Pre-lysis washing improves DNA extraction from a forest soil

A pre-lysis buffer washing procedure was introduced to DNA extraction from a forest soil with high organic matter and iron oxide contents. Sodium phosphate of 0.1 M (pH 7.5) was used as a buffer to wash soil samples when subsequent lysis buffer was phosphate, and 20 mM EDTA (pH 7.5) was used when subsequent lysis buffer included EDTA. Initial experiments were not successful because the DNA extracts could not be amplified by polymerase chain reaction (PCR). The consideration of introducing a pre-lysis washing procedure was based on the idea that the washing should promote soil dispersion and homogeneity, decrease DNA adsorption by soil components (e.g. iron oxides), and remove covalent cations and those easily-dissolving organic compounds from the soil samples. Results revealed that humic substance content decreased by 31%, but DNA yield increased by 24% in the DNA extracts of the pre-lysis washing procedures, compared to the non-washing procedures. DNA extracted by the pre-washing procedure needed less purification for subsequent 18S and 16S rDNA PCR amplifications. It was recommended that the pre-lysis buffer washing should be used for DNA extraction from those difficult environmental samples, such as the forest soil with high contents of organic matter and iron oxides.     

Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants

A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed.     

Partial characterization of peroxidase and polyphenol oxidase activities in blackberry fruits

A partial characterization of peroxidase (POD) and polyphenol oxidase (PPO) activities in blackberry fruits is described. Two cultivars of blackberry (Wild and Thornless) were analyzed for POD and PPO activities. Stable and highly active POD and PPO extracts were obtained using insoluble poly(vinylpyrrolidone) and Triton X-100 in 0.05 M sodium phosphate, pH 7.5, buffer. Blackberry POD and PPO activities have a pH optimum of 6.5, in a reaction mixture of 0.2 M sodium phosphate. Optimal POD activity was found with 3% o-dianisidine. Maximum PPO activity was found with catechol (catecholase activity) followed by 4-methylcatechol. Polyacrylamide gel electrophoresis of blackberry extracts under non-denaturing conditions resolved in various bands. In the POD extracts of Wild fruits, there was only one band with a mobility of 0.12. In the Thornless POD extracts there were three well-resolved bands, with R(f) values of 0.63, 0.36, and 0.09. Both the Wild and Thornless blackberry cultivars produced a single band of PPO, with R(f) values of 0.1 for Wild and 0.06 for Thornless.     

Loss of low molecular weight dissolved organic carbon (DOC) and nitrogen (DON) in H2O and 0.5 M K2SO4 soil extracts

Soil extracts are routinely used to quantify dissolved organic nutrient concentrations in soil. Here we studied the loss and transformation of low molecular weight (LMW) components of DOC (¹⁴C-glucose, 1 and 100 μM) and DON (¹⁴C-amino acid mixture, 1 and 100 μM) during extraction of soil (0-6 h) with either distilled water or 0.5 M K₂SO₄. The extractions were performed at 20 °C, at 4 °C, or in the presence of an inhibitor of microbial activity (HgCl₂ and Na-azide). We showed that both glucose and amino acids became progressively lost from solution with increasing shaking time. The greatest loss was observed in H₂O extracts at 1 μM for both substances (>90% loss after 15 min). Lower temperature (4 °C) and presence of K₂SO₄ both resulted in reduced loss rates. The presence of microbial inhibitors effectively eliminated the loss of glucose and amino acids. We conclude that microbial transformation of LMW-DOC and DON during H₂O or K₂SO₄ extraction of soil may affect the estimation of their concentrations in soil. This finding has significant implications for methods that rely on chemical extractions to estimate LMW-C components of DOC and DON.     

Partial characterization of polyphenol oxidase activity in raspberry fruits.

A partial characterization of polyphenol oxidase (PPO) activity in raspberry fruits is described. Two early cultivars harvested in May/June (Heritage and Autumm Bliss) and two late cultivars harvested in October-November (Ceva and Rubi) were analyzed for PPO activity. Stable and highly active PPO extracts were obtained using insoluble poly(vinylpyrrolidone) (PVP) and Triton X-100 in sodium phosphate, pH 7.0 buffer. Polyacrylamide gel electrophoresis of raspberry extracts under nondenaturing conditions resolved in one band (R(f)()(1) = 0.25). Raspberry PPO activity has pH optima of 8.0 and 5.5, both with catechol (0.1 M). Maximum activity was with D-catechin (catecholase activity), followed by p-coumaric acid (cresolase activity). Heritage raspberry also showed PPO activity toward 4-methylcatechol. Ceva and Autumm Bliss raspberries showed the higher PPO activity using catechol as substrate.     

Effect of Extraction Conditions on Yield,Composition, and Viscosity Stability of Barley β-Glucan Gum

Cereal β-glucan can function as a thickener, but endogenous β-glucanase enzymes of the grain cleave β-glucan, reducing its viscosity. Although different extraction techniques have been developed, the viscosity stability of β-glucan gum has not been reported. The objective of this study was to investigate the effect of extraction treatments on the yield, purity, and viscosity stability of barley β-glucan (BBG) gum. A regular barley cultivar, Condor, and a waxy cultivar blend were extracted at pH 7–10 and 55°C for 0.5 hr. Four extraction conditions were evaluated: 1) extraction at high pH with no additional heat treatment; 2) boiling of extract; 3) prior refluxing of flour with 70% ethanol; and 4) treatment of extract with thermostable α-amylase for purification. Viscosity of extracts was monitored for ≥24 hr at 25°C. The highest β-glucan purities were achieved with a boiled Condor extract at pH 7 (81.3% db, 4.1% yield) and with refluxed waxy barley extracted at pH 8 and treated with α-amylase and (79.3% db, 5.1% yield). Gums extracted without subsequent heat treatment or prior refluxing of flour had high protein (>17%) and starch (>24%) impurities, respectively. The viscosity of gums obtained without heating was unstable. Prior refluxing treatment was not sufficient to stabilize final extracts. Boiling extracts resulted in stable but low viscosity. Reflux followed by purification treatment produced the highest stable viscosity for 0.5% solutions of both Condor (64 mPa sec^-1, pH 7) and waxy (48.8 mPa sec^-1, pH 8) extracts. Stable BBG gum with high viscosity can be obtained using thermal treatments in combination with high pH. The potential use of such gums as thickeners in food systems needs to be assessed.     

Interaction between chloroplast pigments and lipoxygenase enzymatic extract of olives.

Lipoxygenase activity in olive fruits increases considerably when the enzyme is extracted using extraction buffer with ethylenediaminetetraacetate, Triton X-100, dithiothreitol, sodium meta-bisulfite, and hydrated polyvinylpolypyrrolidone. In the absence of these compounds the activity measured of the crude extract is almost negligible, and further effects of the enzymatic reaction, such as bleaching activity on chloroplast pigments, do not appear. Moreover, when the oxidizing level of the medium is increased by the addition of linoleic acid or soybean lipoxygenase with linoleic acid, the crude enzymatic extracts of olives reduce the destructive capacity of soybean lipoxygenase on chlorophylls to one-sixth. These results suggest a double pigment protection against lipoxygenase in the olive crude extract, one inhibiting the enzyme and the other interrupting the chain of oxidative reactions beginning with hydroperoxide formation.     

Liquid extraction of low molecular mass organic acids and hydroxamate siderophores from boreal forest soil

Low molecular mass organic acids (LMMOAs) and hydroxamate siderophores (HS) are molecules secreted by microbes and have previously been found in soil solution and in cultures. Mycorrhizal fungi are suggested to be involved in the nutrient uptake processes of trees and weathering of minerals. In this study soil samples taken from the O and E horizons of a podzol were extracted with 10 mM potassium phosphate buffer at pH 7.2. Variable parameters included addition of methanol to the extraction buffer and the use of ultrasonication or rotary shaking during extraction. LMMOAs and HS content of the soil extracts were determined. Analysis of soil extracts were carried out by liquid chromatography mass spectrometry (LC–MS) and the extraction results compared to results for soil solution samples obtained by centrifugation of the soils sampled. The extraction yields were significantly increased by addition of methanol to the extraction buffer, especially for the O horizon samples. Rotary shaking of the samples for 90 min gave slightly higher yields than ultrasonication for 15 min but the reduction in extraction time makes ultrasonication an attractive option. Of the HSs determined, ferricrocin was found in all samples. Optimal extraction conditions showed citric acid and isocitric acid to be the most abundant organic acids in the O and E horizons, respectively.     

Liquid extraction of low molecular mass organic acids and hydroxamate siderophores from boreal forest soil

Low molecular mass organic acids (LMMOAs) and hydroxamate siderophores (HS) are molecules secreted by microbes and have previously been found in soil solution and in cultures. Mycorrhizal fungi are suggested to be involved in the nutrient uptake processes of trees and weathering of minerals. In this study soil samples taken from the O and E horizons of a podzol were extracted with 10 mM potassium phosphate buffer at pH 7.2. Variable parameters included addition of methanol to the extraction buffer and the use of ultrasonication or rotary shaking during extraction. LMMOAs and HS content of the soil extracts were determined. Analysis of soil extracts were carried out by liquid chromatography mass spectrometry (LC–MS) and the extraction results compared to results for soil solution samples obtained by centrifugation of the soils sampled. The extraction yields were significantly increased by addition of methanol to the extraction buffer, especially for the O horizon samples. Rotary shaking of the samples for 90 min gave slightly higher yields than ultrasonication for 15 min but the reduction in extraction time makes ultrasonication an attractive option. Of the HSs determined, ferricrocin was found in all samples. Optimal extraction conditions showed citric acid and isocitric acid to be the most abundant organic acids in the O and E horizons, respectively.     

Development of an enzyme-linked immunosorbent assay for seven sulfonamide residues and investigation of matrix effects from different food samples

Direct competitive enzyme-linked immunosorbent assays (ELISA) were developed to detect a broad range of sulfonamides in various matrices. Screening for this class of antibiotics in pig muscle, chicken muscle, fish, and egg extracts was accomplished by simple, rapid extraction methods carried out with only phosphate-buffered saline (PBS) buffer. Twenty milliliters of extract solution was added to 4 g of sample to extract the sulfonamide residues, and sample extracts diluted with assay buffer were directly analyzed by ELISA; matrix effects could be avoided with 1:5 dilution of pig muscle, chicken muscle, and egg extracts with PBS and 1:5 dilution of fish extract with 1% bovine serum albumin (BSA)-PBS. For liver sample, the extraction method was a little more complicated; 2 g of sample was added to 20 mL of ethanol, mixed, and then centrifuged. The solvent of 10 mL of the upper liquid was removed, and the residues were dissolved in 10 mL of PBS and then filtered; the filtrate was diluted two-fold with 0.5% BSA-PBS for ELISA. These common methods were able to detect seven sulfonamide residues such as sulfisozole, sulfathiazole, sufameter, sulfamethoxypyridazine, sulfapyridine, sulfamethizole, and sulfachlorpyridazine in pig muscle, liver, chicken muscle, egg, and fish. The assay's detection limits for these compounds were less than 100 microg kg-1. Various extraction methods were tested, and the average recovery (n=3) of 100 microg kg-1 for the matrices was found to range from 77.3 to 123.7%.     

Functional properties of guava seed glutelins

Five guava seed glutelin extracts were obtained with different buffer solutions: Na(2)B(4)O(7) alone (Glut.Bo) or containing SDS (Glut.BoSDS), 2-mercaptoethanol (Glut.Bo2-ME), or a combination of both (Glut.BoSDS2-ME) and NaOH (Glut.Na). All borate buffer solutions were at pH 10. The higher yield of glutelins corresponded to the Glut.BoSDS extract (81.9% dry basis) and the lower to Glut.Bo (6.8%). The functional properties of the five guava seed glutelin extracts were determined. Glut.BoSDS, Glut.BoSDS2-ME, and Glut.Na showed high values for several properties, including surface hydrophobicity (7.7, 10.8, and 0.6, respectively), solubility at pH 10 (91.1, 77.9, and 96.7, respectively), water-holding capacity at pH 3.6 (1.7, 2.5, and 2.8, respectively), emulsifying activity index (pH 10; 503.5, 238.2, and 838.0, respectively), and foaming properties (pH 10; V(0) = 0.14, 0.25, and 0.19, respectively; V(max) = 6.1, 5.59, and 4.51, respectively; t(1/2) = 266, 255.3, and 94 s, respectively). These results suggest that the denaturing reagent (SDS or NaOH) during extraction conferred on the proteins a structure that facilitated the development of their functional properties.     

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.     

Cloud point extraction preconcentration prior to high-performance liquid chromatography coupled with cold vapor generation atomic fluorescence spectrometry for speciation analysis of mercury in fish samples

A cloud point extraction methodology was developed for simultaneous preconcentration of Hg(II), methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury (PhHg) prior to reversed-phase high-performance liquid chromatography (HPLC) on-line coupled with cold vapor atomic fluorescence spectrometry for speciation analysis of mercury in fish. The four mercury species were taken into complexes with ammonium pyrrolidine dithiocarbamate (APDC) in aqueous nonionic surfactant Triton X-114 medium and concentrated in the surfactant-rich phase by bringing the solution to the temperature of 40 degrees C. Baseline separation of the enriched complexes was achieved on an RP-C(18) column with a mixture of methanol, acetonitrile, and water (65:15:20, v/v) containing 200 mmol L(-1) HAc (pH 3.5) as the mobile phase. An on-line postcolumn oxidation of the effluent from HPLC, in the presence of K2S2O8 in HCl, was applied in the system followed by an optimal cold vapor generation of mercury species. The variables affecting the complexation and extraction steps were examined. The preconcentration of 10 mL of solution with 0.08% w/v Triton X-114 and 0.04% w/v APDC at pH 3.5 gave enrichment factors of 29, 43, 80, and 98 for MeHg, EtHg, PhHg, and Hg(II), respectively. Low detection limits (S/N = 3) were obtained, ranging from 2 to 9 ng L(-1) (as Hg) for all species. The developed method was successfully applied to the speciation of mercury in real fish samples.     

Characterization of humus-protease complexes extracted from soil

Pyrophosphate (140 mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: A_I with nominal molecular weight (nmw) > 100 kD, A_II with nmw between 10 kD and 100 kD and R with nmw < 10 kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-l-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-l-phenylalanyl l-leucine (ZPL), specific for carboxypeptidases; and casein, essentially a non-specific substrate. The derivative fractions were also analysed for their amino acid N and humic (HA) and fulvic (FA) acid contents. The organic matter of extracts and derivative fractions obtained from the pasture soil was analysed by isoelectric focusing (IEF) and that of fractions analysed by pyrolysis gas chromatography (Py-GC). Activities of the extract were monitored for their thermal stability and those of the extract and derivative fractions for their optimal pH.Due to the mechanical disintegrating action of sodium pyrophosphate over the humic substances during the fractionation process the amount of total organic C and FA in the fractions was ranked as R > A_II > A_I. The lowest amino acid N/organic C was found in the R fraction, whereas A_II fraction was rich in humic acids, carbohydrates and amino acid N and A_I fraction showed the lowest carbohydrate content. At least 70% of the total BAA- and ZPL-hydrolysing activity was associated to particles with nmw higher than 10 kD and at least 30% of these activities were present in particles with nmw higher 100 kD. Casein-hydrolysing activity was quite evenly distributed among the three fractions (A_I, A_II and R). The extracted protease-organic complexes were resistant to thermal denaturation and some of them showed optimal activity at pH values higher than 10 as a result of the polyanionic characteristics of the humic material surrounding enzyme molecules and of the presence of alkaline protease. Comparison of data obtained in Py-GC analyses and in protease activity suggests that BAA-hydrolysing activity was associated to a highly condensed humic matter and ZPL-hydrolysing activity to less resistant humic substances, while at least some of the extracted casein-hydrolysing activity was present as glyco-proteins not associated to humus. BAA-hydrolysing activity was probably inhibited by fresh organic matter of carbohydrate origin whereas lignin derived organic matter probably inhibited ZPL- and casein-hydrolysing activity.     

Supercritical carbon dioxide extraction of oil and squalene from amaranthus grain

Supercritical carbon dioxide (SC CO(2)) was used for the extraction of oil and squalene from Amaranthus grain. Very small amounts of oil could be extracted by SC CO(2) from undisrupted grains, although SC CO(2) possesses higher diffusivity. Grinding increased the extraction rate and oil yield, and smaller particle size gave higher extraction rate. The oil yield and initial extraction rate increased linearly with the increasing SC CO(2) flow rate from 1 to 2 L/min. Increasing the flow rate of SC CO(2) above 2 L/min resulted in only a slight increase of oil yield and extraction rate. In the pressure range of 150-250 bar, extraction decreased with increasing temperature at a constant pressure, whereas at a pressure of 300 bar, the extraction yield increased with increasing temperature. Possible reasons for this are discussed. Effects of temperature and pressure on squalene yield were different from those on oil yield. A good oil yield (4.77 g of oil/100 g of grain) was obtained at 40 degrees C and 250 bar. The highest squalene yield (0.31 g of squalene/100 g of grain) and concentration (15.3% in extract) were obtained at 50 degrees C and 200 bar, although the oil yield under this condition was low (2.07 g of oil/100 g of grain). The moisture content within 0-10% had little influence on yields of oil and squalene at 40 degrees C and 250 bar. Finally, the oil yield and the squalene concentration in the extracts by SC CO(2) were compared to those by solvent extraction.     

Investigation and optimization of the factors influencing sorghum protein extraction

To optimize the extraction of sorghum proteins, several variables were examined: sample-to-solvent ratio, detergent type and concentration, reducing agent type and concentration, extraction time, and buffer pH and concentration. Samples were quantified and characterized by RP-HPLC, FZCE, and nitrogen analysis. These studies revealed that pH, detergent type, reducing agent type, and sample-to-solvent ratio all had significant effects on the levels of protein extracted. Increasing SDS concentration (2%) and solvent-to-flour ratio (20:1) with multiple 5 min extracts reduced extraction time by 35-80% while still extracting the same levels of total protein relative to the control methodology. Reproducibility using the multiple extractions was found to be excellent with relative standard deviations of <2% for consecutive extractions.     

Rhamnolipid Foam Enhanced Remediation of Cadmium and Nickel Contaminated Soil

Column experiments were conducted to evaluate the feasibility of using a rhamnolipid foam to remove heavy metals (Cd and Ni) from a sandy soil contaminated with Cd (1706 ppm) and Ni (2010 ppm). Best results were obtained from the foam generated by a 0.5% rhamnolipid solution with an initial pH value of 10.0 after flushing with 20-pore-volume of solution. These conditions removed 73.2% of the Cd and 68.1% of the Ni. Removal efficiencies by foam generated by a chemical surfactant, Triton X-100, were investigated as a comparison. It removed 65.5% of the Cd and 57.3% of the Ni under the same conditions. After a 20-pore-volume liquid solution flushing, 0.5% rhamnolipid (initial pH 10.0) without foam generation removed 61.7% of the Cd and 51.0% of the Ni, whereas 0.5% Triton X-100 (initial pH 10.0) removed 52.8% of the Cd and 45.2% of the Ni. Distilled water with adjusted pH only was also used to flush through the contaminated soil column as a control. It removed 17.8% of the Cd and 18.7% of the Ni. This study shows that rhamnolipid foam technology can be an effective means for the remediation of cadmium and nickel contaminated soil.     

Activation of polyphenol oxidase in extracts of bran from several wheat (Triticum aestivum) cultivars using organic solvents, detergents, and chaotropes

Polyphenol oxidase (PPO), known to induce browning in wheat-based products, has been shown to be activatable in wheat (Triticum aestivum) bran extracts by chemical compounds. The activity in the extracts could be increased to varying degrees with acetone, methanol, ethanol, 2-propanol, and n-butanol as additives in the extraction buffer. The most potent alcoholic activator was n-butanol (about a 3-fold increase), followed by 2-propanol and ethanol, whereas methanol had the least effect. Ionic detergents in the extraction buffer were also good activators, with sodium dodecyl sulfate (SDS) being more potent (3-fold increase) than cetyltrimethylammonium bromide (CTAB) that had only half as much effect, whereas the nonionic detergent, Triton X-114, was ineffective. The chaotropes, urea and guanidine x HCl (GND), were the most potent activators of all, increasing the activity over 4-fold. Of the two chaotropes, GND was more effective at lower concentrations (<6 M) than urea. However, the enzyme activity lessened at a higher concentration of GND (6 M), while there was a further increase in the activity with 6 M urea treatment. The activity lessened with higher concentration of GND presumably as a result of extensive denaturation of the enzyme, as GND is known to be a more potent denaturant than urea. It is hypothesized that in wheat PPO exists in an inactive form which may be activated by the presence of activators, hitherto unknown, similar in effect to that elicited by the chemical denaturants in this study.     

Determination of the activity of acidic phytate-degrading enzymes in cereal seeds

Five different methods were compared to elucidate the total activity of the acidic phytate-degrading enzymes present in the seeds of rye, wheat, and barley. Phytate-degrading activity was studied at pH 5.0 by quantifying the liberated phosphate. Rye showed the highest acid phytate-degrading activity among the cereals studied. Using an aqueous extract, only 30-50% of the activity was found (rye, 3443 mU g(-1) of grain; wheat, 1026 mU g(-1) of grain; barley, 1032 mU g(-1) of grain) compared to that found by the direct incubation of the dry-milled cereal grains in a buffered phytate-containing solution (rye, 6752 mU g(-1) of grain; wheat, 2931 mU g(-1) of grain; barley, 2093 mU g(-1) of grain). Extending the extraction time resulted in an increase in extractable phytate-degrading activity by, at maximum, 10-15%. Extraction of phytate-degrading activity is strongly enhanced in the presence of Triton X-100 and the protease inhibitor phenylmethylsulfonyl fluoride (rye, 6536 mU g(-1) of grain; wheat, 2873 mU g(-1) of grain; barley, 2023 mU g(-1) of grain), suggesting at least a partial association with membrane structures and a degradation by proteolytic activity during extraction. In addition, it was shown that determining phytate-degrading activity by quantification of the liberated inorganic phosphate is more robust and precise than determining phytate-degrading activity by quantification of the residual phytate.     

Long-term effects of metal-containing farmyard manure and sewage sludge on soil organic matter in a fluvisol

Our aim was to establish the long-term effects of repeated applications after 20 y of organic amendments (farmyard manure at 10 t ha⁻¹ y⁻¹, and urban sewage sludge at two different rates, 10 t ha⁻¹ y⁻¹ and 100 t ha⁻¹ every 2 y) on the quality of a sandy and poorly buffered soil (Fluvisol, pH 6). Chemical characteristics and biodegradability of the labile organic matter, which is mainly derived from microbial biomass and biodegradation products of organic residues, were chosen as indicators for soil quality. The organic C content had reached a maximal value (30.6 g C kg⁻¹ in the 100 t sludge-treated soil), i.e. about 2.5 times that in the control. Six years after the last application, the organic C content and the microbial biomass content remained higher in sludge-treated soils than in the control. In contrast, the proportion of labile organic matter was significantly lower in sludge-treated soils than in manure-treated and control soils. The labile organic matter of sludge extracts appeared less humified than that of manure-treated and control soils.