Journal of Soil Science and Plant Nutrition The activity and kinetic parameters of oxidoreductases in phaeozem in response to long-term fertiliser management Z.C Shang 1,2,4, L.L Zhang1*, Z.J. Wu1, P. Gong1,4, D.P. Li 1, P. Zhu3 and H.J. Gao3 1Instituite of Applied Ecology, Chinese Academy of Sciences, P. O. Box 417, Shenyang, 110016, China. 2Shanghai Research Institute of Chemical Industry, Shanghai, 200062, China. 3Research Center of Agricultural Environment and Resources, Jilin Academy of Agricultural sciences, P. O. Box 306. Changchun, 130124, China. 4Graduate School of the Chinese Academy of Sciences, Beijing 100039, China.*Corresponding author: [email protected] Abstract This study describes the effects of balanced versus nutrient-deficient fertilisation on soil nutrient content and selected oxidoreductase activity and kinetic parameters in a long-term (28 years) field experiment conducted using a phaeozem type soil in the Jilin Province of northeast China. As compared to no or unbalanced fertilisation, balanced fertilisation improved the overall chemical fertility of the soil and sig- nificantly increased the activities and Vmax values of soil dehydrogenase and cata- lase. Compared with control (CK), unbalanced fertilisation (with the exception of P deficiency) significantly increased the total carbon content and soil dehydrogenase activity but had less of an effect on the Vmax of the enzyme, whereas the soil cata- lase activity and its Vmax were less affected under unbalanced fertilisation condi- tions. The Km value of soil dehydrogenase increased with the application of chemi- cal NPK combined with farmyard manure but decreased under the application of NPK, NP, and PK. The Km value of soil catalase decreased under the application of NK and showed little difference between CK and the other fertilisation treatments. The variations in the activities and kinetic parameters of the enzymes revealed the benefits of long-term balanced fertilisation, particularly the combined application of chemical and organic fertilisers, by improving the chemical and biological fertility of phaeozem. The results also indicated that unbalanced fertilisation with P deficiency (NK) could enhance phaeozem quality, but this effect was limited. Keywords: Enzymatic kinetic parameters, dehydrogenase, catalase, phaeozem, fertilisation. Shang et al. 1. Introduction The dehydrogenase and catalase activities in soil are economical decisions about their fertiliser management mainly associated with soil microbes (García-Gil strategies rather than from the viewpoint of sustainable et al., 2000; Masciandaro et al., 2000; Zhang et al., development (Chu et al., 2007). When the farmer is un- 2009). In general, the activities of these enzymes can der economic stress, the nitrogen and, to a lesser extent, be used to characterise the abundance and metabolic phosphorus levels are usually maintained, but the need activity of soil microbes, whereas their kinetic param- for potassium is either underestimated or ignored. As a eters are used to describe the catalytic activity, ori- result, unbalanced fertilisation remains widespread. It gin, and substrate affinity of the enzymes (García-Gil is, therefore, urgent to formulate a rational fertilisation et al., 2000; Zhang et al., 2009). Many studies have system that can both maintain the farmers’ income and examined the effects of different fertilisation systems improve the quality of the soil. on the activities of these enzymes, but little informa- Phaeozem is an agricultural soil found in approx- tion is available concerning the long-term effects of imately 20.2% of all arable land (Wang et al., 2009) fertiliser management on the activities and kinetic and is the prevalent soil type in the Jilin Province of parameters of these two enzymes (Chu et al., 2007, northeast China, an important grain production area of Borowska and Koper, 2010). the country. To assist the local farmers in developing a Organic and inorganic fertilisers are primarily rational fertilisation strategy, a long-term field experi- used to increase the availability of nutrients; however, ment with maize was conducted in Gongzhuling City. organic fertilisers usually increase the physical and Many studies have revealed that long-term fertilisa- chemical characteristics of the soil and enzymatic ac- tion, whether balanced or unbalanced, influences both tivity (Masto et al., 2006, Chu et al., 2007, Borowska the abundance and composition of the soil microbial and Koper, 2010). It has been reported that inorganic community (Wu et al., 2011; Wertz et al., 2012). Be- fertilisers have a relatively reduced effect on the soil cause the enzymes in the soil originate mainly from microbial activity than organic fertilisers (Hopkins these microorganisms, our hypothesis was that bal- and Shiel, 1996). Masciandaro et al., (2000) found anced or unbalanced long-term fertilisation could that the Vmax value of soil dehydrogenase markedly alter the specific activity and kinetic parameters of increased under organic (manure) versus chemical oxidoreductases to differing degrees. The aim of this fertiliser (urea) but that its Km value did not change, study was to evaluate the changes in the activities and indicating that the use of organic fertiliser caused an kinetic characteristics of selected oxidoreductase un- increase of dehydrogenase in the active microbial der different long-term fertiliser managements. biomass but did not alter its substrate affinity. García- Gil (2000) indicated that the catalase activity was in- 2. Materials and methods creased in soil treated with organic fertiliser due to the stimulation of enzyme biosynthesis by the addition of 2.1 Site description and field experiment organic residues (García-Gil et al., 2000). The balanced supplementation of major nutrient el- The long-term experimental site in Gongzhuling City ements (N, P, K) is usually beneficial for plant growth. (43°39’53”N, 124°59'44"E) in Jilin Province was es- However, in China, farmers are often forced to make tablished in 1979. The mean annual temperature is Journal of Soil Science and Plant Nutrition The activity and kinetic parameters of oxidoreductases in phaeozem 5-6°C, with maximum and minimum temperatures of M NaHCO3 and was measured colorimetrically using 36.6°C and -34.6°C, respectively. This area experi- molybdate (Olsen and Sommers, 1982). The available ences four distinct seasons, with a cold winter and a K content was determined by extraction with ammo- warm summer (June-August). The area has an annual nium acetate and was measured using flame photometry precipitation of approximately 570 mm, with 70% of (Shi, 1976). The soil pH was determined using a glass the total rain falling in June, July, and August. The non- electrode (LICI, PHS-3C). frost period lasts for 120-140 days. Six treatment types were tested: Control (no fertilisation, CK), NK, NP, 2.4 Measurements of the soil enzyme activities PK, NPK, and M+NPK (farmyard manure plus NPK). Each treatment comprised an area of 100 m2 and was The soil dehydrogenase activity (mg TPF per g soil performed in three replicates. The annual amounts of per 24 h) was determined by the spectrophotomet- chemical fertilisers and farmyard manure used were ric measurement of 2,3,5-triphenylformazan (TPF) -2 -2 -2 165 kg hm N, 82.5 kg hm P2O5, 82.5 kg hm K2O, formation after the soil samples were treated with and 23100 kg hm-2 manure. The M+NPK regimen in- 2,3,5-triphenyltetrazolium chloride (TTC) at 37 ºC volved the application of the same N amount as the for 24 h (Tabatabai, 1994). The soil catalase activity other N-containing treatments and included 49.5 kg (mmol H2O2 per g soil per h) was determined by the -2 -2 hm inorganic N and 115.5 kg hm organic N. All of colorimetric measurement of unreacted H2O2 after the the fertilisers were applied as base fertiliser. soil samples were treated with H2O2 at 20ºC for 10 min (Trasar-Cepeda et al., 1999). 2.2 Soil sampling 2.5 Measurements of the soil enzyme kinetic A total of five soil cores (60 cm3) were collected from parameters each treatment replicate after harvesting the maize and were pooled into a composite sample. A portion of the The substrate concentrations for the measurement of sample was air-dried and sieved through a 2 mm mesh the soil dehydrogenase and catalase activities ranged for the analysis of the chemical properties; another por- from 10 to 90 mM and from 2 to 16 mM, respective- tion was kept fresh and sieved (2 mm) for the measure- ly (the series of substrate concentrations are 10, 20, ments of the enzyme activity and kinetic parameters. 30, 50, 70, or 90 mM and 2, 4, 6, 10, 13, or 16 mM, respectively). The kinetic parameters Vmax and Km 2.3 Assays of the soil chemical properties were determined using the Lineweaver-Burk version of the Michaelis-Menten equation, as follows: The total C and N contents were determined using an element analyser (Elementar Vario EL) (Matejovic, 1995), and the total P content was determined using the HClO4 digestion method (Olson and Sommers, 1982). The available N content was extracted from where V is the reaction velocity, Vmax is the maxi- fresh subsamples with 2 M KCl and measured using a mum reaction velocity, Km is the Michaelis constant continuous flow analyser (AAШ German). The avail- (the substrate concentration at half-maximal reaction able P content was determined by extraction with 0.5 velocity) and [S] is the substrate concentration. Journal of Soil Science and Plant Nutrition Shang et al. 2.6 Statistical analysis 3. Results The statistical analysis was performed using Microsoft 3.1 Soil nutrient contents and pH value Excel 2000 and the SPSS 16.0 program for Windows. All of the data shown in the tables and figures represent Compared with CK, all of the fertilisation treat- the mean ± SD of three replicates. A P value less than ments, with the exception of NK, had significantly 0.05 was considered statistically significant.