented that nitrate and nitrite reduction yields ammonia
regardless
of the electron
donor
used
[21.48,491.
Further work will be focused on 16s rDNA sequence analysis in order to reveal the filiation of the
groups involved in H 2S-mediated DNRA.
4. Conclusions
Results presented in this paper show that addition
of NO, to sulfide-containing,
freshwater sediments,
both in natural and laboratory conditions, results in
the oxidation of sulfide and the concomitant accumulation of ammonia. The suggested sulfide-dependent,
ammonia-producing,
dissimilatory
NO;
reduction
pathway represents up to 30% of the total nitrate
reduction.
Electrons coming from either organic carbon or
S,OzP are mostly transferred to NO,. resulting in a
transient accumulation of NO;. When either FeS or
H,S are present, electrons are transferred to NO;,
producing N, and ammonia, respectively.
Apparently, electrons would be driven to either denitrification or DNRA, depending on the initial concentration
of free sulfide. Thus, the oxidation of low KS metalbound sulfides would produce N,, whereas the presence of H,S would cause incomplete denitrification,
mainly to NO and ammonia, through partial inhibition of NO- and N,O-reductase.
R.C. Brunet, L.J. Garcia-Gil/
FEMS Microbiology
These conclusions should be applied when NO;
is considered to be added to aquatic ecosystems for
management purposes. If ammonia is produced, the
electrons remain in the system and therefore what
happens is a transfer of the ‘problem’ from sulfur to
nitrogen. Under an ecological perspective H,S and
NH: are not very different; both of them are electron donors for chemosynthetic
bacteria, toxic for
higher organisms and, even more so, ammonia can
be taken up as a N source by a wealth of organisms
as for example algae and photosynthetic
bacteria.
Gaseous nitrogen, in turn, takes the electrons out,
resulting in an effective oxidation of the whole system.
According to these conclusions, prior removal of
sulfide is strongly recommended
in order to obtain
optimal organic carbon mineralization via denitrification.
Acknowledgements
Authors are indebted to Lluis Baheras, Elisabet
Pinart, Uta Sterr, Juana Rodriguez and Anna M.
Aymerich for their help during field and laboratory
work. Elena Saguer from Dept. EQATA of the University of Girona assisted the authors during chromatographic
analysis.
The project was partially
funded by grants NAT 91-0708 and CIRIT AR-87.
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