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Anaerobic nitrate reduction to ammonium in two strains isolated from coastal marine sediment: A dissimilatory pathway
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
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.
References [I] Payne. W.J. (1973) Reduction of nitrogenous oxides by microorganisms. Bacterial. Rev. 37, 409-352. [Z] Stouthamer, A.H. (19881 Dissimilatory reduction of oxidized nitrogen compounds. In: Biology of Anaerobic Microorganisms (Zehnder, A.J.B., Ed.), pp. 179-24-l. John Wiley. New York. [3] Smith. S. and Zimmerman. K. (1981) Nitrous oxide production by non denitrifying soil nitrate reducers. Soil Sci. Sot. Am. J. 35, 865-871. [4] Koike. I. and Hattori. A. (I 978) Denitrification and ammonia formation in anaerobic coastal sediments. Appl. Environ. Microbial. 35. 278-282. [5] Sorensen, J. ( 1978) Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Appl. Environ. Microbial. 35, 301-305. [6] Nishio. T.. Koike. I. and Hattori. A. (1982) Denitrification. nitrate reduction and oxygen consumption in coastal and marine sediment. Appl. Environ. Microbial. 43. 648-653. [7] Binnerup. S.J.. Jensen, K.. Revsbech. N.P.. Jensen. M.H. and Sorensen. J. ( 19921 Denitrification. disaimilatory nitrate reduction to ammonium and nitrification in a bioturbated estuarine sediment as measured with 15 N and microsensor techniques. Appl. Environ. Microbial. 58, 303-3 13. [8] Tiedje, J.M.. Sextone, S.. Mirold. D.D. and Robinson, J.A. ( 1982) Denitrification: ecological niches, competition and survival. Ant. Van Leeuwenhoeck 48, 261-284. [9] Benin, P., Gilewicz. M. and Bertrand, J.C. (1989) Effect of oxygen on each step of denitrification on P.tr&r~rnonns rrcrrrticrr strain 617. Can. J. Microbial. 35. 1061-1064. [IO] Dunn. G.M.. Herbert, R.A. and Brown, C.M. (1979) Influence of oxygen tension on nitrate reduction by a Klehsielkr sp. growing on chemostrat culture. J. Gen. Microbial. 112, 379-383. [l I] Knowles, R. (1982) Denitrification. Microbial. Rev. 36. 3370. [ 121 Samuelson. M.O. and Ronner. U. (1982) Ammonium production by dissimilatory nitrate reducers isolated from Baltic sea
water. as indicated by ‘sN study. Appl. Environ. Microbial. 34. 1241-1233. 1131MacFarlanne. G.T. and Herbert, R.A. (1982) Nitrate dissimilation by Vlbrio sp. isolated from estuarine sediments. J. Gen. Microbial. 128. 2463-2468. 11‘4 Cole. J.A. and Brown. C.M. (1980) Nitrite reduction to amonia by fermentative bacteria: A short circuit in biological nitrogen cycle. FEMS Microbial. Lett. 7. 65-72. G.T. and Herbert, R.A. (19821 [I51 Keith. S.M.. MacFarlanne. Diasimilatory nitrate reduction by a strain of clostridium isolated from estuarine sediments. Arch. Microbial. 66. 132162. [I61 Keith. S.M. and Herbert, R.A. (1982) Dissimilatory nitrate reduction by a strain of Des@~~ihrio desn!filric(lus. FEMS Microbial. Lett. 18. 55-59. 1171 Dalsgard, T. and Bak. F. (19941 Nitrate reduction in a sulfate reducing bacterium. Drsttfforibrin tlesu~fttricam. isolated from rice paddy soil: Sulfide inhibition, kinetics and regulation. Appl. Environ.Microbiol. 60, 291-294. [I81 Raymond, N.. Benin. P. and Bertrand, J.C. (1992) Comparison of methods for measuring denitrifying activity in marine sediments from the Western mediterranean coast. Oceanologica Acta IS. 137-143. E.. Mille, G. and [I91 Bonin. P.. Gilewicz. M.. Rambeloarisoa. Bertrand, J.C. (1990) Effect of crude oil on denitrification and sulfate reduction in marine sediments. Biogeochemistry 10. 161-173. [201 Baumann. P. and Baumann, L. (1981) The marine gram negative eubacteria: genus Pttorohncferilrrll. Brnrcken. A/t~ro~mwm, Psedmorm md Alcnligetws. In: The Prokaryotes (Starr. M.P.. Stolp. H., Trupper, H.G.. Balows. A. and Schlegel. H.G.. Eds.), pp. 1302- 1330. Springer Verlag, New York. members [211 Stolp. H. and Gadkari. D. ( 198 I) Non-pathogenic of the genus Prrudormm~.~. In: The Prokaryotes (Starr. M.P.. Stolp. H., Trupper. H.G.. Balows. A. and Schlegel, H.G.. Eds.). pp. 719-741. Springer Verlag. New York. [Xl Bonin. P.. Gilewicz. M. and Bertrand. J.C. (1989) Denitrification by a marine bacterium Pse~/omonn.~ ~~outicn strain 617. Ann. Inst. Pasteur Microbioi. 138. 371-383. [Xl Bendschneider. K. and Robinson. R.J. (1972) A new spectrometric method for determination of nitrite in sea water. J. Marine Res. I. 87-963. [W Treguer. P. and Le Corre, P. (1975) Manuel d’analyse des sels nutritif5 dans I’eau de mer. Laboratoire d‘oceanologie chimique. Universite de Bretagne Occidentale. Brest. France. 1IO pp. [I51 Solorzano. L. (I 969) Determination of ammonium in natural waters. the phenol hypochlorite method. Limnol. Oceanogr. 13. 799-801. [261 Chan, Y.K. and Knowles. R. (1979) Measurement of denitrification in fresh-water sediments by an in situ acetylene inhibition method. Appl. Environ. Microbial. 37. 1067-1072. P71 MacAulife. L. (I971 l GC determination of solutes by multiple phase equilibration. Chem. Technol. I. 36-5 I. analysis. In: [781 Hauck. R.D. (19821 Nitrogen-Isotope-ratio Methods of Soil Analysis, Agro. Monogr. 9. pt. 2. 2nd ed.
(Page. A.L. et al.. Eds.). pp. 735-779. Agronomy. [29] Fazzolari,
American Society of
Madison. Wisconsin. E.. Mariotti.
A. and Germond. J.C. (1990)
tt’r ~nni,~er~tv. Can. J. Microbial. W.H.
and Tiedje.
J.M.
Nitrate
36. 779-7X (1980)
I. of
119. 3-17-7-23.
ment. advantage*. und potential problems. In: Denitrification (Revsbech.
N.P.
and Stirenxen. J..
Edh.). pp. 15 I - 166. Plenum Press. New York and London. [3?] Sgrensen. J. (1987)
Nitrate
reduction
in marine sediment:
Pathways and interaction with iron and sulfur cycling. Gr-
I,
omicrobiol. J. 5. 40142
[33] Herbert.
R.A. and Nedwell.
factors in regulating
(I 990)
Role of environmental in intertidal
sedi-
in Soil and Sediment (Revshech.
N.P. and Sorensen. J., Eds.), pp. 77-89. York and London.
(Zehnder.
A.J.B.,
Plenum Press. New
Ed.),
pp.
179-241.
Disaimilatory reduction of NO? to NH:
and N,O by a soil Citrohocter
sp, Appl. Environ. Microbial.
43. 854-860. (1985)
Dissimilatory
nitrate reduction of
nitrite. nitrous oxide and ammonium by Psrudornor~trs pmre,fi/cir~~. Appl. Environ. Microbial. [37] Weimer.
P.J. (1981)
_elucosn fermentation biol. 130. 103-l
Control
50. 811-S 15.
of product formation
during
by Bnci//~.c m(zcelz111.s. .I. Cen. Micro-
Il.
[38] Stewart, V. (1988) Nitrate reduction in relation to facultative metabolism in Enterobacteria. Microbial.
B.
nitrate respiration
men&. In: Denitrification
bic Microorganisms
[36] Samuelson, M.O.
[31] Knowles. R. (1990) Acetylene inhibition technique: Developin Soil and Sediment
Ecology of denitrification and dissimila-
John Wiley and Sons. New York. [35] Smith. M.S. (1987)
The reduction
nitrate to ammonium by a C/~stridi~>t~ sp. isolated from soil. J. Gen. Microbial.
( 1988).
tory nitrate reduction to ammonium. In: Biology of Anaero-
reduction to ammonia: a dissimilatory procesh in Otterohac,[30] Caskey.
[33] Tiedje. J.M.
[39] Rehr, B. and Klemme. between denitrifying
J.H. (1989)
for nitrate
Psrudofr~ontrs .sfuf:rr-i and nitrate am-
monifyinp enterobacteris. 330.
Rev. 6, 190-232.
Competition
FEMS
Microbial.
Ecol. 62. 211-
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
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.
References [I] Payne. W.J. (1973) Reduction of nitrogenous oxides by microorganisms. Bacterial. Rev. 37, 409-352. [Z] Stouthamer, A.H. (19881 Dissimilatory reduction of oxidized nitrogen compounds. In: Biology of Anaerobic Microorganisms (Zehnder, A.J.B., Ed.), pp. 179-24-l. John Wiley. New York. [3] Smith. S. and Zimmerman. K. (1981) Nitrous oxide production by non denitrifying soil nitrate reducers. Soil Sci. Sot. Am. J. 35, 865-871. [4] Koike. I. and Hattori. A. (I 978) Denitrification and ammonia formation in anaerobic coastal sediments. Appl. Environ. Microbial. 35. 278-282. [5] Sorensen, J. ( 1978) Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Appl. Environ. Microbial. 35, 301-305. [6] Nishio. T.. Koike. I. and Hattori. A. (1982) Denitrification. nitrate reduction and oxygen consumption in coastal and marine sediment. Appl. Environ. Microbial. 43. 648-653. [7] Binnerup. S.J.. Jensen, K.. Revsbech. N.P.. Jensen. M.H. and Sorensen. J. ( 19921 Denitrification. disaimilatory nitrate reduction to ammonium and nitrification in a bioturbated estuarine sediment as measured with 15 N and microsensor techniques. Appl. Environ. Microbial. 58, 303-3 13. [8] Tiedje, J.M.. Sextone, S.. Mirold. D.D. and Robinson, J.A. ( 1982) Denitrification: ecological niches, competition and survival. Ant. Van Leeuwenhoeck 48, 261-284. [9] Benin, P., Gilewicz. M. and Bertrand, J.C. (1989) Effect of oxygen on each step of denitrification on P.tr&r~rnonns rrcrrrticrr strain 617. Can. J. Microbial. 35. 1061-1064. [IO] Dunn. G.M.. Herbert, R.A. and Brown, C.M. (1979) Influence of oxygen tension on nitrate reduction by a Klehsielkr sp. growing on chemostrat culture. J. Gen. Microbial. 112, 379-383. [l I] Knowles, R. (1982) Denitrification. Microbial. Rev. 36. 3370. [ 121 Samuelson. M.O. and Ronner. U. (1982) Ammonium production by dissimilatory nitrate reducers isolated from Baltic sea
water. as indicated by ‘sN study. Appl. Environ. Microbial. 34. 1241-1233. 1131MacFarlanne. G.T. and Herbert, R.A. (1982) Nitrate dissimilation by Vlbrio sp. isolated from estuarine sediments. J. Gen. Microbial. 128. 2463-2468. 11‘4 Cole. J.A. and Brown. C.M. (1980) Nitrite reduction to amonia by fermentative bacteria: A short circuit in biological nitrogen cycle. FEMS Microbial. Lett. 7. 65-72. G.T. and Herbert, R.A. (19821 [I51 Keith. S.M.. MacFarlanne. Diasimilatory nitrate reduction by a strain of clostridium isolated from estuarine sediments. Arch. Microbial. 66. 132162. [I61 Keith. S.M. and Herbert, R.A. (1982) Dissimilatory nitrate reduction by a strain of Des@~~ihrio desn!filric(lus. FEMS Microbial. Lett. 18. 55-59. 1171 Dalsgard, T. and Bak. F. (19941 Nitrate reduction in a sulfate reducing bacterium. Drsttfforibrin tlesu~fttricam. isolated from rice paddy soil: Sulfide inhibition, kinetics and regulation. Appl. Environ.Microbiol. 60, 291-294. [I81 Raymond, N.. Benin. P. and Bertrand, J.C. (1992) Comparison of methods for measuring denitrifying activity in marine sediments from the Western mediterranean coast. Oceanologica Acta IS. 137-143. E.. Mille, G. and [I91 Bonin. P.. Gilewicz. M.. Rambeloarisoa. Bertrand, J.C. (1990) Effect of crude oil on denitrification and sulfate reduction in marine sediments. Biogeochemistry 10. 161-173. [201 Baumann. P. and Baumann, L. (1981) The marine gram negative eubacteria: genus Pttorohncferilrrll. Brnrcken. A/t~ro~mwm, Psedmorm md Alcnligetws. In: The Prokaryotes (Starr. M.P.. Stolp. H., Trupper, H.G.. Balows. A. and Schlegel. H.G.. Eds.), pp. 1302- 1330. Springer Verlag, New York. members [211 Stolp. H. and Gadkari. D. ( 198 I) Non-pathogenic of the genus Prrudormm~.~. In: The Prokaryotes (Starr. M.P.. Stolp. H., Trupper. H.G.. Balows. A. and Schlegel, H.G.. Eds.). pp. 719-741. Springer Verlag. New York. [Xl Bonin. P.. Gilewicz. M. and Bertrand. J.C. (1989) Denitrification by a marine bacterium Pse~/omonn.~ ~~outicn strain 617. Ann. Inst. Pasteur Microbioi. 138. 371-383. [Xl Bendschneider. K. and Robinson. R.J. (1972) A new spectrometric method for determination of nitrite in sea water. J. Marine Res. I. 87-963. [W Treguer. P. and Le Corre, P. (1975) Manuel d’analyse des sels nutritif5 dans I’eau de mer. Laboratoire d‘oceanologie chimique. Universite de Bretagne Occidentale. Brest. France. 1IO pp. [I51 Solorzano. L. (I 969) Determination of ammonium in natural waters. the phenol hypochlorite method. Limnol. Oceanogr. 13. 799-801. [261 Chan, Y.K. and Knowles. R. (1979) Measurement of denitrification in fresh-water sediments by an in situ acetylene inhibition method. Appl. Environ. Microbial. 37. 1067-1072. P71 MacAulife. L. (I971 l GC determination of solutes by multiple phase equilibration. Chem. Technol. I. 36-5 I. analysis. In: [781 Hauck. R.D. (19821 Nitrogen-Isotope-ratio Methods of Soil Analysis, Agro. Monogr. 9. pt. 2. 2nd ed.
(Page. A.L. et al.. Eds.). pp. 735-779. Agronomy. [29] Fazzolari,
American Society of
Madison. Wisconsin. E.. Mariotti.
A. and Germond. J.C. (1990)
tt’r ~nni,~er~tv. Can. J. Microbial. W.H.
and Tiedje.
J.M.
Nitrate
36. 779-7X (1980)
I. of
119. 3-17-7-23.
ment. advantage*. und potential problems. In: Denitrification (Revsbech.
N.P.
and Stirenxen. J..
Edh.). pp. 15 I - 166. Plenum Press. New York and London. [3?] Sgrensen. J. (1987)
Nitrate
reduction
in marine sediment:
Pathways and interaction with iron and sulfur cycling. Gr-
I,
omicrobiol. J. 5. 40142
[33] Herbert.
R.A. and Nedwell.
factors in regulating
(I 990)
Role of environmental in intertidal
sedi-
in Soil and Sediment (Revshech.
N.P. and Sorensen. J., Eds.), pp. 77-89. York and London.
(Zehnder.
A.J.B.,
Plenum Press. New
Ed.),
pp.
179-241.
Disaimilatory reduction of NO? to NH:
and N,O by a soil Citrohocter
sp, Appl. Environ. Microbial.
43. 854-860. (1985)
Dissimilatory
nitrate reduction of
nitrite. nitrous oxide and ammonium by Psrudornor~trs pmre,fi/cir~~. Appl. Environ. Microbial. [37] Weimer.
P.J. (1981)
_elucosn fermentation biol. 130. 103-l
Control
50. 811-S 15.
of product formation
during
by Bnci//~.c m(zcelz111.s. .I. Cen. Micro-
Il.
[38] Stewart, V. (1988) Nitrate reduction in relation to facultative metabolism in Enterobacteria. Microbial.
B.
nitrate respiration
men&. In: Denitrification
bic Microorganisms
[36] Samuelson, M.O.
[31] Knowles. R. (1990) Acetylene inhibition technique: Developin Soil and Sediment
Ecology of denitrification and dissimila-
John Wiley and Sons. New York. [35] Smith. M.S. (1987)
The reduction
nitrate to ammonium by a C/~stridi~>t~ sp. isolated from soil. J. Gen. Microbial.
( 1988).
tory nitrate reduction to ammonium. In: Biology of Anaero-
reduction to ammonia: a dissimilatory procesh in Otterohac,[30] Caskey.
[33] Tiedje. J.M.
[39] Rehr, B. and Klemme. between denitrifying
J.H. (1989)
for nitrate
Psrudofr~ontrs .sfuf:rr-i and nitrate am-
monifyinp enterobacteris. 330.
Rev. 6, 190-232.
Competition
FEMS
Microbial.
Ecol. 62. 211-