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Denitrification potential of a salt marsh soil: Effect of temperature, pH and substrate concentration
0038-07 I7/g2/020117-09W3.00/0 Copyright 0 1982 Pergamon Press Ltd
S0il Bbl. Biochrm. Vol. 14. pp. II7 10 125. 1982 Printed in Great Britain. All rights reserved
DENITRIFICATION POTENTIAL OF A SALT MARSH SOIL: EFFECT OF TEMPERATURE, pH AND SUBSTRATE CONCENTRATION USHA
S. GEORGE
ALAN
and
D. ANTOINE
Department of Biochemistry and Microbiology, Rutgers-The State University of New Jersey, New Brunswick, NJ 08903, U.S.A. (Accepted 10 August 1981)
Summary-Denitrification was studied using samples of salt marsh soils collected from the New Jersey coast. The pH, organic matter content, NO; and NO; concentrations were determined on samples from marshes with and without grasses. Denitrification was measured in laboratory studies over a temperature range from 4” to 60°C and a pH range from 5.0 to 9.0 by monitoring NO; reduction, NO; reduction and N2 evolution. Optimum conditions were controlled by a temperature-pH interaction which caused shifts in the pH optima relative to the change in temperature. NO, and NO; were
reduced over a broad range of NO; concentration; whereas, 0.2 mg NO;-N ml- ’ completely inhibited denitrification. The presence of NO; reverses this inhibition. N20 was produced only at low pH values and low NO, concentrations. It was concluded that the NO; disrupted of the three main processes of denitrification.
INTRODUCTION
There is considerable information concerning the major factors which influence the reaction sequence of denitrification (Delwiche and Bryan, 1976; Focht and Verstraete, 1977). The literature is incomplete, however, in its description of how the major factors of temperature, pH, substrate concentration and O2 tension interact with each other to influence the reactions of denitrification. This is of considerable importance since these factors can rapidly change in the environment and influedce selection among populations of denitrifying bacteria (Kaplan et al., 1979). Recent studies on the process of denitrification in salt marsh ecosystems emphasize the anaerobic nature of the soil environment (Kaplan et al., 1979) and the role of the salt marsh grass root-rhizome system in the regulation of denitrifying activity (Sheer and Payne, 1979). Our objective was to characterize salt marsh soils obtained from many of the coastal areas of New Jersey and, using a representative salt marsh soil for inoculation, measure the utilization of NO; by the reactions of denitrtication as influenced by temperature, pH and substrate concentration. The optimum conditions for denitrification obtained from these laboratory studies were compared to the natural conditions present in the marsh soils.
MATERIALS
AND
METHODS
Sample collection
Samples were collected in May, 1976, during low tide at sites along the New Jersey coast from Jenkins Sound, Cape May County in the south to the Raritan Bay, Middlesex County in the north (Fig. 1). Samples were dug with a trowel to a depth of 15 cm and, when available, samples were taken at each site from areas
reducing system was the most easily
with marsh grasses (Spartina and Phragnites spp) and from areas with no vegetation. For growth studies soil samples (5Og) were collected in June, 1976 from areas lacking vegetation in the salt marsh in Cheesequake State Park, Middlesex County, and used within 12 h of collection. Soil analyses
All analyses except the pH determinations were done on air dried samples. The dried samples were ground in a mortar and screened ( < 2 mm) before use. The pH was measured electrometrically on a slurry of log wet soil and 10ml deionized water. For organic matter, 10 g dry soil was incinerated in a muffle furnace at 500°C until no further weight loss was observed. The final weight loss was measured and corrected for bound water (105”C, 12 h). NO; and NO; were extracted from 5 g of an airdried sample which was mixed with 25 ml deionized water, shaken for 30min and filtered through a medium grade filter paper. The filtrate was centrifuged at 10,000 g for 10min and NO; (Jenkins and Medsker, 1964) and NO; (Garrett and Nason, 1969) determined. Growth studies
The growth studies were performed using 250ml Erlenmeyer flasks fitted with rubber stoppers into which two glass tubes were inserted. The larger (5.0 x l.Ocm, o.d.) tube was sealed with a serum vial cap and was used to collect and sample evolved gases. The smaller (4.0 x 0.5 cm, o.d.) tube was plugged with non-absorbant cotton and served as a liquid sampling port and as a vent. A moist soil sample (log) was suspended in 100 ml sterile 0.8% saline and inoculated into the sterile culture flasks as 1% (v/v) of the total flask volume. The experiments were run in triplicate 117
118
and ALAN D. ANTOINE
USHA SGEORGE
Temperature, pH nnd suhstrute tests
The effect of selected temperatures between 4” and 65°C was studied. The pH of the culture medium was pH 7.0 and three replicate flasks were incubated at each temperature. The effect of selected pH values between pH 5 and 8 was studied by varying the relative concentrations of KHzPOl and Na*HPO,. The molarity of the buffer in this experiment was O.~M, twice that of the normal culture medium, in order to maintain constant pH, and the experiments were run at 20” and 37°C. Three replicate flasks were incubated at each pH for both temperatures. The effect of concentrations between 0 and 1Omg NO;-N ml-’ (as KN03) or of con~ntrations between 0 and 1 mg NO;-N ml-’ (as NaNO;) was studied in separate experiments. In addition increasing NO; concentrations (0.02-0.2 mg NO; -N ml- ‘) were also tested in the normal growth medium containing 0.8 mg NO;-N ml- i. These experiments were carried out in duplicate at pH 7.0 and 28°C. NO; and NO; were assayed every 6 or 12 h. Flasks were observed for the onset of gas production and samples were taken with a gas syringe and analyzed. The quantity of gas evolved was not measured and no liquid displacement problem was encountered due to prior liquid subsampling.
I
Fig. 1. Salt marsh sampling sites along the New Jersey coast. 1. Raritan Bay, Cheeseauake State Park (northwest corner), Middiesex County. 2. Barnegat Bay, Barnegat National Wildlife Refuge (northwest corner at route 534), Ocean Countv. 3. Little Eaa Harbor (3 km south of Tuckerton). Ocean County. 4:-Great Bay (northeast shore. 10 km south of Tuckerton), Ocean County. 5. Brigantine Beach (I km north of Brigantine on route 87). Atlantic County. 6. Great Egg Harbor (2 km east of Somers Point on Route 152). Atlantic County. 7. Great Sound (3 km west of Avalon). Cape May County. 8. Jenkins Sound (4 km
southwest of Stone Harbor). Cape May County.
for each soil sample. Flasks were subsampled syringe during incubation.
by
Culture medium The culture medium was a modification of the medium of Sperl and Hoare (1971). Its composition was (per liter): ethanol, 5 ml; KNOa, 5.8 g; KH2FOa. l.36g;Na~HPO~,Z.l3g~MgSO~~7H~O,~.2g:CaCl~~ 2Hz0. 10 mg; FeSOI.7Hz0, 8 mg; MnCl,*4H20, 2.5 mg; NazMoO,, 2.5 mg; ZnSO,.7H,O, 0.1 mg; EDTA, 5 mg. All components except NO; and phosphates were filtered sterilized and then added aseptically to the incomplete autoclaved medium. The final pH of the culture medium was pH 7.0 unless otherwise indicated. The complete medium was added to the inoculated culture flasks by filling to the stopper. The gas sampling tube was filled and capped.
Gus chromutogru~h~ The gases evolved were analyzed by the GC procedures of Payne (1973a,b) and Bollag (1973). The former procedure permits the separation of NzO and CO1 from Nz plus 0,; the latter separates Nz from OZ. Chromatography was done using an F and M model 720 GC equipped with a thermoconductivity detector.
RESULTS The pH, organic matter content, and the NO; and NOz concentrations of the salt marsh soils are shown in Table 1. Spartina marsh grass predominates in a salt water environment whereas Phrugmites marsh grass normally indicates a fresh water inflow. The pH ranged from 3.4 to 7.7 and most of the soils were acidic. The soils were generally more alkaline at the southern sites (Fig. 1). There was correlation between the pH of the soil and the type of vegetation in that the Phragmites marsh grass was most commonly associated with the acidic soils. The organic matter content of the soils ranged from 0.4 to 19.8% and was generally much lower in sites without vegetation in comparison to the sites with vegetation. The one high value was from a site at the edge of a freshwater stream. The soil samples were low in NO; and NO;. NO; concentrations ranged from below the detection limits of the assay to a high of 5.3pg N g-r. NO; was generally highest in areas of Spartina growth and lowest in areas with no vegetation cover. NO; concentrations were generally lower than the corresponding NO; concentrations and averaged 7.3% of the average NO; concentration. While NO; was highest in
Denitrification potential of a salt marsh soil
119
Table 1. Characteristics of New Jersey salt marsh soils
Vegetation
Sampling site
None’
Raritan Bay
Spartiw
P~?agmites PhTagmit~ Spartina Phragmites Spartina Phragmites Spartina Phragmites
Barnegat Bay Little Egg Harbor Great Bay Brigantine Great Egg Harbor
None’ Spartina Phragmites Spartina
Great Sound Jenkins Sound
None None’ Spartim Phr~mite~
Averages
PH
Organic matter (%)
4.8 5.0 5.4 4.8 5.4 6.0 5.0 5.8 6.2 6.2 4.3 7.1 3.4 6.8 5.1 7.7 6.3 6.2 5.6
0.6 0.8 1.8 52 17.6 2.8 15.6 7.0 0.8 19.8 0.4 4.0 5.6 10.4 Il.4 0.6 3.4 4.6 6.2
NO; -N (11814-r)
NOi-N (1Lgg_‘1 0.62
ii
0.11
0:9
0.32 0.13
1.5 5.3 ::!: 2.3 0.d
3.1 2.4 0.9 1.0 % 0:02 2 1:5
Numerical results are the mean of three determinations. ’ Site bordered by Spartina marsh grass. ’ Below limit of detection.
-i
0.8
0.4
0.4
0.2
0.8
0.4
E
z ,‘fl
0.4
0.2
9
i
z z
,lN
B
E . o 0.8
E 0.4
@
0.2
.g
E
d e C 2
the mid-coastal sites, NO; ern sites.
8 0.4
t i 0.9
0.4
0.4
0.2
0
2
4
6
6
IO
12
14
16
16
0
Time , days Fig. 2. The effect of temperature on NO, (B) and NO; (0) reduction. The arrows mark the points at which NO; reduction (N2 evolution) was first observed.
was highest in the north-
Effecct0s re~perurure ~nitrification was measured in growth medium with an initial pH 7.0 using soils from Cheesequake State Park (Raritan Bay). Denitrification occorred between 4” and 60°C but not a 65°C. The time required for the onset and completion of NO; and NO; reduction varied for each temperature (Fig. 2) at this pH. NO; reduction began at 54 h, for 28” or 37’C, at 60 h for 20” or 45”C, and after a minimum of 72 h for So”, 60” and 4C (not shown, but in that order). A similar pattern was observed for the time required to complete the reduction of NO;. All NO; was reduced at every temperature except 4’ and 60°C where reduction was continuing after 2 weeks incubation. NO; reduction (i.e. Ns gas fo~ation) also began earliest at 28” or 37°C. At 4°C and above 45°C (not shown), more time was required for NO; reduction to begin. The pattern of complete NO; reduction followed the pattern of onset for 20’-45°C in that all NO; had been reduced within 14 days for temperatures between 20” and 45°C; 30 days or more were required for the complete removal of NO; at 4”, 50” and 60°C. At 37°C NO; reduction began at the same time as Nz gas was released even though most (85-!@!A) of the NO; was still present in the growth medium (Fig. 2). Slightly less NO; remained at 20” or 28°C when NO; reduction commenced, but at 45’C and above most of the NO; had been reduced before NO; reduction began. Analysis of the gases evolved at 37’C showed that COz was the predominant gas in
120
USHA S. GEORGEand ALAN D. ANTOINE
0
2
4
6
8
IO 12 14 16 18 Time
2
4
6
8
IO 12 14 16 18
0
, days
Fig. 3. The effect of pH on NO; (0) and NO; (0) reduction at 37°C. The arrows mark the points at which NO; reduction (N2 evolution) was first observed.
the mixture during the earlier stages of NO; reduction, however, as NO; reduction continued, N2 rapidly became the major gaseous constituent. A similar pattern was observed at the other temperatures. No N20 was present in the gases evolved at any temperature during growth studies at pH 7.0.
ofpH Denitrification was observed for a pH range from 5.0 to 8.0 with test points at each 0.5 increment. While Effect
NO; and NO; were reduced at every pH tested, the duration of both of these processes depended on the pH of the growth medium. At 37”C, the onset and complete reduction of NO; was most rapidly effected at pH 7.5, while higher or lower pH values delayed the onset and decreased the rate of NO; reduction (Fig. 3) with increasing acidity (pH 6.5 and below) having a greater retarding effect than alkalinity (pH 8.0). NO; reduction began on day 3 and was complete by day 5 for pH 7.5.
0.8
7 iE
0.4
Z I I” 4
0.8
E _ 2
0.4
8 OI E .z 0.8
04
2
4
6
8
IO 12 14 16 18 Time,
2
4
6
8
IO 12 I4
I6
I8
days
Fig. 4. The effect of pH on NO; (0) and NO; (0) reduction at 2o’C. The arrows mark the points at which NO; reduction (N, evolution) was first observed.
121
Denitrification potential of a salt marsh soil Although the time taken for NO; to become completely reduced was prolonged by increased acidity (day 13 for pH 5.0). the time the reaction began did
not vary greatly relative to the optimum under these circumstances. NO; reduction at pH 5.0 began on day 5, the same time as it began at pH 8.0. At pH 5.0 on day 5 little NO; had been reduced to NO;, whereas at pH 8.0 most of the NO; had already been reduced. At lower temperatures, the effect of pH on NO; and NO; reduction was similar to the effect at 37°C in that NO; and NO; became completely reduced at every pH studied. Also, the time required for all NO; and NO; to be reduced was greater at acidic than at alkaline pH values. It is noteworthy, however, that at lower temperatures there was a downward shift in the pH optimum and more time was required for NO; and NO; to be reduced at acidic, but not alkaline pH’s (Fig. 4). At 20°C the pH optimum for both reductions was pH 7.0. Below pH 7.0, relative to the trend at 37°C the time taken for both reductions to begin and reach completion increased significantly. The decreases in the rates of NO; and NO; reduction resulted in a greater accumulation of NO; in the growth medium. Nz and CO1 were the only gases evolved during NO; reduction at neutral and alkaline pH values. Below pH 6.0, traces of NzO were observed in flasks incubated at 37” and 20°C. The evolved NzO was completely reduced to Nz by the time all NO; had been reduced. Eflect of nitrate concentration The effect on growth and denitrification of 10-fold increases in NO; concentration from 1 pg to 10.0 mg N ml-’ was studied at 28’C and pH 7.0. Visible growth with the concomitant reduction of NO; to NO; was observed within 4 days at most NO; concentrations studied. However, there was a 7 day lag in the culture containing 10mg NO;-N ml-‘. All reactions of denitrification were recorded at concentrations of 0.1 and l.Omg NOT-N ml-‘. At concentrations of 1OPg NO;-N ml-’ or below, the NO; formed was reduced, presumably by assimilation, since no gas was formed and NH; could not be detected. A similar effect (i.e. no Nz production) was observed at 1Omg NO;-Nml-‘. but NH: was present in the culture medium. When the concentration range of NO; was limited between 0.1 and 1.5 mg N ml- ‘, increased NO; concentrations delayed the onset of NO; reduction (Fig. SA). Although the delay was greatest at 1.5 mg NO;-Nml-r, the resultant rate of NO; reduction was superior relative to the other concentrations. Nevertheless, NO; was completely reduced within 6 days for all concentrations of NO; tested. For the same range of NO; concentrations, the onset of NO; reduction was also delayed (Fig. 5B). Although NO; accumulated in the media increasing amounts, it was subsequently and completely reduced by the,denitrifying population. Analysis of the gases formed from NO; reduction at different NO; concentrations showed that traces of N20 were produced at a NO; concentration of 0.1 mgN ml-‘. In this instance NsO was completely
(a)
0
(b) 0.25
Time , days Fig. 5. The effect of NO; concentration on NO; (a) and NO; (b) reduction. Initial NO; concentrations (mg N ml- ‘) are indicated for each curve; the arrows (-+) (panel a) mark the points at which NO; reduction (N2 evolution) was first observed.
reduced to N2 before all NO; had been reduced. Gases produced from cultures containing higher initial concentration of NO; consisted of only N2 and cos. Efict of nitrate concentration The effect on growth and N, evolution of tenfold from 0.1 pg to increases in NO; concentration 1.0 mg N ml-’ as the only source of N for growth was studied at 28°C and pH 7.0. Growth was observed in media with NO; concentrations of 0.1 fig to 0.1 mg N ml-‘, but N2 was evolved only at 0.1 mgNml_‘. Below this concentration NO; was utilized by assimilation; no NHf was present in the growth media. Growth was inhibited by NO; concentrations of l.OmgNml-‘. When the NO; concentration range was limited, gas was formed at NO; concentrations from 0.05 to 0.13mgNml-’ (Fig. 6). At 0.02mg NO;-Nml-‘, NO; was utilized but no gas was formed. Under these growth conditions (pH 7.0, 28”C), NO; was used at a maximum rate at 0.1 mg NO;-Nml-r with decreased rates of utilization at higher or lower concentrations. Only CO* and N2 were detected in the gases.
USHA
122
S. GEORGEand
ALAN D. ANTOINE
NOT-N ml-’ slowed the onset and rate of NO; reduction and prolonged NO; reduction. With a NO; concentrations to further increase in 0.09 mg N ml-‘, the onset and rate of NO; reduction and the rate and completion of NO; reduction were markedly accelerated and were essentially equivalent to the nitrite-less control. At higher NO; concentrations, this trend was reversed with increased times observed. It is noteworthy that the onset of gas for-
mation at 0.13 and 0.2mg NO;-Nml-‘, although delayed, was accompanied by a rapid decrease, then an increase in NO; accumulation. Again only COa and N2 were evolved in most cases. When the initial concentration of NO; was 0.2 mg N ml-‘, NaO was formed and was subsequently reduced to N2. DISCUSSION
234567
I
Time,
days
Fig. 6. The effect of NO; concentration on NO; reduction. Initial NO; concentrations (mg N ml-‘) are indicated for each curve; the arrows (-+) mark the points at which NO; reduction (N2 evolution) was first observed.
When the same NO; concentration range was tested in the presence of NO; (initial concentration of 0.8 mg N ml- I), an interesting result was observed (Fig. 7). Initial concentrations of 0.02 and 0.05 mg
Our results indicate that while conditions in the New Jersey salt marsh soils are suboptimal, they do not inhibit denitrification. The concentrations of NO; and NO; are extremely low in these soils, a condition which remains essentially constant from May through September (unpublished results). This suggests that either denitrification proceeds very rapidly and depletes the supply of NO; or that there are suboptimal concentrations of both NO; or NO; to stimulate denitrification. A combination of these two explanations has been established from previous field work. In a study of NO; reduction in the salt marsh of Sapelo Island, Georgia, Payne (1973a) found that little N2 was produced from the endogenous NO; (6CG30 nmol N I-‘) supply present in the soil. with soil the supplementation of On 10 nmol NO; - N I-‘, Nz was evolved within 3 h. Kaplan rt al. (1977) confirmed that at maximum rates of denitrification, NO; in the interstitial water of
0.8 0.4 -
E
z
8 I
2
123456789
3456789 Time
1 I
0
, days
Fig. 7. The effect of NO; concentration on NO; (0) and NO; (0) reduction. The initial concentration of NO; (mg N ml- *) is indicated on each panel. The initial concentration of NO; was 0.8 mg N ml- ‘. The arrows mark the points at which NO; reduction (N2 evolution) was first observed.
Denitrification potential of a salt marsh soil soil can be depleted in less than 7 h. Since NO; and NO; from seawater or freshwater inflows are available to the denitrifying populations of these ecosystems in fluctuating concentrations, a careful marsh
monitoring of this chemical in the field will enable a better evaluation of our results.
Most of the soils were acidic and. thus relatively unfavorable for denitrification to occur. at optimum rates, In laboratory studies high equilibrium concentrations of NO; were observed under acidic conditions of pH 6.0 and below. This was not observed in most of the soil samples obtained along the New Jersey coastline where low NO; concentrations were observed. In addition, certain soil samples had neutral pH values, a condition which would favor denitrification, and which partially explains the presence of low NO; concentrations in samples from the three southern sites. In general it would be expected that due to a low pH reaction in these samples, the predominant end product of denitrification would be N20 rather than Nz. This is suggested by the growth study experiments and confirmed by Van Cleemput et al. (1975) where it was shown that N,O was the major product below pH 6.0 following the addition of NO; to soil plots. Rates of denitrification in soil can be closely correlated to the amount of organic matter present (Burford and Bremner, 1975). Carbon sources for the denitrifying population are primarily supplied by plants either due to tissue decomposition or from living root exudates (Bailey, 1976; Christian et al., 1975). We noted that most sites having substantial amounts of organic matter were from areas with vegetation. NO; concentrations were also relatively high at these sites, How these characteristics correlate to actual rates of denitrification in this case is unknown. It would be expected that the net effect is an increased potential for denitrification due to the production of belowground macroorganic matter by the Spartim, and presumably Phragmites, marsh grasses (Sherr and Payne, 1979). Also, since relatively acidic conditions were found to be associated with most vegetated sites, the modifying influence or organic matter on the effect of pH may also be important (Ekpete and Cornfield, 1965). The rate of denitrification has been assumed to increase with increasing temperature (Focht, 1974). But, it has been shown by Stanford et al. (1975), that in widely differing soil samples, little denitrification occurs between 5” and loOC, while the rate of denitrification increases up to a maximum activity at 37°C and remains at that rate when the temperature is further increased to 45°C. We have found a similar temperature effect in growth studies. An optimum denitritication activity was observed at 28”-37°C; however, denitrification activity decreased with further increases in temperature up to 60°C. Garcia (1974) reported a similar pattern for rice field sediment samples in which the optimum temperature for NzO reduction was 37”C, with a slower reaction at 28” and 45°C. The secondary optimum for N*O reduction observed at 65°C in rice field sediment samples was not observed in the present study; no denitrfication was observed at any temperature higher than 60°C. How these findings relate to the natural ecosystem is difficult to evaluate. The rate of
123
denitrification measured as N2 production throughout the year in a New England salt marsh by Kaplan et al. (1979), did correlate with increasing temperature, however, in this case surface temperatures were not found to exceed 35°C. Surface temperatures approaching 40°C have been observed in many of the New Jersey salt marshes in June through September, while at the same time, temperatures taken at a soil depth of 15’cm rarely exceed 30°C (unpublished observation). Although N,O has been shown by Dowdell and Smith (1974), to accumulate in the soil with seasonal increases in temperature, it is known that this cause and effect is not limited to temperature alone. For example, increased soil aeration increases the amount of NzO produced. This has been attributed by Van Cleemput et al. (1976) to a decreased rate of N,O reduction relative to the rate of NO; reduction. In addition NO; concentration was also found to increase in the study by Dowdell and Smith (1974), which is highly indicative of an active nitrifying population in the same ecosystem. Kaplan et al. (1979) and Focht and Verstraete (1977) discuss this interaction. There are conflicting reports concerning the optimum pH for denitrification. This is partly due to the fact that in natural environments, the effect of one factor cannot be measured with reliability due to the overlapping interactions with other factors. For example, in laboratory studies using a rice field soil, Garcia (1975) determined that the optimum pH for the reduction of NzO was pH 7.0, whereas in field studies the optimum pH was not measurable due to the modifying influence of other factors such as the age of the rice crop, temperature fluctuations and the effect of the rhizosphere. Additional factors can modify the influence of pH in natural environments such as O2 concentrations and metal ions as well as the direct influence resulting from the production of hydroxyl ions during the reactions of denitrification. The effect of pH observed in the present study is similar to that observed by Valera and Alexander (1961) in axenic cultures of bacteria in that the optimum pH was 7.0-7.5 depending on temperature. Acid conditions were found to be more inhibitory to denitrifying activity than alkaline conditions. This is contrary, however, to the results obtained by Garcia (1974) from rice field sediment samples where at 37°C an optimum pH of 7.0 was found, a lower optimum than we found for that temperature, and where alkalinity was more inhibitory than acidity. The temperaturc+pH interaction observed in this study explains the differences in pH optima obtained by various investigators. In particular, it is noteworthy that discussions concerning optimum conditions are consistent for only one given set of parameters. In the present situation, the optimum pH was modified by factors other than temperature, when the incubation temperature was decreased from 37” to 20°C. One obvious reason for the altered temperature-pH interaction would be that different bacterial populations are responsible for denittication at 37°C as compared to 20°C and that the microbial populations have different pH optima. In soils it has been demonstrated by McGarity and Myers (1968) that the rate of NO; reduction increases with increasing NO; concentration. Similarly
124
USHA S. GEORGEand ALAN D. ANTOINE
Garcia (1974) observed increased rates of NzO reduction proportional to increasing N,O substrate concentrations in rice field sediments. We observed that increasing NO; con~ntrations delayed the onset of NO; and NO; reduction, however, the resultant rate of NO; reduction was greatest for the highest concentration tested. A similar pattern has been reported by Chen et al. (1972) for lake sediment samples in which increasing concentrations of NO; decreased the amount of denitrification and reduced the amount of “N-N* evolution. In the absence of NO;, NO; concentrations above 0.2 mg NO;-N ml- ’ completely inhibited denitrification. This was surprising since NO; concentrations up to 0.5 mg NO;N ml-’ have been observed in the growth medium during the course of NO; reduction and, in which case, the NO; formed was further reduced. A similar effect was also observed in experiments performed to determine the effect of increasing NO; concentrations on NO; reduction. During growth in these experiments, NO; and NO; reduction did occur in cultures that had an initial NO; concentration of 0.2 mg NO;-N ml- l, in addition to the initial NO; concentration of 0.8 mgNO;N ml- r. Thus, it is suggested from this evidence that an adequate supply of NO; allows better growth of the organisms and that this increased cell mass can subsequently reduce higher concentrations of NO;. NO; reduction seems to be more resistant to extreme conditions that NO; or N,O reduction. Bollag (1973) also suggested that NO; reduction was more easily disrupted than NO; reduction. Only in the present temperature and pH studies was NO; usually reduced, although the range of pH values studies was rather limited. At 45°C and above, there were long lag periods before NO; reduction commenced and the rate of NO; reduction was very slow. At low pH values (pH 6 and below) NO; reduction proceeded very slowly. The sharp increase in the rate, after the peak NO; concentration was reached, was probably due to the increase in pH due to the production of OH- in the denitrification process. NOT reduction did commence before the pH was significantly altered. The lower pH value appears to have inhibited the reduction of N20 as well as the reduction of NO;. Finally, at high NO; concentrations (10mg NO;-Nml-r), no NO; was observed in 30 days, although NO; reduction did take place after 7 days. In Payne’s (1973) studies with resting cells, NO; was totally reduced to NO; before NO; was reduced to N,O. In our study NO; reduction normally commenced before all of the NO; was reduced. Only at temperatures of 45°C or higher was NO; totally reduced before NO; reduction commenced. Surprisingly, N20 temporarily accumulated at low NO; concentrations, but not at higher concentrations. The latter situation was found by Blackmer and Bremner (1978). It is suggested that our study has selected a denitrifying population normally capable of forming N2, without the accumulation of N20. N20 was also observed during growth at pH 5.0, but not at higher pH values. This is similar to the results obtained by Van Cleemput er al. (1975, 1976) and as postulated by Focht (1974). From this it
appears that NzO will accumulate conditions for denitrihcation.
during suboptimal
Aclinowlrdgrmmrs-This work has been supported by the Rutgers University Research Council, the New Jersey Agricultural Experiment Station. and the Atlantic Foundation.
REFERENCES
BAILEYL. D. (1976) Effect of temperature and roots on denitrification in a soil. Canadian Journai of Soil Science 56, 79-87.
BOLLAGJ. M., DRYZMALAS. and KARDOSL. T. (1973) Biological versus chemical denitrification in soil. Soil Science 116, 4450. BURFORDJ. R. and BREMNERJ. M. (1975) Relationships between denitrification capacities of soils and totai, water-soIubIe, and readily decomposable soil organic matter. Soil Biology 8~ Biochemistry 7, 389-394. BLACKMERA. M. and BREMNERJ. M. (1978) Inhibitory effect of nitrate on reduction of NzO to N, by soil microorganisms. Soil Biology & Biochemistry 10, 187-191. CHEN R. L., KEENEYD. R., KONRADJ. G., HOLDINGA. J. and GRAETZD. A. (1972) Gas production in sediments of Lake Mendota. Journal of E~vironmentul Quaky 1, 1.55-159. CHRISTIANR. R., BANCROFTK. and WIEBE W. J. (1975) Distribution of microbial adenosine triphosphate in salt marsh sediments of Sapelo Island, Georgia. Soil Science 119, 89-97. DELWICHEC. C. and BRYANB. A. (1976) Denitrification. Annual Reviews of ~~c~obioiogy 30, 241-262. DOWDELLR. J. and SMITHK. A. 11974) Field studies of the soil atmosphere. II. Occurrence of nitrous oxide. Journal of Soil Sci&ce 25, 231-238. EKPETED. M. and CORNFIELD A. H. (1965) . , The effect of OH and the addition of organic matter on denitrificatjon losses from soil. Namre 2@&1200. F~CHT D. D. (1974) The effect of temperature, pH and aeration on the production of nitrous oxide and gaseous oxygen. Soil Science 11% 173-179. FOCHTD. D. and VERSTRAETE W. (1977) Biochemical ecology of nitrification and denitrification. Advances in Microbial Ecology 1, 1355214. GARCIAJ. L. (1974) Reduction de I’oxy de nitreaux dans sois de rizeres du Senegal: mesure de l’activite denitrificante. Soif Biologp & B~ochemjsrr~ 6, 78-84. GARCIAJ. L. (1975) Evaluation de la denitr~cation darts les rizeres par la methode de reduction de NzO. Soil Biology & Biochemistry 7, 251-256.
GARRETTR. H. and NASONA. (1969) Further purification and properties of Neurospora nitrate reductase. Journal of Biological Chemistry 244, 2870-2882.
JENKINSD. and MEDSKERL. L. (1964) Brucine method for the determination of nitrate in ocean estuaries and fresh water. Analytical Chemistry 36, 610612. KAPLANW., TEALJ. M. and VAL~ELAI. (1977) Denitrification in salt marsh sediments. Evidence for seasonal temperature selection among populations of denitrifying bacteria. Microbiology and Ecology 3, 193204. KAPLAN,W., VALIELAI. and TEALJ. M. (1979) Denitrification in a salt marsh ecosystem. ~irn~l~y and Oeeanogruphy 24, 726-734.
McG~~rrv J. W. and MYERSR. K. (1968) Denitrifying activity in solodized solonetz soils of eastern Australia. Proceedings. Soil Science Society of America 32, 8 12-8 17. PAYNEW. J. (1973a) Gas chromatographic analysis of denitrification by marine organisms. In Estuarine Microbial Ecology (L. H. Stevenson and R. R. Colewell, Eds), pp. 53-71. University of South Carolina Press, Columbia.
Denitrification potential of a salt marsh soil PAYNEW. J. (1973b) The use of gas chromatography for studies of denitrification in ecosystems. Bulletin fir Ecological Research 263-268.
Communications
(Stockholm)
17,
SHERRB. F. and PAYNEW. J. (1979) Role of the salt marsh grass Spartina altern@ora in the response of soildenitrifying bacteria to glucose enrichment. Applied und En~iro~tal Micr~iof~y 38.747-748. SPERLG. T. and HOARED. S. (1971) ~nitriB~tion with methanol. Selective enrichment for ~y~omicrobtum spcties. Journal of Bacteriology 188, 733-736.. STANFORD G., DZIENA S. and VAN DER POL R. A. (1975) Effect of temperature on denitrification rates in soil.
125
Proceedings. Soil Science Sociery of America 39, 867870.
VALERAC. L. and ALEXANDERM. (1961) Nutrition and physiology of denitrifying bacteria. Plant & Soil IS, 268-280.
VAN CLEEMPU~0, PATRICKW. H. and MC~LHENNYR. C. (1975) Formation of chemical and biological denitrification end products in flooded soil at controlled pH and redox potential. Soii Balky & R~hemistry 7, 329-332. VANCLEU(PUT0.. PATRICKW. H. and MCILHENNYR. C. (1976) Nitrite decomposition in flooded soits under controlled pH and redox potential conditions. Proceedings. Soil Science Society of America 40, 55-60.
S0il Bbl. Biochrm. Vol. 14. pp. II7 10 125. 1982 Printed in Great Britain. All rights reserved
DENITRIFICATION POTENTIAL OF A SALT MARSH SOIL: EFFECT OF TEMPERATURE, pH AND SUBSTRATE CONCENTRATION USHA
S. GEORGE
ALAN
and
D. ANTOINE
Department of Biochemistry and Microbiology, Rutgers-The State University of New Jersey, New Brunswick, NJ 08903, U.S.A. (Accepted 10 August 1981)
Summary-Denitrification was studied using samples of salt marsh soils collected from the New Jersey coast. The pH, organic matter content, NO; and NO; concentrations were determined on samples from marshes with and without grasses. Denitrification was measured in laboratory studies over a temperature range from 4” to 60°C and a pH range from 5.0 to 9.0 by monitoring NO; reduction, NO; reduction and N2 evolution. Optimum conditions were controlled by a temperature-pH interaction which caused shifts in the pH optima relative to the change in temperature. NO, and NO; were
reduced over a broad range of NO; concentration; whereas, 0.2 mg NO;-N ml- ’ completely inhibited denitrification. The presence of NO; reverses this inhibition. N20 was produced only at low pH values and low NO, concentrations. It was concluded that the NO; disrupted of the three main processes of denitrification.
INTRODUCTION
There is considerable information concerning the major factors which influence the reaction sequence of denitrification (Delwiche and Bryan, 1976; Focht and Verstraete, 1977). The literature is incomplete, however, in its description of how the major factors of temperature, pH, substrate concentration and O2 tension interact with each other to influence the reactions of denitrification. This is of considerable importance since these factors can rapidly change in the environment and influedce selection among populations of denitrifying bacteria (Kaplan et al., 1979). Recent studies on the process of denitrification in salt marsh ecosystems emphasize the anaerobic nature of the soil environment (Kaplan et al., 1979) and the role of the salt marsh grass root-rhizome system in the regulation of denitrifying activity (Sheer and Payne, 1979). Our objective was to characterize salt marsh soils obtained from many of the coastal areas of New Jersey and, using a representative salt marsh soil for inoculation, measure the utilization of NO; by the reactions of denitrtication as influenced by temperature, pH and substrate concentration. The optimum conditions for denitrification obtained from these laboratory studies were compared to the natural conditions present in the marsh soils.
MATERIALS
AND
METHODS
Sample collection
Samples were collected in May, 1976, during low tide at sites along the New Jersey coast from Jenkins Sound, Cape May County in the south to the Raritan Bay, Middlesex County in the north (Fig. 1). Samples were dug with a trowel to a depth of 15 cm and, when available, samples were taken at each site from areas
reducing system was the most easily
with marsh grasses (Spartina and Phragnites spp) and from areas with no vegetation. For growth studies soil samples (5Og) were collected in June, 1976 from areas lacking vegetation in the salt marsh in Cheesequake State Park, Middlesex County, and used within 12 h of collection. Soil analyses
All analyses except the pH determinations were done on air dried samples. The dried samples were ground in a mortar and screened ( < 2 mm) before use. The pH was measured electrometrically on a slurry of log wet soil and 10ml deionized water. For organic matter, 10 g dry soil was incinerated in a muffle furnace at 500°C until no further weight loss was observed. The final weight loss was measured and corrected for bound water (105”C, 12 h). NO; and NO; were extracted from 5 g of an airdried sample which was mixed with 25 ml deionized water, shaken for 30min and filtered through a medium grade filter paper. The filtrate was centrifuged at 10,000 g for 10min and NO; (Jenkins and Medsker, 1964) and NO; (Garrett and Nason, 1969) determined. Growth studies
The growth studies were performed using 250ml Erlenmeyer flasks fitted with rubber stoppers into which two glass tubes were inserted. The larger (5.0 x l.Ocm, o.d.) tube was sealed with a serum vial cap and was used to collect and sample evolved gases. The smaller (4.0 x 0.5 cm, o.d.) tube was plugged with non-absorbant cotton and served as a liquid sampling port and as a vent. A moist soil sample (log) was suspended in 100 ml sterile 0.8% saline and inoculated into the sterile culture flasks as 1% (v/v) of the total flask volume. The experiments were run in triplicate 117
118
and ALAN D. ANTOINE
USHA SGEORGE
Temperature, pH nnd suhstrute tests
The effect of selected temperatures between 4” and 65°C was studied. The pH of the culture medium was pH 7.0 and three replicate flasks were incubated at each temperature. The effect of selected pH values between pH 5 and 8 was studied by varying the relative concentrations of KHzPOl and Na*HPO,. The molarity of the buffer in this experiment was O.~M, twice that of the normal culture medium, in order to maintain constant pH, and the experiments were run at 20” and 37°C. Three replicate flasks were incubated at each pH for both temperatures. The effect of concentrations between 0 and 1Omg NO;-N ml-’ (as KN03) or of con~ntrations between 0 and 1 mg NO;-N ml-’ (as NaNO;) was studied in separate experiments. In addition increasing NO; concentrations (0.02-0.2 mg NO; -N ml- ‘) were also tested in the normal growth medium containing 0.8 mg NO;-N ml- i. These experiments were carried out in duplicate at pH 7.0 and 28°C. NO; and NO; were assayed every 6 or 12 h. Flasks were observed for the onset of gas production and samples were taken with a gas syringe and analyzed. The quantity of gas evolved was not measured and no liquid displacement problem was encountered due to prior liquid subsampling.
I
Fig. 1. Salt marsh sampling sites along the New Jersey coast. 1. Raritan Bay, Cheeseauake State Park (northwest corner), Middiesex County. 2. Barnegat Bay, Barnegat National Wildlife Refuge (northwest corner at route 534), Ocean Countv. 3. Little Eaa Harbor (3 km south of Tuckerton). Ocean County. 4:-Great Bay (northeast shore. 10 km south of Tuckerton), Ocean County. 5. Brigantine Beach (I km north of Brigantine on route 87). Atlantic County. 6. Great Egg Harbor (2 km east of Somers Point on Route 152). Atlantic County. 7. Great Sound (3 km west of Avalon). Cape May County. 8. Jenkins Sound (4 km
southwest of Stone Harbor). Cape May County.
for each soil sample. Flasks were subsampled syringe during incubation.
by
Culture medium The culture medium was a modification of the medium of Sperl and Hoare (1971). Its composition was (per liter): ethanol, 5 ml; KNOa, 5.8 g; KH2FOa. l.36g;Na~HPO~,Z.l3g~MgSO~~7H~O,~.2g:CaCl~~ 2Hz0. 10 mg; FeSOI.7Hz0, 8 mg; MnCl,*4H20, 2.5 mg; NazMoO,, 2.5 mg; ZnSO,.7H,O, 0.1 mg; EDTA, 5 mg. All components except NO; and phosphates were filtered sterilized and then added aseptically to the incomplete autoclaved medium. The final pH of the culture medium was pH 7.0 unless otherwise indicated. The complete medium was added to the inoculated culture flasks by filling to the stopper. The gas sampling tube was filled and capped.
Gus chromutogru~h~ The gases evolved were analyzed by the GC procedures of Payne (1973a,b) and Bollag (1973). The former procedure permits the separation of NzO and CO1 from Nz plus 0,; the latter separates Nz from OZ. Chromatography was done using an F and M model 720 GC equipped with a thermoconductivity detector.
RESULTS The pH, organic matter content, and the NO; and NOz concentrations of the salt marsh soils are shown in Table 1. Spartina marsh grass predominates in a salt water environment whereas Phrugmites marsh grass normally indicates a fresh water inflow. The pH ranged from 3.4 to 7.7 and most of the soils were acidic. The soils were generally more alkaline at the southern sites (Fig. 1). There was correlation between the pH of the soil and the type of vegetation in that the Phragmites marsh grass was most commonly associated with the acidic soils. The organic matter content of the soils ranged from 0.4 to 19.8% and was generally much lower in sites without vegetation in comparison to the sites with vegetation. The one high value was from a site at the edge of a freshwater stream. The soil samples were low in NO; and NO;. NO; concentrations ranged from below the detection limits of the assay to a high of 5.3pg N g-r. NO; was generally highest in areas of Spartina growth and lowest in areas with no vegetation cover. NO; concentrations were generally lower than the corresponding NO; concentrations and averaged 7.3% of the average NO; concentration. While NO; was highest in
Denitrification potential of a salt marsh soil
119
Table 1. Characteristics of New Jersey salt marsh soils
Vegetation
Sampling site
None’
Raritan Bay
Spartiw
P~?agmites PhTagmit~ Spartina Phragmites Spartina Phragmites Spartina Phragmites
Barnegat Bay Little Egg Harbor Great Bay Brigantine Great Egg Harbor
None’ Spartina Phragmites Spartina
Great Sound Jenkins Sound
None None’ Spartim Phr~mite~
Averages
PH
Organic matter (%)
4.8 5.0 5.4 4.8 5.4 6.0 5.0 5.8 6.2 6.2 4.3 7.1 3.4 6.8 5.1 7.7 6.3 6.2 5.6
0.6 0.8 1.8 52 17.6 2.8 15.6 7.0 0.8 19.8 0.4 4.0 5.6 10.4 Il.4 0.6 3.4 4.6 6.2
NO; -N (11814-r)
NOi-N (1Lgg_‘1 0.62
ii
0.11
0:9
0.32 0.13
1.5 5.3 ::!: 2.3 0.d
3.1 2.4 0.9 1.0 % 0:02 2 1:5
Numerical results are the mean of three determinations. ’ Site bordered by Spartina marsh grass. ’ Below limit of detection.
-i
0.8
0.4
0.4
0.2
0.8
0.4
E
z ,‘fl
0.4
0.2
9
i
z z
,lN
B
E . o 0.8
E 0.4
@
0.2
.g
E
d e C 2
the mid-coastal sites, NO; ern sites.
8 0.4
t i 0.9
0.4
0.4
0.2
0
2
4
6
6
IO
12
14
16
16
0
Time , days Fig. 2. The effect of temperature on NO, (B) and NO; (0) reduction. The arrows mark the points at which NO; reduction (N2 evolution) was first observed.
was highest in the north-
Effecct0s re~perurure ~nitrification was measured in growth medium with an initial pH 7.0 using soils from Cheesequake State Park (Raritan Bay). Denitrification occorred between 4” and 60°C but not a 65°C. The time required for the onset and completion of NO; and NO; reduction varied for each temperature (Fig. 2) at this pH. NO; reduction began at 54 h, for 28” or 37’C, at 60 h for 20” or 45”C, and after a minimum of 72 h for So”, 60” and 4C (not shown, but in that order). A similar pattern was observed for the time required to complete the reduction of NO;. All NO; was reduced at every temperature except 4’ and 60°C where reduction was continuing after 2 weeks incubation. NO; reduction (i.e. Ns gas fo~ation) also began earliest at 28” or 37°C. At 4°C and above 45°C (not shown), more time was required for NO; reduction to begin. The pattern of complete NO; reduction followed the pattern of onset for 20’-45°C in that all NO; had been reduced within 14 days for temperatures between 20” and 45°C; 30 days or more were required for the complete removal of NO; at 4”, 50” and 60°C. At 37°C NO; reduction began at the same time as Nz gas was released even though most (85-!@!A) of the NO; was still present in the growth medium (Fig. 2). Slightly less NO; remained at 20” or 28°C when NO; reduction commenced, but at 45’C and above most of the NO; had been reduced before NO; reduction began. Analysis of the gases evolved at 37’C showed that COz was the predominant gas in
120
USHA S. GEORGEand ALAN D. ANTOINE
0
2
4
6
8
IO 12 14 16 18 Time
2
4
6
8
IO 12 14 16 18
0
, days
Fig. 3. The effect of pH on NO; (0) and NO; (0) reduction at 37°C. The arrows mark the points at which NO; reduction (N2 evolution) was first observed.
the mixture during the earlier stages of NO; reduction, however, as NO; reduction continued, N2 rapidly became the major gaseous constituent. A similar pattern was observed at the other temperatures. No N20 was present in the gases evolved at any temperature during growth studies at pH 7.0.
ofpH Denitrification was observed for a pH range from 5.0 to 8.0 with test points at each 0.5 increment. While Effect
NO; and NO; were reduced at every pH tested, the duration of both of these processes depended on the pH of the growth medium. At 37”C, the onset and complete reduction of NO; was most rapidly effected at pH 7.5, while higher or lower pH values delayed the onset and decreased the rate of NO; reduction (Fig. 3) with increasing acidity (pH 6.5 and below) having a greater retarding effect than alkalinity (pH 8.0). NO; reduction began on day 3 and was complete by day 5 for pH 7.5.
0.8
7 iE
0.4
Z I I” 4
0.8
E _ 2
0.4
8 OI E .z 0.8
04
2
4
6
8
IO 12 14 16 18 Time,
2
4
6
8
IO 12 I4
I6
I8
days
Fig. 4. The effect of pH on NO; (0) and NO; (0) reduction at 2o’C. The arrows mark the points at which NO; reduction (N, evolution) was first observed.
121
Denitrification potential of a salt marsh soil Although the time taken for NO; to become completely reduced was prolonged by increased acidity (day 13 for pH 5.0). the time the reaction began did
not vary greatly relative to the optimum under these circumstances. NO; reduction at pH 5.0 began on day 5, the same time as it began at pH 8.0. At pH 5.0 on day 5 little NO; had been reduced to NO;, whereas at pH 8.0 most of the NO; had already been reduced. At lower temperatures, the effect of pH on NO; and NO; reduction was similar to the effect at 37°C in that NO; and NO; became completely reduced at every pH studied. Also, the time required for all NO; and NO; to be reduced was greater at acidic than at alkaline pH values. It is noteworthy, however, that at lower temperatures there was a downward shift in the pH optimum and more time was required for NO; and NO; to be reduced at acidic, but not alkaline pH’s (Fig. 4). At 20°C the pH optimum for both reductions was pH 7.0. Below pH 7.0, relative to the trend at 37°C the time taken for both reductions to begin and reach completion increased significantly. The decreases in the rates of NO; and NO; reduction resulted in a greater accumulation of NO; in the growth medium. Nz and CO1 were the only gases evolved during NO; reduction at neutral and alkaline pH values. Below pH 6.0, traces of NzO were observed in flasks incubated at 37” and 20°C. The evolved NzO was completely reduced to Nz by the time all NO; had been reduced. Eflect of nitrate concentration The effect on growth and denitrification of 10-fold increases in NO; concentration from 1 pg to 10.0 mg N ml-’ was studied at 28’C and pH 7.0. Visible growth with the concomitant reduction of NO; to NO; was observed within 4 days at most NO; concentrations studied. However, there was a 7 day lag in the culture containing 10mg NO;-N ml-‘. All reactions of denitrification were recorded at concentrations of 0.1 and l.Omg NOT-N ml-‘. At concentrations of 1OPg NO;-N ml-’ or below, the NO; formed was reduced, presumably by assimilation, since no gas was formed and NH; could not be detected. A similar effect (i.e. no Nz production) was observed at 1Omg NO;-Nml-‘. but NH: was present in the culture medium. When the concentration range of NO; was limited between 0.1 and 1.5 mg N ml- ‘, increased NO; concentrations delayed the onset of NO; reduction (Fig. SA). Although the delay was greatest at 1.5 mg NO;-Nml-r, the resultant rate of NO; reduction was superior relative to the other concentrations. Nevertheless, NO; was completely reduced within 6 days for all concentrations of NO; tested. For the same range of NO; concentrations, the onset of NO; reduction was also delayed (Fig. 5B). Although NO; accumulated in the media increasing amounts, it was subsequently and completely reduced by the,denitrifying population. Analysis of the gases formed from NO; reduction at different NO; concentrations showed that traces of N20 were produced at a NO; concentration of 0.1 mgN ml-‘. In this instance NsO was completely
(a)
0
(b) 0.25
Time , days Fig. 5. The effect of NO; concentration on NO; (a) and NO; (b) reduction. Initial NO; concentrations (mg N ml- ‘) are indicated for each curve; the arrows (-+) (panel a) mark the points at which NO; reduction (N2 evolution) was first observed.
reduced to N2 before all NO; had been reduced. Gases produced from cultures containing higher initial concentration of NO; consisted of only N2 and cos. Efict of nitrate concentration The effect on growth and N, evolution of tenfold from 0.1 pg to increases in NO; concentration 1.0 mg N ml-’ as the only source of N for growth was studied at 28°C and pH 7.0. Growth was observed in media with NO; concentrations of 0.1 fig to 0.1 mg N ml-‘, but N2 was evolved only at 0.1 mgNml_‘. Below this concentration NO; was utilized by assimilation; no NHf was present in the growth media. Growth was inhibited by NO; concentrations of l.OmgNml-‘. When the NO; concentration range was limited, gas was formed at NO; concentrations from 0.05 to 0.13mgNml-’ (Fig. 6). At 0.02mg NO;-Nml-‘, NO; was utilized but no gas was formed. Under these growth conditions (pH 7.0, 28”C), NO; was used at a maximum rate at 0.1 mg NO;-Nml-r with decreased rates of utilization at higher or lower concentrations. Only CO* and N2 were detected in the gases.
USHA
122
S. GEORGEand
ALAN D. ANTOINE
NOT-N ml-’ slowed the onset and rate of NO; reduction and prolonged NO; reduction. With a NO; concentrations to further increase in 0.09 mg N ml-‘, the onset and rate of NO; reduction and the rate and completion of NO; reduction were markedly accelerated and were essentially equivalent to the nitrite-less control. At higher NO; concentrations, this trend was reversed with increased times observed. It is noteworthy that the onset of gas for-
mation at 0.13 and 0.2mg NO;-Nml-‘, although delayed, was accompanied by a rapid decrease, then an increase in NO; accumulation. Again only COa and N2 were evolved in most cases. When the initial concentration of NO; was 0.2 mg N ml-‘, NaO was formed and was subsequently reduced to N2. DISCUSSION
234567
I
Time,
days
Fig. 6. The effect of NO; concentration on NO; reduction. Initial NO; concentrations (mg N ml-‘) are indicated for each curve; the arrows (-+) mark the points at which NO; reduction (N2 evolution) was first observed.
When the same NO; concentration range was tested in the presence of NO; (initial concentration of 0.8 mg N ml- I), an interesting result was observed (Fig. 7). Initial concentrations of 0.02 and 0.05 mg
Our results indicate that while conditions in the New Jersey salt marsh soils are suboptimal, they do not inhibit denitrification. The concentrations of NO; and NO; are extremely low in these soils, a condition which remains essentially constant from May through September (unpublished results). This suggests that either denitrification proceeds very rapidly and depletes the supply of NO; or that there are suboptimal concentrations of both NO; or NO; to stimulate denitrification. A combination of these two explanations has been established from previous field work. In a study of NO; reduction in the salt marsh of Sapelo Island, Georgia, Payne (1973a) found that little N2 was produced from the endogenous NO; (6CG30 nmol N I-‘) supply present in the soil. with soil the supplementation of On 10 nmol NO; - N I-‘, Nz was evolved within 3 h. Kaplan rt al. (1977) confirmed that at maximum rates of denitrification, NO; in the interstitial water of
0.8 0.4 -
E
z
8 I
2
123456789
3456789 Time
1 I
0
, days
Fig. 7. The effect of NO; concentration on NO; (0) and NO; (0) reduction. The initial concentration of NO; (mg N ml- *) is indicated on each panel. The initial concentration of NO; was 0.8 mg N ml- ‘. The arrows mark the points at which NO; reduction (N2 evolution) was first observed.
Denitrification potential of a salt marsh soil soil can be depleted in less than 7 h. Since NO; and NO; from seawater or freshwater inflows are available to the denitrifying populations of these ecosystems in fluctuating concentrations, a careful marsh
monitoring of this chemical in the field will enable a better evaluation of our results.
Most of the soils were acidic and. thus relatively unfavorable for denitrification to occur. at optimum rates, In laboratory studies high equilibrium concentrations of NO; were observed under acidic conditions of pH 6.0 and below. This was not observed in most of the soil samples obtained along the New Jersey coastline where low NO; concentrations were observed. In addition, certain soil samples had neutral pH values, a condition which would favor denitrification, and which partially explains the presence of low NO; concentrations in samples from the three southern sites. In general it would be expected that due to a low pH reaction in these samples, the predominant end product of denitrification would be N20 rather than Nz. This is suggested by the growth study experiments and confirmed by Van Cleemput et al. (1975) where it was shown that N,O was the major product below pH 6.0 following the addition of NO; to soil plots. Rates of denitrification in soil can be closely correlated to the amount of organic matter present (Burford and Bremner, 1975). Carbon sources for the denitrifying population are primarily supplied by plants either due to tissue decomposition or from living root exudates (Bailey, 1976; Christian et al., 1975). We noted that most sites having substantial amounts of organic matter were from areas with vegetation. NO; concentrations were also relatively high at these sites, How these characteristics correlate to actual rates of denitrification in this case is unknown. It would be expected that the net effect is an increased potential for denitrification due to the production of belowground macroorganic matter by the Spartim, and presumably Phragmites, marsh grasses (Sherr and Payne, 1979). Also, since relatively acidic conditions were found to be associated with most vegetated sites, the modifying influence or organic matter on the effect of pH may also be important (Ekpete and Cornfield, 1965). The rate of denitrification has been assumed to increase with increasing temperature (Focht, 1974). But, it has been shown by Stanford et al. (1975), that in widely differing soil samples, little denitrification occurs between 5” and loOC, while the rate of denitrification increases up to a maximum activity at 37°C and remains at that rate when the temperature is further increased to 45°C. We have found a similar temperature effect in growth studies. An optimum denitritication activity was observed at 28”-37°C; however, denitrification activity decreased with further increases in temperature up to 60°C. Garcia (1974) reported a similar pattern for rice field sediment samples in which the optimum temperature for NzO reduction was 37”C, with a slower reaction at 28” and 45°C. The secondary optimum for N*O reduction observed at 65°C in rice field sediment samples was not observed in the present study; no denitrfication was observed at any temperature higher than 60°C. How these findings relate to the natural ecosystem is difficult to evaluate. The rate of
123
denitrification measured as N2 production throughout the year in a New England salt marsh by Kaplan et al. (1979), did correlate with increasing temperature, however, in this case surface temperatures were not found to exceed 35°C. Surface temperatures approaching 40°C have been observed in many of the New Jersey salt marshes in June through September, while at the same time, temperatures taken at a soil depth of 15’cm rarely exceed 30°C (unpublished observation). Although N,O has been shown by Dowdell and Smith (1974), to accumulate in the soil with seasonal increases in temperature, it is known that this cause and effect is not limited to temperature alone. For example, increased soil aeration increases the amount of NzO produced. This has been attributed by Van Cleemput et al. (1976) to a decreased rate of N,O reduction relative to the rate of NO; reduction. In addition NO; concentration was also found to increase in the study by Dowdell and Smith (1974), which is highly indicative of an active nitrifying population in the same ecosystem. Kaplan et al. (1979) and Focht and Verstraete (1977) discuss this interaction. There are conflicting reports concerning the optimum pH for denitrification. This is partly due to the fact that in natural environments, the effect of one factor cannot be measured with reliability due to the overlapping interactions with other factors. For example, in laboratory studies using a rice field soil, Garcia (1975) determined that the optimum pH for the reduction of NzO was pH 7.0, whereas in field studies the optimum pH was not measurable due to the modifying influence of other factors such as the age of the rice crop, temperature fluctuations and the effect of the rhizosphere. Additional factors can modify the influence of pH in natural environments such as O2 concentrations and metal ions as well as the direct influence resulting from the production of hydroxyl ions during the reactions of denitrification. The effect of pH observed in the present study is similar to that observed by Valera and Alexander (1961) in axenic cultures of bacteria in that the optimum pH was 7.0-7.5 depending on temperature. Acid conditions were found to be more inhibitory to denitrifying activity than alkaline conditions. This is contrary, however, to the results obtained by Garcia (1974) from rice field sediment samples where at 37°C an optimum pH of 7.0 was found, a lower optimum than we found for that temperature, and where alkalinity was more inhibitory than acidity. The temperaturc+pH interaction observed in this study explains the differences in pH optima obtained by various investigators. In particular, it is noteworthy that discussions concerning optimum conditions are consistent for only one given set of parameters. In the present situation, the optimum pH was modified by factors other than temperature, when the incubation temperature was decreased from 37” to 20°C. One obvious reason for the altered temperature-pH interaction would be that different bacterial populations are responsible for denittication at 37°C as compared to 20°C and that the microbial populations have different pH optima. In soils it has been demonstrated by McGarity and Myers (1968) that the rate of NO; reduction increases with increasing NO; concentration. Similarly
124
USHA S. GEORGEand ALAN D. ANTOINE
Garcia (1974) observed increased rates of NzO reduction proportional to increasing N,O substrate concentrations in rice field sediments. We observed that increasing NO; con~ntrations delayed the onset of NO; and NO; reduction, however, the resultant rate of NO; reduction was greatest for the highest concentration tested. A similar pattern has been reported by Chen et al. (1972) for lake sediment samples in which increasing concentrations of NO; decreased the amount of denitrification and reduced the amount of “N-N* evolution. In the absence of NO;, NO; concentrations above 0.2 mg NO;-N ml- ’ completely inhibited denitrification. This was surprising since NO; concentrations up to 0.5 mg NO;N ml-’ have been observed in the growth medium during the course of NO; reduction and, in which case, the NO; formed was further reduced. A similar effect was also observed in experiments performed to determine the effect of increasing NO; concentrations on NO; reduction. During growth in these experiments, NO; and NO; reduction did occur in cultures that had an initial NO; concentration of 0.2 mg NO;-N ml- l, in addition to the initial NO; concentration of 0.8 mgNO;N ml- r. Thus, it is suggested from this evidence that an adequate supply of NO; allows better growth of the organisms and that this increased cell mass can subsequently reduce higher concentrations of NO;. NO; reduction seems to be more resistant to extreme conditions that NO; or N,O reduction. Bollag (1973) also suggested that NO; reduction was more easily disrupted than NO; reduction. Only in the present temperature and pH studies was NO; usually reduced, although the range of pH values studies was rather limited. At 45°C and above, there were long lag periods before NO; reduction commenced and the rate of NO; reduction was very slow. At low pH values (pH 6 and below) NO; reduction proceeded very slowly. The sharp increase in the rate, after the peak NO; concentration was reached, was probably due to the increase in pH due to the production of OH- in the denitrification process. NOT reduction did commence before the pH was significantly altered. The lower pH value appears to have inhibited the reduction of N20 as well as the reduction of NO;. Finally, at high NO; concentrations (10mg NO;-Nml-r), no NO; was observed in 30 days, although NO; reduction did take place after 7 days. In Payne’s (1973) studies with resting cells, NO; was totally reduced to NO; before NO; was reduced to N,O. In our study NO; reduction normally commenced before all of the NO; was reduced. Only at temperatures of 45°C or higher was NO; totally reduced before NO; reduction commenced. Surprisingly, N20 temporarily accumulated at low NO; concentrations, but not at higher concentrations. The latter situation was found by Blackmer and Bremner (1978). It is suggested that our study has selected a denitrifying population normally capable of forming N2, without the accumulation of N20. N20 was also observed during growth at pH 5.0, but not at higher pH values. This is similar to the results obtained by Van Cleemput er al. (1975, 1976) and as postulated by Focht (1974). From this it
appears that NzO will accumulate conditions for denitrihcation.
during suboptimal
Aclinowlrdgrmmrs-This work has been supported by the Rutgers University Research Council, the New Jersey Agricultural Experiment Station. and the Atlantic Foundation.
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