Microbial community structure and resource availability drive the catalytic efficiency of soil enzymes under land-use change conditions

The turnover of nutrients bound to organic matter is largely mediated by extracellular hydrolytic enzymes (EHEs) produced by soil microorganisms. However, little is known about the environmental drivers (e.g., soil pH, C content, C:N ratio) of the catalytic properties of EHEs and their functional link to the structure of soil microbial communities. We linked catalytic properties, K_m and V_max, determined by Michaelis–Menten kinetics, to a set of environmental and microbial properties in the soils of a land-use sequence ranging from undisturbed natural forest to pastures of different ages and to secondary succession in the Andes of southern Ecuador. The sensitivity of the substrate affinity constant (K_m) and the maximum rate (V_max) of six EHEs (β-cellobiohydrolase (CBH), β-glucosidase (BG), N-acetylglucosaminidase (NAG), α-glucosidase (AG), xylanase (XYL), acid phosphomonoesterase (AP)) to changing environmental conditions was tested by fluorogenic substrates. We used the V_max-to-K_m ratio (K_a) as a proxy for the catalytic efficiency and the signature membrane phospholipid fatty acids as a proxy of microbial community structure.Microbial communities adapted to environmental changes, selected for enzymes with higher substrate affinity (K_m) and catalytic efficiency (K_a) compared with pure cultures. Along the land-use sequence, catalytic efficiency increased from natural forest to young pasture, while it decreased during long-term pasture use and secondary succession. This is consistent with three to five times faster turnover of tested substrates (estimated based on Michaelis–Menten kinetic parameters) at the young pasture compared with the long-term pasture and secondary succession. Environmental drivers of the K_m were enzyme-specific (e.g., the pH for XYL, the C:N ratio for AP, and the C availability for NAG) and differed from those for V_max. A decoupled response of V_max and K_m to land-use changes observed for AG, BG, CBH, XYL, and AP, implies divers consequences for ecosystem processes mediated by these enzymes. A high abundance of Gram(−) bacteria triggered the catalytic properties (K_m and/or K_a) of enzymes decomposing cellulose, hemicellulose, starch, and monophosphoesters. The importance of climatic factors for catalytic properties of EHEs was emphasized by the K_a values extracted from the literature and demonstrated good correspondence of K_a between soils from geographically distinct experimental plots.     

Method for the estimation of nutrient uptake kinetic parameters using nonparametric statistics

The uptake of many plant nutrients has been shown to follow Michaelis‐Menten enzyme kinetics, and as a result several methods of collecting and analyzing uptake data have been developed. The method proposed here consists of a continuously flowing hydroponic system and a method of data analysis that estimates a value of K_mand V_max for each plant. The method is nondestructive; it does not require large amounts of space; and the plants are near steady‐state uptake. In simulated experiments with various assumptions about variability in the data, the nonparametric statistical analysis of the results provided estimates as good as or better than regression analysis estimates of the two parameters of the Michaelis‐Menten equation.     

Parameter estimation for models of ligninolytic and cellulolytic enzyme kinetics

While soil enzymes have been explicitly included in the soil organic carbon (SOC) decomposition models, there is a serious lack of suitable data for model parameterization. This study provides well-documented enzymatic parameters for application in enzyme-driven SOC decomposition models from a compilation and analysis of published measurements. In particular, we developed appropriate kinetic parameters for five typical ligninolytic and cellulolytic enzymes (β-glucosidase, cellobiohydrolase, endo-glucanase, peroxidase, and phenol oxidase). The kinetic parameters included the maximum specific enzyme activity (V_max) and half-saturation constant (K_m) in the Michaelis–Menten equation. The activation energy (Ea) and the pH optimum and sensitivity (pH_opt and pH_sen) were also analyzed. pH_sen was estimated by fitting an exponential-quadratic function. The V_max values, often presented in different units under various measurement conditions, were converted into the same units at a reference temperature (20 °C) and pH_opt. Major conclusions are: (i) Both V_max and K_m were log-normal distributed, with no significant difference in V_max exhibited between enzymes originating from bacteria or fungi. (ii) No significant difference in V_max was found between cellulases and ligninases; however, there was significant difference in K_m between them. (iii) Ligninases had higher Ea values and lower pH_opt than cellulases; average ratio of pH_sen to pH_opt ranged 0.3–0.4 for the five enzymes, which means that an increase or decrease of 1.1–1.7 pH units from pH_opt would reduce V_max by 50%. (iv) Our analysis indicated that the V_max values from lab measurements with purified enzymes were 1–2 orders of magnitude higher than those for use in SOC decomposition models under field conditions.     

Ferric reduction by geum urbanum: A kinetic study

The reduction capacity for ferric chelates of Geum urbanum L. showed a marked increase when plants were grown under conditions of iron‐shortage. Ferric ethylenediaminetetraacetate (FeEDTA) was reduced with a pH optimum between 5 and 6. The reaction exhibited a low substrate affinity with a K_m much higher than the expected concentration range of soluble iron in the soil. Analysis of the saturation plots conform to Michaelis‐Menten kinetics. Both V_max and K_m values varied to a broad extent with changing assay and plant culturing conditions. Ferricyanide was reduced at significantly higher rates than FeEDTA and inhibited the reduction of FeEDTA compete‐tively. The kinetic characteristics of iron reduction by plants will be discussed in terms of ecological significance as part of an adaptation to the soil conditions.     

Effect of different treatments with crop residues on soil phosphatase activity

Summary Phosphomonoesterase (both acid and alkaline) and phosphodiesterase activity was either activated or inhibited in a soil treated with different crop residues. Phosphotriesterase activity remained unaffected. The kinetic parameters (V _max and K _m) of treated soil samples were modified in the same way: Increases or decreases in the V _max values corresponded to increases or decreases in the K _m values. The V _max values, rather than the K _m values, were found to have a predominant effect on phosphatase activity, thus indicating a fundamental role for the enzyme concentration. A positive and generally significant correlation was found between the activity of each phosphatase, which suggests an unspecific source of these enzymes. The values of the determination coefficients (R ² × 100) show that a low percentage of the variability may be ascribed to interactions among phosphatase activities.     

Iron and zinc absorption characteristics and copper inhibitions in cucurbitaceae

Iron and Zn absorption, interactions, and Cu inhibitions were characterized in cucumber (Cucumis sativus L.), watermelon (Citrullus lanatus Thunb.), and pumpkin (Cucurbita moschata Poir.) by kinetic parameters V_max and K_m. Influx and V_max values for Fe and Zn absorption decreased in each species as plant age increased. For the Michaelis constant, K_m, Fe values in cucumber and watermelon and Zn values in watermelon and pumpkin were relatively unchanged with increased plant age. K_m values for Zn absorption in cucumber and Fe absorption in pumpkin decreased as plant age increased. Among species, watermelon appeared to have a particularly effective uptake mechanism for Zn at low solution concentrations. Non‐competitive inhibition of Zn absorption by Fe (20, 50 uM) was indicated in each species. Iron uptake in pumpkin was inhibited non‐competitively by Zn (5, 10 uM), however no significant effects of Zn on Fe absorption were evident in either watermelon or cucumber. Copper (0.5, 1, 5 uM) inhibited uptake of Fe non‐competitively and Zn competitively in each species.     

A microplate fluorimetric assay for the study of enzyme diversity in soils

A fluorimetric microplate enzyme assay has been developed to study enzyme diversity in soil as an approach to understanding functional diversity. The microplate assay allows a large number of soil samples and/or enzymes to be analysed in a short time. The substrates used are conjugates of the highly fluorescent compounds 4-methylumbelliferone (MUB) and 7-amino-4-methyl coumarin (AMC). The main advantage of using fluorimetrically-labelled substrates is that product formation can be measured directly in the microplate without previous extraction and purification of the product. A detailed protocol for this new technology is presented and some potential applications are outlined. A comparative study was carried out between the new microplate fluorimetric assay and a standard colorimetric enzyme assay based on p-nitrophenyl substrates. The kinetics of β-glucosidase and phosphatase activities were investigated in soils with different fertilizer backgrounds. Both methods generated similar values for V_max (maximum rate of activity) whereas the affinity of β-glucosidase and phosphatase for their respective substrates (as indicated by K_m values, Michaelis–Menten constant) was up to two orders of magnitude greater for the 4-methylumbelliferyl substrates compared to the p-nitrophenyl substrates.     

Influence of natural and synthetic humic substances on the activity of urease

The activity of a purified urease, obtained from Bacillus pasteurii, was inhibited by humic and fulvic acids obtained from an agricultural soil. Enzyme kinetic studies showed that the humic substances affected the affinity of the enzyme for its substrate (K_m) and the maximum velocity of the reaction (V_max). The V_max was inhibited to the same extent by both humic (HA) and fulvic (FA) acids, the precise effect depending on the pH and concentration of humic substance. At pH 4.0, HA concentrations of 25 pg cm^?3 and 10 μg cm^?3 inhibited the V_max by 38.5% and 20% respectively. HA and FA had similar effects on the K_m but in this case the lowering of the affinity of the enzyme for its substrate was not concentration dependent in the range 0–25 μg cm^?3 of humic substance. Typically, the affinity was decreased from a K_M of 50 mM in the control to 67 mM in the presence of HA and FA. The effects were not due primarily to the ash or N contents of the humic substances because de-ashed humic acid and synthetic model humic (made from catechol, guaiacol, pyrogallol, resorcinol and protocatechuic acid) and fulvic acid (made from polymaleic acid), containing virtually no ash or N, were equally as effective. The effect was not related to the phenolic monomers which, before polymerization, had no effect on urease activity.     

Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle-size fractions

The location of extracellular enzymes within the soil architecture and their association with the various soil components affects their catalytic potential. A soil fractionation study was carried out to investigate: (a) the distribution of a range of hydrolytic enzymes involved in C, N and P transformations, (b) the effect of the location on their respective kinetics, (c) the effect of long-term N fertilizer management on enzyme distribution and kinetic parameters. Soil (silty clay loam) from grassland which had received 0 or 200 kg N ha⁻¹ yr⁻¹ was fractionated, and four particle-size fractions (>200, 200-63, 63-2 and 0.1-2 μm) were obtained by a combination of wet-sieving and centrifugation, after low-energy ultrasonication. All fractions were assayed for four carbohydrases (β-cellobiohydrolase, N-acetyl-β-glucosaminidase, β-glucosidase and β-xylosidase), acid phosphatase and leucine-aminopeptidase using a microplate fluorimetric assay based on MUB-substrates. Enzyme kinetics (V_max and K_m) were estimated in three particle-size fractions and the unfractionated soil. The results showed that not all particle-size fractions were equally enzymatically active and that the distribution of enzymes between fractions depended on the enzyme. Carbohydrases predominated in the coarser fractions while phosphatase and leucine-aminopeptidase were predominant in the clay-size fraction. The Michaelis constant (K_m) varied among fractions, indicating that the association of the same enzyme with different particle-size fractions affected its substrate affinity. The same values of K_m were found in the same fractions from the soil under two contrasting fertilizer management regimes, indicating that the Michaelis constant was unaffected by soil changes caused by N fertilizer management.     

Characterization of extracellular phosphatase activities in periphytic biofilm from paddy field

Periphytic biofilms exist widely in paddy fields, but their influences on the hydrolysis of organic phosphorus(P) have rarely been investigated. In this study,a periphytic biofilm was incubated in a paddy soil solution, and hydrolysis kinetic parameters(half-saturation constant(Km) and maximum catalytic reaction rate(Vmax)), optimal environmental conditions, substrate specificity, and response to different P regimes of the phosphatase activities in the periphytic biofilm were determined, in order to characterize extracellular phosphatase activities in periphytic biofilms from paddy fields. The results indicated that the periphytic biofilm could produce an acid phosphomonoesterase(PMEase), an alkaline PMEases, and a phosphodiesterase(PDEase). These three phosphatases displayed high substrate affinity, with Km values ranging from 141.03 to 212.96 μmol L^-1. The Vmax/Km ratios for the phosphatases followed the order of alkaline PMEase > acid PMEase > PDEase, which suggested that the PMEases, especially the alkaline PMEase, had higher catalytic efficiency. The optimal pH was 6.0 for the acid PMEase activity and 8.0 for the PDEase activity, and the alkaline PMEase activity increased with a pH increase from 7.0 to 12.0. The optimal temperature was 50℃ for the PMEases and 60℃ for the PDEase. The phosphatases showed high catalytic efficiency for condensed P over a wide pH range and for orthophosphate monoesters at pH 11.0, except for inositol hexakisphosphate at pH 6.0. The inorganic P supply was the main factor in the regulation of phosphatase activities. These findings demonstrated that the periphytic biofilm tested had high hydrolysis capacity for organic and condensed P,especially under P-limited conditions.     

Ermittlung kinetischer Parameter der sauren Phosphatasen intakter Zuckerrübenwurzeln bei variierter Phosphaternährung

Determination of kinetic parameters of acid phosphatases in intact sugar beet roots of variable phosphorus nutrition Organically bound phosphorus has to be hydrolysed before its P can be taken up by plants. Both microbes and plant roots possess phosphatases, which could be of importance especially in soils with low concentrations of inorganic phosphorus in the soil solution. This could be the reason why nutrient uptake models underestimate the P-uptake by plants when P-mobilization by the phosphatases of roots is not taken into consideration. Therefore the activity of acid phosphatases (P_ase) was determined to answer the following questions: 1) To which extent does the root bound acid phosphatase (P_ase) follow Michaelis-Menten kinetics? 2) By which of the four linear transformations of the Michaelis-Menten equation (Lineweaver/Burk, Hanes, Eadie/Hofstee, Eisenthal/Cornish-Bowden) can plausible values of V_max and K_m be determined? 3) Which effect has the P nutrition of the plant on these kinetic parameters? Sugar beet plants were grown in full nutrient solution containing 1 and 100 μM P respectively. The P_ase activity of the intact roots was measured at pH 5 using p-nitrophenylphosphate (25—15000 μM p-NPP). V_max values were calculated per m root length. Acid phosphatase activity principally followed Michaelis-Menten kinetics. Transformations and calculations of V_max and K_m after Eadie/Hofstee and Eisenthal/Cornish-Bowden suggested the existence of at least two enzyme systems (P_{ase 1}, P_{ase 2}). The following kinetic parameters were found: P_{ase 1}: P deficient plants: V_max: 43—45 nmol m^—1 min^—1, K_m: 31—37 μM NPP; P sufficient plants: V_max: 7 nmol m^—1 min^—1, K_m: 47—53 μM NPP. P_{ase 2}: P deficient plants: V_max: 230—293 nmol m^—1 min^—1, K_m: 1579—3845 μM NPP; P sufficient plants: V_max: 123—171 nmol m^—1 min^—1, K_m: 3027—7000 μM NPP. Thus plants with sufficient P nutrition have a lower affinity to P_org and a lower hydrolysis of P_org. For P nutrition of crops P_{ase 1} might be the most important enzyme.     

Correlations between Kinetic Parameters of Phosphate Uptake and Internal Phosphorus Concentrations in Maize Plants: Making it Possible to Estimate the Status of Phosphate Uptake according to Shoot Phosphorus Concentrations

Abstract

The relationship between internal phosphorus (P) concentration [P] and kinetics of phosphate (Pi) uptake was investigated in maize seedlings grown hydroponically at different Pi concentrations (0.1–1,000 µM) and in the phase of Pi deprivation (0–10 d). The results indicated when the internal [P] was higher than 85 µmol g^?1 dw, apparent K_m, C_min, and V_max were significantly (P<0.01) related to [P]s in shoots and roots; when the internal [P] was lower than 85 µmol g^?1 dw, K_m and C_min were small and only V_max was significantly related (P<0.01) to internal [P]s. Three equations were deduced from the linear regressions of the kinetic parameters and [P]s in shoots. Using these equations, the values of apparent K_m, C_min, and V_max of Pi uptake of seedlings grown in different circumstances were calculated according to [P]s in shoots. In all the circumstances involved, for K_m and C_min, there was a parallel relationship between the values estimated by [P]s in shoots and by the Pi‐depletion technique; for V_max, the values estimated by [P]s in shoots were consistent with those obtained from Pi‐depletion experiments except the period of supplying Pi to the Pi‐starved seedlings over several days. These results indicated it is possible to estimate the Pi‐uptake status according to shoot P concentrations in maize plants under experimental conditions, which might be helpful to estimate in‐season status of Pi uptake of maize plants in the field.     

The effect of soil pH and high urea concentrations on urease activity in soil

The rate of hydrolysis of urea in soil over the wide range of concentrations, up to 10 moles N per dm³ soil solution, found in fertilizer practice, was examined in Begbroke sandy loam adjusted to different pH values. On rewetting air-dry soil, urease activity increased rapidly, reached a maximum within the first 24 h and then decreased slowly to level off after about 4 days. Pretreatment of the soil with urea or ammonium had no effect on the urease activity. Urease activity increased with substrate concentration, reached an optimum value and then decreased with rising urea concentration. The results could be explained by substrate inhibition at higher urea concentrations, and the data are well described by a modified Michaelis-Menten equation involving three parameters, V_max, K_m and K_i where K_i is an inhibition constant. K_m decreased linearily with rise in pH whereas K_i increased slightly between pH 4.9 and 7.0 and steeply between 7.0 and 8.4. V_max increased with rise in pH, reached a maximum value at pH 6.0 and then declined at higher pHs. There was a further reaction, reaching a maximum rate at a urea concentration of about 0.2 molar N in the soil solution, that followed Michaelis-Menten kinetics. K_m for this high affinity reaction increased up to pH 7.2 and then decreased at higher pH values; V_max increased up to pH 6.8 and then decreased. The contribution of the high affinity reaction was small except at low concentrations of urea.     

Kinetic behaviour of alkaline phosphatase desorbed from different clay minerals

The kinetic (K_m, V_max) of alkaline phosphatase (AP) desorbed from different Ca-homoionic clay minerals (montmorillonite, illite, and kaolinite) by extraction with Tris-Malate-Citrate buffer solution (pH 9.6) was studied in model experiments. After extraction (shaking for 15 min.) the K_m and V_max were measured in the extract, the remaining sediment and in the whole set-up. With kaolinite and illite, V_max of the desorbed AP was lower than that of the sediment. However, with montmorillonite, V_max of AP in the extract and whole system increased if compared to the control, but decreased in the sediment. The K_m of desorbed AP increased from 4.3 × 10^?3 (control) to 5.0 × 10^?3 M (illite), 5.4 × 10^?3M (kaolinite), and 5.5 × 10^?3M (montmorillonite). These values were lower than those obtained with the various sediments and whole experimental systems. An aberrant behaviour was recorded with the illite sorbed AP which showed an increase in affinity towards the substrate. Generally speaking, AP desorbed from clays may be reduced in its affinity towards the substrate p-nitrophenylphosphate by residual inhibitor and/or conformational change of the enzyme.     

Feldmethode zur Bestimmung der substrat‐induzierten Bodenatmung

A field method for the measurement of substrate‐induced soil respiration A novel method for in situ measurements of microbial soil activity using the CO₂ efflux combined with kinetic analysis is proposed. The results are compared with two conventional, laboratory methods, (1) substrate‐induced respiration using a ’︁Sapromat’ and (2) dehydrogenase activity. Soil respiration was measured in situ after addition of aqueous solutions containing 0 to 6 g glucose kg^—1 soil. The respiration data were analysed using kinetic models to describe the nutritional status of the soil bacteria employing few representative parameters. The two‐phase soil respiration response gave best fit results with the Hanes' or non‐parametric kinetic model with Michaelis‐Menten constants (K_m) of 0.05—0.1 g glucose kg^—1 soil. The maximum respiration rates (V_max) were obtained above 1 g glucose. Substrate‐induced respiration rates of the novel in situ method were significantly correlated to results of the ’︁Sapromat’ measurements (r² = 0.81***). The in situ method combined with kinetic analysis was suitable for the characterisation of microbial activity in soil; it showed respiration rates lower by 59% than measured in the laboratory with disturbed samples.     

Fluctuations of formamide hydrolase activity in a barley field soil after drying and rewetting

Formamide hydrolase activity in a barley field soil was described by a two-component, rather than a one-component model of Michaelis–Menten kinetics. The two-component model had both a high-affinity (K_m values of 0.5–1.0 mM) and a low-affinity (K_m values of 30–60 mM) activity. Rapid and transient increases in both the overall heterotrophic activity (CO₂ production) and the high-affinity component of formamide hydrolase activity were observed in rewetted soil samples, suggesting that the high-affinity component represented enzyme activity of active microorganisms. Short-term field variations in the high-affinity component during a barley growth season were consistent with rapid, transient responses of microbial activity to precipitation and soil wetting. An assay of the high-affinity component of formamide hydrolase activity as approximated by rate determination at a single low (1 mM) formamide concentration can provide an easy and rapid indication of enzyme activity related directly to active microorganisms in the soil.     

Nitrate assay by multiple wavelength spectroscopy and analysis of kinetic parameters of net nitrate uptake by barley seedlings

A method for determination of nitrate concentration and estimation of kinetic parameters of nitrate uptake by spectroscopy based on absorbances at multiple wavelengths has been developed to estimate nitrate uptake by barley (Hordeum vulgare L. cv. Steptoe) seedlings. Nitrate concentration in the nutrient solution was determined from the slope of the linear regression line of the absorbances to nitrate absorption coefficients at 12 wavelengths. Interference by root exudates was only due to an absorption component changing with wavelength in correlation with nitrate absorption. The standard error of the determination decreased in reverse proportion to the square root of the number of the wavelengths. A linear form of the net uptake equation, NUR = I_maxC/K_m+C‐E, could be expressed as NUR = (I_max ‐ E) ‐ K_m NUR/C ‐ K_mE I/C where NUR is net uptake rate, Imax is maximum influx, C is concentration, Km is the Michaelis constant, and E is an efflux constant. The method described here was used to determine the time course of nitrate depletion by barley seedlings from their nutrient solution. The isotherm of net nitrate uptake rates derived from the time course was analyzed after modifications based on the linear form of the net uptake equation. The analysis yielded highly significant results (P<0.0001).     

A Practical Spectrophotometric Approach for the Determination of Lipoxygenase Activity of Durum Wheat

In this study, a practical spectrophotometric approach was used to determine the hydroperoxidation activity of durum wheat lipoxygenase (LOX). As stated in the related literature, the buffered linoleic acid solution used as the reaction medium is not optically clear enough at neutral and lower pH values due to its limited solubility. In our study, the optical clarity was obtained by the formation of sodium-salt of unreacted linoleic acid just before absorbance measurement. The durum wheat LOX was characterized in terms of pH and temperature optima as well as kinetic parameters. The maximum linoleic acid hydroperoxidation activities were determined at pH 5.0 and 6.5 and at 40°C This result can be considered as evidence for the presence of at least two LOX isoforms with respective optima at pH 5.0 and 6.5 in the crude durum wheat extract. The Michaelis constant (K_m) and maximum hydroperoxidation) activity rate (V_max) of durum wheat LOX for linoleic acid were estimated) to be 0.131 ± 0.019 mM and 42.37 ± 3.32 units/mg of protein/min, respectively. The method seems to be useful for the determination of LOX activity in durum wheat and its milling fractions.     

Fire interacts with season to influence soil respiration in tropical savannas

Soil respiration (R_s) is the second-largest source of CO₂ to the atmosphere in terrestrial systems. In tropical savannas seasonal moisture availability and frequent fires drive ecosystem dynamics and may have a considerable impact on soil carbon (C) cycling, including R_s. In order to test the effect of fire on soil C cycling we measured R_s in annually burnt and unburnt plots in wet and dry seasons at a long-term fire experiment established in savanna woodlands of northern Australia. There was a significant interaction between season and fire, with highest rates of daily R_s (722 mmol CO₂ m⁻² d⁻¹) observed in the wet season on unburnt, leaf litter patches. The three fold higher R_s rate on unburnt plots in the wet season was due to greater root-derived respiration (R_root: 356 mmol CO₂ m⁻² d⁻¹), while smaller changes to soil-derived respiration (R_soil: 51 mmol CO₂ m⁻² d⁻¹) were simply the result of C moving through decomposition rather than combustion pathways. Relationships between instantaneous R_s and soil temperature showed hysteresis with variable direction, suggesting that season and fire treatment also influence the soil depth at which CO₂ is produced. We suggest that (1) changes to fire regimes, through active management or climate change, in tropical savannas could have an impact on R_s, and (2) the direct effect of fire on soil C cycling is limited to the removal of aboveground litter inputs.     

A comparison of methods for estimating phosphorus uptake kinetics under steady‐state conditions 1

Michaelis‐Menten kinetic parameters (I_max and K_M) are useful for describing nutrient uptake by plants. This paper compares two methods for estimating the kinetics of P uptake. Both methods employed a steady‐state hydroponic system to measure P uptake by wheat (Triticum aestivum L.) seedlings. In one method, uptake was measured from two P concentrations in nutrient solution, with I_max and K_M estimated by direct linear plot (DLP). In an alternate, multiple concentration (MC) method, uptake was measured from five P concentrations, and kinetic parameters were estimated by either nonlinear regression or the Hanes plot. The I_max and K_M, estimates obtained by the DLP method were compared to those obtained by the MC method. The MC method offered practical advantages. Unlike the DLP, it allowed estimation of the external P concentration at which net influx = 0 (C_min), and did not require a priori estimates of K_M and C_min. The MC method provided more precise median parameter estimates as indicated by smaller nonparametric confidence intervals. Using the median C_min value of 1.9 μM, the best estimates of I_max and K_M (and 96% confidence intervals) derived by nonlinear regression were 2.2 (1.6 to 2.8) nmol P g^‐1s^‐1, and 11 (10.6 to 12.9) μM, respectively.