J.N. Ladd

Short-term assays of soil proteolytic enzyme activities using proteins and dipeptide derivatives as substrates

Abstract Conditions are described for the rapid and precise assay of soil proteases, using proteins and dipeptide derivatives as substrates in the absence of added bacteriostatic agents. The rate of substrate hydrolysis was proportional to the soil concentration; the release of amino compounds per unit weight of soil was directly related to the incubation time. The soils investigated varied widely in their pH, texture, organic matter content, and total exchangeable cations, but nevertheless exhibited optimal protease activity near pH 8.0 and 60°C, and were consistent in their preferential hydrolysis of dipeptide derivatives containing amino acids with hydrophobic side chains. However, soils varied widely in their relative activities towards a given dipeptide derivative and towards benzoyl arginine amide (BAA), a cationic substrate used in the assay of ‘trypsin-like’ enzymes. Benzyloxycarbonyl (Z) phenylalanyl leucine was hydrolysed most rapidly by all soils investigated. Activities towards Z-phenylalanyl leucine far exceeded those towards protein substrates and were highly correlated with the clay contents of the soils but not well correlated with the organic matter contents.

Turnover of carbon and nitrogen through the microbial biomass in a sandy loam and a clay soil incubated with [14C(U)]glucose and [15N](NH4)2So4 under different moisture regimes

Abstract Two soils, one a sandy loam and the other of relatively high clay content, were incubated with [14C(U)]gtucose and [15N](NH4)2SO4 for 101 days, either under continuously moist conditions, or with intermittent drying of soils. Rates of evolution of 14CO2, decline in residual organic 14C, and net immobilization and mineralization of N and 15N in the sandy loam soil were more rapid than in the clay soil. First order decay rates for the decomposition of residual 14C, after 10 days, were consistently twice as fast in the sandy loam soil. By contrast, the efficiency with which glucose was utilized within the first few days, and the amounts of C, 14C, N and 15N present as soil biomass throughout the incubation, were greater in the clay soil than in the sandy loam. Biomass 14C as a percentage of residual organic 14C, was consistently 1.5 times greater in the clay soil. Compared with soils held continuously moist, soils which were intermittently dried and remoistened contained smaller amounts of isotope-labelled biomass C and N, but overall similar amounts of total residual organic 14C and 15N. Remoistening of dried soils caused a temporary (4 days) flush in C and N mineralization rates. A simulation model describes C and N behaviour in the two soils. Three features of the model are proposed to expain short-term differences between soils in the rates of C and N turnover, viz. the clay soil (a) has a greater capacity to preserve biomass C and N (b) holds a higher proportion of microbial decay products in the near vicinity of surviving cells, and, to a lesser extent, (c) utilizes glucose and metabolic products more efficiently for biosynthetic reactions.

Decomposition of 14C-labelled glucose and legume material in soils: Properties influencing the accumulation of organic residue C and microbial biomass C

Abstract Relationships were established between the properties of 23 soils. Particularly identified were those soil properties correlated with (i) native biomass C, with (ii) the extent of decomposition of added [ 14 C]glucose and [ 14 Medicago littoralis plant residues after 44 and 66 weeks incubation respectivelyand with (iii) the concentrations of substrate-derived C found in the microbial biomass and non-biomass residues. The native biomass C and biomass 14 C from glucose and M. littoralis were highly correlated with each other and with soil clay content and other related soil properties, e.g. CEC and total soil pore space. For glucose-amended soils total residual 14 C was also correlated with soil clay content. Differences between soils in the concentration of total residual organic 14 C were due entirely to differences in the amounts of 14 C present in the microbial biomass. Thus, statistically non-biomass 14 C accounted for a constant proportion of input 14 C. In contrast with glucose decomposition, total residual organic 14 C from M. littoralis decomposition was not significantly correlated with clay content and related properties except when the statistical analyses were confined to soils of neutral to alkaline pH. Soils of mildly acidic pH retained more residual non-biomass 14 C than did neutral to alkaline soils of similar clay contents. The close direct correlations between biomass 14 Cand biomass 14 C from glucose and plant material metabolismand soil properties indicated that soil charge or structure or both are important factors influencing microbial biomass accumulation in soils. These factors may override such influences as substrate type, concentration and efficiency of utilisation in determining biomass C concentration in soils after long (1 yr) incubation.

Microbial biomass formed from 14C, 15N-labelled plant material decomposing in soils in the field

Abstract The decomposition of 14C, 14N-labelled medic (Medicago littoralis) material and the net formation and decay of isotope-labelled biomass have been measured in four South Australian soils in the field over 4 yr. The field sites were in similar climatic zones but two sites received about twice as much rainfall as the others. The soils were calcareous and of similar pH, but differed in texture and organic matter content. The decomposition of the organic-14C and organic-15N residues were, for a given site, similar. Initially, the concentrations of labelled residues decreased rapidly, then very slowly. Decomposition rates in a heavy clay soil were significantly less than in the other soils during the first 16 weeks after incorporation of plant material, but thereafter, rates of decomposition in all soils were similar, despite differences in soil texture and climate. More than 50% of the medic-14C had disappeared from all soils after 4 weeks of decomposition and only 15–20% of the medic-14C remained as organic residues after 4 yr. Of the medic-15N 60–65% remained as organic residues after 32 weeks decomposition; the percentage decreased to 45–50% after 4 yr. The amounts of 14C, 14N-labelled biomass, formed from decomposing plant material, were maximal 4–8 weeks after incorporation of plant material into the soils. In samples taken at 8 weeks from the sandy Roseworthy soil, biomass-14C and -15N accounted for 14 and 22% respectively of the total organic-14C and -15N residues present. Thereafter in this soil, the concentrations of biomass-14C and -15N decreased, rapidly at first then more slowly. Nevertheless, throughout most of the decomposition the rates of decrease in the concentrations of biomass-14C and -15N exceeded those of the non-biomass, labelled organic residues. The proportions of 14C, 15N-labelled materials accounted for in the labelled biomass varied between soils. Soils of higher clay content generally retained higher proportions of residual organic-14C and -14N in the biomass, even though the net rates of decomposition of total labelled residues did not differ significantly between soils during most of the decomposition.

Carbon and nitrogen mineralization from two soils of contrasting texture and microaggregate stability: Influence of sequential fumigation, drying and storage

Two top soils (an Alfisol and a Vertisol) of contrasting cation exchange capacity, micro-porosityand microaggregate stability were sampled from the same climatic region and were incubated for 4 weeks with 14C-labelled plant material. Each soil was then subjected to combinations of two of the following treatments: (1) drying (40°C), remoistening and incubation for 10 days at 25°C, (2) fumigation with chloroform vapour and incubationand (3) storage (4°C) and incubation. The amounts of CO2 and 14CO2 evolved and inorganic N released during each incubation period were measured. Also, during drying and after remoistening of soils, concentrations of biomass C and 14C were monitored using a fumigation extraction technique. Biomass C and 14C decreased by 26–30% during desiccation and increased to 77–84% of untreated control soils during incubation after rewetting. The relative decline in biomass during soil drying was of similar magnitude for both soils and for 14C-labelled and total biomass C, indicating that factors other than soil properties had determined the extent of decline. The evolution of microbial populations exposed frequently to high temperatures and to extreme desiccation in the natural field environment has been proposed to explain the similar responses of biomass C to the imposed drying regime. Previous fumigation-incubation of soils to destroy the majority of the microbial biomass had little effect on the sizes of the C mineralization flushes obtained when the soils were subsequently dried, rewetted and incubated. The specific activities of the CO2-C flushes after drying were much lower than those from fumigated or stored control soils respectively. This was especially evident for CO2-C flushes from the well-aggregated Vertisol. From the magnitude of the flushes of CO2 and 4CO2 after the various combinations of treatmentsand from their specific activities, we have deduced that microbial cells killed by soil desiccation had made only a minor contribution to the C and N mineralization flushes after soil rewetting and incubation. The larger contribution had come from other sources, the relative importance of which appears to be influenced by soil characteristics, possibly cation exchange capacity and microporosity.

Distribution and recovery of nitrogen from legume residues decomposing in soils sown to wheat in the field

Abstract Unground 15 N-labelled medic material ( Medicago littoralis ) was mixed with topsoils at 3 field sites in South Australia, allowed to decompose for about 8 months before sowing wheat, and then for a further 7 months until crop maturity. The site locations were chosen to permit comparisons of recoveries and distribution of 15 N in soils (organic N and inorganic N to 90 cm depth) and wheat (grain, straw and roots to 20 cm depth) in areas where rainfall (and wheat yields) differed greatly. Soils differed also in their texture and organic matter contents. Recoveries of applied 15 N in wheat plus soil were 93.1% from a sandy loam (Caliph) and 92.3% from a sandy soil (Roseworthy) despite differences in rainfall and extent of leaching of the 15 NO 3 − formed from the decomposing medic residues. From a heavy clay soil (Northfield), which received the highest rainfall, the 15 N recovery was 87.7%. The loss of 15 N at this site was not due to leaching, as judged by 15 NO 3 − distribution in the soil profile at seeding and crop maturity. Wheat plants took up only 10.9–17.3% of the 15 N added as legume material. Percentage uptakes of 15 N were not related to grain yields. The proportions of wheat N derived from decomposing medic residues were 9.2% at Caliph (input medic, N, 38 kg N ha −1 ), 10.5% at Roseworthy (input medic N, 57 kg N ha −1 ), and only 4.6% at Northfield (input medic N, 57 kg N ha −1 ). Most (51–70%) of the 15 N recovered in wheat was accounted for in the grain. Inorganic 15 N in the soil profiles was depleted during the cropping phase, and at wheat harvest represented from 0.6 to 3.1% only of 15 N inputs. The major 15 N pool was soil organic 15 N accounting for 71.9–77.7% of 15 N inputs. We conclude that, in the context of N supply from decomposing medic tissues to wheat crops, the main value of the legume is long-term, i.e. in maintaining soil organic N concentrations to ensure adequate delivery of N to future cereal crops. The N of the wheat was not uniformly labelled, root N being generally of the highest atom% enrichmensts, and straw N of the lowest. Nevertheless, at the Roseworthy site, the enrichments of wheat N were similar to those of NO 3 − N in the profile at seeding, indicating that the proportions of 14 N and 15 N in the inorganic N pool did not change appreciably during the cropping period. By assuming equilibrium at this site, we calculate that during 15 months decomposition the soil plus legume delivered about 189 kg N ha −1 , of which 93.2 kg ha −1 (49.3%) was taken up by the wheat, 37.2 kg ha −1 (19.7%) was immobilized or remained as fine root residues, and 17.3 kg ha −1 (9.2%) remained as inorganic N in the soil profile; 41.7 kg ha −1 (22.1%) was unaccounted for in the soil-plant system, and was probably lost via inorganic N. Thus about 6.5 kg inorganic N ha −1 was supplied by the soil plus medic residues per 100 kg dry matter ha −1 removed as wheat grain.

Microbial biomass responses to seasonal change and imposed drying regimes at increasing depths of undisturbed topsoil profiles

Concentrations of microbial biomass C and extractable ninhydrin-reactive N in increasing depth layers of an Alfisol topsoil were determined periodically over 13 months for soils under two contrasting tillage regimes. Concentrations decreased rapidly with depth to 10 cm, halving approximately 2.5 cm with each. Organic C and total N concentrations also decreased with depth of topsoil, those of the surface layer (0–2.5 cm) being 1.5–2.1 times and 1.3–2.4 times those respectively of the 2.5–7.5 cm layer. Tillage management affected the relative concentrations of biomass C, total organic C and total N in surface and lower depths of topsoil, but only through its influence on surface soil concentrations. Respective concentrations at 0–2.5 cm depths for soils under a direct drill tillage regime were on average 24, 28 and 50% higher than for soils subjected annually to district cultivation practices. Highest values for microbial biomass C were recorded for moist Winter-sampled soils, and the lowest in soils sampled at times of severe desiccation after prolonged hot dry periods in Summer. Seasonal trends in biomass contents were not significantly influenced by tillage practice. Averaged for both tillage regimes, the seasonal decrease in biomass C was 28% for the surface soils and 23% for the subsurface (2.5–7.5 cm) soils. Biomass C concentrations of soils sampled dry from the field, moistened, and assayed without prior incubation were about 10% higher than those of the same samples incubated moist for 2 weeks before assay. We suggest that severe soil desiccation was likely the main cause of the biomass C decline, and that the decline may be underestimated, due (1) in the case of soils moistened without incubation before assay, to restricted hydrolysis-deamination of killed cell protein under the field dry conditions, but not under assay conditions, and (2) in the case of soils moistened and incubated before assay, to growth of cells on substrates from killed cells and non-biomass sources during the preincubation period. In contrast to soil cores sampled seasonally from the field, biomass C concentrations at different depths of the Alfisol topsoil were not significantly affected by controlled drying of undisturbed cores under constant or fluctuating temperature regimes. Neither were the inorganic N contents of the total cores, nor the C and N mineralization activities of soils sampled from different core depths, influenced by the imposed drying treatments, which resulted in gradual and graded losses of soil moisture. During soil drying, nitrate-N accumulated in the surface layer of the cores, but became redistributed on remoistening of the cores. Thus gradual drying of intact soil cores at moderate temperatures did not per se significantly influence the availability for decomposition of organic substrates, which has implications for modelling C and N mineralization in natural environments with intermittent soil drying.

Utilization by wheat crops of nitrogen from legume residues decomposing in soils in the field

Abstract Ground 15N-labelled legume material (Medicago littoralis) was mixed with topsoils in confined microplots in the field, and allowed to decompose for 7 and 5 months in successive years (1979, 1980) before sowing wheat. The soil cropped in 1979 (and containing 15N-labelled wheat roots and legume residues) was cropped again in 1980. The results support evidence that ungrazed legume residues, incorporated in amounts commonly found in southern Australian wheat growing regions, contribute only a little to soil available N and to crop N uptake, even in the first year of their decomposition. Thus mature first crops of wheat, although varying greatly in dry matter yield (2.9-fold) and total N uptake (2.4-fold), took up only 27.8 and 20.2% of the legume N applied at 48.4 kg ha−1, these corresponding to 6.1 and 10.8% of the N of the wheat crops. The availability of N from medic residues to a second wheat crop declines to The proportional contributions of medic N to soil inorganic N, N released in mineralization tests, and to wheat crop N, differed between seasons and soils, but for a given crop did not significantly differ between tillering, flowering and maturity. In both years, grain accounted for 52–65% of the total 15N of first crops, roots for

Effect of substrate location in soil and soil pore-water regime on carbon turnover

Introduction of 14C-labelled glucose into soil pores of two different size classes (<6 and 6–30 μm neck dia) of a Vertisol, and subsequent comparison of 14C flow at two matric potentials (— 50 and −10 kPa), enabled an evaluation of the effects of substrate location and pore-water regime on carbon turnover. Based on 14CO2 evolution and biomass 14C concentrations in soil, turnover of added substrate carbon during a 4-week incubation was found to be greater when substrate was located in the larger pores, and particularly when such amended soil was held at the lower soil water matric potential. Observed differences in carbon turnover highlighted the importance of spatial compartmentalization of substrates and decomposers (primary and secondary) in soil due to both pore size exclusion (microhabitats) and the degree to which pores are water-filled.

Studies of nitrogen immobilization and mineralization in calcareous soils—I: Distribution of immobilized nitrogen amongst soil fractions of different particle size and density

Abstract 15NO−3 was immobilized in a calcareous sandy soil and a calcareous clay soil each incubated with glucose and wheat straw. Net mineralization of organic-15N was more rapid in the sandy soil, irrespective of C amendment, and in soils amended with glucose. Intermittent drying and wetting of soils during incubation stimulated mineralization of 15N-labelled and native soil organic-N in all treatments. The availability (percentage mineralization) of recently-immobilized 15N consistently exceeded that of the native soil N. Ratios of the availability of labelled and unlabelled N were similar in the sandy and clay soils but varied according to C amendment, drying and wetting cycle and incubation period. Changes in the distribution of immobilized N amongst soil extracts and soil fractions of different particle size and density were determined during periods of net N mineralization. In straw-amended soils, the organic-15N of a light fraction, sp.gr. Drying and rewetting of soils hastened or magnified changes occurring in the organic-15N of soil fractions, but qualitatively, the pattern of change was similar to that observed with soils incubated under uniformly-moist conditions. The percentage distribution of labelled and unlabelled N suggested that in the long term, the silt fraction will accumulate an increasing proportion of the more stable nitrogenous residues.