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Soil Organic Carbon, Black Carbon, and Enzyme Activity Under Long-Term Fertilization
Journal of Integrative Agriculture 2014, 13(3): 517-524
March 2014
RESEARCH ARTICLE
Soil Organic Carbon, Black Carbon, and Enzyme Activity Under LongTerm Fertilization SHAO Xing-hua and ZHENG Jian-wei Department of Life Science, Shangrao Normal University, Jiangxi 334001, P.R.China
Abstract The present study aims to understand the effects of long-term fertilization on soil organic carbon (SOC), black carbon (BC), enzyme activity, and the relationships among these parameters. Paddy field was continuously fertilized over 30 yr with nine different fertilizer treatments including N, P, K, NP, NK, NPK, 2NPK (two-fold NPK), NPK+manure (NPKM), and CK (no fertilization), N, 90 kg urea-N ha-1 yr-1; P, 45 kg triple superphosphate-P2O5 ha-1 yr-1; K, 75 kg potassium chloride-K2O ha-1 yr-1; and pig manure, 22 500 kg ha-1 yr-1. Soil samples were collected and determined for SOC, BC content, and enzyme activity. The results showed that the SOC in the NPKM treatment was significantly higher than those in the K, P, and CK treatments. The lowest SOC content was found in the CK treatment. SOC content was similar in the N, NP, NK, NPK, 2NPK, and NPKM treatments. There was no significant difference in BC content among different treatments. The BC-to-SOC ratios (BC/SOC) ranged from 0.50 to 0.63, suggesting that BC might originate from the same source. Regarding enzyme activity, NPK treatment had higher urease activity than NPKM treatment. The urease activity of NPKM treatment was significantly higher than that of 2NPK, NP, N, P, K, CK, and NPKM treatment which produced higher activities of acid phosphatase, catalase, and invertase than all other treatments. Our results indicated that long-term fertilization did not significantly affect BC content. Concurrent application of manure and mineral fertilizers increased SOC content and significantly enhanced soil enzyme activities. Correlation analysis showed that catalase activity was significantly associated with invertase activity, but SOC, BC, and enzyme activity levels were not significantly correlated with one another. No significant correlations were observed between BC and soil enzymes. It is unknown whether soil enzymes play a role in the decomposition of BC. Key words: organic carbon, black carbon, enzyme activity, fertilization
INTRODUCTION Soil fertility and productivity depend on soil organic matter (OM), which represents the dominant reservoir of plant nutrients such as N, P and S (Steiner et al. 2007). The soil organic carbon pool in 1 m depth is 3.3 times of the atmospheric and 4.5 times of the biotic carbon pool (Lal 2004). As the largest carbon pool, changes in the soil carbon pool may considerably influence
global carbon balance (Lal 2008). Agricultural soil currently accounts for 12% of the total terrestrial land area (Leff et al. 2004). Agricultural activities such as tillage, land-use conversion, fertilization, and biomass alteration lead to fundamental changes in terrestrial carbon pools and fluxes (McLauchlan 2006). Numerous studies have described how fertilization changes the soil organic carbon (SOC) pool (Li et al. 2006; Xie et al. 2012; Wang et al. 2013). Specifically, fertilization with inorganic fertilizer combined with organic manure
Received 9 October, 2013 Accepted 18 December, 2013 Correspondence SHAO Xing-hua, Mobile: 13361731892, E-mail: [email protected]
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60707-8
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seems to have better results in improving soil OM than inorganic fertilization alone (Shao et al. 2011; Zhu et al. 2012). The SOC pool has two main components, i.e., the inert or recalcitrant component and the labile or active fraction (Lal 2006). Black carbon (BC), the main form of the inert or recalcitrant component of SOC pool, is widely distributed in atmosphere, hydrosphere, biosphere, and lithosphere. Black carbon is mainly produced by incomplete combustion of organics such as plant materials or fossil fuels (Kuhlbusch 1998; Schmidt and Noack 2000). The ubiquity of BC is related to its extensive production in combustion processes and its recalcitrant nature (Muri et al. 2002). BC is a continuum of fixed carbon (C) forms rather than a particular compound. The refractory nature of BC enables it to act as a significant sink of atmospheric CO 2 and slows the rate at which photosynthetic C is returned to the atmosphere (Woolf et al. 2010). Photochemical abiotic oxidation and microbial decomposition have been suggested to be the two major mechanisms for BC degradation (Goldberg 1985). Liang et al. (2008) suggested that BC decomposes extremely slowly so that chemical changes in samples cannot be detected. Zimmerman et al. (2010) measured the release of CO2 over one year from microbial and sterile incubations of biochars made of a range of biomass types or prepared under various combustion conditions and found that carbon release from abiotic incubations was 50-90% of that from microbe-inoculated incubations. Kuzyakov et al. (2009) reported that BC decomposition rate was very low under natural soil conditions but increased dramatically up to 5 times after glucose was added to boost microbial growth, suggesting that high soil microbial activity greatly accelerated BC decomposition. However, how BC content is affected by long-term fertilization practices under field conditions is largely unclear. Enzymes in soil affect soil composition by catalyzing a number of reactions. Many studies have shown that soil enzymes indicate changes in soil ecological system function and effects of pollution and agricultural practices on soil physical and chemical properties (Ren and Stefano 2000). Urease, acid phosphatase, and invertase are enzymes
involved in the N, P and C cycles (Bremner and Mulvaney 1978; Amador et al. 1997) and they control mineralization and transformation of soil N, P, and C compounds, which provide essential nutrition for plants. Protection from activated oxygen species such as hydrogen peroxide can be achieved using catalase (Liu et al. 2008). Decomposition of OM in soil depends on substance properties and accessibility of microorganisms and their enzymes (Ekschmitt et al. 2008). It is believed that enzyme activities also affect BC stability in soils. However, only a few studies have examined effects of increased soil enzyme activity on BC decomposition. In the present study, we aimed to investiagte the difference in SOC and BC contents and enzyme activity among the different fertilization threatments in soils and assess the correlations among these parameters. We speculated that SOC, BC, and enzyme activity in soil and their correlations would be affected by long-term fertilization practices.
RESULTS SOC and BC contents SOC contents in the nine treatments ranged from 15.9 to 17.9 g kg-1 with a mean of 17.1 g kg-1 (Table 1). The SOC content in NPKM did not significantly differ from those in N, NP, NK, NPK, and 2NPK treatments. BC contents in the nine treatments varied between 8.9 and 10.6 g kg-1 with an average of 9.66 g kg-1. Statistical analyses revealed that the difference in BC content among different treatments was not significant. Thus, the BC content in all fertilizer treatments did not markedly vary from that in CK by fertilization. This phenomenon can be attributed to intrinsic characteristics of BC that resists to microbial decomposition and chemically transformation. The BC content changes were too small to be detected for the long-term (>30 yr) fertilization in this study.
BC/SOC ratio BC/SOC ratios ranged from 0.5 to 0.63, similar in all the treatments. Thus it is believed that the soil BC was mainly from atmospheric deposition and fertilization
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Soil Organic Carbon, Black Carbon, and Enzyme Activity Under Long-Term Fertilization
Table 1 SOC and BC contents under different fertilization treatments Treatment CK N P K NP NK NPK 2NPK NPKM
SOC (g kg-1) 15.9±0.18 d 17.2±0.67 abc 16.4±0.86 cd 16.7±0.23 bcd 17.2±0.50 abc 17.4±0.32 ab 17.1±0.37 bc 17.7±0.36 a 17.9±0.35 a
BC (g kg-1) 8.9±0.26 a 10.3±0.16 a 10.3±0.17 a 9.2±0.20 a 9.7±0.27 a 10.6±0.18 a 9.1±0.11 a 9.8±0.19 a 9.0±0.15 a
BC/SOC 0.56±0.05 a 0.60±0.10 a 0.63±0.09 a 0.55±0.03 a 0.57±0.01 a 0.61±0.02 a 0.54±0.11 a 0.55±0.05 a 0.50±0.03 a
SOC, soil organic carbon; BC, black carbon; BC/SOC, BC-to-carbonic ratio. Values are menas±SD. Values in the same column marked by the same letters were not significantly different (P>0.05).
since each block was isolated from others. Different soil fertilization treatments over 30 yr had not significant impacts on the proportion of BC in SOC. BC content.
Enzyme activity We found that acid phosphatase activity in NPKM was significantly higher than those in CK, P, K, NP, NK, NPK, and 2NPK treatments (Fig.). Acid phosphatase activity in NK was significantly higher than those in NP, NPK, and 2NPK treatments, whereas the activity in 2NPK was higher than those in NP and NPK treatments. The lowest acid phosphatase activity was found in NP and NPK, followed by CK, P, K, and 2NPK treatments. These results indicated that concurrent application of chemical fertilizer and organic manure in soils was the most effective treatment in enhancing acid phosphatase activity. We also found that acid phosphatase activity was lower if no soil fertilizer was applied, K fertilizer alone was applied, or P fertilizer was concurrently applied with other chemical fertilizers. The urease activity in the NP treatment was significantly lower than those in all other treatments. In addition, the urease activity in NPKM significantly differed from those in CK, N, P, K, NP, and 2NPK treatments. Significant differences were also found between CK and NP treatments. The catalase activity in NPKM was significantly higher than those in all other treatments. In addition, the catalase activity in NK significantly differed from those in N, P, and NPK treatments. No statistical significances in catalase activity were observed between any other two
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treatments. Finally, the invertase activity in NPKM treatment was significantly higher than those in all other treatments.
Interrelation between SOC, BC, and enzyme activity The SOC was insignificantly correlated with BC and invertase (Table 2). Meanwhile, BC had weak negative correlations with urease, invertase and catalase, but positive with acid phosphatase. A significant correlation was observed between invertase and catalase. Table 2 Correlation coefficients between soil organic carbon (SOC), black carbon (BC), and enzyme activity SOC BC Urease Acid phosphatase Invertase Catalase
SOC BC Urease Acid phosphatase 1 0.16 1 0.14 -0.31 1 0.23 0.23 0.07 1 0.58 -0.32 0.39 0.53 0.55 -0.18 -0.04 0.49
Invertase Catalase
1 0.76**
1
**
, highly significant correlation (P<0.01).
DISCUSSION The SOC content in CK treatment was lower than that of the initial soil, whereas SOC contents in all other eight treatments were higher than that of the initial soil. The NPKM treatment obtained the highest SOC content, whereas the lowest SOC contents were found in the P and K treatments among all fertilization treatments. A plausible explanation for these findings is that changes in SOC content depend on the balance between gains and losses of C. When organic matter input exceeds organic matter loss, soil organic carbon would increase, otherwise soil organic carbon would decrease. In the present study, nutrient input was mainly fertilization and root residue, nutrient output was mainly biomass production. We found that NPKM treatment produced the highest grain yield (10 098 kg ha -1 ) followed by 2NPK (9 487 kg ha -1 ) and NPK (8 102 kg ha -1 ). Yields of CK, N, P, K, NP and NK treatments were 5 950, 6 422, 6 459, 5 571, 6 825, and 6 008 kg ha-1, respectively. Differences in yields between CK, N, P, K, NP, and NK treatments were not significant
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Urease (mg g-1 d-1)
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B
a cd
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Fig. Enzyme activities under different fertilization treatments. A, urease activity. B, acid phosphatase activity. C, catalase activity. D, invertase activity. N, P, and K indicate chemical fertilizers of ureaine, potassium dihydrogen phosphate, and potassium chloride; NP, NK, and NPK treatments indicate different combinations of chemical fertilizers; 2NPK indicates chemical fertilizers N, P, and K twice the amount used in the NPK treatment; NPKM indicates chemical fertilizer N, P, and K plus organic matter (pig manure). Error bars were means±SD (n=3). The Fisher’s least significant difference value was plotted to scale significant mean separation (P<0.05).
(P>0.05). The difference between NPKM and 2NPK treatments was not significant either. The correlation coefficient between SOC and yield was 0.723** (n=9). Comparing with CK, fertilization with NPK, 2NPK, and NPKM significantly improved grain production by 36, 59, and 70%, respectively. Higher yields indicate greater nutrient output. Although NPKM treatment produced the highest grain yield, our previous study showed that nutrient supplies in NPKM-fertilized soil were still higher than those in mineral-fertilized soil and CK (Shao et al. 2012). Our findings were in agreement with previous reports that concurrent NPK and organic manure application improved stubble content in fields (Li et al. 2003), and single chemical fertilizer application improved production and enhanced nutrient output (Regmi et al. 2002). In the present work, nutrient output was probably higher than nutrient input when no fertilizer was applied (CK), leading to lower yield and SOC content in CK treatment compared with the initial soil.
SOC contents in treatments, i.e., N, NP, NK, NPK, 2NPK, and NPKM, did not significantly differ from each other in this study, agreeing with previous report of effects of different long-term fertilizations on SOC content (Zhang et al. 2009). These results highlighted the importance of nitrogenous fertilizer in enhancing the SOC pool (Ouédraogo et al. 2006). BC has attracted increased attention as a research subject because of its important role in global climate and environmental system, especially in global biogeochemical cycle (Han and Cao 2005). The initial soils for all treatments were located in the same experiment site and were assumed to have similar traits at the beginning of the experiment. All treated soils were also exposed to similar external environment through the 30 yr of treatment. Thus, the only factor that varied between soils was the fertilization treatment. Our data showed that BC contents in all treated soils were statistically similar, and indicating that fertilization over 30 yr did not affect BC content. As a matter of fact, due to BC inertness for biological
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Soil Organic Carbon, Black Carbon, and Enzyme Activity Under Long-Term Fertilization
and chemical reactions, very long periods would be necessary to obtain measurable transformations. The BC/SOC ratio is related to the BC source and human activity (Muri et al. 2002). The BC/ SOC ratios of P and NK treatments were 0.63 and 0.61, respectively, whereas the BC/SOC ratios of other treatments (CK, N, K, NP, NPK, 2NPK, and NPKM) varied between 0.5 and 0.6. He and Zhang (2006) found the BC/OC ratio of roadside soil, park soil, residential soil and suburb soil had apparently difference, and he indicated this difference may be related with the source of BC and SOC. In this study the BC/SOC ratios of all treatments had little differences. Hence, we speculated that BC originated from the same source. Activities of acid phosphatase, catalase, and invertase in soils treated with chemical fertilizer and OM were higher than those in soils treated with chemical fertilizers alone or no fertilizer. Acid phosphatase is the dominant enzyme involved in mineralizing P in acidic soils (Olander and Vitousek 2000). In our previous reports, we found that P fertilizer treatments (P, NP, NPK, and 2NPK) resulted in P accumulation which perhaps inhibited acid phosphatase activity (Shao et al. 2012). Clarholm (1993) found that when the biological demand for P increased, P application in soil enhanced the activity of acid phosphatase. However, when P supplies from chemical P fertilizer exceeded biological demand for P, the activity of acid phosphatase was inhibited by P fertilizer. As expected, biomass was rather low in CK and K treatments together with low acid phosphatase activity. The catalase activity of all fertilization treatments did not show significant difference. It has been reported previously that different fertilization managements induced minimal changes in catalase activity (Xu et al. 2004). The urease activity in NPK was higher than that in NPKM, which was higher than those in treatments with chemical fertilizer alone or no fertilizer. Zhang et al. (2010) reported in a longterm rice fertilization experiment that three kinds of fertilization treatments NPK, pig manure and straw manure significantly increased soil enzyme activities, and pig manure had stronger effects on urease and acid phosphatase activities than pure chemical fertilizers. Zheng et al. (2008) also found that organic manure
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fertilizer enhanced urease, acid phosphatase, catalase, dehydrogenase and invertase activities in upland red soil in a long-term fertilization experiment. It is believed that organic manure, which is rich in various enzymes itself, provides nutrition and ideal environment for microbial growth and soil enzyme production (Dick et al. 1988). Application of organic manure combined with inorganic NPK produced higher acid phosphatase, catalase and invertase activity than that by chemical fertilizer alone or CK, suggesting that this specific combination was the optimum method to enhance soil enzyme activities. Correlation analysis indicated that BC content was negatively correlated with enzyme activity. Although BC is relatively resistant to decomposition under biotic and abiotic conditions (Schmidt and Noack 2000), it is clear that it will be oxidized and finally mineralized to CO2 over long timescale and a significant oxidation by microbes can not be excluded (Cheng et al. 2006), especially under relatively higher microbe activity, the decomposition ratio of BC might be enhanced (kuzyakov et al. 2009). At the early stage of decomposition, chemical oxidation plays an important role. Oxygen-containing functional groups are formed on BC surface during chemical oxidation process, making BC susceptible to decomposition by soil microbes (Cheng et al. 2006; Joseph et al. 2010). Soil enzyme activities are related to microbe quantity (Acosta-Martínez et al. 2011). In the present study, urease, invertase, and catalase activities were found to be negatively correlated with BC content in soil although not at levels of statistical significance. Therefore, further research is required in order to identify whether soil enzymes play a role in the decomposition of BC or not. Huang et al. (2012) studied effects of human activity on the accumulation of BC in surface dusts and found that BC content in surface dusts was proportional to the accumulation of SOC. He suggested BC in surface dusts mainly originated from atmospheric decomposition, and dust easily integrated with BC and generally carrying BC diffusion in the atmosphere; therefore, BC content in surface dusts is generally higher than that in agricultural soil. In our study, all experimental soils were exposed to the
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same external environment and thus had identical atmospheric decomposition of BC. Meanwhile, fertilization did not significantly affect the BC content but increased the SOC content. Consequently, no correlation was found between BC and SOC in our study. We concluded that BC content in agricultural soil was related to the biological process of surface soil and largely depended on biomass mineralization.
CONCLUSION
to generate one composite sample, air dried, and sieved through a 2-mm stainless steel mesh screen to remove plant material, soil macrofauna, and stones. The soil samples were then stored in well-sealed polythene bags at 4°C until analysis. Table 3 Fertilization treatments1) Fertilizer N P K OM 1)
Our study showed that fertilization, especially NPKM treatment had greater benefits in increasing SOC, improving the activities of soil acid phosphatase, catalase, invertase and urease. But fertilization did not significantly affect BC. It is not certain if there is any links between enzyme activity and BC concentration in soil.
MATERIALS AND METHODS Study site and soil sampling Soil samples were collected from experimental fields that had been fertilized since 1981 at the Institute of the Red Soil in Jinxian County (116°17´E, 28°35´N), Jiangxi Province, China. The soil parents was quaternary red clay and the soil type was waterlogged paddy soil. Experimental fields were planted with rice twice a year, and fertilizers were applied before each rice planting. The rice species were changed every 5 yr. The 30-yr average annual precipitation was 1 400 mm, and the mean annual temperature was 17.7°C. Physical and chemical traits of the arable layer before fertility experiments were: soil pH=6.9 (1:2.5, soil/water); organic carbon (OC)=16.3 g kg -1; total nitrogen=1.49 g kg -1 ; total phosphorus=0.44 g kg -1 ; total potassium= 10.39 g kg -1 ; alkaline nitrogen=144 mg kg -1 ; available phosphorus=9.5 mg kg -1; available potassium=81.2 mg kg-1; cation exchange capacity (CEC)=27 cmol kg-1; clay=241 (<0.002 mm g kg-1). A fully randomized plot design was used in the experiment with treatments including (1) CK (no fertilizer), (2) N, (3) P, (4) K, (5) NP, (6) NK, (7) NPK, (8) 2NPK, and (9) NPKM (pig manure). Each treatment was carried out in three plots. Fertilizers were applied during fall and spring each year (one-third in fall and two-thirds in spring), and the detailed fertilizer application rates were presented in Table 3. Soil samples were collected in July 2010 from six cores within each treatment plot from the top 0-20 cm soil layer using a hand auger. The six soil cores were mixed well
CK N 90 -
P 45 -
Treatments (kg ha-1 yr-1) K NP NK - 90 90 - 45 75 75 -
NPK 90 45 75 -
2NPK 180 90 150 -
NPKM 90 45 75 22 500
N, urea-N; P, triple superphosphate-P; K, potassium chloride-K; OM, pig manure; -, no fertilizer.
Analyses Chemical analysis Soil organic carbon content was determined using the potassium dichromate-sulfuric acid digestion method at digestion temperature of 175-180°C (Lu 1999). The calculated value of SOC content was denoted as M. BC content was determined by the 0.1 N potassium dichromate-sulfuric acid digestion method described by Liu and Zhang (2010). 25 mL potassium dichromate (0.6 mol L -1 ) and sulfuric acid (2 mol L -1 ) was added to the soil sample in a centrifugal tube. The mixture was subjected to ultrasonic disintegration for 30 min followed by incubation at 55°C for 12 h during which the oxidation of BC took place. This process was repeated five times to allow a total of 60 h incubation at 55°C for BC oxidation. The sample was subsequently centrifuged and the supernatant containing titrated residue of potassium dichromate was collected. The content of oxidizable OC in the supernatant was calculated and denoted as N. The BC content was calculated from the difference between M and N. All soil analyses were performed in triplicates unless otherwise specified. Enzyme assays Activities of urease, invertase, and catalase were assessed using <1 mm air-dried samples at optimal pH values for enzyme activity. Each enzymatic assay was performed in triplicates with a standard positive control. Enzymatic activities were expressed on a moisture-free basis according to the method of Guan (1986). In the urease (EC 3.5.1.5) assay, the soil sample was incubated with urea (10% w/v) for 24 h at 37°C and the NH3 released was measured colorimetrically at 578 nm. In the invertase (EC 3.2.1.26) assay, the soil sample was incubated with sucrose for 24 h at 37°C and the hydrolysis of sucrose was monitored colorimetrically at 508 nm. In the catalase (EC 1.11.1.6) assay, H2O2 decomposed by the soil sample was measured by back-titration with KMnO4, and the calculated activity was expressed as milliliter H2O2 decomposed by 1 g of soil. Acid phosphatase (EC 3.1.3.2) activity was determined according to the method of Xiao et al. (2008),
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Soil Organic Carbon, Black Carbon, and Enzyme Activity Under Long-Term Fertilization
in which enzyme-catalyzed p-nitrophenol formation was determined colorimetrically at 420 nm on a Hitachi U-2000 spectrophotometer.
Statistical analysis Results were expressed as means calculated over triplicates. Means separated with P<0.05 in the Tukey-Kramer test were considered statistically different. Correlations between variables were assessed by the Spearman correlation coefficient and a test of significance. All statistical analyses were conducted with the SAS software package for Windows (SAS 2002).
Acknowledgements This work was supported by the National Natural Science Foundation of China (41261074) and the Foundation of Educational Department of Jiangxi Province, China (GJJ12605).
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affected by long-term land use regimes and fertilization in manural loess soil. Plant Nutrition and Fertilizer Science, 19, 404-412. (in Chinese) Woolf D, Amonette J E, Street-Perrott F A, Lehmann J, Joseph S. 2010. Sustainable biochar to mitigate global climate change. Nature, 56, 1-9. Xiao T, Chang S X, Richard K. 2008. Soil compaction and forest floor removal reduced microbial biomass and enzyme activities in a boreal aspen forest soil. Biology and Fertility of Soils, 44, 471-479. Xie L J, Wang B R, Xu M G, Peng C, Liu H. 2012. Changes of soil organic carbon storage under longterm fertilization in black and grey-desert soils. Plant Nutrition and Fertilizer Science, 18, 98-105. (in Chinese) Xu F L, Liang Y L, Zhang C E, Du S N, Chen Z J. 2004. Effects of fertilization on cucumber growth and soil biological characteristics in sunlight green house. Chinese Journal of Applied Ecology, 15, 1227-1230. (in Chinese) Zhang L, Zhang W J, Xu M G, Cai Z J, Peng C, Wang B R, Liu H. 2009. Effects of long-term fertilization on change of labile organic carbon in three typical upland soils of China. Scientia Agricultura Sinica, 42, 1646-1655. (in Chinese) Zhang Q C, Wang X Q, Shi Y N, Wang G H. 2010. Effects of different fertilizer treatments on ecological characteristics of microorganism in chemical fertilizer omission paddy soil. Plant Nutrition and Fertilizer Science, 16, 118-123. (in Chinese) Zheng Y, Gao Y S, Zhang L M, He Y Q, He J Z. 2008. Effects of long-term fertilization on soil microorganisms and enzyme activities in an upland red soil. Plant Nutrition and Fertilizer Science, 14, 316-321. (in Chinese) Zhu L Q, Yang M F, Xu M L, Zhang W Y, Bian X M. 2012. Effects of different fertilization modes on paddy field topsoil organic carbon content and carbon sequestration duration in South China. Chinese Journal of Applied Ecology, 23, 87-95. (in Chinese) Zimmerman A R. 2010. Abiotic and microbial oxidation of laboratory-produced black carbon (biochar). Environmental Science & Technology, 44, 1295-1301. (Managing editor SUN Lu-juan)
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March 2014
RESEARCH ARTICLE
Soil Organic Carbon, Black Carbon, and Enzyme Activity Under LongTerm Fertilization SHAO Xing-hua and ZHENG Jian-wei Department of Life Science, Shangrao Normal University, Jiangxi 334001, P.R.China
Abstract The present study aims to understand the effects of long-term fertilization on soil organic carbon (SOC), black carbon (BC), enzyme activity, and the relationships among these parameters. Paddy field was continuously fertilized over 30 yr with nine different fertilizer treatments including N, P, K, NP, NK, NPK, 2NPK (two-fold NPK), NPK+manure (NPKM), and CK (no fertilization), N, 90 kg urea-N ha-1 yr-1; P, 45 kg triple superphosphate-P2O5 ha-1 yr-1; K, 75 kg potassium chloride-K2O ha-1 yr-1; and pig manure, 22 500 kg ha-1 yr-1. Soil samples were collected and determined for SOC, BC content, and enzyme activity. The results showed that the SOC in the NPKM treatment was significantly higher than those in the K, P, and CK treatments. The lowest SOC content was found in the CK treatment. SOC content was similar in the N, NP, NK, NPK, 2NPK, and NPKM treatments. There was no significant difference in BC content among different treatments. The BC-to-SOC ratios (BC/SOC) ranged from 0.50 to 0.63, suggesting that BC might originate from the same source. Regarding enzyme activity, NPK treatment had higher urease activity than NPKM treatment. The urease activity of NPKM treatment was significantly higher than that of 2NPK, NP, N, P, K, CK, and NPKM treatment which produced higher activities of acid phosphatase, catalase, and invertase than all other treatments. Our results indicated that long-term fertilization did not significantly affect BC content. Concurrent application of manure and mineral fertilizers increased SOC content and significantly enhanced soil enzyme activities. Correlation analysis showed that catalase activity was significantly associated with invertase activity, but SOC, BC, and enzyme activity levels were not significantly correlated with one another. No significant correlations were observed between BC and soil enzymes. It is unknown whether soil enzymes play a role in the decomposition of BC. Key words: organic carbon, black carbon, enzyme activity, fertilization
INTRODUCTION Soil fertility and productivity depend on soil organic matter (OM), which represents the dominant reservoir of plant nutrients such as N, P and S (Steiner et al. 2007). The soil organic carbon pool in 1 m depth is 3.3 times of the atmospheric and 4.5 times of the biotic carbon pool (Lal 2004). As the largest carbon pool, changes in the soil carbon pool may considerably influence
global carbon balance (Lal 2008). Agricultural soil currently accounts for 12% of the total terrestrial land area (Leff et al. 2004). Agricultural activities such as tillage, land-use conversion, fertilization, and biomass alteration lead to fundamental changes in terrestrial carbon pools and fluxes (McLauchlan 2006). Numerous studies have described how fertilization changes the soil organic carbon (SOC) pool (Li et al. 2006; Xie et al. 2012; Wang et al. 2013). Specifically, fertilization with inorganic fertilizer combined with organic manure
Received 9 October, 2013 Accepted 18 December, 2013 Correspondence SHAO Xing-hua, Mobile: 13361731892, E-mail: [email protected]
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60707-8
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seems to have better results in improving soil OM than inorganic fertilization alone (Shao et al. 2011; Zhu et al. 2012). The SOC pool has two main components, i.e., the inert or recalcitrant component and the labile or active fraction (Lal 2006). Black carbon (BC), the main form of the inert or recalcitrant component of SOC pool, is widely distributed in atmosphere, hydrosphere, biosphere, and lithosphere. Black carbon is mainly produced by incomplete combustion of organics such as plant materials or fossil fuels (Kuhlbusch 1998; Schmidt and Noack 2000). The ubiquity of BC is related to its extensive production in combustion processes and its recalcitrant nature (Muri et al. 2002). BC is a continuum of fixed carbon (C) forms rather than a particular compound. The refractory nature of BC enables it to act as a significant sink of atmospheric CO 2 and slows the rate at which photosynthetic C is returned to the atmosphere (Woolf et al. 2010). Photochemical abiotic oxidation and microbial decomposition have been suggested to be the two major mechanisms for BC degradation (Goldberg 1985). Liang et al. (2008) suggested that BC decomposes extremely slowly so that chemical changes in samples cannot be detected. Zimmerman et al. (2010) measured the release of CO2 over one year from microbial and sterile incubations of biochars made of a range of biomass types or prepared under various combustion conditions and found that carbon release from abiotic incubations was 50-90% of that from microbe-inoculated incubations. Kuzyakov et al. (2009) reported that BC decomposition rate was very low under natural soil conditions but increased dramatically up to 5 times after glucose was added to boost microbial growth, suggesting that high soil microbial activity greatly accelerated BC decomposition. However, how BC content is affected by long-term fertilization practices under field conditions is largely unclear. Enzymes in soil affect soil composition by catalyzing a number of reactions. Many studies have shown that soil enzymes indicate changes in soil ecological system function and effects of pollution and agricultural practices on soil physical and chemical properties (Ren and Stefano 2000). Urease, acid phosphatase, and invertase are enzymes
involved in the N, P and C cycles (Bremner and Mulvaney 1978; Amador et al. 1997) and they control mineralization and transformation of soil N, P, and C compounds, which provide essential nutrition for plants. Protection from activated oxygen species such as hydrogen peroxide can be achieved using catalase (Liu et al. 2008). Decomposition of OM in soil depends on substance properties and accessibility of microorganisms and their enzymes (Ekschmitt et al. 2008). It is believed that enzyme activities also affect BC stability in soils. However, only a few studies have examined effects of increased soil enzyme activity on BC decomposition. In the present study, we aimed to investiagte the difference in SOC and BC contents and enzyme activity among the different fertilization threatments in soils and assess the correlations among these parameters. We speculated that SOC, BC, and enzyme activity in soil and their correlations would be affected by long-term fertilization practices.
RESULTS SOC and BC contents SOC contents in the nine treatments ranged from 15.9 to 17.9 g kg-1 with a mean of 17.1 g kg-1 (Table 1). The SOC content in NPKM did not significantly differ from those in N, NP, NK, NPK, and 2NPK treatments. BC contents in the nine treatments varied between 8.9 and 10.6 g kg-1 with an average of 9.66 g kg-1. Statistical analyses revealed that the difference in BC content among different treatments was not significant. Thus, the BC content in all fertilizer treatments did not markedly vary from that in CK by fertilization. This phenomenon can be attributed to intrinsic characteristics of BC that resists to microbial decomposition and chemically transformation. The BC content changes were too small to be detected for the long-term (>30 yr) fertilization in this study.
BC/SOC ratio BC/SOC ratios ranged from 0.5 to 0.63, similar in all the treatments. Thus it is believed that the soil BC was mainly from atmospheric deposition and fertilization
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Soil Organic Carbon, Black Carbon, and Enzyme Activity Under Long-Term Fertilization
Table 1 SOC and BC contents under different fertilization treatments Treatment CK N P K NP NK NPK 2NPK NPKM
SOC (g kg-1) 15.9±0.18 d 17.2±0.67 abc 16.4±0.86 cd 16.7±0.23 bcd 17.2±0.50 abc 17.4±0.32 ab 17.1±0.37 bc 17.7±0.36 a 17.9±0.35 a
BC (g kg-1) 8.9±0.26 a 10.3±0.16 a 10.3±0.17 a 9.2±0.20 a 9.7±0.27 a 10.6±0.18 a 9.1±0.11 a 9.8±0.19 a 9.0±0.15 a
BC/SOC 0.56±0.05 a 0.60±0.10 a 0.63±0.09 a 0.55±0.03 a 0.57±0.01 a 0.61±0.02 a 0.54±0.11 a 0.55±0.05 a 0.50±0.03 a
SOC, soil organic carbon; BC, black carbon; BC/SOC, BC-to-carbonic ratio. Values are menas±SD. Values in the same column marked by the same letters were not significantly different (P>0.05).
since each block was isolated from others. Different soil fertilization treatments over 30 yr had not significant impacts on the proportion of BC in SOC. BC content.
Enzyme activity We found that acid phosphatase activity in NPKM was significantly higher than those in CK, P, K, NP, NK, NPK, and 2NPK treatments (Fig.). Acid phosphatase activity in NK was significantly higher than those in NP, NPK, and 2NPK treatments, whereas the activity in 2NPK was higher than those in NP and NPK treatments. The lowest acid phosphatase activity was found in NP and NPK, followed by CK, P, K, and 2NPK treatments. These results indicated that concurrent application of chemical fertilizer and organic manure in soils was the most effective treatment in enhancing acid phosphatase activity. We also found that acid phosphatase activity was lower if no soil fertilizer was applied, K fertilizer alone was applied, or P fertilizer was concurrently applied with other chemical fertilizers. The urease activity in the NP treatment was significantly lower than those in all other treatments. In addition, the urease activity in NPKM significantly differed from those in CK, N, P, K, NP, and 2NPK treatments. Significant differences were also found between CK and NP treatments. The catalase activity in NPKM was significantly higher than those in all other treatments. In addition, the catalase activity in NK significantly differed from those in N, P, and NPK treatments. No statistical significances in catalase activity were observed between any other two
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treatments. Finally, the invertase activity in NPKM treatment was significantly higher than those in all other treatments.
Interrelation between SOC, BC, and enzyme activity The SOC was insignificantly correlated with BC and invertase (Table 2). Meanwhile, BC had weak negative correlations with urease, invertase and catalase, but positive with acid phosphatase. A significant correlation was observed between invertase and catalase. Table 2 Correlation coefficients between soil organic carbon (SOC), black carbon (BC), and enzyme activity SOC BC Urease Acid phosphatase Invertase Catalase
SOC BC Urease Acid phosphatase 1 0.16 1 0.14 -0.31 1 0.23 0.23 0.07 1 0.58 -0.32 0.39 0.53 0.55 -0.18 -0.04 0.49
Invertase Catalase
1 0.76**
1
**
, highly significant correlation (P<0.01).
DISCUSSION The SOC content in CK treatment was lower than that of the initial soil, whereas SOC contents in all other eight treatments were higher than that of the initial soil. The NPKM treatment obtained the highest SOC content, whereas the lowest SOC contents were found in the P and K treatments among all fertilization treatments. A plausible explanation for these findings is that changes in SOC content depend on the balance between gains and losses of C. When organic matter input exceeds organic matter loss, soil organic carbon would increase, otherwise soil organic carbon would decrease. In the present study, nutrient input was mainly fertilization and root residue, nutrient output was mainly biomass production. We found that NPKM treatment produced the highest grain yield (10 098 kg ha -1 ) followed by 2NPK (9 487 kg ha -1 ) and NPK (8 102 kg ha -1 ). Yields of CK, N, P, K, NP and NK treatments were 5 950, 6 422, 6 459, 5 571, 6 825, and 6 008 kg ha-1, respectively. Differences in yields between CK, N, P, K, NP, and NK treatments were not significant
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Urease (mg g-1 d-1)
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Fig. Enzyme activities under different fertilization treatments. A, urease activity. B, acid phosphatase activity. C, catalase activity. D, invertase activity. N, P, and K indicate chemical fertilizers of ureaine, potassium dihydrogen phosphate, and potassium chloride; NP, NK, and NPK treatments indicate different combinations of chemical fertilizers; 2NPK indicates chemical fertilizers N, P, and K twice the amount used in the NPK treatment; NPKM indicates chemical fertilizer N, P, and K plus organic matter (pig manure). Error bars were means±SD (n=3). The Fisher’s least significant difference value was plotted to scale significant mean separation (P<0.05).
(P>0.05). The difference between NPKM and 2NPK treatments was not significant either. The correlation coefficient between SOC and yield was 0.723** (n=9). Comparing with CK, fertilization with NPK, 2NPK, and NPKM significantly improved grain production by 36, 59, and 70%, respectively. Higher yields indicate greater nutrient output. Although NPKM treatment produced the highest grain yield, our previous study showed that nutrient supplies in NPKM-fertilized soil were still higher than those in mineral-fertilized soil and CK (Shao et al. 2012). Our findings were in agreement with previous reports that concurrent NPK and organic manure application improved stubble content in fields (Li et al. 2003), and single chemical fertilizer application improved production and enhanced nutrient output (Regmi et al. 2002). In the present work, nutrient output was probably higher than nutrient input when no fertilizer was applied (CK), leading to lower yield and SOC content in CK treatment compared with the initial soil.
SOC contents in treatments, i.e., N, NP, NK, NPK, 2NPK, and NPKM, did not significantly differ from each other in this study, agreeing with previous report of effects of different long-term fertilizations on SOC content (Zhang et al. 2009). These results highlighted the importance of nitrogenous fertilizer in enhancing the SOC pool (Ouédraogo et al. 2006). BC has attracted increased attention as a research subject because of its important role in global climate and environmental system, especially in global biogeochemical cycle (Han and Cao 2005). The initial soils for all treatments were located in the same experiment site and were assumed to have similar traits at the beginning of the experiment. All treated soils were also exposed to similar external environment through the 30 yr of treatment. Thus, the only factor that varied between soils was the fertilization treatment. Our data showed that BC contents in all treated soils were statistically similar, and indicating that fertilization over 30 yr did not affect BC content. As a matter of fact, due to BC inertness for biological
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Soil Organic Carbon, Black Carbon, and Enzyme Activity Under Long-Term Fertilization
and chemical reactions, very long periods would be necessary to obtain measurable transformations. The BC/SOC ratio is related to the BC source and human activity (Muri et al. 2002). The BC/ SOC ratios of P and NK treatments were 0.63 and 0.61, respectively, whereas the BC/SOC ratios of other treatments (CK, N, K, NP, NPK, 2NPK, and NPKM) varied between 0.5 and 0.6. He and Zhang (2006) found the BC/OC ratio of roadside soil, park soil, residential soil and suburb soil had apparently difference, and he indicated this difference may be related with the source of BC and SOC. In this study the BC/SOC ratios of all treatments had little differences. Hence, we speculated that BC originated from the same source. Activities of acid phosphatase, catalase, and invertase in soils treated with chemical fertilizer and OM were higher than those in soils treated with chemical fertilizers alone or no fertilizer. Acid phosphatase is the dominant enzyme involved in mineralizing P in acidic soils (Olander and Vitousek 2000). In our previous reports, we found that P fertilizer treatments (P, NP, NPK, and 2NPK) resulted in P accumulation which perhaps inhibited acid phosphatase activity (Shao et al. 2012). Clarholm (1993) found that when the biological demand for P increased, P application in soil enhanced the activity of acid phosphatase. However, when P supplies from chemical P fertilizer exceeded biological demand for P, the activity of acid phosphatase was inhibited by P fertilizer. As expected, biomass was rather low in CK and K treatments together with low acid phosphatase activity. The catalase activity of all fertilization treatments did not show significant difference. It has been reported previously that different fertilization managements induced minimal changes in catalase activity (Xu et al. 2004). The urease activity in NPK was higher than that in NPKM, which was higher than those in treatments with chemical fertilizer alone or no fertilizer. Zhang et al. (2010) reported in a longterm rice fertilization experiment that three kinds of fertilization treatments NPK, pig manure and straw manure significantly increased soil enzyme activities, and pig manure had stronger effects on urease and acid phosphatase activities than pure chemical fertilizers. Zheng et al. (2008) also found that organic manure
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fertilizer enhanced urease, acid phosphatase, catalase, dehydrogenase and invertase activities in upland red soil in a long-term fertilization experiment. It is believed that organic manure, which is rich in various enzymes itself, provides nutrition and ideal environment for microbial growth and soil enzyme production (Dick et al. 1988). Application of organic manure combined with inorganic NPK produced higher acid phosphatase, catalase and invertase activity than that by chemical fertilizer alone or CK, suggesting that this specific combination was the optimum method to enhance soil enzyme activities. Correlation analysis indicated that BC content was negatively correlated with enzyme activity. Although BC is relatively resistant to decomposition under biotic and abiotic conditions (Schmidt and Noack 2000), it is clear that it will be oxidized and finally mineralized to CO2 over long timescale and a significant oxidation by microbes can not be excluded (Cheng et al. 2006), especially under relatively higher microbe activity, the decomposition ratio of BC might be enhanced (kuzyakov et al. 2009). At the early stage of decomposition, chemical oxidation plays an important role. Oxygen-containing functional groups are formed on BC surface during chemical oxidation process, making BC susceptible to decomposition by soil microbes (Cheng et al. 2006; Joseph et al. 2010). Soil enzyme activities are related to microbe quantity (Acosta-Martínez et al. 2011). In the present study, urease, invertase, and catalase activities were found to be negatively correlated with BC content in soil although not at levels of statistical significance. Therefore, further research is required in order to identify whether soil enzymes play a role in the decomposition of BC or not. Huang et al. (2012) studied effects of human activity on the accumulation of BC in surface dusts and found that BC content in surface dusts was proportional to the accumulation of SOC. He suggested BC in surface dusts mainly originated from atmospheric decomposition, and dust easily integrated with BC and generally carrying BC diffusion in the atmosphere; therefore, BC content in surface dusts is generally higher than that in agricultural soil. In our study, all experimental soils were exposed to the
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same external environment and thus had identical atmospheric decomposition of BC. Meanwhile, fertilization did not significantly affect the BC content but increased the SOC content. Consequently, no correlation was found between BC and SOC in our study. We concluded that BC content in agricultural soil was related to the biological process of surface soil and largely depended on biomass mineralization.
CONCLUSION
to generate one composite sample, air dried, and sieved through a 2-mm stainless steel mesh screen to remove plant material, soil macrofauna, and stones. The soil samples were then stored in well-sealed polythene bags at 4°C until analysis. Table 3 Fertilization treatments1) Fertilizer N P K OM 1)
Our study showed that fertilization, especially NPKM treatment had greater benefits in increasing SOC, improving the activities of soil acid phosphatase, catalase, invertase and urease. But fertilization did not significantly affect BC. It is not certain if there is any links between enzyme activity and BC concentration in soil.
MATERIALS AND METHODS Study site and soil sampling Soil samples were collected from experimental fields that had been fertilized since 1981 at the Institute of the Red Soil in Jinxian County (116°17´E, 28°35´N), Jiangxi Province, China. The soil parents was quaternary red clay and the soil type was waterlogged paddy soil. Experimental fields were planted with rice twice a year, and fertilizers were applied before each rice planting. The rice species were changed every 5 yr. The 30-yr average annual precipitation was 1 400 mm, and the mean annual temperature was 17.7°C. Physical and chemical traits of the arable layer before fertility experiments were: soil pH=6.9 (1:2.5, soil/water); organic carbon (OC)=16.3 g kg -1; total nitrogen=1.49 g kg -1 ; total phosphorus=0.44 g kg -1 ; total potassium= 10.39 g kg -1 ; alkaline nitrogen=144 mg kg -1 ; available phosphorus=9.5 mg kg -1; available potassium=81.2 mg kg-1; cation exchange capacity (CEC)=27 cmol kg-1; clay=241 (<0.002 mm g kg-1). A fully randomized plot design was used in the experiment with treatments including (1) CK (no fertilizer), (2) N, (3) P, (4) K, (5) NP, (6) NK, (7) NPK, (8) 2NPK, and (9) NPKM (pig manure). Each treatment was carried out in three plots. Fertilizers were applied during fall and spring each year (one-third in fall and two-thirds in spring), and the detailed fertilizer application rates were presented in Table 3. Soil samples were collected in July 2010 from six cores within each treatment plot from the top 0-20 cm soil layer using a hand auger. The six soil cores were mixed well
CK N 90 -
P 45 -
Treatments (kg ha-1 yr-1) K NP NK - 90 90 - 45 75 75 -
NPK 90 45 75 -
2NPK 180 90 150 -
NPKM 90 45 75 22 500
N, urea-N; P, triple superphosphate-P; K, potassium chloride-K; OM, pig manure; -, no fertilizer.
Analyses Chemical analysis Soil organic carbon content was determined using the potassium dichromate-sulfuric acid digestion method at digestion temperature of 175-180°C (Lu 1999). The calculated value of SOC content was denoted as M. BC content was determined by the 0.1 N potassium dichromate-sulfuric acid digestion method described by Liu and Zhang (2010). 25 mL potassium dichromate (0.6 mol L -1 ) and sulfuric acid (2 mol L -1 ) was added to the soil sample in a centrifugal tube. The mixture was subjected to ultrasonic disintegration for 30 min followed by incubation at 55°C for 12 h during which the oxidation of BC took place. This process was repeated five times to allow a total of 60 h incubation at 55°C for BC oxidation. The sample was subsequently centrifuged and the supernatant containing titrated residue of potassium dichromate was collected. The content of oxidizable OC in the supernatant was calculated and denoted as N. The BC content was calculated from the difference between M and N. All soil analyses were performed in triplicates unless otherwise specified. Enzyme assays Activities of urease, invertase, and catalase were assessed using <1 mm air-dried samples at optimal pH values for enzyme activity. Each enzymatic assay was performed in triplicates with a standard positive control. Enzymatic activities were expressed on a moisture-free basis according to the method of Guan (1986). In the urease (EC 3.5.1.5) assay, the soil sample was incubated with urea (10% w/v) for 24 h at 37°C and the NH3 released was measured colorimetrically at 578 nm. In the invertase (EC 3.2.1.26) assay, the soil sample was incubated with sucrose for 24 h at 37°C and the hydrolysis of sucrose was monitored colorimetrically at 508 nm. In the catalase (EC 1.11.1.6) assay, H2O2 decomposed by the soil sample was measured by back-titration with KMnO4, and the calculated activity was expressed as milliliter H2O2 decomposed by 1 g of soil. Acid phosphatase (EC 3.1.3.2) activity was determined according to the method of Xiao et al. (2008),
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Soil Organic Carbon, Black Carbon, and Enzyme Activity Under Long-Term Fertilization
in which enzyme-catalyzed p-nitrophenol formation was determined colorimetrically at 420 nm on a Hitachi U-2000 spectrophotometer.
Statistical analysis Results were expressed as means calculated over triplicates. Means separated with P<0.05 in the Tukey-Kramer test were considered statistically different. Correlations between variables were assessed by the Spearman correlation coefficient and a test of significance. All statistical analyses were conducted with the SAS software package for Windows (SAS 2002).
Acknowledgements This work was supported by the National Natural Science Foundation of China (41261074) and the Foundation of Educational Department of Jiangxi Province, China (GJJ12605).
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