Microbial biomass in soil: Effects of some experimental variables on biochemical estimations

The effects of experimental variables on estimates of biomass C and mineral-N (Min-N) flush by the chloroform fumigation technique were determined in near neutral to slightly alkaline topsoil samples of a Typic Haplaquoll taken at three different times under grazed grass-clover pastures. The variables were soil mesh size ( < 3.3 and < 2 mm), water content [50 and 60% of water-holding capacity (WHC)], the use of samples that were “fresh” or that had been previously incubated (7 days at 50% of WHC at 25°C), and, for the biomass C estimates, various incubation periods for measuring CO₂C production.Estimates of biomass C were most strongly influenced by the incubation period selected for CO₂C production by unfumigated soil. The effects of soil mesh size and water content were significant for some samples, but were not consistent. Prior incubation lowered all biomass C estimates significantly, except for some samples where a 0–10 day period was used for measuring CO₂C production by unfumigated soil; (the presence or absence of soda-lime during incubation had no influence on subsequent rates of CO₂ production by the unfumigated samples).Min-N flush was not consistently influenced by these variables, although some significant treatment effects occurred. Biomass C-to-Min-N flush ratios were predictably dependent upon the biomass C estimates used. They averaged 9.0 in “fresh” samples and 6.0 in incubated samples, when the incubation periods for CO₂C production by unfumigated samples were 10–20 and 0–10 days respectively.Results indicate that care should be taken in interpreting and comparing biochemical indices of microbial biomass, and their ratios, obtained by different or modified experimental procedures.     

Microbial biomass estimations in soils from tussock grasslands by three biochemical procedures

Microbial biomass was determined by three biochemical procedures in nine topsoils from a climosequence in tussock grasslands. The pH values of the samples ranged from 4.4 to 6.2 and organic C contents from 2.5 to 20.0%. When determined by a chloroform-fumigation procedure, contents of biomass C and mineral-N (Min-N) flush ranged from 530–2780 and 59–167 μgg^?1 dry soil respectively. Adenosine 5'-triphosphate (ATP) content ranged from 2.2 to 10.7 μg g^?1 dry soil. All three estimates were significantly correlated with each other and with several soil properties, including organic C and total N contents and CO₂ production. They were not significantly correlated with any climatic factor.In spite of these significant correlations, the ratios of the biomass estimates varied appreciably in the different soils. The ratios of biomass C/Min-N flush ranged from 7.8 to 22.8 (average 12.5), biomass C/ATP from 163 to 423 (average 248) and Min-N flush/ATP from 12 to 35 (average 22). These ratios were mostly higher than those found elsewhere for Australian and English soils. The high biomass C/ATP and Min-N flush/ATP ratios did not appear to originate from inefficient extraction of “native” ATP or from the soils' P status. Based on these results, care in the use of factors for obtaining soil microbial biomass content from Min-N flush or ATP values is indicated.     

Adenosine triphosphate (ATP) and microbial biomass in soil: Effects of storage at different temperatures and at different moisture levels

Abstract

On air‐drying, the ATP contents of two moist soils fell to about one quarter of their original values. When a freshly‐sampled soil (field temperature 5.5°C) was stored moist (43% water holding capacity) for 7 days at 25°C the ATP content increased from 4.54 to 7.84 μg ATP g^‐1 soil. Storage at 10°C caused a smaller increase; to 5.39 μg g^‐1 soil. Microbial biomass C also increased on storage but the relative increase was less than that of ATP. Thus the biomass C/ATP ratio fell from 234 in the freshly sampled soil to 168 in the soil stored moist for 7 days at 25°C. The ATP content declined to less than half its starting value if storage was under waterlogged conditions.

The ATP method for determining microbial biomass in soil depends on the use of a constant factor (5.85 mg ATP g^‐1 biomass C) for converting ATP content to biomass C. This factor came from work on soils that had been stored moist at 25°C for several days before biomass C and ATP measurements were made: it is only applicable to soils that have been stored in this way.     

Mineralization of bacteria and fungi in chloroform-fumigated soils

It has been suggested by others that the size of the flush of mineralization caused by CHC1₃ fumigation can be used to estimate the amount of microbial biomass in soils. Calculation of biomass from the flush requires that the proportion of CHCl₃-killed cell C mineralized be known. To determine this proportion, 15 species of [¹⁴C]labelled fungi and 12 species of [¹⁴C]labelled bacteria were added to four types of soil and these were fumigated for 24 h with CHC1₃, reinoculated with unfumigated soil, and incubated at 22°C for 10 days. The average percentage mineralization of the fungi was 43.7 ± 5.3, while the average for the bacteria was 33.3 ± 9.9. Using a 1:3 ratio for distribution of total biomass between the bacterial and fungal populations, respectively, it was calculated that the average mineralization of both types of cells was 41.1%. In experiments conducted to determine if CHC1₃ vapour alters stabilized microbial metabolites or dead microbial cells in a manner which makes them more susceptible to degradation, it was found that both fumigated and unfumigated dead fungal materials mineralized to the same extent in soil during 10 days of incubation.     

Correlation between four methods to estimate total microbial biomass in stored,air-dried and glucose-amended soils

Correlation between the microbial volume, chloroform fumigation (CO₂-C flush), substrateinduced respiration (SIR) and ATP content methods to estimate microbial biomass was assessed on three New Zealand soils (two grassland, one arable) under three different treatments (stored, air-dried and glucose-amended). There were significant, positive correlations between all methods, r = 0.69–0.88, which were improved, r = 0.71–0.96, if the data for air-dried or glucose-amended soils were excluded from the analyses. The best agreement was between CO₂-C flush and ATP and the worst between CO₂-C flush and microbial volume. Exclusion of air-dried soil data improved these correlations.Estimates of microbial biomass for each soil often differed significantly between the four methods, when conversion factors cited in the literature were used. Ratios (i.e. conversion factors) between CO₂-C flush and ATP or SIR, or SIR and volume, were different to those cited in the literature, and only similar if specific data were excluded.We recommend that a minimum of two and preferably three methods be used to quantify the microbial population of soil, and that emphasis should be placed on the relative differences within and between soils using data which have not been converted to biomass C. Conversion of data to biomass C may result in substantial errors.     

Physical factors influencing decomposition of organic materials in soil aggregates

Glucose or starch labelled with ¹⁴C was mixed thoroughly into slurried soils. Aggregates of different sizes were obtained from the soils as they dried. The labelled substrates were considered to be distributed in both micro- and macropores in the aggregates. Control samples (labelled substrates in macropores only) were prepared by adding the labelled carbohydrates after the formation of the aggregates. The various samples were sterilized by γ-irradiation and stored at ?15°C.Samples were wetted to about ?20kPa, inoculated with soil organisms, and incubated for 4 weeks at 28°C in closed systems, which enabled regular measurement of ¹⁴CO₂ released.Based on the ¹⁴CO₂ released, it was concluded that starch was protected from microbial attack when present in micropores in aggregates made from fine sandy loam.After incubation samples were dried and rewetted. The flush of ¹⁴CO₂ released was twice as big for samples containing labelled starch compared with glucose, showing that disruption of aggregates, containing residual starch, and rearrangements of soil components are as important as chemical and biological factors in causing the flush of CO₂ resulting from wetting a soil. Mechanical disruption of the aggregates resulted in a similar flush of ¹⁴CO₂.     

Fluctuations in microbial biomass indices at different sampling times in soils from tussock grasslands

Microbial biomass in four topsoils from New Zealand tussock grasslands was estimated by three biochemical procedures at five sampling times over a 15 month period. In Conroy, Cluden and Tima soils, biomass C content was high in two sets of March (summer-autumn) samples and low in October (early spring) samples; in Carrick soil from a wetter, cooler environment, it was similar at all sampling times. Significant time-of-sampling variations occurred with Min-N flush in Tima and Carrick soils, and with adenosine 5'-triphosphate (ATP) content in three of the soils. Generally, the ratios of these biomass indices also varied significantly at some sampling times. Because of this variability, common factors could not validly be used with these soils for estimating biomass C contents from Min-flush or ATP values.The contribution of bacteria and fungi to the respiratory activity of the microbial biomass was unsuccessfully investigated using streptomycin and actidione as differential inhibitors of anabolic metabolism in the presence of added glucose. In three of the soils, rates of O₂ uptake did not generally increase significantly during incubation, even with added N, P, K and S or prior incubation overnight. In Conroy soil, rates did increase significantly, but the effects of the antibiotics separately and together could not be satisfactorily balanced.     

Microbial biomass and activity indices after organic substrate addition to a selenium-contaminated soil

High concentrations of Se in soil might have negative effects on microorganisms. For this reason, the effect of organic substrate addition (glucose + maize straw) on Se volatilisation in relation to changes in microbial biomass and activity indices was investigated using an artificially Se-contaminated soil. Microbial biomass N was reduced on average by more than 50% after substrate addition, but adenylate energy charge (AEC) and metabolic quotient qCO₂ were both increased. The Se content decreased by nearly 30% only with the addition of the organic substrate at 25°C. No significant Se loss occurred without substrate at 25°C or with substrate at 5°C. In the two treatments with substrate addition, the substrate-derived CO₂ evolution was about 30% lower with Se addition than without. In contrast, Se had no effect on any of the other soil microbial indices analysed, i.e. microbial biomass C, microbial biomass N, adenosine triphosphate (ATP), AEC, ATP-to-microbial biomass C, and qCO₂.     

Interference from plant roots in the estimation of soil microbial ATP,C, N and P

Excised, solution-grown roots of maize or ryegrass added to two pasture soils at the rate of 6.0mg g^?1 and 13.8 mg g^?, respectively, increased the flush (fumigated minus control values) of CO₂-C by up to 1.89-fold, KCl extractable N by up to 1.88-fold, and NaHCO₃ extractable P by 3.28-fold. The ATP content of the soil was increased by up to 1.42-fold. Because of high variability the effect of the roots on the C and N flushes was not significant at P < 0.05.Incubation of the root-amended soils for 7 days at 25°C prior to fumigation much decreased the contribution from the roots to the C and N flush, and to the ATP content. There was, however, still a large significant effect of the roots on the P-flush, this being up to 3 times greater than the equivalent soil without roots.In soil samples with a high viable root density (> 6mg g^?1) such as may occur in dense pastures, greenhouse pot experiments or rhizosphere soil samples, it is recommended that they be incubated for 7 days prior to fumigation and analyses. Without such prior incubation there is the risk that root material may be included in the “microbial” biomass estimations.     

A simple method for quantitative determination of ATP in soil

A simple method to measure soil ATP by the luciferin-luciferase system is described. The ATP is extracted from the soil by vigorous shaking with a sulfuric acid-phosphate solution for 15 min. An aliquot of the soil suspension is neutralized with a Tris-EDTA solution and mixed with a special ATP releasing reagent (NRB). ATP is measured after a 10 s exposure to the NRB reagent, followed by addition of luciferin-luciferase and integration over 10 s in a Lumacounter M 2080. The ATP content in soils which had been stored at 5°C for 90 days and then incubated at 25°C for 5 days, ranged from 0.37 to 7.52 μg ATP g^?1 dry wt, with standard deviations less than 10%. There was a close (r = 0.96) linear relationship between ATP content and biomass C determinated by fumigation for this group of soils. The soil biomass contained 4.2–7.1 μg ATP mg^?1 biomass C. The ATP content of the biomass declined during storage at 5°C for 210 days.     

Microbial biomass dynamics in recently air-dried and rewetted soils compared to others stored air-dry for up to 103 years

Our aim was to compare the soil microbial biomass concentration and its activity (measured as CO₂-C evolved) following the rewetting and aerobic incubation of soils which have previously been stored air-dry for different periods. Some of the soils have been stored in the Rothamsted sample archive for 103 years, others were comparable freshly sampled soils following air-drying and rewetting and other soils were stored air-dry for 2 years then rewetted for the work described here. Following air-drying, soil ATP concentrations were variable in recently air-dried soil, comprising about 10-35% of the initial ATP concentrations in fresh soil. Following rewetting, the percentage recovery of ATP increased in all soils by 7 days, then declined to between 73% and 87% of the original ATP concentration in the air-dried soils by day 12. Storage of air-dried soils decreased the ability of the microbial biomass to restore its ATP concentrations. For example, the ATP concentration in a soil sampled from stubbed (i.e. tree seedling, saplings and bushes cut frequently to ground level) grassland of the Broadbalk continuous wheat experiment at Rothamsted then air-dried for 2 years was only about 14% of that in the fresh soil at 2 days after rewetting. In other soils from the Hoosfield Barley Experiment, also at Rothamsted, previously given NPK or FYM since 1852, and sampled then stored air-dry for between 13 and 83 years, from 52% to 57% of the ATP in the comparable fresh soils was measured at two days after rewetting. The soil ATP concentration then changed little more up to 12 days. One of the most interesting findings was that while the microbial biomass ATP concentration in the above NPK soils only ranged from about 2 to 4 μmol ATP g⁻¹ biomass C, in the FYM soil the microbial biomass ATP concentrations (range 11.5-13.6 μmol ATP g⁻¹ biomass C) were the same as we repeatedly measure in fresh moist aerobic soil. We do not yet know the reasons for this. More than twice as much CO₂-C was evolved from the long-term stored soils than from freshly sampled ones. However, the specific respiration of the microbial biomass did not change much after the first 12 years of storage, indicating that loss of viability mainly occurred in the earlier years.     

The effects of biocidal treatments on metabolism in soil—I. Fumigation with chloroform

This series of five papers is a study of how biocidal treatments influence metabolism in soil, directed particularly towards the flush of decomposition caused by fumigation, and designed to see if the size of this flush can be used as a measure of the soil biomass.Chloroform fumigation caused an immediate increase in the amounts of ammonium and organic C extracted from a soil by 1 N K₂SO₄. When the CHCl₃-treated soil was then inoculated with fresh soil and incubated for 10 days. it consumed 2·8 times more O₂, evolved 2·2 times more CO₂ and mineralised 7·3 times more N than an unfumigated soil. Extractable organic C decreased by about 40% when the fumigated soil was incubated for 10 days. A second fumigation given immediately after the first produced no further increase in the flush, but some recovery occurred if the soil was incubated between fumigations. However, this recovery was slow and incomplete; a second fumigation given 53 days after the first gave a flush only one-seventh the size of the first. Glucose (or ryegrass) added to the soil and allowed to decompose before fumigation increased the size of the flush. After a 52-day incubation, 29% of the C originally added as ¹⁴C labelled glucose remained in the soil; fumigation on the 52nd day increased the evolution of labelled CO₂ during the subsequent 10-day period by a factor of 8. Fumigation of a soil that had already been sterilized by 2·5 Mrads of gamma radiation increased the flush slightly; the amount of O₂ consumed in 10 days increased from 123 to 137 mg/100 g soil. It is proposed that the flush of decomposition following CHCl₃ fumigation is caused by the decomposition of killed organisms by the survivors (or by organisms added in the inoculum) and that organisms are more rapidly and completely attacked after exposure to CHCl₃ than after irradiation. On this hypothesis. 10% of the glucose C originally added to the soil was located in the soil biomass after 52 days.     

Relationships of soil respiration to microbial biomass, substrate availability and clay content

The roles of microbial biomass (MBC) and substrate supply as well as their interaction with clay content in determining soil respiration rate were studied using a range of soils with contrasting properties. Total organic C (TOC), water-soluble organic carbon, 0.5 M K₂SO₄-extractable organic C and 33.3 mM KMnO₄-oxidisable organic carbon were determined as C availability indices. For air-dried soils, these indices showed close relationship with flush of CO₂ production following rewetting of the soils. In comparison, MBC determined with the chloroform fumigation-extraction technique had relatively weaker correlation with soil respiration rate. After 7 d pre-incubation, soil respiration was still closely correlated with the C availability indices in the pre-incubated soils, but poorly correlated with MBC determined with three different techniques—chloroform fumigation extraction, substrate-induced respiration, and chloroform fumigation-incubation methods. Results of multiple regression analyses, together with the above observations, suggested that soil respiration under favourable temperature and moisture conditions was principally determined by substrate supply rather than by the pool size of MBC. The specific respiratory activity of microorganisms (CO₂-C/MBC) following rewetting of air-dried soils or after 7 d pre-incubation was positively correlated with substrate availability, but negatively correlated with microbial pool size. Clay content had no significant effect on CO₂ production rate, relative C mineralization rate (CO₂-C/TOC) and specific respiratory activity of MBC during the first week incubation of rewetted dry soils. However, significant protective effect of clay on C mineralization was shown for the pre-incubated soils. These results suggested that the protective effect of clay on soil organic matter decomposition became significant as the substrate supply and microbial demand approached to an equilibrium state. Thereafter, soil respiration would be dependent on the replenishment of the labile substrate from the bulk organic C pool.     

Relation between respiration,ATP content,and Adenylate Energy Charge (AEC) after incubation at different temperatures and after drying and rewetting

Temperature, drying, and rewetting are important climatic factors that control microbial properties. In the present study we looked at the respiration rates, adenosine 5′‐triphosphate (ATP) content, and adenylate energy charge (AEC) as a measure for energy status of microbial biomass in the upper 5 cm of mineral soils of three beech forests at different temperatures and after rewetting. The soils differed widely in pH (4.0 to 6.0), microbial biomass C (92 to 916 μg (g DW)^—1) and ATP content (2.17 to 7.29 nmol ATP (g DW)^—1). The soils were incubated for three weeks at 7 °C, 14 °C, and 21 °C. After three weeks the microbial properties were determined, retaining temperature conditions. The temperature treatment did not significantly affect AEC or ATP content, but respiration rates increased significantly with increasing temperature. In a second experiment the soils were dried for 12 hours at 40 °C. Afterwards the soils were rewetted and microbial properties were monitored for 72 hours. After the drying, respiration rates dropped below the detection limit, but within one hour after rewetting respiration rates increased above control level. Drying reduced AEC by 16 % to 44 % and ATP content by 47 % to 78 %, respectively. Rewetting increased AEC and ATP content significantly as compared to dry soil, but after 72 hours the level of the controls was still not reached. The level of AEC values indicated dormant cells, but ATP content increased. These results indicate that the microbial carbon turnover was not directly linked to microbial growth or microbial energy status. Furthermore our results indicate that AEC may describe an average energy status but does not reflect phases of growing, dormant, or dying cells in the complex microbial populations of soils.     

Microbial Degradation of Dimethylsilanediol in Soil

Dimethylsilanediol (DMSD) is the ultimate hydrolysis product of silicone (polydimethylsiloxane = PDMS) polymer in soil. Our previous paper showed that it would volatilize from soil, and the present study investigates the importance of microbial degradation in removing DMSD from soil. DMSD (¹⁴C-labeled) was thus incubated (1 mg kg^-1) for 30 wk at 25 °C in soils from a permanent grass field, a corn field, a deciduous woodland, and a pine woodland. Release of¹⁴ CO₂ varied from 0.4 to 1.6% wk^-1. For 3 of the soils, ¹⁴CO₂ increased with higher microbial biomass, while organisms in the deciduous woodland soil were more active in degrading DMSD than organisms in the other soils. After 30 weeks, most of the remaining ¹⁴C in the soil had moved from freely available water extractable to less available acid and base extractable fractions. Similar incubations with 2% plant litter showed extensive transfer of the DMSD into the litter layer. Incubations with a microbial inhibitor showed less DMSD degradation, while cold storage of soils almost completely stopped degradation. These results suggest that microbial degradation is an important mechanism of DMSD loss from soil.     

The effects of biocidal treatments on metabolism in soil—V: A method for measuring soil biomass

A new method for the determination of biomass in soil is described. Soil is fumigated with CHCl₃ vapour, the CHCl₃ removed and the soil then incubated. The biomass is calculated from the difference between the amounts of CO₂ evolved during incubation by fumigated and unfumigated soil. The method was tested on a set of nine soils from long-term field experiments. The amounts of biomass C ha^?1 in the top 23 cm of soil from plots on the Broadbalk continuous wheat experiment were 530 kg (unmanured plot), 590 (plot receiving inorganic fertilizers) and 1160 (plot receiving farmyard manure). Soils that had been fallowed for 1 year contained less biomass than soils carrying a crop. A calcareous woodland soil contained 1960 kg biomass C ha^?1, and an unmanured soil under permanent grass 2020. The arable soils contained about 2% of their organic C in the biomass; uncultivated soils a little more—about 3%.     

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.     

Straw decomposition and nitrogenase activity (C2H2 reduction): Effects of soil moisture and temperature

The effects of moisture and temperature on straw decomposition (CO₂ production) and nitrogenase activity (C₂H₂ reduction) were measured in laboratory experiments to evaluate the potential for nitrogenase activity at various times of the year in soils with added wheat straw. Soils collected from two areas (Gunnedah and Cowra) representative of large areas of the wheat belt of New South Wales, Australia, were examined. Straw decomposed over a wide range of temperatures (20–50°C, Gunnedah; 15–45°C, Cowra) and moistures (0.3–2.0 times ?10 kPa water content). Microbial populations that fix nitrogen were adapted to a broad range of temperatures (10–45°C, Gunnedah; 4–40°C; Cowra). However, nitrogenase activity with added glucose occurred at much lower soil moisture contents in the Gunnedah soil (0.5–1.75 times ?10 kPa water content) than in the Cowra soil (1.0–2.5 times ?10 kPa water content). Despite the differences between the soils the results show that there is potential for straw decomposition and nitrogenase activity throughout most of the year.     

Changes in Microbial Functional Diversity and Activity in Paddy Soils Irrigated with Industrial Wastewaters in Bandung, West Java Province, Indonesia

Characteristics, such as microbial biomass, basal respiration, and functional diversity of the microbial communities, were investigated in paddy soils located in Bandung, West Java Province, Indonesia, that have been heavily polluted by industrial effluents for 31 years. Paddy soil samples (10?C20 cm) were taken from two sites: polluted soils and unpolluted soils (as control sites). The polluted soils contained higher salinity, higher sodicity, higher nutrient contents, and elevated levels of heavy metals (Cr, Mn, Ni, Cu, and Zn) than unpolluted soils. Soil physicochemical properties, such as maximum water holding capacity, exchangeable sodium percentage, sodium adsorption ratio, and swelling factor, in polluted soils were much greater than those in unpolluted soils (P?<?0.05). Changes in the physical and chemical soil properties were reflected by changes in the microbial communities and their activities. BIOLOG analysis indicated that the functional diversity of the microbial community of polluted soils increased and differed from that of unpolluted soils. Likewise, the average rate of color development (average well color development), microbial biomass (measured as DNA concentration), and the soil CO₂ respiration were higher in polluted soils. These results indicate that major changes in the chemical and physical properties of paddy soils following the application of industrial wastewater effluents have had lasting impacts on the microbial communities of these soils. Thus, the increased activity, biomass, and functional diversity of the microbial communities in polluted soils with elevated salinity, sodicity, and heavy metal contents may be a key factor in enhancing the bioremediation process of these heavily polluted paddy soils.     

Effects of copper on the decomposition of plant residues,microbial biomass,and β-glucosidase activity in soils

Abstract

Two types of soils (Brown Lowland soil and Ando soil), which were artificially enriched with different amounts of Cu, were incubated with or without pulverized orchard grass for 12 weeks at 25°C. For both soils with and without orchard grass amendment, the amount of CO₂ evolved over the 12-week period of incubation decreased by the enrichment with Cu at a concentration exceeding 1,000 mg kg^?1 soil. The decrease of the mineralization of added orchard grass in the Cu-enriched soil was conspicuous especially during the initial period of incubation. The amount of microbial biomass C at the end of the incubation was significantly reduced by the Cu enrichment regardless of the amendment with orchard grass. The relative decrease of the soil microbial biomass was much greater than that of the soil respiration. The amount of biomass C was negatively correlated with the amount of 0.1 M CaCl₂-extractable Cu as a logarithmic function. On the other hand, the β-glucosidase activity at the end of the incubation was not significantly affected by the presence of Cu in the soils without orchard grass amendment and increased with the increase in the amount of enriched Cu in the orchard grass-amended soils.