An automated colorimetric method for the determination of urease activity in soil and plant material

Abstract

An automated colorimetric method Cor determining urease activity in soils and plant material is described. The method, using ammonium determination by the nitroprusside‐catalysed indophenol reaction, is Caster and more precise than a colorimetric measurement of urea.     

Conductimetric determination of soil urease activity

Abstract

A fast and sensitive method to determine urease activity in soils is described. It involves extraction of NH₄‐N with 1 N. KC1 : 0.01 N HC1 and measurement of NH₄ concentration by electroconductivity using a steady state flow system across a membrane. Sampling was done directly from the extracts with an automatic sampler at 40 samples per hour. The detection limit for NH₄‐N was below 0.05 ppm in the extract. Extracts could be stored at room temperature for 17 hours before measuring NH₄. No post‐treatment hydrolysis of urea occured.     

Nitrification results in underestimation of soil urease activity as determined by ammonium production rate

The majority of soil urease activity measurements have been based on the rate of ammonium production under optimal conditions. However, such procedures do not exclude ammonium consumption by the nitrification process. The purpose of this study was to determine the percentage of soil urease activity that is underestimated due to soil nitrification. Six soils with diverse properties were incubated using a standard procedure for measuring soil urease activity. The dynamics of nitrite and nitrate were observed during the incubation. Our results showed that the percentage of underestimation ranged from 7.38% to 15.97%, depending on soil types and whether or not a buffer was used. We recommend that nitrification be taken into account when soil urease activity is assayed by the ammonium production rate method.     

Influence of soil properties on urease activity under different agro-ecosystems

Urease activity (by buffer and non-buffer method) of soils of different agro-ecosystems in alluvial soil was studied. Urease activity by buffer method records a higher value than the non-buffer method. Both the methods showed significant positive correlation (0.99**) between each other. Urease activity by both methods showed positive correlation with organic matter (0.88** and 0.89**, buffer method) and clay content (0.91** and 0.83*, non-buffer method)) of the soils. Multiple regression analysis showed that the stabilization of urease activity in the soils studied was due to an organic matter?–?enzyme complex. Among the organic matter humus C plays a dominating role to control the urease activity of the soils.     

Production of urease by microbial activity in soils under aerobic and anaerobic conditions

Summary Several workers have reported that O₂ has little, if any, effect on hydrolysis of urea by soil urease, but others have reported that it has a marked effect, hydrolysis being significantly faster in soils under aerobic conditions than in O₂-depleted soils. In studies to account for these divergent results, we found that whereas plant residues and other readily decomposable organic materials markedly stimulated microbial production of urease in soils under aerobic conditions, they did not greatly stimulate production of urease in soils under anaerobic conditions. We also found that although anaerobic conditions retarded production of urease by soil microorganisms, they did not inhibit hydrolysis of urea by soil urease. These observations suggest that the divergent findings concerning the effect of O₂ on hydrolysis of urea by soil urease may have resulted from differences in the amounts of readily decomposable organic materials in the soils studied.     

Regulation of urease production in soil by microbial assimilation of nitrogen

Summary Studies of the effects of different forms of N on urease production in soils amended with organic C showed that although microbial activity, as measured by CO₂ production, was stimulated by the addition of NH₄ ⁺ or NO₃ ^- to C-amended soils (200 mol glucose-C g^–1 soil), urease production was repressed by these forms of N. The addition of L-methionine sulfoximine, an inhibitor of inorganic N assimilation by microorganisms, relieved the NH₄ ⁺ and NO₃ ^- repression of urease production in C-amended soil. The addition of sodium chlorate, an inhibitor of NO₃ ^- reduction to NH₄ ⁺ by microorganisms, relieved the NO₃ ^- repression of urease production, but did not eliminate the repression associated with NH₄ ⁺. These observations indicate that microbial production of urease in C-amended soils is not directly repressed by NH₄ ⁺ or NO₃ ^-, but by products formed by microbial assimilation of these forms of N. This conclusion is supported by our finding that the biologically active L-isomers of alanine, arginine, asparagine, aspartate, and glutamine, repressed urease production in C-amended soil, whereas the D-isomers of these amino acids had little or no influence on urease production. This work suggests that urease synthesis by soil microorganisms is controlled by the global N regulon.     

Short- and long-term effects of heavy metals on urease activity in soils

Summary The inhibitory effects of cadmium, chromium, copper, lead, nickel and zinc on urease activity of five different soils during two different periods were investigated, in order to obtain information on the change in heavy metal toxicity with time. The results are presented graphically as logistic dose-response curves. When the ecological dose range was used as a measure of toxicity this value decreased significantly only for copper in the sandy soil. Considering toxicity as the ecological dose-50% (ED50) value, toxicity tended to increase over 1 1/2 years for cadmium, copper and zinc. For nickel and lead, however, the toxicity stabilized in all soils, except in sand and clay. The average ED 50 value of zinc varied between 100 and 300 mg kg^–1 and its toxicity was highest. It is emphasized that these data may help to set limits for the heavy-metal pollution of soils.     

Factors affecting l-asparaginase activity in soils

Summary Studies on the distribution of l-asparaginase in soil profile samples revealed that its activity generally decreases with sample depth and is accompanied by a decrease in organic C content. Statistical analyses indicated that l-asparaginase activity was significantly correlated (^** P<0.01) with organic C (r=0.86^**) and total N (r=0.78^**) in the 26 surface soil samples examined. There was no significant relationship between l-asparaginase activity and the percentage of clay or sand. There was, however, a significant correlation between l-asparaginase activity and amidase (r=0.82^**) and urease (r=0.79^**) activities in the surface samples studied. The effects of 21 trace elements, 12 herbicides, 2 fungicides, and 2 insecticides on l-asparaginase activity in soils showed that most of the trace elements and pesticides, at the concentrations used, inhibited the reaction catalyzed by this enzyme. The degree of inhibition varied among soils. When the trace elements were compared, at the rate of 5 mol g^-1 soil, the average inhibition of l-asparaginase in three soils showed that Ag(I), Cd(II), Hg(II), Ni(II), Pb(II), and V(IV) were the most effective inhibitors (average inhibition 20%). The least effective inhibitors (average 10%) included Cu(I), Ba(II), Co(II), Sn(II), Zn(II), Al(III), Se(IV), As(V), and Mo(VI). Other trace elements that inhibited l-asparaginase activity in soils were Cu(II), Mn(II), As(III), B(III), Cr(III), Fe(III), Ti(IV), and W(VI). When the pesticides were compared, at the rate of 10 g active ingredient g^-1 soil, the average inhibition of l-asparaginase activity in three soils ranged from 4% with Merpan to 46% with Malaspray. Other pesticides that inhibited l-asparaginase activity in soils (average inhibition in parentheses) were Aatrex (17%), Alanap (21%), Amiben (18%), Banvel (12%), Bladex (24%), 2,4-D (17%), Dinitramine (19%), Eradicane (16%), Lasso (40%), Paraquat (33%), Sutan (39%), treflan (7%), Menesan (18%), and Diazinon (33%).     

豆科牧草根际土壤脲酶活性的研究

研究结果表明:土壤脲酶活性与养分含量呈极显著相关,可作为评价土壤肥力的生物学指标;脲酶活性的垂直分布随土层深度而递减;土壤脲酶活性数量随作物生长与根系生物量的增强而增加,与土壤微生物数量显著相关;根际土壤脲酶活性高于近根际土壤;不同种类豆科牧草,根际土壤脲酶活性存在明显差异。深根豆科牧草苜蓿、草木樨可留给深层土壤大量根系残留物,从而有利于提高土壤深层脲酶活性与综合供肥能力。     

杀灭菊酯对亚热带果园红壤脲酶活性的生态毒理效应

通过模拟方法,系统研究杀灭菊酯对亚热带果园红壤脲酶活性的影响。结果表明:杀灭菊酯对土壤脲酶活性表现出先激活后抑制趋势,处理前3 d土壤脲酶活性升高,处理3 d后土壤脲酶活性受到抑制,7 d后低于对照水平;激活、抑制程度与处理浓度呈正相关。底物(尿素)浓度饱和前,土壤脲酶活性随尿素浓度增加而升高。模型U=β0 β1×C能较好拟合土壤脲酶活性(U)和尿素浓度(C)、杀灭菊酯浓度(C)之间关系,揭示出土壤酶促反应过程存在吸附-解吸的机制,证实不同肥力土壤脲酶活性受杀灭菊酯影响差异较大,高肥力土壤对土壤脲酶活性具有一定的缓冲作用,受杀灭菊酯影响较小。     

Significance of soil microorganisms for the mobilization of nonexchangeable ammonium

To estimate the availability of nonexchangeable NH _inf4 ^sup+ –N for soil microorganisms four incubation experiments were conducted under controlled conditions. The following results were obtained: Incorporating glucose as a source of readily oxidizable organic material favored the release of nonexchangeable NH _inf4 ^sup+ –N. Mobilization of NH _inf4 ^sup+ from the interlayers of the clay minerals was decreased by the application of K⁺⁺, while Ca²⁺, which is supposed to expand the lattice of the clay minerals, had no influence on the release of NH _inf4 ^sup+ . Soil temperature had no effect on microbiological mobilization of NH _inf4 ^sup+ . It is assumed that, generally, the influence of nitrifying bacteria on the mobilization of nonexchangeable NH _inf4 ^sup+ –N is negligible. However, in soils with abundant amounts of available carbon promoting the activity of heterotrophic soil microorganisms, the release of NH _inf4 ^sup+ from clay minerals is favored under fallow conditions.     

Suitability of dehydrogenase activity assay as an index of soil biological activity

Summary Dehydrogenase activity was studied in typical soils of the Northwest German Lower Plains in order to test some criticisms raised by nannipieri et al. (1990) about methodology. We found that in the same soils with the sampe crop, dehydrogenase activity varies significantly. In the field dehydrogenase activity was dependent on the soil type and not on the cropping system. There is no adequate explanation of these findings. Dehydrogenase activity is a parameter of soil microbiological activity and is influenced by many factors. Either purely accidental the results are or dehydrogenase activity is affected by unknown ecological interactions and by the composition of soil microflora. We conclude that without simultaneous investigations of other microbiological parameters (microbial biomass, ATP levels, enzyme activities, etc.) the measurement of dehydrogenase activity creates confusion and may impede valid ecological comparisons of soils.Dedicated to Prof. H.-P. Blume, Kiel, on the occasion of his 60th birthday     

氨固定对土壤微生物生物量氮矿化测定的影响

Two soils with relatively high(Soil 1)and low(Soil 2) ammonium fixation capacities were used in this study to examiune the effect of ammonium fixation on the determination of N mineralized from soil microbial biomass.Organism suspension was quantitatively introduced to Soil 1 at various rates.Both fumigation-incubation (FI) and fumigation-extraction (FE) methods were used to treat the soil.The amount of fixed NH4^ increased with increasing rate of organism-N addition.A close correlation was found between the amount of fixed ammonium and th rate of organism-m addition.The net increases of fixed NH4^ -N were equivalent to 38% and 12% of the added organism-N for FI and FE treatments,rspectively,in this specific soil.To provide isotopic evidence,^15N-labelled organism-N was added to Soils 1 and 2 at 121.4 mg N kg^-1.In FI treatment,22 and 3mg N kg^-1 of labelled N were found in the fraction of fixed NH4^ -N in Soile 1 and 2 respectively;while in FE treatment,9 mg N kg^-1 of labelled N was found in the fraction of fixed NH4^ -N in Soil 1 only.There was no labelled N in the fraction of fixed NH4^ -N in Soil 2.In all of the unfumigated (check) soils,there was little or no labelled N in the fixed fractions,probably because the organism-N added was easily mineralized and nitrified.A mean of 0.64 for KN value,the fraction of N mineralized in the killed microbial biomass,as obtained with inclusion of the net increase of fixed NH4^ -N,The corresponding value calculated with exclusion of the net increase of ficed NH4^ -N was 0.46 ,It was concluded that ammonium fixation was a problem in deterination of KN,particularly for soils with a high ammonium fixation capacity, Results also showed that microbial biomass N measurement by FFE method was less affected by ammonium process than that by FI method.     

Application of the Berthelot reaction to the determination of ammonium‐N in soil extracts and soil digests

Abstract

The phenol‐hypochlorite‐ammonium reaction of Berthelot can be utilized in manual procedures for the analysis of NH₄‐N in a variety of soils applications, including total N measurement in soils, particle size separates and soluble organic matter fractions, and in measuring NH₄‐N in soil extracts. A simple, convenient, and versatile procedure is described.     

Effect of urease inhibitors on urea hydrolysis and ammonia volatilization

Summary Two laboratory incubation experiments were conducted to study the effects of the urease inhibitors hydroquinone (HQ), phenyl phosphorodiamidate (PPDA), and N-(n-butyl) thiophosphoric triamide (NBPT) in retarding the hydrolysis of urea, in the evolution of mineral N, and in reducing NH₃ loss through volatilization, under aerobic and waterlogged conditions, both at 25°C. NBPT generally exceeded PPDA and HQ in the ability to delay urea hydrolysis and NH _inf4 ^sup+ accumulation under aerobic conditions, whereas PPDA retarded these activities more effectively under anaerobic conditions. HQ was less effective than the other two urease inhibitors. Under aerobic conditions, 20% of the applied urea was lost through NH₃ volatilization after 5 days in the system without an inhibitor. With the addition of HQ and PPDA, the volatilization was delayed by 1 day but not eliminated. NBPT effectively decreased the NH₃ loss, from 20 to 3% of the applied urea. A more severe N loss (40%) occurred in the waterlogged system. HQ had little effect on NH₃ volatilization. PPDA decreased the NH₃ loss from 40 to less than 20% of the applied urea. The effectiveness of NBPT decreased under anaerobic conditions. It was concluded that urease inhibitors can reduce NH₃ volatilization following the application of urea. However, environmental conditions might have an important influence on the effectiveness of these inhibitors.     

A high sample volume procedure for the colorimetric determination of soil organic matter

Abstract

A modified procedure for routine determination of soil organic matter is described. The procedure employs a wet combustion method of reducing the carbon in soil organic matter with dichromate in an acid media. The modification will permit 700–800 organic matter determinations per day with samples containing less than 8% 0M without undue difficulty.     

Enzyme activity and carbon dioxide evolution from upland and wetland rice soils under three agricultural practices in hilly regions

Summary Dehydrogenase activity (a measure of microbial biomass), urease activity and CO₂ evolution were measured in soils planted to rice (Oryza sativa L.) under three different agricultural practices prevalent in hill regions. The effects of hill slope, terrace and valley agriculture were investigated for two cropping seasons. The valleys and terraces were kept flooded during each cropping season while the hill slopes were cultivated with dryland practices. The type of agricultural practice and the date of observation had a significant influence on enzyme activity and CO₂ evolution. A positive correlation was observed between dehydrogenase and urease activity and soil moisture content. Dehydrogenase and urease activity and soil respiration were positively correlated among themselves. The activity of both enzymes and CO₂ evolution were highest in valley soils followed by terrace and hill-slope sites.     

Different approaches to evaluating soil quality using biochemical properties

Soil biochemical properties are indicators of soil quality, but there is still no consensus as to how they should be used. We review the trends in their use over the last decade. Generally, biochemical properties related to the biocycles of the elements (C, N, P and S) are used to diagnose soil quality. These properties include both general biochemical parameters (i.e. microbial biomass C, dehydrogenase activity and N mineralization potential) and specific biochemical parameters (i.e. the activity of hydrolytic enzymes, such as phosphatase, urease and β-glucosidase). Biochemical properties can be used both individually, as simple indices, or in combination using complex equations derived from mathematical combinations or the application of statistical programs. The results described in the literature for both are contradictory and question the validity of the use of biochemical properties as quality indicators. Complex expressions, in which different properties are combined, are thought to be highly suitable for estimating soil quality, although their use is limited to the area and situation in which they have been described. Generally, the greatest problems posed by the use of biochemical properties as soil quality indicators include the lack of reference values, the contradictory behaviour shown by these properties when a soil is degraded, and the regional variations in expression levels. Most of these problems are derived from the scarce information available on the biochemical properties of soil. For this reason, obtaining soil quality indicators of general use will require a coordinated effort from the international scientific community to standardise the analytical methods and to compile databases of biochemical properties from soils under diverse geographic conditions and with different uses and management.     

An evaluation of some manual colorimetric methods for the determination of inorganic nitrogen in soil extracts

Abstract

A number of manual colorimetric methods for the determination of inorganic nitrogen in 1 M KCl soil extracts were investigated to find techniques that were inexpensive, rapid, versatile and suitable for laboratories with limited analytical equipment. Three colorimetric methods for No^? ₃‐N determination were evaluated and only the copperised/cadmium reduction technique suffered no significant interference from the Cl^? present in the extracting solution. A phenol‐hypo‐chlorite (Berthelot) procedure for NH⁺ ₄‐N determination and the Griess‐Ilosvay method for NO^? ₂‐N determination were both found suitable for N determination in 1M KC1 soil extracts. The reliability and accuracy obtainable with the manual colorimetric methods described was shown to be comparable with that obtained from colorimetric analyses performed using an AutoAnalyser.     

Effects of soil properties and trace metals on urease activities of calcareous soils

A study was conducted to investigate the relationship between urease activity and some physical, chemical, and microbiological properties of soils from central Iran. Inhibitory effects of Cr, Cd, and Pb on urease activity were also studied. Results indicated that no significant difference was observed between urease activity of field-moist and air-dried soils. Soil organic C and total N correlated highly significantly with urease activity, with r values of 0.899*** and 0.797***, respectively. There was also a significant correlation between urease activity and the number of bacteria grown on urea-agar media, with r value of 0.470*. A significant negative correlation (r =–0.492*) was observed between urease activity and electrical conductivity of saturation paste extracts. There were no significant correlations between urease activity and soil textural properties, pH, calcium carbonate equivalent, cation exchange capacity, and populations of soil bacteria on nutrient agar and population of soil fungi on potato dextrose agar. Both Cd and Pb inhibited urease activity to a similar extent and to a greater extent than did Cr.