Study the Changes in Soil Organic Carbon of Rice-Maize Cropping System in the Top Layer of Alluvisol Soil in Dan Phuong: A Study of C-13 Stable Isotope Composition ( δ 13 C)

In this study, the experiments on field were conducted to examine the change in the content of soil organic carbon (SOC), its C-13 stable isotope composition (δ 13 C) and some main physical, chemical parameters (soil moisture, pH, soil density, content of humic, fulvic, total N, total P, total K) in alluvial soil of Dan Phuong region—Vietnam at a depth of 0 - 30 cm when we changed the regime from 2 maize -1 rice crop to 2 rice - 1 maize crop per 1 year. In addition to analyzing the main parameters in soil, C content and its δ 13 C value in parts of rice and maize (root, stem and leaf) were also analyzed to assess the contribution of plant residues on soil organic carbon content after harvest. The experiment was carried out in 2016-2017 on the field with the tradi-tional farming method of local farmers along with the tropical monsoon weather conditions of the North-Vietnam. The results showed that SOC had positive correlation with total N, total P parameters and negative correlation with δ 13 C values of soil samples at two layers (0 - 15 cm and 15 - 30 cm). The average of total dry biomass (stem, stump + roots and leaf parts) per 1 rice and 1 maize crop was 10.64 Mg/ha and 9.09 Mg/ha, respectively. The average of δ 13 C value of rice (C 3 plant) was −29.78‰ and its value of maize (C 4 plant) was −12.61‰. The new plant (rice) contributes to the total soil organic carbon content from 11.31% to 44.14% at the 0 - 15 cm layer and from 6.55% to 11.31% at the 15 - 30 cm layer in one-year experiment period.


Introduction
In soil science, soil organic carbon (SOC) plays a very important role in soil structure, soil chemical and physical characteristics and soil fertility [1] [2]. In soil-crop ecosystems, SOC storage reflects the net balance between ongoing carbon (C) accumulation resulting from inputs of crop biomass (aboveground, belowground) and/or exogenous organic matter (e.g., manure, straw) and soil C decomposition processes due to microbial oxidation [3] [4].
Stable isotope ratio of 13 C/ 12 C in SOC expressed in the delta notation (δ 13 C SOC )-a natural tracer, is interested in many areas of research on environmental processes such as carbon sinks and photosynthetic mechanisms of plants [5] [6], assessing the carbon reservoir turnover times and soil carbon dynamic in agroforestry ecosystems, methods of fixation and storage of carbon dioxide in soils [1] [6] [7] [8] [9] or exploring soil mineralization processes [10]. Plant species (C 3 , C 4 and CAM plants) differ in their isotopic 13 C values due to isotopic discrimination by their photosynthetic enzymes and the regulation of the diffusion resistance of their stomata. C 3 plants (rice, wheat, soybean …) have δ 13 C average value of −27‰ (−35‰ to −20‰), C 4 plants (sugarcane, corn …) have δ 13 C average value of −13‰ (−15‰ to −7‰) [11] [12]. For topsoil calculations, SOM has stable carbon isotopic composition comparable to that of the source plant material and changes in the proportion of C 3 and C 4 plants will result in a corresponding change in the δ 13 C SOC . Therefore, δ 13 C value of SOM can be used to study C turnover in soils in areas where C 3 vegetation was replaced by C 4 plants or vice versa [11].
Furthermore, the cultivation models such as intensive farming, intercropping, crop rotation (C 3 , C 4 plant) and agricultural practices will affect the total SOC budget, nutrient cycling, soil-water relationships, erosion processes and carbon sequestration. Such information is important to determine impacts of land use on the sustainability of cropping systems, and its feedbacks to future climate changes.
The use of stable 13 C isotope techniques to study above issues is not new in the world but it is still quite new in Vietnam. Therefore, in this paper, we applied the 13 C stable isotope technique to study changes in soil organic carbon at 0 -30 cm depth from maize-rice-maize crop system to rice-rice-maize crop system of alluvial soil in Dan Phuong area. This is a type of soil that accounts for a large proportion of Vietnam agricultural soil which has high C sequestration potential and reduces greenhouse gas (CO 2 , CH 4 ) in agro-ecosystem.

Crop Management
The experimental site was implemented on agricultural soil at Dan Phuong district (21˚06'21.0''N, 105˚39'45.0''E)-a suburban area of Hanoi city, Vietnam for 2 years (2016 and 2017). This area has been growing crop sequence of maizemaize-rice for approximately 10 years.
The experiment site has a tropical monsoon climate with an average annual precipitation of 1721 mm, an average annual temperature of 25.14˚C, atmosphere humidity of 75.58% and a mean annual sunshine hour of 1207 h (according to the Report of Center for Agricultural Meteorological Research, Institute of Hydrometeorology and Climate Change). The soil profile of the experimental site was classified as alluvial soil of the Red river or Eutric Fluvisol (FAO, 1990).
Rice-rice-maize or maize-rice-maize cropping system was the most important cropping system in the region.

Sampling and Sample Preparation
Soil samples were collected at the beginning and before harvesting crops. Soil samples were taken using a core sampler (6 cm i.d.) to a depth of 30 cm and then it was divided into two layers: 0 -15 cm and 15 -30 cm depth. This depth was chosen because it contains most of the root apparatus of rice and maize species.
For each crop, three replicates were taken according to a completely randomized experimental design. Each replicate was composed of three subsamples, which were combined. Then, the soil samples were sieved (<4 mm), visible plant debris was removed, and the soils were then air-dried. On the same date, separate samples (50 mm diameter) of undisturbed soil were taken for a bulk density analysis.
The samples are spread on stainless steel trays with the use of a stainless-steel spatula, air-dried at room temperature or dried at 40˚C -50˚C in a ventilated oven for two days. Dried soils were homogenized in ceramic mortar and sieved through 1 mm mesh sieve to remove bricks, stones, gravel and roots. Samples were then ground and sieved through 100 μm mesh sieve, dried the specimen at 50˚C for 24 hours. Finally, samples were subdivided into subsamples for determination pH, soil organic carbon, total N, total P, total K, and carbon stable isotope components in SOC ( 13 C/ 12 C ratio -δ 13 C SOC ).
The plant samples were collected at harvesting time for each crop. Random choosing of 10 maize plants or 10 rice clusters of each treatment, rinse and subdivide into 4 parts: root, stem, leave and seeds. Each plant part was cut into 0.5 -1.0 cm, dried at 40˚C -50˚C in a ventilated oven until unchanged weight. Dry mass of each part was determined by weight. The dried plant samples were then grounded and sieved through 100 μm mesh sieve for carbon stable isotope ratio and percent of C.

Physiochemical Soil Annalysis
The soil samples were characterized using common methods. The content of clay, silt and sand in the fine soil samples (grains < 2 mm in diameter) was

Isotope Analysis of δ 13 C in Soil and Plants by EA-IRMS
Stable carbon isotopic composition-δ 13 C values (the proportion of 13 C with respect to 12 C), measured for soil and plant samples were used to determine the contribution of plant carbon source to soil organic carbon. The rice and maize plant samples and those of the soil samples were dried at 60˚C, ball-milled, passed through a 100-μm sieve, and fumigated with 12 N HCl overnight to re-move carbonates [13] [14]. The δ 13 C of plant and soil samples were analyzed using isotope ratio mass-spectrometer (IRMS, Micromass GV Instrument, UK) equipped with an elemental analyzer (EA, EuroVector, Italy) at the Isotopes Hydrology Laboratory, Institute for Nuclear Sciences and Technology, Vietnam. The 13 C/ 12 C isotope ratio is expressed in the delta notation (δ 13 C) that was expressed as: where: With respect to the different cropping treatments, the δ 13 C values of the SOM were used to calculate the proportion of new C (f new , i.e., the C derived from current maize/rice residues) and old C(f old = 1 − f new , soil C present prior to tillage, i.e., C in the initial soil) using a mass balance equation [11]: where: δ new represents the δ 13 C values of organic C in soil fractions after 1years of tillage, δ old represents the δ 13 C values of organic C in the initial soil, i.e., the soil samples prior to tillage, δ veg represents the δ 13 C values of the mixed samples, including plant materials (maize and/or rice leaves, stems and roots) in both cropping treatment.

Statistical Analysis
Pearson's test was used to evaluate the correlation coefficient and differences in means of the soil characteristics: soil humidity, bulk density, pH, humic, fulvic, SOC, TN, TP, TK and δ 13 C from two depths (0 -15 and 15 -30 cm) among three treatments. The statistical analyses were done with SPSS 20.0 software.

The Yield and Carbon Content of the Crops and δ 13 C Values in Plants Samples
Results of seasonal analysis of maize and rice samples (stump + roots, stem and leaf parts) in the study area for 2 years from 2016 to 2017 were showed in Table  1. The average of total dry biomass (stem, stump + roots and leaf parts) per 1 rice crop of 3 models was 10.64 Mg/ ha for 2 years. The average of total dry biomass (stem, root and leaf parts) per 1 maize crop of 3 models was 9.09 Mg/ha for Journal of Environmental Protection 2 years. The average of δ 13 C values of rice and maize parts were −29.78‰ and −12.61‰, respectively. These results are completely suitable with the world research on the δ 13 C value of C 3 plants-rice and C 4 plants-maize. The different in δ 13 C value of these plants is the signature for carbon sequestration study.

Soil Properties of Two Layers
We conducted sampling and analyzing some soil properties according to the depth before and after harvest of some maize-maize-rice and rice-rice-maize cultivation models. The average analysis results of soils in 2016 and 2017 at 2 depths (0 -15 cm and 15 -30 cm) of the MRM, RRM-NR and RRM-R models were presented in Table 2 and Table 3. Results showed that there were minor changes in the parameters of pH, SOC, TN, TK and δ 13     Using SPSS 20.0 software for statistical analysis of the correlation between soil moisture content, density, pH, humic, fulvic, SOM, TN, TP, TK and δ 13 C. Results of soil analysis at a depth of 0 -15 cm (Table 4) showed that the OC content was positively correlated with the parameters of TN, TP, and fulvic and inversely correlated with δ 13 C and pH parameters. The results in Table 5 also showed the positive correlation between OC and TN, TP parameters, and inversely correlated with the δ 13 C parameter. This correlation was consistent with the trend of shifting crops from cultivating of 2 corn crops + 1 rice crop/1 year to cultivating of 2 rice crops + 1 corn crop/1 year.

The Soil Organic Carbon and Its Relation with δ 13 C Values
The variation of SOC content of 3 models (MMR, RRM-NR, RRM-R) in 2 soil layers in 2017 was presented in Figure 1 and Figure 2. The results showed that the SOC content tends to decrease during the time of winter-spring season in both depths 0 -15 cm and 15 -30 cm and gradually increase during summer-autumn season and reach the highest value in September. Figure 1 and The SOM content and its relation with δ 13 C in SOM was shown in Figure 3 in. The results showed that SOM content in the 0 -15 cm soil layer was higher from 2 to 2.5 times than SOM content of the 15 -30 cm layer and the δ 13 C value of upper layer was depleted than the δ 13 C value from 2‰ to 3‰. The depleted δ 13 C value and the increasing SOC content of the topsoil were due to SOC being decomposed from rice straw and rice roots after harvest (the average δ 13 C value of rice was −29.78‰). At a depth of 15 -30 cm, the concentration of SOC was lower than that of the surface layer and the δ 13 C value of SOC was enriched because this layer was not replenished regularly with plant matter except deep plant roots. In addition, many published works on SOC kinetics show that δ 13 C values tend to be enriched at deeper layer due to the natural isotope fractionation of the microbial decomposition process of microorganisms and the evaporation process of 12 C light isotope of gases such as CO 2 and CH 4 .

Carbon Sequestration Potential of Rice-Maize Crop Systems
Applying the formula (2) to calculate the amount of C accumulated in the soil layers when shifting the 2 maize -1 rice crop model to 2 rice -1 maize crop model with no plant return and plant return in Dan Phuong alluvial soil, the results are obtained in Table 6.  The δ 13 C SOMnew value decreased from 1‰ to 1.5‰ when we changed from 2 maize -1 rice cultivation to 2 rice -1 maize cultivation in 2017. For the RRM-NR model, the results showed that the average contribution rate of new plants (rice) was about 11.31% at 0 -15 cm depth, and 6.55% at 15 -30 cm depth and the total amount of SOM accumulated after 1 year of cultivation on both layers was 2.259 MgC/ha. When leaving rice straw in the field (RRM-R), the average contribution rates of new plants to the total amount of SOM of 0 -15 cm and 15 -30 cm layers were accounted for 44.14% and 16.46%, respectively. The average cumulative amount of SOM in the 0 -30 cm layer was 9.505 MgC/ha at the RRM-R model. This value was 4 times higher than that of the RRM-NR model (2.259 MgC/ha). Thus, if farmers leave plant residues (straw) after each harvest on the field, the amount of C from new plants accumulated in the soil in the form of SOM will increase significantly. This is a way to get more CO 2 from the air into plant tissues and store CO 2 as SOC in farmland.

Conclusion
Our research on soil organic carbon, C-13 stable isotope and some main characteristics of the alluvial soil at Dan Phuong district-Vietnam was conducted in 2016-2017 when we changed from 2 maize + 1 rice crop model to 2 rice + 1 maize crop model. The annual average of total dry biomass of rice and maize samples (stem, root, leaf parts) was 10.64 Mg/ha and 9.09 Mg/ha, respectively. The average δ 13 C value of Khang Dan rice was −29.78‰ (C 3 plant) and that value of HN88 maize (C 4 plant) was −12.61‰. The amount of C accumulated in the soil in the form of SOM increases with time, the OC content is positively correlated with the parameters of TN, TP, fulvic and inversely correlated with δ 13 C parameter (0 -30 cm layer). The top soil layers were regularly provided plant residues, so the δ 13 C value of SOM was changed corresponding to the δ 13 C value of plant. The average cumulative amount of SOM in the 0 -30 cm layer was from 2.259 MgC/ha to 9.505 MgC/ha. This is a nature stable isotope method that can be used to relatively accurately quantify the C sequestration ability of the popu-lar agricultural farming models in Vietnam and around the world. We need to conduct long-term and deeper research for many years to estimate SOC sequestration in some popular agricultural farming models and find the ways to minimize the number of greenhouse gases in the atmosphere.