Advances in Animal Biosciences (2013), 4:s1, pp 42–49 & The Animal Consortium 2013 doi:10.1017/S2040470013000307
advances in animal biosciences
Effects of inorganic v. organic copper on denitrification in agricultural soil Q. Wang1, M. Burger2, T. A. Doane2, W. R. Horwath2, A. R. Castillo3 and F. M. Mitloehner11
Department of Animal Science, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA; 2Department of Land, Air and Water Resources, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA; 3University of California Cooperative Extension Merced County, Merced, CA 95341, USA
Nitrous oxide reductase (N2OR), the enzyme responsible for the reduction of N2O to N2 in denitrification, uses copper (Cu) as its cofactor. Its activity is lowered under conditions of Cu deficiency. In general, high organic matter (OM) soil decreases Cu availability. The present study investigated different Cu forms, namely organic (ORG) v. inorganic (INO), and associated concentrations (750, 550, 125, 60 mg Cu/g soil) for their efficacy in affecting denitrification and especially N2OR activity in high OM peat soil in a water saturated anaerobic condition for 24 h. Gas and liquid samples were taken every 8 h and analyzed for NO32, NO22, N2O and N2. Inorganic Cu treatments did not affect N transformation rates and N2OR activity among the different treatments ( P . 0.05) throughout the incubation compared with the control (CON). The ORG Cu treatments increased NO32 ( P , 0.05), NO22 ( P , 0.05) and N2O ( P , 0.05) transformation rates compared with CON. These changes were ORG Cu dose dependent. N2OR activity increased first in the 750 mg ORG Cu treatment ( P , 0.05) during 8 to 16 h followed by the other ORG Cu treatments ( P , 0.05) during 16 to 24 h compared with CON. These results highlight the importance of Cu form and concentration on N transformation rate during denitrification. The findings can potentially be applied to systems like soil, wastewater, constructed wetlands, etc., in which reactions of the denitrification pathway are manipulated. Keywords: inorganic copper, organic copper, denitrification, nitrous oxide reductase
Implications The present study is the first that has focused explicitly on the role of copper in the denitrification process in agricultural soil, at concentrations that are not excessive but instead promote the reactions that convert NO32 to NO22, NO, N2O, and ultimately to N2. The results from the present study have potential application in designing and optimizing procedures in which reduction of N2O and other reactions of denitrification pathway are manipulated in systems like wetlands, wastewater treatment, etc.
Introduction Denitrification refers to the respiratory reduction of nitrate (NO32) and nitrite (NO22), to the gaseous products nitric oxide (NO), nitrous oxide (N2O), and nitrogen gas (N2), which are linked to electron transport phosphorylation activities. Several processes other than denitrification produce N2O, such as nitrification, nitrifier denitrification and dissimilatory NO32 reduction to ammonium (NH41) (Bleakley and Tiedje, 1982; Stevens et al., 1998; Pathak, 1999; Mathieu et al., 2006; Burgin and Hamilton, 2007; Baggs, 2008). However, -
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denitrification is the only biological process that has the capacity to reduce N2O to N2. Each reduction step in the denitrification pathway involves a metalloenzyme that uses a redox active metal cofactor (Kroneck et al., 1988; Scott et al., 1989; Carr and Ferguson, 1990; Godden et al., 1991; Glockner et al., 1993; Butler et al., 2002; Richardson et al., 2009). The enzyme responsible for the reduction of N2O to N2 is nitrous oxide reductase (N2OR). Copper (Cu) is the cofactor in N2OR, with one enzyme containing 12 Cu atoms (Cambillau et al., 2000; Thomson et al., 2000 and 2002; Haltia et al., 2003; Hasnain et al., 2006). Another denitrification enzyme that uses Cu as a cofactor is NO22 reductase, responsible for the reduction of NO22 to NO (Liu et al., 1986; Libby and Averill, 1992). Much research has been conducted in aquatic and pure culture systems demonstrating the importance of Cu to the denitrification process, especially to N2OR activity (Iwasaki et al., 1980; Iwasaki and Terai, 1982; CatalanSakairi et al., 1997; Cervantes et al., 1998; Granger and Ward, 2003). Considerably less research has been conducted on the effects of Cu on N2OR activity in soils and sediments. Inadequate amounts or low availability of Cu will reduce the activity of N2OR in soils. Soil with high organic matter (OM) or clay content often have low Cu bioavailability (Lucas, 1948; Gilbert, 1952; Elliott et al., 1986; LHerroux et al., 1997; Bolan et al., 2004; Gerzabek et al., 2006; Lair et al., 2007).
Effects of inorganic v. organic copper on denitrification Copper and molybdenum form strong coordinate linkages with organic ligands found in OM. Organic (ORG) Cu forms strong chelation complexes with organic molecules within the OM. The chelation complex contains two or more coordinate positions around the metal ion, forming a complex ring structure. The formation of more than one bond between the metal and the organic molecule usually imparts high stability (Stevenson, 1994). The stability of the metalchelate process is determined by a host of factors including the number of atoms forming the organic ligand, number of ring structures, concentration of metal ions and pH. Cu forms the strongest stability complex of the primary transition series metals. For these reasons, Cu can be a limiting essential nutrient, especially in soils of high OM. Addressing the limiting factors affecting the complete reduction of inorganic N to N2 in soils and sediments is important to reducing N2O emissions. This is especially important in agricultural soils, the primary source of N2O emission globally (Oenema et al., 2005). The aim of this study is to investigate the effects of Cu form and concentration on denitrification, in a low Cu, high OM contents peat soil, with a focus on N2OR activity. Material and methods
Soil Peat soil was collected from an agricultural field on Twitchell Island in California’s Sacramento-San Joaquin River Delta. The soil is classified as a Rindge mucky silt loam (euic, thermic Typic Medisaprist). The peat soil was chosen for this study because of its high NO32 and OM contents and low Cu concentration. These soil conditions are conducive to a large denitrification potential. In addition, the high OM content lowers available Cu through strong OM complexion reactions and high Cu chelating capacity. Soil OM content determined by loss on ignition was 38%; Total C and N contents determined by direct combustion/gas chromatograph analysis were 18% and 1.15%; dissolved organic carbon (DOC), NH41-N, NO32-N, (extracted by 0.5 M K2SO4 at an extractant to soil ratio 10 to 1) was 720 mg DOC/g, and 1.4 mg NH41-N and 305 mg NO32-N/g soil, respectively. Available Cu as measured by diethylenetriaminepentaacetic acid extraction (Lindsay and Norvell, 1978) was 9 mg/g soil and the pH (1 : 1 H2O) was 5.2. The soil was air-dried and sieved (,1 mm) to remove stones and plant residues, such as roots. Soil incubation Cu source. The ORG Cu source was the soluble fraction of a proprietary Cu-peptide used in animal feed additive R formulations (Bioplex , Alltech, Nicholasville, KY, USA). This product was chosen as a typical source of Cu that might reach the environment via application of animal manure. The INO Cu source was Cu sulfate (CuSO4). Incubation. Twenty-seven soil samples (6.3 g/sample, air dry) were weighed into 60-ml glass serum vials. Nine treatments
with different concentrations of either INO Cu or ORG Cu or no Cu (CON) were randomly assigned to the vials. The experiment was arranged in a completely randomized design with three replications per treatment. Preliminary experiments were conducted to identify the approximate concentration of Cu at which denitrification would be affected. The concentrations of INO and ORG Cu chosen were 750, 550, 125, and 60 mg Cu/g soil (abbreviated as INO 750, INO 550, INO 125, INO 60, ORG 750, ORG 550, ORG 125, ORG 60). These treatments were applied to soils in 9 ml of deionized water. The ORG Cu treatments increased the DOC content of the soil by 90, 65, 10 and 5 mg/g soil in the 750, 550, 125 and 60 treatments, respectively. Each vial then received 1 ml of a solution of 99 atom% K15NO3 resulting in an overall 15N isotopic enrichment of the NO32 pool of 10.0 atom% 15N. The vials were sealed with rubber septa and aluminum seals. In order to create an anaerobic environment in the vials, each vial was evacuated and flushed with helium (He) for 10 min. This process was repeated three times. The vials were shaken in an orbital shaker/incubator (Model C25, New Brunswick Scientific, NJ, USA) at 150 r.p.m. and 358C to facilitate denitrification and diffusion of gaseous products. The incubation period lasted 24 h.
Sample collection. Every 8 h, 1 ml gas and 0.5 ml soil solution were removed from each vial. A gas tight syringe with a two-way stopcock was used for gas sampling. The syringe was flushed with He before sampling to avoid introducing air from the needle into the incubation vials. The gas samples were transferred to 12 ml evacuated gas tight vials (Labco, Lampeter, UK) until further analysis. The soil solution samples were immediately centrifuged to remove soil and the supernatants were stored at 2208C until further analysis. After sampling, vials were returned to the incubator to continue the incubation. Gas analysis Concentration of N2 and N2O and 15N isotope ratios of these gases were measured on a ThermoFinnigan GasBench 1 PreCon trace gas concentration system interfaced to a ThermoScientific Delta V Plus isotope-ratio mass spectrometer (IRMS; ThermoFinnigan, Bremen, Germany). Gas samples were purged from vials through a double-needle sampler into a He carrier stream (20 ml/min). Nitrogen gas was sampled by a rotary eight-port valve and passed to the IRMS through a molecular sieve 5A GC column (15m 3 0.53 mm ID, 258C, 3 ml/min). The remaining gas sample passed through a CO2 scrubber (Ascarite) and N2O was trapped and concentrated in two liquid nitrogen cryotraps. N2O was carried by He to the IRMS via a Poroplot Q GC column (25m 3 0.53 mm, 258C, 1.8 ml/min). Analysis of soil solution Concentrations of NO32 and NO22 in the soil solution were determined spectrophotometrically (Verdouw et al., 1978; Doane and Horwath, 2003). 43
Wang, Burger, Doane, Horwath, Castillo and Mitloehner
Calculations Determination of NO32 to N2O and N2. The amount of N2 present at each time point was calculated from the measured concentration, converted from molar ratio units to mass per volume units assuming ideal gas relations, and multiplied by the volume of the head space. The amount of N2 and N2O derived from NO32 was calculated as: mg N2 N=g soil ðatom% 15 N excess of N2 Þ=
ð1Þ
ðatom% 15 N excess of soil NO3 Þ mg N2 O N=g soil ðatom% 15 N excess of N2 OÞ=
ð2Þ
ðatom% 15 N excess of soil NO3 Þ
Results
The percent of soil NO32 converted to N2 and N2O was calculated as: 100 amount of N2 derived from NO3 ðmg N=g soilÞ = mg NO3 Ninitial =g soil
ð3Þ
100 amount of N2 O derived from NO3 ðmg N=g soilÞ= mg NO3 Ninitial =g soil
Statistics All data were analyzed using SAS v. 9.3 (SAS Institute nc., Cary, NC). Accumulation rate of NO32, NO22, N2O and N2, among different treatments and time periods and the percentage of NO32 converted to N2O and N2 among different treatments and time periods were analyzed using an ANOVA within the Proc Mixed Model. Treatment, time and their interactions were fixed effects and replication the random effect. Means among treatments within each time period and among time periods within each treatment were compared using least significant differences. Differences were considered significant if P , 0.05.
ð4Þ
The amount of N2O in solution was estimated using the Bunsen coefficient (Bleakley and Tiedje, 1982), which was added to the amount in the headspace to give total N2O.
Reaction rates. The rates of accumulation of NO32, NO22, N2O and N2 (Table 1) were calculated as the difference in the concentrations (in mg N/g soil) of a given species between the initial and final time points (expressed per hour) of the given interval divided by the duration of the interval.
Effects of inorganic Cu on denitrification Inorganic Cu did not affect N transformation rates in the denitrification pathway (Table 1). The NO32 consumption rate was not different for any INO Cu treatments within each time period of the incubation. Within each INO Cu treatment, the NO32 consumption rate did not differ among time periods except for the 8 to 16 h period in the INO 550 treatment. No accumulation of NO22 was observed in any of the INO Cu treatments. The N2O and N2 accumulation rates did not differ among INO Cu treatments within time periods, nor did they differ within INO Cu treatments over time periods. In the INO Cu treatments over time periods, the NO32 recovery percentages as N2O and N2 were not different than the control (Figure 1). Effect of ORG Cu on denitrification NO32 reduction. In the ORG Cu treatments, the rates of NO32 reduction increased with increasing Cu concentrations. From 0 to 16 h, NO32 was completely consumed in the ORG 750 and ORG 550 treatments, whereas approximately onethird and two-thirds of the NO32 remained in the soil of ORG
Table 1 Effects of different concentrations of inorganic and organic Cu and incubation times on NO32, NO22, N2O and N2 accumulation Accumulation ratea NO32 Treatments
0 to 8 h
NO22
N2O
N2
8 to 16 h 16 to 24 h 0 to 8 h 8 to 16 h 16 to 24 h 0 to 8 h 8 to 16 h 16 to 24 h 0 to 8 h 8 to 16 h 16 to 24 h
22.37dA INO 750 25.34bcd* A** d INO 550 20.80A 24.16cd B d INO 125 20.69A 23.90cd A INO 60 24.51bcd 26.33cd A A ORG 750 213.58aB 227.19aC ORG 550 28.97abc 225.40aC B ab ORG C125 210.32B 215.05bB bcd ORG 60 26.60 A 27.73cA Cu 0 23.69cd 23.34cd A A
25.42ab A 23.65ab A 23.28ab A 21.8ab A 20.19aA 20.73aA 28.48bA 24.49ab A 26.31ab A
0.03dA 0dA 0dA 0dA 19.82aA 14.48bA 5.46cA 2.29dA 0.03dA
0cA 0cA 0cA 0cA 19.82aB 13.59bB 0.14cB 1.04cB 0.03cA
0aA 0aA 0aA 0aA 0aC 20.89bC 21.51cB 20.87bB 0aA
1.44cA 0.61aA 20.11aA c 1.07A 0.47aA 20.10aA c 0.89A 20.08aA 20.04aA 1.23cA 20.27aA 20.19aA 12.84aA 211.42bC 21.42ab B 10.85ab 1.73aC 212.57bB A 8.66ab 2.48aB 22.67ab A C bc 5.29A 3.32aA 22.12ab B 1.14cA 1.29aA 20.80aA
0.07bA 0.12bA 0bA 0bA 1.01ab C 1.26ab C 1.82aA 0.31bA 0.04bA
0cA 0.06cA 0.09cA 0.13cA 32.84aA 15.14bB 2.59cA 0.84cA 0.24cA
0.04bA 0.03bA 0bA 0.02bA 4.10bB 25.40aA 3.64bA 0.33bA 0.25bA
NO32 5 nitrate; NO22 5 nitrite; N2O 5 nitrous oxide; N2 5 nitrogen gas; INO 5 inorganic, ORG 5 organic values are in mg Cu/g soil. For a given N species, means designated with the same letter (lower letter or capital letter) are not significantly different (P . 0.05). n 5 3. a mg/g soil/h. Positive numbers indicate accumulation; negative ones indicate consumption. *Means of a given N species among different treatments within the same time period followed by the same lower case letter are not significantly different. **Means of a given N species during different time periods within a treatment followed by the same capital letter are not significantly different.
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Effects of inorganic v. organic copper on denitrification
Figure 1 Percent of initial NO32 converted to N2O and N2, and measured concentrations of NO32 and NO22 in inorganic Cu treatments. Standard error of the mean shown as the line bars, n 5 3.
125 and ORG 60 treatments, respectively. The rate of NO32 disappearance was higher from 8 to 16 h (27.19 and 25.40 mg/g soil/h) than that from 0 to 8 h (13.58 and 8.97 mg/g soil/h) at the two highest ORG Cu concentrations (ORG 750 and 550 treatments).
NO22 accumulation and reduction. From 0 to 8 h, NO22 accumulated among all ORG Cu treatments (19.82, 14.48, 5.46 and 2.29 mg/g soil/h for ORG 750, 550, 125 and 60 Cu treatments). From 8 to 16 h, NO22 was quickly reduced in the ORG 750 and ORG 550 treatments (19.82 and 13.59 mg/g soil/h for ORG 750 and 550 Cu treatments) to the base concentration (the NO22 concentration in the soil before incubation started), but in the ORG 125 and ORG 60 treatments the rates of NO22 consumption was lower (0.14 ad 1.04 mg/g soil/h for ORG 125
and 60 Cu treatments) than the rates of accumulation during 0 to 8 h. From 16 to 24 h, NO22 decreased in all the treatments.
N2O accumulation and reduction. N2O and N2 were expressed as percentage conversion of NO32 (Figure 2). In the ORG 750 treatment, N2O peaked at 8 h (31% conversion from NO32) and was reduced from 8 to 16 h with a concomitant increase of N2. In the ORG 550 treatment, N2O peaked at 16 h (30.6% conversion of NO32) and disappeared between 16 and 24 h. In the ORG 125 treatment the highest N2O concentration was recorded at 16 h (31% conversion of NO32), and the reduction of N2O to N2 was slower than at the two greater ORG Cu concentrations as there was only a 6.4% decrease of N2O from 16 to 24 h. In the ORG 60 treatment N2O was lower than in the higher Cu concentration treatments and 45
Wang, Burger, Doane, Horwath, Castillo and Mitloehner
Figure 2 Percent of initial NO32 converted to N2O and N2, and measured concentrations of NO32 and NO22 in organic Cu treatments. Standard error of the mean shown as the line bars, n 5 3.
peaked at 16 h (20.9% conversion of NO32). In the following 8 h, N2O was reduced by only 5.1%. In the ORG 750 treatment, N2O decreased simultaneously with NO22 from 8 to 16 h. However, in the ORG 550 treatment, N2O increased as NO22 decreased, indicating that N2OR activity was lower in the ORG 550 than in the ORG 750 treatment. In ORG 125 and ORG 60 treatments, N2O increased as NO22 decreased, which suggested lower N2OR activity in these treatments. Discussion
Cu availability In the present study ORG Cu was more effective in accelerating denitrification activity than INO Cu. This difference is 46
likely due to greater bioavailability of ORG Cu in soil. Cu is easily bound to, and complexes with, soil OM, more than any other transition metal in soil. Adriano (2001) compared the affinity of a series of metals to OM and reported that Cu is the metal most strongly complexed with OM. Kornegay et al. (1976) found Cu to be immobile in soil with high OM due to the complexation of Cu by OM. Several other researchers noted that Cu in organic soils is held tenaciously by the OM (Gilbert, 1952; Jones, 1967; Gigliotti et al., 2009; He et al., 2011). Therefore, when the OM content is high, Cu bioavailability is likely low (Kornegay et al., 1976; LHerroux et al., 1997; Bolan et al., 2004; Gerzabek et al., 2006; Lair et al., 2007). ORG Cu in the present study was in the form of a copper-peptide complex. In this complex, Cu may form bidenate, terdenate, tetradenate and pentadenate
Effects of inorganic v. organic copper on denitrification with the peptide, which imparts high stability to the complex. As a result, ORG Cu may be more stable in soil solution (less reaction with OM) and accessible to denitrifiers than INO Cu, which is easily bound to ligands containing either oxygen or N, P, S as donor atoms in the soil.
Importance of Cu to N2OR In the present study, N2O reduction rate was significantly increased in ORG 750 and ORG 550 treatments, demonstrating a threshold concentration for Cu on N2OR activity under the present study conditions and emphasizing the importance of Cu form and concentration on N2OR activity. In addition, in the present study N2OR activity reached its maximum from 8 to 16 h in ORG 750 treatment while in ORG 550 treatment, the enzyme activity was at its optimum from 16 to 24 h. This result indicates enzyme induction is dependent on Cu concentration. The importance of adequate Cu to induce N2OR activity in denitrifiying bacteria has been demonstrated in previous studies. Iwasaki and Terai (1982) cultured denitrifying bacteria in Cu-depleted and replete media under anaerobic conditions and found that under Cu deficient conditions, N2O accumulated with only slight accumulation of N2, whereas in the presence of sufficient Cu (5.5 mM), N2O did not appear and N2 was the main gaseous product. Granger and Ward (2003) studied the effect of Cu on N2OR by culturing denitrifiers in artificial seawater medium under trace mineral deficient conditions and found that decreasing Cu concentrations (,11 nM) in the culture medium lead to the accumulation of N2O. The author also reported that N2OR activity was higher in Cu replete cultures than that in Cu depleted cultures. However, Labbe et al. (2003) conducted an experiment to examine the effect of different metals (Fe, Mo, Cu, Mo, Zn) on denitrifying bacteria activity in the artificial seawater denitrifying system and reported that when CuSO4 was added alone to the system it had no impact on the denitrification process. This finding is in contrast to the present results in which ORG Cu had a significant impact on the denitrification process and on N2OR activity. This might be explained by other metal deficiencies, which might have masked the effects of Cu on N2OR, that is, that the availability of other metals were deficient in their experimental system, for example, Mo for NO32 reductase, Fe for NO22 reductase, which masked the effects of Cu on N2OR. In the present experiment, these metals were likely present in adequate amounts while Cu was the deficient one and, therefore, Cu supplementation had a significant effect on denitrification and N2OR. Another possibility for the different results between these two studies could be due to the difference in the Cu form (CuSO4 v. Cu peptide) applied to the experimental systems. Cu effects on NO32, NO22 and N2OR reductases In the denitrification pathway, Cu is a cofactor for both NO22 reductase and N2OR and, therefore, affects their activity (Matsubara et al., 1982; Viebrock and Zumft, 1988; Kukimoto et al., 1994). The effects of Cu on NO32, NO22 and N2OR were investigated by Ho et al. (1993) who isolated denitrifying enzymes from denitrifiers and supplemented the enzymes
with diethyldithiocarbamate (DDC), a Cu chelating agent, for 5 min. The activity of NO22 reductase activity was totally inhibited whereas NO32 reductase and N2OR activity were affected to a lesser extent, indicating the importance of Cu to NO22 reductase. Several other researchers have reported the inhibitory effect of DDC on NO22 reductase and the importance of Cu to NO22 reductase activity (Shapleigh and Payne, 1985; Coyne et al., 1989). In the present study, the observation that ORG Cu accelerated NO22 reduction compared with INO Cu and CON could be due to its impact on NO22 reductase activity. In the present study, ORG Cu increased NO32 reductase activity compared with INO Cu and CON treatments. NO32 reductase requires a molybdenum cofactor (Moco) (Kuper et al., 2004). Biosynthesis of Moco has been reported to require Cu to protect the dithiolene moiety of Moco before the insertion of molybdenum (Morrison et al., 2007). In the present study, ORG Cu exerted a significant effect on increasing NO32 reductase activity, which could be attributed to ORG Cu being more available. NO32 reductase activity has been reported to be inhibited by NO22 (Knowles, 1982; Glass and Silverstein, 1998). The increase in NO32 reductase activity in the ORG Cu 750 and 550 treatments during 8 to 16 h compared with 0 to 8 h period is more readily explained as inhibitory effect of NO22 build-up in the soil from 0 to 8 h. The Cu concentration needed to promote N2OR activity is likely much higher than the Cu concentration required by NO32 and NO22 reductase. The N2O accumulation rate did not differ in the ORG 750, 550 and 125 treatments from 0 to 8 h. However, N2O consumption in the ORG 750 treatment started during the 8 to 16 h period, whereas in the ORG 550 and 125 treatments, N2O decreased from 16 to 24 h. During this period, N2O consumption occurred at a lower rate in the ORG 125 and 60 treatments than in the ORG 750 and ORG 550 treatments. Cu availability may have influenced the kinetics of N2O accumulation because N2OR has a relatively high Cu requirement compared with that of the enzyme(s) leading to N2O production. NO22 reductase, a trimer with a di-copper complex found in each monomer (Paul and Karlin, 1991), requires only six Cu atoms to maintain its function whereas 12 Cu atoms are required for N2OR to reach optimal function. The Cu requirement of NO22 reductase and N2OR has been investigated by Cervantes et al. (1998), who observed both NO22 and N2O accumulation in an anaerobic reactor at low Cu (Cu applied in this study was CuCl2) concentration (,28 mg Cu/l). However, at Cu concentration .28 mg Cu/l, NO22 accumulation stopped while N2O accumulation continued (the highest Cu concentration applied in that study was 56 mg Cu/l). The author concluded that Cu was the limiting factor for NO22 reductase activity but not for N2OR activity and that the accumulation of N2O was not due to the lack of Cu but the fact that N2OR is slower than the preceding enzymes in the denitrification process. In contrast to those findings, the present study showed that N2OR activity increased at higher Cu concentrations. A reason for the divergent results in these two studies could be 47
Wang, Burger, Doane, Horwath, Castillo and Mitloehner that the Cu concentration (56 mg Cu/l) applied in Cervantes’ study was not high enough to activate N2OR or that the inorganic Cu (CuCl2) was not readily available to the denitrifiers in Cervantes’ anaerobic incubation system. NO22 accumulation in the present study may have occurred because the synthesis of NO22 reductase requires more time than that of NO32 reductase (Williams et al., 1978). Betlach and Tiedje (1981) explained that NO22 accumulation in three pure cultures of denitrifiers was due to differences in their relative reduction rates. A substantial accumulation of N2O, followed by its rapid reduction took place in the present study, which suggests that N2OR synthesis was slower than that of the enzymes leading to N2O production. The lag of N2OR synthesis compared with other enzymes in denitrification has been reported by other workers (Betlach and Tiedje, 1981; Cervantes et al., 1998). Firestone and Tiedje (1979) also demonstrated that synthesis of enzymes in the denitrifying pathway is staggered and that NO32 reductase required the least amount of time for synthesis. It seems that, often, accumulation of N2O as a first response to anaerobic conditions is inevitable because the synthesis of denitrifying enzymes occurs at varying rates (Firestone and Tiedje, 1979). However, ORG Cu studied in the present study showed a potential to shorten the lag phase of N2OR synthesis and, therefore, accelerated N2O reduction to N2.
Significance The present study is the first that has focused explicitly on the role of Cu in the denitrification process in soil, at concentrations that are not excessive but instead promote the reactions that convert NO32 to NO22, NO, N2O, and ultimately to N2. Two different forms of Cu were compared for their effectiveness in enhancing the successive reduction steps in the denitrification pathway, with an emphasis on the final step, reduction of N2O. While peat soil has a higher OM content than other soil types, the data presented here provide reference values for the amount of Cu needed to affect various reactions in an environment with high Cu chelating potential. Our results, therefore, have potential application in designing and optimizing procedures in which reduction of N2O and other reactions of denitrification pathway are manipulated in systems like wetlands, wastewater treatment, etc. Further information The Dairy Solutions Symposium is a biennial event that covers a wide variety of themes and topics of relevance and importance to the dairy industry. The aim is to provide high level, up-to-date information and research to dairy professionals, technologists and scientists. In 2012, the theme addressed the biggest challenge facing all those involved in dairy production: Optimizing production efficiency while lowering environmental impact. For more information, please visit www.dairycowsolutions.com or contact
[email protected]. 48
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