A microplate assay to measure soil microbial biomass phosphorus

Quantification of phosphorus (P) concentrations in microbial biomass is required to better understand how P immobilization and turnover in soils are controlled by environmental and anthropogenic factors. Soil microbial biomass P (MBP) is generally extracted using the chloroform fumigation-direct extraction procedure and then analysed for P using the ammonium molybdate-ascorbic acid method on a flow injection analysis (FIA) system. Our objective was to determine whether a microscale malachite green method on a microplate system would provide as accurate MBP analysis as the ascorbic acid method on an FIA system. Twelve soils were collected from agricultural fields in southwestern Quebec, fumigated with chloroform and extracted with 0.5 M NaHCO₃ (pH 8.5). The dissolved inorganic phosphorus (DIP) concentration in fumigated soils was not affected by the method of analysis, and results from the two systems of analysis were significantly correlated (r =0.998, P <0.05). The MBP concentrations in these agricultural soils were between 0.36 and 60.05 g P g^–1, consistent with other published values. Our results indicate that MBP can be assessed equally well with the malachite green method using a microplate system as with the ascorbic acid method on an FIA system. The microplate system is rapid and requires smaller volumes of samples and reagents than the FIA system, thus reducing the quantity of waste produced. We conclude that the microscale malachite green method could be applied to measure the MBP concentration in a wide range of soils with good sensitivity, reproducibility and accuracy.     

Bench scale and microplate format assay of soil enzyme activities using spectroscopic and fluorometric approaches

Method standardization and validation are essential for meaningful data comparison and interpretation. The objective of this study was to compare bench scale and microplate format assays of soil enzyme activities using spectroscopic (p-nitrophenyl or pNP) and fluorometric (4-methylumbelliferyl or MUF) based approaches. Three assay approaches, including pNP-bench, pNP-microplate and MUF-microplate, were compared. Data from microplate format assays in the presence of soil suspension suggested that MUF-based assays were about 14 times more sensitive than pNP-based assays. MUF detection provided measures as low as 50 pmol of MUF in a microplate well. However, the pNP bench scale assay was the most sensitive of the three assays in quantifying enzyme activities. The limit of quantification (LOQ) values expressed as corresponding enzyme activities using the protocols tested were 0.049, 0.242, and 0.0936 mmol kg⁻¹ h⁻¹ for pNP bench, pNP microplate, and MUF microplate assays, respectively. Of the microplate assays, the MUF-based assay was more precise than the procedure using pNP. The presence of soil suspension increased standard errors, which more than doubled for the detection of pNP in microplate assays, but showed little effect on the standard error for the detection of MUF. For meaningful data interpretation, we suggest that LOQ values be calculated for enzyme assays and caution be exercised when interpreting data below LOQ values. Based on evaluations of three different enzymes in 16 diverse soils, the activities measured by the three protocols were often significantly different, but were in the same order of magnitude and significantly correlated, suggesting that the same pool of isoenzymes were measured by the different protocols.     

土壤微生物多样性研究方法综述

Soil microorganisms play a central role in decomposing organic matter, in determining the release of mineral nutrients, and in nutrient cycling. Recently, extensive studies have focused on soil microbial diversity. However, understanding the diversity of this complex microbial community in the soil environment is a challenging task. Thus, it is important to master and comprehend appropriate methods for studying soil microbial diversity. Concepts of soil microbial diversity and major methods of study are briefly introduced in this paper. Then, the application of biochemical-based and molecular-based techniques in this area, and their advantages and disadvantages are evaluated. Based on recent related research, perspectives for studying microbial diversity in soils are presented.     

Community level functional diversity and enzyme activities in paddy soils under different long-term fertilizer management practices

Soil microbial community structure and function are commonly used as indicators for soil quality and fertility. The present study deals with the effect of different long-term fertilizer management practices on community-level physiological profiles (CLPP) and soil enzyme activities of paddy soils. Since 1954, chemical fertilizers have been applied in the fields as N–P₂O₅–K₂O, and compost has been added as rice straw at 0, 7.5, 22.5, and 30.0 Mg ha⁻¹ in NPK, NPKC750, NPKC2250, and NPKC3000 treatments, respectively. Community-level functional diversity was significantly enhanced in the plots treated with both chemical fertilizer and compost as compared to only chemical fertilizer and untreated control plots. Average well color development (AWCD) obtained by the Biolog Eco plate indicates that there were few differences among soil samples. Shannon diversity and evenness indices were the highest in NPKC750-treated soil and the lowest in chemically fertilized soil. Dehydrogenase, cellulose, β-glucosidase, and acid and alkaline phosphomonoesterase activities were significantly increased depending on the amount of added compost with inorganic fertilizers; the alkaline phosphomonoesterase activity was the most sensitive to treatments. Our results demonstrated that enzyme activities can be used as sensitive and liable indicators in long-term managed rice-paddy ecosystems.     

Multiple-substrate enzyme assays: a useful approach for profiling enzyme activity in soils?

This study focuses on the applicability of multiple-substrate enzyme assays to simultaneously determine various soil enzyme activities within one assay. Mineral soils from agricultural field sites differing in soil properties and management were used to optimise substrate composition and concentration of 4-methylumbelliferone and 7-amino-4-methylcoumarin derivatives as model substrates. In contrast to conventional assays, enzyme activity was measured at soil pH since optimum pH is not more applicable using a multiple-substrate approach. Furthermore, enzyme activity was not calculated from the product formed but from substrate decrease. After incubation the added substrates were re-extracted, separated by high-performance liquid chromatography and quantified by UV-absorption at 320 nm. This approach allows simultaneous measurement of the activity of β-d-glucosidase, N-acetyl-β-d-glucosaminidase, β-d-glucuronidase, β-d-xylosidase, phosphomonoesterase, sulfoesterase and leucine-aminopeptidase within one assay and with sufficient accuracy. However, incomplete re-extraction due to adsorption of substrates to the soil matrix was observed. In addition, certain competitive inhibition effects due to chemically similar substrates were found. Compared to conventional methods, no distinct differences in enzyme activity profile were detected, with both assays—conventional and multiple-substrate approach—leading to similar differentiation among the investigated soils. In conclusion, the multiple-substrate approach may serve as time-saving alternative to standard enzyme assays in mineral soils. Certainly, since the multiple-enzyme assay is conducted at soil pH, the procedure leads to reduced comparability of soils with contrasting pH values.     

A comparative study of methods for the analysis of total lead in soils

Several methods, nominally for the analysis of total Pb in soils, have been comparatively evaluated. The efficiencies of different acid reagents for extraction of Pb have been found to vary greatly, and it is recommended that a HNO₃/HF mixture be used, together with the method of standard additions.     

Structural diversity and enzyme activity of volcanic soils at different stages of development and response to experimental disturbance

We investigated the phospholipid fatty acid (PLFA) diversity and enzyme activities in soils from the volcano, Mt. Etna (Sicily). The soils were at sites which have been developing for different periods of time and have formed in volcanic lava of differing ages that have been supplemented with volcanic ejecta from subsequent eruptions. However, the plant communities indicated a marked successional difference between the sites and we have used this as a proxy for developmental stage. We have compared the structural and functional properties of the microbial communities in soils from the two sites and tested experimentally the hypothesis that the more diverse community was more resistant and resilient to disturbance. The experimental disturbance imposed was heating (60 °C for 48 h) and the recovery of enzyme activities (β-glucosidase, acid phosphatase and arylsulfatase) and structural properties (PLFA profiles) were then followed over six months. The microbial community in the soil from the older site was more structurally diverse and had a larger total PLFA concentration before disturbance than that of the soil from the younger site. The older soil community was not more resistant and resilient following an environmental disturbance as the younger soil community was equally or more resistant and resilient for all parameters. Changes in enzyme activities following disturbance were almost entirely attributable to changes in biomass (total PLFA).     

Effect of pH on enzyme stability in soils

The pH stability of urease, acid phosphatase, alkaline phosphatase and phosphodiesterase in soils was investigated by first incubating a soil sample at the indicated pH (1–13) for 24 h and then measuring the activity at its optimal pH under standardized conditions. Generally, the decline in enzyme activity in a pH-profile near the optimum pH range was due to a reversible reaction that involved ionization or deionization of acidic or basic groups in the active centre of the enzyme-protein. Irreversible inactivation of the enzyme was particularly evident at the lower and higher ranges of acidic and alkaline conditions. Results showed that the pH stability of soil enzymes was highly dependent on the soils being assayed. The variation among soils may be attributed to the diversity of vegetation, micro-organisms and soil fauna as sources contributing to the enzyme activity and to the protective sites which allowed entrapment of the enzyme within colloidal humus and organo-mineral complexes. Adherence of the enzyme-protein to the humic-clay fractions would allow some resistance to pH denaturation.     

Nitrogen availability decreases prokaryotic diversity in sandy soils

In this study, the interrelation between nitrogen availability and prokaryotic diversity are studied using a well-characterised system from a long-term field experiment on a loamy sandy soil. The prokaryotic potential functional diversity and community composition were assessed using community-level physiological profiling (CLPP), and their phylogenetic diversity was analysed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) in combination with sequencing analysis. Highest prokaryotic potential functional diversity was measured in the control soil receiving no N fertilisation, indicating an efficient as well as versatile utilisation of the substrates in this soil. Both substrate utilisation richness and substrate utilisation evenness, the two constituents of the functional diversity, were decreased with increasing N supply. Furthermore, distinct prokaryotic community compositions were generated in N-enriched soils compared to unfertilised control soils. These differences suggest a dominance of populations adapted to utilising readily available substrates. We demonstrated that the shift in prokaryotic functional diversity was connected to a shift in the phylogenetic structure of the bacterial and archaeal communities. Taken together, our data clearly show that, for the sandy soil system, prokaryotic diversity and N availability were interrelated.     

土壤总体酶活性指标的初步研究

土壤酶作为土壤生态系统的主要组成部分之一,在营养物质转化、肥力水平提高、污染物清除等方面发挥着重要作用。本文采用长期定位试验地土样,测定了三大水解酶活性,并构建了土壤酶总体活性指标。结果显示:土壤酶活性可明显反映培肥效果的优劣;土壤化学性质与酶活性间呈显著或极显著正相关,揭示出土壤脲酶、转化酶和碱性磷酸酶在一定程度上均可表征土壤肥力水平;构建的土壤总体酶活性指标(Et=sum Xi/X,from i=1 to n Total enzyme activity index)的优点是消除了不同土样酶活性的量纲及大小的影响,且无量纲的最终参数便于比较;计算获得的土壤总体酶活性参数,可更好表征土壤中总体酶活性和肥力水平高低,结果与主成分、聚类分析的相同。     

Inoculation of Pseudomonas sp. GHD-4 and mushroom residue carrier increased the soil enzyme activities and microbial community diversity in Pb-contaminated soils

Journal of Soils and Sediments - To remedy the heavy metal polluted soils, microbe-assisted phytoextraction was employed to remove Pb from soils. The Pb contents, soil enzyme activities, and...     

Functional diversity of microbial communities in saline soils of the semidesert zone

The health status of microbial communities in soils of the Sulak Lowland (Dagestan) was estimated on the basis of data on their functional diversity. The health status of the microbial communities decreased in the following soil sequence: typical meadow soil > meadow-chestnut soil > dark chestnut soil > saline soil (solonchak). The low concentration of soluble salts (<1 meq/100 g of soil) had a positive effect on the functioning of the microbial communities. The health status of the microbial communities also depended on the soil humus content and pH conditions.     

不同植烟年限土壤pH和酶活性的变化

随着烟草连作年限的延长，烟田土壤的某些性质往往发生不同程度的恶化，从而对烟叶产质量造成不良的影响，这已成为当前我国烟叶生产可持续发展的重要限制因子。目前我国关于连作烟田土壤障碍因子的研究多集中在土壤养分方面怯引，而对土壤酶活性等其他可能性因子的研究则报道较少。为此，我们对不同植烟年限土壤的pH和酶活性的变化进行了调查研究，希望为更加有效地调控连作烟田土壤障碍提供服务，改善烟叶产质量，促进烟叶生产的可持续发展。     

Genetic diversity of rhizobia associated with alfalfa in Serbian soils

We have evaluated the genetic diversity and phylogeny of alfalfa rhizobia, originating from different types of soils in Serbia and their ability to establish an effective symbiosis with alfalfa (Medicago sativa L.). A collection of 65 strains isolated from root nodules of alfalfa were characterized by rep-PCR analysis, partial and complete 16S rDNA gene and recA gene sequencing, as well as atpD gene sequencing and DNA–DNA hybridizations. The results of the sequence analyses revealed that Sinorhizobium meliloti is the dominant species in alfalfa nodules. Only one strain was identified as Sinorhizobium medicae, two strains as Rhizobium tibeticum and one strain as Rhizobium sp. Despite the fact that the majority of strains were identified as S. meliloti, a high genetic diversity at strain level was detected. Almost all isolates shared the ability to nodulate and fix nitrogen with M. sativa, except 11 of them, which were incapable of fixing nitrogen with this species. About 50% of the isolates showed values of symbiotic effectiveness (SE) above 50%, while 10% of the strains were highly effective with SE values above 70%. Some of the strains which were highly effective in nitrogen fixation at the same time could intensively solubilize phosphates, offering a possibility for multipurpose inoculum development. This was the first genetic study of rhizobia isolated from this region and also the first report of natural presence of R. tibeticum in root nodules of M. sativa.     

A study of argillic horizons in some soils in Morocco

Whether some soils observed in Morocco prior to the 1966 conference on Mediterranean soils held in Madrid had argillic horizons was the subject of active discussions. Textural differences between A- and B-horizons were obvious. Consequently, individual horizons of a number of profiles were sampled for micromorphological study. No clay films were observed in thin sections representing the B-horizons free in carbonate. Peds had pressure faces and internal striations in the matrix. Microchurning and swelling and shrinking are believed to have obliterated evidence of clay illuviation in the B-horizons, but clay coatings were observed at greater depth in well-developed B_ca-horizons.     

Use of tween 20 as a substrate for assay of lipase activity in soils

Abstract

A method for assaying the soil lipase activity is described. It involves the titrimetric estimation of the amount of lauric acid released by the lipase activity when the soil is incubated with Tween 20 in the presence of toluene at 30°C for 18 h under agitation. The method is simple and precise and incubation without agitation is also possible. The method has been applied to six different kinds of soils. The lipase activity in the cultivated soils ranged from 22.5 to 75.5 mmol min^?1 g^?1 of dried soil. The K _m value for Tween 20 was 1.8 × 10^?4 m. The optimum pH was approximately 7.5. The hydrolysis of liveen 20 in soil was inhibited by glycerol which was the essential moiety of glyceride. The inhibition by glycerol was found to be competitive. These results indicate that Tween 20 is a potential substrate for the assay of the glyceride hydrolytic activity in soils.     

Problems with toluene and the determination of extracellular enzyme activity in soils

Six of 7 fungi imperfecti, 7 of 13 basidiomycetes, and 6 of 14 bacteria tested were able to grow with 0.1 or 0.05% toluene as a sole source of carbon indicating an invalid use of toluene as a biostatic agent for analysis of extracellular enzymes in soil. Analysis of laccase and peroxidase activities in soil under controlled laboratory conditions revealed significant variability, indicating even greater difficulty in evaluating these soil enzyme activities under natural conditions.     

Factors regulating acetylene reduction assay for measuring heterotrophic nitrogen fixation in water-logged soils

In the C₂H₂-C₂H₄ assay for measurement of heterotrophic N₂ fixation in water-logged soils, the diffusion of C₂H₂ into the soil and the recovery of C₂H₄ from it are critical factors regulating the assay result. To establish an C₂H₂-C₂H₄ assay technique suitable for waterlogged soils, the C₂H₂-reducing activities (ARA), assayed by varying the method of assay gas filling, the pC₂H₂ of the assay gas, the duration of assay incubation and of soil vibration before the gas sampling, were compared.

A maximum ARA was measured when the following set of procedures were applied to the soil sample in assay flasks: 1) a 4-fold repetition of I-min evacuation under 0.01 atmospheric pressure and the subsequent I-min filling under 1 atmospheric pressure with assay gas at pC₂H₂ of 0.1 atm, 2) an assay incubation for 3 hr, and 3) a sampling of an aliquot of the headspace gas after strongly vibrating the flask for 1 min.

The ARA measured by this technique was several times larger than those measured by the techniques hitherto applied, and corresponded to an almost 80% of the V _max of the sample. This technique was, therefore, proposed for the assay of heterotrophic N₂ fixation in waterlogged soils.

A striking depression of ARA in the soil sample prepared with agitation indicated that a microbial ecosystem established in the soil should be kept as undisturbed as possible throughout the C₂H₂-C₂H₄ assay.     

Assessment of the bacterial diversity in soils: Evolution of approaches and methods

This review analyzes the publications of Russian and foreign microbiologists presenting new approaches and methods for assessing the bacterial diversity of soils in the last twenty years. Using the example of peat soils, it is shown how the concepts of the diversity of the bacterial communities changed in conformity with the evolution of the analytical methods—from the traditional cultural to the molecular-biological ones. The data on the new phylotypes, genera, and species of bacteria adapted to growth in the acid medium and low temperatures characteristic of bog ecosystems are presented. Presently, one of the principal problems of soil microbiology is the necessity of the transfer from the databases on the microbial diversity constructed on the basis of molecular-biological methods to the analysis of the ecological functions of soil microorganisms. The prospects of the ecological evaluation of the bacterial diversity in soils based on the integration of different methods are discussed.