Soil microbial biomass and fluorescein diacetate hydrolytic activity in Japanese acidic tea field soils

The soil microbial biomass (SME) content and fluorescein diacetate (FDA) hydrolytic activity in 21 acidic tea field soils in Japan were determined. SM 3 content in the tea field soils was quantitatively similar to that in 13 arable soils with neutral soil pH previously reported. However, the ratio of the SMB content to organic matter content in the tea field soils classified as red-yellow soil, brown forest soil, and lithosol was clearly lower than that in the neutral arable soils classified as non-volcanic ash soil. FDA hydrolytic activity in the tea field soils was higher than the activity in various soils with neutral soil pH and showed a negative relationship with the soil pH.     

Non-enzymatic hydrolysis of fluorescein diacetate (FDA) in a Mediterranean oak (Quercus ilex L.) litter

We show the presence of interfering substances when the total microbial activity in litter samples is measured with fluorescein diacetate (FDA), and we propose some methodological modifications to avoid such interference. Three distinct litter layers (the OhLn, the OhLv and the OhLf) of evergreen oak (Quercus ilex L.) were characterized by ¹³C CPMAS NMR and the spectra show that the recalcitrant aromatic and phenolic compounds increase with the degree of degradation of litter. A wide range of sources of interference in the hydrolysis of FDA was found. To understand the origin of this interference, sterilized litter materials (i.e. γ‐irradiated or autoclaved) and a wide range of organic substances (i.e. amino acids, glucose, sorbitol and organic humic acids) were investigated. Insignificant differences on the FDA hydrolysis activity (FDA activity) were found in the γ‐irradiated and non‐irradiated OhLn litter, indicating that γ‐irradiation does not destroy enzymes. Conversely, after heat‐sterilization of litter, samples showed FDA activity corresponding to 60, 34.8 and 30.8% (in the OhLn, the OhLv and the OhLf layers, respectively) of that of control litters. This indicates the presence of non‐enzymatic interfering substances in the FDA assays. As the humification and litter depth increased, hydrolysis of FDA due to interferences decreased, indicating degradation and/or chelation of interfering substances. We hypothesize that lysine, arginine, histidine and cysteine are mainly responsible for the hydrolysis of FDA. We suggest that the use of phosphate buffer (50 mm , pH 7.0) with incubation < 30 minutes, in combination with a temperature between 30 and 40°C, produces insignificant interference in the determination of the final FDA activity in litter samples.     

An improved method of fluorescein diacetate determination for assessing the effects of pollutants on microbial activity in urban river sediments

Journal of Soils and Sediments - The fluorescein diacetate (FDA) method has been widely used to quantify microbial activity rapidly and sensitively. However, due to high silt–clay ratios and...     

A physiological method for the quantitative measurement of microbial biomass in soils

A method is described for the rapid and objective estimation of the amount of carbon in the living, non-resting microbial biomass of soils. The method, which is based on the initial respiratory response of microbial populations to amendment with an excess of a carbon and energy source, was quantified using an expanded version of Jenkinson's technique.The simultaneous application of the two methods to 50 soil samples showed a highly significant correlation (r = 0.96) between both. From this correlation it could be deduced that at 22°C, a substrate-induced maximal respiratory rate of 1 ml CO₂· h^?1 corresponds to c. 40 mg microbial biomass C. Evidence supporting these results was obtained from pure culture studies. The various soil types investigated were collected from agricultural as well as forest sites and they contained between 15 and 240 mg microbial C·100g dry soil^?1. The respiratory method provides reproducible estimates of biomass size within 1–3 h after soil amendment. It can be combined without difficulty with a selective inhibition method for determination of bacterial and fungal contributions to soil metabolism.     

Evaluation of a rapid chromic acid method for determination of total nitrogen in soils

Abstract

A chromic acid method proposed for rapid determination of total nitrogen (N) in soils was evaluated by comparing its results with those obtained by a Kjeldahl method commonly used for total N analysis of soils. Analyses of 12 surface soils selected so that they differed markedly in texture and organic carbon content showed that the chromic acid method recovered only 87.5% to 94.1% (average, 90.5%) of the soil N recovered by the Kjeldahl method. The recovery of N from ammonium sulfate and soils by the chromic acid method decreased with increase in time of digestion with chromic acid from 1 minute to 15 minutes (the recommended digestion time). This indicates that the low recovery of soil N by the chromic acid method was at least partly due to oxidation of ammonium to nitrate and/or nitrite by chromic acid and subsequent gaseous loss of these forms of N. Support for this conclusion was provided by analyses showing that about half of the N that could not be recovered as ammonium after digestion of ammonium sulfate with chromic acid for 15 minutes was in the form of nitrate.     

Effect of novaluron on microbial biomass,respiration, and fluorescein diacetate-hydrolyzing activity in tropical soils

The aim of this study was to assess the potential harmful effects of novaluron on soil microbiological parameters in clay loam alluvial soil (Typic udifluvent) and coastal saline soil (Typic endoaquept) under controlled laboratory tests. The applications of novaluron were made at or above the recommended rates, which includes field rate (FR), two times (2FR), and ten times (10FR) the FR. The laboratory incubation study was carried out at 60% of maximum water holding capacity of soils and at 30°C. Novaluron application rate even up to 10FR resulted in a short-lived and transitory toxic effect on soil microbial biomass C and fluorescein diacetate-hydrolyzing activity. Microbial metabolic quotient changed but for a short period. It can be concluded that novaluron had a transient and negligible harmful effect on the soil microbiological parameters studied at higher rates than those usually used in the field.     

Effects of plant species on microbial biomass phosphorus and phosphatase activity in a range of grassland soils

Soil P transformations are primarily mediated by plant root and soil microbial activity. A short-term (40 weeks) glasshouse experiment with 15 grassland soils collected from around New Zealand was conducted to examine the impacts of ryegrass (Lolium perenne) and radiata pine (Pinus radiata) on soil microbial properties and microbiological processes involved in P dynamics. Results showed that the effect of plant species on soil microbial parameters varied greatly with soil type. Concentrations of microbial biomass C and soil respiration were significantly greater in six out of 15 soils under radiata pine compared with ryegrass, while there were no significant effects of plant species on these parameters in the remaining soils. However, microbial biomass P (MBP) was significantly lower in six soils under radiata pine, while there were no significant effects of plant species on MBP in the remaining soils. The latter indicated that P was released from the microbial biomass in response to greater P demand by radiata pine. Levels of water soluble organic C were significantly greater in most soils under radiata pine, compared with ryegrass, which suggested that greater root exudation might have occurred under radiata pine. Activities of acid and alkaline phosphatase and phosphodiesterase were generally lower in most soils under radiata pine, compared with ryegrass. The findings of this study indicate that root exudation plays an important role in increased soil microbial activities, solubility of organic P and mineralization of organic P in soils under radiata pine.     

Rapid and sensitive determination of microbial activity in soils and in soil aggregates by dimethylsulfoxide reduction

Summary Based on the reduction of dimethylusulfoxide (DMSO) to dimethylsulfide (DMS) by microorganisms, a simple, rapid, sensitive and inexpensive method for the determination of microbial activity in soil samples was developed. When DMSO was added to samples, DMS appeared immediately in the gas phase, which was quantitatively analyzed by gas chromatography. The DMS liberation rate was constant for several hours. The reaction immediately starts and its linearity indicate that neither the physiological state nor the number of organisms were changed by the assay. DMSO reduction is widespread among microorganisms; out of 144 strains tested (both fungi and bacteria) only 5 were unable to carry out this reaction. The reaction in soil samples was strongly inhibited by toluene, cyanide, azide, or by fumigation, but was considerably stimulated by glucose. These findings demonstrate that the reaction was due to the activity of microorganisms. The DMSO reduction in different soil samples was significantly correlated with arginine ammonification and heat output (r>0.9). A good correlation was observed with the organic-matter content (r = 0.74), but not with microbial numbers, clay content, or the pH of the soil. Standard deviations of less than 10% were routinely found. Furthermore, the method is sufficiently sensitive to allow measurements of activity in very small samples (< 0.1 g). For example, a microbial activity profile can be established for a single soil aggregate, revealing marked differences in activity on the outside and in the interior.     

Arginine ammonification,a simple method to estimate microbial activity potentials in soils

A simple, rapid and inexpensive method to determine microbial activity potentials, based on ammonification of arginine, was developed and tested on bacterial cultures and soil samples. The results are highly reproducible and correlate well with respiratory activities. Ammonification starts immediately after the addition of arginine and is linear for more than l h. Both properties show that physiological status and number of microorganisms remain stable during the assay.     

Development of a rapid and sensitive SPE-LC-ESI MS/MS method for the determination of chloramphenicol in seafood

A method based on liquid chromatography-tandem mass spectrometry was developed and validated for the qualitative and quantitative detection of chloramphenicol (CAP) in seafood samples. The analysis of CAP residues in seafood is important because CAP can cause serious acute reactions in humans, including aplastic anemia and leukemia. The proposed methodology includes a cleanup solid-phase extraction procedure with high recovery efficiency (>90%). Chromatographic separation of CAP and the internal standard (IS) was carried out on a C(18) column, followed by mass spectrometric detection using electrospray ionization in the negative-ion mode. The precursor/product ion transitions 321-->257 (CAP) and 354-->290 (IS) were monitored. Statistical evaluation of this multiple reaction monitoring mass spectrometric procedure reveals good linearity, accuracy, and inter- and intraday precisions. The limit of detection was 0.1 ng/mL, and the limit of quantification for CAP in seafood samples is 0.02 microg/kg. Application in seafood samples allowed the detection of CAP in low parts per billion levels.     

A rapid chloroform-fumigation extraction method for measuring soil microbial biomass carbon and nitrogen in flooded rice soils

 A chloroform-fumigation extraction method with fumigation at atmospheric pressure (CFAP, without vacuum) was developed for measuring microbial biomass C (C_BIO) and N (N_BIO) in water-saturated rice soils. The method was tested in a series of laboratory experiments and compared with the standard chloroform-fumigation extraction (CFE, with vacuum). For both methods, there was little interference from living rice roots or changing soil water content (0.44–0.55 kg kg^–1 wet soil). A comparison of the two techniques showed a highly significant correlation for both C_BIO and N_BIO (P<0.001) suggesting that the simple and rapid CFAP is a reliable alternative to the CFE. It appeared, however, that a small and relatively constant fraction of well-protected microbial biomass may only be lysed during fumigation under vacuum. Determinations of microbial C and N were highly reproducible for both methods, but neither fumigation technique generated N_BIO values which were positively correlated with C_BIO. The range of observed microbial C:N ratios of 4–15 was unexpectedly wide for anaerobic soil conditions. Evidence that this was related to inconsistencies in the release, degradation, and extractability of N_BIO rather than C_BIO came from the observation that increasing the fumigation time from 4 h to 48 h significantly increased N_BIO but not C_BIO. The release pattern of C_BIO indicated that the standard fumigation time of 24 h is applicable to water-saturated rice soils. To correct for the incomplete recovery of C_BIO, we suggest applying the k _C factor of 2.64, commonly used for aerobic soils (Vance et al. 1987), but caution is required when correcting N_BIO data. Until differences in fumigation efficiencies among CFE and CFAP are confirmed for a wider range of rice soils, we suggest applying the same correction factor for both methods. Received: 1 June 1999     

Ratios between estimates of microbial biomass content and microbial activity in soils

The content levels and activities of the microbiota were estimated in topsoils and in one soil profile at agricultural and forest sites of the Bornhöved Lake district in northern Germany. Discrepancies between data achieved by fumigation-extraction (FE) and substrate-induced respiration (SIR), both used for the quantification of microbial biomass, were attributed to the composition of the microbial populations in the soils. In the topsoils, the active, glucose-responsive (SIR) versus the total, chloroform-sensitive microbial (FE) biomass decreased in the order; field maize monoculture (field-MM)>field crop rotation (field-CR) and dry grassland>beech forest. This ratio decreased within the soil profile of the beech forest from the litter horizon down to the topsoil. Differences between microbial biomass and activities suggested varying biomass-specific transformation intensities in the soils. The metabolic quotient (qCO₂), defined as the respiration rate per unit of biomass, indicates the efficiency in acquiring organic C and the intensity of C mineralization, while biomass-specific arginine-ammonification (arginine-ammonification rate related to microbial biomass content) seems to be dependent on N availability. The qCO₂, calculated on the basis of the total microbial biomass, decreased for the topsoils in the same order as did the ratio between the active, glucose-responsive microbial biomass to the total, chloroform-sensitive microbial biomass, in contrast to qCO₂ values based on the glucose-responsive microbial biomass, which did not. There was no difference between the levels of biomass-specific arginine-ammonification in topsoils of the fertilized field-CR, fertilized field-MM, fertilized dry grassland and eutric alder forest, but levels were lower in the beech forest, dystric alder forest, and unfertilized wet grassland topsoils. Ratios between values of different microbiological features are suggested to be more useful than microbiological features related to soil weight when evaluating microbial populations and microbially mediated processes in soils.     

Criteria for measurement of microbial growth and activity in soil

Changes in CO₂ evolution, phosphatase and urease activity and ATP contents were related to bacterial and fungal biomass determined microscopically during glucose mineralization at different concentrations of mineral nutrients. Similar results were obtained in a sandy loam and a clay soil except that in the clay the increase in microbial and enzyme activities were delayed. Higher initial rates of CO₂ evolution were noted after the addition of P to a glucose and N amended soil at C:P ratios greater than 30:1. Increases in phosphatase activity coincided with increases in bacterial and fungal populations only in treatments without inorganic P. Peak rates of CO₂ evolution preceded biomass production by 18–24 h, therefore, CO₂ evolution rates did not show a correlation on normal regression analysis with biomass. Soil ATP content was influenced by P concentrations and soil type. ATP was therefore not a specific indicator of biomass in the detailed studies where P concentrations and sequential growth of bacteria and fungi were major factors. Soil urease increased with bacterial and fungal populations. It did not respond to P other than through microbial biomass and was highly correlated with microbial biomass. The results show that no one measurement of microbial biomass or activity is sufficient to interpret microbial growth in the soil system. Each of the criteria measured were sensitive to specific conditions affecting biomass and activity.     

Improved amperometric method for the rapid and quantitative measurement of lipoxygenase activity in vegetable tissue crude homogenates

An improved amperometric method for rapid (2 min) quantitative determination of lipoxygenase (LOX) activity in vegetable tissue crude homogenates is presented. Measured LOX activity was linear (R(2) > 0.99) throughout the entire activity range for green bean and for corn below 70% activity. The resolution was 0.4% or 1.11 micromol L(-1) s(-1) of oxygen. The limit of detection was 3.43 micromol L(-1) s(-1) of oxygen. The amperometric method was improved by encapsulating linoleic acid (LA) in beta-cyclodextrin (CD) resulting in a stable substrate-buffer solution at a pH below 8.0. Ethanol and Tween 20 were not effective in solubilizing high LA concentrations required by the assay. A prototype benchtop instrument with the potential for use in an industrial environment is also presented.     

A rapid dichromate procedure for routine estimation of total nitrogen in soils

Abstract

A simple procedure for routine estimation of total nitrogen (N) in soils is described. It involves (a) digestion of the soil sample with an acidified dichromate (K₂Cr₂O₇‐H2SO₄) solution for 45 minutes in a Pyrex digestion tube in a 40‐tube block digester preheated to 170°C, (b) titrimetric determination of the ammonia‐N liberated by direct steam distillation of the digest with alkali (NaOH), and (c) calculation of the total‐N value by application of a correction factor (1.1) to the measured NH₃‐N value. Studies using 14 soils selected to obtain a wide range in total N content and other properties showed that the uncorrected recovery of N by the procedure described ranged from 88.5% to 92.3% and averaged 90.5%. When a correction factor of 1.1 was applied to the results, the recovery of N ranged from 97.4% to 101.5% and averaged 99.6%.

The dichromate procedure described is rapid and precise, and it permits at least 80 analyses in a normal working day. The only equipment required for its use is equipment commonly used for total N analysis of soils by the Kjeldahl method (40‐tube block digester and steam distillation unit), and the acidified dichromate solution used for digestion of the soil is, the reagent commonly used for determination of organic carbon in soil by a modified Mebius procedure. Besides being simpler and more rapid than the Kjeldahl method, the procedure described has the advantage that it does not require the expensive fume disposal system needed for Kjeldahl digestion.     

A new rapid and accurate method for the determination of carbon in soils

The most reliable method for determining carbon in soils is the dry combustion method, the technique of which was established for the first time by JUSTUS von LIEBIG in 1831 and for nearly a century his combustion technique, essentially unmodified save for improvements in materials, constituted the standard method for determination of carbon and hydrogen. PREGL (1), in his publication on organic microanalyses in 1916, reported an excellent method in which a carbon determination could be carried out in an hour with a sample of only a few milligrams. After that, his method was improved in many respects by many investigators (2,3). Since these methods require, however, both complicated and expensive apparatus as well as trained personnel and are time-consuming, they are not considered to be suitable for practical use in soil research laboratories at all.     

A rapid method of evaluating different compounds as inhibitors of urease activity in soils

A simple method of studying the abilities of different compounds to inhibit urease activity in soils is described. It involves determination of the effect of the test compound on the amount of urea hydrolyzed by incubation of soils with urea and toluene at 37°C for 5 hr, urea hydrolysis being estimated by colorimetric determination of urea in the extracts obtained by treatment of the incubated samples with 2 M KC1 containing a urease inhibitor (phenylmercuric acetate). The method is rapid and precise, and it is readily adapted for studies of the rate of inactivation of urease inhibitors in soils.     

Application of respiration and FDA hydrolysis measurements for estimating microbial activity during composting processes

Olive-tree leaves (OL) were mixed with olive press cake (OPC) and extracted olive press cake (EPC) at 1:1 dw/dw ratios to prepare two composting mixtures (OL+OPC and OL+EPC). Both CO₂–C evolution and fluorescein diacetate (FDA) hydrolysis, determined as estimates of the microbial activity during composting, were related to temperature fluctuations in the compost piles, showing greater values at the temperature peaks, compared to the end, of each thermophilic phase. This, however, was only shown after handling and incubating samples at the temperatures of the compost mixtures at the sampling times and not at a low standard temperature. Incubating samples from thermophilic phases at low standard temperatures resulted in underestimation of the microbial activity occurring during composting. The effect of incubation temperature was less dramatic for FDA hydrolysis compared to CO₂–C evolution measurements, probably reflecting the reduced dependence of enzymes involved in FDA hydrolysis on the respective temperatures. However, FDA hydrolysis was a less sensitive indicator of microbial activity, probably due to extracellular cleavage of fluorescein by persistent esterases, at lowered microbial activity phases. Total microbial biomass, estimated by the fumigation–extraction method, was not consistently related to temperature fluctuations during composting and showed a clear increase at the end of composting, probably resulting from a large slow-growing mycelial community colonising the end products. Since high temperatures did not induce significant non-microbial CO₂–C release and FDA degradation, we propose the performance of microbial activity measurements during thermophilic composting phases at the actual temperatures evolving in the composts.