D. Barraclough

The use of 15N pool dilution and enrichment to separate the heterotrophic and autotrophic pathways of nitrification

Abstract The techniques of 15 N pool dilution and enrichment were used to separate autotrophic and heterotrophic nitrification in an acid woodland soil. Results from laboratory incubations indicated that a maximum of 8% of the observed nitrification could be the result of heterotrophic nitrifiers oxidizing organic nitrogen to nitrate without passing through the exchangeable soil ammonium pool. Autotrophic nitrification rates ranged from 0.96 to 2.64 μg N g −1 d −1 , depending on the time of the year the soil was sampled. Heterotrophic nitrification rates ranged from 0.08 to 0.12 μg N g −1 d −1 and appeared unaffected by the nitrification inhibitor N-Serve.

The direct or MIT route for nitrogen immobilization: A 15N mirror image study with leucine and glycine

Abstract Current models of nitrogen mineralization implicitly assume that all the N in organic substrates is mineralized to NH4+ + before assimilation by growing microorganisms — the so-called MIT hypothesis. In theory, however, N mineralization during the decomposition of low molecular weight substrates such as amino acids can occur by a direct route in which the substrate is assimilated into the cell, deaminated, and only the surplus released into the soil NH4+ pool. The decomposition in soil of the amino acids leucine and glycine was studied, using the techniques of 15N isotope dilution and enrichment, to determine whether the direct route of N assimilation was occurring in soil. The results showed clearly that the conventional MIT route was not operative and were consistent with the direct route. The relative proportions of N from leucine and glycine mineralized into the NH4+ pool were in close agreement with predictions based on the direct hypothesis. Results from other published work on amino acid decomposition, reinterpreted using these techniques, were also consistent with the direct route.

Gross rates of N mineralization associated with the decomposition of plant residues

Abstract Nitrogen-15 isotope dilution was used to determine gross rates of N mineralization immediately following the incorporation of crop residues in soil. Laboratory and field experiments were performed. Two crop residues were used: oil-seed rape stems and winter wheat straw. Incorporation of oil-seed rape residues into the soil resulted in an immediate increase in gross N mineralization rate from 0.87 to 2.2 mg kg−1 d−1. After 12 d, gross N mineralization in the presence of the residues had dropped to 0.79 mg kg−1 d−1, compared to 0.72 mg kg−1 d−1 in the control. Of the N in the oil-seed rape, 24% was mineralized in 12 d. In contrast, incorporation of winter wheat residues caused only a small immediate increase in gross N mineralization to 0.92 mg kg−1 d−1. Of the N in the winter wheat residues, 12% was mineralized in the 12 d following incorporation. Longer-term measurements indicated that the residue incorporation was still influencing N mineralization 54 d after incorporation with 19 and 11% of the mineralization flux resulting from oil-seed rape and winter wheat decomposition, respectively. Field measurements showed that 5 months after incorporation, oil-seed rape residues were contributing 13% of the mineralization flux while winter wheat residues were contributing 9%.

Soil N dynamics in a natural calcareous grassland under a changing climate

Abstractu2002This paper reports the results from a medium-term field scale investigation into the effects of simulated climate change on soil N mineralisation in a semi-natural calcareous grassland in southern England. The experiment utilised soil warming cables, automatic rainshelters and a watering system to examine two climate change scenarios: warmer winters with summer drought and warmer winters with enhanced summer rainfall. Gross N mineralisation rates in treated plots were determined, using 15N pool dilution techniques, at 6-weekly intervals over a 3-year period. Results from control plots showed a strong seasonality of mineralisation with highest rates in autumn and winter and lowest rates in summer. They suggest that water availability is the main constraint on microbial processes and plant growth. Unexpectedly, additional summer rainfall had no direct effect on N mineralisation at the time of application (summer). The treatment did, however, significantly (<0.05%) reduce rates in subsequent autumn and winter months. In contrast, summer drought significantly increased N mineralisation rates in autumn and winter. Winter warming similarly had no direct effect on N mineralisation in winter but decreased rates in spring. We hypothesise that the observed treatment effects result from changes in organic C and N input, in plant litter, resulting from the direct impact of climatic manipulation on perennial plant growth, death and senescence. This paper compares and contrasts the response to climate manipulation in the grassland system with results from other ecosystem types such as northern forests.

Contributions to gross N mineralization from 15N-labelled soil macroorganic matter fractions during laboratory incubation

Abstract We examined the role of macroorganic matter contributions to rates of gross nitrogen mineralization measured during a laboratory incubation at 15°C. Two 15N-labelling techniques were employed. The first, referred to as the direct method, involved a mirror-image approach whereby labelled or unlabelled macroorganic matter from two different soils was incorporated into soil, and the contribution of this material to gross mineralization was measured using a combination of isotope dilution and pool enrichment procedures. Macroorganic matter was extracted from a sandy loam (Sonning soil) and an organic loam (Black soil). The second technique, a difference method, involved comparing measured gross rates of mineralization in soil with or without the incorporated unlabelled macroorganic matter. During the 66 day incubation, approx. 2.4 and 10.8% of the incorporated macroorganic matter was released from the Sonning and Black soil macroorganic matter fractions, respectively. Following correction to the amounts of macroorganic matter normally recovered in the soils, it was estimated that the Black macroorganic matter fraction contributed from 3 to 21.9% of the gross N mineralization, with a mean of 12%. The contribution of the Sonning macroorganic matter ranged from 0% initially to approx. 14% at the end of the incubation, with a mean of 4.5%. Agreement between the two techniques was reasonable, although the variation encountered in the difference method was considerably less than that observed in the direct method.

Gross mineralisation of nitrogen during the decomposition of leaf protein I (ribulose 1,5-diphosphate carboxylase) in the presence or absence of sucrose

Abstract Ribulose 1,5-diphosphate carboxylase (Rubisco) comprises 35–40% of the protein in plant leaves. It is an ideal analogue for the nitrogenous part of leaf material. We have used 15N isotope dilution to study the mineralisation of nitrogen and carbon during Rubisco decomposition in a sandy soil in the presence or absence of sucrose. The results showed that gross N mineralisation was only slightly affected by the addition of sucrose but that gross N immobilisation increased markedly. They confirmed earlier work showing that the decomposition was effected by bacteria and that direct assimilation of peptides or amino acids occurred without prior mineralisation. One interpretation of these findings is that there are two functional groups of bacteria in this soil: one which uses protein for energy and C and N and another which uses carbohydrate as an energy and C source and NH4+ mineralised by the first group as an N source. They may provide a link between bacterial functionality in terms of nutritional requirements and the two main N transformations in soil-mineralisation and immobilisation. The implications of these conclusions in terms of current models of the soil N cycle are explored and a modified soil N cycle is proposed.

Comparison of 15N labelling methods to measure gross nitrogen mineralisation

Abstract A laboratory study was conducted to compare methods of labelling the soil NH4+ pool with 15N as a prerequisite to the measurement of gross N mineralisation. Composite soil (0–12 cm) was collected from 12 sites differing in land use and soil texture. Three methods were used to apply 15N to the soil: (i) addition of a 15NH3–air gas mixture to the headspace above the soil (gas jar), (ii) injection of a 15NH3–air gas mixture into re-packed soil cores (gas injector) and (iii) addition of a (15NH4)2SO4 solution pipetted onto the soil surface (solution). 15N isotopic dilution was determined between 24 and 72 h after 15N application. Of the applied 15N 52 to 93% was recovered as inorganic N 24 h after application. Less 15N was recovered in the clay loam compared to the sandy loam and silty clay loam for all methods. Recovery from the gas injector was lower than from the gas jar or solution treatments in the sandy loam and silty clay loam. No difference in the recovery of applied 15N between methods was observed in the clay loam. 15N isotopic dilution was less in the gas jar compared to the gas injector and solution treatments. This resulted in significantly lower estimates of gross N mineralisation rates and NH4+ consumption for the gas jar treatment. The non-uniform distribution of 15N through the soil probably caused these differences as a consequence of 15NH3 dissolving quickly in the surface of the soil. In seven of the 12 soils, gross rates of N mineralisation were not significantly different when 15N was applied as solution or by gas injection. In the remaining five soils, estimates of gross N mineralisation rates were greater in the solution treatment. Ammonium consumption was significantly different between the solution and gas injector treatments in three soils. Where there were differences in values for gross N mineralisation and NH4+ consumption rates between methods, the effects were not associated with a particular soil texture or land use. Greater nitrification of 15NH4+ to 15NO3− and shorter pseudo-residence times of the NH4+ pool were observed in the solution treatment compared to gas injector. This suggests a difference in the distribution of 15N within soil microsites, with the 15N applied in the solution treatment being more accessible for microbial consumption. Nevertheless, application of 15N as an NH4+ solution, or injection of 15NH3 gas into soil, resulted in estimates of a similar magnitude for gross N mineralisation rates and identified the same differences between soil type and land use.

Nitrogen and carbon mineralization in soil amended with d- and l-leucine

Abstract The rates of gross C mineralization over short-term (0–6 h) and longer-term (0–6 d) incubation of soil amended with the l - and d -enantiomers of leucine have been determined from the CO 2 production. The rates of N mineralization over 6 d incubations of similarly amended soils were determined by 15 N isotope pool dilution for gross N mineralization and both α-amino N disappearance and NH 4 + accumulation for net N mineralization. The rates of net and gross N mineralization and of C mineralization from d -leucine were all significantly less than those from l -leucine and we present the first data for mineralization of N from a d -amino acid in soil. The hypothesis that the onset of rapid utilization of d -leucine by microorganisms was delayed whilst deamination occurred to yield an achiral keto-acid was not supported because the pulse in gross N mineralization following d -leucine addition to the soil was coincident with that of C mineralization and occurred later than the pulse of gross N mineralization in soil amended with l -leucine. By the time all the added amino acid had disappeared from the extractable pool, 26% of the N added as l -leucine had been taken up and possibly assimilated by the soil microorganisms compared with 47% for d -leucine. Based on these estimates of N assimilation, the C:N ratio of the microorganisms assimilating l -leucine could have been nearly twice that of the microorganisms assimilating d -leucine.

Contributions to N mineralization from soil macroorganic matter fractions incorporated into two field soils

Abstract The contribution of soil macroorganic matter N (macro-OM, d −3 ) to gross rates of N mineralization was examined in two grassland field soils in England using three 15 N-labelling techniques, referred to as the difference, mirror image and net recovery methods. The difference method involved measuring gross mineralization rates in soils with or without incorporated unlabelled macro-OM, using isotope dilution of added 15 NH 4 + . The mirror image approach involved measuring the isotopic enrichment of the ammonium pool in soil to which 15 N-labelled macro-OM had been incorporated. The net recovery method was a net N mineralization estimate based upon the recovery of label in plants and soil to which 15 N-labelled macro-OM had been incororated. The difference method provided the most accurate estimates of N mineralization from the incorporated macro-OM. Estimates made using the direct and net recovery methods were more variable and confounded by movement of the macro-OM away from the original site of incorporation. Approximately 2.4 and 13.7% of the N in the incorporated macro-OM isolated from the different soils was mineralized over the 66 d following incorporation. This represented approximately 3.4 and 2.3% of the cumulative gross N mineralization when corrected for background amounts of soil macro-OM. This low contribution suggests that most of the N mineralized in grazed grasslands is derived from other forms of soil organic matter associated with mineral particles.

Nitrogen mineralisation and plant nitrogen acquisition in a nitrogen-limited calcareous grassland

Abstract A field study measured the rate of soil mineral N supply and its effects on plant biomass and N accumulation in a 13-year-old, naturally regenerating, calcareous grassland. Gross rates of N mineralisation (2 μg g −1 day −1 , i.e. 0.69 kg ha −1 day −1 ), assessed using 15 N pool dilution, were at the lower end of the range previously reported for grasslands. Weekly additions of liquid N fertiliser ([NH 4 ] 2 SO 4 , NH 4 NO 3 or KNO 3 ) and, to a lesser extent the addition of water, increased plant growth substantially, demonstrating that the primary constraint to plant growth was low N availability. In plants that had received NO 3 − , the activity of the inducible enzyme nitrate reductase in shoots initially increased in proportion to the amount of NO 3 − supplied. However, as above-ground herbage accumulated, nitrate reductase activity declined to similar low levels in all treatments, despite the continuance of the constant NO 3 − additions. The decline in NR specific activity reflected declining tissue NO 3 − concentrations, although total plant NRA may have remained constant during the period of study. The study has shown that plant growth is limited by low N mineralisation rates and indeed the soil is a sink for much added N. Low water availability provides an additional constraint on N mineralisation in this calcareous grassland soil. Any disturbances in the N cycle which increase the availability of mineral N will result in a substantial increase in plant growth within this ecosystem.