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Biochar application changed arylsulfatase activity, kinetic and thermodynamic aspects
European Journal of Soil Biology 95 (2019) 103134
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Biochar application changed arylsulfatase activity, kinetic and thermodynamic aspects
T
Allahyar Khadema,∗, Hossein Besharatib, Mohammad Ali Khalajc a
Department of Soil Science and Engineering, Faculty of Agriculture, Shahrekord University, P.O. Box 115, Shahrekord, Iran Agricultural Research, Education and Extension Organization (AREEO), Soil and Water Research Institute, Karaj, Iran c Department of Technology and Production Management, Ornamental Plants Research Center (OPRC), HSIR, AREEO, Iran b
A R T I C LE I N FO
A B S T R A C T
Handling editor: Bryan Griffiths
The aim of this research was to evaluate the response of soil arylsulfatase (ARS) kinetic and thermodynamic parameters to biochar application. The kinetic parameters including Vmax, Km and catalytic efficiency (Vmax Km−1) as well as thermodynamic ones (Ea, ΔHa and Q10) were determined in two different textured clayey and sandy loam soils amended with 0.5 and 1.0% (w/w) of unpyrolyzed maize residue (UMR) or its biochars prepared at 200 and 600 °C (B200 and B600, respectively). The results showed that the application of amendments to sandy loam soil increased enzymatic activity (16–106%), Km (37–113%) and the enzyme concentration (Vmax) (13–39%), whereas the Vmax Km−1 (38–86%), Ea (6–21%) and Q10 (8–11%) were decreased in the biocharamended soils compared to the unamended control (CK). In clayey soil, biochar addition increased ARS activity (16–150%) and decreased the Vmax (10–41%), Vmax Km−1 (63–355%), Ea (20–63%) and Q10 (10–32%) and had no significant effect on Km. Generally, the effects of the amendments application were greater at 1% than 0.5% application rate and in clayey than sandy loam soil. Soil texture is a determinant factor in soil arylsulfatase response (activity, kinetics and thermodynamics) to biochar application.
Keywords: Biochar Arylsulfatase Kinetic parameters Thermodynamic
1. Introduction Soil microbial metabolic processes are mediated by the enzymes [1] who's their activities reflect the soil health [2]. In fact, the soil enzyme activities have strong correlation with soil fertility [3], microbial activity and cycling of elements in soil [4] and fluctuates with management practice [5]. Arylsulfatase is a vital soil enzyme because of its function in sulfur cycling and sulfate availability for plant uptake [6]. This enzyme catalysis the hydrolysis of aromatic sulfate esters (R-OSO3) to phenols and release the sulfate for uptake by plants or microbes [7,8]. Biochar, the product of the pyrolysis of C-based feedstocks has attracted many interests as ‘soil conditioner’ in recent years [9,10]. It is well proved that biochar can affect soil physical, chemical and biological properties [9,11] and hence the soil enzyme activity [10,12]. However, the effects of biochar application on arylsulfatase activity was weak and depended on type of soil and biochar application rate [5,13]. Although there are numerous researches on the effects of biochar on soil enzyme potential activity, the main focus of these studies was on the apparent activity of enzymes so, its effect on the kinetic and thermodynamic indices of enzymes is scarcely studied and remained as research priority [9,10,14].
∗
Kinetic parameters of soil enzymes including maximum velocity (Vmax) and Michaeilis-Menthen constant (Km) indicate the splitting velocity of enzyme-substrate complexes into enzyme and the reaction products and reflect the conjunction affinity between enzyme and substrate, respectively [15]. These parameters make a linkage between the enzymatic reaction rate and substrate concentration [16–19]. The study of these parameters can promote our knowledge regarding the status of the active enzyme and catalytic reaction, because of providing important information on the nature, stability of the enzyme and enzyme-substrate complex [18]. Km is an indication of the affinity of the enzyme for its substrate and enzyme efficiency. The changes in Km (i.e., high or low substrate affinity) can indicate an increase or decrease in the overall enzyme function [19]. The Vmax parameter represents the total concentration of an enzyme [16,18,19]. The results of previous studies indicated that the Vmax and Km values for soil enzymes changed with management practice including the application of organic amendments such as crop residues [16] and biochar Jin, 2010; [20]. To our knowledge, there is no study regarding the effects of biochar on soil arylsulfatase kinetic parameters. The better understanding of the thermodynamic parameters of soil enzymes including activation energy (Ea) and temperature coefficient
Corresponding author. E-mail address: [email protected] (A. Khadem).
https://doi.org/10.1016/j.ejsobi.2019.103134 Received 8 May 2019; Received in revised form 24 September 2019; Accepted 26 September 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
European Journal of Soil Biology 95 (2019) 103134
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produced at 200 °C (B200) and 600 °C (B600) were used in two soil types (sandy loam and clayey texture). A sample of each soil type without UMR and maize biochars was also included as the negative control (CK). The air-dried soils (300 g) were mixed with UMR and its biochars at 0.5 and 1% (w/w) thoroughly in plastic jars. Deionized water was used to adjust soil moisture content at 70% water holding capacity. The jars were incubated for 90 days at 25 ± 1 °C. The soil moisture content was maintained constant.
(Q10) has necessity in studying the temperature sensitivity of soil enzymes following temperature fluctuations and global warming [21,22]. In addition, thermodynamic parameters provide an insight regarding the thermal and proteolytic stability of enzymes, as well their resistance to environmental changes [23,24]. At present, there is scarce information on how biochar affects the thermodynamic characteristics of soil enzymes. In the only one related study [25], studied the effects of biochar on thermodynamic parameters of soil enzymes and reported that biochar application reduced Ea and Q10 values for several soil hydrolases. Their observed results were ascribed to the release of different isozymes or the conformation changes due to enzyme adsorption on biochar surfaces. Hence, the main objectives of the current study were to (1) quantify the effects of maize biochars produced at 200 °C and 600 °C on the activity, kinetic and thermodynamic parameters of soil arylsulfatase, (2) compare the ARS activity, kinetic and thermodynamic characteristics between the soils amended with uncharred feedstock and its biochars; and (3) establish whether biochar effects on enzyme activity, kinetic and thermodynamic parameters would depend on soil texture.
2.3. Soil chemical and microbial analysis
2. Materials and methods
The soil organic carbon (SOC) content were determined according to the method of [27]. Soil microbial biomass carbon (MBC) determined as a measure of total microbial biomass using the fumigation-incubation method and a Kc value of 0.45 [28]. Substrate-induced respiration (SIR), as indicator of metabolically active microbial biomass, was determined using 1% (w/w) glucose solution as the substrate [29]. Total microbial population determined according to the method introduced by Ref. [30] basis on serial dilution and enumerating the colony forming unit (CFU) on plate culture by most probable number (MPN) tables.
2.1. Soil and biochar analysis
2.4. Enzyme activity, kinetic and thermodynamic parameters
Two sampling sites were selected at 50° 15′ 1.2″ N, 35°44′ 11.7″ E and 50° 42′ 27.2″ N, 35° 49′ 4.03″ E in Alborz province in Iran. The soils classified as Typic Haplocalcid and has not been cultivated or fertilized for three years. The mentioned soil characteristics was reported by Ref. [26]. The biochar was produced from maize stalks over a 2 h period by slow pyrolysis process at 200 and 600 °C in a thermal furnace. The results of soils and biochars analysis is presented in Table 1.
The activity of arylsulfatase (EC 3.1.6.1) determined according to the procedure described by Ref. [31] based on the colorimetric determination of the p-nitrophenol (PNP) released in the presence of disodium p-nitrophenyl sulfate (PNPS) as substrate. In brief, 1 g of the soil was incubated for 1 h at 37 °C with 0.25 ml of toluene, 1 ml of 0.25 mM PNPS and 4 ml of acetate buffer (pH 5.8). After the incubation time 1 ml of CaCl2 (0.5 M) and 4 ml of NaOH (0.5 M) was added, shaken, filtered and optical density measured at 400 nm. Soil enzyme activity expressed as μmol PNP released g−1 dry soil h−1. For estimation of the kinetic parameters, eight concentrations of PNPS (0, 0.5, 1.0, 5, 10, 15, 25 and 50 mM) were used as enzyme substrate and kinetic parameters, Km and Vmax values, calculated using the conventional Michaelis–Menten equation [17]:
2.2. Soil incubation test Soil incubation experiment was arranged as a completely randomized factorial design with four replicates. Three soil amendments including Unpyrolyzed maize residue (UMR) and maize biochars
v0 =
Table 1 Selected characteristics of the soils and biochars used in the study [23,24]. Variables
pH EC Ash V.M Fixed C BET Surface Area C O N H C:N O:C H:C H:O (O + N):C CEC Sand Silt Clay
Unit
dS m−1 % % % m2 g−1 % % % %
cmol (+) kg−1 g kg−1 g kg−1 g kg−1
Amendment (B200)
(B600)
Clayey
Sandy Loam
5.67 3.68 8.00 81.0 11.0 5.62 40.8 29.7 1.07 6.65 32.7 0.971 0.0136 0.014 1.00 21.2
6.25 3.67 14.0 68 18 12.4 48.7 29.4 1.61 6.06 25.9 0.805 0.0104 0.0129 0.84 18.6
11.2 4.75 39.0 24 37 88.4 63.0 4.83 1.61 1.76 33.5 0.102 0.0023 0.0228 0.13 11.7
7.68 0.33
8.32 0.37
(1) −1
−1
where ʋo is the initial reaction velocity (μmol PNP g soil h ), [S] is the PNPS concentration (mM), Km is the PNPS concentration at half maximal velocity (mM), and Vmax is the maximal velocity (μmol PNP g−1 soil h−1). The catalytic efficiency factor (ratio between Vmax and Km) also was calculated [15]. Arylsulfatase thermodynamic parameters were estimated using the Arrhenius equation [17]:
Soil Texture
(UMR)
Vmax [S ] Km [S ]
E k = A × exp ⎛− a ⎞ ⎝ RT ⎠ 0.423
0.220
0.034
0.022
12.5
10.0
26.5
8.80
10 380 520
80 100 100
(2)
The linear form of Arrhenius equation was used as follow:
ln k = lnA −
Ea RT
(3) −1
−1
where k is the enzyme activity (μmol PNP g h ) at a given temperature, A is the pre-exponential factor, R is the gas constant (8.314 J K−1 mol−1), Ea is activation energy (kJ K−1 mol−1) and T is the absolute temperature (Kelvin). The potential enzyme activity at temperatures 17, 27, 37 and 47 °C (T), plotted versus 1/T for estimating the Ea. These temperatures were chosen to have 4-point plot for reliable estimation of Ea. In addition, these temperatures contain the standard temperature for enzyme activity determination (37 °C), two lower temperatures (17 and 27 °C) and higher one (47 °C) which approximately reflect the soil temperature fluctuation in warm and cold season. The following equations were used for estimating the activation enthalpy values (ΔHa) and temperature coefficients or sensitivities (Q10),
UMR, Unpyrolysed Maize Residue; B200, Biochar prepared at 200 °C; B600, Biochar prepared at 600 °C; EC, Electrical conductivity; V.M, Volatile Matter; C, Carbon; O, Oxygen; N, Nitrogen; H, Hydrogen; CEC, Cation Exchange Capacity; BET (Brunaer-Emmett-Teller) surface area with N2 adsorption isotherm. 2
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respectively [17,22]: ΔHa = Ea − RT
Q10 =
kT + 10 kT
Table 2 Effects of biochar amendments on soil organic matter (SOM), microbial biomass carbon (MBC), substrate-induced respiration (SIR) and total microbial population (TMP) in the clayey and sandy loam soils.
(4) (5)
Variable
Amendment
Clayey Soil
Sandy Loam Soil
SOC (%)
CK UMR B200 B600 CK UMR B200 B600 CK UMR B200 B600 CK UMR B200 B600
0.48 ± 0.009 D 0.70 ± 0.024 C 0.76 ± 0.016 B 0.89 ± 0.055 A 291.7 ± 6.94 D 699.9 ± 26.4 A 658.6 ± 26.0 B 530.0 ± 14.3 C 23.4 ± 1.06 D 53.0 ± 3.45 A 46.7 ± 2.40 B 36.3 ± 0.68 C 15.0 ± 1.25 C 31.0 ± 1.26 A 24.0 ± 1.14 B 17.0 ± 1.02 C
0.27 ± 0.012 d 0.42 ± 0.020 c 0.57 ± 0.020 b 0.74 ± 0.060 a 206.0 ± 19.2 d 532.3 ± 22.0 a 506.7 ± 19.5 b 438.3 ± 15.6 c 14.4 ± 0.71 c 39.4 ± 2.41 a 37.5 ± 2.38 a 29.4 ± 1.08 b 2.00 ± 0.07 d 13.0 ± 1.15 a 11.0 ± 0.61 b 6.00 ± 0.66 c
2.5. Adsorption of enzyme and its product by biochar To interpret the results of the enzyme activity, the adsorption of arylsulfatase and the product of its activity p-nitrophenol (PNP) on biochar surface was measured by the method described by Refs. [23,32]. The biochar and purified enzyme (Sigma, S9626) mixture was prepared by shaking 10 mg biochar with 4 ml of acetate buffer (MUB, pH 5.8) and 50 μg ml−1 ARS for 15 min, and the mixture was centrifuged at 10000 g for 1 min. The pellet was re-suspended and washed three times with 1 ml of the appropriate buffer to remove any weakly associated ARS, and then re-suspended in 50 ml of buffer solution. The supernatant fractions and washings were collected and the concentration of enzyme in the solution was determined using the Bradford method described by Ref. [33]. For PNP adsorption test, a similar procedure was carried out and the concentration of PNP in the solution was determined as common procedure in enzyme activity determination (Section 2.4). The adsorbed ARS or PNP on biochar surface was measured by subtracting the ARS or PNP that recovered from that added initially.
MBC (mg C kg−1soil)
SIR (mg C–CO2 kg−1soil h−1)
TMP (Colony gr−1) × 106
Each value is mean (n = 4) ± standard errors. Different lowercase letters for the clayey soil and uppercase letters for the sandy loam soil represent significant differences between treatments by LSD's test at α = 0.05. CK, control; UMR, uncharred maize residue; B200, maize biochar obtained at 200 °C; B600, maize biochar obtained at 600 °C.
CK, respectively (Fig. 1). The increase of the enzyme activity in clayey soil were 0.76, 0.16, 0.51-fold at 0.5% and 1.3, 0.54, and 0.55-fold at 1% application rate for UMR, B200 and B600, respectively.
2.6. Statistical analysis The non-linear ligand binding with one site saturation regression equation were used to calculate the enzyme kinetic parameters using the software Sigma Plot 10 for Windows. All data were analyzed for normality by the Shapiro–Wilk test before statistical comparisons with 4 replicates. Significant differences between the treatments were calculated using three factor analysis of variance (ANOVA) and means were separated by least significant difference (LSD) test, at P < 0.05. All statistical tests were carried out using the Minitab 17 software for Windows.
3.3. Enzyme adsorption on biochar surface The results indicated that increasing pyrolysis temperature enhanced the adsorption capacity for ARS (Fig. 2). The ARS adsorption by B200 and B600 biochars was 22 and 24 μg g−1 biochar, that is 3.1 and 3.4 times higher than the UMR (7 μg g−1 biochar), respectively. This result confirms that the sorption of ARS to biochar may cause a partial reduction of its activity, which to some extent is observed in the determination of ARS activity (Fig. 2). In addition, there was no significant difference between the B200 and B600 regarding to their capacity for ARS adsorption (Fig. 2).
3. Results 3.1. Changes in soil chemical and biological properties
3.4. Changes in enzyme kinetic parameters Biochar application significantly increased SOC compared with CK treatments (Table 2). The highest SOC concentration was recorded in the B600 treatment whereas the lowest was in the CK treatment, in both clayey and sandy loam soils. In addition, the SOC content increased with increasing the pyrolysis temperature. The changes in soil microbial attributes were similar for MBC, SIR and TMP in response to the UMR and biochars applied. The amendment addition increased soil microbial attributes over the control by 82–158%, 55–174% and 15–550% for MBC, SIR and TMP, respectively. Increasing the pyrolysis temperature has negative effects on all measured microbial properties, in that way, the increase was smaller in higher temperature biochars (Table 2).
The relationship between the reaction rate and the substrate concentration was hyperbolic for ARS in both soils which indicate that saturation kinetics fitted well the conventional Michaelis-Menten equation (Fig. 3). The interaction of soil texture × amendment type and amendment type × application rate was significant for the Km (Table 3, Fig. 4 a, b). The addition of UMR and biochars to clayey soil had no significant effect on Km. In sandy loam soil, however, the UMR and B200 increased the Km (113% and 37%, respectively) over the CK treatment. Additionally, no significant change was observed in the Km with increasing pyrolysis temperature in clayey soils, whereas in sandy loam soil, B200 and B600 caused significant decrease (35% and 75%, respectively) in Km relative to UMR. The interaction of experimental factors had significant effects on the maximum velocity (Vmax) (Table 3). The addition of UMR and its biochar increased Vmax (10–41%), and the increase was approximately equal in clayey (10–41%) and sandy loam soil (13–39%) and greater at 0.5% (10–44%) than 1% (10–39%) application rate (Fig. 5 a). In general, there was no clear trend in Vmax by increasing the pyrolysis temperature. In clayey soil and at 0.5% application rate, only B600 resulted in an increase (41%) in the Vmax compared to UMR, whereas at 1% application rate, there was no significant difference
3.2. Arylsulfatase activity The ANOVA results showed that the interaction of soil texture, amendments and rate was significant on ARS activity (p < 0.05; Table 3). The addition of UMR, B200 and B600 increased the enzyme activity (0.16–1.5-fold) and the increase was greater in clayey (0.16–1.5) than sandy loam soil (0.16–1.06) and at 1% (0.37–1.5) than 0.5% (0.16–1.06) application rate (Fig. 1). The results showed that in sandy loam soil the addition of UMR, B200 and B600 increased the ARS activity by 1.6, 0.16 and 0.51-fold at 0.5% and 1.5, 0.37 and 0.8-fold at 1% application rate compared with 3
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Table 3 Analysis of Variance (ANOVA) for the effects of soil texture, amendment type and rate and its interactions on activity, kinetic and thermodynamic parameters of soil arylsulfatase. variables
D.F. ARS Activity Vmax Km Vmax Km−1 Ea ΔH Q10
Main Effects
Interactions
Soil Texture (T)
Rate (R)
Amendment (A)
T×R
T×A
A×R
T×A×R
1 12.5 (0.86) ** 2196.9 (90.2) ** 41.6 (26.9) ** 12129.2 (85.9) ** 281.2 (26.3) ** 253.2 (9.54) ** 0.1586 (33.9) **
1 197.3 (13.6) ** 21.5 (0.88) ** 2.40 (1.55) ns 277.4 (1.97) ** 2.51 (0.23) ns 18.7 (1.48) ns 0.0007 (0.11) ns
2 556.2 (76.5) ** 6.62 (0.54) ** 8.52 (11.0) * 78.9 (1.12) ** 100.1 (18.7) ** 1326.4 (68.5) ** 0.0579 (21.9) **
1 0.009 (0.00) ns 4.19 (0.17) * 4.47 (2.89) * 188.1 (1.33) ** 18.6 (3.48) ns 43.9 (1.13) ns 0.0117 (15.8) ns
2 7.09 (0.98) * 1.21 (0.10) ns 9.96 (12.9) * 117.5 (1.66) ** 15.8 (2.96) ns 22.1 (1.14) ns 0.0079 (2.58) ns
2 24.7 (3.40) ** 54.5 (4.48) ** 3.18 (4.11) * 338.3 (4.79) ** 9.10 (30.7) ** 433.4 (17.1) * 0.0026 (0.86) ns
2 8.21 (1.13) * 30.7 (2.52) ** 0.91 (1.18) ns 118.5 (1.68) ** 178.5 (16.7) ** 6.93 (0.36) ns 0.1033 (24.9) *
Error
C.V. (%)
R2 (%)
36 1.46 (3.62) 0.73 (1.08) 1.70 (39.5) 5.90 (1.50) 4.78 (0.89) 4.31 (1.8) 0.0052 (3.8)
– 24.8 25.6 57.9 21.4 12.1 13.7 6.60
– 95.3 98.6 48.5 98.1 69.3 78.2 69.2
D.F., Degree of freedom; ARS, Arylsulfatase; C.V, coefficient of variation; R2, coefficient of determination. **, * and ns are significant at the 1 and 5%, and non-significant, respectively. The numbers in parentheses represent the relative contribution of each variable source in each parameter (that is, the ratio of the sum of squares for that source to the total sum of squares multiplied by 100).
and B600 (13%) reduced the Vmax compared with UMR. The results showed that the interaction of all tested factors was significant for arylsulfatase catalytic efficiency (VmaxKm−1) (Table 3). The addition of UMR and its biochars in sandy loam soil (except for B600 at 1% application rate) decreased (38–86%) the catalytic efficiency compared with the control treatment (Fig. 4 b). In clayey soil, however, the amendment addition increased (63–355%) the VmaxKm−1 and the changes were greater at 1% (63–355%) than 0.5% (33–182%) application rate. 3.5. Changes in thermodynamic parameters The temperature dependence of ARS activity followed the Arrhenius model up to the point of enzyme inactivation (47 °C) (Fig. 6). The results of ANOVA showed that the amendment had significant effects on the activation energy (Ea) of arylsulfatase (p < 0.05) (Table 3). The addition of UMR and B200 to sandy loam soil decreased the Ea (6–21%) and the decrease was greater at 0.5% (17% and 21%) than 1% (6% and 14%) application rate (Fig. 4c.). The addition of biochar to clayey soil increased the activation energy (20–63%), and the increase was greater at 1% (20–63%) than 0.5% (25–45%). The results of ANOVA (Table 3) showed that the only interaction between the amendment type and rate was significant for activation enthalpy (ΔHa) (p < 0.05). The addition of UMR and B600 significantly increased ΔHa, and the increase was greater at 1% than 0.5% application rate (Fig. 3c). In addition, by increasing pyrolysis temperature B600 increased the ΔHa compared to UMR, and the increase was greater at 1% (36%) than 0.5% (18%). The results indicated that interaction of three factors was significant for temperature coefficient (Q10) of arylsulfatase (Table 3). In sandy loam soil, the addition of B200 at 0.5% and 1% and UMR at 0.5% decreased the Q10 by 10%, 8% and 11%, compared to CK, respectively. In clayey soil, the amendments increased Q10 (10–32%), and the increase was greater at 1% (10–32%) than 0.5% (12–22%) (Table 4).
Fig. 1. Potential activity values of aryl sulfatase (ARS) in unamended and amended soils. Values are mean (n = 4) ± standard errors. Different uppercase letters represent significant differences between unamended (CK) and amended soils by LSD's test at α = 0.05. Different lowercase letters represent significant differences between the treatment by LSD's test at α = 0.05. CK, control; UMR, uncharred maize residue; B200; maize biochar obtained at 200 °C; B600; maize biochar obtained at 600 °C.
4. Discussion 4.1. Biochar effects on soil chemical and microbiological properties
Fig. 2. Adsorption of aryl sulfatase (ARS) onto unpyrolysed maize residue and its biochars obtained at 200 and 600 °C. Values are mean (n = 4) ± standard errors. Different letters represent significant differences between biochars by LSD's test at α = 0.05.
Our results indicate the stimulation effects of UMR and its biochars prepared at 200 and 600 °C on SOC and microbial attributes. Biochar, due to its large specific surface area and porous structure, provides suitable microenvironment for colonization of soil microbial communities [14]. Volatile compounds are low molecular weight compounds in biochar which believed as an indicator of the labile components [14]. The presence of volatile compounds and abundant nutrients in the biochars might be involved in the microbial stimulation of soil
between the treatments. The results showed that, the Vmax increased with increasing the application rate of UMR and B200, while the reverse trend was observed for B600. In sandy loam soil at 0.5% application rate, the B200 (17%), and at 1% application rate B200 (11%) 4
European Journal of Soil Biology 95 (2019) 103134
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Fig. 3. Michaelis–Menten plots showing aryl sulfatase activity as a function of substrate concentration measured in unamended and amended soils. Values are mean (n = 4) ± standard errors. CK, control; UMR, uncharred maize residue; B200, maize biochar obtained at 200 °C; B600, maize biochar obtained at 600 °C.
microbes and the carbon molecules [7]. Soil organic amendments such as UMR and biochar could enhance soil enzyme activities by increasing soil organic matter content and therefore, the microbial biomass [9,10]). However, the results of [36] showed a decrease in ARS activity due to biochar application in a period of 90 days and then its activity increased. The initial reduction in enzyme activity was ascribed to the presence of sulfite in soil and its inhibitory effect on the enzyme activity. The increase in enzyme activity after 90 day attributed to the conversion of sulfite to sulfate and reduce its inhibitory effect. The ARS activity showed a distinct change by increasing the pyrolysis temperature. The results indicated that the addition of B200 and B600 reduced the enzyme activity in both clayey (14–38%) and sandy loam (24–46%) soils compared with UMR. This observation indicates that the conversion of raw residues to biochar could have great effects on ARS activity. This finding corresponds with the previous results obtained for alkaline phosphatase [26] and other enzymes [12]. Several potential mechanisms proposed for the declined activity of soil enzyme with increasing pyrolysis temperature which includes structural and chemical changes of biochar [9] and changes in soil microbial composition, soil enzymes immobilization on biochar surface area (Jin, 2010; [14].
following biochar application [26]. Volatile matter content of maize feedstock decreased with increasing pyrolysis temperature, and was 81% for the UMR, 68% for B200 and 24% for the B600 (Table 1). Higher temperature biochars has low volatile matter and high aromaticity, which cause high resistance to microbial decomposition and providing smaller labile organic C for microbial activity and growth [11,26,32].
4.2. Biochar effects on ARS activity The mean ARS activity ranged between 13 and 33 μg PNPg−1h−1 in control and biochar treated soils, which corresponds to the value reported by Ref. [13]. The positive effect of biochar on ARS activity in the current study is in agreement with [34] who reported increases in ARS activity of soils amended with biochar. The possible mechanism behind the increase in enzyme activities following the addition of biochar is attributed to the change in soil physiochemical properties including increased OM content and soil pH, stimulating soil microorganisms to producing extracellular enzymes, co-location of enzyme and substrate as well as soil microbial community changes Jin, 2010; [9,14,26,34]. The higher ARS activity as result of biochar application could be direct effect of increased microbial biomass and activity, because there was a strong correlation between the MBC (r = 0.70, P < 0.001) and SIR (r = 0.74, P < 0.001) values and the ARS activity (Table 5). This correlation suggests that this enzyme is primarily of microbial origin [35]. In addition, there was significant correlation between ARS activity and SOM content (r = 0.34, P < 0.001). SOM plays an important role in maintaining soil enzymes in active forms. This correlation reflected the connection between enzyme substrates, the available energy for soil
4.3. Biochar effects on enzyme kinetic parameters One of the current research objectives was to determine the effect of UMR and biochar on the kinetic indices of soil ARS. The measured Km values (0.5–4.0 mM) are indicative of different soil ARS pools and location, as Km represents sources of the enzymes. The measured values of Km in this study are within the limits reported by Tabatabaei and Bermaner (1971), who suggested that the enzyme affinity to its substrate was partially influenced by the linkage between the enzyme and 5
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Fig. 4. Michaelis-Menten constant (Km) and Activation enthalpy (ΔHa) of aryl sulfatase (ARS) in unamended and amended soils. Values are mean (n = 8) ± standard errors. Different uppercase letters represent significant differences between unamended (CK) and amended soils by LSD's test at α = 0.05. Different lowercase letters represent significant differences between the treatment by LSD's test at α = 0.05. CK, control; UMR, uncharred maize residue; B200; maize biochar obtained at 200 °C; B600; maize biochar obtained at 600 °C.
Fig. 5. Maximum velocity (Vmax), Catalytic efficiency (Vmax/Km) and Activation energy (Ea) of aryl sulfatase (ARS) in unamended and amended soils. Values are mean (n = 4) ± standard errors. Different uppercase letters represent significant differences between unamended (CK) and amended soils by LSD's test at α = 0.05. Different lowercase letters represent significant differences between the treatment by LSD's test at α = 0.05. CK, control; UMR, uncharred maize residue; B200; maize biochar obtained at 200 °C; B600; maize biochar obtained at 600 °C.
organic and inorganic components of soil. The Km value of ARS in clayey soil was smaller than that of sandy loam, indicating the effect of organic matter and clay content on maintaining and increasing the availability of substrate for the enzyme. The increase in Km after the addition of biochar was in agreement with the results of [16] who showed that the Km of ARS significantly changed with the addition of plant residue to soil. According to Quiampix et al. (2002) enzyme adsorption on biochar surface can alter enzyme or active site conformation, and consequently decrease the enzyme affinity to its substrate and increase the Km. Quiquampix et al. (2002) reported that the
immobilization of soil enzymes on biochar can result in change of enzyme conformation and cause a decline of the substrate affinity and increases in Km values. Accordingly, the adsorption of enzyme and its substrate on biochar surface can depending on the enzyme properties, increase the contact between the enzyme and substrates and so lead to an increase in the affinity of the enzyme for substrate (Rao et al., 2000). This result shows that despite enzyme adsorption, there is a strong affinity of ARS for substrate (lower Km) in the clayey soil. The arylsulfatase Vmax was higher in clayey than sandy loam soil, 6
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Fig. 6. Arrhenius equation plot of aryl sulfatase (ARS) in unamended and amended soils. CK, control; UMR, uncharred maize residue; B200, maize biochar obtained at 200 °C; B600, maize biochar obtained at 600 °C.
which indicates more enzymes concentration in clayey soil. The higher concentrations of enzymes in clayey soil can be due to higher content of organic matter and clay, which binds to the enzyme and adsorb it and thus protect the enzyme against degradation by proteases [37]. The results showed that Vmax had a significant correlation with soil organic matter content (r = 0.001, p < 0.001), microbial biomass (r = 0.67, p < 0.001) and microbial population (r = 0.81, p < 0.001). In addition, the higher content of organic matter increased the soil microbial population and biomass due to the provision of the nutritional requirement of microbial community, resulting in more enzyme production in clayey soil. Also, the results of the enzyme adsorption showed that the biochars adsorbed up to 25 μg g−1 purified enzyme. Increasing the Vmax, despite the adsorption of the enzyme, indicating no changes in the enzyme configuration. In addition, there was a significant correlation between the ARS activity and the Vmax values (r = 0.34, P < 0.001), whereas no correlation found between ARS activity and Km, which indicating the prevalence of the Vmax rather than the Km on ARS activity, similar to what previously observed for alkaline phosphatase [26]. The catalytic efficiency of soil enzymes is affected by soil physicochemical properties, substrate accessibility, soil organic matter content, soil microbial stimulation and enzyme production [38]. In general, the addition of biochar resulted in an increase of the Km and the Vmax of sandy soil, but the rate of the increase in maximum velocity was not sufficiently large to compensate the increase of the Km, and therefore decreased the catalytic efficiency of ARS. Therefore, it can be said that in sandy loam soil, the Km has a greater role in the catalytic activity of the enzyme and the contribution of the Vmax is lower. In clayey soil, however, the addition of biochar had no significant effects on Km, while the adsorption of the enzyme or substrate on clay and organic matter, as well as the effect of these soil colloid on microbial population increased the catalytic efficiency of ARS. Overally, the increase of Vmax, Km and catalytic efficiency of arylsulfatase by addition of UMR, B200 and B600 indicates that biochar has no inhibitory effect on enzyme activity and cannot be classified as enzyme inhibitors.
Table 4 Effect of biochar amendment, application rate (0.5 and 1%) and soil texture on soil the temperature coefficient (Q10) of arylsulfatase. Treatment
Sandy Loam Soil
Clayey Soil
0.5%
1%
CK UMR
1.77 (0.047) A 1.57 (0.025) Bd
B200
1.60 (0.047) Bcde 1.69 (0.034) Acd 0.13
1.77 1.71 Abc 1.63 Bde 1.82 Aab
B600 LSD0.05
0.5% (0.047) A (0.053) (0.027) (0.020)
1.56 1.83 Aab 1.75 Ab 1.91 Aa
1% (0.040) B (0.023) (0.044) (0.045)
1.56 1.77 Ab 1.71 Abc 1.75 Ab
(0.040) B (0.040) (0.091) (0.041)
Each data point is mean value of four replications with standard errors. Different uppercase letters in the same column for each application rate represent significant differences between unamended (CK) and amended soils by LSD's test at α = 0.05. Different lowercase letters represent significant differences for two-way interaction terms by LSD's test at α = 0.05. UMR, uncharred corn residue; B200 and B600 corn biochars produced at 200 and 600 °C. Table 5 Pearson correlation coefficients (r) between the kinetic and thermodynamic parameters of ARS and soil attributes (n = 32). Variable
ARS activity
Vmax
Km
Ea
ΔHa
Q10
soil pH SOM Mic POP SIR MBC Vmax Km
−0.36** 0.34** 0.53*** 0.74*** 0.70*** 0.34** 0.19ns
−0.73*** 0.58*** 0.83*** 0.60*** 0.59*** – −0.40**
0.14ns 0.36** −0.24ns 0.02ns 0.01ns −0.37** –
0.05ns 0.33** 0.17ns 0.16ns 0.22* 0.27* −0.24*
0.25* 0.56*** 0.05ns 0.14ns 0.21* 0.26* −0.28*
0.04ns 0.33** 0.17ns 0.15ns 0.21* 0.28* −0.24*
ARS, arylsulfatase; SOM, soil organic matter; Mic POP, microbial population; SIR, substrate-induced respiration; MBC, microbial biomass carbon; Vmax, maximal velocity; Km, Michaelis–Menten constant; Ea, activation energy. ΔHa, activation enthalpy; Q10, temperature coefficient. *P < 0.05. **P < 0.01. ***P < 0.001.
4.4. Biochar effects on enzyme thermodynamic parameters The temperature would affect the enzyme and substrate diffusion, and thus the Km, and Vmax of enzyme reactions. Temperature changes affects the production of enzymes by microbes, as well as the rate of 7
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Declaration of competing interest
enzyme degradation. Activation energy acts as a barrier in the enzyme reaction, and its change indicates surface adsorption by soil particles. The Ea values measured in the current research is within the range of reported by Refs. [15,25]; and smaller than that of [21]. It is concluded from the results of the present study that the addition of biochar to clayey soil with high clay content results in the more stabilization of ARS. However, in sandy loam soil, the addition of UMR and B200 reduced the Ea [39]. attributed the Ea changes to enzyme stabilization by soil organic and inorganic colloids. Their results showed that the greater the enzyme protection due to its association with organic and mineral complexes of the soil, the greater energy barrier that should be overcome during the formation of the enzyme-substrate complex. Also, the results of arylsulfatase adsorption showed that this enzyme was adsorbed on biochar surface, which is consistent with increasing Ea and the catalytic efficiency of the enzyme. On the other hand [25], attributed the decrease in the Ea of arylsulfatase to the production of isozymes, changing the composition of the microbial community, and changing the soil characteristics such as pH, which is consistent with the results obtained in sandy loam soil. In the present study, there was a weak correlation between the Ea and Vmax (r = 0.27 p < 0.01) and Km (r = −0.24, p < 0.01). The addition of the amendment to clayey soil, did not change Km, whereas the Vmax increased. In sandy loam soils, the amendments increased the Km, Vmax and Ea. The increase in Vmax, despite of the increase in Ea, can be attributed to the enzyme stabilization by organic amendments that protect the enzyme from proteases, as well as increase the microbial biomass and the production of enzymes. Higher ΔHa value obtained in B600 treatment suggests more activation energy required for the formation of the transition state and for the products to form [20]. reported increases in ΔHa values for denitrifying enzyme activity with biochar application due to low substrate affinity [26]. found similar results for alkaline phosphomonoesterase. Our finding suggests that higher Ea and ΔHa values with B600 addition would lead to a lower rate of ARS reaction [26]. ascribed the increase in thermodynamic parameters of the enzyme to the adsorption of enzyme to biochar surface. The higher Q10 values in biochar-treated clayey soils indicate that biochar additions would increase the temperature sensitivity and thermal stability of soil ARS. The Q10 values could be attributed to the release of different isoenzymes and a modification of the enzyme conformation, particularly in the enzyme active site essential for binding and catalysis, following enzyme adsorption on biochar surfaces [26]. [25] studied the effects of biochar on the thermodynamic indices of arylsulfatase and reported a decrease in Q10 which corresponds to the situation observed in sandy loam soil. They ascribed the Q10 reduction to the changes in microbial community composition, production of isoenzymes, active site deformation due to biochar adsorption and to some extent the change in soil reaction.
The authors declare that there is no conflict of interests regarding the publication of this paper. References [1] A.D. McLaren, Soil as a system of humus and clay immobilized enzymes, Chem. Scripta 8 (1975) 97–99. [2] S.K. Das, A. Varma, Role of enzymes in maintaining soil health, in: G. Shukla, A. Varma (Eds.), Soil Enzymology. Soil Biology 22, Springer-Verlag Berlin Heidelberg, 2011. [3] E.B. Utobo, L. Tewari, Soil enzymes as bioindicators of soil ecosystem status, Appl. Ecol. Environ. Res. 13 (2015) 147–169. [4] D.J. Burke, M.N. Weintrau, C.R. Hewins, S. Kalisz, Relationship between soil enzyme activities, nutrient cycling and soil fungal communities in a northern hardwood forest, Soil Biol. Biochem. 43 (2011) 795–803. [5] M. Niemi, J. Pöyry, I. Heiskanen, V. Uotinen, M. Nieminen, K. Erkomaa, K. Wallenius, Variability of soil enzyme activities and vegetation succession following boreal forest surface soil transfer to an artificial hill, Nat. Conserv. 8 (2014) 1–25. [6] Y. Jin, X. Liang, M. He, Y. Liu, G. Tian, J. Shi, Manure biochar influence upon soil properties, phosphorus distribution and phosphatase activities: a microcosm incubation study, Chemosphere 142 (2016) 128–135. [7] M.A. Tabatabai, Soil enzymes, in: R.W. Weaver, J.S. Angle, P.S. Bottomley (Eds.), Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties, Soil Science Society of America, Madison, WI, 1994, pp. 775–833. [8] M.A. Kertesz, P. Mirleau, The role of soil microbes in plant sulfur nutrition, J. Exp. Bot. 55 (2004) 1939–1945. [9] A. Khadem, F. Raiesi, Influence of biochar on potential enzyme activities in two calcareous soils of contrasting texture, Geoderma 308 (2017) 149–158. [10] A. Khadem, F. Raiesi, Responses of microbial performance and community to corn biochar in calcareous sandy and clayey soils, Appl. Soil Ecol. 114 (2017) 16–27. [11] J. Paz-Ferreiro, S. Fu, A. Méndez, G. Gascó, Interactive effects of biochar and the earthworm (Pontoscolex) on plant productivity and soil enzyme activities, J. Soils Sediments 14 (2014) 483–494. [12] X. Wang, D. Song, G. Liang, Q. Zhang, C. Ai, W. Zhou, Maize biochar addition rate influences soil enzyme activity and microbial community composition in a fluvoaquic soil, Appl. Soil Ecol. 96 (2015) 265–272. [13] Z. Sun, E.W. Bruun, E. Arthur, L.W. de Jonge, P. Moldrup, H. Hauggaard-Nielsen, L. Elsgaard, Effect of biochar on aerobic processes, enzyme activity, and crop yields in two sandy loam soils, Biol. Fertil. Soils 50 (2014) 1087–1097. [14] J. Lehmann, M.C. Rillig, J. Thies, C.A. Masiello, W.C. Hockaday, D. Crowley, Biochar effects on soil biota: a review, Soil Biol. Biochem. 43 (2011) 1812–1836. [15] Y.L. Zhang, L.J. Chen, C.X. Sun, Z.J. Wu, Z.H. Chen, G.H. Dong, Soil hydrolase activities and kinetic properties as affected by wheat cropping systems of northeastern China, Plant Soil Environ. 56 (2010) 526–532. [16] P. Perucci, L. Scarponi, Arylsulphatase activity in soils amended with crop residues: kinetic and thermodynamic parameters, Soil Biol. Biochem. 16 (1984) 605–608. [17] M. Tabatabai, W. Dick, Enzymes in soil: research and developments in measuring activities, in: R.G. Burns, R.P. Dick (Eds.), Enzymes in the Environment: Activity, Ecology an Applications, Marcel Dekker, New York, 2002, pp. 567–596. [18] M.C. Marx, E. Kandeler, M. Wood, N. Wermbter, S.C. Jarvis, Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle size fractions, Soil Biol. Biochem. 37 (2005) 35–48. [19] D.P. German, M.N. Weintraub, A.S. Grandy, C.L. Lauber, Z.L. Rinkes, S.D. Allison, Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies, Soil Biol. Biochem. 43 (2011) 1387–1397. [20] R. Chintala, R.K. Owen, T.E. Schumacher, K.A. Spokas, L.M. McDonald, K. Kumar, D.E. Clay, D.D. Malo, B. Bleakley, Denitrification kinetics in biomass- and biocharamended soils of different landscape positions, Environ. Sci. Pollut. Res. 22 (2015) 5152–5163. [21] C. Trasar-Cepeda, F. Gil-Sotres, M.C. Leiros, Thermodynamic parameters of enzymes in grassland soils from Galicia, NW Spain, Soil Biol. Biochem. 39 (2007) 311–319. [22] L. Menichetti, A.L. Reyes Ortigoza, N. García, L. Giagnoni, P. Nannipieri, G. Renella, Thermal sensitivity of enzyme activity in tropical soils assessed by the Q10 and equilibrium model, Biol. Fertil. Soils 51 (2015) 299–310. [23] D.D. Allison, Soil minerals and humic acids alter enzyme stability: implications for ecosystem processes, Biogeochemistry 81 (2006) 361–373. [24] P. Nannipieri, L. Giagnoni, L. Landi, G. Renella, Role of phosphatase enzymes in soil, in: E. Bünemann, A. Oberson, E. Frossard (Eds.), Phosphorus in Action, Springer-Verlag, Heidelberg, 2011, pp. 215–243. [25] J. Paz-Ferreiro, S. Fu, A. Mendez, G. Gasco, Biochar modifies the thermodynamic parameters of soil enzyme activity in a tropical soil, J. Soils Sediments 15 (2015) 578–583. [26] A. Khadem, F. Raiesi, Response of soil alkaline phosphatase to biochar amendments: changes in kinetic and thermodynamic characteristics, Geoderma 337 (2019) 44–54. [27] D.W. Nelson, L.E. Sommers, Total carbon, organic carbon and organic matter, in: A.L. Page, et al. (Ed.), Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties, American Society of Agronomy, 1982, pp. 539–577. [28] D.S. Jenkinson, J.N. Ladd, Microbial biomass in soil. Measurement and turnover, in: E.A. Paul, J.N. Ladd (Eds.), Soil Biochemistry. 5, Marcel Dekker, NY, 1981, pp.
5. Concluding remarks To our knowledge, this is the first document reporting the relationship between the kinetic and thermodynamic parameters of ARS and biochar amendment in two different textured calcareous soils. The findings of the present study provide helpful insight with regards to the S cycling and its availability and the underlying mechanisms of enzyme responses in biochar-amended soils. The results of the present study demonstrated that the biochar application significantly increased the arylsulfatase activity through modifying soil chemical and microbiological attributes. The changes in enzyme activity induced by enzyme adsorption on biochar surface and so the corresponding changes in kinetic (Vmax and Km) and thermodynamic (Ea, Q10 and ΔHa) attributes. Pyrolysis temperature has negative effects on enzyme activity. The changes in arylsulfatase kinetic and thermodynamic parameters is soil specific and depends on the applied rate.
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sewage sludge biochar to soil, Biol. Fertil. Soils 48 (2012) 511–517. [35] F. Raiesi, A. Beheshti, Soil specific enzyme activity shows more clearly soil responses to paddy rice cultivation than absolute enzyme activity in primary forests of northwest Iran, Appl. Soil Ecol. 75 (2014) 63–70. [36] S. Jain, D. Mishra, P. Khare, V. Yadav, Y. Deshmukh, A. Meena, Impact of biochar amendment on enzymatic resilience properties of mine spoils, Sci. Total Environ. 544 (2016) 410–421. [37] A.F. Plante, R.T. Conant, E.A. Paul, K. Paustian, J. Six, Acid hydrolysis of easily dispersed and microaggregate‐derived silt‐and clay‐sized fractions to isolate resistant soil organic matter, Eur. J. Soil Sci. 57 (2006) 456–467. [38] S. Loeppmann, E. Blagodatskaya, J. Pausch, Yakov Kuzyakov, Substrate quality affects kinetics and catalytic efficiency of exo-enzymes in rhizosphere and detritusphere, Soil Biol. Biochem. 92 (2016) 111–118. [39] Y.H. Juan, Z.H. Chen, L.J. Chen, Z.J. Wu, R. Wang, W.T. Sun, Y.L. Zhang, Kinetic and thermodynamic behaviors of soil urease as affected by urease inhibitors, J. Soil Sci. Plant Nutr. 10 (2010) 1–11.
415–471. [29] V.L. Bailey, J.L. Smith, H. Bolton, Novel antibiotics as inhibitors for the selective respiratory inhibition method of measuring fungal: bacterial ratios in soil, Biol. Fertil. Soils 38 (2003) 154–160. [30] H.J. Lorch, G. Benckieser, J.C.G. Ottow, Basic methods for counting microorganism in soil and water, in: K. Alef, P. Nannipieri (Eds.), Methods in Applied Soil Microbiology and Biochemistry, Academic Press, London, 1995, pp. 146–161. [31] K. Alef, P. Nannipieri, Methods in Applied Soil Microbiology and Biochemistry, Academic Press, 1995. [32] V.L. Bailey, S.J. Fansler, J.L. Smith, H. Bolton, Reconciling apparent variability in effects of biochar amendment on soil enzyme activities by assay optimization, Soil Biol. Biochem. 43 (2011) 296–301. [33] N.J. Kruger, The Bradford method for protein quantitation, in: J.M. Walker (Ed.), The Protein Protocols Handbook, Humana Press, New Jersey, 2002, pp. 15–21. [34] J. Paz-Ferreiro, G. Gasco, B. Gutierrez, A. Mendez, Soil biochemical activities and the geometric mean of enzyme activities after application of sewage sludge and
9
Contents lists available at ScienceDirect
European Journal of Soil Biology journal homepage: www.elsevier.com/locate/ejsobi
Biochar application changed arylsulfatase activity, kinetic and thermodynamic aspects
T
Allahyar Khadema,∗, Hossein Besharatib, Mohammad Ali Khalajc a
Department of Soil Science and Engineering, Faculty of Agriculture, Shahrekord University, P.O. Box 115, Shahrekord, Iran Agricultural Research, Education and Extension Organization (AREEO), Soil and Water Research Institute, Karaj, Iran c Department of Technology and Production Management, Ornamental Plants Research Center (OPRC), HSIR, AREEO, Iran b
A R T I C LE I N FO
A B S T R A C T
Handling editor: Bryan Griffiths
The aim of this research was to evaluate the response of soil arylsulfatase (ARS) kinetic and thermodynamic parameters to biochar application. The kinetic parameters including Vmax, Km and catalytic efficiency (Vmax Km−1) as well as thermodynamic ones (Ea, ΔHa and Q10) were determined in two different textured clayey and sandy loam soils amended with 0.5 and 1.0% (w/w) of unpyrolyzed maize residue (UMR) or its biochars prepared at 200 and 600 °C (B200 and B600, respectively). The results showed that the application of amendments to sandy loam soil increased enzymatic activity (16–106%), Km (37–113%) and the enzyme concentration (Vmax) (13–39%), whereas the Vmax Km−1 (38–86%), Ea (6–21%) and Q10 (8–11%) were decreased in the biocharamended soils compared to the unamended control (CK). In clayey soil, biochar addition increased ARS activity (16–150%) and decreased the Vmax (10–41%), Vmax Km−1 (63–355%), Ea (20–63%) and Q10 (10–32%) and had no significant effect on Km. Generally, the effects of the amendments application were greater at 1% than 0.5% application rate and in clayey than sandy loam soil. Soil texture is a determinant factor in soil arylsulfatase response (activity, kinetics and thermodynamics) to biochar application.
Keywords: Biochar Arylsulfatase Kinetic parameters Thermodynamic
1. Introduction Soil microbial metabolic processes are mediated by the enzymes [1] who's their activities reflect the soil health [2]. In fact, the soil enzyme activities have strong correlation with soil fertility [3], microbial activity and cycling of elements in soil [4] and fluctuates with management practice [5]. Arylsulfatase is a vital soil enzyme because of its function in sulfur cycling and sulfate availability for plant uptake [6]. This enzyme catalysis the hydrolysis of aromatic sulfate esters (R-OSO3) to phenols and release the sulfate for uptake by plants or microbes [7,8]. Biochar, the product of the pyrolysis of C-based feedstocks has attracted many interests as ‘soil conditioner’ in recent years [9,10]. It is well proved that biochar can affect soil physical, chemical and biological properties [9,11] and hence the soil enzyme activity [10,12]. However, the effects of biochar application on arylsulfatase activity was weak and depended on type of soil and biochar application rate [5,13]. Although there are numerous researches on the effects of biochar on soil enzyme potential activity, the main focus of these studies was on the apparent activity of enzymes so, its effect on the kinetic and thermodynamic indices of enzymes is scarcely studied and remained as research priority [9,10,14].
∗
Kinetic parameters of soil enzymes including maximum velocity (Vmax) and Michaeilis-Menthen constant (Km) indicate the splitting velocity of enzyme-substrate complexes into enzyme and the reaction products and reflect the conjunction affinity between enzyme and substrate, respectively [15]. These parameters make a linkage between the enzymatic reaction rate and substrate concentration [16–19]. The study of these parameters can promote our knowledge regarding the status of the active enzyme and catalytic reaction, because of providing important information on the nature, stability of the enzyme and enzyme-substrate complex [18]. Km is an indication of the affinity of the enzyme for its substrate and enzyme efficiency. The changes in Km (i.e., high or low substrate affinity) can indicate an increase or decrease in the overall enzyme function [19]. The Vmax parameter represents the total concentration of an enzyme [16,18,19]. The results of previous studies indicated that the Vmax and Km values for soil enzymes changed with management practice including the application of organic amendments such as crop residues [16] and biochar Jin, 2010; [20]. To our knowledge, there is no study regarding the effects of biochar on soil arylsulfatase kinetic parameters. The better understanding of the thermodynamic parameters of soil enzymes including activation energy (Ea) and temperature coefficient
Corresponding author. E-mail address: [email protected] (A. Khadem).
https://doi.org/10.1016/j.ejsobi.2019.103134 Received 8 May 2019; Received in revised form 24 September 2019; Accepted 26 September 2019 1164-5563/ © 2019 Elsevier Masson SAS. All rights reserved.
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produced at 200 °C (B200) and 600 °C (B600) were used in two soil types (sandy loam and clayey texture). A sample of each soil type without UMR and maize biochars was also included as the negative control (CK). The air-dried soils (300 g) were mixed with UMR and its biochars at 0.5 and 1% (w/w) thoroughly in plastic jars. Deionized water was used to adjust soil moisture content at 70% water holding capacity. The jars were incubated for 90 days at 25 ± 1 °C. The soil moisture content was maintained constant.
(Q10) has necessity in studying the temperature sensitivity of soil enzymes following temperature fluctuations and global warming [21,22]. In addition, thermodynamic parameters provide an insight regarding the thermal and proteolytic stability of enzymes, as well their resistance to environmental changes [23,24]. At present, there is scarce information on how biochar affects the thermodynamic characteristics of soil enzymes. In the only one related study [25], studied the effects of biochar on thermodynamic parameters of soil enzymes and reported that biochar application reduced Ea and Q10 values for several soil hydrolases. Their observed results were ascribed to the release of different isozymes or the conformation changes due to enzyme adsorption on biochar surfaces. Hence, the main objectives of the current study were to (1) quantify the effects of maize biochars produced at 200 °C and 600 °C on the activity, kinetic and thermodynamic parameters of soil arylsulfatase, (2) compare the ARS activity, kinetic and thermodynamic characteristics between the soils amended with uncharred feedstock and its biochars; and (3) establish whether biochar effects on enzyme activity, kinetic and thermodynamic parameters would depend on soil texture.
2.3. Soil chemical and microbial analysis
2. Materials and methods
The soil organic carbon (SOC) content were determined according to the method of [27]. Soil microbial biomass carbon (MBC) determined as a measure of total microbial biomass using the fumigation-incubation method and a Kc value of 0.45 [28]. Substrate-induced respiration (SIR), as indicator of metabolically active microbial biomass, was determined using 1% (w/w) glucose solution as the substrate [29]. Total microbial population determined according to the method introduced by Ref. [30] basis on serial dilution and enumerating the colony forming unit (CFU) on plate culture by most probable number (MPN) tables.
2.1. Soil and biochar analysis
2.4. Enzyme activity, kinetic and thermodynamic parameters
Two sampling sites were selected at 50° 15′ 1.2″ N, 35°44′ 11.7″ E and 50° 42′ 27.2″ N, 35° 49′ 4.03″ E in Alborz province in Iran. The soils classified as Typic Haplocalcid and has not been cultivated or fertilized for three years. The mentioned soil characteristics was reported by Ref. [26]. The biochar was produced from maize stalks over a 2 h period by slow pyrolysis process at 200 and 600 °C in a thermal furnace. The results of soils and biochars analysis is presented in Table 1.
The activity of arylsulfatase (EC 3.1.6.1) determined according to the procedure described by Ref. [31] based on the colorimetric determination of the p-nitrophenol (PNP) released in the presence of disodium p-nitrophenyl sulfate (PNPS) as substrate. In brief, 1 g of the soil was incubated for 1 h at 37 °C with 0.25 ml of toluene, 1 ml of 0.25 mM PNPS and 4 ml of acetate buffer (pH 5.8). After the incubation time 1 ml of CaCl2 (0.5 M) and 4 ml of NaOH (0.5 M) was added, shaken, filtered and optical density measured at 400 nm. Soil enzyme activity expressed as μmol PNP released g−1 dry soil h−1. For estimation of the kinetic parameters, eight concentrations of PNPS (0, 0.5, 1.0, 5, 10, 15, 25 and 50 mM) were used as enzyme substrate and kinetic parameters, Km and Vmax values, calculated using the conventional Michaelis–Menten equation [17]:
2.2. Soil incubation test Soil incubation experiment was arranged as a completely randomized factorial design with four replicates. Three soil amendments including Unpyrolyzed maize residue (UMR) and maize biochars
v0 =
Table 1 Selected characteristics of the soils and biochars used in the study [23,24]. Variables
pH EC Ash V.M Fixed C BET Surface Area C O N H C:N O:C H:C H:O (O + N):C CEC Sand Silt Clay
Unit
dS m−1 % % % m2 g−1 % % % %
cmol (+) kg−1 g kg−1 g kg−1 g kg−1
Amendment (B200)
(B600)
Clayey
Sandy Loam
5.67 3.68 8.00 81.0 11.0 5.62 40.8 29.7 1.07 6.65 32.7 0.971 0.0136 0.014 1.00 21.2
6.25 3.67 14.0 68 18 12.4 48.7 29.4 1.61 6.06 25.9 0.805 0.0104 0.0129 0.84 18.6
11.2 4.75 39.0 24 37 88.4 63.0 4.83 1.61 1.76 33.5 0.102 0.0023 0.0228 0.13 11.7
7.68 0.33
8.32 0.37
(1) −1
−1
where ʋo is the initial reaction velocity (μmol PNP g soil h ), [S] is the PNPS concentration (mM), Km is the PNPS concentration at half maximal velocity (mM), and Vmax is the maximal velocity (μmol PNP g−1 soil h−1). The catalytic efficiency factor (ratio between Vmax and Km) also was calculated [15]. Arylsulfatase thermodynamic parameters were estimated using the Arrhenius equation [17]:
Soil Texture
(UMR)
Vmax [S ] Km [S ]
E k = A × exp ⎛− a ⎞ ⎝ RT ⎠ 0.423
0.220
0.034
0.022
12.5
10.0
26.5
8.80
10 380 520
80 100 100
(2)
The linear form of Arrhenius equation was used as follow:
ln k = lnA −
Ea RT
(3) −1
−1
where k is the enzyme activity (μmol PNP g h ) at a given temperature, A is the pre-exponential factor, R is the gas constant (8.314 J K−1 mol−1), Ea is activation energy (kJ K−1 mol−1) and T is the absolute temperature (Kelvin). The potential enzyme activity at temperatures 17, 27, 37 and 47 °C (T), plotted versus 1/T for estimating the Ea. These temperatures were chosen to have 4-point plot for reliable estimation of Ea. In addition, these temperatures contain the standard temperature for enzyme activity determination (37 °C), two lower temperatures (17 and 27 °C) and higher one (47 °C) which approximately reflect the soil temperature fluctuation in warm and cold season. The following equations were used for estimating the activation enthalpy values (ΔHa) and temperature coefficients or sensitivities (Q10),
UMR, Unpyrolysed Maize Residue; B200, Biochar prepared at 200 °C; B600, Biochar prepared at 600 °C; EC, Electrical conductivity; V.M, Volatile Matter; C, Carbon; O, Oxygen; N, Nitrogen; H, Hydrogen; CEC, Cation Exchange Capacity; BET (Brunaer-Emmett-Teller) surface area with N2 adsorption isotherm. 2
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respectively [17,22]: ΔHa = Ea − RT
Q10 =
kT + 10 kT
Table 2 Effects of biochar amendments on soil organic matter (SOM), microbial biomass carbon (MBC), substrate-induced respiration (SIR) and total microbial population (TMP) in the clayey and sandy loam soils.
(4) (5)
Variable
Amendment
Clayey Soil
Sandy Loam Soil
SOC (%)
CK UMR B200 B600 CK UMR B200 B600 CK UMR B200 B600 CK UMR B200 B600
0.48 ± 0.009 D 0.70 ± 0.024 C 0.76 ± 0.016 B 0.89 ± 0.055 A 291.7 ± 6.94 D 699.9 ± 26.4 A 658.6 ± 26.0 B 530.0 ± 14.3 C 23.4 ± 1.06 D 53.0 ± 3.45 A 46.7 ± 2.40 B 36.3 ± 0.68 C 15.0 ± 1.25 C 31.0 ± 1.26 A 24.0 ± 1.14 B 17.0 ± 1.02 C
0.27 ± 0.012 d 0.42 ± 0.020 c 0.57 ± 0.020 b 0.74 ± 0.060 a 206.0 ± 19.2 d 532.3 ± 22.0 a 506.7 ± 19.5 b 438.3 ± 15.6 c 14.4 ± 0.71 c 39.4 ± 2.41 a 37.5 ± 2.38 a 29.4 ± 1.08 b 2.00 ± 0.07 d 13.0 ± 1.15 a 11.0 ± 0.61 b 6.00 ± 0.66 c
2.5. Adsorption of enzyme and its product by biochar To interpret the results of the enzyme activity, the adsorption of arylsulfatase and the product of its activity p-nitrophenol (PNP) on biochar surface was measured by the method described by Refs. [23,32]. The biochar and purified enzyme (Sigma, S9626) mixture was prepared by shaking 10 mg biochar with 4 ml of acetate buffer (MUB, pH 5.8) and 50 μg ml−1 ARS for 15 min, and the mixture was centrifuged at 10000 g for 1 min. The pellet was re-suspended and washed three times with 1 ml of the appropriate buffer to remove any weakly associated ARS, and then re-suspended in 50 ml of buffer solution. The supernatant fractions and washings were collected and the concentration of enzyme in the solution was determined using the Bradford method described by Ref. [33]. For PNP adsorption test, a similar procedure was carried out and the concentration of PNP in the solution was determined as common procedure in enzyme activity determination (Section 2.4). The adsorbed ARS or PNP on biochar surface was measured by subtracting the ARS or PNP that recovered from that added initially.
MBC (mg C kg−1soil)
SIR (mg C–CO2 kg−1soil h−1)
TMP (Colony gr−1) × 106
Each value is mean (n = 4) ± standard errors. Different lowercase letters for the clayey soil and uppercase letters for the sandy loam soil represent significant differences between treatments by LSD's test at α = 0.05. CK, control; UMR, uncharred maize residue; B200, maize biochar obtained at 200 °C; B600, maize biochar obtained at 600 °C.
CK, respectively (Fig. 1). The increase of the enzyme activity in clayey soil were 0.76, 0.16, 0.51-fold at 0.5% and 1.3, 0.54, and 0.55-fold at 1% application rate for UMR, B200 and B600, respectively.
2.6. Statistical analysis The non-linear ligand binding with one site saturation regression equation were used to calculate the enzyme kinetic parameters using the software Sigma Plot 10 for Windows. All data were analyzed for normality by the Shapiro–Wilk test before statistical comparisons with 4 replicates. Significant differences between the treatments were calculated using three factor analysis of variance (ANOVA) and means were separated by least significant difference (LSD) test, at P < 0.05. All statistical tests were carried out using the Minitab 17 software for Windows.
3.3. Enzyme adsorption on biochar surface The results indicated that increasing pyrolysis temperature enhanced the adsorption capacity for ARS (Fig. 2). The ARS adsorption by B200 and B600 biochars was 22 and 24 μg g−1 biochar, that is 3.1 and 3.4 times higher than the UMR (7 μg g−1 biochar), respectively. This result confirms that the sorption of ARS to biochar may cause a partial reduction of its activity, which to some extent is observed in the determination of ARS activity (Fig. 2). In addition, there was no significant difference between the B200 and B600 regarding to their capacity for ARS adsorption (Fig. 2).
3. Results 3.1. Changes in soil chemical and biological properties
3.4. Changes in enzyme kinetic parameters Biochar application significantly increased SOC compared with CK treatments (Table 2). The highest SOC concentration was recorded in the B600 treatment whereas the lowest was in the CK treatment, in both clayey and sandy loam soils. In addition, the SOC content increased with increasing the pyrolysis temperature. The changes in soil microbial attributes were similar for MBC, SIR and TMP in response to the UMR and biochars applied. The amendment addition increased soil microbial attributes over the control by 82–158%, 55–174% and 15–550% for MBC, SIR and TMP, respectively. Increasing the pyrolysis temperature has negative effects on all measured microbial properties, in that way, the increase was smaller in higher temperature biochars (Table 2).
The relationship between the reaction rate and the substrate concentration was hyperbolic for ARS in both soils which indicate that saturation kinetics fitted well the conventional Michaelis-Menten equation (Fig. 3). The interaction of soil texture × amendment type and amendment type × application rate was significant for the Km (Table 3, Fig. 4 a, b). The addition of UMR and biochars to clayey soil had no significant effect on Km. In sandy loam soil, however, the UMR and B200 increased the Km (113% and 37%, respectively) over the CK treatment. Additionally, no significant change was observed in the Km with increasing pyrolysis temperature in clayey soils, whereas in sandy loam soil, B200 and B600 caused significant decrease (35% and 75%, respectively) in Km relative to UMR. The interaction of experimental factors had significant effects on the maximum velocity (Vmax) (Table 3). The addition of UMR and its biochar increased Vmax (10–41%), and the increase was approximately equal in clayey (10–41%) and sandy loam soil (13–39%) and greater at 0.5% (10–44%) than 1% (10–39%) application rate (Fig. 5 a). In general, there was no clear trend in Vmax by increasing the pyrolysis temperature. In clayey soil and at 0.5% application rate, only B600 resulted in an increase (41%) in the Vmax compared to UMR, whereas at 1% application rate, there was no significant difference
3.2. Arylsulfatase activity The ANOVA results showed that the interaction of soil texture, amendments and rate was significant on ARS activity (p < 0.05; Table 3). The addition of UMR, B200 and B600 increased the enzyme activity (0.16–1.5-fold) and the increase was greater in clayey (0.16–1.5) than sandy loam soil (0.16–1.06) and at 1% (0.37–1.5) than 0.5% (0.16–1.06) application rate (Fig. 1). The results showed that in sandy loam soil the addition of UMR, B200 and B600 increased the ARS activity by 1.6, 0.16 and 0.51-fold at 0.5% and 1.5, 0.37 and 0.8-fold at 1% application rate compared with 3
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Table 3 Analysis of Variance (ANOVA) for the effects of soil texture, amendment type and rate and its interactions on activity, kinetic and thermodynamic parameters of soil arylsulfatase. variables
D.F. ARS Activity Vmax Km Vmax Km−1 Ea ΔH Q10
Main Effects
Interactions
Soil Texture (T)
Rate (R)
Amendment (A)
T×R
T×A
A×R
T×A×R
1 12.5 (0.86) ** 2196.9 (90.2) ** 41.6 (26.9) ** 12129.2 (85.9) ** 281.2 (26.3) ** 253.2 (9.54) ** 0.1586 (33.9) **
1 197.3 (13.6) ** 21.5 (0.88) ** 2.40 (1.55) ns 277.4 (1.97) ** 2.51 (0.23) ns 18.7 (1.48) ns 0.0007 (0.11) ns
2 556.2 (76.5) ** 6.62 (0.54) ** 8.52 (11.0) * 78.9 (1.12) ** 100.1 (18.7) ** 1326.4 (68.5) ** 0.0579 (21.9) **
1 0.009 (0.00) ns 4.19 (0.17) * 4.47 (2.89) * 188.1 (1.33) ** 18.6 (3.48) ns 43.9 (1.13) ns 0.0117 (15.8) ns
2 7.09 (0.98) * 1.21 (0.10) ns 9.96 (12.9) * 117.5 (1.66) ** 15.8 (2.96) ns 22.1 (1.14) ns 0.0079 (2.58) ns
2 24.7 (3.40) ** 54.5 (4.48) ** 3.18 (4.11) * 338.3 (4.79) ** 9.10 (30.7) ** 433.4 (17.1) * 0.0026 (0.86) ns
2 8.21 (1.13) * 30.7 (2.52) ** 0.91 (1.18) ns 118.5 (1.68) ** 178.5 (16.7) ** 6.93 (0.36) ns 0.1033 (24.9) *
Error
C.V. (%)
R2 (%)
36 1.46 (3.62) 0.73 (1.08) 1.70 (39.5) 5.90 (1.50) 4.78 (0.89) 4.31 (1.8) 0.0052 (3.8)
– 24.8 25.6 57.9 21.4 12.1 13.7 6.60
– 95.3 98.6 48.5 98.1 69.3 78.2 69.2
D.F., Degree of freedom; ARS, Arylsulfatase; C.V, coefficient of variation; R2, coefficient of determination. **, * and ns are significant at the 1 and 5%, and non-significant, respectively. The numbers in parentheses represent the relative contribution of each variable source in each parameter (that is, the ratio of the sum of squares for that source to the total sum of squares multiplied by 100).
and B600 (13%) reduced the Vmax compared with UMR. The results showed that the interaction of all tested factors was significant for arylsulfatase catalytic efficiency (VmaxKm−1) (Table 3). The addition of UMR and its biochars in sandy loam soil (except for B600 at 1% application rate) decreased (38–86%) the catalytic efficiency compared with the control treatment (Fig. 4 b). In clayey soil, however, the amendment addition increased (63–355%) the VmaxKm−1 and the changes were greater at 1% (63–355%) than 0.5% (33–182%) application rate. 3.5. Changes in thermodynamic parameters The temperature dependence of ARS activity followed the Arrhenius model up to the point of enzyme inactivation (47 °C) (Fig. 6). The results of ANOVA showed that the amendment had significant effects on the activation energy (Ea) of arylsulfatase (p < 0.05) (Table 3). The addition of UMR and B200 to sandy loam soil decreased the Ea (6–21%) and the decrease was greater at 0.5% (17% and 21%) than 1% (6% and 14%) application rate (Fig. 4c.). The addition of biochar to clayey soil increased the activation energy (20–63%), and the increase was greater at 1% (20–63%) than 0.5% (25–45%). The results of ANOVA (Table 3) showed that the only interaction between the amendment type and rate was significant for activation enthalpy (ΔHa) (p < 0.05). The addition of UMR and B600 significantly increased ΔHa, and the increase was greater at 1% than 0.5% application rate (Fig. 3c). In addition, by increasing pyrolysis temperature B600 increased the ΔHa compared to UMR, and the increase was greater at 1% (36%) than 0.5% (18%). The results indicated that interaction of three factors was significant for temperature coefficient (Q10) of arylsulfatase (Table 3). In sandy loam soil, the addition of B200 at 0.5% and 1% and UMR at 0.5% decreased the Q10 by 10%, 8% and 11%, compared to CK, respectively. In clayey soil, the amendments increased Q10 (10–32%), and the increase was greater at 1% (10–32%) than 0.5% (12–22%) (Table 4).
Fig. 1. Potential activity values of aryl sulfatase (ARS) in unamended and amended soils. Values are mean (n = 4) ± standard errors. Different uppercase letters represent significant differences between unamended (CK) and amended soils by LSD's test at α = 0.05. Different lowercase letters represent significant differences between the treatment by LSD's test at α = 0.05. CK, control; UMR, uncharred maize residue; B200; maize biochar obtained at 200 °C; B600; maize biochar obtained at 600 °C.
4. Discussion 4.1. Biochar effects on soil chemical and microbiological properties
Fig. 2. Adsorption of aryl sulfatase (ARS) onto unpyrolysed maize residue and its biochars obtained at 200 and 600 °C. Values are mean (n = 4) ± standard errors. Different letters represent significant differences between biochars by LSD's test at α = 0.05.
Our results indicate the stimulation effects of UMR and its biochars prepared at 200 and 600 °C on SOC and microbial attributes. Biochar, due to its large specific surface area and porous structure, provides suitable microenvironment for colonization of soil microbial communities [14]. Volatile compounds are low molecular weight compounds in biochar which believed as an indicator of the labile components [14]. The presence of volatile compounds and abundant nutrients in the biochars might be involved in the microbial stimulation of soil
between the treatments. The results showed that, the Vmax increased with increasing the application rate of UMR and B200, while the reverse trend was observed for B600. In sandy loam soil at 0.5% application rate, the B200 (17%), and at 1% application rate B200 (11%) 4
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Fig. 3. Michaelis–Menten plots showing aryl sulfatase activity as a function of substrate concentration measured in unamended and amended soils. Values are mean (n = 4) ± standard errors. CK, control; UMR, uncharred maize residue; B200, maize biochar obtained at 200 °C; B600, maize biochar obtained at 600 °C.
microbes and the carbon molecules [7]. Soil organic amendments such as UMR and biochar could enhance soil enzyme activities by increasing soil organic matter content and therefore, the microbial biomass [9,10]). However, the results of [36] showed a decrease in ARS activity due to biochar application in a period of 90 days and then its activity increased. The initial reduction in enzyme activity was ascribed to the presence of sulfite in soil and its inhibitory effect on the enzyme activity. The increase in enzyme activity after 90 day attributed to the conversion of sulfite to sulfate and reduce its inhibitory effect. The ARS activity showed a distinct change by increasing the pyrolysis temperature. The results indicated that the addition of B200 and B600 reduced the enzyme activity in both clayey (14–38%) and sandy loam (24–46%) soils compared with UMR. This observation indicates that the conversion of raw residues to biochar could have great effects on ARS activity. This finding corresponds with the previous results obtained for alkaline phosphatase [26] and other enzymes [12]. Several potential mechanisms proposed for the declined activity of soil enzyme with increasing pyrolysis temperature which includes structural and chemical changes of biochar [9] and changes in soil microbial composition, soil enzymes immobilization on biochar surface area (Jin, 2010; [14].
following biochar application [26]. Volatile matter content of maize feedstock decreased with increasing pyrolysis temperature, and was 81% for the UMR, 68% for B200 and 24% for the B600 (Table 1). Higher temperature biochars has low volatile matter and high aromaticity, which cause high resistance to microbial decomposition and providing smaller labile organic C for microbial activity and growth [11,26,32].
4.2. Biochar effects on ARS activity The mean ARS activity ranged between 13 and 33 μg PNPg−1h−1 in control and biochar treated soils, which corresponds to the value reported by Ref. [13]. The positive effect of biochar on ARS activity in the current study is in agreement with [34] who reported increases in ARS activity of soils amended with biochar. The possible mechanism behind the increase in enzyme activities following the addition of biochar is attributed to the change in soil physiochemical properties including increased OM content and soil pH, stimulating soil microorganisms to producing extracellular enzymes, co-location of enzyme and substrate as well as soil microbial community changes Jin, 2010; [9,14,26,34]. The higher ARS activity as result of biochar application could be direct effect of increased microbial biomass and activity, because there was a strong correlation between the MBC (r = 0.70, P < 0.001) and SIR (r = 0.74, P < 0.001) values and the ARS activity (Table 5). This correlation suggests that this enzyme is primarily of microbial origin [35]. In addition, there was significant correlation between ARS activity and SOM content (r = 0.34, P < 0.001). SOM plays an important role in maintaining soil enzymes in active forms. This correlation reflected the connection between enzyme substrates, the available energy for soil
4.3. Biochar effects on enzyme kinetic parameters One of the current research objectives was to determine the effect of UMR and biochar on the kinetic indices of soil ARS. The measured Km values (0.5–4.0 mM) are indicative of different soil ARS pools and location, as Km represents sources of the enzymes. The measured values of Km in this study are within the limits reported by Tabatabaei and Bermaner (1971), who suggested that the enzyme affinity to its substrate was partially influenced by the linkage between the enzyme and 5
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Fig. 4. Michaelis-Menten constant (Km) and Activation enthalpy (ΔHa) of aryl sulfatase (ARS) in unamended and amended soils. Values are mean (n = 8) ± standard errors. Different uppercase letters represent significant differences between unamended (CK) and amended soils by LSD's test at α = 0.05. Different lowercase letters represent significant differences between the treatment by LSD's test at α = 0.05. CK, control; UMR, uncharred maize residue; B200; maize biochar obtained at 200 °C; B600; maize biochar obtained at 600 °C.
Fig. 5. Maximum velocity (Vmax), Catalytic efficiency (Vmax/Km) and Activation energy (Ea) of aryl sulfatase (ARS) in unamended and amended soils. Values are mean (n = 4) ± standard errors. Different uppercase letters represent significant differences between unamended (CK) and amended soils by LSD's test at α = 0.05. Different lowercase letters represent significant differences between the treatment by LSD's test at α = 0.05. CK, control; UMR, uncharred maize residue; B200; maize biochar obtained at 200 °C; B600; maize biochar obtained at 600 °C.
organic and inorganic components of soil. The Km value of ARS in clayey soil was smaller than that of sandy loam, indicating the effect of organic matter and clay content on maintaining and increasing the availability of substrate for the enzyme. The increase in Km after the addition of biochar was in agreement with the results of [16] who showed that the Km of ARS significantly changed with the addition of plant residue to soil. According to Quiampix et al. (2002) enzyme adsorption on biochar surface can alter enzyme or active site conformation, and consequently decrease the enzyme affinity to its substrate and increase the Km. Quiquampix et al. (2002) reported that the
immobilization of soil enzymes on biochar can result in change of enzyme conformation and cause a decline of the substrate affinity and increases in Km values. Accordingly, the adsorption of enzyme and its substrate on biochar surface can depending on the enzyme properties, increase the contact between the enzyme and substrates and so lead to an increase in the affinity of the enzyme for substrate (Rao et al., 2000). This result shows that despite enzyme adsorption, there is a strong affinity of ARS for substrate (lower Km) in the clayey soil. The arylsulfatase Vmax was higher in clayey than sandy loam soil, 6
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Fig. 6. Arrhenius equation plot of aryl sulfatase (ARS) in unamended and amended soils. CK, control; UMR, uncharred maize residue; B200, maize biochar obtained at 200 °C; B600, maize biochar obtained at 600 °C.
which indicates more enzymes concentration in clayey soil. The higher concentrations of enzymes in clayey soil can be due to higher content of organic matter and clay, which binds to the enzyme and adsorb it and thus protect the enzyme against degradation by proteases [37]. The results showed that Vmax had a significant correlation with soil organic matter content (r = 0.001, p < 0.001), microbial biomass (r = 0.67, p < 0.001) and microbial population (r = 0.81, p < 0.001). In addition, the higher content of organic matter increased the soil microbial population and biomass due to the provision of the nutritional requirement of microbial community, resulting in more enzyme production in clayey soil. Also, the results of the enzyme adsorption showed that the biochars adsorbed up to 25 μg g−1 purified enzyme. Increasing the Vmax, despite the adsorption of the enzyme, indicating no changes in the enzyme configuration. In addition, there was a significant correlation between the ARS activity and the Vmax values (r = 0.34, P < 0.001), whereas no correlation found between ARS activity and Km, which indicating the prevalence of the Vmax rather than the Km on ARS activity, similar to what previously observed for alkaline phosphatase [26]. The catalytic efficiency of soil enzymes is affected by soil physicochemical properties, substrate accessibility, soil organic matter content, soil microbial stimulation and enzyme production [38]. In general, the addition of biochar resulted in an increase of the Km and the Vmax of sandy soil, but the rate of the increase in maximum velocity was not sufficiently large to compensate the increase of the Km, and therefore decreased the catalytic efficiency of ARS. Therefore, it can be said that in sandy loam soil, the Km has a greater role in the catalytic activity of the enzyme and the contribution of the Vmax is lower. In clayey soil, however, the addition of biochar had no significant effects on Km, while the adsorption of the enzyme or substrate on clay and organic matter, as well as the effect of these soil colloid on microbial population increased the catalytic efficiency of ARS. Overally, the increase of Vmax, Km and catalytic efficiency of arylsulfatase by addition of UMR, B200 and B600 indicates that biochar has no inhibitory effect on enzyme activity and cannot be classified as enzyme inhibitors.
Table 4 Effect of biochar amendment, application rate (0.5 and 1%) and soil texture on soil the temperature coefficient (Q10) of arylsulfatase. Treatment
Sandy Loam Soil
Clayey Soil
0.5%
1%
CK UMR
1.77 (0.047) A 1.57 (0.025) Bd
B200
1.60 (0.047) Bcde 1.69 (0.034) Acd 0.13
1.77 1.71 Abc 1.63 Bde 1.82 Aab
B600 LSD0.05
0.5% (0.047) A (0.053) (0.027) (0.020)
1.56 1.83 Aab 1.75 Ab 1.91 Aa
1% (0.040) B (0.023) (0.044) (0.045)
1.56 1.77 Ab 1.71 Abc 1.75 Ab
(0.040) B (0.040) (0.091) (0.041)
Each data point is mean value of four replications with standard errors. Different uppercase letters in the same column for each application rate represent significant differences between unamended (CK) and amended soils by LSD's test at α = 0.05. Different lowercase letters represent significant differences for two-way interaction terms by LSD's test at α = 0.05. UMR, uncharred corn residue; B200 and B600 corn biochars produced at 200 and 600 °C. Table 5 Pearson correlation coefficients (r) between the kinetic and thermodynamic parameters of ARS and soil attributes (n = 32). Variable
ARS activity
Vmax
Km
Ea
ΔHa
Q10
soil pH SOM Mic POP SIR MBC Vmax Km
−0.36** 0.34** 0.53*** 0.74*** 0.70*** 0.34** 0.19ns
−0.73*** 0.58*** 0.83*** 0.60*** 0.59*** – −0.40**
0.14ns 0.36** −0.24ns 0.02ns 0.01ns −0.37** –
0.05ns 0.33** 0.17ns 0.16ns 0.22* 0.27* −0.24*
0.25* 0.56*** 0.05ns 0.14ns 0.21* 0.26* −0.28*
0.04ns 0.33** 0.17ns 0.15ns 0.21* 0.28* −0.24*
ARS, arylsulfatase; SOM, soil organic matter; Mic POP, microbial population; SIR, substrate-induced respiration; MBC, microbial biomass carbon; Vmax, maximal velocity; Km, Michaelis–Menten constant; Ea, activation energy. ΔHa, activation enthalpy; Q10, temperature coefficient. *P < 0.05. **P < 0.01. ***P < 0.001.
4.4. Biochar effects on enzyme thermodynamic parameters The temperature would affect the enzyme and substrate diffusion, and thus the Km, and Vmax of enzyme reactions. Temperature changes affects the production of enzymes by microbes, as well as the rate of 7
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Declaration of competing interest
enzyme degradation. Activation energy acts as a barrier in the enzyme reaction, and its change indicates surface adsorption by soil particles. The Ea values measured in the current research is within the range of reported by Refs. [15,25]; and smaller than that of [21]. It is concluded from the results of the present study that the addition of biochar to clayey soil with high clay content results in the more stabilization of ARS. However, in sandy loam soil, the addition of UMR and B200 reduced the Ea [39]. attributed the Ea changes to enzyme stabilization by soil organic and inorganic colloids. Their results showed that the greater the enzyme protection due to its association with organic and mineral complexes of the soil, the greater energy barrier that should be overcome during the formation of the enzyme-substrate complex. Also, the results of arylsulfatase adsorption showed that this enzyme was adsorbed on biochar surface, which is consistent with increasing Ea and the catalytic efficiency of the enzyme. On the other hand [25], attributed the decrease in the Ea of arylsulfatase to the production of isozymes, changing the composition of the microbial community, and changing the soil characteristics such as pH, which is consistent with the results obtained in sandy loam soil. In the present study, there was a weak correlation between the Ea and Vmax (r = 0.27 p < 0.01) and Km (r = −0.24, p < 0.01). The addition of the amendment to clayey soil, did not change Km, whereas the Vmax increased. In sandy loam soils, the amendments increased the Km, Vmax and Ea. The increase in Vmax, despite of the increase in Ea, can be attributed to the enzyme stabilization by organic amendments that protect the enzyme from proteases, as well as increase the microbial biomass and the production of enzymes. Higher ΔHa value obtained in B600 treatment suggests more activation energy required for the formation of the transition state and for the products to form [20]. reported increases in ΔHa values for denitrifying enzyme activity with biochar application due to low substrate affinity [26]. found similar results for alkaline phosphomonoesterase. Our finding suggests that higher Ea and ΔHa values with B600 addition would lead to a lower rate of ARS reaction [26]. ascribed the increase in thermodynamic parameters of the enzyme to the adsorption of enzyme to biochar surface. The higher Q10 values in biochar-treated clayey soils indicate that biochar additions would increase the temperature sensitivity and thermal stability of soil ARS. The Q10 values could be attributed to the release of different isoenzymes and a modification of the enzyme conformation, particularly in the enzyme active site essential for binding and catalysis, following enzyme adsorption on biochar surfaces [26]. [25] studied the effects of biochar on the thermodynamic indices of arylsulfatase and reported a decrease in Q10 which corresponds to the situation observed in sandy loam soil. They ascribed the Q10 reduction to the changes in microbial community composition, production of isoenzymes, active site deformation due to biochar adsorption and to some extent the change in soil reaction.
The authors declare that there is no conflict of interests regarding the publication of this paper. References [1] A.D. McLaren, Soil as a system of humus and clay immobilized enzymes, Chem. Scripta 8 (1975) 97–99. [2] S.K. Das, A. Varma, Role of enzymes in maintaining soil health, in: G. Shukla, A. Varma (Eds.), Soil Enzymology. Soil Biology 22, Springer-Verlag Berlin Heidelberg, 2011. [3] E.B. Utobo, L. Tewari, Soil enzymes as bioindicators of soil ecosystem status, Appl. Ecol. Environ. Res. 13 (2015) 147–169. [4] D.J. Burke, M.N. Weintrau, C.R. Hewins, S. Kalisz, Relationship between soil enzyme activities, nutrient cycling and soil fungal communities in a northern hardwood forest, Soil Biol. Biochem. 43 (2011) 795–803. [5] M. Niemi, J. Pöyry, I. Heiskanen, V. Uotinen, M. Nieminen, K. Erkomaa, K. Wallenius, Variability of soil enzyme activities and vegetation succession following boreal forest surface soil transfer to an artificial hill, Nat. Conserv. 8 (2014) 1–25. [6] Y. Jin, X. Liang, M. He, Y. Liu, G. Tian, J. Shi, Manure biochar influence upon soil properties, phosphorus distribution and phosphatase activities: a microcosm incubation study, Chemosphere 142 (2016) 128–135. [7] M.A. Tabatabai, Soil enzymes, in: R.W. Weaver, J.S. Angle, P.S. Bottomley (Eds.), Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties, Soil Science Society of America, Madison, WI, 1994, pp. 775–833. [8] M.A. Kertesz, P. Mirleau, The role of soil microbes in plant sulfur nutrition, J. Exp. Bot. 55 (2004) 1939–1945. [9] A. Khadem, F. Raiesi, Influence of biochar on potential enzyme activities in two calcareous soils of contrasting texture, Geoderma 308 (2017) 149–158. [10] A. Khadem, F. Raiesi, Responses of microbial performance and community to corn biochar in calcareous sandy and clayey soils, Appl. Soil Ecol. 114 (2017) 16–27. [11] J. Paz-Ferreiro, S. Fu, A. Méndez, G. Gascó, Interactive effects of biochar and the earthworm (Pontoscolex) on plant productivity and soil enzyme activities, J. Soils Sediments 14 (2014) 483–494. [12] X. Wang, D. Song, G. Liang, Q. Zhang, C. Ai, W. Zhou, Maize biochar addition rate influences soil enzyme activity and microbial community composition in a fluvoaquic soil, Appl. Soil Ecol. 96 (2015) 265–272. [13] Z. Sun, E.W. Bruun, E. Arthur, L.W. de Jonge, P. Moldrup, H. Hauggaard-Nielsen, L. Elsgaard, Effect of biochar on aerobic processes, enzyme activity, and crop yields in two sandy loam soils, Biol. Fertil. Soils 50 (2014) 1087–1097. [14] J. Lehmann, M.C. Rillig, J. Thies, C.A. Masiello, W.C. Hockaday, D. Crowley, Biochar effects on soil biota: a review, Soil Biol. Biochem. 43 (2011) 1812–1836. [15] Y.L. Zhang, L.J. Chen, C.X. Sun, Z.J. Wu, Z.H. Chen, G.H. Dong, Soil hydrolase activities and kinetic properties as affected by wheat cropping systems of northeastern China, Plant Soil Environ. 56 (2010) 526–532. [16] P. Perucci, L. Scarponi, Arylsulphatase activity in soils amended with crop residues: kinetic and thermodynamic parameters, Soil Biol. Biochem. 16 (1984) 605–608. [17] M. Tabatabai, W. Dick, Enzymes in soil: research and developments in measuring activities, in: R.G. Burns, R.P. Dick (Eds.), Enzymes in the Environment: Activity, Ecology an Applications, Marcel Dekker, New York, 2002, pp. 567–596. [18] M.C. Marx, E. Kandeler, M. Wood, N. Wermbter, S.C. Jarvis, Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle size fractions, Soil Biol. Biochem. 37 (2005) 35–48. [19] D.P. German, M.N. Weintraub, A.S. Grandy, C.L. Lauber, Z.L. Rinkes, S.D. Allison, Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies, Soil Biol. Biochem. 43 (2011) 1387–1397. [20] R. Chintala, R.K. Owen, T.E. Schumacher, K.A. Spokas, L.M. McDonald, K. Kumar, D.E. Clay, D.D. Malo, B. Bleakley, Denitrification kinetics in biomass- and biocharamended soils of different landscape positions, Environ. Sci. Pollut. Res. 22 (2015) 5152–5163. [21] C. Trasar-Cepeda, F. Gil-Sotres, M.C. Leiros, Thermodynamic parameters of enzymes in grassland soils from Galicia, NW Spain, Soil Biol. Biochem. 39 (2007) 311–319. [22] L. Menichetti, A.L. Reyes Ortigoza, N. García, L. Giagnoni, P. Nannipieri, G. Renella, Thermal sensitivity of enzyme activity in tropical soils assessed by the Q10 and equilibrium model, Biol. Fertil. Soils 51 (2015) 299–310. [23] D.D. Allison, Soil minerals and humic acids alter enzyme stability: implications for ecosystem processes, Biogeochemistry 81 (2006) 361–373. [24] P. Nannipieri, L. Giagnoni, L. Landi, G. Renella, Role of phosphatase enzymes in soil, in: E. Bünemann, A. Oberson, E. Frossard (Eds.), Phosphorus in Action, Springer-Verlag, Heidelberg, 2011, pp. 215–243. [25] J. Paz-Ferreiro, S. Fu, A. Mendez, G. Gasco, Biochar modifies the thermodynamic parameters of soil enzyme activity in a tropical soil, J. Soils Sediments 15 (2015) 578–583. [26] A. Khadem, F. Raiesi, Response of soil alkaline phosphatase to biochar amendments: changes in kinetic and thermodynamic characteristics, Geoderma 337 (2019) 44–54. [27] D.W. Nelson, L.E. Sommers, Total carbon, organic carbon and organic matter, in: A.L. Page, et al. (Ed.), Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties, American Society of Agronomy, 1982, pp. 539–577. [28] D.S. Jenkinson, J.N. Ladd, Microbial biomass in soil. Measurement and turnover, in: E.A. Paul, J.N. Ladd (Eds.), Soil Biochemistry. 5, Marcel Dekker, NY, 1981, pp.
5. Concluding remarks To our knowledge, this is the first document reporting the relationship between the kinetic and thermodynamic parameters of ARS and biochar amendment in two different textured calcareous soils. The findings of the present study provide helpful insight with regards to the S cycling and its availability and the underlying mechanisms of enzyme responses in biochar-amended soils. The results of the present study demonstrated that the biochar application significantly increased the arylsulfatase activity through modifying soil chemical and microbiological attributes. The changes in enzyme activity induced by enzyme adsorption on biochar surface and so the corresponding changes in kinetic (Vmax and Km) and thermodynamic (Ea, Q10 and ΔHa) attributes. Pyrolysis temperature has negative effects on enzyme activity. The changes in arylsulfatase kinetic and thermodynamic parameters is soil specific and depends on the applied rate.
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sewage sludge biochar to soil, Biol. Fertil. Soils 48 (2012) 511–517. [35] F. Raiesi, A. Beheshti, Soil specific enzyme activity shows more clearly soil responses to paddy rice cultivation than absolute enzyme activity in primary forests of northwest Iran, Appl. Soil Ecol. 75 (2014) 63–70. [36] S. Jain, D. Mishra, P. Khare, V. Yadav, Y. Deshmukh, A. Meena, Impact of biochar amendment on enzymatic resilience properties of mine spoils, Sci. Total Environ. 544 (2016) 410–421. [37] A.F. Plante, R.T. Conant, E.A. Paul, K. Paustian, J. Six, Acid hydrolysis of easily dispersed and microaggregate‐derived silt‐and clay‐sized fractions to isolate resistant soil organic matter, Eur. J. Soil Sci. 57 (2006) 456–467. [38] S. Loeppmann, E. Blagodatskaya, J. Pausch, Yakov Kuzyakov, Substrate quality affects kinetics and catalytic efficiency of exo-enzymes in rhizosphere and detritusphere, Soil Biol. Biochem. 92 (2016) 111–118. [39] Y.H. Juan, Z.H. Chen, L.J. Chen, Z.J. Wu, R. Wang, W.T. Sun, Y.L. Zhang, Kinetic and thermodynamic behaviors of soil urease as affected by urease inhibitors, J. Soil Sci. Plant Nutr. 10 (2010) 1–11.
415–471. [29] V.L. Bailey, J.L. Smith, H. Bolton, Novel antibiotics as inhibitors for the selective respiratory inhibition method of measuring fungal: bacterial ratios in soil, Biol. Fertil. Soils 38 (2003) 154–160. [30] H.J. Lorch, G. Benckieser, J.C.G. Ottow, Basic methods for counting microorganism in soil and water, in: K. Alef, P. Nannipieri (Eds.), Methods in Applied Soil Microbiology and Biochemistry, Academic Press, London, 1995, pp. 146–161. [31] K. Alef, P. Nannipieri, Methods in Applied Soil Microbiology and Biochemistry, Academic Press, 1995. [32] V.L. Bailey, S.J. Fansler, J.L. Smith, H. Bolton, Reconciling apparent variability in effects of biochar amendment on soil enzyme activities by assay optimization, Soil Biol. Biochem. 43 (2011) 296–301. [33] N.J. Kruger, The Bradford method for protein quantitation, in: J.M. Walker (Ed.), The Protein Protocols Handbook, Humana Press, New Jersey, 2002, pp. 15–21. [34] J. Paz-Ferreiro, G. Gasco, B. Gutierrez, A. Mendez, Soil biochemical activities and the geometric mean of enzyme activities after application of sewage sludge and
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