Dai Jian-jun, Liu Li-zhi, Wang Xiao-chun, Fang Qiu-na, Cheng Ye-ru, Wang Dan-ni, and Peng Xian-long

College of Resources and Environmental Science, Northeast Agricultural University, Harbin 150030, China

Abstract: The effects of different amounts of carbon and nitrogen sources on the soil microbial biomass carbon, dissolved organic carbon and related enzyme activities were studied by the simulation experiment of rice straw returning to the field, and the mechanism of the decomposition of rice straw returning to the field was discussed.Completely randomized experiment of the two factors of the three levels was designed, and a total of nine treatments of indoor soil incubation tests were conducted.Full amount of rice straw was applied to the soil in this simulation experiment and different amounts of brown sugar and urea were added in the three levels of 0 (no carbon source and nitrogen source), 1 (low levels of carbon and nitrogen sources) and 2 (high levels of carbon and nitrogen sources), respectively.The results showed that the addition of different amounts of carbon and nitrogen sources to the rice straw could increase the soil carbon content.Compared with T0N0, the microbial biomass carbon of T2N2 was increased significantly by 170.48%; the dissolved organic carbon content of T1N2 was significantly increased by 58.14% and the free humic acid carbon contents of T0N2, T1N1 and T2N0 were significantly increased by 56.16% and 45.55% and 47.80%, respectively; however, there were no significant differences among those of treatments at later incubation periods.The addition of different carbon and nitrogen sources could promote the soil enzyme activities.During the incubation period, all of the soil enzyme activities of adding sugar and urea were higher than those of T0N0 treatment.Therefore, the addition of different amounts of carbon and nitrogen sources to rice straw returning could improve soil microbial biomass carbon content, dissolved organic carbon and soil enzyme activities.

Key words: rice straw returning, carbon and nitrogen sources, microbial biomass carbon, dissolved organic carbon, soil enzyme activity

Introduction

Northeast China is the main production base of commercial grains of China.The crop straw resources are very rich.In 2015, northeast China collected about 159 million tons of straw output, but the amount of straw waste and incineration was as high as 21% (Louet al., 2011; Panet al., 2013).The annual production of rice straw in the United States is about 450 million tons, but the amount of rice straw is returned to the field accounts for more than 60% of the total rice straw production.Britain returned 73% of rice straw directly to the fields (Shen and Chen, 2009; Liuet al., 2008).With the rapid development of agricultural economy in northeast China, farmers' living conditions and changes in rural fuel structure, crop straws have gradually become waste of agricultural products and environmental problems, such as severe haze weather caused by straw burning has become an urgent social issue that needs to be resolved (Fanget al., 2017; Xieet al., 2010).Straw returning is an essential source of soil organic carbon on farmland (Sunet al., 2012).Previous studies have shown that straw returning can significantly increase soil organic carbon content and improve soil organic carbon activity and quality (Liuet al., 2008).Reasonable straw returning measures are beneficial to strengthen carbon and nitrogen transformation activity in soil (Li and Wu, 2006).Straw returning can not only provide abundant carbon sources for microorganisms in the soil, stimulate microbial activity, improve soil fertility (Yanget al., 2012), but also reduce greenhouse gas generated by straw burning (Guoet al., 2016), which is considered as effective farmland.Fertilization measure is also an economical and sustainable way to utilize straw resources.

However, from the autumn harvest to the next year spring in the northeastern region, the temperature is low and the C/N ratio of rice straw is high, which causes slow decomposition of straws in natural condition.There are few active carbon and nitrogen in the straw that can be directly used by soil microorganisms, which limits the proliferation of soil microorganisms.Microorganisms participating in the decomposition of organic matter in straws must secrete extracellular enzymes to the outside, degraded macromolecular organic matter into small molecular organic matter through enzymatic catalysis, and "infiltrate" from the outside of the microorganism into the body for absorption and utilization.Therefore, the straw decomposing after returning to the field is a series of enzyme-catalyzed biochemical processes carried out by a series of microorganisms secreting various functions of extracellular enzymes (Liet al., 2017; Liu and Li, 2010).To accelerate soil microbial growth and increase soil enzyme activity, the active carbon and nitrogen sources should be applied, which microorganisms can be directly used, thereby the decomposition of rice straw should be accelerated.Studies have shown that after the straw returning to the soil, the soil enzyme activities and carbon turnover are affected by the microbial decomposition process (Li, 1992; Qianet al., 2012; Yanet al., 2015).Jianget al.(2016) believed that crop straw combined with exogenous carbon can promote the mineralization and decomposition of soil organic carbon (Blagodatskaya and Kuzyakov, 2008), increase soil microbial biomass and soil enzyme activity.It has different effects on soil microbial biomass and enzyme activity, when the crop straws combined with nitrogen fertilizer are returned to the fields (Wanget al., 2014).

At present, there are a lot of researches on the application of nitrogen fertilizer to rice straw returning to the field, but fewer studies on addition of active carbon and nitrogen sources to rice straw returning in northeastern China.The aim of this paper was to study the effects of adding exogenous carbon and nitrogen on soil microbial biomass carbon, soil dissolved organic carbon (DOC) and soil enzyme activities under the conditions of rice straw returning to the field through indoor simulated cultivation experiments, to investigate the influence mechanism of exogenous carbon and nitrogen on the degradation of rice straw, which would provide a theoretical basis for rice straw returning to the field in northeastern China.

Materials and Methods

Experiment materials

Test materials of rice straw and soil samples were both taken from rice fields in Wuchang City (44°4'N, 126°33'E), which located in Harbin, China.The total amount of rice straw returning to the field was 7 500 kg ? hm-2and the rice straw contained 42.3% carbon and 0.72% nitrogen, and C : N was 59 : 1.The soil to be tested was meadow paddy soil.The total nitrogen of the soil was 1.74 g ? kg-1, the total phosphorus was 1.46 g ? kg-1, the available nitrogen content was 142.90 mg ? kg-1, the available phosphorus was 16.04 mg ? kg-1, the available potassium content was 135.62 mg ? kg-1, the organic matter content was 36.63 g ? kg-1and pH was 5.26; brown sugar (40% carbon contented) and urea (46% nitrogen contented, 20% carbon contented) were used for the active carbon and nitrogen materials.

Experiment design

A 50-day laboratory incubation experiment was conducted in Northeast Agricultural University in October, 2018 (from October to November, the indoor temperature was 16-22℃).The test was set two factors of carbon and nitrogen, and carbon and nitrogen sources levels (brown sugar and urea) were 0 (no carbon and nitrogen source), 1 (low-level pure carbon 30 kg ? hm-2and pure nitrogen 60 kg ? hm-2) and 2 (high levels of pure carbon 60 kg ? hm-2and pure nitrogen 120 kg ? hm-2), completely randomized trial design.A total of nine treatments in triplicate is shown in Table 1.The treatments were T0N0, T0N1, T0N2, T1N0, T1N1, T1N2, T2N0, T2N1 and T2N2.The 1.67 g crushed rice straw powder was thoroughly mixed with urea (N), brown sugar, (T) and soil, and 500 g of the air-dried soil sample was weighed for each treatment, and placed in a 1 000 mL jar.Distilled water was added and adjusted to about two centimeters of immersed soil.Each jar was treated as a treatment which was repeated three times.The 100 g of fresh soil was removed from each 1 000 mL jar, on days of 0, 10, 30 and 50, after the beginning of incubation and stored in a cryogenic refrigerator for the determination of soil enzyme activities and microbial carbon contents; the soil-soluble organic carbon and free humic acid carbon were determined by air-dried soil.

Table 1 Test design

Measurement items and methods

The determination of soil dissolved organic carbon (DOC) and free humic acid carbon was described inSoil Agrochemical Analysisby Lu (2000): determination of soil DOC used 0.1 mol ? L-1K2Cr2O7oxidation and 0.2 mol ? L-1FeSO4titration; determination of free humic acid in soil leached by sodium pyrophosphate, oxidated with 0.8 mol ? L-1K2Cr2O7and titrated with 0.2 mol ? L-1FeSO4.Soil microbial biomass carbon was determined by chloroform fumigation-K2SO4extraction and TOC method.Soil cellulase was determined by 3, 5-dinitrosalicylic acid colorimetric method (after 72 h incubation at 37℃), and the activity was expressed in milligrams of glucose in 10 g soil after 72 h; the soil urease was determined by indophenol colorimetry (after 24 h incubation at 37℃), and the activity was expressed by the number of NH3-N in 1 g of soil after 24 h; the soil invertase was determined by 3, 5-dinitrosalicylic acid colorimetric method (after 24 h incubation at 37℃).The activity was expressed as milligrams of glucose in 1 g of soil after 24 h (Guan, 1986).

Data processing and analysis

The experimental data was sorted and tabulated by Excel 2010 software.SPSS 22.0 software was used for the analysis of variance (ANOVA).The significance level of the test data was tested by Duncan method (p<0.05).

Results

Effects of additions of carbon and nitrogen on soil microbial biomass carbon

Soil microbial biomass carbon (MBC) content was an active part of the soil carbon pool, and its sensitivity to soil texture was much higher than the total soil organic carbon.It could be seen from Fig.1 that during the whole incubation period, the changing trend of the amount of MBC in the treatment of straw increased slowly after the initial increase, and the treatment increased from the 0th to the 30th day and reached the highest on the 30th day.MBC content of T2N2 treatment was significantly different from that of other treatments.T2N2 was significantly higher than T0N0 treatment by 170.48%.T0N0 was significantly low compared with other treatments in the absence of carbon source treatment; all the treatments showed a downward trend from the 30th to the 50th day.

Fig.1 Effects of application quantity of different carbon and nitrogen sources on soil microbial biomass carbonDifferent letters indicate significant differences (p<0.05).

Effects of soil addition of carbon and nitrogen on soil dissolved organic carbon (DOC)

It could be seen from Table 2 that with the increase of incubation time, the soil DOC content of the treatments with carbon source increased rapidly from the 0th to the 10th day, and on the 10th day, the soil DOC content of each treatment was the highest and the relationship was in the order of T1N2>T2N0>T2N2>T1N0>T2N1>T1N1.The treatment of T1N2 was significantly different from the treatments of T1N1 and T2N1; the content of DOC in each treated soil decreased rapidly from the 10th to the 30th day, and slowly falling from the 30th to the 50th day.DOC content of the soil in the treatment without adding carbon source showed a downward trend from the 0th to the 50th day, and the difference between treatments was not significant.It could be seen from Table 3 that the content of free humic acid carbon in each treatment showed a trend of increasing first, and then declined with the increase of incubation time.Within the incubation of the 0th to the 10th day, the free humic acid carbon of treatments showed an upward trend.On the 10th day of incubation, the content of free humic acid carbon reached the maximum.T1N1, T2N0 and T0N2 were significantly different from other treatments, and T2N0 was significantly higher than T1N2 treatment by 69%, and T1N1 and T2N0 were significantly different from other treatments in the process of adding carbon source.T0N2 was significantly different from other treatments in the absence of carbon source treatment; all the treatments showed a downward trend from the 10th to the 50th day.

Table 2 Effect of soil application of carbon and nitrogen on content of dissolved organic carbon in soil (g C ? kg-1)

Table 3 Effect of soil application of carbon and nitrogen on soil free humic acid carbon (g C ? kg-1)

Effects of soil addition of carbon and nitrogen on soil enzyme activity

Urease was one of the main enzymes in the soil.It was an amidase widely distributed in bacteria, fungi and higher plants.It played a key role in the urea conversion process.In addition, urease was closely related to the soil organic matter content and the number of microorganisms.The urease activity reflected the level of available nitrogen supply in the soil.It could be seen from Fig.2 that the urease activity of each treatment showed a trend of increasing first, and then decreased, during the incubation time.On the 10th day of incubation, the urease activity of each treatment reached the maximum.The activity sequence of urease among that of treatments was in the order of T0N1>T1N2>T1N1>T1N0>T0N2>T2N1>T2N0>T0N0>T2N2.Urease activity of T0N1 was significantly higher than that of T0N0, T0N2, T1N0, T2N0, T2N1 and T2N2 by 97.40%, 67.57%, 20.30%, 91.47%, 81.40% and 111.84%, respectively.From the 30th to the 50th day of incubation, the urease activity of each treatment decreased.

Fig.2 Effect of soil application of carbon and nitrogen on soil urease activityDifferent letters indicate significant differences (p<0.05).

Invertase was an enzyme involved in the soil organic carbon cycle and was one of the critical indicators for measuring soil fertility.Under normal conditions, soils with better fertility and higher organic matter had higher invertase activity.As shown in Fig.3, the invertase activity of each treatment was increasing from the 0th to the 10th day of the incubation, and declining from the 10th to the 50th day.On the 10th day of incubation, invertase activity of each treatment reached the maximum, and the activity was in the order of T2N1>T2N2>T1N1>T1N0>T1N2>T0N1>T0N2>T0N0>T2N0, in which the invertase activity of T2N0 treatment was significantly higher than that of other treatments.

Fig.3 Effect of soil application of carbon and nitrogen on soil invertase activityDifferent letters indicate significant differences (p<0.05).

Soil cellulase was an essential enzyme in the carbon cycle.It was mainly involved in the hydrolysis of cellulose, which hydrolyzed cellulose to cellobiose, whose activity was inhibited by degradation products (Zhenget al., 2009; Du and Zhong, 2005).As could be seen from Fig.4, during the incubation period, the cellulase activity of each treatment increased from the 0th to the 10th day and decreased from the 10th to the 50th day.On the 10th day of incubation, the cellulase activity of each treatment reached the maximum and the activity was in the order of T1N0>T2N1>T2N0>T0N2>T0N0>T2N2>T0N1>T1N2>T1N1, and the cellulase activity of T1N0 was significantly higher than that of other treatments.On the 50th day of incubation, the cellulase activity of each treatment declined to the lowest.

Fig.4 Effect of soil application of carbon and nitrogen soil cellulase activityDifferent letters indicate significant differences (p<0.05).

Multi-factor analysis of soil microbial biomass carbon and soil enzyme activities with added carbon and nitrogen

It could be seen from Table 4 that except for the treatment of carbon on the 10th day and nitrogen on the 50th day, the effects of carbon or nitrogen addition alone on soil MBC were significant or extremely significant, while the interaction effects of carbon and nitrogen on soil microbial biomass carbon were all extremely significant.Except for the treatment of carbon on the 0th day and nitrogen on the 50th day, the effects of carbon or nitrogen on soil invertase activity were significant or extremely significant, while the interaction effects of carbon and nitrogen on soil invertase activity were significant or extremely significant except for the treatments of the 0th day.Except for the treatment of carbon on the 0th and the 50th day and nitrogen on the 50th day, the effects of carbon or nitrogen addition alone on soil urease activity were significant or extremely significant; except for the treatment on the 30th day, the interaction effects of carbon and nitrogen on soil urease activity were significant or extremely significant.Except for the treatment of carbon on the 0th day and nitrogen on the 10th day, the effects of carbon or nitrogen addition alone on soil cellulase activity were significant or extremely significant; the interaction effects of carbon and nitrogen on soil cellulase activity were significant or extremely significant.

Table 4 Multi-factor analysis of soil microbial biomass carbon and soil enzyme activities with added carbon and nitrogen

Discussion

Rice straw returning significantly affected soil dissolved carbon content and its stability.It was generally believed that the larger the molecular weight of DOC and the more complex the molecular structure was, the more stable the organic matter was (Li, 1992).When the soil was incubated to the 10th day, compared with T0N0, the soil free humic acid carbon and MBC of those treatments added carbon and nitrogen to the straw returning to the field significantly increased, which might be attributed to the addition of carbon and nitrogen source providing an initial nutrients for soil microorganism, increasing microbial activities, and then promoting straw decomposition and improving soil fertility.However, the difference of soil DOC was not significant at later incubation period, which might be related to DOC itself, accounting for a small part of soil organic carbon and of a small molecular weight, which could be directly used by soil microorganisms (Li and Wu, 2006; Genget al., 2013).In this study, compared to T0N0, the addition of carbon and nitrogen to rice straw returning significantly affected the soil DOC content.

Soil enzymes were a class of catalytically active biological substance (Doddet al., 2000), whose activity reflected the direction and intensity of carbon and nitrogen decomposition in soil (Jastrowet al., 2000).It could promote soil metabolism and affect crop growth by participating in the cycling of soil C, N, P and other elements, soil mineralization and complex biochemical processes (Frankenberger and Dick, 1983; Liet al., 2005; Zhanget al., 2009).The addition of carbon and nitrogen to the straw returning field provided the soil microorganisms with directly usable active substances, which in turn enhanced the life activities of the microorganisms (Guan, 1989), and promoted the decomposition of sucrose accelerating the carbon cycle of the soil.In this experiment, it could be found that the addition of carbon and nitrogen sources to the rice straw returning significantly increased soil invertase activity.The sugar in the early incubation stage decomposed rapidly and the activity of invertase increased rapidly.On the 10th day of incubation, the invertase activity reached a peak.The invertase activity of T2N0 treatment was significantly high, and gradually decreased slightly at the later stages, which was consistent to that of Huang (2012) and Heet al(2009).Urease could catalyze organic matter decomposition in soil, and its enzymatic product, ammonia, was one of the plant nitrogen source (Zhou and Ding, 2007).After adding carbon and nitrogen sources to rice straw, it provided a direct available carbon and nitrogen sources for urease-secreting microorganisms in the soil, in order to increase the quantity and activity of urease.The increase of urease activity meant soil organic matter content increasing and a positive accumulation of nitrogen in the soil.The results of this experiment indicated that after the addition of carbon and nitrogen sources to the rice straw returning, the urease activity of each treatment tended to have few change at the early and later stages.On the 10th day of incubation, the urease activity reached the maximum value, and the urease activity of T0N1 treatment was significantly higher than that of other treatments.However, it did not reach a significant level, which was consistent with the results of the study on the effect of adding straw on red soil (Zhaiet al., 2013).Soil cellulase was an important enzyme in the carbon cycle.It was mainly involved in the hydrolysis of cellulose, which hydrolyzed cellulose to cellobiose, whose activity was induced by substrate and inhibited by degradation products (Zhenget al., 2009; Du and Zhong, 2005).The cellulase activity increased after adding carbon and nitrogen sources to rice straw returning field.This might be due to the fact that a large amount of cellulose in the straw entered the soil with the returning treatment, the soil microbial metabolism activity was activated, and the microorganism was induced to secrete a large amount of cellulase by the substrate.On the 10th day of incubation, the cellulase activity of T1N0 was significantly higher than that of other treatments.Results of Shi (2003) and Chen (2007) had shown that the decomposition rate of straw in the first three months reached 50%, and cellulose decomposition was mainly carried out in the early stage of returning to the field, which was consistent with the result of this experiment.

The addition of carbon and nitrogen sources to rice straw returning increased the above three soil enzyme activities and the enzyme activities were higher than those of treatment T0N0.The changes of these three enzyme activities might further prove that the addition of carbon and nitrogen sources to rice straw returning field was beneficial to increase the number of soil microorganisms, soil enzyme activities and to accelerate the decomposition of rice straw.

Conclusions

The addition of carbon and nitrogen sources to rice straw returning to the field could increase soil MBC, DOC content, soil urease, invertase and cellulase activities.The effects of carbon or nitrogen alone, and the interaction effects of carbon and nitrogen on soil microbial biomass carbon and soil sucrase, urease and cellulase activities all reached significant or extremely significant level in most incubation periods.Thus, the addition of carbon and nitrogen sources to rice straw might promote soil MBC, DOC and the three soil enzymes (soil urease, sucrase and cellulase) activities.In conclusion, this study revealed that adding carbon and nitrogen sources to rice straw might increase the number of soil microorganisms and improve soil enzyme activity, thus accelerating the decomposition of the rice straw.