Effect of Digestate and Straw Combined Application on Maintaining Rice Production and Paddy Environment

Few studies have focused on the combined application of digestate and straw and its feasibility in rice production. Therefore, we conducted a two-year field experiment, including six treatments: without nutrients and straw (Control), digestate (D), digestate + fertilizer (DF), digestate + straw (DS), digestate + fertilizer + straw (DFS) and conventional fertilizer + straw (CS), to clarify the responses of rice growth and paddy soil nutrients to different straw and fertilizer combinations. Our results showed that digestate and straw combined application (i.e., treatment DFS) increased rice yield by 2.71 t ha−1 compared with the Control, and digestate combined with straw addition could distribute more nitrogen (N) to rice grains. Our results also showed that the straw decomposition rate at 0 cm depth under DS was 5% to 102% higher than that under CS. Activities of catalase, urease, sucrase and phosphatase at maturity under DS were all higher than that under both Control and CS. In addition, soil organic matter (SOM) and total nitrogen (TN) under DS and DFS were 20~26% and 11~12% higher than that under B and DF respectively, suggesting straw addition could benefit paddy soil quality. Moreover, coupling straw and digestate would contribute to decrease the N content in soil surface water. Overall, our results demonstrated that digestate and straw combined application could maintain rice production and have potential positive paddy environmental effects.


Introduction
China is the largest consumer and producer and a major importer of chemical fertilizer [1]. In spite of its vital importance for food security, the overuse of chemical fertilizer has caused environmental problems globally, such as greenhouse gas (GHG) emissions and groundwater pollution [2]. As renewable resources with high efficiency and rich nutrients, straw and digestate have a great potential to substitute chemical fertilizer and promote sustainable agriculture development in China [3,4].
Agricultural wastewater, such as husbandry wastewater, aquaculture wastewater and liquid digestate, is easy to collect and utilize. The valuable resources in agricultural wastewater should be recycled and reused for environmental sustainability. Digestate is a kind of residue liquid of biogas produced by anaerobic fermentation with livestock and poultry manure as the main raw materials [5]. Valorization of livestock and poultry manure by anaerobic digestion has been considered as a standout option for bioenergy production in terms of energy efficiency and environmental impact [6]. Digestate can act as soil conditioner and provide valuable nutrients to plants [7,8], or digestate can be directly used as a nutrient source for soilless cultivation [9]. However, it is more often widely used

Experiment Treatments
Our field experiment was conducted for two years at the Suzhong Dadi Agricultural Technology Company in Gaoyou, Jiangsu Province, China. The experiment site is characterized by a subtropical warm monsoon climate, with an annual mean precipitation of 1000 mm and an annual mean temperature of 14.8 • C. The frost-free period lasts 217 days. The digestate was obtained from a local large-scale pig farm (Xingmu Pig Farm), with the following basic properties: pH was 7.76; total nitrogen (TN), available nitrogen (AN) and available phosphorus (AP) were 1012, 551 and 753 mg L −1 , respectively. Six treatments ( Table 1) were included in our study: without nutrients and straw (Control), digestate (D), digestate + fertilizer (DF), digestate + straw (DS), digestate + fertilizer + straw (DFS) and conventional fertilizer + straw (CS). For CS, the management was based on local farming habits. Each treatment had two plots (5 m × 4 m) as replicates. The irrigation and drainage time, frequency and quantity were identical for all treatments. Seedlings were raised by mechanical plastic plate and planted by artificial simulation machine at 18-20 days after seedling.

Sampling and Analytical Procedures
Plant and soil samples (0-20 cm) were collected from each plot at tillering, jointing, heading and mature stages. Soil samples were air-dried after plant residues and stones were removed. Plant samples were oven-dried at 105 • C for 30 min and then dried at 80 • C until a constant weight was reached. The N content in rice organs was determined by semi-micro-Kjeldahl method [31]. Soil catalase, alkaline phosphatase, urease and sucrase activities were determined using the permanganate titration method [32], disodium benzene phosphate colorimetry method, sodium phenoxide colorimetry and DNS colorimetry (3,5-initrosalicylic acid) [33], respectively. TN, AN, AP, AK and soil organic matter (SOM) contents in soil were tested by the semi-micro-Kjeldahl method, alkali disintegration spread-sodium bicarbonate method, sodium bicarbonate method [34], a flare photometer and potassium dichromate method [35], respectively. We randomly collected five water samples as replicates in each plot for detecting TN, NO 3 − -N and NH 4 + -N concentrations in paddy surface water at 1, 2, 3, 5 and 7 days after every application of digestate. TN content was measured with alkaline potassium persulfate [36], NO 3 − -N content was measured with UV spectrophotometry [37] and NH 4 + -N content was measured with Nessler s reagent [38].

Statistical Analysis
Statistical analysis was performed using SPSS 16.0 Statistical Software (IBM, Chicago, IL, USA). One-way ANOVA was used to determine differences between treatments. Means were compared using least significant difference (LSD), and significance was determined at p < 0.05. Figures were drawn in OriginPro 8.5.1 (OriginLab, Northampton, MA, USA).

Plant N Distribution
In all treatments, N distribution in the stalk significantly increased from tillering to heading and decreased from heading to maturity, with translocation to grain occurring (Figure 1). At tillering stage, compared to the CS and Control, the digestate can promote the N distribution to leaf. At jointing stage, the trend of N distribution to leaf was the same as that at tillering stage, with DS being the highest and the control being the lowest, but the difference was not significant. The highest N distribution in leaf was found in DS both at tillering and jointing stages. At heading stage, N distribution to leaf was DF > CS > DFS > D > DS > Control; the highest value was 52.40% under DF, and the lowest was 45.08% under Control (Figure 1), with a significant difference, while the other four treatments had no significant difference. At maturity, N distribution to grain was in the decreasing order of D > DF > DS > DFS > CS > Control; the highest value was 62.18% under D, and the lowest was 57.87% under Control ( Figure 1).

Plant N Distribution
In all treatments, N distribution in the stalk significantly increased from tillering to heading and decreased from heading to maturity, with translocation to grain occurring (Figure 1). At tillering stage, compared to the CS and Control, the digestate can promote the N distribution to leaf. At jointing stage, the trend of N distribution to leaf was the same as that at tillering stage, with DS being the highest and the control being the lowest, but the difference was not significant. The highest N distribution in leaf was found in DS both at tillering and jointing stages. At heading stage, N distribution to leaf was DF > CS > DFS > D > DS > Control; the highest value was 52.40% under DF, and the lowest was 45.08% under Control (Figure 1), with a significant difference, while the other four treatments had no significant difference. At maturity, N distribution to grain was in the decreasing order of D > DF > DS > DFS > CS > Control; the highest value was 62.18% under D, and the lowest was 57.87% under Control ( Figure 1).
The results showed that when digestate was utilized as organic fertilizer, compared with conventional fertilizer, N distribution to leaf was higher at tillering stage, which laid a good foundation for growth at the early stage. More N was transferred to the grain at maturity, which had the advantage of N distribution to grain.  The results showed that when digestate was utilized as organic fertilizer, compared with conventional fertilizer, N distribution to leaf was higher at tillering stage, which laid a good foundation for growth at the early stage. More N was transferred to the grain at maturity, which had the advantage of N distribution to grain.

The Degree of Straw Decomposition
After the straw was buried, with the increase in soil depth, the decay degree of each treatment showed a downward trend. We found that on the 15th day after the straw was buried, the decomposition degrees at 0, 10 and 20 cm soil depth under DS were 2.02, 1.87 and 1.10 times higher than that under CS. The differences in decomposition rates between treatments decreased with the increase in soil depth ( Figure 2). On the 40th day, the decomposition degree at 0 cm soil depth under DS was still higher than that under CS, while the difference was not significant at 10 and 20 cm soil depths. On the 70th day, the decomposition degrees at 0 and 10 cm soil depths under DS were just slightly higher than those under CS. The above showed that compared with chemical fertilizer, digestate had a better effect of promoting straw decomposition, especially at the early stage; when decomposing surface straw, this effect was more obvious.

The Degree of Straw Decomposition
After the straw was buried, with the increase in soil depth, the decay degree of each treatment showed a downward trend. We found that on the 15th day after the straw was buried, the decomposition degrees at 0, 10 and 20 cm soil depth under DS were 2.02, 1.87 and 1.10 times higher than that under CS. The differences in decomposition rates between treatments decreased with the increase in soil depth ( Figure 2). On the 40th day, the decomposition degree at 0 cm soil depth under DS was still higher than that under CS, while the difference was not significant at 10 and 20 cm soil depths. On the 70th day, the decomposition degrees at 0 and 10 cm soil depths under DS were just slightly higher than those under CS. The above showed that compared with chemical fertilizer, digestate had a better effect of promoting straw decomposition, especially at the early stage; when decomposing surface straw, this effect was more obvious.

Soil Enzymatic Activities
Soil catalase can catalyze the decomposition of hydrogen peroxide generated from the metabolism of the soil and living organisms into oxygen and water, so as to protect organisms from damages caused by hydrogen peroxide. Catalase activity was significantly higher at all periods under DS than under D. Catalase activity under DS increased by 8.46%, 8.72%, 7.69% and 8.29% at tillering, jointing, heading and mature stages, respectively, compared to that under B. Soil catalase activity under DS treatment increased by 11.57%, 27.17%, 21.76% and 14.28% at tillering, jointing, heading and mature stages, respectively, compared to that under CS (Table 3). Our results showed that the returning of digestate and straw could contribute to the improvement of soil catalase activity.
Soil alkaline phosphatase can catalyze the mineralization and hydrolysis of soil organic phosphorus and can help the plants to absorb phosphorous. During the tillering and jointing periods, alkaline phosphatase activity was significantly decreased in DS compared to D and in DFS compared to DF. At maturity, alkaline phosphatase activity in DS and DFS increased by 22.39% and 33.50%, respectively (Table 3). This indicates that the coupling of straw with digestate restrained the effect of alkaline phosphatase at the early growth stage but would contribute to the improvement of alkaline phosphatase activity at maturity.
Soil urease catalyzes the hydrolysis of amide N compounds into inorganic N compounds that can be directly absorbed by plants. There were no significant differences in urease activity at the early growth stage. At maturity, soil urease activity under DS was 25.65%, 32.03%, 34.13% and 48.90% higher than that under B, DF, DFS and CS treatments, respectively (Table 3). This indicates that the coupling of straw with digestate could improve the soil urease activity, especially at maturity.
Soil sucrase plays an important role in the increase in soil soluble nutrients and is related to SOM, phosphorus, microbial number and soil respiration. Soil sucrase activity under B and DS was significantly higher than that under control at all periods; thus, either digestate alone or the combination of straw and digestate will contribute to the improvement of soil sucrase activity ( Figure 3D).

Soil Enzymatic Activities
Soil catalase can catalyze the decomposition of hydrogen peroxide generated from the metabolism of the soil and living organisms into oxygen and water, so as to protect organisms from damages caused by hydrogen peroxide. Catalase activity was significantly higher at all periods under DS than under D. Catalase activity under DS increased by 8.46%, 8.72%, 7.69% and 8.29% at tillering, jointing, heading and mature stages, respectively, compared to that under B. Soil catalase activity under DS treatment increased by 11.57%, 27.17%, 21.76% and 14.28% at tillering, jointing, heading and mature stages, respectively, compared to that under CS (Table 3). Our results showed that the returning of digestate and straw could contribute to the improvement of soil catalase activity.
Soil alkaline phosphatase can catalyze the mineralization and hydrolysis of soil organic phosphorus and can help the plants to absorb phosphorous. During the tillering and jointing periods, alkaline phosphatase activity was significantly decreased in DS compared to D and in DFS compared to DF. At maturity, alkaline phosphatase activity in DS and DFS increased by 22.39% and 33.50%, respectively (Table 3). This indicates that the coupling of straw with digestate restrained the effect of alkaline phosphatase at the early growth stage but would contribute to the improvement of alkaline phosphatase activity at maturity.
Soil urease catalyzes the hydrolysis of amide N compounds into inorganic N compounds that can be directly absorbed by plants. There were no significant differences in urease activity at the early growth stage. At maturity, soil urease activity under DS was 25.65%, 32.03%, 34.13% and 48.90% higher than that under B, DF, DFS and CS treatments, respectively (Table 3). This indicates that the coupling of straw with digestate could improve the soil urease activity, especially at maturity.
Soil sucrase plays an important role in the increase in soil soluble nutrients and is related to SOM, phosphorus, microbial number and soil respiration. Soil sucrase activity under B and DS was significantly higher than that under control at all periods; thus, either digestate alone or the combination of straw and digestate will contribute to the improvement of soil sucrase activity ( Figure 3D).

Soil Organic Matter and Nutrient Contents
We found that the response of SOM and soil nutrients to treatments differed. The SOM decreased in the order of DFS > DS > DF > D > CS > Control at maturity (Figure 4).
Concretely, the SOM under DS was 11.93% higher than that under B, and the SOM under DFS was 15.50% higher than that under DF ( Table 4). The results showed that straw addition could benefit paddy soil quality after digestate application. The highest TN content was also found in DFS, increased by 12.02% compared with DF, and the TN content in DS was 8.14% higher than that in B. Table 4. Soil organic matter (SOM), total nitrogen (TN), available nitrogen (AN), available phosphorus (AP) and available potassium (AK) contents at maturity under the different treatments.    The highest AN, AP and AK contents were all found in DS. The AN content in DS increased by 17.72% compared with D, and that in DFS was 9.04% higher than that in DF. The AP contents of D, DF, DS and DFS were 15.58%, 16.38%, 39.72% and 18.86% higher, respectively, than that under CS. Moreover, the AK content under DS was 10.71% and 31.90% higher than that under CS and Control, respectively (Table 4). Our results indicate that the digestate and straw combined application was beneficial to increase the nutrient content of paddy soil.

TN, NO 3 − -N and NH 4 + -N Contents in Soil Surface Water
Our results showed that TN, NO 3 − -N and NH 4 + -N concentrations in soil surface water all decreased by over 50% in the first 3 days after digestate addition, regardless of treatments and applied periods ( Figure 5).

TN, NO3 − -N and NH4 + -N Contents in Soil Surface Water
Our results showed that TN, NO3 − -N and NH4 + -N concentrations in soil surface water all decreased by over 50% in the first 3 days after digestate addition, regardless of treatments and applied periods ( Figure 5). When digestate was irrigated as base fertilizer, the TN concentration under D, DF, DS and DFS treatment decreased in the third day compared to that in the first day, and the decrease under DS and DFS tended to be faster than that under D and DF. Seven days later, the TN concentration under DS and DFS showed no significant difference with that under CS. This indicates that when digestate was irrigated as base fertilizer and coupled with straw, it would contribute to the absorption of N within 1-3 days. When digestate was irrigated as panicle fertilizer, the TN concentrations under D, DF, DS and DFS treatments within 1-3 days were significantly higher than those under Control and CS. On the 7th day, the TN concentrations of all treatments showed no significant differences, except DF was slightly higher ( Figure 5A,B). Our results showed that after the digestate was irrigated as panicle fertilizer, despite being coupled with straw, the TN concentration was fairly high within 1-3 days; measures should be taken to avoid environmental water loss risk.
When digestate was irrigated as base fertilizer, the NO3 − -N concentration under DF and DFS decreased by 65.44% and 74.65%, respectively, on the 3rd day compared to the 1st day. The NO3 − -N concentration under DFS decreased faster than that under DF. When digestate was irrigated as panicle fertilizer, the NO3 − -N concentration under DF and DFS decreased by 42.12% and 64.21%, respectively, on the 7th day compared to the 1st day. The NO3 − -N concentration under DFS decreased faster than that under DF, similar to the situation where digestate was irrigated as base fertilizer ( Figure 5C,D). This indicates that straw addition could definitely reduce the NO3 − -N concentration of surface water.
When digestate was irrigated as base fertilizer, the NH4 + -N concentrations of all treatments tended to become similar. Over time, they tended to decrease. On the 7th day, the NH4 + -N concentration under DS and DFS was slightly higher than that under CS, but not significantly. This indicates that the NH4 + -N concentration of surface water was high at the beginning but decreased to a relatively safe concentration after 7 days. When digestate When digestate was irrigated as base fertilizer, the TN concentration under D, DF, DS and DFS treatment decreased in the third day compared to that in the first day, and the decrease under DS and DFS tended to be faster than that under D and DF. Seven days later, the TN concentration under DS and DFS showed no significant difference with that under CS. This indicates that when digestate was irrigated as base fertilizer and coupled with straw, it would contribute to the absorption of N within 1-3 days. When digestate was irrigated as panicle fertilizer, the TN concentrations under D, DF, DS and DFS treatments within 1-3 days were significantly higher than those under Control and CS. On the 7th day, the TN concentrations of all treatments showed no significant differences, except DF was slightly higher ( Figure 5A,B). Our results showed that after the digestate was irrigated as panicle fertilizer, despite being coupled with straw, the TN concentration was fairly high within 1-3 days; measures should be taken to avoid environmental water loss risk.
When digestate was irrigated as base fertilizer, the NO 3 − -N concentration under DF and DFS decreased by 65.44% and 74.65%, respectively, on the 3rd day compared to the 1st day. The NO 3 − -N concentration under DFS decreased faster than that under DF. When digestate was irrigated as panicle fertilizer, the NO 3 − -N concentration under DF and DFS decreased by 42.12% and 64.21%, respectively, on the 7th day compared to the 1st day. The NO 3 − -N concentration under DFS decreased faster than that under DF, similar to the situation where digestate was irrigated as base fertilizer ( Figure 5C,D). This indicates that straw addition could definitely reduce the NO 3 − -N concentration of surface water. When digestate was irrigated as base fertilizer, the NH 4 + -N concentrations of all treatments tended to become similar. Over time, they tended to decrease. On the 7th day, the NH 4 + -N concentration under DS and DFS was slightly higher than that under CS, but not significantly. This indicates that the NH 4 + -N concentration of surface water was high at the beginning but decreased to a relatively safe concentration after 7 days. When digestate was irrigated as panicle fertilizer, the NH 4 + -N concentrations under B, DF, DS and DFS on the 3rd day decreased by 32.99%, 22.92%, 53.77% and 54.27%, respectively, compared to those on the 1st day ( Figure 5E,F). NH 4 + -N concentration under DS and DFS treatment decreased faster than that under B and DF. When digestate was irrigated as panicle fertilizer, coupling with straw could obviously reduce the NH 4 + -N concentration.
Some NH 4 + is oxidized to NO 3 − in aerobic microsites in the soil surface water and is quickly lost by denitrification as it diffuses into anaerobic microsites [39].

Discussion
Our results show that higher N distribution in leaves at tillering stage under combined application of digestate and straw provided a good foundation for rice growth in the early stage. More N could be transferred to the grains at maturity, leading to better production [40], which was supported by the theoretical yields in our study. However, due to the very high input of chemical fertilizer, conventional management practice might still have higher rice yield than digestate input only, in agreement with previous studies [41]. In addition, digestate could promote straw decomposition based on our results, which supported that digestate addition in preprocess of straw decomposition might be the best promoter [42]. Our results show that the decomposition degree of DS was higher than that of DFS. This may be because higher rates of N fertilization inhibited soil enzyme activities and functional diversity of microbial communities [43]. Further efforts should be made to determine why negative effects appeared when chemical fertilizer was added.
Our finding that digestate and straw combined application increased soil urease activity at maturity agreed with the results in rice-wheat and wheat-maize rotation systems in previous studies [44][45][46]. In addition, digestate application has been proved to increase the soil sucrase and phosphatase activities but significantly reduce the activity of soil catalase [47]. Our results suggest the same trends for sucrase and phosphatase activities during the key stages, though these trends were not significant; in contrast, digestate application likely increased catalase activity.
There are plenty of studies demonstrating that digestate application can increase not only SOM contents but also soil nutrients, such as TN, total phosphorus, AN and AP, with or without straw retention [48]. Concretely, we found that combined addition of digestate and straw caused a greater increase in SOM and nutrient (i.e., TN, AN, AP and AK) contents than digestate applied alone. This could possibly be explained by digestate being characterized as rich in nutrients and thus being able to supply the nutrients directly; it could also increase soil nutrients indirectly through promoting enzymatic activities [49]. In agreement with previous studies, N concentration in soil surface water increased after using digestate as base fertilizer or panicle fertilizer in this study [50,51]; however, the greater differences in TN, NO 3 − -N and NH 4 + -N concentrations between treatments after digestate addition as base fertilizer compared to digestate addition as panicle fertilizer were likely the result of N saturation in soil surface water. Furthermore, after using digestate as base fertilizer, NO 3 − -N contents under D and DF were higher than those under DS and DFS, but the NH 4 + -N contents under D and DF were lower than those under DS and DFS. This might because that NO 3 --N mainly came from the nitrification of NH 4 + -N [52]. When straw decomposed, oxygen was consumed, thereby inhibiting nitrification and promoting denitrification.

Conclusions
Digestate addition maintained the rice production and had some positive effects on paddy soil and water properties in our study. Digestate application significantly increased rice yield compared with Control; combined with straw addition, it could distribute more N to rice grains, which benefited rice production. Moreover, digestate promoted straw decomposition compared with CS. Activities of catalase, urease, sucrase and phosphatase at maturity under DS were all higher than those under Control and CS. Thus, among the six treatments, DS is the best application. Above all, our findings could provide some evidence for the possibility of using digestate to replace chemical fertilizer in rice production. We also suggest that further efforts should be made to explore the mechanisms of the combined application of digestate and straw so that suggestions can be provided for the better management of digestate applied in the future.