Phosphomonoesterase Activities, Kinetics and Thermodynamics in a Paddy Soil After Receiving Swine Manure for Six Years

Pedosphere 25(2): 294–306, 2015 ISSN 1002-0160/CN 32-1315/P c 2015 Soil Science Society of China ° Published by Elsevier B.V. and Science Press

Phosphomonoesterase Activities, Kinetics and Thermodynamics in a Paddy Soil After Receiving Swine Manure for Six Years LI Liang1 , LIANG Xin-Qiang1,∗ , LI Hua2 , JI Yuan-Jing3 , LIU Jin3 , YE Yu-Shi3 , TIAN Guang-Ming1 , CHEN Ying-Xu1 and LUO Yong-Ming4 1 Institute

of Environmental Science and Technology, College of Environmental and Resources Sciences, Zhejiang University, Hangzhou 310058 (China) 2 Zhejiang Academy of Agricultural Sciences, Hangzhou 310021 (China) 3 Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou 310058 (China) 4 Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003 (China) (Received May 19, 2014; revised January 16, 2015)

ABSTRACT Soil phosphomonoesterase plays a critical role in controlling phosphorus (P) cycling for crop nutrition, especially in P-deficient soils. A 6-year field experiment was conducted to evaluate soil phosphomonoesterase activities, kinetics and thermodynamics during rice growth stages after consistent swine manure application, to understand the impacts of swine manure amendment rates on soil chemical and enzymatic properties, and to investigate the correlations between soil enzymatic and chemical variables. The experiment was set out in a randomized complete block design with three replicates and five treatments including three swine manure rates (26, 39, and 52 kg P ha−1 , representing low, middle, and high application rates, respectively) and two controls (no-fertilizer and superphosphate at 26 kg P ha−1 ). The results indicated that the grain yield and soil chemical properties were significantly improved with the application of P-based swine manure from 0 to 39 kg P ha−1 ; however, the differences between the 39 (M39 ) and 52 kg P ha−1 treatments (M52 ) were not significant. The enzymatic property analysis indicated that acid phosphomonoesterase was the predominant phosphomonoesterase in the tested soil. The M39 and M52 treatments had relatively high initial velocity (V0 ), maximal velocity (Vmax ), and activation grade (lgNa ) but low Michaelis constant (Km ), temperature coefficient (Q10 ), activation energy (Ea ), and activation enthalpy (∆H), implying that the M39 and M52 treatments could stimulate the enzyme-catalyzed reactions more easily than all other treatments. The correlation analysis showed that the distribution of soil phosphomonoesterase activities mainly followed the distributions of total C and total N. Based on these results, 39 kg P ha−1 could be recommended as the most appropriate rate of swine manure amendment. Key Words:

activation energy, activation enthalpy, enzyme-catalyzed reaction, maximal velocity, Michaelis constant

Citation: Li, L., Liang, X. Q., Li, H., Ji, Y. J., Liu, J., Ye, Y. S., Tian, G. M., Chen, Y. X. and Luo, Y. M. 2015. Phosphomonoesterase activities, kinetics and thermodynamics in a paddy soil after receiving swine manure for six years. Pedosphere. 25(2): 294–306.

INTRODUCTION Phosphorus (P) is a limiting nutrient to sustain crop yields in most agro-ecological zones over the world (Khan and Joergensen, 2009; Ramaekers et al., 2010) since some of them are bound in the detritus as organic P and others are bound with Ca2+ , Fe3+ , and Al3+ , remaining inaccessible to plants (Miller and Fox, 2011; Huang et al., 2012). Farmers usually add organic manures and mineral P fertilizers to remedy P deficiency (Ayaga et al., 2006; Malik et al., 2012). Mineral P fertilizer provides available P for plants growth but does not contribute to the improvement of soil physical conditions. Organic manure application can improve soil chemical and biological properties (Liu et al., 2013). Swine manure is a relatively inexpensive form ∗ Corresponding

author. E-mail: [email protected].

of organic manure and has been one of the most commonly applied fertilizers in organic rice production in developed agricultural regions such as the Taihu Lake region of southeastern China. Actually, the lower cost of production make organic paddies more profitable than conventional mineral fertilizer paddies (Liang et al., 2013). Swine manure presents management challenges due to its inconsistent nutrient content and rate of release (Guo et al., 2004; Liang et al., 2013). Generally, swine manure contains a relatively high total P content but the P availability is quite low in manure applied rice fields. Long-term high application rate of swine manure can lead to the increase of P precipitation in topsoil and P loss from farmland runoff to rivers and streams (Koopmans et al., 2007; Xavier et al., 2009).

PHOSPHOMONOESTERASE IN PADDY SOIL

A large part of P has been found in organic P such as phosphoinositide, phosphorus esters and nucleotides in organic matter-amended soils (N`eble et al., 2007). However, microorganisms and plants can only assimilate mineral orthophosphate P in the soil (Rao et al., 1996). Most of organic P must be hydrolyzed into effective P forms via phosphatase which mostly originates from soil microorganisms and plant root exudates (Criquet and Braud, 2007; Saha et al., 2008; Albrecht et al., 2010). Soil phosphatase plays a pivotal role in the overall process of mineralizing organic P. Phosphomonoesterases are considered as the predominant phosphatases in most soils and are divided into acid phosphomonoesterase (ACP) (pH 4–6) and alkaline phosphomonoesterase (ALP) (pH 8–10) according to their pH optima in the enzymatic reactions (Tabatabai, 1994; Lu, 2000; Criquet et al., 2004; Kunito et al., 2012). Soil phosphomonoesterase is involved in soil P cycling and catalyzes the conversion of soil P from unavailable to available forms. Therefore, it is imperative to discover the mechanism of swine manure effect on soil phosphomonoesterase activities and kinetics in the process of organic P transformation. A lot of studies were focused on soil phosphomonoesterase activity changes in a single sampling date and ignored the dynamics of phosphomonoesterase relative to other soil chemical factors over time. Previous researches have pointed out soil phosphomonoesterase activity was directly and indirectly affected by substrate concentration, reactive time and temperature (Koch et al., 2007; Wallenstein et al., 2009; Stone et al., 2012). The paddy soils were sampled during rice growth stages to study the kinetic and thermodynamic properties of phosphomonoesterase by measuring p-nitrophenol released in reactions when the substrate concentration, reactive time and temperature changed, so as to figure out the kinetic and thermodynamic parameters. Soil phosphomonoesterase kinetic parameter Vmax indicates the maximal velocity of enzyme-substrate complexes decomposing into enzyme and reaction products and parameter Michaelis constant Km reflects the binding affinity between enzyme and substrate (German et al., 2011). Thermodynamic parameter Q10 (temperature coefficient) is the factor by which a biological process changes in response to a 10 ◦ C temperature increase. Activation energy (Ea ) is the energy differential between reactants and transitional substances that subsequently decompose into products. The value of activation enthalpy (∆H) is related to the events necessary to the formation of transition state. Activation grade (Na ) is used to describe the activation level of

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substance (per mole) transformed into the activated state in the enzymatic reaction. Our primary objectives were i) to evaluate soil phosphomonoesterase activities, kinetics and thermodynamics during the rice growth stages after 6 years of consistent swine manure applications, ii) to understand the impacts of swine manure amendment rates on soil chemical and enzymatic properties, and iii) to investigate the correlations between soil enzymatic and chemical variables. MATERIALS AND METHODS Experiment The study was carried out at the experimental farm of ShuangQiao, located in Jiaxing City (120◦ 400 E, 30◦ 500 N) in the Taihu watershed, Zhejiang Province of China. It has the typical characteristics of subtropical monsoon climate. The mean annual temperature and rainfall were 15.7 ◦ C and 1 200 mm, respectively. The soil type is gleyed paddy soil (clay loam), which is the typical soil in the Taihu Lake region of Southeast China. The experimental site was established in 2005. The cropping system was rice (Oryza sativa L.)-oilseed rape (Brassica napus L.) rotation pattern. Generally, the rice season was from June to November and the oilseed rape season from November to May of the following year. Fifteen plots in dimension 4 m × 5 m were laid out two parallel rows and barriers were constructed between them with concrete. Five fertilization treatments in a randomized complete block design with three replicates consisted of: no-fertilizer control (P0 ), 26 kg P ha−1 used as the conventional fertilizer superphosphate (12% P2 O5 ) (P26 ), and 26 (low), 39 (middle), and 52 (high) kg P ha−1 used as different P-based swine manure (M26 , M39 , and M52 , respectively). The swine manure contained organic matter 150 g kg−1 , N 5.6 g kg−1 , P 4.3 g kg−1 , and K 4.0 g kg−1 . The superphosphate and swine manure were applied as basal fertilizer in June and the urea fertilizer as topdressing in July and August. Generally, a typical rice growth stage includes before plowing (BP), seedling stage (SS), tillering stage (TS), heading stage (HS) and maturity stage (MS). Soil surface layer (0–20 cm) samples were collected from a 6-year field over rice growth stages. Fifteen soil cores were taken randomly from each plot and pooled together as a composite sample. After sampling, the visible leaves and roots were removed from the fresh soil samples and then the samples were stored in refrigerator at 4 ◦ C for enzymatic analysis. Half of the

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soil samples were air dried and passed through a 2-mm sieve for soil pH and organic C determination, through a 1-mm sieve for soil Olsen P determination, through a 0.15-mm sieve for soil total C, total N, total P and organic P determination. All of the sub-samples were stored at room temperature until chemical analysis. Soil chemical analysis Soil pH was measured in soil:water suspension (1:5, w/v) with a glass electrode. Total C and total N were analyzed by dry combustion using a vario MAX CNS elemental analyzer (Elementar, Hanau, Germany). Organic C was also determined by dry combustion using an Elemental analyzer (Elementar, Hanau, Germany) after removing inorganic C with 0.1 mol L−1 HCl (Cheng et al., 2008). Total P was determined by the H2 SO4 -HClO4 heating digestion and then PO3− 4 forms were analyzed by the molybdenum-blue method. The organic P was measured by combustion method (maintained 1 h under 550 ◦ C) and then dissolution using 1 mol L−1 H2 SO4 as described by Walker and Adams (1958). Soil Olsen P was extracted by 0.5 mol L−1 NaHCO3 solution (pH 8.5) at soil to solution ratio of 1:20 (w/v) (Olsen and Sommers, 1982), and the filtrate was analyzed for P with the molybdate blue method (Kuo, 1996). Enzymatic analysis ACP and ALP activities were assayed as described by Tabatabai (1994). Using 1 mL of 0.5 mol L−1 pnitrophenyl phosphate (analytical reagent, Sigma, St. Louis, USA) as substrate, 1 g fresh soil was placed into an Erlenmeyer flask, with 0.2 mL toluene and 4 mL modified universal buffer (MUB, pH = 6.5 and 11.0 for ACP and ALP activities, respectively), and incubated for 1 h in a water bath at 37 ◦ C. After incubation, 1 mL of 0.5 mol L−1 CaCl2 and 4 mL of 0.5 mol L−1 NaOH were added to terminate the enzymatic reaction and extract the produced p-nitrophenyl, and the solution was filtered quickly, then the p-nitrophenol was determined spectrophotometerically at 405 nm. The same procedures in ACP and ALP activities measurements were followed for the controls, but the 1 mL 0.5 mol L−1 p-nitrophenyl phosphate was added after (instead of before) the incubation. ACP kinetic and thermodynamic parameters were calculated from the data obtained at different substrate concentrations and temperatures. There were 10, 20, 30, 40, and 50 mmol L−1 p-nitrophenyl phosphate used as substrate, and incubation temperatures were 17, 27, 37, 47, and 57 ◦ C, respectively.

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Calculations Kinetic parameters (V0 , Vmax , and Km ) were determined using the Michaelis-Menten equation as follow: V0 = Vmax ·S0 /(Km + S0 )

(1)

where V0 is the initial velocity (mg kg−1 h−1 ). S0 is the substrate concentration (mmol L−1 ). Vmax is the maximal velocity (mg kg−1 h−1 ) when all enzymes are substrate-saturated. Km is a Michaelis constant, reflecting the binding affinity of substrate and enzyme and being equal to the substrate concentration at half-maximal velocity. Vmax and Km values are calculated using linear regression and Lineweaver-Burk transformed (Stone et al., 2012). Thermodynamic parameters (Q10 , Ea , ∆H, and lgNa ) can be calculated by the following equations. Q10 = VT2 /VT1

(2)

where temperature coefficient Q10 is the factor by which a biological process changes in response to a 10 ◦ C temperature increase, and VT1 and VT2 are the reaction velocities under the set temperature T1 and the higher temperature (T2 ) by increasing 10 ◦ C (T2 = T1 + 10), respectively. Ea = 0.23R·lgQ10 ·T1 ·T2 /(T2 − T1 )

(3)

where Ea is the energy differential (kJ mol−1 ) between reactants and transitional substances that subsequently decompose into products, and R is the gas constant (8.314 J mol−1 K−1 ). ∆H = Ea − R·T

(4)

where ∆H is the activation enthalpy (J mol−1 ), related to the events necessary to the formation of transition state, and the temperature T is equal to (T1 + T2 )/2. lgNa = 23.78 − Ea /2.303R·T

(5)

where Na is the activation grade, describing the activation level of per mole of substance transformed into the activated state in the enzymatic reaction. Statistical analysis Each sample was analyzed in triplicate and the values were then averaged. Statistical analyses were performed using Excel 2010 (Microsoft Co., Seattle, USA) and the SPSS software package version 16.0 (SPSS Inc., Chicago, USA). Statistically significant differences were identified using analysis of variance and Duncan’s multiple comparison test. Data were compared

PHOSPHOMONOESTERASE IN PADDY SOIL

among treatments using the least significant difference test at the 0.05 or 0.01 probability levels. RESULTS Rice yields There was a clear effect of manure-applied P on rice grain yield (mean of six years) (Fig. 1). The annual average grain yield of the control was 5 813 kg ha−1 , which is at par with the local farmers’ average. In addition, the plot receiving superphosphate showed noteworthy improvement in rice grain yield, up to 7 117 kg ha−1 . The P applications at low, middle and high rates (as swine manure) produced rice yields of about 7 283, 8 455 and 8 587 kg ha−1 , respectively. The superphosphate application increased the rice yield by 22%, while the P applications at low, middle and high rates (as manure) increased the rice yields by 25%, 45% and 48%, respectively, compared with control. However, the differences between the P26 and M26 treatments were not significant. There were also no significant differences between the M39 and M52 treatments.

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the high values of total C, total N, total P, and organic P were observed in the growth stages of SS and TS. Obviously, organic P content accounted for 10% to 23% of total P content under the P0 treatment and for 16% to 32% under the P26 treatment. This proportion reached up to 40%, 60%, and 67% under the M26 , M39 , and M52 treatments, respectively. The Olsen P in all treatments was not significantly influenced by P additions. The high values of C/N occurred in the M39 and M52 treatments. The soil chemical properties of M52 and M39 showed no significant differences at various rice growth stages. Soil ACP and ALP activities Soil ACP and ALP activities were relatively higher in the M26 treatment compared to the P26 treatment over the whole rice growth stages (Fig. 2). Soil ACP activity ranged from 115.00 to 454.83 mg p-nitrophenol kg−1 h−1 , while soil ALP activity from 24.41 to 172.20 mg p-nitrophenol kg−1 h−1 . Moreover, there was declining tendency for ALP activity with the decrease of swine manure P rate from 52 to 0 kg P ha−1 . Soil ACP activity was 2.53 to 5.06 times as high as the corresponding ALP activity. However, swine manure amendment had no significant effect on soil ACP and ALP activities from 39 to 52 kg P ha−1 . Soil ACP and ALP activities showed sharply increase in the TS stage for all treatments. Soil ACP kinetic parameters

Fig. 1 Grain yields of late-season rice under 5 P fertilizer treatments: no-fertilizer control (P0 ), 26 kg P ha−1 applied as conventional fertilizer superphosphate (P26 ), and 26, 39, and 52 kg P ha−1 applied as P-based swine manure (M26 , M39 , and M52 , respectively). Vertical bars represent the standard deviations of the means (n = 3). Bars with the same letter are not significantly different at P < 0.05.

Soil chemical properties Changes of soil chemical properties responded to the fertilizer applications were observed at various rice growth stages (Table I). Soil pH changed from 6.18 to 7.08, and it increased with the swine manure application, but decreased with the superphosphate application in comparison with the control. The total C, total N, total P, and organic P decreased in the same order of M52 > M39 > M26 > P26 > P0 . Generally,

Soil ACP initial velocity (V0 ) of the catalytic reaction was significantly affected by temperature and substrate concentration and a peak value appeared in the TS stage for all treatments (Fig. 3). The V0 value was much higher in the M39 or M52 treatment compared with the other treatments. Simultaneously, the M26 treatment almost improved V0 value when compared with P26 treatment at the same temperature and substrate concentration conditions. Information on Vmax and Km responses to superphosphate and swine manure applications under different temperatures during rice growth (Fig. 4) showed that Vmax decreased in the following order at the same temperature: M39 > M26 > P26 > P0 . Generally, the value of Vmax did not significantly increase with increasing swine manure application rates from 39 to 52 kg P ha−1 . The highest Vmax value appeared in the TS stage for all treatments. In the TS stage, the Vmax reached the maximum value of 167.59, 171.08, 187.96, 192.34, and 189.36 mg kg−1 h−1 , respectively, in the P0 , P26 , M26 , M39 , and M52 treatments at 57 ◦ C. The Km value ranged from 18.82

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TABLE I Some chemical properties of paddy soil at various rice growth stages under 5 P fertilizer treatments: no-fertilizer control (P0 ), 26 kg P ha−1 applied as the conventional fertilizer superphosphate (P26 ), and 26, 39, and 52 kg P ha−1 applied as P-based swine manure (M26 , M39 , and M52 , respectively) Growth stagea) BP

P0 P26 M26 M39 M52 P0 P26 M26 M39 M52 P0 P26 M26 M39 M52 P0 P26 M26 M39 M52 P0 P26 M26 M39 M52

SS

TS

HS

MS

a) BP

Treatment

pH (H2 O) 6.53ab) 6.48a 6.77a 7.01a 7.05a 6.58a 6.43a 6.90a 7.06a 7.08a 6.39a 6.45a 6.43a 6.56a 6.61a 6.20a 6.18a 6.25a 6.41a 6.44a 6.66a 6.50a 6.69a 7.02a 7.07a

Total C 18.21b 19.02b 20.39b 27.14a 27.30a 17.98d 22.48c 26.25b 32.05a 33.01a 17.23d 22.76c 25.91b 31.34ab 33.09a 16.71d 21.32c 24.89b 29.72a 30.87a 18.16b 19.22b 22.29ab 24.31a 25.62a

Total N

Total P

Organic P

Olsen P

C/N ratio

2.15b 2.24b 2.35b 3.00a 3.03a 2.09b 2.29b 2.65b 3.24a 3.30a 2.03c 2.52b 2.86b 3.33a 3.58a 1.97b 2.39b 2.77b 3.29a 3.33a 2.24a 2.33a 2.67a 2.83a 2.93a

g kg−1 0.75a 0.99a 1.04a 1.20a 1.31a 0.73c 1.24b 1.32b 2.15a 2.42a 0.67b 1.04b 1.32b 1.90a 2.06a 0.39b 0.53b 0.83b 1.45a 1.49a 0.38b 0.71b 0.79b 1.45a 1.54a

0.09a 0.16a 0.24a 0.56a 0.68a 0.07b 0.20b 0.53b 1.29a 1.44a 0.08b 0.21b 0.50b 1.01a 1.37a 0.09b 0.17b 0.27b 0.75a 0.82a 0.05a 0.19a 0.22a 0.56a 0.61a

0.07a 0.09a 0.12a 0.15a 0.16a 0.10a 0.18a 0.10a 0.19a 0.20a 0.12a 0.13a 0.15a 0.19a 0.22a 0.10a 0.11a 0.13a 0.17a 0.19a 0.09a 0.12a 0.12a 0.17a 0.18a

8.42b 8.48b 8.66b 9.04a 9.00a 8.59b 9.81a 9.89a 9.97a 9.99a 8.48b 9.01a 9.05a 9.40a 9.24a 8.48b 8.90a 8.97a 9.02a 9.26a 8.10a 8.23a 8.32a 8.57a 8.73a

= before plowing; SS = seedling stage; TS = tillering stage; HS = heading stage; MS = maturity stage. followed by the same letter(s) within each column are not significantly different for each rice growth stage at P < 0.05.

b) Means

to 143.22 mmol L−1 and its lowest value appeared in the M39 or M52 treatment while its highest value appeared in the P0 treatment. During the experiment, the change of Vmax basically showed an opposite trend with the change of Km . Soil ACP thermodynamic parameters The thermodynamic parameters such as temperature coefficient (Q10 ), activation energy (Ea ), activation enthalpy (∆H) and activation grade (lgNa ) of soil ACP in the temperature range of 17–27 ◦ C are summarized in Table II. The values of Q10 , Ea , and ∆H in the 5 treatments followed the order of P0 > P26 > M26 > M39 > M52 at each rice growth stage except for the TS. The Q10 , Ea , and ∆H values appeared a sharp decline in the TS, and increased slightly in the HS and MS for all treatments. The Q10 value varied from 1.39 to 3.36. In the TS, Ea had decreased to 3.12, 3.00, 2.83, and 2.89 kJ mol−1 for the P26 , M26 , M39 , and M52 treatments, respectively, compared with 3.29 kJ mol−1 for the P0 treatment. Simultaneously, ∆H decreased to 499, 385, 212, and 270 J mol−1 for the P26 , M26 , M39 , and M52 treatments, respectively,

compared with 666 J mol−1 for the P0 treatment. The change of lgNa had an opposite trend with the changes of Q10 , Ea , and ∆H at 17–27 ◦ C. Correlations between soil chemical and enzymatic variables The correlation analysis between soil chemical properties and enzymatic parameters indicated that soil ACP and ALP activities developed significant and positive correlations with the following variables: total C (r = 0.984 and 0.944 for ACP and ALP, respectively), total N (r = 0.989 and 0.945), total P (r = 0.968 and 0.977), organic P (r = 0.936 and 0.999), and Olsen P (r = 0.929 and 0.998) (Table III). All the chemical variables measured, except for pH, were significantly (P < 0.05) correlated with V0 with r values ranging from 0.820 to 0.983. There were significantly (P < 0.01) positive relationships between Vmax and all chemical variables measured except for pH, with r values ranging from 0.912 to 0.994. Whereas, Km value did not show any significant relationship with chemical variables, with r values ranging from −0.335 to −0.706.

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Fig. 2 Acid phosphomonoesterase (ACP) and alkaline phosphomonoesterase (ALP) activities of paddy soil at various rice growth stages under 5 P fertilizer treatments: no-fertilizer control (P0 ), 26 kg P ha−1 applied as conventional fertilizer superphosphate (P26 ), and 26, 39, and 52 kg P ha−1 applied as P-based swine manure (M26 , M39 , and M52 , respectively). Vertical bars represent the standard deviations of the means (n = 3). Bars with the same letter within a given growth stage are not significantly different among different fertilization treatments at P < 0.05. PNF = p-nitrophenol; BP = before plowing; SS = seedling stage; TS = tillering stage; HS = heading stage; MS = maturity stage.

Q10 , Ea , and ∆H were negatively correlated with chemical variables. In particular, the Q10 , Ea , and ∆H were significantly correlated with total C, total N, and C/N ratio. lgNa values were positively correlated with all chemical variables (Table III). DISCUSSION Effect of swine manure on rice yield and soil P status The results of this study confirmed the findings of others that the crop yield with organic manure was higher than that with superphosphate fertilizer at the same level of P addition (Damodar Reddy et al., 2000). This may be caused by two reasons. In part, some nutrients necessary for plant growth may be provided through swine manure. Furthermore, addition of swine manure is widely known to favorably affect soil microbiological and chemical properties (Guo et al., 2004). Rice yield significantly (P < 0.05) increased with the P rates from 0 to 39 kg P ha−1 . However, swine manure amendment had no significant effect on grain yields from 39 to 52 kg P ha−1 . The reason might

be that 39 kg P ha−1 of the applied swine manure was sufficient completely for rice growth. Previous research showed that the farmers of this region annually applied 50 kg P ha−1 swine manure to ensure high yields (Wang et al., 2001). In this study, the swine manure amendment rate of 39 kg P ha−1 is recommended to improve soil chemical and biological conditions while maintaining the maximum potential for rice production of this region. As shown previously, swine manure contributed more to total P and organic P compared with superphosphate fertilizer (Table I). There are three main reasons for the positive effects of swine manure on soil P status. First, swine manure could supply organic carbon source for microbial growth (Nayak et al., 2007; Ge et al., 2009), resulting in the improvement of transformation efficiency of soil organic P (Marinari et al., 2000). Second, swine manure contains a large number of phosphomonoesterase, which accelerates P transformation from organic form to an inorganic form (Guan, 1989). Third, some of high-molecular-weight compounds, such as humic and fulvic acids derived

Fig. 3 Initial velocity (V0 ) of soil acid phosphomonoesterase, affected by soil temperature (T ) and substrate concentration (C), at various rice growth stages under 5 P fertilizer treatments: no-fertilizer control (P0 ), 26 kg P ha−1 applied as conventional fertilizer superphosphate (P26 ), and 26, 39, and 52 kg P ha−1 applied as P-based swine manure (M26 , M39 , and M52 , respectively). BP = before plowing; SS = seedling stage; TS = tillering stage; HS = heading stage; MS = maturity stage.

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Fig. 3

(Continued)

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Fig. 4 Maximal velocity (Vmax ) and Michaelis constant (Km ) of soil acid phosphomonoesterase at different soil temperatures and various rice growth stages under 5 P fertilizer treatments: no-fertilizer control (P0 ), 26 kg P ha−1 applied as conventional fertilizer superphosphate (P26 ), and 26, 39, and 52 kg P ha−1 applied as P-based swine manure (M26 , M39 , and M52 , respectively). Vertica bars represent the standard deviations of the means (n = 3). Bars with the same letter within a given temperature are not significantly different among different fertilization treatments at P < 0.05. BP = before plowing; SS = seedling stage; TS = tillering stage; HS = heading stage; MS = maturity stage.

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TABLE II Thermodynamic parametersa) (Q10 , Ea , ∆H, lgNa ) of soil acid phosphomonoesterase at 17–27 ◦ C at various rice growth stages under 5 P fertilizer treatments: no-fertilizer control (P0 ), 26 kg P ha−1 applied as conventional fertilizer superphosphate (P26 ), and 26, 39, and 52 kg P ha−1 applied as P-based swine manure (M26 , M39 , and M52 , respectively) Growth stageb)

Treatment

BP

P0 P26 M26 M39 M52 P0 P26 M26 M39 M52 P0 P26 M26 M39 M52 P0 P26 M26 M39 M52 P0 P26 M26 M39 M52

SS

TS

HS

MS

Q10

Ea

∆H

lgNa

3.36ac) 2.28b 2.16b 2.04b 1.99b 1.77a 1.60a 1.58a 1.48a 1.47a 1.49a 1.46a 1.43a 1.39a 1.42a 2.94a 2.17a 2.07a 1.95ab 1.87b 3.06a 2.92a 2.84ab 2.75ab 2.68b

kJ mol−1 9.98a 6.79b 6.34b 5.87c 5.67c 4.70a 3.87b 3.77b 3.23b 3.17b 3.29a 3.12a 3.00a 2.83b 2.89b 8.88a 6.38b 5.99b 5.50bc 5.16c 9.21a 8.83b 8.60b 8.33b 8.12b

J mol−1 7 365a 4 171b 3 725bc 3 255c 3 050c 2 085a 1 253b 1 149b 611b 555b 666a 499a 385a 212a 270a 6 265a 3 763b 3 375b 2 883c 2 538c 6 595a 6 209a 5 980a 5 715ab 5 502b

22.12a 22.65a 22.73a 22.81a 22.84a 23.00a 23.14a 23.16a 23.24a 23.25a 23.24a 23.26a 23.28a 23.31a 23.30a 22.31a 22.72a 22.79a 22.87a 22.93a 22.25a 22.32a 22.35a 22.40a 22.43a

a) Q 10 = temperature coefficient; Ea = activation energy; ∆H = activation enthalpy; Na = b) BP = before plowing; SS = seedling stage; TS = tillering stage; HS = heading stage; MS

activation grade. = maturity stage. c) Means followed by the same letter(s) within each column are not significantly different for each rice growth stage at P < 0.05. TABLE III Pearson correlation coefficients between soil chemical properties and enzymatic parametersa) Chemical property

ACP activity

ALP activity

V0

Vmax

Km

Q10

Ea

∆H

lgNa

pH Total C Total N Total P Organic P Olsen P C/N ratio

0.818 0.984** 0.989** 0.968* 0.936* 0.929* 0.943*

0.920 0.944* 0.945* 0.977** 0.999** 0.998** 0.868

0.820 0.976** 0.983** 0.972* 0.959* 0.952* 0.923*

0.912 0.981** 0.977** 0.994** 0.979** 0.981** 0.954*

−0.706 −0.385 −0.366 −0.483 −0.567 −0.587 −0.335

−0.782 −0.938* −0.936* −0.889 −0.802 −0.797 −0.974*

−0.803 −0.952* −0.950* −0.908 −0.826 −0.822 −0.981**

−0.804 −0.952* −0.950* −0.908 −0.826 −0.822 −0.981**

0.804 0.951* 0.950* 0.908 0.826 0.822 0.981**

*, **Significant at P < 0.05 and P < 0.01, respectively. = acid phosphomonoesterase; ALP = alkaline phosphomonoesterase; V0 = initial velocity; Vmax = maximal velocity; Km = Michaelis constant; Q10 = temperature coefficient; Ea = activation energy; ∆H = activation enthalpy; Na = activation grade. a) ACP

from swine manure, occupy part of the iron and aluminum oxide adsorption sites, which inhibits the adsorption of soil P (Xavier et al., 2009; Wang et al., 2012). Effect of swine manure on soil ACP and ALP activities The results clearly showed that soil ACP and ALP activities in the swine manure addition plots were relatively higher than those in the superphosphate ad-

dition plots (Fig. 2), which confirmed the findings of others that organic manure stimulated soil enzyme activities (Elfstrand et al., 2007; Tao et al., 2009). Guan (1989) concluded that organic manure contained phosphomonoesterase itself, which helps to transform organic P into inorganic P. In this study, soil ACP significantly (P < 0.05) increased with P application from 0 to 39 kg P ha−1 . However, swine manure amendment had no significant effect on ACP activity from 39 to

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52 kg P ha−1 . As some studies mentioned that the presence of increased P concentrations might decrease phosphatase activities (Olander and Vitousek, 2000; Dilly and Nannipieri, 2001). As reported by Lu (2000), ACP activity generally exceeded ALP activity in the low pH soils. Our studies found that ACP activity was strikingly higher than ALP activity in all treatments. In other words, ACP was the predominant in maintaining and controlling P cycling in the soil with pH from 6.18 to 7.08. Interestingly, plant roots are main producers of ACP, but do not secrete ALP. ALP originates from soil fauna, bacteria and fungi (Kr¨amer and Green, 2000). This showed that rice roots play a vital role in P mineralization through the secretion of ACP in the tested soil. Both ACP and ALP activities appeared a peak in the TS stage for all treatments. It is suggested that in this rice growth stage, various organic matter were decomposed into small molecule compounds, humus and auxin substances, stimulated the metabolism of rice roots and soil microbe, and finally resulted in the increase of ACP and ALP secretion. Effect of swine manure on soil ACP kinetics As anticipated, compared with the superphosphate treatment, swine manure increased the V0 and Vmax values and decreased the Km value over the whole rice growth stages (Figs. 3 and 4). These results agreed with the previous research by Zaman et al. (1999), who found that soil organic matter addition had more influence on soil kinetics compared with superphosphate addition at the same P level. At the low substrate concentration, the V0 increased sharply and leveled off gradually with the increasing of substrate concentration in all treatments, and these results accorded with the characteristics of general enzymatic kinetic (Zhu, 2011). The Vmax increased because the calorific movement of enzyme molecules hurried and the collisions between enzyme and substrate sped up, resulting in more substrate transformed (Zhang et al., 2010). The low Km value represented the strong affinity between substrate and soil enzyme. The lowest Km value appeared in the M39 or M52 treatment while its highest value appeared in the P0 treatment, implying that the affinity increased markedly in the M39 or M52 treatment and unobviously increased in the other treatments compared with the P0 treatment. Generally, the Km of pure enzyme or soil enzyme decreased with increasing temperature because of enhanced translocation and dissolution of the substrates (Zhang et al., 2010). However, in this study, the strongest affinity between substrate and

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soil enzyme occurred at 37 ◦ C due to soil heterogeneity. The change of Km had an opposite trend with the change of the corresponding Vmax , implying that the affinity between phosphomonoesterase and substrate had positive relationship with phosphomonoesterase amounts, which actually agrees with the result found by Zhu (2011). Effect of swine manure on soil ACP thermodynamics The thermodynamic parameters (Q10 , Ea , ∆H, and lgNa ) of soil ACP at various rice growth stages under 17–27 ◦ C were studied (Table II). The Q10 value of the enzyme-catalyzed reaction is approximately equal to 2 (Trasar-Cepeda et al., 2007; Zhang et al., 2010). However, in this study, the Q10 value ranged from 1.39 to 3.36. Although there were few reports that the value was higher than 2, the high Q10 of swine manure applied soils indicated a strong thermodynamic effect on soil ACP velocity. The Ea value for enzyme-catalyzed reactions is usually lower than that for non-enzymatic reactions, because enzymes can reduce the energy barrier that must be overcome before the reaction can take place. The high value of ∆H implies a large amount of stretching, squeezing or even breaking of chemical bonds occurred before its transition state (Lai and Tabatabai, 1992). The Q10 value followed the order of P0 > P26 > M26 > M39 > M52 at each rice growth stage except for the TS. The values of Ea and ∆H had the similar tendency with Q10 , implying that the plots amended with swine manure had a certain buffer effect on enzyme activity with temperature change. Swine manure application could stimulate the enzymecatalyzed reactions more easily than superphosphate application by decreasing the Ea and ∆H values in the temperature range of 17–27 ◦ C. Soil enzymatic parameters related to chemical properties The analysis results showed positive and significant relationships between phosphomonoesterase activities and total C, and similar results have been observed between phosphomonoesterase activities and total N (Table III), implying that the distributions of ACP and ALP activities mainly followed the distributions of total C and total N. The high total C and total N contents of the M39 and M52 treatments may explain the high levels of ACP and ALP activities observed at various rice growth stages. Therefore, the phosphomonoesterase activity can be an indicator of the changes in soil amendments (Kr¨amer and Green, 2000; Sardans et al., 2008). Xie et al. (2011) reported that the low concentration but high proportion of organic P

PHOSPHOMONOESTERASE IN PADDY SOIL

developed high activities of phosphomonoesterase. In this study, ACP and ALP activities had significant and positive correlations with organic P. This is not consistent with the previous report (Albrecht et al., 2010) where negative correlation between phosphomonoesterase activities and organic P was found. Some studies also reported that Km was affected by soil organic matter content, because soil organic C trapped soil phosphomonoesterase, prevented the phosphomonoesterase from binding with substrate, and thus increased the Km value (Tietjen and Wetzel, 2003; Zhang et al., 2010). However, the data analysis did not show any relationship between Km and chemical variables in this experiment. The Km , Q10 , Ea , and ∆H were all negatively correlated with chemical variables, indicating that the better soil nutrition condition, the lower Km , Q10 , Ea , and ∆H values would be. The relatively low Km , Q10 , Ea , and ∆H values can cause the enzymatic reactions more likely to occur in the soil. CONCLUSIONS After 6-year of field experimentation, swine manure application significantly increased rice yield, and improved soil chemical and enzymatic properties compared with superphosphate application (26 kg P ha−1 ). Simultaneously, the grain yield and soil chemical properties were significantly improved with the application of P-based swine manure from 0 to 39 kg P ha−1 . The M39 and M52 treatments had high V0 , Vmax , and lgNa values but low Km , Q10 , Ea , and ∆H values, and there were no significant differences between M39 and M52 treatments at various rice growth stages. The correlation analysis showed that the distributions of ACP and ALP activities mainly followed the distributions of total C and total N. The Km , Q10 , Ea , and ∆H were all negatively correlated with chemical variables, indicating that the better the soil nutrition condition, the lower the Km , Q10 , Ea and ∆H values would be. Therefore, 39 kg P ha−1 could be recommended as the most appropriate rate of swine manure application, since the 39 kg P ha−1 swine manure treatment significantly increased rice grain yield and improved soil chemical and enzymatic properties while maintaining the most efficient usage of swine manure. Further study should pay more attention to the dynamics of soil phosphomonoesterase relative to soil temperature change over the course of rice growth stages. ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (Nos. 21077088, 41271314

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and 51008107). The authors would like to thank Prof. Maria Arlene ADVIENTO-BORBE from the University of California at Davis, USA for English revising. The authors are also grateful to anonymous reviewers for their helpful comments and suggestions on this manuscript. REFERENCES Albrecht, R., Le Petit, J., Calvert, V., Terrom, G. and P´ erissol, C. 2010. Changes in the level of alkaline and acid phosphatase activities during green wastes and sewage sludge cocomposting. Bioresource Technol. 101: 228–233. Ayaga, G., Todd, A. and Brookes, P. C. 2006. Enhanced biological cycling of phosphorus increases its availability to crops in low-input sub-Saharan farming systems. Soil Biol. Biochem. 38: 81–90. Cheng, C. H., Lehmann, J., Thies, J. E. and Burton, S. D. 2008. Stability of black carbon in soils across a climatic gradient. J. Geophys. Res. 113: G02027, doi: 10.1029/2007JG000642. Criquet, S., Braud, A. and N` eble, S. 2007. Short-term effects of sewage sludge application on phosphatase activities and available P fractions in Mediterranean soils. Soil Biol. Biochem. 39: 921–929. Criquet, S., Ferre, E., Farnet, A. M. and Le Petit, J. 2004. Annual dynamics of phosphatase activities in an evergreen oak litter: Influence of biotic and abiotic factors. Soil Biol. Biochem. 36: 1111–1118. Damodar Reddy, D., Subba Rao, A. and Rupa, T. R. 2000. Effects of continuous use of cattle manure and fertilizer phosphorus on crop yields and soil organic phosphorus in a Vertisol. Bioresource Technol. 75: 113–118. Dilly, O. and Nannipieri, P. 2001. Response of ATP content, respiration rate and enzyme activities in an arable and a forest soil to nutrient additions. Biol. Fert. Soils. 34: 64–72. Elfstrand, S., B˚ ath, B. and M˚ artensson, A. 2007. Influence of various forms of green manure amendment on soil microbial community composition, enzyme activity and nutrient levels in leek. Appl. Soil Ecol. 36: 70–82. Ge, G. F., Li, Z. J., Zhang, J., Wang, L. G., Xu, M. G., Zhang, J. B., Wang, J. K., Xie, X. L. and Liang, Y. C. 2009. Geographical and climatic differences in long-term effect of organic and inorganic amendments on soil enzymatic activities and respiration in field experimental stations of China. Ecol. Complex. 6: 421–431. German, D. P., Marcelo, K. R. B., Stone, M. M. and Allison, S. D. 2011. The Michaelis-Menten kinetics of soil extracellular enzymes in response to temperature: A cross-latitudinal study. Glob. Change Biol. 18: 1468–1479. Guan, S. Y. 1989. Studies on the factors influencing soil enzyme activities. I. Effects of organic manures on soil enzyme activities and N, P transformation. Acta Pedol. Sin. (in Chinese). 26: 72–78. Guo, H. Y., Zhu, J. G., Wang, X. R., Wu, Z. H. and Zhang, Z. 2004. Case study on nitrogen and phosphorus emissions from paddy field in Taihu region. Environ. Geochem. Hlth. 26: 209–219. Huang, W. J., Zhang, D. Q., Li, Y. L., Lu, X. K., Zhang, W., Huang, J., Otieno, D., Xu, Z. H., Liu, J. X., Liu, S. Z. and Chu, G. W. 2012. Responses of soil acid phosphomonoesterase activity to simulated nitrogen deposition in three forests of subtropical China. Pedosphere. 22: 698–706. Khan, K. S. and Joergensen, R. G. 2009. Changes in microbial

306

biomass and P fractions in biogenic household waste compost amended with inorganic P fertilizers. Bioresource Technol. 100: 303–309. Koch, O., Tscherko, D. and Kandeler, E. 2007. Temperature sensitivity of microbial respiration, nitrogen mineralization, and potential soil enzyme activities in organic alpine soils. Global Biogeochem. Cy. 21: GB4017, doi:10.1029/2007GB002983. Koopmans, G. F., Chardon, W. J. and McDowell, R. W. 2007. Phosphorus movement and speciation in a sandy soil profile after long-term animal manure applications. J. Environ. Qual. 36: 305–315. Kr¨ amer, S. and Green, D. M. 2000. Acid and alkaline phosphatase dynamics and their relationship to soil microclimate in a semiarid woodland. Soil Biol. Biochem. 32: 179–188. Kunito, T., Tobitani, T., Moro, H. and Toda, H. 2012. Phosphorus limitation in microorganisms leads to high phosphomonoesterase activity in acid forest soils. Pedobiologia. 55: 263–270. Kuo, S. 1996. Phosphorus. In Sparks, D. L. (ed.) Methods of Soil Analysis. Part 3. Chemical Methods. SSSA Book Series No. 5. SSSA and ASA, Madison, WI. pp. 869–919. Lai, C. M. and Tabatabai, M. A. 1992. Kinetic parameters of immobilized urease. Soil Biol. Biochem. 24: 225–228. Liang, X. Q., Li, L., Chen, Y. X., Li, H., Liu, J., He, M. M., Ye, Y. S., Tian, G. M. and Lundy, M. 2013. Dissolved phosphorus losses by lateral seepage from swine manure amendments for organic rice production. Soil Sci. Soc. Am. J. 77: 765– 773. Liu, Y. R., Li, X., Shen, Q. R. and Xu, Y. C. 2013. Enzyme activity in water-stable soil aggregates as affected by longterm application of organic manure and chemical fertiliser. Pedosphere. 23: 111–119. Lu, R. K. 2000. Analytical Methods of Soil Agrochemistry (in Chinese). China Agricultural Science and Technology Press, Beijing. Malik, M. A., Marschner, P. and Khan, K. S. 2012. Addition of organic and inorganic P sources to soil—Effects on P pools and microorganisms. Soil Biol. Biochem. 49: 106–113. Marinari, S., Masciandaro, G., Ceccanti, B. and Grego, S. 2000. Influence of organic and mineral fertilisers on soil biological and physical properties. Bioresource Technol. 72: 9–17. Miller, B. W. and Fox, T. R. 2011. Long-term fertilizer effects on oxalate-desorbable phosphorus pools in a typic paleaquult. Soil Sci. Soc. Am. J. 75: 1110–1116. Nayak, D. R., Jagadeesh Babu, Y. and Adhya, T. K. 2007. Longterm application of compost influences microbial biomass and enzyme activities in a tropical Aeric Endoaquept planted to rice under flooded condition. Soil Biol. Biochem. 39: 1897– 1906. N` eble, S., Calvert, V., Le Petit, J. and Criquet, S. 2007. Dynamics of phosphatase activities in a cork oak litter (Quercus suber L.) following sewage sludge application. Soil Biol. Biochem. 39: 2735–2742. Olander, L. P. and Vitousek, P. M. 2000. Regulation of soil phosphatase and chitinase activity by N and P availability. Biogeochemistry. 49: 175–190. Olsen, S. R. and Sommers, L. E. 1982. Phosphorus. In Page, A. L., Miller, R. H. and Deeney, D. R. (eds.) Methods of Soil Analysis. Part 2. ASA, Madison, WI. pp. 403–430. Ramaekers, L., Remans, R., Rao, I. M., Blair, M. W. and Vanderleyden, J. 2010. Strategies for improving phosphorus acquisition efficiency of crop plants. Field Crop. Res. 117: 169– 176. Rao, M. A., Gianfreda, L., Palmiero, F. and Violante, A. 1996. Interactions of acid phosphatase with clays, organic molecules and organo-mineral complexes. Soil Sci. 161: 751–760.

L. LI et al.

Saha, S., Mina, B. L., Gopinath, K. A., Kundu, S. and Gupta, H. S. 2008. Relative changes in phosphatase activities as influenced by source and application rate of organic composts in field crops. Bioresource Technol. 99: 1750–1757. Sardans, J., Pe˜ nuelas, J. and Ogaya, R. 2008. Experimental drought reduced acid and alkaline phosphatase activity and increased organic extractable P in soil in a Quercus ilex Mediterranean forest. Eur. J. Soil Biol. 44: 509–520. Stone, M. M., Weiss, M. S., Goodale, C. L., Beth Adams, M., Fernandez, I. J., German, D. P. and Allison, S. D. 2012. Temperature sensitivity of soil enzyme kinetics under Nfertilization in two temperate forests. Glob. Change Biol. 18: 1173–1184. Tabatabai, M. A. 1994. Soil enzymes. In Weaver, R. W., Angle, J. S. and Bottomley, P. S. (eds.) Methods of Soil Analysis. Part 2. Microbiological and Biochemical Properties. SSSA Book Series No. 5. Soil Science Society of America, Madison, WI. pp. 775–833. Tao, J., Griffiths, B., Zhang, S. J., Chen, X. Y., Liu, M. Q., Hu, F. and Li, H. X. 2009. Effects of earthworms on soil enzyme activity in an organic residue amended rice-wheat rotation agro-ecosystem. Appl. Soil Ecol. 42: 221–226. Tietjen, T. and Wetzel, R. G. 2003. Extracellular enzyme-clay mineral complexes: Enzyme adsorption, alteration of enzyme activity, and protection from photodegradation. Aquat. Ecol. 37: 331–339. Trasar-Cepeda, C., Gil-Sotres, F. and Leir´ os, M. C. 2007. Thermodynamic parameters of enzymes in grassland soils from Galicia, NW Spain. Soil Biol. Biochem. 39: 311–319. Walker, T. W. and Adams, A. F. R. 1958. Studies on soil organic matter: I. Influence of phosphorus content of parent materials on accumulations of carbon, nitrogen, sulfur, and organic phosphorus in grassland soils. Soil Sci. 85: 307–318. Wallenstein, M. D., Mcmahon, S. K. and Schimel, J. P. 2009. Seasonal variation in enzyme activities and temperature sensitivities in Arctic tundra soils. Glob. Change Biol. 15: 1631– 1639. Wang, K., Zhang, Z. J., Zhu, Y. M., Wang, G. H., Shi, D. C. and Christie, P. 2001. Surface water phosphorus dynamics in rice fields receiving fertiliser and manure phosphorus. Chemosphere. 42: 209–214. Wang, S. X., Liang, X. Q., Chen, Y. X., Luo, Q. X., Liang, W. S., Li, S., Huang, C. L., Li, Z. Z., Wang, L. L., Li, W. and Shao, X. X. 2012. Phosphorus loss potential and phosphatase activity under phosphorus fertilization in long-term paddy wetland agroecosystems. Soil Sci. Soc. Am. J. 76: 161–167. Xavier, F. A. S., de Oliveira, T. S., Andrade, F. V. and de S´ a Mendon¸ca, E. 2009. Phosphorus fractionation in a sandy soil under organic agriculture in Northeastern Brazil. Geoderma. 151: 417–423. Xie, C. S., Zhao, J., Tang, J., Xu, J., Lin, X. Y. and Xu, X. H. 2011. The phosphorus fractions and alkaline phosphatase activities in sludge. Bioresource Technol. 102: 2455–2461. Zaman, M., Di, H. J. and Cameron, K. C. 1999. A field study of gross rates of N mineralization and nitrification and their relationships to microbial biomass and enzyme activities in soils treated with dairy effluent and ammonium fertilizer. Soil Use Manage. 15: 188–194. Zhang, Y. L., Chen, L. J., Sun, C. X., Li, D. P., Wu, Z. J. and Duan, Z. H. 2010. Kinetic and thermodynamic properties of hydrolases in Northeastern China soils affected by temperature. Agrochimica. 54: 232–244. Zhu, M. E. 2011. Soil Enzyme Kinetics and Thermodynamics (in Chinese). Science Press, Beijing.