FEMS Microbiology Ecology 13 (1993) 85-92
© 1993 Federation of European Microbiological Societies 0168-6496/93/$06.00
Published by Elsevier
85
FEMSEC 00486
Kinetics of FeS-mediated denitrification
in sediments from the Camargue
(Rhone delta, southern France)
L.J. Garcia-Gil
a
and H.L. Golterman
b
a Institute of Aquatic Ecology, University of Girona, Girona, Spain, and b Station Biologique de la Tour du Valat, Aries, France
(Received 19 January 1993; revision received 29 July 1993; accepted 24 August 1993)
Abstract." Denitrification rates were measured in sediments after the addition of different concentrations of FeS. A decrease of the
denitrification rate was observed when high concentrations of ferrous iron ( > 10 raM) were present. In the experiments with no
significant concentrations of free Fe 2+, the relationship between NO 3 reduction and FeS concentration followed Michaelis and
Menten kinetics. The maximum rate was 0.273 mmol 1- i d -x, 6 times as much as the basal rate 0.046 mmol 1-1 d -~, which was
attributed to organic matter; the K s was 1.45 mM FeS. The stoichiometry of the overall reaction involving NO 3 reduction and the
concomitant S2- oxidation was also investigated. Measured A S / A N ratios ranged between 0.55 and 0.64, with 2H2S+SO4zchanging less than 10%. These values agree with the theoretically expected value of 0.56.
Key words: Sediment; Denitrification; FeS; Kinetics
Introduction
T h e b i o g e o c h e m i c a l cycles o f iron, s u l p h u r a n d
n i t r o g e n , a r e i m p o r t a n t in s e d i m e n t s a n d waters,
c o n t r o l l i n g t h e c a r b o n cycle t h r o u g h t h e i r influe n c e on p r i m a r y p r o d u c t i o n a n d m i n e r a l i z a t i o n .
T h e r e l a t i o n s h i p s b e t w e e n s u l p h u r a n d iron a r e
well k n o w n b o t h in s e d i m e n t s a n d anoxic w a t e r s
[1-4]. T h e r e are, however, few p u b l i s h e d works
on t h e r e l a t i o n s h i p b e t w e e n n i t r o g e n a n d b o t h
t h e iron a n d s u l p h u r cycles. It is k n o w n t h a t
Correspondence to: L.J. Garcia-Gil, Institute of Aquatic Ecol-
ogy, University of Girona, Hospital 6, E-17071 Girona, Spain.
p r o c e s s e s involving t h e s e e l e m e n t s t a k e p l a c e at
d i f f e r e n t E h [5,6]. Thus, d u r i n g m i n e r a l i z a t i o n
processes, w h e n O 2 c o n c e n t r a t i o n is low ( < 4 m g
1-1) N O 3 m a y p a r t i a l l y r e p l a c e 0 2 as e l e c t r o n
a c c e p t o r [7,8]. O n c e N O 3 has b e e n e x h a u s t e d ,
first F e 3+ a n d e v e n t u a l l y SO4z - a r e u s e d as elect r o n a c c e p t o r s by d i f f e r e n t k i n d s o f m i c r o o r g a n isms. This series of r e d u c t i o n s is set in an ' a p p a r e n t ' r e d o x s e q u e n c e t h a t is r e l a t e d to t h e
d e c r e a s i n g t h e r m o d y n a m i c a n d m e t a b o l i c effectiveness o f m i n e r a l i z a t i o n as t h e E h o f t h e envir o n m e n t b e c o m e s lower [6]. T h e r e f o r e , p r o c e s s e s
such as d e n i t r i f i c a t i o n a n d iron a n d s u l p h a t e red u c t i o n o f t e n o c c u r s e q u e n t i a l l y in t i m e a n d s p a c e
[9]. G e n e r a l i n f o r m a t i o n a b o u t d e n i t r i f i c a t i o n as
86
well as environmental conditions affecting denitrification is discussed in [10] and [11] respectively.
FeS is a compound resulting from the chemical
precipitation of reduced iron and sulphur, which
are commonly found in sediments of many aquatic
environments [12-14]. Although FeS has been
traditionally though to be a very insoluble and
stable compound, it can be readily oxidized under
aerobic conditions by a number of lithotrophic
bacteria such as Thiobacillus spp. Moreover, under anaerobic conditions, FeS is a good electron
donor for anoxygenic photosynthesis of Chlorobium spp. [15] as well as denitrification by facultative anaerobic lithotrophs. In previous work [16]
it was shown that the addition of FeS to sediments considerably increased the denitrification
rate. Some bacteria of the genus Thiobacillus are
able to carry out denitrification chemoautotrophically, using reduced compounds of sulphur as
electron donors [17]. The best known is Thiobacillus denitrificans, which can use H2S, S ° or $ 2 0 ~as reducing agents for dissimilatory nitrate reduction [18,19].
The aim of this work was to study the kinetics
of FeS-mediated denitrification and to determine
the stoichiometry of the overall process.
Materials and Methods
Mud was collected from different sites in the
Camargue, in the Rh6ne river delta (southern
France) selecting the more oxidized (yellowish
colored) to ensure that the initial FeS concentration was zero.
For the kinetics experiments seven bottles of
0.5 l each were filled with sediment previously
m i x e d w i t h some water and sieved through a 0.5
m m mesh size. Different quantities of FeS were
obtained by adding FeCl 2 • 4 H 2 0 and N a 2 S - 9
H 2 0 to each bottle containing mud as described
by G o l t e r m a n [16]. A control bottle was left without any addition (FeS c o n c e n t r a t i o n - - 0 ) . For
analysis, 20-ml samples were taken daily after
vigorously shaking the slurry, and centrifuged for
15 rain at 4000 rpm. T h e clear water in the
supernatant was used for the chemical analyses.
Nitrate was determined by Nesslerization after
100% reduction with TiCI 3 to N H 3 [20]. Nitrite
was determined using the I.B.P. Nr. 8 method
5.4.1. [21]. Sulphate was analyzed colorimetrically
using a mixed B a / C r O 4 solution as described by
Golterman and De G r a a f Bierbrauwer-Wiirtz [22].
The total S 2- concentration was determined colorimetrically with A s 2 0 3 as reductant, after removing the H2S from the sediment with O2-free
N 2 and trapping in 1% Zn acetate. All experiments were carried out at room temperature.
Kinetic parameters were calculated using a
derivative-free non-linear regression, from B M D P
statistical package software.
Results
In a first experiment, FeC12 was added in
excess to ensure that all the sulphide was combined in the form of FeS. Figure 1 shows the
nitrate reduction rate as a function of the FeS
concentration in the first experiment in which an
excess of Fe 2+ was added in all bottles. A decrease of the denitrification rate was found at
high concentrations of FeS in the sediment. In a
second experiment in which all Fe 3 ÷ was reduced
and where the Fe 2+ concentration was about 0.1
mM, no denitrification took place. These observations suggest a possible inhibitory effect of
0.8 F ~ - '
~
0.7 i
.
0.6
~
0.5
g O.4
~ 0.3
/
. . . . . . . . . . .
~ O.2
0.1 !
i
0
4
8
,
i ~
12
16
_
_
20
24
28
32
36
40
FeS (mmol S 14)
Fig. 1. Kinetics of nitrate reduction as a function of sulphide
concentration (FeS) in the sediment. In this experiment an
excess of Fe 2+ was added (see text).
87
Table 1
0.3
J
t
Nitrite and ammonia concentrations at different FeS concentrations, as function of time in the first experiment (see text
for explanation)
E~ 0.2
Days
0
FeS (mM)
0
1.25
2.5
5
10
20
40
0
1.25
2.5
5
10
20
40
3
5
7
Nitrite (~M)
n.d.
2.6
n.d.
9.5
n.d.
10.4
n.d.
68.7
n.d.
4.9
n.d.
32.7
n.d.
n.d.
0.4
1.7
9.0
54.6
4.0
7.6
n.d.
1.3
1.3
4.9
53.6
12.2
4.9
n.d.
Ammonia
n.d.
n.d.
n.d.
n.d.
46.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
166.0
300.0
60.0
n.d.
n.d.
n.d.
144.0
112.7
n.d.
90.0
(gM)
n.d.
n.d.
n.d.
28.8
n.d.
n.d.
n.d.
~
o.1
4
Z
do
t
J
0
L
I
2
4
!
6
FeS (rnrnolS 14)
I
/
8
10
Fig. 2. Michaelis curve showing the relationship between the
denitrification rate and the concentration of S2--FeS. do:
basal organic matter denitrification = 0.046 mmol NO~- 1-1
day - l . dmax = 0.273 mmol NO 3 1-1 day -1. K s = 1.45 mmol
Sl-k
n.d. = not detectable,
F e 2÷. B o t h n i t r i t e a n d a m m o n i a c o n c e n t r a t i o n s
w e r e h i g h e r a t t h e e n d o f t h i s e x p e r i m e n t , as
s h o w n i n T a b l e 1.
I n a t h i r d e x p e r i m e n t , n o f r e e F e 2÷ w a s allowed to be present in the sediment. In this case,
the denitrification rates were found to follow the
M i c h a e l i s a n d M e n t e n k i n e t i c s (Fig. 2) a c c o r d i n g
to:
( d m a x -d =do+
do)S
K s +S
Table 2
Nitrite and ammonia concentrations at different FeS concentrations, as function of time
FeS (mM)
0.00
1.25
2.50
3.75
5.00
7.50
10.0
0.00
1.25
2.50
3.75
5.00
7.50
10.0
n.d. = not detectable.
Day
0
1
Nitrite (/zM)
32.0
27.3
33.9
29.8
27.5
36.8
48.6
41.6
15.0
34.3
19.2
17.8
34.1
51.2
43.6
8.0
11.7
8.4
7.2
6.4
9.2
46.48
5.75
2.23
1.83
n.d.
n.d.
n.d.
44.4
5.8
4.2
4.5
3.7
4.2
3.1
45.4
11.9
n.d.
n.d.
n.d.
n.d.
n.d.
Ammonia (/~M)
133.9
15.9
102.2
25.9
127.1
23.2
119.1
29.7
120.0
11.0
151.8
26.8
n.d.
39.1
85.2
122.8
115.1
143.6
142.0
155.7
102.7
133.3
123.8
103.3
104.1
133.5
145.6
186.9
101.5
94.6
177.7
114.0
209.5
121.6
166.4
143.5
80.9
74.1
2
3
4
5
118.2
68.8
114.3
124.7
88
3.0 [
where d is the denitrification rate at each FeS
concentration, S is the substrate (FeS) concentration, d o is the basal denitrification (due to organic matter) and dma× and K S are the maximum
denitrification rate and the half-saturation constant respectively. The calculated values for these
parameters were d 0=0.046 mmol 1-~ d -~ of
NO3-, d,~,x = 0.273 mmol 1-1 d-1 of NOj- and
K S = 1.45 mmol 1-1 of S 2-. Nitrite concentrations
were higher at the beginning of the experiments
with values up to 50 /xM. When the experiment
was finished, nitrite was only present in the bottle
with sediment containing no FeS (Table 2). The
ammonia concentration did not change significantly and remained between about 100 and 200
/xM.
The overall increase of sulphate concentration
after five days incubation was proportional to the
decrease in nitrate concentration during the same
period (Fig. 3). At the end of the experiment, the
higher the amount of nitrate reduced, the higher
the concentration of SO42" found. Thus, a quantitative relationship between the oxidation of FeS
and the reduction of NO 3 can be deduced. The
stoichiometry of the FeS-dependent denitrification was then measured using two kinds of sediments, one with low organic matter ( < 2%) and
another 'rich' in organic matter ( > 5%). Both the
depletion of NO 3 and the production of SO 2were measured over 14 days in the first sediment
7
I N N "1 .[NC} "1
2.5
E
~
2.0
1.5
g
1.0
0.5
o
o
1.25
2.5
5
1o
20
FeS (mmol S I~)
Fig. 3. Initial (i) and final (f) concentrations of NO 3 and
SO 2 ( × 1 0 ) after five days incubation, with respect to the
concentration of total sulphur (FeS) in the experimental sedi ments.
and 9 days in the second. The measured A S / A N
ratios were fairly consistent with the theoretical
value 0.56 (Table 3), whereas that of denitrification in the control sediment without FeS was
considerably lower. In sediments containing organic matter, the A S / A N ratios measured after
the correction for organic carbon denitrification
ranged between 0.54 and 0.66. The denitrification
observed in the control without FeS was due to
the higher content of organic matter of this sediment as compared to the previous one. I f the
Table 3
Quantitative relationship between the oxidation of FeS ( a s o 2 - ) and the reduction of nitrate ( A N O j - ) in two experiments with
sediments containing low ( < 2%) and high ( > 5%) organic matter respectively. Concentrations in mmol 1- I
FeS
ANO 3
5 days
AS/aN
aSO 214 days
5 days
14 days
4.43
4.71
5.93
5.86
0
0
3.17
3.11
0.37
0.38
3.85
3.81
> 5% organic matter
2.50
2.50
+
6.14
7.00
+
7.57
7.86
(after correction for "org-C" denitrification:
0
2.40
2.40
0.37
2.80
2.80
< 2% organic matter
2.36
1.71
+
5.24
+
4.86
0.55-0.64
0.54-0.64
0.46-0.40
0.32-0.40
0.66-0.63
0.58-0.53
89
values for A S / A N were corrected for the heterotrophic denitrification, values of about 0.6
were found.
Only the denitrification rate in the experiment
with the high organic C concentration was corrected as an increase was found in the H C O 3
concentration. This was not the case in the experiment with the low organic C concentration. The
increase of H C O 3 cannot be used for the calculation of heterotrophic denitrification because of
the complexity of the Ca2+/HC03/CO 2- system and the lack of knowledge about the nature
of the organic substrate.
Discussion
The kinetic experiments reported here support
the idea [16] that FeS in sediments quantitatively
enhances dissimilatory denitrification. For a given
concentration of NO~-, the denitrification rate
increased with the FeS concentration, until saturation was reached. This rate is limited by the
number of bacteria, assumed to be of the genus
Thiobacillus denitrificans among others. T h e increase of sulphate as an oxidation product of FeS
as well as the concomitant decrease of ,NO~concentration are stoichiometrically related. This
means that, under the experimental conditions
tested, all the electrons used in the reduction of
nitrate were transferred from sulphide and Fe z+ .
Organic carbon denitrification seems to be less
efficient when FeS is present.
As far as the production of both nitrite and
ammonia is concerned, the results varied depending on whether free Fe 2+ was absent or present.
It seems that there exists no dependence between
these nitrogen compounds and the FeS concentration in sediment. When no free iron was present, nitrite was formed in the beginning of the
experiments but rapidly disappeared and became
undetectable. The differences in nitrite concentration in the different experiments could reflect
the selection and enrichment of various metabolic
groups of denitrifying bacteria. Although the differences in the initial rates of nitrate and nitrite
reduction should be considered, the factors determining nitrite accumulation still remain uncer-
tain. Accumulation of ammonia could be attributed to other metabolic pathways related to
sulphide oxidation and nitrate reduction such as
D N R A (Dissimilatory Nitrate Reduction to Ammonia) which is performed by a number of facultative anaerobic bacteria such as Desulfouibrio
gigas [23]. A more detailed review on this subject
can be found in [24]. Some ammonia is released
by the mineralization of organic matter. The inhibitory effect of high concentrations of free Fe 2+
as well as the unpredictable appearance of both
nitrite and ammonia, remain unexplained, and
are being investigated.
There is not much literature dealing with denitrification and the oxidation of reduced sulphur
compounds especially in the form of insoluble
FeS. Haider et al. [25] carried out some laboratory experiments on denitrification using reduced
compounds of iron and sulphur, especially Fe z+
and FeS z (pyrite). An inhibitory effect of HzS on
denitrification has been reported by several authors [26-29]. FeS acts as a non-toxic store of
suitable electrons for many lithotrophic bacteria.
The increasing FeS concentrations strongly increased the total amount of NO 3 reduced and
the rate at which this process took place. As we
succeeded in getting enrichment cultures (but no
pure cultures) of Thiobacillus denitrificans with
solid FeS from the experimental flasks, we think
that the activity of these bacteria was responsible
for the enhanced denitrification rate by the oxidation of FeS. This explains the accumulation o f
SO42-. The denitrification rate is proportional to
FeS content of the sediment until saturation rates
are reached. Such observations are supported by
accurate stoichiometrical measurements. Therefore it can be concluded that the presence of FeS
in sediment increases the denitrification rate following Michaelis and Menten kinetics. Nitrate
reduction can be up to six times higher in sediments containing 10 mmol S 1-1 than in those
without FeS.
In eutrophic ecosystems NO 3 and FeS may
often occur together. The high nutrient concentrations cause a high primary production finally
leading to anoxic sediments. The NO 3 in the
water entering the ecosystem will then be rapidly
denitrified. In natural ecosystems nitrate is rarely
90
I
m-. Fe3,
~
Fe 2+
~
~
OH4
002
Fig. 4. Scheme showing the main respiratory electron transfers occurring during the mineralization of organic matter in
sediments. The processes indicated are arranged sequentially
starting with 1 through 5. When nitrate is present, electrons of
end-products as Fe 2+ and H2S are re-introduced in the
sequence through the biological oxidation of FeS.
found together with sulphide. This is because, in
the sequence of biological redox processes occurring in sediments, nitrate is already reduced before sulphate-reduction takes place. However,
electron flow in sediments must be interpreted
more dynamically to understand the mineralization of organic matter in anoxic environments.
FeS-mediated denitrification may result in the
recirculation of the electrons coming from organic matter that are stored in the form of FeS
and then react with the constantly entering nitrate. Therefore, electrons are lost from the system in the form of N 2 by flowing from sulphide to
nitrate (Fig. 4). Thus, FeS in sediments should be
considered as an active participant in biological
processes rather than an insoluble, stable and
inert compound whose transformations are quantitatively negligible.
Further research should include physiological
experiments using pure cultures of Thiobacillus
denitrificans to do comparative studies between
organic matter and FeS denitrification as well as
the effect of other metal sulphides. The production of nitrite, as noticed by Golterman [16] must
also be studied with pure cultures of this bacterium to understand the reason for this unexpected intermediate.
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