MICROBIOLOGY
ECOLOGY
ELSEVIER
FEMS Microbiology
Ecology
19 (1996) 27-38
Anaerobic nitrate reduction to ammonium in two strains isolated
from coastal marine sediment: A dissimilatory pathway
Patricia Bonin
Received
13 September
1995; revised 9 October
1995: accepted 9 October
I995
Abstract
A total of 28 nitrate-reducing
bacteria were isolated from marine sediment (Mediterranean coast of France) in which
dissimilatory reduction of nitrate to ammonium (DRNA) was estimated as 80% of the overall nitrate consumption. Thirteen
isolates were considered as denitrifiers and ten as dissimilatory ammonium producers. 15N ammonium production from ‘.5N
nitrate by an Etzrerrrbacter
sp. and a Vibrio sp.. the predominant bacteria involved in nitrate ammonification in marine
sediment, was characterized in pure culture studies. For both strains studied, nitrate-limited culture (I mM) produced
ammonium as the main product of nitrate reduction (> 90%) while in the presence of 10 mM nitrate, nitrite was
accumulated in the spent media and ammonia production was less efficient. Concomitantly with the dissimilation of nitrate
to nitrite and ammonium the molar yield of growth on glucose increased. Metabolic products of glucose were investigated
under different growth conditions. Under anaerobic conditions without nitrate, ethanol was formed as the main product; in
the presence of nitrate. ethanol disappeared and acetate increased concomitantly with an increased amount of ammonium.
These results indicate that nitrite reduction to ammonium allows NAD regeneration and ATP synthesis through acetate
formation, instead of ethanol formation which was favoured in the absence of nitrate.
Ke~wor%s; Nitrate reduction;
Ammonium:
Marine bacterium;
Dissimilatory
1. Introduction
Many bacteria can utilize nitrate instead of oxygen as the terminal electron acceptor. Depending on
the end products, two different pathways of dissimilatory nitrate reduction
can be distinguished:
(i)
firstly. nitrate can be reduced to gaseous products
(N,O or N?) during denitrification
constituting a net
loss of nitrogen for the ecosystem [I]; (ii) altematively, nitrate can be converted into ammonium in a
dissimilatory
process called nitrate ammonification
or dissimilatory
reduction of nitrate to ammonium
(DRNA) [2]. This pathway keeps nitrogen as ammo-
pathway
nium available for the food chain, although part of
the nitrogen can be lost (about 10%/o), since Smith
and Zimmermann
(198 1) [3] have reported that N,O
is also produced during nitrate ammonification.
So
far. for marine coastal sediment, the focus of most
field studies has been on the measurement of denitrification; more recently, evidence has been provided
for nitrate ammonification
[4-61 using various methods. First, the rate of nitrate ammonification
has
been estimated from the difference between the overall NO,
reduction and denitrification
from ‘the
acetylene inhibition technique’ [5] or from the rate of
“NZ production after addition of an ‘5N0,
tracer
0168-6496/96/$15.00 lb 1996 Federation of European Microbiological Societies. All rights reserved
SSD/ 0168-6496(95)00075-5
[6]. A more accurate method has been reported by
Koike and Hattori [4], who determined the “N?.
“NH:
and ‘“N-particulate organic nitrogen (PON)
produced from ” NO,. Since the efficiency of the
C2H2 blockage was reduced at low NO, concentration in the presence of H,S, determination of nitrate
ammonification
based on an extended version of the
acetylene inhibition technique is open to criticism,
and the occurrence of nitrate ammonification
is controversial [7]. The two reduction pathways (denitrification and nitrate ammonification)
can occur under
similar environmental
conditions
[8]. which are
anaerobiosis
or occur in presence of low oxygen
concentrations
[9,10] with nitrate. More knowledge
of the microorganisms
involved in both processes is
necessary for a better understanding
of their regulation processes. While denitrification is carried out by
a large variety of microorganisms
with roughly the
same well-known
biochemical
pathway [I I]. relatively little attention has been focused on bacteria
taking part in nitrate reduction to ammonium.
Our
knowledge of the organisms following this pathway
is scanty and is focused on only a few strains.
Furthermore, nitrate ammonification
seems to be a
more complex process which is not confined to
facultative anaerobic bacteria [ 12- 141 but also occurs in strict anaerobes such as Clostl-idimz [ 151 or
Deszdfor~ibrio [ 16,171. and the pathways followed by
the various strains are very different.
The purpose of this study was (i) to isolate organisms responsible for reduction of nitrate to ammonium from sediments in which we have estimated the
nitrate ammonifying
activity using an extended
acetylene inhibition technique, and (ii) to seek evidence of this pathway on the isolates using ‘sN
isotopic methods. We also describe the role of nitrate
as an alternative electron acceptor in two isolates and
show how the availability of carbon substrate and
nitrate correlates with cell yield, the products of
nitrate reduction and glucose fermentation.
2. Material
and methods
2.1. Sediment studies
2.1. I. Study area
The sediments used were sampled at Carteau cove
in the Gulf of Fos. located I? miles east of Marseille
near the mouth of the river Rhone. on the French
Mediterranean
coast. Undisturbed
sediment cores
were taken by hand in Plexiglas tubes at 5 m water
depth.
-3.1.2. Nitrate wductiorl and denitrjfication in sediment slurries
The top 10 cm of sediment cores were sectioned
into 2 cm thick segments. 4 ml of each segment were
distributed
into 13 ml tubes containing
4 ml of
natural sea water. Denitrifying activities were measured in sediment slurries as described previously
using the acetylene block method [18]. The tubes
were sealed with rubber stoppers. and anaerobic
conditions were obtained by flushing N2 through the
tube. Acetylene (20 kPa), which inhibits the reaction
from N,O to N,. was distributed into the tube. Two
tubes were taken for analysis after 0, 0.25, 0.5. I. I .5
and 2 h incubation at 20°C. The linear initial rate of
N20 accumulation is considered as a measure of in
situ denitrification
activity. The nitrite reduction
(RNO,) was estimated in the tubes which were used
to determine denitrification
after NO< and NO,
analysis. The rate of nitrite reduction, the second
step of denitrification,
can be determined from the
difference between the rate of nitrate disappearance
and the net rate of nitrite production [ 191.
2.2. Strain sti&e.s
2.2.1. Pqmlatiorl densi?, isolatior~ arid chmvacterixrtiori of’ flitrate-redwirlg bmTetY0
The number of denitrifying
and nitrate-reducing
bacteria was determined using a modified MPN procedure. We used two different culture media prepared with artificial sea water (ASW) [30] in which
ammonium chloride was omitted: (i) a minimal glucose medium (P) containing glucose (5 g . I-’ ) and
potassium-nitrate
( I g . I- ’ ) and (ii) a medium (D)
generally used for denitrifying bacteria enumeration
containing
lactate (I g I-’ ). acetate (I g . I- ’ 1,
succinate ( I g 1_ ’ ) and potassium nitrate ( I g 1~’ 1.
The media were dispensed in 9 ml aliquots into
Hungate tubes, flushed with N, and sterilized. A
series of ten-fold dilution of sediment slurries ( I ml
sediment) was prepared in Hungate tubes containing
ASW (9 ml). Using syringes. 1 ml of suspended
inocula of each dilution was transferred into 3 tubes
P. Bmirt
/ FEM.5 Microhiolo,~y
of each medium (P and Dl. In order to block the
N1O reduction, 10 kPa of acetylene was added in D
medium. The tubes were incubated for one week at
30°C and the presence of denitrifying bacteria was
established from analysis of N,O in D medium. The
presence of nitrate ammonifying
bacteria was established from analysis of ammonium in P medium.
Bacterial strains were purified from nitrate agar
plates (bio-soyase 5 g I-‘, trypcase soja 5 g I- ‘,
agar 15 g . ll’. KNO, I g I-’ dissolved in ASW)
which had been streaked with the most diluted suspension of positive tubes from the MPN assays.
The ability of each isolate to reduce NO; to
NO;, N20 or NH: was determined by inoculating
Hungate tubes containing D or P media in the presence of NH,Cl and 10 kPa C? H, to block N20
reduction. Cultures were incubated for 48 h under
anaerobic conditions, then samples were assayed for
NO; reduction to NO;. N,O and NH:. Each isolate
was also characterized by Gram-strain. catalase, oxidase and the fermentation oxidation test using methods described by Stolp and Gadkari [21].
2.3. Media arid culture
ofpure strains
The bacteria were put on agar plates, and stock
cultures were prepared as described by Bonin et al.
La.
Anaerobic cultures were incubated at 30°C in a
150 ml serum flask containing
100 ml of medium
sealed with rubber stoppers. For bacterial cultures,
ASW was used as base medium; ammonium (NH,Cl
1 g . 1-l 1 was supplemented
as a nitrogen source.
Glucose. Na-L-lactate, Na-acetate, and potassium nitrate were added as indicated in the experiments
previously described. Anaerobic conditions were obtained by flushing nitrogen through the flask for 20
min. Acetylene (10 kPa) was injected in the flask to
block the nitrous oxide reductase activity. “N nitrate
(99.8% in excess) was added in some cultures and
corresponded to 5% of the initial N-nitrate content.
An inoculum of 1 ml of preculture growth on the
same medium was added just before sealing the
flask. At different sampling times, 5 ml of suspension and 2.5 ml of gas (in anaerobic flasks) were
sampled to analyze the different forms of nitrogen.
At the same time, growth was monitored by measuring optical density at 450 nm (Shimatzu UV 240
Ecology
19 (IYY6) 27-38
spectrophotometer).
Bacterial biomass
mined by dry-weight measurements.
29
was
deter-
2.4. Chemical analysis
Nitrate and nitrite concentrations
were determined
by continuous flow photometry with an autoanalyser
(Technicon
AA II): NO_ as described by BendSchneider and Robinson [23], NO; after reduction
by Cu-Cd column using the method of TrCguer and
Lecorre [24]. Ammonium was analyzed according to
the procedure of Solorzano [25].
The N20 in the headspace was sampled using a
preevacuated venoject tube. Extraction of N,O from
the liquid phase was carried out by the procedure of
Chan and Knowles [26] modified by the technique of
multiple equilibrium [27]. Nitrous oxide was determined using a gas chromatograph (Girdel series 301.
equipped with an electron capture detector as previously described [22].
To determine the isotopic excess of NH:
for
was separated by a diffusion
each sample, NH:
procedure. 2 ml of spent culture medium was treated
in a stoppered flask with mild alkali (MgO); conversion was carried out at 60°C during one week.
Evolved NH, was collected as N-NH:
in an acidified (50 ml of 0.5 N H2SOJ) disk cut from GF/D
filter (Whatmann)
suspended on the stopper [28].
The isotopic analyses were performed with a mass
spectrometer (ANCA-MS, Europa Scientific).
Glucose, lactate, acetate and ethanol were determined enzymatically using a test-kit from Boehringer.
Mannheim, Germany.
3. Results
3. I. Nitrate reduction in sediment slurries
Sediment samples were collected during the winter of 1992 at Carteau cove in the Gulf of Fos,
located 12 miles east of Marseille near the mouth of
the river Rh6ne. The station was at 5 m depth. The
sediments consisted of muddy sand. In sediment
interstitial water, the nitrate and ammonia concentrations ranged from 3.7 PM to 17.81 PM and from
130 to 280 PM, respectively (Table 1). Measurements of nitrite reduction and denitrification
were
undertaken. The aim of this part of the work was to
Table
I
Abiotic parameters, bacterial enumerations and activities of marine sediment versus depth
perform the pathway
this sediment sample.
of nitrate
ammonification
Depth (cm)
O-2
2-J
4-6
6-X
3.2. Isolrrtiot~ atld charucterizatiot~
NO;
(/.LM)
3.7
17.8
3.78
S.69
itzg-bacteria
NO;
(FM)
0.87
1.12
0.8
0.72
0.002I
0.0023
0.0028
170
280
130
100
+ 150
- 160
- I.50
- 230
68.8
I52
39.6
30.8
10.29(17)
11.17(7)
10.53C?l)
7.ll(19)
6.5 IO’
9.5 IO5
2 IO’
2.5 lOi
2 IO ’
1.5 10”
4.5 IO-’
1.6 IO’
N,O C/.LM)
0.004
NH:
Redox (mV)
NO;
reduction
I
pmol.l-‘-d-l
Denitrification
~mol.l-‘,d-‘(S)
Denitrifiers
(bact. ml-’
)
Nitrate ammonifiers
(bact. ml-’
)
investigate if the sediment showed a capacity for
dissimilatory NO, reduction to NH:, before undertaking the isolation of strains associated with this
process. For all samples, denitrification
ranged from
7-l 1 pmol . 1-I . d-’ and nitrite reduction
proceeded at velocities several times higher than denitrification. Although the use of the acetylene blockage
method could lead to underestimation
of denitrification in some ecosystems, we have chosen to estimate
the nitrate ammonification
capacity using S@rensen’s
procedure [5] because it is simple and does not
require any addition to the natural nitrate pool. This
procedure is based on the assumption that: (i) N,O
reduction and nitrification are completely blocked by
acetylene under anaerobiosis, and (ii) in marine sediments where ammonium concentration
exceeds 200
PM. nitrate assimilation processes are insignificant.
So dissimilatory
nitrate reduction can be estimated
from overall nitrite reduction minus denitrification.
According to this hypothesis, the percentage of nitrite transformed in NzO by denitrification
ranged
from 7 to 2 1% of the nitrite reduction, indicating that
nitrate ammonification
can be taken as the major
pathway of nitrate dissimilation in these sediments.
The most probable number enumeration
gives
values ranging from lo5 bacteria. ml-’ to 9.5 IO5
bacteria . ml ’ and from 1.6 IO3 bacteria. ml-’ to
4.5 10’ bacteria. ml-’ for denitrifiers and ammonium producers, respectively. These results undoubtedly demonstrate the presence of bacteria able to
qf nitrate
in
reduc-
Strains able to grow under anerobiosis with nitrate
as an electron acceptor were isolated from P medium
because of its considerable glucose content and its
ability to support fermentative growth. After incubation for two weeks under anaerobic conditions. media inoculated with the most diluted suspensions
showing growth were spread on plates of nitrate
agar. After 48 h of incubation,
28 colonies were
selected. This isolation procedure is used to select
the strains which were most numerous in the sediment sample. Fourteen isolates were purified from
the 2-4 cm thick segment where the presumed ntrate ammonification
capacity is greatest (strain numbers 14 to 39) and a total of 14 strains from the other
slices combined.
The ability of the strains to reduce nitrate to NzO
(in the presence of C,H,) or ammonium was examined with P medium under anaerobic conditions with
or without ammonium amendment (1 g. 1-l 1 as a
nitrogen source for growth. For the isolates (numbers
6, 9, 11, 12 and 13), which showed a slight growth
with glucose as a source of carbon and energy.
glucose was replaced by the same amount of Nalactate.
During the experiment
where ammonium
was
omitted. the cells grew very slowly. Contrary to the
results reported by Fazzolari et al. [29] or Samuelson
and Ronner [ 121, after 3 days of incubation,
the
accumulation
of ammonium as a product of nitrate
ammonification
was insignificant
or not observed.
The rate of ammonium
assimilation
seems to be
higher than that of production by nitrate ammonification.
Based on the hypothesis that 1 g . 1-l NH: was
sufficient to block nitrate assimilation
[I], we attempted to investigate
if ammonium
is produced
from nitrate by comparing the amount of nitrite plus
nitrous oxide produced in the presence of acetylene
and the amount of nitrate consumed. If the nitrogen
budget deficit was higher than 30% (3 mM), strains
were presumed to be nitrate ammonifiers. Except for
the ammonium producers, the average recovery of N
P. Bark / FEMS Microbiulogy Ecology I9 (19961 27-38
(NO;, NO; and N,O 1 was good (about 90%) (Fig.
1). Among the isolates, three groups of bacteria were
identified with regard to the end products: (i) nitrite
producers which transformed the overall nitrate reduced to nitrite: strains 33. 34, 37, 38 and 41; (ii)
denitrifiers which reduced nitrate to gaseous products (N,O and N?): strains 2, 5, 6, 8, 10, 11, 12, 13,
14, 17, 18, 19 and 21; and (iii) nitrate ammonifiers:
strains 1, 7, 9, 15, 16, 20. 22. 39, 40 and 45. For the
latter strains. the sum of the nitrate reduction products ( NO; plus N,O) accounted for less than 70%
of the consumed nitrate; these strains are presumed
to be nitrate ammonifiers.
Except for strain numbers
16 and 17, for all
denitrifiers, the nitrous oxide production decreased
when the cells were incubated in the absence of
acetylene; these two strains are not able to reduce
nitrous oxide reductase to molecular nitrogen.
With nitrate reducers and ammonifiers,
nitrous
oxide was produced independently of the presence of
acetylene. This could be correlated with the nitrite
reductase activity on nitrate as reported by Smith and
Zimmermann
[3].
3.3. Physiology
of nitrate reduction
Five isolates (strains 2, 5, 20, 39 and 45) were
selected for further studies together with a bacterium
31
previously isolated in our laboratory and identified
as the denitrifier, Pseudomonas nautica IP 617 [22].
The preliminary
experiments
indicated that strains
20, 39 and 45 were presumed ammonium producers
and strains 2 and 5 denitrifiers.
A 15N study was undertaken to verify that the
produced NH l was derived from NO;. Measurements were made during anaerobic
growth on
medium containing glucose (1 g . l-l), Na-lactate (1
g .l-‘)
and KNO, (1 g .l-‘).
The medium was
enriched with 500 pmol of l5 NO; (99% in excess)
per I. In order to block nitrous oxide reduction, 10
kPa acetylene were added. Fig. 2 correlates nitrogen
budget patterns and optical density. With regard to
denitrifiers, for Ps. nautica (Fig. 2a), when NO,
disappeared, NO;
concentration
increased with a
transient accumulation
(less than 1 mM) at the end
of the exponential phase of growth, whereas strains 2
and 5 accumulated a much lower concentration
of
nitrite at the midexponential
phase (Fig. 2b, c). The
production of nitrous oxide was-observed at up to 4.5
mM, as early as the beginning of the growth, nitrate
was stoichiometrically
converted to N,O. The N20
accumulation
was acetylene-dependent
(data not
shown). The “N analysis performed on NH: and
organic nitrogen fraction revealed that “NO,
was
not transformed to 15NHT. Thus, the nitrogen budget
18
16
14
i
strain
Fig. I. Products of nitrate reduction by the isolated strains grown
NO,: shaded bars. NO;: solid bars N,O.
number
in P medium
under anaerobic
conditions
with acetylene.
Striped bars,
33
P. Bonito / FEMS Microhiolog~
patterns confirm that these three strains are denitrifying bacteria.
In contrast, the same 15N experiments indicated
the presence of three isolates (strains 20, 39 and 45)
able to perform the dissimilation of nitrate to ammonium (Fig. 2d-f). The growth pattern on nitrate was
the same for the three strains. All NO_; that had
disappeared, is accumulated as NO, until the end of
the exponential phase of growth with a maximum
NO; concentration of 8.53 mM, 10.05 mM and 9.13
mM for strains 20, 39 and 45, respectively. Between
Ecology
IY f lYY6) 27-38
I l-20% of the NO, added was converted to N,O.
This N,O production was also observed in the absence of acetylene. “N-ammonia
production began
at the end of the log phase. After depletion of NO;,
the accumulated NO; was reduced to ammonium
which reached. after 48 h, a maximum concentration
of I. I4 mM. 2.5 mM and 1.92 mM for strains 20. 39
and 45. respectively. After 32 h, part of the “NO,;
reduced to free “NH:
has been incorporated into
organic matter. Only about I PM of NO, has been
converted to N-organic matter via NH: production.
1
d
.l cj
I
.Ol
0
10
30
20
40
50
60
0
lo
20
40
30
Time(h)
'rime(h)
b
2
‘Ibe
Time (h)
(hl
.Ol
0
10
20
30
TimeIh)
40
50
60
0
10
20
30
40
50
60
Time(h)
Fig. 2. Growth of the selected strains under anaerobic conditions with acetylene. (a) Psrudon~ottn.t r~cruricu: (b) strain number 2; (c) strain
number 5: Cd) strain number 20: (e) strain number 39: (f) strain number 45. Striped bars, NO;; shaded bars, NO;; solid bars, N,O; white
bars. NH:.
P. Bonin / FEMS Microbiology
3.4. Growth characteristics
and Vibrio sp. 45
of Enterobacter
Ecoloy~
19 (19%)
33
27-38
mentative metabolism. Enterobacter are non-motile
coccobacillus oxidase positive and Vibrio are mobile
incurved rods oxidase negative.
In the following study the selected strains are
called Enterobacter sp. 39 and Vibrio sp. 45.
The effects of media composition on the products
of NO; reduction by the two selected strains were
examined (Fig. 3 and Table 2). Cell growth, glucose
consumption, acetate and ethanol production and the
sp. 39
Strains 39 and 45 were chosen to represent the
isolated ammonium producers. A limited number of
morphological
and biochemical
tests permit inclusion of these two strains in the genera Enterobacter
and Vibrio, respectively. Both genera group species
that are Gram-negative,
catalase positive with fer-
a : Enterobacter
sp. 39
Growth conditions
G20
G4Nl
Nl
G4NlO
G20
NlO
0
48
BPNIO
120t--
0
12
48
78
12
78
Time (h)
b : Vlrio
sp. 45
Growth conditions
G4Nl
‘0
48
12
78
G20
Nl
G4NlO
G20
NlO
I
BPNlO
0
Time (h)
Fig. 3. Percentage of nitrate reduction products by Entrrobrrcrer
sp. 39 and Vibrio sp, 45 grown with various nitrate or glucose
concentrations.
G4 and G?O correspond to glucose concentrations of 4 and 20 mM: Nl and NlO to nitrate concentration (KNO,) of 1 and 10
mM respectively; BP: bactopeptone. Striped bars. NO;: shaded bars, NO;: solid bars. N,O: white bars. NH:.
34
P. Bunk
/ FEMS Microbiology
nitrogen compound patterns were followed under
aerobic and anaerobic conditions in the presence (1
or 10 mM) or absence of nitrate and with two
glucose concentrations
(4 or 20 mM). The assimilation of nitrate was inhibited by ammonium (1 g . 1-l )
[Il.
For both strains, 15N ammonium
was produced
under all growth conditions. Results obtained from
nitrogen limited culture (I mM nitrate) in the presence of 4 mM or 20 mM of glucose show that Vibrio
sp. 45 (Fig. 3b) transformed the totality (100%) of
nitrate reduced to ammonium, whereas Enterobucter
sp. 39 (Fig. 3a) tended to produce slightly less.
Enterobacter
sp. 39, which increased the nitrate
concentration
to IO mM, produced a culture with a
less efficient utilization of nitrate for growth. About
50% and 20% of nitrate utilized was excreted as
ammonium for 4 and 20 mM glucose, respectively.
Vibrio sp. 45, in the medium containing
20 mM
glucose and IO mM nitrate, produced significantly
more NO; and only 10% of nitrate was reduced to
NH j. In bactopeptone medium (5 g . l_ ’ ), the amount
of ammonium produced seems smaller, presumably
because this substrate was less fermentative.
In the presence of 10 mM KNO,, glucose addi-
Ecolo<gy 19 (IYY6) 27-38
tion increased the apparent production of nitrite at
the end of the exponential phase of growth, probably
causing growth inhibition. This is consistent with
results presented in Table 2; in the presence of 10
mM nitrate no increase in glucose consumption was
observed when the glucose concentration
was increased from 4 to 20 mM. Moreover, under anaerobic conditions, when the glucose concentration
increased from 4 to 20 mM, both strains exhibited a
lower yield of cells per mol glucose utilized.
For Enterobucter
sp. 39, the nitrate addition (I
mM and IO mM) augmented the molar yield on
glucose by 2 and 3 times than estimated in the
absence of nitrate (Table 2). For Vibrio sp. 45, the
effect of NO, addition on the growth yields was less
marked than on Esterobacter
sp. 39. Caskey and
Tiedje [30] observed the same effect and concluded
that NO, allows more efficient utilization of the
energy derived from glucose metabolism and that the
increase of the YATp could only be explained if NO;
was being reduced by dissimilatory mechanisms.
In the aerobic culture only acetate was transiently
found. Nitrate had no appreciable effect on growth;
no nitrate was consumed and neither nitrite nor
ammonium were accumulated even at high density
Table 2
Influence of culture media on the growth yield. on the use of glucose and the production
Vibrio sp. 45 under anaerobic conditions
Growth
conditions
Enterobuctrr
Glucose
consumption
YX/S
tmMl
I
6.46
G4NIO
3.87
G20NIO
2.17
37.5
33.9
80.0
40.2
134.0
93.0
G4NO
2.89
73.0
G20NO
6.83
55.6
G4N 1
4.79
82.0
3.68
G20NO
5.07
I
4.00
G20N
)
Acetate
produced (mMl
6.92
5.79
0.46
0.69
0.09
0.3 I
6.42
6.81
0.5 I
0.43
0.06
0.07
0
0
1.29
I .02
3.96
4.2 I
sp. 45
Vibrio
I
8.73
69.0
G4NlO
4.19
93.0
G20N IO
4.53
70.9
G20N
Ethanol
produced (mM
sp. 39 and
sp, 39
G4NO
G4N
of ethanol and acetate by Entrrobncrrr
Yx/s is expressed in g cell dry weight. mmol~ ’ glucose consumed.
Cl4 and Cl20 corresponds
to glucose concentrations
of 3 and 30 mM. NI and NIO to nitrate
respectively.
0.66
0.44
I.50
I .88
3.75
3.34
concentration
(KNO,)
I and 10 mM
P. Benin / FEMS Microbiology
of cells at the end of culture (data not shown). This
fact was additional evidence supporting the dissimilatory function of ammonium production.
For both strains tested, in anaerobic culture without nitrate the main product of glucose fermentation
is ethanol (Table 2). In the presence of nitrate. the
formation of ethanol declined and acetate was produced instead (Table 2); the higher the NO, concentration, the greater was the acetate production and
the lower the ethanol production.
4. Discussion
In the studied site, the two major pathways of
NO; dissimilation
have been observed: denitrification and nitrate ammonification
(DRNA). The latter
pathway seems to be most dominant in the Carteau
cove. Both the acetylene inhibition technique and
15N isotope assay can be used to determine the
distribution
of dissimilatory
nitrate reduction. The
15N isotope assay has the obvious advantage of
directly measuring 15NH: production; but in sediments where the nitrate concentration
is very low
(only a few FM), such as those studied, the addition
of “NO_; can stimulate the activities; the increase of
NO,
concentration
can favour denitrification.
In
contrast to this method, the extended version of the
acetylene inhibition technique may result in underestimation of denitrification.
Indeed, the efficacy of
this method could be reduced at low NO, concentration and under strongly reducing conditions
[31].
Although the C2H? method has disadvantages,
it
allows estimation of DRNA activity in such a sediment where nitrate ammonification
can account for
most of the NO, consumption
(about 80%). These
results are in accordance
with those obtained in
estuarine sediments, showing that nitrate ammonification was 65-9010 of the overall nitrate consumption [5.32].
Although dissimilatory nitrate reduction to ammonium is recognized as important in anaerobic habitats
such as marine sediment [4,5], this process has not
been systematically
studied at the microbiological
level. Enumerations
of denitrifiers and nitrate ammonifiers have also been undertaken. Whereas the
nitrate ammonification
is presumed to be a major
pathway of nitrate dissimilation in these sediments, it
Ecology
IY
ClYY6127-3X
35
is surprising that the number of ammonium producers is IOO-fold lower than that of the denitrifiers.
There are various possible explanations:
(i) perhaps
not all denitrifiers are active in the sediment, (ii)
nitrate ammonifiers are more active than denitrifiers,
or (iii> the culture medium used is not the most
suitable medium for enumerating
dissimilatory
nitrate reducers to ammonium
in marine sediment.
Indeed, direct plating methods for the isolation of
nitrate-respiring
bacteria select for the growth of the
fastest growing members which are not necessarily
important in situ. Herbert and Nedwell [33] have
reported that using direct plating methods on nutrient-rich media, Aerornonas/ Vibrio spp. (DRNA)
were numerically
dominant, whereas chemostat enrichments
yielded
primarily
oxidative
bacteria
(Pseudomonas
and Acinetobacter
with acetate as
C-source) or fermentative types such as nitrate ammonifiers (Klebsiella
and Vibrio with glycerol as
C-source). A total of 28 strains were isolated from P
medium. Although the isolation procedure was different and the number of isolates studied was small,
it is interesting to note that in marine sediment, the
ratio denitrifiers/ammonium
producers (1.8: 1) obtained in this study is close to those reported with
soil samples by Fazzolary et al. [29] (1.5: 11, or Smith
and Zimmerman [3] (1.7: 1). These authors observed
that among 214 soil bacterial isolates able to reduce
NO,, 209 produced N,O and only 46 were denitrifiers. Moreover, they reported that under adequate
growth conditions most non-denitrifying
N,O producers were capable of fermentative dissimilation of
NO,
to NH,+. At the present time, there is no
evidence to support the classification of nitrite producers with ammonium producers.
Two strains with DRNA capacity, belonging to
Enterobucter and Vibrio genera, were selected. Their
glucose metabolism and growth were studied in the
presence or absence of NO,. The only data in
literature on the population ecology of the dissimilatory nitrate reduction to ammonium are reported by
Cole and Brown [14] and Herbert and Nedwell [33].
These authors found that Aeromonas/Vibrio
spp.
are the most prevalent nitrate reducers followed by
Enterobacteriaceae.
The Vibrio spp. isolated by Cole
and Brown are able to accumulate ammonium under
only NO;
limiting conditions,
while the species
selected by the latter authors did not reduce nitrate to
creased thereby could result from the increase in
ATP synthesis
during the formation
of acetate,
NADH being the major source of electrons for nitrite
reduction as it has been previously described for E.
coli [38]. Thus energy metabolism and the regulatory
hierarchy with respect to the use of electron acceptors were very similar to those known from E. coli.
The NOj seems to act as an electron sink allowing
the reoxidation of NADH produced during glycolysis. Pyruvate formed led to the production of acetate
and ATP via acetyl CoA and acetylphosphate. In the
absence of NO;, the NADH reduced acetyl CoA to
ethanol without ATP (Fig. 4). Whereas Rehr and
Klemme [39] reported that the ratio of the fermentation products, ethanol and acetate, was strongly affected by C/N ratio, our results clearly showed that
while the C/N
ratio was 5-fold higher, the
acetate/ethanol
ratio remained constant (Table 2).
Nevertheless.
the different values of the ratio of
fermentation products were correlated to the limiting
or not limiting nitrate supplies (I mM or 10 mM).
For both strains studied, the comparison of growth
conditions
indicates that such a process is more
ammonium. From these limited data the DRNA capacity of Enterobacteriaceae
and Vibrio may be even
more widespread than hitherto recognized [34]. According to the authors cited and judging from the
facility to isolate Eilterobacter sp. 39 and Vibrin sp.
45 from the most diluted suspension
of positive
tubes from the MPN assays for nitrate reducer enumeration, these strains may be the most common
nitrate ammonifiers
in marine sediments. The process of dissimilatory nitrate reduction to ammonium
by both isolates was demonstrated using ‘“N experiments. The growth kinetics and the nitrogen compound patterns are consistent with data obtained with
Citrobacter sp. [35], Ps. putrefaciens [36] and Enterobacter amnigenus [29]. In glucose fermentation,
the products of fermentation
(acetate, ethanol and
formate) are produced in amounts roughly equivalent
to the glucose consumed [37]. The type and relative
amounts of the products were significantly
affected
by the presence of NOj
and OZ. Whatever the
strain, physiological
studies show that for both isolates, when nitrate was added, more acetate and less
ethanol were accumulated.
The growth yield in-
alcohol
dehydrogenase
1 Ethanol+
1 Acetaldehyde
aldehyde
dehydrogenase
+
1 Acetyl CoA
t
*
2 NAD+
pyruvate formate lyase
2 NADH
1 Glucose-,
2 Pyruvate
*
)
1 Acetyl CoA
1 Formate
5 H+
lNO2-
1 NH4++3C02+2H20
1 NADH
1
-
2 Formate
formate nitrite
reductase
phosphotransacetylae
NH4+
+
1
NO2-
NADH nitrite oxidoreductase
1 Acetyl P
acetate kinase
1 Acetate
Fig. 1. Glucose fermentation
with or without nitrate
efficient in nitrate limited culture and apparently
depends on the nature and the concentration
of the
available carbon.
Acknowledgements
This work was partly supported by funds from the
‘Programme National d’OcCanologie Cot&-e’. I am
grateful to Dr. G. Slawyk for his interest in the work.
and M. Paul for his careful reading. I wish to thank
N. Garcia and D. Raphel for I5 ammonium
and
nitrate analyses, respectively.
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