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Relationships between the denitrification capacities of soils and total, water-soluble and readily decomposable soil organic matter
Sod
BwI.
Biuchcwtz.
Vol.l.,pp. 3X9-394. PergamonPress1975. Printed inGreatRrltain
RELATIONSHIPS BETWEEN THE DENITRIFICATION CAPACITIES OF SOILS AND TOTAL, WATER-SOLUBLE AND READILY DECOMPOSABLE SOIL ORGANIC MATTER J. R.
BURFORD*
and J. M.
BREMNER
Department of Agronomy. Iowa State University. Ames, IA 50010. U.S.A. (Accepted 17 April
1975)
Summary-The relationships between the denitrification capacities of 17 surface soils and the amounts of total organic carbon, mineralizable carbon, and water-soluble organic carbon in these soils were investigated. The soils used differed markedly in pH, texture. and organic-matter content. Denitrifi~tion capacity was assessed by determining the N evolved as N2 and N,O on anaerobic incubation of nitrate-treated soil at 20°C for 7 days, and mineralizable carbon was assessed by determining the C evolved as CO, on aerobic incubation of soil at 20°C for 7 days. The denitrification capacities of the soils studied were significantly correlated (r = @77***) with total organic carbon and very highly correlated (r = @99***) with water-soluble organic carbon or mineralizable carbon. The amount of nitrate N lost on anaerobic incubation of nitrate-treated soils for 7 days was very closely related jr = @999***) to the amount of N evolved as N, and N20. The work reported indicates that denitrification in soils under anaerobic conditions is controlled largely by the supply of readily decomposable organic matter and that analysis of soils for mineralizable carbon or water-soluble organic carbon provides a good index of their capacity for denitrification of nitrate. INTRODUCTION
in the presence of nitrate or under aerobic conditions Recent concern about the potential adverse effects of in the presence of any suitable source of nitrogen. nitrate on environmental quality and public health Although they can utilize both nitrate and oxygen has drawn attention to the need for research on the as hydrogen acceptors, there is good evidence that they will utilize nitrate only when the supply of contribution of soils and fertilizers to nitrate enrichment of water resources and the factors affecting deni- oxygen is not adequate for their demand (see Bremner trification of nitrate in soils and waters. The need and Shaw, 195s; Broadbent and Clark. 1965). There for more information concerning the factors influenc- are reports in the literature that denitrification can occur in soils under apparently aerobic conditions, ing dcnitrification in soils also has been emphasized but it is now generally accepted that the denitri~~by the increased cost and shortage of fertilizer nitrogen and by the consequent need to develop prac- tion observed under such conditions occurred at anaerobic sites and that denitrification does not occur tices that will reduce gaseous loss of fertilizer nitrogen from soils through denitrification of nitrate derived in soils until the oxygen supply is so restricted that the denitrifying bacteria cannot obtain enough from nitrogen fertilizers. Although the term denitrifi~tion has been used to oxygen to meet their requirements (Broad~nt and Clark, 1965). These is good reason to believe that describe various processes leading to loss of nitrate, the supply of readily decomposable organic matter the proper use of this term is to refer to the biological is also a critical factor for denitrification of nitrate reduction of nitrate or nitrite to gaseous forms of nitrogen. Most investigations of denitrification have in soils because it is well established that denitrification in waterlogged soils can be greatly promoted by shown that the nitrogen volatilized by biological reduction of nitrate is in the form of nitrous oxide addition of organic materials. Bremner and Shaw (19.58) found that the effects of organic materials on (N,O) and (or) molecular nitrogen (N2), but evolution denitrification in waterlogged soils varied with their of nitric oxide (NO) has been detected. resistance to decomposition, easily decomposable The denitrifying bacteria responsible for reduction substances such as glucose, mannitol and sucrose havof nitrate to gaseous forms of nitrogen are facultative ing much greater effects than di~cultly decomposable anaerobes that have the ability to use both oxygen and nitrate (or nitrite) as hydrogen acceptors. Pro- materials such as lignin and sawdust. They also found vided that an oxidizable substrate is present in the that the ability of wheat or oat straw to promote denitrification in waterlogged soils was greatly medium, they can grow under anaerobic conditions reduced when these materials were extracted with water or allowed to decompose before use as energy * Present address: Department of Soit Science, University of Reading. London Road, Reading, Berks RGI 5AQ. sources for denitrifying bacteria. These observations indicated that denitrification in soils is controlled larEngland. Journal Paper No. J-8101 of the Iowa Agriculture & gely by the supply of water-soluble or readily decomHome Economics Experiment Station, Ames, Iowa. Prqiect posable organic matter. The purpose of the work reported here was to test the validity of this concluNo. 1835.
390
J. R. BURFORU and J. M.
sion and to determine if analysis of soils for watersoluble or readily decomposable organic matter provides a good index of their ability to denitrify nitrate under anaerobic conditions. The soils used were selected to obtain a wide range in pH, texture, and organic-matter content. Their ability to denitrify nitrate under anaerobic conditions was assessed by determining the N, and N,O evolved when they were incubated (20°C) under helium for 7 days after treatment with KNO,, and their content of water-soluble organic matter was assessed by determining the organic carbon extracted by water at 23°C. Their content of readily decomposable organic matter was assessed by determining the C evolved as COZ when they were incubated (20°C) aerobically for 7 days. The carbon thus estimated is referred to as mineralizable carbon. MATERIALS
AND METHODS
The soils used (Table 1) were surface (n-1 5 cm) samples selected to obtain a wide range in pH (5%7.8), texture (2-94x sand, l-39% clay), and organic-matter content (0.3&5.95% organic carbon). Before use, each sample was air-dried and ground to pass a 2-mm screen. Total organic carbon in the soils was determined by the method of Mebius (1960). The other soil analyses reported in Table 1 were performed as described by Nelson and Bremner (1969). Water-soluble organic carbon in the soils studied was estimated by analyzing extracts obtained by gently shaking log of soil with 20ml of water for 15 min in a stoppered 50-ml polyethylene centrifuge tube and by centrifuging and filtering the resulting soil suspension. Centrifugation was performed at 19,500g with a Sorval Type SS-1 centrifuge, and the supernatant from centrifugation for 5 min was decanted into another 50-ml polyethylene centrifuge tube and centrifuged for a further 60 min. The’ clear supernatant from the second centrifugation was filtered with suction through a 47 mm dia 0.2 pm Metriccl membrane filter (Gelman Instrument Co., Ann Arbor, Michigan) previously washed with lOOm1 of water, and organic carbon in the filtrate was estimated by a modification of the dichromate oxidation method of Mebius (1960). In this modification, 5 ml of extract were treated with 5ml of 0.06~ K2CrZ07 and 15ml of concentrated H2S04 in a 125 ml ErlenTable I. Analyses of
soils
BKEMNIS
meyer flask fitted with a reflux condenser, and the mixture was boiled under reflux for 30 min. Residual dichromate in the cooled digest was determined by titration with 0.03 N Mohr salt [(NH,)2S0,.FeSO,‘6H,O]. Mineralizable carbon in the soils studied was estimated by determining the amount of carbon evolved as CO, on incubation of 5g of soil with 1.5ml of water at 20°C for 7 days. Incubations were performed in 12Xml n~~rr(~w-lno~lti~bottles scaled with rubber septa (cat. no. 2330, Arthur H. Thomas Co.. Philadelphia, Pennsylvania), and the CO? evolved was determined by the gas chromatographic procedure described by Burford and Bremner (1972), which involves use of krypton as an internal standard and of a column of Porapak Q for isothermal (25°C) separation of COZ and Kr from other gases. The denitrification capacities of the soils studied were assessed by estimating the amount of N evolved as NZ and N@ on anaerobic incubation (helium atmosphere) at 2OY? for 7 days of 7.5 g of soil treated with 15ml of KN03 solution containing 3OOO~gof nitrate N (4OOjdgof nitrate N/g of soil). Incubations were performed in V-ml narrow-mouth bottles sealed with rubber septa (cat. no. 2330. Arthur H. Thomas Co.). Before incubation, the air in the bottles containing the nitrate-treated soil samples was replaced with helium by a procedure similar to that used by McCarity (1961) to replace air with argon for manomctric estimation of the N gases evolved on anaerobic incubation of nitrate-treated soils. In this procedure, 2S-gauge hypodermic needles (length, 16mm) were inserted through the rubber septa used to seal the incubation bottles (to allow passage of gas to and from the bottles), and the bottles were placed in a vacuum desiccator. The bottles and their contents were evacuated to I mm (Hg) pressure and bank-flushed to room pressure with pure helium. This procedure was repeated three times, and, in the final flushing with helium, the pressure was increased to slightly in excess of room pressure. The desiccator lid was detached, and the hypodermic needles were removed rapidly from the septa to seal the bottles. An accurately measured amount (250~1) of pure krypton was then injected into each bottle, and, after 15 min, 10~1 samples of the gas phase in each bottle were analyzed for Nz and Kr by gas chromatography (any bottles containing more than trace amounts of N, were rejected). Immediately after removal of these gas samples, the shallow well on the top of each septum was sealed with molten paraffin wax, and the sealed bottles were placed in an incubator maintained at ZO’C. After 7 days. the Nz and N,O evolved on incubation were determined by gas chromatographic analysis of 10~1 samples of the gas phase in each bottle for N,, N,O and Kr. This analysis was performed with a Beckman GC-4 gas ChroI~atog~dph equipped with a helium-ionization detector connected to a I mV linear recorder and fitted with a 5.5m x I.59 mm (i.d.) column of Porapak Q operated at 25°C. The operational details were identical to those of the procedure described by Burford and Bremner (1972) for gas chromatographic determination of CO, evolved from soils in closed systems using Kr as an internal standard. The N,, N,O and Kr peaks on the gas chromatograms obtained with Porapak Q at
Denitrification in soils 25°C were completely resolved from each other and from the peaks obtained with CO,, CH,, C,H, and other gases known to be evolved from soils under anaerobic conditions, and the N, peak (retention time, 1.60 min) was resolved from the composite peak obtained with O2 and Ar (retention time, I.70 min). The NJ, N,O and Kr peak heights on the gas chromatograms of the gas samples analyzed were measured. and the amounts of N, and N,O evolved on incubation were calculated from these measurements by techniques similar to those described by Burford and Bremner (1972), the only difference being that the calibration standards used were Kr-N,-N,@He mixtures instead of Kr-W-air mixtures. The gases employed in the methods used to assess mineralizable carbon and denitrification capacity were purified compressed products of the Matheson Company, Joliet, Illinois. The gas syringes used in these methods were Pressure-Lok syringes supplied by Precision Sampling Corporation, Baton Rouge, Louisiana. Exchangeable ammonium, nitrite, and nitrate were determined by the steam distillation methods described by Bremner and Keeney (1966), and hydroxylamine was determined by the calorimetric method described by Csaky (1948). Analyses for these forms of N were performed on extracts obtained by shaking the soil samples under analysis with 2 M KC1 (lOml/g of soil) and filtering the resulting suspensions as described by Bremner and Keeney (1966). Analyses of these extracts for nitrite by the calorimetric method described by Bremner (1965) showed that the results obtained by this method agreed closely with those obtained by the steam distillation method. All analyses and experiments reported were performed in duplicate or triplicate. RESULTS
AND
DISCUSSION
The technique adopted for assessment of the denitrification capacities of the soils studied was based on experiments involving determination of the (N,O + N,)-N evolved when these soils were incubated anaerobically (helium atmosphere) at 20°C for various times after treatment with KNO, (4OOpg of nitrate N/g of soil). These experiments showed that, with all soils studied, the amount of (N,O + N1)-N evolved in 7 days was not much greater than the amount evolved in 5 days and that the rate of production of (N,O + N,)-N after 7 days was much slower than the corresponding rate during the first 3 days of incubation. Also, nitrate analyses performed in these experiments showed that the amount of nitrate N lost in 7 days was not much greater than the amount lost in 5 days. Figures l-3 show that the denitrification capacities of the 17 soils studied were significantly correlated (1. = 0.77***) with total organic carbon (Fig. I) and very highly correlated (r = 0.99***) with water-soluble organic carbon (Fig.2) or mineralizable carbon (Fig. 3). The amounts of water-soluble organic carbon in these soils ranged from 9 to 259 pg/g of soil (average, 97 pg/g) and represented 0.1%0.77% (average, 0.40%) of the total organic carbon (Table 2). The amounts of mineralizable carbon ranged from 35 to 441 /*g/g of soil (average, 187 pg/g) and represented
400 . Y=5OOx+62 t
Fig. I. Relationship between denitrification capacity and total organic carbon (17 soils). 0.35 to 1.270/, (average, 0.78%) of the total organic carbon (Table 2). Total organic carbon was significantly correlated with water-soluble organic carbon (r = 0.76***) and with mineralizable carbon (r = 0.82***), and there wasa highly significant correlation (r = 0.97***) between mineralizable carbon and water-soluble organic carbon (Fig. 4). Table 3 shows the amounts of nitrate N lost and the amounts of N evolved as N, and N20 when the soils studied were incubated anaerobically at 20°C for 7 days after treatment with nitrate (400,~g of nitrate N/g of soil). The amount of nitrate N lost ranged from 16 to 395 pg/g of soil (average, 143 pg/g), and the amount of N evolved as N,O and N2 ranged from 14 to 385 pg/g of soil (average, 137 pg/g). The
400-
= '6 Y) % 3000 ; " z" gb s," 20028 '5p pz z 'g t IOOsz 0% P
Y-I-563x-174 . . /
. / P .r
q 0
I 100 Water-soluble
I 200 organic
C
I 300 (pg/g
of soil)
Fig. 2. Relationship between denitrification capacity and water-soluble organic carbon (17 soils).
392
J. R. BURFORUand f. M. r-
1
BREMNER
Table 2. Amounts of mineralizable carbon and watersoluble organic carbon in soils studied
4oC. )-
t-
,-
I-”
.
I
I 0
I
300
200
100
Mweralizable
C (pQ/Q
I
I
400
500
of soil)
Fig. 3. Relationship between denitrification capacity and mineralizable carbon (17 soils).
Table 3. Amounts of nitrate N lost and of N evolved as Nz and N,O on anaerobic incubation (20°C) of nitratetreated soils for 7 days
and N, accounted for 8X-990,:, (average, 95%) of the nitrate N lost, and the N recovered as N,O represented 2-790/, (average, 39%) of the (N,O + N2)-N evolved. The amount of nitrate
N evolved as N,O
N lost was very closely related (r = 0.999***) to the amount of N evolved as N, and N&I (Fig. 5). Several products besides N2 and N,O are known (or suspected) to be formed by microbial reduction of nitrate under anaerobic conditions, but very little of the nitrate N lost in the incubation experiments reported in Table 3 could be accounted for by analyses for these products after 7 days. Nitric oxide was detected in the atmospheres of several soils after 7
500 Y= 1~914x+@7 .
400 c 9 z p
*
l
300-
2 0 % 200-
~
Nitrate-N 0
I 100
I 200
Water-~lubu~
WQO”iC c
lost
(~Q/Q
of soil)
I 300 $&Q/Q Of S&f
Fig. 4. Retationship between mineralizable carbon water-soluble organic carbon (17 soils).
and
Fig. 5. Relationship between (N2 + N,O)-N evolved and nitrate-N lost on anaerobic incubation (20°C. 7 days) of soils treated with 400 pptn of nitrate N as KNO, (I7 soils).
Denitrification in soils
days, but the amounts of NO-N detected did not represent more than 0.1% of the nitrate N lost. Nitrite was detected in the Buckner, Hayden, and Primghar soils. The amount of nitrite N detected in the Buckner soil represented about 6% of the ni’trate N lost, whereas the amount detected in the Hayden and Primghar soils accounted for less than 1% of the nitrate N lost. comparison of the amounts of ammonium present in the nitrate-treated soils and the corresponding untreated soils after anaerobic incubation for 7 days indicated that some reduction of nitrate to ammonium occurred during incubation of the nitrate-treated soils. Except with the Buckner soil, however, very little (< 2%) of the nitrate N lost on incubation of the nitrate-treated soils could be accounted for as ammonium N. With the Buckner soil, about 6% of the nitrate N lost could be accounted for as ammonium N. No trace of hydroxylamine could be detected in any of the nitrate-treated soils after 7 days. The following equations have been used to calculate the amount of available carbon required for microbial reduction of nitrate to N,O or N,: 4(CH,O) + 4N0,
+ 4H + = 4C0, + 2N20 + 6H,O
5(CHz0) + 4N0,
+ 4H ’ = 5C0, + 2Nz + 7H,O
According to these equations, 1 pg of available carbon is required for production of 1.17pugof N as N,O or of 0.99 /~g of N as NZ. Calculations from these values show that 097 pg of available carbon is needed to produce 1 ,ug of (NzO + N&N in which the ratio of N,O-N to N,-N is the average of the ratios observed in the experiments reported in Table 3. The average amount of N evolved as (N,O + NJ-N from the 17 soils used in these experiments was 137 pg/g of soil (Table 3), and the average amounts of total organic carbon, mineralisable carbon, and water-soluble organic carbon in these soils were 26170, 187 and 97 pg/g of soil, respectively (Table 2). Calculations from these values show that the average amounts of total organic carbon, mineralizable carbon, and water-soluble organic carbon per pg of N evolved as N,O or N, were 191, 1.36 and 0.71 btg/g of soil, respectively. Since 0.99pg of available carbon is needed to prodvce 1 pg of (N,O + N&N in which the ratio of N,O-N to N,-N is the average of the ratios observed in the experiments reported in Table 3, it can be calculated that, on the average, the amounts of total organic carbon, mineralizable carbon, and water-soluble organic carbon were, respectively, 193, 1.36, and 0.72 times the amount needed for the observed production of N,O and Nz~ In other words, the amount of carbon as total organic carbon far exceeded the amount needed for the denitri~catiol~ observed, whereas the amount as mineralizable carbon was not much greater than the amount needed, and the amount as water-soluble organic carbon was less than the amount required. If it is assumed that the water-soluble organic carbon was completely utilized by the denitrifying bacteria, it can be deduced that, on the average, 28% of the carbon utilized by these bacteria for reduction of nitrate to N, and N,O
393
was obtained from water-insoluble forms of organic carbon. Several investigations have indicated that the ratio of N,O to N, produced by denitrification of nitrate in soils can vary widely and is affected by pH, temperature, and the ratio of ammonium to nitrate in the system (e.g. Wijler and Delwiche, 1954; Nitmmik, 19.56; Schwar~beck, MacGregor and Schmidt, 1961). Statistical analysis of the data obtained in our work showed that the ratio of NzO to N, was not closely correlated with any of the soil properties listed in Table 1 or with the amount of mineralizable carbon or water-soluble organic carbon. It is evident from Tables 1 and 3, however, that this ratio tended to decrease with increase in soil pH. Wijler and Delwiche (1954) found that the ratio of N,O to N, depended on the soil pH and concluded that reduction of N,O to N, was strongly inhibited beIow pH 7. However, Cady and Bartholomew (1960) observed essentially complete reduction of N,O to N, in a study of denitrification in an acidic sandy loam (pH 4.5 in M KCl). With the soils used in our work, the average ratio of N,O-N to N,-N for soils in the pH range 5.8-6.6 (1.40: I) was markedly higher than the corresponding ratio for soils in the pH range 6.7-7.8 (038: 1). Since estimation of mineralizable carbon provides an estimate of readily decomposable organic matter, the highly significant relationship observed in our work between denitr~fication capacity and mineralizable carbon (Fig. 3) supports the conclusion that denitrification in soils under anaerobic conditions is controlled largely by the supply of readily decomposable organic matter. The highly significant relationship between denitri~~tion capacity and water-soluble organic carbon (Fig. 2) provides additional evidence for this conclusion because there is very little doubt that the water-soluble fraction of soil organic matter is particularly susceptible to decomposition. Assuming that mineralizable carbon includes water-soluble organic carbon, calculations from the analyses reported in Table 2 show that, on the average, watersoluble organic matter accounted for slightly more than half of the readily decomposable organic matter in the soils studied. To confirm our conclusion that denitrifi~tion in soils under anaerobic conditions is controlled largely by the supply of readily decomposable organic matter for microbial reduction of nitrate, we studied the effect of glucose on denitrification in six soils that exhibited a low capacity for denitrification of nitrate (Buckner, Thurman, Ida, Belinda, Marshall, and Grundyf. When incubated anaerobically at 20°C for 7 days after treatment with nitrate (0.4mg of nitrate N/g of soil), these soils denitrified only 4-17x of the added nitrate (Table 3). When incubated under the same conditions after treatment with both nitrate and glucose (0.4mg of nitrate N and 2mg of glucose C/g of soil), they denitrified all the added nitrate (no nitrate N was detected after 7 days, and 9699% of the nitrate N lost was recovered as N, and NzO). The finding that the denitrification capacities of the 17 soils studied were very highly correlated with water-soluble organic carbon (Fig. 2) and with mineralizable carbon (Fig. 3) indicates that analysis of soils for these forms of carbon will provide a good index
394
of their capacity
J. R. BURFORDand J. M. BREWER
for denitrification
of nitrate.
It
should be noted, however, that the soils used in our work were air-dried samples of surface soils. Further work is needed to determine if similar rest&s are obtained with field-moist samples of surface. soils and subsoils.
Acknowledgrmerrts-Journal Paper No. J-8101 of the Iowa Agriculture and Home Economics Experiment Station. Ames, Iowa. Projects 1835 and 1845. This work was supported in part by the Rockefeller Foundation.
REFERENCES BREMNERJ. M. (1965) Inorganic forms of nitrogen. In Methods of Soil Analysis, Part 2 (C. A. Black, Ed.). pp. 1179-1237, American Society of Agronomy, Madison, Wisconsin. BREMNERJ. M. and KEENEYD. R. (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils. 3. Exchangeable ammonium, nitrate and nitrite by extraction-distillation methods. Proc. Soil Sci. Sot. Am. 30, 577-582. BREMNER J. M. and SHAWK. (1958) Denitrification in soit --II: Factors affecting denitrifi~tion. J. Agric. Sci. 51, 40-52.
BROADBENT F. E. and CLARKF. E. (1965) Denitrification. In Soil Nitrogen (W. V. Bartholomew and F. E. Clark, Eds.), pp. 344-359. American Society of Agronomy, Madison, Wisconsin. BURFORDJ. R. and BREMNERJ. M. (1972) Gas chromatographic determination of carbon dioxide evolved from soils in closed systems. Soil Biol. Biochem. 4. 191-197. CADY F. B. and BARTHOLOMEW W. V. (1960) Seauential products of anaerobic denitrification bin Norfolk soil material. Proc. Soil Sci. Sot. Am. 24. 477-482. CSAKYT. Z. (1948) On the estimation of bound hydroxylamine in biological materials. Acta Chem. Stand. 2, 45t3454. MCGAR~TYJ. W. (1961) Denitrification studies on some South Australian soils. PI. Soil 14, l-21. MEBIUSL. J. (1960) A rapid method for the determination of organic carbon in soil. Analyt. Chim. Acta 22, 120124. NELSON D. W. and BREMNER J. M. (1969) Factors affecting chemical transformations of nitrite in soils. Soif Biof. Biochem. 1, 229-239. N~MMIKH. (1956) Investigation on denitrification in soil. Acta Agric. Scand. 6, 195-228. SCHWARTZBECK R. A., MACGREGORJ. M. and SCHMIDT E. L. (1961) Gaseous nitrogen losses from nitrogen fertilized soils measured with infrared and mass spectroscopy. Prac. Soil Sci. Sot. Am. 25, 186-192. WIJLER J. and DELW~CHEC. C. (1954) Investi~tions on the denitrifying process in soil. PI. Soil 5, 155-169.
BwI.
Biuchcwtz.
Vol.l.,pp. 3X9-394. PergamonPress1975. Printed inGreatRrltain
RELATIONSHIPS BETWEEN THE DENITRIFICATION CAPACITIES OF SOILS AND TOTAL, WATER-SOLUBLE AND READILY DECOMPOSABLE SOIL ORGANIC MATTER J. R.
BURFORD*
and J. M.
BREMNER
Department of Agronomy. Iowa State University. Ames, IA 50010. U.S.A. (Accepted 17 April
1975)
Summary-The relationships between the denitrification capacities of 17 surface soils and the amounts of total organic carbon, mineralizable carbon, and water-soluble organic carbon in these soils were investigated. The soils used differed markedly in pH, texture. and organic-matter content. Denitrifi~tion capacity was assessed by determining the N evolved as N2 and N,O on anaerobic incubation of nitrate-treated soil at 20°C for 7 days, and mineralizable carbon was assessed by determining the C evolved as CO, on aerobic incubation of soil at 20°C for 7 days. The denitrification capacities of the soils studied were significantly correlated (r = @77***) with total organic carbon and very highly correlated (r = @99***) with water-soluble organic carbon or mineralizable carbon. The amount of nitrate N lost on anaerobic incubation of nitrate-treated soils for 7 days was very closely related jr = @999***) to the amount of N evolved as N, and N20. The work reported indicates that denitrification in soils under anaerobic conditions is controlled largely by the supply of readily decomposable organic matter and that analysis of soils for mineralizable carbon or water-soluble organic carbon provides a good index of their capacity for denitrification of nitrate. INTRODUCTION
in the presence of nitrate or under aerobic conditions Recent concern about the potential adverse effects of in the presence of any suitable source of nitrogen. nitrate on environmental quality and public health Although they can utilize both nitrate and oxygen has drawn attention to the need for research on the as hydrogen acceptors, there is good evidence that they will utilize nitrate only when the supply of contribution of soils and fertilizers to nitrate enrichment of water resources and the factors affecting deni- oxygen is not adequate for their demand (see Bremner trification of nitrate in soils and waters. The need and Shaw, 195s; Broadbent and Clark. 1965). There for more information concerning the factors influenc- are reports in the literature that denitrification can occur in soils under apparently aerobic conditions, ing dcnitrification in soils also has been emphasized but it is now generally accepted that the denitri~~by the increased cost and shortage of fertilizer nitrogen and by the consequent need to develop prac- tion observed under such conditions occurred at anaerobic sites and that denitrification does not occur tices that will reduce gaseous loss of fertilizer nitrogen from soils through denitrification of nitrate derived in soils until the oxygen supply is so restricted that the denitrifying bacteria cannot obtain enough from nitrogen fertilizers. Although the term denitrifi~tion has been used to oxygen to meet their requirements (Broad~nt and Clark, 1965). These is good reason to believe that describe various processes leading to loss of nitrate, the supply of readily decomposable organic matter the proper use of this term is to refer to the biological is also a critical factor for denitrification of nitrate reduction of nitrate or nitrite to gaseous forms of nitrogen. Most investigations of denitrification have in soils because it is well established that denitrification in waterlogged soils can be greatly promoted by shown that the nitrogen volatilized by biological reduction of nitrate is in the form of nitrous oxide addition of organic materials. Bremner and Shaw (19.58) found that the effects of organic materials on (N,O) and (or) molecular nitrogen (N2), but evolution denitrification in waterlogged soils varied with their of nitric oxide (NO) has been detected. resistance to decomposition, easily decomposable The denitrifying bacteria responsible for reduction substances such as glucose, mannitol and sucrose havof nitrate to gaseous forms of nitrogen are facultative ing much greater effects than di~cultly decomposable anaerobes that have the ability to use both oxygen and nitrate (or nitrite) as hydrogen acceptors. Pro- materials such as lignin and sawdust. They also found vided that an oxidizable substrate is present in the that the ability of wheat or oat straw to promote denitrification in waterlogged soils was greatly medium, they can grow under anaerobic conditions reduced when these materials were extracted with water or allowed to decompose before use as energy * Present address: Department of Soit Science, University of Reading. London Road, Reading, Berks RGI 5AQ. sources for denitrifying bacteria. These observations indicated that denitrification in soils is controlled larEngland. Journal Paper No. J-8101 of the Iowa Agriculture & gely by the supply of water-soluble or readily decomHome Economics Experiment Station, Ames, Iowa. Prqiect posable organic matter. The purpose of the work reported here was to test the validity of this concluNo. 1835.
390
J. R. BURFORU and J. M.
sion and to determine if analysis of soils for watersoluble or readily decomposable organic matter provides a good index of their ability to denitrify nitrate under anaerobic conditions. The soils used were selected to obtain a wide range in pH, texture, and organic-matter content. Their ability to denitrify nitrate under anaerobic conditions was assessed by determining the N, and N,O evolved when they were incubated (20°C) under helium for 7 days after treatment with KNO,, and their content of water-soluble organic matter was assessed by determining the organic carbon extracted by water at 23°C. Their content of readily decomposable organic matter was assessed by determining the C evolved as COZ when they were incubated (20°C) aerobically for 7 days. The carbon thus estimated is referred to as mineralizable carbon. MATERIALS
AND METHODS
The soils used (Table 1) were surface (n-1 5 cm) samples selected to obtain a wide range in pH (5%7.8), texture (2-94x sand, l-39% clay), and organic-matter content (0.3&5.95% organic carbon). Before use, each sample was air-dried and ground to pass a 2-mm screen. Total organic carbon in the soils was determined by the method of Mebius (1960). The other soil analyses reported in Table 1 were performed as described by Nelson and Bremner (1969). Water-soluble organic carbon in the soils studied was estimated by analyzing extracts obtained by gently shaking log of soil with 20ml of water for 15 min in a stoppered 50-ml polyethylene centrifuge tube and by centrifuging and filtering the resulting soil suspension. Centrifugation was performed at 19,500g with a Sorval Type SS-1 centrifuge, and the supernatant from centrifugation for 5 min was decanted into another 50-ml polyethylene centrifuge tube and centrifuged for a further 60 min. The’ clear supernatant from the second centrifugation was filtered with suction through a 47 mm dia 0.2 pm Metriccl membrane filter (Gelman Instrument Co., Ann Arbor, Michigan) previously washed with lOOm1 of water, and organic carbon in the filtrate was estimated by a modification of the dichromate oxidation method of Mebius (1960). In this modification, 5 ml of extract were treated with 5ml of 0.06~ K2CrZ07 and 15ml of concentrated H2S04 in a 125 ml ErlenTable I. Analyses of
soils
BKEMNIS
meyer flask fitted with a reflux condenser, and the mixture was boiled under reflux for 30 min. Residual dichromate in the cooled digest was determined by titration with 0.03 N Mohr salt [(NH,)2S0,.FeSO,‘6H,O]. Mineralizable carbon in the soils studied was estimated by determining the amount of carbon evolved as CO, on incubation of 5g of soil with 1.5ml of water at 20°C for 7 days. Incubations were performed in 12Xml n~~rr(~w-lno~lti~bottles scaled with rubber septa (cat. no. 2330, Arthur H. Thomas Co.. Philadelphia, Pennsylvania), and the CO? evolved was determined by the gas chromatographic procedure described by Burford and Bremner (1972), which involves use of krypton as an internal standard and of a column of Porapak Q for isothermal (25°C) separation of COZ and Kr from other gases. The denitrification capacities of the soils studied were assessed by estimating the amount of N evolved as NZ and N@ on anaerobic incubation (helium atmosphere) at 2OY? for 7 days of 7.5 g of soil treated with 15ml of KN03 solution containing 3OOO~gof nitrate N (4OOjdgof nitrate N/g of soil). Incubations were performed in V-ml narrow-mouth bottles sealed with rubber septa (cat. no. 2330. Arthur H. Thomas Co.). Before incubation, the air in the bottles containing the nitrate-treated soil samples was replaced with helium by a procedure similar to that used by McCarity (1961) to replace air with argon for manomctric estimation of the N gases evolved on anaerobic incubation of nitrate-treated soils. In this procedure, 2S-gauge hypodermic needles (length, 16mm) were inserted through the rubber septa used to seal the incubation bottles (to allow passage of gas to and from the bottles), and the bottles were placed in a vacuum desiccator. The bottles and their contents were evacuated to I mm (Hg) pressure and bank-flushed to room pressure with pure helium. This procedure was repeated three times, and, in the final flushing with helium, the pressure was increased to slightly in excess of room pressure. The desiccator lid was detached, and the hypodermic needles were removed rapidly from the septa to seal the bottles. An accurately measured amount (250~1) of pure krypton was then injected into each bottle, and, after 15 min, 10~1 samples of the gas phase in each bottle were analyzed for Nz and Kr by gas chromatography (any bottles containing more than trace amounts of N, were rejected). Immediately after removal of these gas samples, the shallow well on the top of each septum was sealed with molten paraffin wax, and the sealed bottles were placed in an incubator maintained at ZO’C. After 7 days. the Nz and N,O evolved on incubation were determined by gas chromatographic analysis of 10~1 samples of the gas phase in each bottle for N,, N,O and Kr. This analysis was performed with a Beckman GC-4 gas ChroI~atog~dph equipped with a helium-ionization detector connected to a I mV linear recorder and fitted with a 5.5m x I.59 mm (i.d.) column of Porapak Q operated at 25°C. The operational details were identical to those of the procedure described by Burford and Bremner (1972) for gas chromatographic determination of CO, evolved from soils in closed systems using Kr as an internal standard. The N,, N,O and Kr peaks on the gas chromatograms obtained with Porapak Q at
Denitrification in soils 25°C were completely resolved from each other and from the peaks obtained with CO,, CH,, C,H, and other gases known to be evolved from soils under anaerobic conditions, and the N, peak (retention time, 1.60 min) was resolved from the composite peak obtained with O2 and Ar (retention time, I.70 min). The NJ, N,O and Kr peak heights on the gas chromatograms of the gas samples analyzed were measured. and the amounts of N, and N,O evolved on incubation were calculated from these measurements by techniques similar to those described by Burford and Bremner (1972), the only difference being that the calibration standards used were Kr-N,-N,@He mixtures instead of Kr-W-air mixtures. The gases employed in the methods used to assess mineralizable carbon and denitrification capacity were purified compressed products of the Matheson Company, Joliet, Illinois. The gas syringes used in these methods were Pressure-Lok syringes supplied by Precision Sampling Corporation, Baton Rouge, Louisiana. Exchangeable ammonium, nitrite, and nitrate were determined by the steam distillation methods described by Bremner and Keeney (1966), and hydroxylamine was determined by the calorimetric method described by Csaky (1948). Analyses for these forms of N were performed on extracts obtained by shaking the soil samples under analysis with 2 M KC1 (lOml/g of soil) and filtering the resulting suspensions as described by Bremner and Keeney (1966). Analyses of these extracts for nitrite by the calorimetric method described by Bremner (1965) showed that the results obtained by this method agreed closely with those obtained by the steam distillation method. All analyses and experiments reported were performed in duplicate or triplicate. RESULTS
AND
DISCUSSION
The technique adopted for assessment of the denitrification capacities of the soils studied was based on experiments involving determination of the (N,O + N,)-N evolved when these soils were incubated anaerobically (helium atmosphere) at 20°C for various times after treatment with KNO, (4OOpg of nitrate N/g of soil). These experiments showed that, with all soils studied, the amount of (N,O + N1)-N evolved in 7 days was not much greater than the amount evolved in 5 days and that the rate of production of (N,O + N,)-N after 7 days was much slower than the corresponding rate during the first 3 days of incubation. Also, nitrate analyses performed in these experiments showed that the amount of nitrate N lost in 7 days was not much greater than the amount lost in 5 days. Figures l-3 show that the denitrification capacities of the 17 soils studied were significantly correlated (1. = 0.77***) with total organic carbon (Fig. I) and very highly correlated (r = 0.99***) with water-soluble organic carbon (Fig.2) or mineralizable carbon (Fig. 3). The amounts of water-soluble organic carbon in these soils ranged from 9 to 259 pg/g of soil (average, 97 pg/g) and represented 0.1%0.77% (average, 0.40%) of the total organic carbon (Table 2). The amounts of mineralizable carbon ranged from 35 to 441 /*g/g of soil (average, 187 pg/g) and represented
400 . Y=5OOx+62 t
Fig. I. Relationship between denitrification capacity and total organic carbon (17 soils). 0.35 to 1.270/, (average, 0.78%) of the total organic carbon (Table 2). Total organic carbon was significantly correlated with water-soluble organic carbon (r = 0.76***) and with mineralizable carbon (r = 0.82***), and there wasa highly significant correlation (r = 0.97***) between mineralizable carbon and water-soluble organic carbon (Fig. 4). Table 3 shows the amounts of nitrate N lost and the amounts of N evolved as N, and N20 when the soils studied were incubated anaerobically at 20°C for 7 days after treatment with nitrate (400,~g of nitrate N/g of soil). The amount of nitrate N lost ranged from 16 to 395 pg/g of soil (average, 143 pg/g), and the amount of N evolved as N,O and N2 ranged from 14 to 385 pg/g of soil (average, 137 pg/g). The
400-
= '6 Y) % 3000 ; " z" gb s," 20028 '5p pz z 'g t IOOsz 0% P
Y-I-563x-174 . . /
. / P .r
q 0
I 100 Water-soluble
I 200 organic
C
I 300 (pg/g
of soil)
Fig. 2. Relationship between denitrification capacity and water-soluble organic carbon (17 soils).
392
J. R. BURFORUand f. M. r-
1
BREMNER
Table 2. Amounts of mineralizable carbon and watersoluble organic carbon in soils studied
4oC. )-
t-
,-
I-”
.
I
I 0
I
300
200
100
Mweralizable
C (pQ/Q
I
I
400
500
of soil)
Fig. 3. Relationship between denitrification capacity and mineralizable carbon (17 soils).
Table 3. Amounts of nitrate N lost and of N evolved as Nz and N,O on anaerobic incubation (20°C) of nitratetreated soils for 7 days
and N, accounted for 8X-990,:, (average, 95%) of the nitrate N lost, and the N recovered as N,O represented 2-790/, (average, 39%) of the (N,O + N2)-N evolved. The amount of nitrate
N evolved as N,O
N lost was very closely related (r = 0.999***) to the amount of N evolved as N, and N&I (Fig. 5). Several products besides N2 and N,O are known (or suspected) to be formed by microbial reduction of nitrate under anaerobic conditions, but very little of the nitrate N lost in the incubation experiments reported in Table 3 could be accounted for by analyses for these products after 7 days. Nitric oxide was detected in the atmospheres of several soils after 7
500 Y= 1~914x+@7 .
400 c 9 z p
*
l
300-
2 0 % 200-
~
Nitrate-N 0
I 100
I 200
Water-~lubu~
WQO”iC c
lost
(~Q/Q
of soil)
I 300 $&Q/Q Of S&f
Fig. 4. Retationship between mineralizable carbon water-soluble organic carbon (17 soils).
and
Fig. 5. Relationship between (N2 + N,O)-N evolved and nitrate-N lost on anaerobic incubation (20°C. 7 days) of soils treated with 400 pptn of nitrate N as KNO, (I7 soils).
Denitrification in soils
days, but the amounts of NO-N detected did not represent more than 0.1% of the nitrate N lost. Nitrite was detected in the Buckner, Hayden, and Primghar soils. The amount of nitrite N detected in the Buckner soil represented about 6% of the ni’trate N lost, whereas the amount detected in the Hayden and Primghar soils accounted for less than 1% of the nitrate N lost. comparison of the amounts of ammonium present in the nitrate-treated soils and the corresponding untreated soils after anaerobic incubation for 7 days indicated that some reduction of nitrate to ammonium occurred during incubation of the nitrate-treated soils. Except with the Buckner soil, however, very little (< 2%) of the nitrate N lost on incubation of the nitrate-treated soils could be accounted for as ammonium N. With the Buckner soil, about 6% of the nitrate N lost could be accounted for as ammonium N. No trace of hydroxylamine could be detected in any of the nitrate-treated soils after 7 days. The following equations have been used to calculate the amount of available carbon required for microbial reduction of nitrate to N,O or N,: 4(CH,O) + 4N0,
+ 4H + = 4C0, + 2N20 + 6H,O
5(CHz0) + 4N0,
+ 4H ’ = 5C0, + 2Nz + 7H,O
According to these equations, 1 pg of available carbon is required for production of 1.17pugof N as N,O or of 0.99 /~g of N as NZ. Calculations from these values show that 097 pg of available carbon is needed to produce 1 ,ug of (NzO + N&N in which the ratio of N,O-N to N,-N is the average of the ratios observed in the experiments reported in Table 3. The average amount of N evolved as (N,O + NJ-N from the 17 soils used in these experiments was 137 pg/g of soil (Table 3), and the average amounts of total organic carbon, mineralisable carbon, and water-soluble organic carbon in these soils were 26170, 187 and 97 pg/g of soil, respectively (Table 2). Calculations from these values show that the average amounts of total organic carbon, mineralizable carbon, and water-soluble organic carbon per pg of N evolved as N,O or N, were 191, 1.36 and 0.71 btg/g of soil, respectively. Since 0.99pg of available carbon is needed to prodvce 1 pg of (N,O + N&N in which the ratio of N,O-N to N,-N is the average of the ratios observed in the experiments reported in Table 3, it can be calculated that, on the average, the amounts of total organic carbon, mineralizable carbon, and water-soluble organic carbon were, respectively, 193, 1.36, and 0.72 times the amount needed for the observed production of N,O and Nz~ In other words, the amount of carbon as total organic carbon far exceeded the amount needed for the denitri~catiol~ observed, whereas the amount as mineralizable carbon was not much greater than the amount needed, and the amount as water-soluble organic carbon was less than the amount required. If it is assumed that the water-soluble organic carbon was completely utilized by the denitrifying bacteria, it can be deduced that, on the average, 28% of the carbon utilized by these bacteria for reduction of nitrate to N, and N,O
393
was obtained from water-insoluble forms of organic carbon. Several investigations have indicated that the ratio of N,O to N, produced by denitrification of nitrate in soils can vary widely and is affected by pH, temperature, and the ratio of ammonium to nitrate in the system (e.g. Wijler and Delwiche, 1954; Nitmmik, 19.56; Schwar~beck, MacGregor and Schmidt, 1961). Statistical analysis of the data obtained in our work showed that the ratio of NzO to N, was not closely correlated with any of the soil properties listed in Table 1 or with the amount of mineralizable carbon or water-soluble organic carbon. It is evident from Tables 1 and 3, however, that this ratio tended to decrease with increase in soil pH. Wijler and Delwiche (1954) found that the ratio of N,O to N, depended on the soil pH and concluded that reduction of N,O to N, was strongly inhibited beIow pH 7. However, Cady and Bartholomew (1960) observed essentially complete reduction of N,O to N, in a study of denitrification in an acidic sandy loam (pH 4.5 in M KCl). With the soils used in our work, the average ratio of N,O-N to N,-N for soils in the pH range 5.8-6.6 (1.40: I) was markedly higher than the corresponding ratio for soils in the pH range 6.7-7.8 (038: 1). Since estimation of mineralizable carbon provides an estimate of readily decomposable organic matter, the highly significant relationship observed in our work between denitr~fication capacity and mineralizable carbon (Fig. 3) supports the conclusion that denitrification in soils under anaerobic conditions is controlled largely by the supply of readily decomposable organic matter. The highly significant relationship between denitri~~tion capacity and water-soluble organic carbon (Fig. 2) provides additional evidence for this conclusion because there is very little doubt that the water-soluble fraction of soil organic matter is particularly susceptible to decomposition. Assuming that mineralizable carbon includes water-soluble organic carbon, calculations from the analyses reported in Table 2 show that, on the average, watersoluble organic matter accounted for slightly more than half of the readily decomposable organic matter in the soils studied. To confirm our conclusion that denitrifi~tion in soils under anaerobic conditions is controlled largely by the supply of readily decomposable organic matter for microbial reduction of nitrate, we studied the effect of glucose on denitrification in six soils that exhibited a low capacity for denitrification of nitrate (Buckner, Thurman, Ida, Belinda, Marshall, and Grundyf. When incubated anaerobically at 20°C for 7 days after treatment with nitrate (0.4mg of nitrate N/g of soil), these soils denitrified only 4-17x of the added nitrate (Table 3). When incubated under the same conditions after treatment with both nitrate and glucose (0.4mg of nitrate N and 2mg of glucose C/g of soil), they denitrified all the added nitrate (no nitrate N was detected after 7 days, and 9699% of the nitrate N lost was recovered as N, and NzO). The finding that the denitrification capacities of the 17 soils studied were very highly correlated with water-soluble organic carbon (Fig. 2) and with mineralizable carbon (Fig. 3) indicates that analysis of soils for these forms of carbon will provide a good index
394
of their capacity
J. R. BURFORDand J. M. BREWER
for denitrification
of nitrate.
It
should be noted, however, that the soils used in our work were air-dried samples of surface soils. Further work is needed to determine if similar rest&s are obtained with field-moist samples of surface. soils and subsoils.
Acknowledgrmerrts-Journal Paper No. J-8101 of the Iowa Agriculture and Home Economics Experiment Station. Ames, Iowa. Projects 1835 and 1845. This work was supported in part by the Rockefeller Foundation.
REFERENCES BREMNERJ. M. (1965) Inorganic forms of nitrogen. In Methods of Soil Analysis, Part 2 (C. A. Black, Ed.). pp. 1179-1237, American Society of Agronomy, Madison, Wisconsin. BREMNERJ. M. and KEENEYD. R. (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils. 3. Exchangeable ammonium, nitrate and nitrite by extraction-distillation methods. Proc. Soil Sci. Sot. Am. 30, 577-582. BREMNER J. M. and SHAWK. (1958) Denitrification in soit --II: Factors affecting denitrifi~tion. J. Agric. Sci. 51, 40-52.
BROADBENT F. E. and CLARKF. E. (1965) Denitrification. In Soil Nitrogen (W. V. Bartholomew and F. E. Clark, Eds.), pp. 344-359. American Society of Agronomy, Madison, Wisconsin. BURFORDJ. R. and BREMNERJ. M. (1972) Gas chromatographic determination of carbon dioxide evolved from soils in closed systems. Soil Biol. Biochem. 4. 191-197. CADY F. B. and BARTHOLOMEW W. V. (1960) Seauential products of anaerobic denitrification bin Norfolk soil material. Proc. Soil Sci. Sot. Am. 24. 477-482. CSAKYT. Z. (1948) On the estimation of bound hydroxylamine in biological materials. Acta Chem. Stand. 2, 45t3454. MCGAR~TYJ. W. (1961) Denitrification studies on some South Australian soils. PI. Soil 14, l-21. MEBIUSL. J. (1960) A rapid method for the determination of organic carbon in soil. Analyt. Chim. Acta 22, 120124. NELSON D. W. and BREMNER J. M. (1969) Factors affecting chemical transformations of nitrite in soils. Soif Biof. Biochem. 1, 229-239. N~MMIKH. (1956) Investigation on denitrification in soil. Acta Agric. Scand. 6, 195-228. SCHWARTZBECK R. A., MACGREGORJ. M. and SCHMIDT E. L. (1961) Gaseous nitrogen losses from nitrogen fertilized soils measured with infrared and mass spectroscopy. Prac. Soil Sci. Sot. Am. 25, 186-192. WIJLER J. and DELW~CHEC. C. (1954) Investi~tions on the denitrifying process in soil. PI. Soil 5, 155-169.