Scientific registration number : 804 Symposium number : 13A Presentation : oral. MURPHY Daniel, WILLISON Toby, BAKER Julie, GOULDING Keith

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1 Scientific registration number : 804 Symposium number : 13A Presentation : oral Gross nitrification to gross immobilisation ratios offer a means of assessing potential N loss from soils Le rapport de la nitrification brute à l immobilisation brute fournit un moyen d évaluer les pertes en azote potentielles des sols MURPHY Daniel, WILLISON Toby, BAKER Julie, GOULDING Keith Department of Soil Science, IACR-Rothamsted, Harpenden, Herts., AL52JQ, U.K. Introduction Leaching of nitrate is both environmentally and economically undesirable. Understanding the factors and mechanisms which influence nitrate supply is necessary if nitrogen is to be used efficiently in agro-ecosystems. It is generally assumed that in soils where nitrogen cycling is highly conservative and ammonium is limited that plant uptake and microbial immobilisation are more competitive for ammonium than nitrifying bacteria (Tietema and Wessel, 1992). This suggests that ammonium supply is the key factor regulating nitrification (Robertson, 1982; Donaldson and Henderson, 1990). Traditionally it has been difficult to determine the relative importance of ammonium consumptive processes due to a lack of methodology which enables pathways within the nitrogen cycle to be separated. Most techniques have required the measurement of a product or the disappearance of a substrate and only measure net fluxes. Problems of overestimation of net rates of nitrogen transformations when a substrate is supplied are well documented (Frisk and Schmidt, 1996; Willison et al., 1998b). 15 N isotopic dilution enables gross rates of N transformations to be determined without the priming effect of more traditional methods (Hart et al., 1994). Although the theory of 15 N isotopic dilution was established in 1954 (Kirkham and Bartholomew, 1954) it has only been in the last 10 years that the actual methodology (Nishio et al., 1985; Davidson et al., 1991; Stark and Firestone, 1995; Murphy et al., 1997) and interpretative understanding (Myrold and Tiedje, 1986; Bjarnason, 1988; Barraclough, 1991; Wessel and Tietema, 1992) has existed. Examining ratios calculated from 15 N isotopic dilution studies may provide greater understanding than just measuring gross rates. Tietema and Wessel (1992) described the use of the ratio of gross nitrification to ammonium immobilisation as a means of quantifying competition between organotrophic microbes and nitrifiers for ammonium. This ratio quantifies the capacity of a soil to over produce nitrate and can be used to assist in determining the N saturation status of a soil (Aber, 1992). Tietema and Wessel (1992) and Murphy et al. (1998) have applied the ecological 1

2 concept of pseudo-residence time (Frissel, 1981) to 15 N isotopic dilution data to examine the relationship between the size of the ammonium or nitrate pool and the rate of removal of nitrogen from these pools. In addition, microbial nitrogen efficiency, defined as the gross amount of nitrogen immobilised per unit of carbon respired (Aber and Melillo, 1980), and can be used to determine if the active microbial population is efficient/inefficient at cycling nitrogen. However, the use of these ratios have not been evaluated across a range of land uses and the value for each ratio which indicates if a soil is nitrogen saturated or nitrogen limited is not well defined. We aimed to measure gross nitrogen transformations and to calculate the ratios under land uses where conventional wisdom would suggest that soils were either nitrogen saturated or nitrogen limited. Materials and Methods Soils were collected from unfertilised areas of the Broadbalk Continuous Wheat Experiment, the Park Grass Continuous Hay Experiment, and Knott Wood (woodland soil) on Rothamsted Experimental Station (Hertfordshire, United Kingdom). Broadbalk was sampled from Plot 3 Section 3 which has received no fertiliser since 1843 and has been under a 5 year rotation of fallow, potatoes, wheat, wheat, wheat since 1986 and under arable crops since The area on Park Grass has been cut for hay every year since Knott Wood is a mixed deciduous woodland which has been managed only by infrequent coppicing for the last 300 years. Soils were also collected from a drained fenland peat (Suffolk, United Kingdom) which had been under either agricultural cropping (arable site) for the last 50 years or under poplars (woodland site) for the last 30 years (Willison et al., 1998a). 15 N isotopic dilution experiments were conducted as described by Willison et al., 1998a,b). Fresh soil (35.0 g dry weight) was weighed into glass jars so that the soil depth was less that 2.0 cm. To each jar 3.0 ml of solution containing 5.0 mg N kg -1 dry soil was applied dropwise to the surface of the soil as either ( 15 NH 4 ) 2 SO 4 or K 15 NO 3 at 10.3 atom % 15 N. Jars were covered with parafilm containing pin holes to enable gas exchange and incubated at 20 C for either 0, 1, 2, 3, or 7 days. Soil (3 replicates) was then extracted with 60 ml of 2.0 M KCl and filtered. Ammonium and nitrate concentrations were determined colorimetrically on an Alpkem analyser. The 14 N: 15 N ratio of ammonium and nitrate was determined on an isotope ratio mass spectrometer linked to a combustion analyser (ANCA-MS Europa Scientific) after diffusion onto acidified glass fiber disks (Brooks et al., 1989). Gross rates of mineralisation, ammonium consumption, nitrification and nitrate consumption were calculated using the formula of Kirkham and Bartholomew (1954). Ammonium immobilisation was estimated as the difference between ammonium consumption and gross nitrification. Carbon dioxide evolution was determined after 0 h, 3 h, 6 h, 18 h and 24 h on soil (200 g dry weight equivalent, 5 replicates) which was incubated in 1 l glass preserving jars. Gas was analysed by gas chromatography using an electron capture detector. 2

3 Results The gross nitrogen transformation rates, nitrogen pool sizes and ratios determined in this study provide a contrasting picture of five different land uses (Table 1). Broadbalk Plot 3 Section 3 is an atypical arable soil since it does not receive any nitrogen fertiliser. The soil has a low rate of gross nitrogen mineralisation, a very low rate of gross nitrification, immobilisation dominates over nitrification and the ammonium pseudo-residence time is short. This soil is nitrogen limited and unlikely to lose nitrogen. Park Grass has a high rate of gross nitrogen mineralisation and virtually no nitrification. Nitrogen immobilisation dominates over nitrification. This soil is unlikely to lose nitrogen due to the rapid cycling of nitrogen between mineralisation and immobilisation. Knott Wood has a higher nitrification rate which is reflected by the larger nitrate pool size. Ammonium consumption is split equally between nitrification and immobilisation. The pseudoresidence times are long for both the ammonium and nitrate pools which suggests a soil with an excess of nitrogen which could potentially be lost. We suspect that Knott Wood is moving towards nitrogen saturation status probably due to atmospheric nitrogen inputs. For this area annual nitrogen inputs from atmospheric deposition are on average 50 kg N ha -1 (Goulding et al., 1998) and probably exceed plant and microbial demand. The peat soils are different to the mineral soils at Rothamsted with high organic matter contents, comparatively high gross mineralisation and gross nitrification rates. In the peat woodland gross nitrification dominates over ammonium immobilisation. In the peat soils nitrate pool sizes are large and nitrate pool pseudo-residence times are long suggesting that these soils are likely to lose nitrogen. Microbial nitrogen efficiency was higher for the arable and grassland soils compared to the woodland soils suggesting tighter cycling of nitrogen and less likelihood of nitrogen loss. Discussion Both conceptual and numerical approaches have been developed to estimate the nitrogen saturation status in forest soils and the nitrogen fertiliser requirement of grassland and arable soils. However, it is still difficult to quantify the success of these approaches or to provide quantitative predictions of nitrogen supply. The application of 15 N isotopic dilution techniques is likely to assist with these difficulties. In addition, determining ratios, instead of comparing gross rates, will enable an index of nitrogen status to be applied across land uses. However, at present the range of values for each ratio which defines a nitrogen saturation status are not known and there is a lack of published results for comparison. The question is how well can the methodology and understanding be applied across a range of land uses. A further development of this approach will be to better define the range of values for a given nitrogen status. However, the ratios can only provide an indication of the potential for nitrogen loss since actual loss will also be influenced by factors such as nitrogen pool sizes, soil physical properties and climatic conditions. The ratios are therefore likely to be of most use when determined alongside other soil characteristics and incorporated into decision support systems or used as soil diagnostics in model development. 3

4 Conclusion Ratios calculated from gross nitrogen transformation data were used to determine if soils which differed in land use were nitrogen limited or nitrogen saturated. Soils were then characterised according to whether they had a low, medium or high potential for nitrate loss. The ratios identified differences in the potential for nitrate loss between soils under different land use and management. It may be possible to use these ratios as indicators of nitrate loss although the actual amount of nitrate loss will also depend on the size of the nitrate pool, soil physical properties, climatic conditions and, in the case of agroecosystems, any direct loss from fertiliser or manure. Acknowledgments- We would like to thank the Royal Society for the Protection of Birds (RSPB) for permission to sample the peat soils. This work was funded by the UK Ministry of Agriculture, Fisheries and Food. IACR-Rothamsted receives grant-aided support from the Biotechnology and Biological Sciences Research Council. References Aber, J. D. (1992). Nitrogen cycling and nitrogen saturation in temperate forest ecosystems. Tree 7, Aber, J. D. and Melillo, J. M. (1980). Litter decomposition: Measuring relative contributions of organic matter and nitrogen to forest soils. Canadian Journal of Botany 58, Barraclough, D. (1991). The use of mean pool abundance to interpret 15 N tracer experiments. I. Theory. Plant and Soil 131, Bjarnason, S. (1988). Calculation of gross nitrogen immobilisation and mineralisation in soil. Journal of Soil Science 39, Brooks, P. D., Stark, J. M., McInteer, B. B. and Preston, T. (1989). Diffusion method to prepare soil extracts for automated nitrogen-15 analysis. Soil Science Society of America Journal 53, Davidson, E. A., Hart, S. C., Shanks, C. A. and Firestone, M. K. (1991). Measuring gross nitrogen mineralization, immobilization and nitrification by 15 N isotopic dilution of intact soil cores. Journal of Soil Science 4, Donaldson, J. M. and Henderson, G. S. (1990). Nitrification potential of secondary succession upland Oak forests: I. Mineralization and nitrification during laboratory incubations. Soil Science of America Journal 54, Fisk, M. C. and Schmidt, S. K. (1996). Microbial responses to nitrogen additions in alpine tundra soil. Soil Biology & Biochemistry 28, Goulding, K. W. T., Bailey, N. J., Bradbury, N. J., Hargreaves, P., Howe, M., Murphy, D. V., Poulton, P. R. and Willison, T. W. (1998). Nitrogen deposition and its contribution to nitrogen cycling and associated aoil processes. New Phytologist (in press). Hart, S. C., Stark, J. M., Davidson, E. A. and Firestone, M. K. (1994). Mineralization, immobilization and nitrification. In: methods of Soil Analysis Part 2 (R. W. Weaver ed.) SSSA Book Ser. 5. SSSA, Madison, WI. pp

5 Kirkham, D. and Bartholomew, W. V. (1954). Equations for following nutrient transformations in soil, utilizing tracer data. Soil Science Society of America Proceedings 18, Murphy, D. V., Fillery, I. R. P. and Sparling, G. P. (1997). Method to label soil cores with 15 NH 3 gas as a prerequisite for 15 N isotopic dilution and measurement of gross N mineralization. Soil Biology & Biochemistry 29, Murphy, D. V., Fillery, I. R. P. and Sparling, G. P. (1998). Seasonal fluctuations in gross N mineralisation, ammonium consumption, and microbial biomass in a Western Australian soil under different land uses. Australian Journal of Agricultural Research 49, Myrold, D. D. and Tiedje, J. M. (1986). Simultaneous estimation of several nitrogen cycle rates using 15 N: Theory and application. Soil Biology & Biochemistry 18, Nishio, T., Kanamori, T. and Fujimoto, T. (1985). Nitrogen transformations in an aerobic soil as determined by a 15 NH + 4 dilution technique. Soil Biology & Biochemistry 17, Robertson, G. P. (1982). Nitrification in forested ecosystems. Philosophical Transactions of the Royal Society of London 296, Stark, J. M. and Firestone, M. K. (1995). Isotopic labelling of soil nitrate pools using nitrogen-15-nitric oxide gas. Soil Science of America Journal 59, Tietema, A. and Wessel, W. W. (1992). Gross nitrogen transformations in the organic layer of acid forest ecosystems subjected to increased atmospheric nitrogen input. Soil Biology & Biochemistry 24, Wessel, W. W. and Tietema, A. (1992). Calculating gross N transformation rates of 15 N pool dilution experiments with acid forest litter: Analytical and numerical approaches. Soil Biology & Biochemistry 14, Willison, T. W., Baker, J. C. and Murphy, D. V. (1998a). Nitrogen dynamics and methane fluxes from a drained fenland peat. Biology and Fertility of Soils (in press). Willison, T. W., Baker, J. C., Murphy, D. V. and Goulding, K. W. T. (1998b). Comparison of a wet and dry 15 N isotopic dilution technique as a short term nitrification assay. Soil Biology & Biochemistry (in press). Keywords : nitrification, immobilisation, nitrification immobilisation ratio, N loss, soil, 15 N, isotopic dilution technique Mots clés : nitrification, immobilisation, rapport nitrification immobilisation, perte en azote, sol, 15 N, technique de dilution isotopique 5

6 Table 1. Inorganic nitrogen pool sizes (mg N kg -1 ), gross nitrogen transformation rates (mg N kg -1 d -1 ), gross nitrification to ammonium immobilisation ratio, pseudo-residence times (days), microbial nitrogen efficiency ratio and the potential for nitrate loss from soils under a range of land uses. Rothamsted soils Peat soils Characteristic N-limited N-saturated Broadbalk Park Grass Knott Wood Arable Woodland NH + 4 pool size Small Large NO 3 - pool size Small Large Gross mineralisation Gross immobilisation Gross nitrification Ratio: nitrification to Low High >100 NH + 4 immobilisation <1 >1 NH + 4 pool residence Fast Slow turnover turnover NO - 3 pool residence Fast Slow > >100 >100 turnover turnover Microbial efficiency High ratio Low ratio Potential for N loss Low High Low Low Medium Medium High 6