Sorption–desorption of phenolic acids as affected by soil properties

Phenolic acids have been implicated in the process of allelopathy and are, therefore, of interest in plant management as a basis for new herbicide structures. The potential bioavailability of phenolic acids is controlled by sorption–desorption processes in soil. Sorption–desorption of p-coumaric acid (4-hydroxycinnamic acid), ferulic acid (3-methoxy-4-hydroxycinnamic acid), veratric acid (3,4-dimethoxybenzoic acid), vanillic acid (3-methoxy-4-hydroxybenzoic acid), and p-hydroxybenzoic acid (4-hydroxybenzoic acid) was characterized on soils with varying physicochemical properties. The phenolic acids sorbed quickly (<8 h) and in high proportions to the amount applied (average 84% of applied was sorbed). Sorption was irreversible with the batch desorption method used (0.01 N CaCl₂ extraction). Pretreatment of soils to remove organic matter and free metal oxides from the soils decreased sorption, particularly in soils with free oxides removed. Statistical analysis suggested that sorption of p-coumaric and ferulic acids was correlated with soil clay content and veratric acid sorption was correlated with several soil factors. In contrast, no consistent relationship between soil characteristics and vanillic and p-hydroxybenzoic acid sorption was found. Based on the results of these experiments, i.e. the high reactivity of the phenolic acids, it is unlikely that these chemicals would be transported far from their point of origin, limiting their range of influence. It appears that, for phenolic acids to have allelopathic effects on plants, they would have to persist in the soil for long periods, resulting in a buildup of the chemical to high enough levels so that sufficient chemical would be in solution to cause the allelopathic effects, even though they may be strongly sorbed at lower concentrations.     

Extractant ph and the release of phenolic compounds from soils,plant roots and leaf litter

The influence of pH was examined, over the range from 6 to 14, on the amounts of p-hydroxybenzoic, vanillic, p-coumaric, ferulic and syringic acids, p-hydroxybenzaldehyde and vanillin, extracted from four soils and associated roots or leaf litter. Adjustment of pH was obtained by the addition of graded amounts of Ca(OH)₂ to water or by 2 m NaOH. The roots associated with three of the soils were from permanent pasture, perennial ryegrass and red clover, while the leaf litter associated with the fourth soil was from beech.The amounts of each phenolic compound extracted increased continuously with increasing pH, from a “threshold” value which varied between pH 7.5 and 10.5. The amounts extracted by water alone from the soil under permanent pasture, at pH 5.8, were equivalent to concentrations in the soil solution ranging from 1.4 μm for p-hydroxybenzoic acid to < 10 nm for ferulie acid. Amounts of up to 2000 times greater than these were extracted by 2 m NaOH. Similar effects of extractant pH were found with the other soils.Comparisons of the amounts of the phenolic compounds extracted from the soils, with the amounts extracted from the associated roots or leaf litter, suggested that substantial proportions of the soil phenolic compounds were either derived from organic residues more than 4 yr old or were the result of microbial synthesis.     

Acid pyrophosphate extraction of soil fulvic acids

A simple three step method is described for isolation of soil fulvic acids in high yield. The complexing agent H₂P₂O₇^2? (at pH 2) is used to release soil-bound fulvic acids. Extraction of humic acids is minimal. Selective separation of the protonated fulvic acids from the ionic extractant is achieved on a non-ionic polyacrylate resin (Amberlite XAD-7); after washing the resin, fulvic acids were retrieved in >98% yield by adjusting the pH to 6.5. Two problems associated with the classical alkali extraction method are avoided: possible alkaline oxidation of phenolic components, and their oxidation by Fe³⁺ under the acidic conditions employed to precipitate humic acids. The product typically has an ash weight of <0.6% after one XAD treatment. The method has been applied to three soils and one IHSS peat sample.     

Extractability of 2,4-D,Dicamba and MCPP from Soil

The adsorption of herbicides on soil colloids is a major factor determining their mobility, persistence, and activity in soils. Solvent extraction could be a viable option for removing sorbed contaminants in soils. This study evaluated the extractability of three herbicides: 2,4 dichlorophenoxy-acetic acid (2,4-D), 4-chloro-2-methylphenoxypropanoic acid (mecoprop acid or MCPP), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). Three solvents (water, methanol, and iso-propanol) and three methods of extraction (column, batch, and soxhlet) were compared for their efficiencies in removing the herbicides from three soils (loamy sand, silt loam, and silty clay). Both linear and non-linear Freundlich isotherms were used to predict sorption intensity of herbicides on soils subjected to various extraction methods and conditions. High K_dand K_fr, and low N values were obtained for all herbicides in silty clay soil by batch extraction. Methanol was the best solvent removing approximately 97% of all added herbicides from the loamy sand either by column or soxhlet extraction method. Isopropanol ranked second by removing over 90% of all herbicides by soxhelet extraction from all three soils. However, water was ineffective in removing herbicides from any of the soils using any of the three extracting procedures used in this study. In general, the extent of herbicide removal depended on soil type, herbicide concentration, extraction procedure, solvent type and amount, and extraction time.     

Interaction of the Mixture of Phenolic Acids with Modified Kaolinite under Batch and Dynamic Conditions

Phenolic acids play an important role in the formation of soil profiles, however their cooperative sorption by mineral phases under environmentally relevant concentrations is poorly studied. In the present work the sorption of an equimolar mixture of phenolic acids by kaolinite modified with amorphous aluminum hydroxide has been studied under both batch and continuous-flow conditions. It has been found that the sorption of gallic and protocatechuic acids containing OH groups in the ortho position exceeds the sorption of p-hydroxybenzoic and methoxylated acids (vanillic, syringic, and ferulic) by an order of magnitude. The study of sorption under dynamic (continuous-flow) conditions has shown the competition of acids for binding sites, while the active centers of kaolinite-Al(OH)_x are being occupied. The sorbed gallic acid displaces the other acids, which pass into solution in the following order: p-hydroxybenzoic acid > vanillic acid > syringic acid ? ferulic acid > protocatechuic acid. The revealed regularities indicate potentially important role of ortho-substituted hydroxybenzoic acids in the formation of soil organic matter, while p-hydroxybenzoic, vanillic, and syringic acids can be more important for the composition of soil solutions and natural waters.     

Fractionation of dissolved organic matter on the XAD resin

Analytical fractionation conditions on the resin XAD-7H of organic substances from gravitational soil moisture influence the proportion and the yield of the extracted fractions. Increasing the column capacity factor k’ is accompanied by a decreased yield of hydrophobic fraction and the change in its composition. The sorption column capacity factor of k’ = 30 is sufficient for the extraction of all hydrophobic components from a soil solution with a concentration of C_DOM of ～25 mg/L.     

Bound phenolic compounds in water extracts of soils,plant roots and leaf litter

Seven soils were examined for their contents of p-hydroxybenzoic, vanillic, p-coumaric and ferulic acids, p-hydroxybenzaldehyde and vanillin. Water-soluble forms, both “free” and “bound” of the phenolic compounds accounted for less than 0.7% of the total amount of each acid or aldehyde as determined by extraction of the soil with 2 M NaOH. In most instances, more than 50% of the water-soluble compounds were in the bound form, which was estimated after conversion to the free form by treatment of the water extract with NaOH. Water-soluble forms, both free and bound, of each compound also occurred in roots associated with six of the soils, and in beech litter associated with the seventh.     

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.     

Macronutrients and metals released from soils by solutions of naturally occurring phenols

Phenolic compounds produced by plants enter the soil by leaching and litter decomposition. The goal of this work is to determine the effect of phenolic compounds on solubility of plant macronutrients and metals in agroforestry systems. Soils from forest and pasture systems were repeatedly extracted with water (control) or phenolic solutions and then compared to a Mehlich 3 reference. The phenolics were aqueous solutions of tannic acid or β –1,2,3,4,6‐penta‐O‐galloyl‐D‐glucose (PGG) (hydrolyzable tannins), procyanidin (condensed tannin), or small phenolics catechin, gallic acid, or methyl gallate. The concentration of the macronutrients Ca, Mg, K, P, and S, and the metals Fe, Al, Mn, and Zn in the supernatants was determined by inductively‐coupled plasma spectroscopy. Cumulative extraction of macronutrients was generally similar to or less than the amount obtained by the Mehlich 3 extraction with the lowest recoveries obtained with the water control, PGG, and procyanidin. Metals tended to be somewhat more extractable from forest soil, especially with gallic acid, tannic acid or PGG treatments. Three mechanisms affected extraction of analytes by phenol‐containing solutions: (1) pH‐driven dissolution (Ca and Mg), (2) chelation of the metal (Al) by the polyphenol, or (3) reduction of the metal (Fe and Mn). Relatively low extraction of nutrients by some polyphenols is attributed to the tendency of some phenols to sorb to soil. This study demonstrates that tannins and related compounds change the solubility of macronutrients and metals in soils by a complex process that is not easily predictable from simple chemical properties of the phenolics.     

Environmental behaviors of phenolic acids dominated their rhizodeposition in boreal poplar plantation forest soils

Purpose

The deposition of phenolic acids in soils is of ecological and environmental importance for growth of trees and nutrient cycling of soil. The objective of this study was to investigate the relationship between environmental behaviors of phenolic acids and their rhizodeposition in forest soils. The results could provide more information regarding the ecological process of root exudates at the plant-soil interface.

Materials and methods

The amounts of four types of phenolic acids (i.e., p-hydroxybenzoic acid, benzoic acid, cinnamic acid, and vanillin acid) in the rhizosphere and bulk soils of two plantation forests were measured and compared using HPLC (Thermo Electron Corp., USA). The root exudates of poplar saplings under three nutrient conditions (normal (CK), nitrogen stress (low-N), and phosphorus stress (low-P)) were collected via adsorption resins (Amberlite XAD-16, USA). The phenolic acids in root exudates were then quantified by external standards. The adsorption and degradation dynamics of the phenolic acids in soils were monitored by exogenous application in the lab. Several models were employed to reveal the environmental behavior properties of phenolic acids after they entered into soils. Meanwhile, the enumeration of the culturable bacteria and fungi was conducted using soil dilution plate method to measure the abundance variation of the microbial community along with the degradation of the phenolic acids.

Results and discussion

More phenolic acids deposited in the rhizosphere soils than in the bulk soils of the poplar plantations. The difference was significant (p?<?0.05). Under normal nutrient conditions, poplar roots could secrete phenolic acids. However, in low-N and low-P conditions, poplar roots would significantly increase the secretion (p?<?0.05). Phenolic acids were quickly adsorbed by the soil within 24 h. These four phenolic acids showed varied adsorption capacities by soil, but they are generally more than 1500 μg g soil^?1. Phenolic acids were degraded over time with half-lives around 1.29–4.24 days. Among them, p-hydroxybenzoic acid showed the highest secretion capability and the longest persistence, indicating that p-hydroxybenzoic acid had higher deposition potential in soils. Environmental behaviors should be responsible for the rhizodepostion of phenolic acids in poplar plantations.

Conclusions

Roots could release a certain amount of phenolic acids into the rhizosphere soil, especially under nutrient stress. Phenolic acids could be easily adsorbed onto soil colloids. However, the degradation amount of phenolic acids was positively related with the abundance of the fungi community. Thus, the rhizodeposited phenolic acids were the trade-off between those produced by root exudation and those consumed by microbial degradation.

     

Effect of soil organic matter on peroxidase activity of wheat roots

Some soil organic matter fractions inhibited the peroxidase activity of wheat-root filtrates, but the effect was related to the enzyme hydrogen donor used. In the presence of o-dianisidine as the hydrogen donor, humic acid and the fractions obtained from it by water or acid refluxing inhibited the enzyme activity. The greatest effects were produced by those fractions which were insoluble after such treatments. The inhibitory effects were reversible and non-competitive, the Michaelis constant of the enzyme being only slightly affected, and independent of pH of the assay media. When guaiacol was used as the enzyme substrate the inhibitory effects of the humic acid and its fractions were variable and less marked.p-Coumaric and p-hydroxybenzoic acids inhibited peroxidase activity when o-dianisidine was used as substrate, but stimulated the enzyme in the presence of guaiacol. Polymaleic acid which is thought to have a similar structure to fulvic acid, inhibited peroxidase in the presence of both hydrogen donors. whereas fulvic acid was considerably less effective.     

Determination of phosphorus in calcareous soils by mehlich 3, mehlich 2, cal,and egner extractants

Abstract

Acid extractants were found to be a problem when determining phosphorus in some soils especially in soils with a higher content of carbonates. Carbonate content, soil pH or extractable calcium cannot exactly predict the behavior of a particular soil. On the other hand, measurement of pH after extraction reflects the ability of soil to change the properties of an extractant. The objective of this study was to find the relationships between pH changes during the extraction and the extraction of phosphorus for Mehlich 3, Mehlich 2, CAL, and Egner extractants. Twelve soil samples from various regions of the Czech Republic were taken for investigation. The pH/KCl of the samples was in the range 4.9–7.3. Calcium carbonate was added into the extracting bottles to the soil samples before extraction. The CaCO₃/soil ratio was from 0 to 0.5. The content of phosphorus and pH were measured in filtrates after extraction. The ratio P₁ =P_meas /P_O was calculated for all measurements, all additions of calcium carbonate and all soils. P_meas is the measured value of phosphorus after extraction with the addition of calcium carbonate and P_o is the value of phosphorus determined after the extraction without addition of calcium carbonate. The ratio P_r reflects the changes in the extractability of phosphorus and it is independent of the absolute content of phosphorus in soil. The relationships between P_r and pH after extraction for CAL, Mehlich 2, and Mehlich 3 extractants were found. No such relationship was found for Egner extractant. Correction equations (x=pH after extraction, y=P_r):     

Effect of pH of ammonium oxalate extracting solutions on prediction of plant available molybdenum in soil

Abstract

Molybdenum (Mo) is an essential element of plants and animals and is of concern from human nutrition and environmental standpoints. Rational applications to soil of Mo in fertilizers, sewage sludges, or other soil amendments requires information of the concentrations of Mo in soils and plants. Two greenhouse experiments were conducted at Lexington, Kentucky, using surface samples of 12 soils (11 soil types) derived from diverse parent materials in Kentucky with soil pH ranging from 5.18 to 7.46. Molybdenum (Na₂MoO₄.2H₂O) was added at rates equivalent to 0, 0.3, and 0.6 mg Mo kg^‐1 soil. Tobacco (Nicotiana tabacum L., cv. Ky14) and soybean [Glycine max. (L.) Merrill cv. McCall] were grown to provide plant Mo data for Mo soil test correlations and comparisons. The primary purpose of these investigations was to determine the effect of pH of NH₄‐oxalate extractant solution on the relationship of soil Mo and Mo uptake by tobacco and soybeans, and to evaluate the automated KI‐H₂O₂ procedure for use in determining Mo in soil extracting solutions. The mean dry weight and Mo concentration of tobacco and soybean were increased by applications of Mo fertilizer to soil in the greenhouse. Dry matter of tobacco was increased 11 to 25% and concentration of Mo from 40 to 82% by each increment of added Mo fertilizer. The results of this study suggest that many soils in Kentucky are not meeting the requirements for Mo sufficiency for tobacco and soybean plants. The average amount of soil Mo extracted by NH₄‐oxalate decreased with increasing pH of extractant. Regression estimates for the relationship of Mo uptake by tobacco or soybean and extractable soil Mo show that the slope and the coefficients of determination increased with pH of NH₄‐oxalate solution from pH 3.3 to 6.0 and then decreased again at pH 6.4. The greatest amounts of variation in Mo uptake by plants (67% and 20%, respectively, for tobacco and soybean) were accounted for by the soil Mo data at pH 6.0. Soil Mo values for the NH₄‐oxalate extractant (pH 6.0) were related to values for anion exchange resin extractant (r² = 0.61**), but not soil pH. However, values for anion exchange resin were more closely related to Mo uptake by tobacco (r² = 0.86**) and soybean (r² = 0.60**) than were values for NH₄‐oxalate (r² = 0.65** and r² = 0.27**, respectively). Results of this study indicate that the automated KI‐H₂O₂ method used previously in analysis for plant Mo can be used to analyze Mo in soil extracts. Other instrumentation such as GFAAS and ICP may be effective in the analysis of extracts obtained by the NH₄‐oxalate (pH 6.0) or by anion exchange resin procedures when the Mo concentration of extracts falls within the detection limits of the instrument.     

Tannic acid reduces recovery of water-soluble carbon and nitrogen from soil and affects the composition of Bradford-reactive soil protein

Tannins are plant-derived polyphenolic compounds that precipitate proteins, bind to metals and complex with other compounds. Solutions of tannic acid, or other phenolic compounds, were added to soil samples to determine if they would affect recovery of soluble soil carbon (WSC) or –nitrogen (WSN) or influence the extraction and composition of Bradford-reactive soil protein (BRSP), associated with glomalin. Tannic acid-C added with water was not completely recovered from samples and the amount of total net WSC and WSN recovered was reduced, suggesting formation of insoluble complexes. By comparison, non-tannin phenolics like gallic acid, or methyl gallate, had little effect on extraction of WSC or WSN while a simple gallotannin derived from tannic acid, 1,2,3,4,6-penta-O-galloyl-d-glucose (PGG), inhibited extraction most. The C and N concentrations in BRSP increased when soil samples were treated with tannic acid or PGG before extraction, a procedure that includes autoclaving. Increases were greatest in the 10–20 cm compared to 0–5 cm depth. Accompanying these were declines in the ratio of absorbance at 465 and 665 nm (E4/E6 ratio) of BRSP extracts suggesting formation of larger or heavier molecules. In contrast, C and N composition in lyophilized BRSP was unaffected or even slightly reduced when tannic acid or PGG were added to the BRSP extract solution after the extraction process. We conclude that some tannins can reduce the solubility of labile soil C and N, at least temporarily and given unpredictability of response associated with phenolic substances, the Bradford assay should not be relied on to quantify pools or composition of soil proteins like glomalin.     

Quantifying microbial biomass phosphorus in acid soils

 This study aimed to validate the fumigation-extraction method for measuring microbial biomass P in acid soils. Extractions with the Olsen (0.5 M NaHCO₃, pH 8.5) and Bray-1 (0.03 M NH₄F–0.025 M HCl) extractants at two soil:solution ratios (1 : 20 and 1 : 4, w/v) were compared using eight acid soils (pH 3.6–5.9). The data indicated that the flushes (increases following CHCl₃-fumigation) of total P (P_t) and inorganic P (P_i) determined by Olsen extraction provided little useful information for estimating the amount of microbial biomass P in the soils. Using the Bray-1 extractant at a soil:solution ratio of 1 : 4, and analysing P_i instead of P_t, improves the reproducibility (statistical significance and CV) of the P flush in these soils. In all the approaches studied, the P_i flush determined using the Bray-1 extractant at 1 : 4 provided the best estimate of soil microbial biomass P. Furthermore, the recovery of cultured bacterial and fungal biomass P added to the soils and extracted using the Bray-1 extractant at 1 : 4 was relatively constant (24.1–36.7% and 15.7–25.7%, respectively) with only one exception, and showed no relationship with soil pH, indicating that it behaved differently from added P_i (recovery decreased from 86% at pH 4.6 to 13% at pH 3.6). Thus, correcting for the incomplete recovery of biomass P using added P_i is inappropriate for acid soils. Although microbial biomass P in soil is generally estimated using the P_i flush and a conversion factor (k _P) of 0.4, more reliable estimates require that k _P values are best determined independently for each soil. Received: 3 February 2000     

Degradation of certain aromatic compounds by rhizobia

The ability of 22 strains of Rhizobium to degrade catechol, protocatechuic acid, p-hydroxybenzoic acid and salicylic acid all at 1 mm concentration was examined. In the presence of 4.8 mm Na-glutamate, all rhizobia tested degraded catechol (99–100%), p-hvdroxybenzoic acid (79–99%), protocatechuic acid (81 97%) and salicylic acid (20–83%).The concentration of Na glutamate in the medium affected the degradation of the phenolic compounds at 1 mm concentration. Increased glutamate favoured degradation of p-hydroxybenzoic and salicylic acids but had little effect on catechol. Degradation of protocatechuic acid was inhibited by increased glutamate concentration.Rhizobium phaseoli 405 grown with 8.0 mM Na-glutamate, directly cleaved catechol and protocatechuic acid. p-Hydroxybenzoic acid was converted to protocatechuic acid before ring cleavage. Salicylic acid was converted to gentisic acid before further oxidation. O₂ uptake experiments showed that R. phaseoli 405 grown with p-hydroxybenzoic acid was adapted to this compound and protocatechuic acid. A lag of 30 min was required for catechol and salicylic acid.     

Efficiency of methanol:Water solutions for metribuzin extraction from selected soils

Abstract

Extraction efficiency for metribuzin [4‐amino‐6‐(1,1‐dimethyl‐ethy 1)‐3‐(methylthio)‐l,2,4‐triazin‐5(4H)‐one] from soil was evaluated using four solutions of methanol:water at ratios of 1:1, 7:3, 4:1, and 1:0 v/v. Two concentrations of metribuzin (O.216 and 2.44 ug/ g soil, unlabeled and ^I4C‐metribuzin, respectively) were added to surface and subsurface samples of a Dundee silt loam soil. Although the differences in extraction efficiencies were slight due to differences in methanol:water ratios, the metribuzin recovery varied with soil depth. Less metribuzin was recovered from surface soil extracted with 1:1 and 1:0 methanol:water solutions when compared to 7:3 and 4:1 solutions (80 and 81% vs 89 and 88% recoveries, respectively). About equal quantities, 81 to 85%, were recovered from the subsurface soil. Three extraction shaking times (0.5 h followed by another 0.5 h; 4 h:4 h; and 24 h:24 h) were also evaluated using the 4:1 extractant. No recovery differences were observed between the 0.5 h and 4 h shaking times. However, significantly higher recoveries occurred in both the surface and subsurface soil with 24 h shaking. The efficiency of this method was also determined on Eustis loamy sand, Sharkey clay, and Dorovan muck soils of diverse physical and chemical properties. The 4:1 extracting solution consistently yielded among the highest metribuzin recovery (74 to 97%) from the four soil types, and two soil depths of a Dundee soil.     

Development of a New Soil Extractant for Simultaneous Phosphorus,Ammonium, and Nitrate Analysis

Abstract

A new soil extractant (H³A) with the ability to extract NH₄, NO₃, and P from soil was developed and tested against 32 soils, which varied greatly in clay content, organic carbon (C), and soil pH. The extractant (H³A) eliminates the need for separate phosphorus (P) extractants for acid and calcareous soils and maintains the extract pH, on average, within one unit of the soil pH. The extractant is composed of organic root exudates, lithium citrate, and two synthetic chelators (DTPA, EDTA). The new soil extractant was tested against Mehlich 3, Olsen, and water for extractable P, and 1 M KCl and water‐extractable NH₄ and NO₂/NO₃. The pH of the extractant after adding soil, shaking, and filtration was measured for each soil sample (5 extractants×2 reps×32 soils=320 samples) and was shown to be highly influential on extractable P but has no effect on extractable NH₄ or NO₂/NO₃. H³A was highly correlated with soil‐extractable inorganic N (NH₄, NO₂/NO₃) from both water (r=0.98) and 1 M KCl (r=0.97), as well as being significantly correlated with water (r=0.71), Mehlich 3 (r=0.83), and Olsen (r=0.84) for extractable P.     

Extracting soil microarthropods with olive oil: A novel mechanical extraction method for mesofauna from sandy soils

Many soil ecologists still encounter practical difficulties when extracting microarthropods from the soil. Methods using a humidity gradient, established by the use of a heat source, for collecting soil animals appeared not sufficiently efficient in case of sandy soils. For such type of soils, flotation techniques proved more suitable. The use of toxic or aggressive flotation fluids like kerosene, dibromo ethane, carbon tetrachloride and heptane, however, makes these methods less favourable. To circumvent this problem, a novel technique has been developed based on the flotation principle but using olive oil. The method uses a pumping system that injects olive oil at the base of a Perspex column in which the soil sample is suspended in water using a propeller. In this way, intensive contact between oil droplets and the organisms is established, increasing extraction efficiency. After stopping the propeller, animals can easily be collected from the oil floating to the water surface. Adding coloured mites to soil samples was used to determine extraction efficiency of the method. Average (±SD) recoveries of 82.7 ± 7.4% (n = 34) and 89.7 ± 10.0% (n = 10), respectively were obtained when extracting storage mites and predatory mites from a sandy soil.     

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.