Influence of pH,dispersion methods,and different compounds on adenosine triphosphate recovery from soil

We studied the recovery of ATP from soil. A soil-water suspension was prepared by two different methods (simple stirring or ballottini mill treatment) at different pH levels and in the presence of different chemicals [Na₂SO₄, Na₃PO₄, Na₅P₃O₁₀, adenosine, ethylenediaminetetra-acetic acid (EDTA), TRIS]. The ATP recovery was evaluated by adding [³H]-8 ATP to the solution and comparing the values obtained by radioactivity measurements with those obtained by an enzymatic assay. Strongly acidic (pH lower than 1.0) or alkaline (pH 10.0) extractions yielded the best ATP recoveries compared with intermediate pH values. At pH 10.0, the addition of Na₃PO₄ or Na₅P₃O₁₀ gave a high level of ATP recovery, 68 and 96%, respectively. No ATP hydrolysis occurred under alkaline extraction conditions. Under acidic extraction conditions, the addition of adenosine, EDTA, Na₂SO₄, or Na₅P₃O₁₀ improved ATP recovery but it was never higher than 34%. The results were discussed in terms of the effects of different physical and chemical conditions on cell disruption, ATP stability, ATP interactions with soil components, and ATP solubilization.     

Adenylates as an estimate of microbial biomass C in different soil groups

Adenylate (i.e. adenosine tri- (ATP), di- (ADP) and monophosphates (AMP)) and microbial biomass C data were collected over a wide range of sites including forest floor layers and forest, grassland and arable soils. Microbial biomass C was measured by fumigation extraction and adenylates after alkaline Na₃PO₄/DMSO/EDTA extraction and HPLC detection. Our aims were (1) to test whether the sum of adenylates is a better estimate for microbial biomass than the determination of ATP, (2) to compare our conversion values with those proposed by others, and (3) to analyse whether soil properties or land use form affect the relationships between ATP, adenylates and microbial biomass C. A close relationship was found between microbial biomass C and ATP (r=0.96), but also with the sum of adenylates (r=0.96) within all appropriately conditioned soil samples (n=112). In the mineral soil (n=98), the geometric means of the ATP-to-microbial biomass C ratio and the adenylates-to-microbial biomass C ratio were 7.4 and 11.4 μmol g⁻¹, respectively. The mean ratios did not differ significantly between the different texture classes and land use forms. In the forest floor, the ATP-to-microbial biomass C ratio and the adenylates-to-microbial biomass C ratio were both roughly two-thirds of those of the mineral soil. The average adenylate energy charge (AEC) of all soil samples was 0.79 and showed a strong negative relationship with the soil pH (r=−0.69). However, the AEC is presumably only indirectly affected by the soil pH.     

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.     

Remediation of Lead Contaminated Soil by EDTA. I. Batch and Column Studies

Extraction using ethylenediaminetetraacetic acid (EDTA), and other chelates has been demonstrated to be an effective method of removal of Pb from many contaminated soils. However, column leaching of Pb from alkaline soils with EDTA has been problematic due to extremely low soil permeability. The first purpose of this study was to develop batch extraction procedures and methods of analysis of batch extraction data to provide Pb solubility information which can be used to model the column extraction of Pb from soils. The second purpose was to determine the effect of the addition of KOH and CaCl₂ to K₂H₂EDTA extract solution on both hydraulic conductivity and Pb removal. A Pb-contaminated soil sample was collected from an abandoned battery recycling facility. Both batch shaker extractions and column leaching experiments were completed using 5 different EDTA extract solutions. When only CaCl₂ was added to EDTA no change in the amount of Pb removed by batch extraction was observed. As expected, lead solubility was observed to decrease as pH was increased by the addition of KOH. However, Pb solubility was only slightly decreased by the addition of both CaCl₂ and KOH. The amount of time required to leach 6.0 L of extraction solution through the soil columns varied from 2 to 33 days. The addition of CaCl₂ and/or KOH resulted in increased soil hydraulic conductivity relative to the EDTA-only solution. The hydraulic conductivity was related to residual calcium carbonate content, suggesting that dissolution of CaCO₃ and subsequent production of CO₂ gas in the soil pores was partially responsible for the observed reductions in soil permeability. However, Pb removal was diminished with the addition of CaCl₂ and KOH because of the decreased Pb solubility and also kinetic limitations associated with the shorter residence time of the extract solution in the column.     

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.     

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.     

Survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in various soil particles: importance of the attached bacterial phenotype

The risk of enteropathogens to food and water is highly dependent on their survival in soil environments. Here, the effects of soil type, particle size, the presence of natural organic matter (NOM) or Fe/Al (hydro)oxides on pathogenic Escherichia coli O157:H7 survival in sterilized soil particles were assessed through survival, attachment, metabolic activity, and qRT-PCR analyses. The abundance of inoculated E. coli O157:H7 in Brown soil (Alfisol) particles increased 0.6–1.4 log₁₀ CFU/g within 3 days (except for NOM-stripped clay), while that in Red soil (Ultisol) particles decreased rapidly in 8 days post-inoculation. Additionally, survival of bacteria was significantly enhanced when Fe/Al (hydro)oxides had been removed from Red soil particles. For the two soils, E. coli O157:H7 survived the longest in NOM-present clays and the bacterial adenosine 5′-triphosphate (ATP) levels were 0.7–2.0 times greater in clays than in sands and silts on day 8. Moreover, clays were more effective than silts and sands in binding cells and changing the expressions of acetate pathway-associated genes (pta and ackA). For silts and sands, E. coli O157:H7 decayed more rapidly in the presence of NOM and similar trends of bacterial ATP levels were observed between NOM-stripped and NOM-present soil particles, indicating that the primary role of NOM was not as a nutrient supply. These findings indicate that soil particles function mainly through attachment to change the metabolic pathway of E. coli O157:H7 and ultimately impact the survival of bacterial pathogens in soils.     

Soil organic phosphorus in tropical forests: an assessment of the NaOH–EDTA extraction procedure for quantitative analysis by solution 31P NMR spectroscopy

The extraction of soil organic phosphorus by the NaOH–EDTA procedure was assessed in detail for a tropical forest soil (clay‐loam, pH 4.3, total carbon 2.7%). Optimum conditions for the quantification of soil organic phosphorus and characterization of its composition by solution ³¹P NMR spectroscopy were extraction in a solution containing 0.25 m NaOH and 50 mm Na₂EDTA in a 1:20 solid to solution ratio for 4 hours at ambient laboratory temperature. Replicate analyses yielded a coefficient of variation of 3% for organic phosphorus as a proportion of the spectral area. There was no significant difference in total phosphorus extraction from fresh and air‐dried soil, although slightly more organic phosphorus and less paramagnetic ions were extracted from dried soil. The procedure was not improved by changing the concentration of NaOH or EDTA, extraction time, or solid to solution ratio. Pre‐extraction with HCl or Na₂EDTA did not increase subsequent organic phosphorus extraction in NaOH–EDTA or improve spectral resolution in solution ³¹P NMR spectroscopy. Post‐extraction treatment with Chelex resin did not improve spectral resolution, but removed small concentrations of phosphorus from the extracts. Increasing the pH of NaOH–EDTA extracts (up to 1.0 m NaOH) increased the concentration of phosphate monoesters, but decreased DNA to an undetectable level, indicating its hydrolysis in strong alkali. The standardized NaOH–EDTA extraction procedure is therefore recommended for the analysis of organic phosphorus in tropical forest soils.     

Decomposition of Zn-rich Arabidopsis halleri Litter in Low and High Metal Soil in the Presence and Absence of EDTA

Hyperaccumulating plants are increasingly investigated in combination with EDTA addition to soil for phytoremediation of heavy metal contaminated soils. A 60-day incubation experiment was carried out to investigate the effects of heavy metal release during the decomposition of Zn-rich (15.7 mg g^?1 dry weight) Arabidopsis halleri litter on C mineralization, microbial biomass C, biomass N, ATP, and adenylate energy charge (AEC). These effects were investigated in two soils with different Zn, Cu, and Pb levels, with and without EDTA addition to soil. The sole addition of Zn-rich A. halleri litter to the two soils did not increase the contents of NH₄NO₃ extractable Zn, only with the combined additions of EDTA and litter was there a considerable increase, being equivalent to three times the added amount in the low metal soil and to 50% in the high metal soil. Litter amendment increased the CO₂ evolved; being equivalent to 44% of the added C in the two soils, but EDTA addition had no significant effect on CO₂ evolution. Litter amendment resulted also in an 18% increase in microbial biomass C, 27% increase in ATP and 6% increase in AEC in the two soils, but EDTA had again no effect on these indices at both metal levels. In contrast, the sole addition of litter had no effect on microbial biomass N, but EDTA addition increased microbial biomass N on average by 49%. The application of EDTA for chelate-assisted phytoextraction should in the future consider the risk of groundwater pollution, which is intensified by resistance of EDTA to microbial decomposition.     

Effects of humic and fulvic acids on release of fixed potassium

Release of potassium fixed by expanding silicate clays is considered of practical importance in soil fertility. Humic and fulvic acids are expected to play a definite role in liberating this fixed K, because of their chelating power, but not much is known in this respect. The following investigation was conducted to study release of fixed K by montmorillonite and illite, using humic and fulvic acids isolated from the surface horizon of a Cecil soil (Typic Hapludult, Red Yellow Podzolic soil) as extractants. For comparison, extraction was also done with 1 N neutral NH₄ -acetate, a mixture of 0.05 N HCl and 0.025 N H₂SO₄ or H₂ O. Supporting analyses of clays were carried out with X-ray diffraction to establish changes, if any, in crystal structure due to fixation, release of K, or adsorption of humic compounds. The results indicated that humic and fulvic acids released some of the K fixed by montmorillonite or illite. In terms of percentage of the total K fixed, 9 to 28% were released by the various extractants. The percentages K released by humic and fulvic acids were similar from both montmorillonite and illite, but based on absolute values, humic and fulvic acids extracted less K (mg/100 g) from illite than montmorillonite. Although statistically significant at the 5% level of probability, the capacity of humic compounds to liberate fixed K was not different markedly from those of NH₄ -acetate and the double acid mixture. Differences in pH of humic solutions had no influence on extraction of fixed K. X-ray diffraction analysis yielded curves showing an increase in spacing from 10.4 Å for K-montmorillonite to 13.2 Å as a result of extraction with the double acid mixture.     

A method for measuring adenosine triphosphate in soil

A method was devised for the extraction and measurement of adenosine 5'-triphosphate (ATP) in soil that minimizes sorption of ATP on the soil colloids. Soil was ultrasonified for 1 min with a solution containing trichloracetic acid (0.5 m). disodium hydrogen orthophosphate (0.25 m) and paraquat dichloride (0.1 m). The ATP content of the filtered extract was determined without further treatment in a scintillation spectrometer by the firefly luciferin-luciferase system. Recovery of added ATP was greater using the extratant containing trichloracetic acid, orthophosphate and paraquat than with trichloracetic acid alone or with a sulphuric acid extradant. Recoveries of added ATP ranged from 45% to 84% in thirteen different soils; ATP contents from 0.64 to 9.03 μg g^?1 soil.     

A rapid method to determine cation exchange capacity and exchangeable bases in calcareous,gypsiferous, saline and sodic soils

Summary

A simple, single‐step extraction with LiEDTA for the estimation of CEC and exchangeable bases in soils has been developed. Multivalent cations are stripped from the soil adsorption sites by the strongly chelating agent EDTA, and are replaced by Li. In soils without CaCO₃ or water soluble salts, exchangeable divalent cations (Ca, Mg) are chelated by EDTA and exchangeable monovalent cations (Na, K) are replaced in a single extraction step using 0.25–2.5 g of soil and 10.0 ml of extractant.

In calcareous soils the CEC can be determined in the same way, but for the extraction of exchangeable Ca and Mg, another separate extraction is needed because dissolution of calcite by EDTA is unavoidable. This extraction is done with as much NaEDTA as needed to extract only exchangeable Ca and Mg in a 1:2 (m/V) soil/alkaline‐50% (V/V) aethanolic solution to minimize dissolution of calcite.

In gypsiferous soils gypsum is transformed into insoluble BaSO₄ and soluble CaEDTA by LiBaEDTA thus avoiding interference of Ca from dissolution of gypsum, which renders the traditional methods for determining CEC unsuitable for such soils. To determine exchangeable Ca and Mg, Na₄EDTA is used as for calcareous soils.

In saline/sodic soils replacement of Na by Li is incomplete but the Na/Li‐ratio at the complex after extraction is proportional to the molar Na/Li‐ratio in the extracts, so that the CEC and original exchangeable sodium (ESP) content can be calculated. Additional analysis of Cl and, if necessary, SO₄ in the extracts of saline soils can be used to correct for the effect of dissolution of the salts on the sum of exchangeable cations.

This new method is as convenient as the recently developed AgTU (silverthiourea), but is better suitable for calcareous and gypsiferous soils.     

化学萃取修复尾渣土壤的金属形态变化特征

The efficiency of EDTA, HNO3 and CaCl2 as extractants to remove Pb, Zn and Cu from tailing soils without varying soil pH was investigated with distributions of Pb, Zn and Cu being determined before and after extraction using the sequential extraction procedure of the optimized European Community Bureau of Reference （BCR）. Results indicated that EDTA and HNO3 were both effective extracting agents.The extractability of extractants for Pb and Zn was in the order EDTA 〉 HNO3 〉 CaCl2, while for Cu it was HNO3 〉 EDTA 〉 CaCl2. After EDTA extraction, the proportion of Pb, Zn and Cu in the four fractions varied greatly, which was related to the strong extraction and complexation ability. Before and after extraction with HNO3 and CaCl2, the percentages of Pb, Zn and Cu in the reducible, oxidizable and residual fractions changed little compared to the acid-extractable fraction. The lability of metal in the soil and the kinds of extractants were the factors controlling the effects of metal extraction.     

Sequential exhaustive extraction of a Mollisol soil, and characterizations of humic components, including humin, by solid and solution state NMR

A comprehensive sequential extraction procedure was applied to isolate soil organic components using aqueous solvents at different pH values, base plus urea (base‐urea), and finally dimethylsulfoxide (DMSO) plus concentrated H₂SO₄ (DMSO‐acid) for the humin‐enriched clay separates. The extracts from base‐urea and DMSO‐acid would be regarded as ‘humin’ in the classical definitions. The fractions isolated from aqueous base, base‐urea and DMSO‐acid were characterized by solid and solution state NMR spectroscopy. The base‐urea solvent system isolated ca. 10% (by mass) additional humic substances. The combined base‐urea and DMSO‐acid solvents isolated ca. 93% of total organic carbon from the humin‐enriched fine clay fraction (<2 μm). Characterization of the humic fractions by solid‐state NMR spectroscopy showed that oxidized char materials were concentrated in humic acids isolated at pH 7, and in the base‐urea extract. Lignin‐derived materials were in considerable abundance in the humic acids isolated at pH 12.6. Only very small amounts of char‐derived structures were contained in the fulvic acids and fulvic acids‐like material isolated from the base‐urea solvent. After extraction with base‐urea, the 0.5 m NaOH extract from the humin‐enriched clay was predominantly composed of aliphatic hydrocarbon groups, and with lesser amounts of aromatic carbon (probably including some char material), and carbohydrates and peptides. From the combination of solid and solution‐state NMR spectroscopy, it is clear that the major components of humin materials, from the DMSO‐acid solvent, after the exhaustive extraction sequence, were composed of microbial and plant derived components, mainly long‐chain aliphatic species (including fatty acids/ester, waxes, lipids and cuticular material), carbohydrate, peptides/proteins, lignin derivatives, lipoprotein and peptidoglycan (major structural components in bacteria cell walls). Black carbon or char materials were enriched in humic acids isolated at pH 7 and humic acids‐like material isolated in the base‐urea medium, indicating that urea can liberate char‐derived material hydrogen bonded or trapped within the humin matrix.     

Comparison of soil tests to determine micronutrients status in Argentina soils

Abstract

Soil extraction techniques to measure the status of available micronutrients for plants are important in the diagnosis of deficiency or toxicity. Mehlich 3 (M3), EDTA (pH=8.2), DTPA‐TEA, and Soltanpour and Schwab (SS) solutions were confronted for their ability to extract simultaneously copper (Cu), zinc (Zn), manganese (Mn), and iron (Fe). Argentinean soils from different taxonomic orders with widely varying properties were investigated. The values obtained showed that DTPA‐TEA and SS solutions extracted similar amounts of Zn, Fe, and Mn, while EDTA dissolved comparatively higher amounts of Fe and Mn. Mehlich 3 yielded the highest extractions for the four micronutrients. Soil pH not only affected the extraction of Mn by DTPA‐TEA, SS, and EDTA extractions, but also the extraction of Fe by EDTA. The organic carbon affected the determination of Fe and Zn in all cases. The correlations of the different tests for Cu, Zn, Mn, and Fe were significant. The results suggest that for the determination of the bioavailable status of micronutrients, any of the studied tests could be applied using the soil edaphic properties as factors to improve the correlations between them and standardize the methods.     

Extraction kinetics of copper,zinc, iron,and manganese from contaminated sediment using Disodium Ethylenediaminetetraacetate

One technique for cleansing heavy metal contaminated soils is to wash the excavated soil with an extraction solution of a chelating agent. The rate of extraction is an important parameter when considering the length of time needed for soil clean-up and the amount and concentration of wash solution required. The extraction kinetics of copper, zinc, iron and manganese from a contaminated sediment of the Clark Fork River in western Montana, U.S.A., with Disodiun Ethylenediaminetetraacetate (Na₂EDTA) as the extraction agent, were investigated. The results showed the extraction process consisted of rapid extraction in the first minutes followed by much slower extraction for the remainder of the experiment. The rate of extraction, particularly in the rapid phase, demonstrated clear pH dependence: the lower the pH, the faster the extraction rate. In the EDTA concentration range of 0.01 M to 0.05 M, the effect of the EDTA concentration on the extraction rate was not important compared with that of the solution pH. Extraction kinetics for different size particles were similar, although in the first few minutes, EDTA extracted more metals from clay and silt than sand. The two reaction, diffusion, and two-constant kinetic models were compared to experimental results. The two reaction model did not fit any of the data well, and only iron extraction could be described with a simple diffusion model. In general the extraction rates can be well described by the two-constant model, C=A t ^B, up to 600 minutes and under different conditions such as solution pH, EDTA concentration, and different sediment particle size.     

A critical assessment of methods for determining organic phosphorus in savanna soils

 Two newly introduced extraction techniques for determining total organic P (P₀) were compared with the standard high-temperature ignition method in selected savanna soils of Nigeria. The two extraction techniques were: (1) concentrated H₂SO₄ and dilute base sequential extraction (18 N H₂SO₄ and 0.5 N NaOH) and (2) basic EDTA method (0.25 M NaOH plus 0.05 M Na₂EDTA). The concentrated H₂SO₄ and dilute base method extracted significantly higher total P₀ than the high-temperature ignition method and the basic EDTA extraction. The high-temperature ignition and the basic EDTA extraction gave similar total P₀ values (mean=91 mg kg^–1 for ignition and 90 mg kg^–1 for basic EDTA). The precision of the methods, determined by coefficients of variation (CV, %) associated with each P₀ determination method in the soils, was better for the concentrated H₂SO₄ and dilute base extraction method (CV=13%) than the ignition method (CV=18%) and the basic EDTA method (CV=15%). The high C : P₀ ratios determined for the high-temperature ignition and basic EDTA extraction indicated that the two methods underestimated total P₀ in the soils. The concentrated H₂SO₄ and dilute base sequential extraction appears to be suitable for the rapid determination of P₀ in savanna soils because the method can be simplified to a single-step analysis. Received: 14 November 1997     

Extraction,purification and properties of soil hydrolases

Invertase, cellulase, phosphatases, protease and β-glucosidase were extracted from permanent pasture soil with 0.2 M phosphate buffer (pH 8) in the presence of 0.2 M EDTA. This extract was further treated with ammonium and salmine sulphates. Attempts were made to fractionate these enzyme activities by gel and anion-exchange chromatography. Specific activities were estimated in all fractions and some characteristics of the purified enzymes (optimum pH, temperature and substrate concentration, and K_m and V_max) were investigated. The results indicated that extracted enzyme activities occurred partly in soil as a carbohydrate-enzyme complex and partly as a humo-carbohydrate complex.     

Specific measurement of soil microbial ATP

A direct procedure to extract and determine microbial adenosine 5'-triphosphate (ATP) in soil has been worked out. The soil is homogenized in cold Tris-EDTA-NaN₃ (TEA) buffer. The ATP content of 100 μl of a 1/1000 suspension is directly determined in a photoncountcr after addition of a detergent (NRB^®) extractant and the firefly luciferin-luciferase system. The method was tested on a wide variety of organisms and soils and compared to several other methods to determine ATP in soils. The method gives ATP recoveries of a minimum of 80% upon addition of cultures to soils.Furthermore, it is rapid, applicable to all soils examined, and most of all strictly specific for microbial biomass.     

Influence of electrolytes on EDTA extraction of Pb from polluted soil

Because the economics of soil extraction processes depend on conservation and reuse of costly chelating agents, the ability of various electrolytes to modify EDTA extraction of Pb from a grossly-contaminated soil (Pb_T=21%) was investigated using batch equilibration experiments. In the absence of added electrolyte, a single 5-hr. extraction with 0.04 M EDTA (corresponding to 1∶1 Pb_T to EDTA ratio) released 65% of Pb_T over the pH 5 to 9 range. Under these conditions, Na⁺-, Li⁺-, and NH₄ClO₄ salts at 0.5 M increased Pb desorption to nearly 80%, probably from exchange displacement of soilbound Pb²⁺ and increased solubility of Pb-containing phases at higher ionic strength. Because Cl^? and ClO₄ ^? salts were equally effective, chlorocomplex formation was insignificant. Under slightly acidic conditions, Ca(ClO₄)₂ and Mg(ClO₄)₂ at 0.167 M caused roughly the same elevation in Pb recovery as 0.5M of the monovalent electrolytes. However, with progressively higher pH, Ca, and to a lesser extent Mg, suppressed Pb solubilization by competitive chelation of EDTA. Pb recovery by EDTA soil washing could be enhanced by addition of Ca salts at pH 4 to 6. Subsequent pH elevation in the presence of Ca would promote decomposition of Pb-EDTA complexes and separation of Pb as a hydroxide precipitate.