Adenosine triphosphate and microbial biomass in soil

Two methods for measuring adenosine 5'-triphosphate (ATP) in soil were compared, one based on extraction with NaHCO₃-CHCl₃ and thel other on extraction by a trichloracetic acid-phosphate-paraquat reagent. Recoveries of added ATP were greater with the NaHCO₃-CHCl₃ reagent but the extraction of “native” soil ATP by NaHCO₃-CHCl₃ was only about a third of that by TCA-phosphate-paraquat.Microbial biomass C and ATP were measured in 8 contrasting English soils, using the fumigation method to measure biomass C and the TCA-phosphate-paraquat method to measure ATP. Except in one acid woodland soil, the ratio (ATP content of the soil)/(biomass C content of the soil) was relatively constant, with a mean of 7.3 mg ATP g^?1 biomass C for the different soils. This value is very similar to that obtained earlier in a range of 11 grassland and arable soils from Australia. Taking the English and Australian grassland and arable soils together, there is a close (r = 0.975) linear relationship between ATP and microbial biomass C that holds over a wide range of soils and climates. From this relationship, the soil biomass contains 7.25 mg ATP g^?1 biomass C, equivalent to an ATP-to-C ratio of 138, or to 6.04 μmoles ATP g^?1 dry biomass.The acid woodland soil (pH 3.9) contained much less biomass C, as measured by the fumigation method, than would have been expected from this relationship. This, and other evidence, suggests that the fumigation method for measuring microbial biomass C breaks down in strongly acid soils.The ATP content of the biomass did not depend on the P status of the soil, as indicated by NaHCO₃-extractable P.     

Effects of phosphorus addition and energy supply on acid phosphatase production and activity in soils

To find out how acid phosphatase activity and production in some Alberta soils may be related to soil properties and past fertilizer history, soils of varying organic matter content, extractable P and P fertilization history were assayed for acid phosphatase using p-nitrophenyl phosphate as substrate. The effect of solution P concentration during the phosphatase assay was examined. The effect of P on the production of new phosphatase was examined in soils incubated with an added energy supply or orthophosphate.Phosphatase activity was influenced by P fertilization practices during the 5 yr before sampling. In a Black Chernozemic soil (Malmo SiCL) with a high organic matter content and high initial phosphatase activity, P fertilization at 27 or 54 kg P ha^?1 y^?1 for 5 yr reduced phosphatase activity by about 20%. However, in a Grey Luvisolic soil (Cooking Lake L) with low organic matter and initial phosphatase, P fertilization at 54 kg P ha^?1y^?1 for 5 yr tended to increase activity, probably by increasing plant root growth and organic matter additions.Assay solutions containing orthophosphate at 0.55 mM reduced activity by 25% and 47% in a Malmo SiCL and Maleb L (Orthic Brown Chernozem) soil respectively. Further increases of phosphate concentration to 5.5 mM reduced phosphatase activity by 50% and 76% in the Malmo and Maleb L soils respectively.Phosphatase activity was increased up to 6-fold by incubation of soil with glucose and NH₄NO₃. Addition of P to produce an added C: added P ratio of 20:1 completely prevented synthesis of phosphatase by proliferating organisms and had a slight inhibitory effect on phosphatase already present. Similarly, addition of P without C in a 6-week incubation had only a small effect on phosphatase activity and maintained P concentrations in the assay solutions slightly below 0.55 mM. It was concluded that the effect of phosphate on soil phosphatase operates more through its effect on phosphatase synthesis than on activity of existing phosphatase.     

A 31P nuclear magnetic resonance study of the phosphorus species in alkali extracts of soils from long-term field experiments

The different forms of phosphorus (P) in 0.5 m sodium hydroxide extracts of soils from long-term field experiments at Rothamsted were characterized by ³¹P-nuclear magnetic resonance spectroscopy (NMR). The extract from an old grassland soil (pH 4.6) from a plot of the Park Grass Continuous Hay Experiment that had received no fertilizer or lime for at least 125 years contained the following forms of P: inorganic orthophosphate (22% of the extracted P), orthophosphate monoesters (49%), orthophosphate diesters (14%), phosphonates (3%), pyrophosphate (4%) and two unidentified forms of P (7%). The soil extract from a Park Grass plot given inorganic phosphate fertilizer (35 kg P ha^?1) annually for 121 years contained the same forms of P and, in addition, a small amount of polyphosphate. There was also evidence of an increase in the orthophosphate monoester fraction. Another old grassland soil, of pH 6.1, contained more total and organic P than Park Grass but the extract contained fewer forms of P: inorganic orthophosphate (14% of the extracted P), orthophosphate monoesters (39%), orthophosphate diesters (34%) and an unidentified form (13%). An area of this grassland that had been ploughed up 20 years previously, and kept bare since, contained less organic P. The extract contained less of the phosphate diesters but the more stable monoesters remained relatively unchanged.     

Effects of glucose and rhizodeposits (with or without cysteine-S) on immobilized-35S, microbial biomass-35S and arylsulphatase activity in a calcareous and an acid brown soil

Our aim was to study the effects of C (as glucose and artificial rhizodeposits) on S immobilization, in relation to microbial biomass‐S and soil arylsulphatase (ARS) activity, in contrasting soils (a calcareous and an acid brown soil). The glucose‐C and artificial rhizodeposit‐C with or without cysteine were added at six rates (0, 100, 200, 400, 600 and 800 mg kg^?1 soil) to the two soils and then incubated with Na₂³⁵SO₄ for 1 week prior to analysis. The percentages of ³⁵S immobilized increased when C as glucose and rhizodeposit (without cysteine) were added to both soils. With cysteine‐containing rhizodeposit, the percentages of ³⁵S immobilized remained relatively stable (23.5% to 29.9%) in the calcareous soil, but decreased in the acid brown soil (52.7% to 31.5%). For both soils, cysteine‐containing rhizodeposit additions showed no significant correlation between immobilized‐³⁵S and microbial biomass‐³⁵S, suggesting that microorganisms immobilized cysteine‐S preferentially instead of ³⁵S from the tracer (Na₂³⁵SO₄). In the calcareous soil, a positive and significant correlation was found between ARS activity and microbial biomass‐³⁵S (r = 0.85, P < 0.05) when glucose was added. We also saw this correlation in the acid brown soil when rhizodeposit‐C without cysteine was added (r = 0.90, P < 0.05). Accordingly, the results showed the presence of extracellular arylsulphatase activity of 48.7 mg p‐nitrophenol kg^?1 soil hour^?1 in the calcareous soil and of 27.0 mg p‐nitrophenol kg^?1 soil hour^?1 in the acid brown soil.     

Adenylate energy charge measurements in soil

Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphatc (ADP) and adenosine 5'-monophosphate (AMP) were extracted from soil with either a solution of trichloroacetic acid, paraquat and phosphate (TCA reagent) or a mixture of chloroform, sodium hydrogen carbonate, phosphate and adenosine (NaHCO₃ reagent). Standard enzymic procedures were used to convert ADP and AMP to ATP, which was measured by the fire-fly luciferin-luciferase system. The measured quantities of nucleotides were corrected for incomplete extraction using the percentage recoveries of added ATP, ADP and AMP. The adenylate energy charge ratio (AEC) was calculated from the formula AEC = ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]).Measurements were made on a grassland soil, following a conditioning incubation at 15°C and 50% WHC for 7 days. Additional measurements were made on the same soil after a further 50- or 100-day incubation at 25°C and 50% WHC, with or without an amendment of 1100 μg ryegrass Cg⁻¹ soil, added at the end of the conditioning incubation. Biomass-ATP concentration, measured in TCA extracts, changed little, even on prolonged incubation, and was maintained at a level comparable to that observed in earlier work (about 10 p mol ATP g⁻¹ biomass C). AEC values in TCA soil extracts were high (0.8–0.9) for all soil treatments and independent of substrate addition or length of incubation.In contrast, AEC was low (0.4) in fresh soil extracted with NaHCO₃ reagent, but increased to 0.6 when ryegrass was incubated with the soil for 50 days. Although the total adenine nucleotide pool (i.e. [ATP] + [ADP] + [AMP]) was similar as measured in NaHCO₃ and in TCA soil extracts, both energy charge and ATP content were lower in the NaHCO₃ extracts. It was therefore concluded that the main reason for the lower AECs observed with the NaHCO₃ reagent was that microbial ATPases were still active during extraction and caused appreciable hydrolysis of microbial ATP to ADP and AMP. In contrast, the TCA reagent rapidly inactivates ATPases and is therefore preferable for extracting adenine nucleotides from soil.The results indicate that the soil microbial biomass, although a mainly dormant population, maintains both AEC and ATP at levels characteristic of exponentially growing organisms in vitro, even during prolonged incubation without fresh substrate. It was also concluded that roots make a negligible contribution to total ATP extracted from fresh sieved soil.     

Evaluation of substitutes for paraquat in soil microbial ATP determinations using the trichloroacetic acid based reagent of

The trichloroacetic acid (TCA) based reagent proposed by Jenkinson and Oades (1979) fits all the criteria required to measure soil microbial biomass ATP (biomass ATP). Amongst other components it contains paraquat (0.1 M 1,1′ dimethyl-4,4′ bipyridylium dichloride), usually extracted from the herbicide Gramoxone. Paraquat is added as an analogue of ATP. It is tightly fixed to soil and so decreases ATP fixation. However, Gramoxone is now banned as an agricultural chemical in both Europe and North America. Our aim was to find an effective replacement for paraquat to measure biomass ATP. The best replacement was 0.6 M imidazole in 1.10 M TCA containing 0.25 M P (termed the TIP reagent). Biomass ATP concentrations were not significantly different in five soils extracted by either reagent 10.37 and 11.17 μmol ATP g⁻¹ biomass C, respectively; standard error of differences of means = 0.36; p = 0.091.     

The adenylate energy charge of the soil microbial biomass

A method was developed for measuring adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) in soil. All three adenine nucleotides were extracted from soil with a solution of trichloroacetic acid, paraquat and phosphate. ATP was measured in the neutralised (pH 7.4) soil extracts by the fire-fly luciferin-luciferase system. ADP was measured as ATP after incubating the neutralised extracts with pyruvate kinase (PK) and phosphoenolpyruvate (PEP) to convert ADP to ATP. AMP was converted to ATP by incubation with the coupled PK-PEP-myokinase system and measured as ATP. The quantities of nucleotides present in the extracts were corrected for incomplete extraction from soil by measuring the percentage recovery of added ATP, ADP and AMP. The adenylate energy charge (AEC) was calculated from the formula AEC = [[ATP] + 0.5[ADP]]/[[ATP] + [ADP] + [AMP]]. Measurements were made on (1) fresh soil, extracted as soon as possible after field sampling (2) soil stored air-dry at 5°C for 18 days and (3) soil stored air-dry at 5°C for 57 days and then rewetted to the original field moisture content and incubated aerobically for 2.5 h at 10°C before extraction.In moist soil the biomass maintains both ATP and AEC at levels close to those of activity growing cells, even though little of the biomass in soil can be in active growth at any given time. ATP accounted for 77% of the total adenine nucleotides (A_T) in the fresh soil, with an AEC of 0.85 (a value comparable to that found in microorganisms undergoing active growth in vitro. In contrast, ATP only accounted for 28% of A_T in the air-dried soil, with an AEC of 0.46. When the air-dried soil was rewetted, ATP increased to 66% of A_T and the AEC increased to 0.76. However, A_T in the air-dried soil (7.65 nmol g^?1 soil) was of the same order as that in rewetted soil (6.70 nmol g^?1) even though the AEC's were very different.These results show that the soil microbial biomass does not maintain a high AEC when air-dried. Once remoistened, the population tends to restore its AEC to the original value. This restoration occurs so rapidly that it cannot be due to the formation of a new biomass.     

Modification and application of a soil ATP determination method

Accurate estimation of microbial adenosine 5′-triphosphate (ATP) is a pre-requisite to quantify the impact of varying environments on microbial activity of soil. We investigated the effectiveness of a high efficiency soil ATP determination method (PA) [Webster, J.J., Hampton, G.J., Leach, F.R., 1984. ATP in soil: a new extractant and extraction procedure. Soil Biology & Biochemistry 4, 335-342] in 10 Ontario (Canada) soils collected along a 100 m transect and spanning a textural class gradient ranging from a sandy loam to clay loam with increasing organic matter. Modifications of the method involved using an extract of autoclaved soil to make the standard curve, as it was found that the light emitted by ATP luciferin-luciferase bioluminescence reaction in the pure extractant was different from that in the extracts. Replacing Tricine with Tris buffer in the assay significantly improved the light emission. On an average, the internal standard calibration method (ISM) measured a smaller amount of extracted ATP (1199 ng ATP g⁻¹ soil) and a lower recovery of ATP spike (82.4±7.2%) than did the standard curve method (SCM) (1246 ng ATP g⁻¹ soil and 91.2±4.5%, respectively) (P<0.05 for both comparisons). However, the average total estimated ATP was higher with ISM (1474±102 ng ATP g⁻¹ soil) than with SCM (1373±88 ng ATP g⁻¹ soil) (P<0.07). While the recovery rates determined using SCM were consistent among the soils tested, the rates measured using ISM was negatively correlated with soil clay and organic matter content, implying that the latter assay was affected by the soil properties. Our results confirmed that the recovery rates obtained by the PA method were the highest among those reported, when only SCM was used.     

Phosphorus Composition and Phosphatase Activities in Soils Affected by Long-Term Application of Pig Manure and Inorganic Fertilizers

The long-term (25 years) effect of using chemical fertilizers and animal manure on soil phosphorus (P) composition and phosphatase activities was investigated in this study. Results showed that pig manure applications significantly increased soil total P, Olsen P, and phosphatase activities, whereas chemical fertilizers had no significant effects on soil chemical properties and phosphatase activities. Manure applications doubled or tripled the orthophosphate concentrations as compared to chemical fertilizers. Analysis of solution ³¹P nuclear magnetic resonance (NMR) spectroscopy showed that P composition in sodium hydroxide (NaOH)–ethylenediamenetetraacetic acid (EDTA) extracts was dominated by orthophosphate (59–84%), followed by phosphomonoesters (15–40%). More organic P (P_o), especially myo-inositol hexakisphosphate, was observed in soil treated with manure as compared with soil treated with chemical fertilizer.     

Differential tolerances of two old world bluestem genotypes to excess aluminum in acid soil and nutrient solution

Two genotypes of Old world bluestems from the species Bothriochloa intermedia (R. Br.), A. Camus, shown earlier to differ in tolerance to acid, Al‐toxic Tatum subsoil at pH 4.1, were characterized further with respect to growth in pots of Tatum soil over a wider pH range and tolerance to Al in nutrient solutions. The two genotypes studied were acid‐soil tolerant P. I. 300860 (860) and acid soil sensitive P. I. 300822 (822).

The soil experiment confirmed earlier rankings of acid soil tolerance in these two genotypes. For example, with 0, 375 or 750 ug CaCO₃ g^‐1 soil (final pH 4.0, 4.3 and 4.6), the 860 genotype produced significantly more dry top weight than 822, but these differences were precluded with 1500 or 3000 ug g^‐1 CaCO₃ added (pH 4.7 and 5.4). At pH 4.3 and 4.6, the root dry weights of the two genotypes were also significantly different and weights were equalized at pH 4.7 and 5.4. The 860 genotype made fairly good top growth (67% of maximum) at pH 4.3 and a soil Al saturation of 63%; this situation was lethal for 822. When grown in greenhouse pots, the acid‐soil tolerant 860 genotype required only about one fourth as much CaCO₃ as 822 to produce good growth of forage on acid Tatum subsoil. If confirmed under field conditions, such a difference could be economically significant in reclaiming acidic marginal land and in producing forage at low cost.

Differential Al tolerance in the two genotypes was confirmed in nutrient solutions. For example, with 8 mg Al L^‐1 added, both top and root dry weights of 860 were significantly higher than those of 822, but with no Al added, these growth differences disappeared.

Mineral analyses of plants did not shed much light on mechanisms of differential acid soil or Al tolerance. For example, Al concentrations in plant tops associated with toxicity varied from 33–43 ug g^‐1 in nutrient solutions containing Al to 119–283 ug g^‐1 in acid soil It appears that elucidation of Al‐adaptive mechanisms will require physiological and biochemical studies at the cellular level.     

An improved method for determination of adenosine triphosphate (ATP) in soil

Different methods for extraction of ATP from soil were examined. The methods were compared with respect to the efficiency of extraction of ATP from soil, and the content of ATP in the extracts was measured by the luciferin-luciferase system. The following extraction procedure was found to be the most efficient:Shaking of the soil with sulphuric acid, followed by filtration, cation exchange of the extract on Na⁺ resin, and adjustment to pH 9.8 with ethanolamine.The method is simpler, faster and more convenient for processing large numbers of samples, than the earlier-described acid extraction methods. Furthermore, ATP levels of 5ngATPg^?1 dry wt soil can be measured and results can be reproduced within ± 5%.The respiration rates of the soils were also measured, and a high degree of correlation was found between carbon dioxide production and ATP levels determined by extraction with sulphuric acid or dimethylsulfoxid.     

ATP in soil: A new extractant and extraction procedure

An extraction mixture comprised of 0.67 m H₃PO₄, 2 m urea, 20% DMSO, 1.8 mg of adenosine, 20 mM EDTA, and 1% Zwittergent 3,10 and a procedure to extract ATP from soil have been developed. The reagents and method were tested on six different Oklahoma soils and yielded a recovery of 99% of the ATP from added Escherichia coli cells. The extraction mixture was designed to minimize interference from soil-derived materials. The phosphoric acid provides acid to extract ATP and to inactivate proteins, and phosphate to saturate phosphate-binding sites. It also complexes or precipitates metal ions. The EDTA chelates metal ions, prevents inhibition of luciferase, and aids lysis of bacterial cells. The adenosine serves to saturate ATP binding-sites. Urea denatures proteins and prevents hydrogen bonding of the released ATP. DMSO, the Polytron treatment, and Zwittergent 3,10 remove cells from surfaces and lyse them. An internal standard of E. coli cells is used to determine efficiency of extraction and assay. When compared with the 12 best methods suggested by previous studies, the newly-formulated extractant and procedure yielded the greatest amount of ATP from soil.     

Isolating the influence of pH on the amounts and forms of soil organic phosphorus

Soil pH influences the chemistry, dynamics and biological availability of phosphorus (P), but few studies have isolated the effect of pH from other soil properties. We studied phosphorus chemistry in soils along the Hoosfield acid strip (Rothamsted, UK), where a pH gradient from 3.7 to 7.8 occurs in a single soil with little variation in total phosphorus (mean ± standard deviation 399 ± 27 mg P kg^?1). Soil organic phosphorus represented a consistent proportion of the total soil phosphorus (36 ± 2%) irrespective of soil pH. However, organic phosphorus concentrations increased by about 20% in the most acidic soils (pH < 4.0), through an accumulation of inositol hexakisphosphate, DNA and phosphonates. The increase in organic phosphorus in the most acidic soils was not related to organic carbon, because organic carbon concentrations declined at pH < 4.0. Thus, the organic carbon to organic phosphorus ratio declined from about 70 in neutral soils to about 50 in strongly acidic soils. In contrast to organic phosphorus, inorganic phosphorus was affected strongly by soil pH, because readily‐exchangeable phosphate extracted with anion‐exchange membranes and a more stable inorganic phosphorus pool extracted in NaOH–EDTA both increased markedly as soil pH declined. Inorganic orthophosphate concentrations were correlated negatively with amorphous manganese and positively with amorphous aluminium oxides, suggesting that soil pH influences orthophosphate stabilization via metal oxides. We conclude that pH has a relatively minor influence on the amount of organic phosphorus in soil, although some forms of organic phosphorus accumulate preferentially under strongly acidic conditions.     

Can ATP be measured in soils treated with industrial oily waste?

Soil samples were collected from a field with a long‐term (10 yr) oily wastewater application history, containing 70 mg g^–1 of oil and grease and an accumulation of heavy metals, and also from a short‐term (1 yr) wastewater application involving different rates of waste, tillage, and nitrogen (N) fertilization. Prior to ATP extraction, the soils were incubated at 22 °C and a water potential of –60 kPa for 21 d and 2 d for the long‐ and short‐term trials, respectively. The light emitted from the bioluminescence reaction was partly quenched in the extract of steam‐sterilized long‐term waste‐treated soil, and curvilinearly responded to the addition of ATP at concentrations higher than 4 ng ATP per assay in contrast to the linear response from the pure extractant and the extract of control soil. Calibration curves developed from the extracts of steam‐sterilized soils were used for calculating ATP in that given soil. ATP determined in the long‐term treated soil was as high as 3201 ng (g soil)^–1. Still, residual oil to ATP concentration ratio was about an order of magnitude higher in the long‐ than in the short‐term waste‐treated soil, reflecting the accumulation of recalcitrant material. In the short‐term treated soils, ATP ranged from 355 to 760 ng (g soil)^–1 and responded to the rate of waste application, tillage, and fertilization. The use of ATP measurement has potential for assessing land management effects and developing tillage and fertilization recommendations for enhanced biodegradation of the oil waste.     

Inhibition of phosphorus sorption to goethite, gibbsite, and kaolin by fresh and decomposed organic matter

The direct effects of dissolved organic matter (DOM) on the sorption of orthophosphate onto gibbsite, goethite, and kaolin were examined using a one-point phosphorus sorption index and the linear Tempkin isotherm model. DOM extracted from fresh and decomposed agricultural residues, as well as model organic and humic acids, were used. Changes in the chemical and sorptive characteristics of the DOM in the absence and presence of added orthophosphate (50 mg l⁻¹) were also determined. For residue-derived materials, DOM sorption to all minerals correlated well with percent hydrophobicity, apparent molecular weight, and phenolic acidity in the absence of added orthophosphate. Sorption of DOM to goethite and gibbsite was significantly decreased in the presence of added P. The correlation coefficient values of percent hydrophobicity, apparent molecular weight, and phenolic acidity to sorption also declined in the presence of added P. Thus, the addition of P substantially lowered fractionation of DOM after sorption to goethite and gibbsite. In contrast, few significant P sorption-induced differences were observed in the kaolin system. According to one-point P sorption results, DOM in the form of Aldrich humic acid, oxalate, and decomposed clover and corn residue, significantly inhibited P sorption to goethite at concentrations of 50 and 200 mg total soluble carbon (C_TS l⁻¹). Phosphorus sorption to gibbsite was significantly inhibited by 50 mg C_TS l⁻¹ derived from decomposed corn residue, fresh dairy manure residue, and oxalate solution. At 200 mg C_TS l⁻¹, all DOM solutions were found to inhibit P sorption to gibbsite. This study suggests that DOM inhibition of P sorption depends on the chemical properties of both the sorbent and the DOM itself. In general, DOM from decomposed organic materials inhibited P sorption to a greater extent than did DOM derived from fresh materials. This stronger inhibition highlights the importance of microbial processes in the release of soluble soil P, a key determinant of P availability to plants.     

Influence of aggregate size on phosphorus changes in a soil cultivated intermittently: analysis by <Superscript>31</Superscript>P nuclear magnetic resonance

A pot trial using wet-sieved soil aggregates (>4, 4–2, 2–1, 1–0.5, 0.5–25, and remaining soil <0.25 mm) from a soil that had been cultivated out of permanent pasture and used for winter forage crops for 2 years examined changes in P forms before and after 35 weeks when resown with perennial ryegrass. Soil analyses showed that P was depleted after 35 weeks growth. Changes in P forms were analyzed by ³¹P nuclear magnetic resonance of soil NaOH-EDTA extracts, which removed 98–96% of total P (about 1,080 mg kg⁻¹ in unsieved soil before pasture growth). This indicated that aggregate size influenced the concentrations and forms of P probably via a combination of physical protection and moisture status: orthophosphate, monoesters, diesters and pyrophosphate increased with decreasing size, while phosphonates and polyphosphates were unaffected. The increase in pyrophosphate was attributed to fungal growth, while decreases in orthophosphate and labile organic P (diesters) decreased due to either leaching or mineralisation and plant uptake. The largest decrease was associated with orthophosphate, which could be replenished by fertiliser. However, given the soil’s high potential for P loss, this should only be done to meet conditions for optimal plant growth as any excess would increase the risk of loss. To further minimise P loss without affecting pasture yield, management should maintain or improve soil structure.     

The acid buffer capacity of some finnish forest soils: Results of acid addition laboratory experiments

Batch acid addition experiments were carried out to determine the acid buffer capacities (amount of acid required to lower soil pH by one unit) of forest soils. Samples of O, E, B (or BC), and C horizons taken from 29 podzolic profiles in southern Finland were used in the experiments. Subsamples of soil were equilibrated for 24 h with NaCl solution containing additions of HCl acid. Cation exchange, mineral dissolution (weathering), and the protonation of organic matter all appeared to have been involved in the buffering of the acid additions. For the O horizon samples, most of the cations released in response to the acid additions were base cations. For the mineral soil samples, most of the cations released were Al³⁺ ions. With the exception of a few samples, the added acid was not fully neutralised and pH was lowered even with the lowest addition treatment. However, the acid addition treatments corresponded to many times the regional annual acid deposition load (1.6–2.0 cmol(c) m^?2). Calculated acid buffer capacities (cmol(c) kg^?1 pH^?1) ranged from 9.8 to 40.8 for O horizon soil samples and from 0.1 (C horizon) to 5.2 (E horizon) for the mineral soil samples. Total acid buffer capacities for a profile (to a depth of 50 cm) ranged from 500 to 2349, with a mean value of 1091 cmol(c) m^?2 pH^?1. It is concluded that, in addition to CEC and base saturation, acid buffer capacity is a useful measure to describe the ecological effects of acid deposition on soil.     

Preferential phosphorus leaching from an irrigated grassland soil

Intact lysimeters (50 cm diameter, 70 cm deep) of silt loam soil under permanent grassland were used to investigate preferential transport of phosphorus (P) by leaching immediately after application of dairy effluent. Four treatments that received mineral P fertilizer alone (superphosphate at 45 kg P ha^?1 year^?1) or in combination with effluent (at ～ 40–80 kg P ha^?1 year^?1) over 2 years were monitored. Losses of total P from the combined P fertilizer and effluent treatments were 1.6–2.3 kg ha^?1 (60% of overall loss) during eight drainage events following effluent application. The rest of the P lost (40% of overall loss) occurred during 43 drainage events following a significant rainfall or irrigation compared with 0.30 kg ha^?1 from mineral P fertilizer alone. Reactive forms of P (mainly dissolved reactive P: 38–76%) were the dominant fractions in effluent compared with unreactive P forms (mainly particulate unreactive P: 15–56%). In contrast, in leachate following effluent application, particulate unreactive P was the major fraction (71–79%) compared with dissolved reactive P (1–7%). The results were corroborated by ³¹P nuclear magnetic resonance analysis, which showed that inorganic orthophosphate was the predominant P fraction present in the effluent (86%), while orthophosphate monoesters and diesters together comprised up to 88% of P in leachate. This shows that unreactive P forms were selectively transported through soil because of their greater mobility as monoesters (labile monoester P and inositol hexakisphosphate) and diesters. The short‐term strategies for reducing loss of P after application of dairy effluent application should involve increasing the residence time of applied effluent in the soil profile. This can be achieved by applying effluent frequently in small amounts.     

A simple method for quantitative determination of ATP in soil

A simple method to measure soil ATP by the luciferin-luciferase system is described. The ATP is extracted from the soil by vigorous shaking with a sulfuric acid-phosphate solution for 15 min. An aliquot of the soil suspension is neutralized with a Tris-EDTA solution and mixed with a special ATP releasing reagent (NRB). ATP is measured after a 10 s exposure to the NRB reagent, followed by addition of luciferin-luciferase and integration over 10 s in a Lumacounter M 2080. The ATP content in soils which had been stored at 5°C for 90 days and then incubated at 25°C for 5 days, ranged from 0.37 to 7.52 μg ATP g^?1 dry wt, with standard deviations less than 10%. There was a close (r = 0.96) linear relationship between ATP content and biomass C determinated by fumigation for this group of soils. The soil biomass contained 4.2–7.1 μg ATP mg^?1 biomass C. The ATP content of the biomass declined during storage at 5°C for 210 days.     

Extraktion eines kupferbelasteten Bodens unter Verwendung eines aminosäurehaltigen Reststoffhydrolysats. 2. Elutionsdynamik und Bilanzierung der Aminosäuren nach kontinuierlichem Eintrag in Bodensäulen

Extraction of a copper contaminated soil material by the percolation of an amino acid containing residue hydrolysate. 2. Time course of amino acid elution and input/output balance of amino acids During 16 days an amino acid containing blood meal hydrolysate (amino acid concentration: 188 mMol·L^?1) was percolated through a column packed soil material (soil content per column: 4.1 kg dry weight, four parallels). The copper contaminated material (soil type: Typic Udifluvent, soil texture: sandy loam, loamy sand) was sampled from an area formerly used for cultivation of hop (Humulus lupus). Besides the investigation of the copper liberation the experiments aimed to determine the elution dynamic and input/output balance of amino acids (time span for amino acids balance 14 days). In total 11.7 L of hydrolysate, containing 2.2 Mol of amino acids, were introduced into each column. The mean amino acid output with the column effluent was 1.13 Mol. This corresponds to an elution degree of 51.2%, related to the sum of applicated amino acids, and to a mean substance specific elution degree of 48.4% reflecting the elution of 15 compounds. The substance specific elution ranged from 9.6% (serine) to 75.5% (valine). The highest concentrations of serine and threonine were determined in the effluents after two days, whereas the histidine concentration was highest at the last sampling. The differences in the percolation properties of the amino acids are discussed in terms of important retention and elimination processes (biodegradation, ad-/desorption, intercalation).