Effects of flushing alkaline thermal hydrolysis liquid to promote Brassica chinensis yield and nitrogen invertase activity mechanistic research
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摘要: 污泥通过碱性热水解工艺(ATH)提取的富含多肽、蛋白质类液体(污泥热碱液)已被证实无毒性且可运用于农业生产中, 并显著促进作物生长。为探究污泥热碱液对小青菜氮素吸收及氮代谢调控机制的影响, 本试验以小青菜为研究对象, 采用盆栽试验, 以不施氮肥为对照, 研究5个污泥热碱液处理土壤中分别施入0 mg·kg−1、20.19 mg·kg−1、40.38 mg·kg−1、60.57 mg·kg−1、80.76 mg·kg−1污泥热碱液, 探讨小青菜植株氮素吸收、氮代谢关键酶活性等的变化。结果表明, 随着施用量的增加, 各指标均呈先升高后下降的趋势, 当施用量为40.38 mg·kg−1时, 小青菜收获后氮素累积量、产量和品质等达较高水平, 硝酸盐含量最低。通过对小青菜氮素吸收量及产量进行拟合, 得出121.48~127.59 kg·hm−2为该污泥热碱液对小青菜的最佳施用量。施用量为40.38 mg·kg−1时, 小青菜中硝酸还原酶(NR)、亚硝酸还原酶(NiR)、谷氨酸脱氢酶(GDH)、谷氨酸合成酶(GOGAT)、谷氨酰胺合成酶(GS)均保持较高的活性, 在小青菜定苗后第2周、第4周和第6周与其他处理相比, NR活性增加56.56%~183.43%、16.55%~150.36%和7.86%~293.25%, NiR活性增加24.70%~348.17%、1.06%~71.24%和7.62%~286.59%, GDH活性增加9.91%~149.21%、37.52%~308.35%和16.08%~123.12%, GS活性增加4.13%~17.82%、5.23%~122.27%和9.91%~121.21%, GOGAT活性增加31.31%~288.16%、9.63%~351.69%和28.45%~1274.32%。冗余分析表明小青菜中GOGAT是决定产量、氮素利用率、氮素吸收率的主要因素, 与产量呈显著正相关。施用适量的污泥热碱液会提高小青菜氮素相关转化酶活性, 促进对氮素吸收及产量的增加。热碱液可作为新型肥料施用, 不仅可以解决污泥资源化问题, 还可以提高小青菜产量及养分吸收。Abstract: The polypeptide-rich liquid extracted from alkaline thermal hydrolysis (ATH) sludge has proven to be non-toxic and usable in agricultural production, resulting in a significant increase in crop growth. To explore the effect of ATH hydrolysate on nitrogen uptake and nitrogen metabolism regulation mechanism of Brassica chinensis, B. chinensis was flushed with ATH hydrolysate (0 mg·kg−1, 20.19 mg·kg−1, 40.38 mg·kg−1, 60.57 mg·kg−1, and 80.76 mg·kg−1) treatments, with no nitrogen fertilizer as the control in a pot experiment. The results showed that as the application rate of flushed ATH hydrolysate increased, all indices first increased and then decreased. When the ATH hydrolysate was flushed at 40.38 mg·kg−1, the nitrogen accumulation, yield, and quality of B. chinensis were higher than those of the control after harvest, and the nitrate content was the lowest. By fitting the nitrogen uptake and yield of B. chinensis, the optimal application amount of ATH hydrolysate was determined to be 121.48−127.59 kg·hm−2. Under the condition of 40.38 mg·kg−1 for potted B. chinensis, nitrate reductase (NR), nitrite reductase (NiR), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), and glutamine synthetase (GS) activities could maintain high activity. In the 2nd, 4th, and 6th weeks after final singling of seedlings, the NR activity increased by 56.56%−183.43%, 16.55%−150.36% and 7.86%−293.25%, NiR activity increased by 24.70%−348.17%, 1.06%−71.24% and 7.62%−286.59%, GDH activity increased by 9.91%−149.21%, 37.52%−308.35% and 16.08%−123.12%, GS activity increased by 4.13%−17.82%, 5.23%−122.27% and 9.91%−121.21%, and GOGAT activity increased by 31.31%−288.16%, 9.63%−351.69% and 28.45%−1274.32%. Redundancy analysis showed that GOGAT in B. chinensis was the main factor determining the yield, nitrogen utilization rate, and nitrogen absorption rate, with a significant positive correlation. The ATH hydrolysate solution had a significant influence on the yield of B. chinensis and the activities of enzymes related to nitrogen absorption and transformation under different flushing rates. An appropriate amount of ATH hydrolysate increases the activity of nitrogen-related invertase in B. chinensis, thereby improving the absorption and utilization of nitrogen and yield. At the same time, it also showed that ATH solution can be used as a new type of fertilizer, which not only solves the problem of sludge but also improves the yield and nutrient absorption of B. chinensis.
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图 1 不同热碱液施用量对各时期小青菜氮素累积量(A)、干生物量(B)的影响
图中竖杠为LSD值, P<0.05。CK为不施氮肥对照, T1、T2、T3、T4和T5分别为施氮肥基础上施用热碱液0 mg·kg−1、20.19 mg·kg−1、40.38 mg·kg−1、60.57 mg·kg−1和80.76 mg·kg−1。The upper vertical bar is the LSD value (P<0.05). CK is the control without nitrogen ferilizer. T1, T2, T3, T4 and T5 are treatments of applicaiton of alkaline thermal hydrolysis liquid of 0 mg·kg−1, 20.19 mg·kg−1, 40.38 mg·kg−1, 60.57 mg·kg−1 and 80.76 mg·kg−1 based on nitrogen application, respectively.
Figure 1. Effects of different alkaline thermal hydrolysis liquid treatments on nitrogen accumulation (A) and dry biomass (B) of Brassica chinensis in different periods
图 3 不同热碱液施用量处理对各时期小青菜硝酸还原酶(NR) (A)与亚硝酸还原酶(NiR) (B)活性的影响
不同小写字母表示处理间在P<0.05水平差异显著。CK为不施氮肥对照, T1、T2、T3、T4和T5分别为施氮肥基础上施用热碱液0 mg·kg−1、20.19 mg·kg−1、40.38 mg·kg−1、60.57 mg·kg−1和80.76 mg·kg−1。Different lowercase letters indicate significant differences among treatments at P<0.05 level. CK is the control without nitrogen ferilizer. T1, T2, T3, T4 and T5 are applicaiton of alkaline thermal hydrolysis liquid of 0 mg·kg−1, 20.19 mg·kg−1, 40.38 mg·kg−1, 60.57 mg·kg−1 and 80.76 mg·kg−1 based on nitrogen application, respectively.
Figure 3. Nitrate reductase (NR) (A) and nitrite reductase (NiR) (B) activities in Brassica chinensis at different growth stages under different alkaline thermal hydrolysis liquid treatments
图 4 不同热碱液施用量处理中各时期小青菜中谷氨酸脱氢酶(GDH)(A)、谷氨酸合成酶(GOGAT)和谷氨酰胺合成酶(GS)(B)活性
不同小写字母表示处理间在P<0.05水平差异显著。CK为不施氮肥对照, T1、T2、T3、T4和T5分别为施氮肥基础上施用热碱液0 mg·kg−1、20.19 mg·kg−1、40.38 mg·kg−1、60.57 mg·kg−1和80.76 mg·kg−1。Different lowercase letters indicate significant differences among treatments at P<0.05 level. CK is the control without nitrogen ferilizer. T1, T2, T3, T4 and T5 are applicaiton of alkaline thermal hydrolysis liquid of 0 mg·kg−1, 20.19 mg·kg−1, 40.38 mg·kg−1, 60.57 mg·kg−1 and 80.76 mg·kg−1 based on nitrogen application, respectively.
Figure 4. Glutamate dehydrogenase (GDH, A), glutamate synthase (GOGAT) and glutamine synthase (GS) (B) activities in Brassica chinensis at different stages under different alkaline thermal hydrolysis liquid flush treatments
图 5 不同时期(A: 第2周; B: 第4周; C: 第6周)不同处理下氮素相关酶与产量、氮素吸收率、氮素利用率的冗余分析(RDA)
CK为不施氮肥对照, T1、T2、T3、T4和T5分别为施氮肥基础上施用热碱液0 mg·kg−1、20.19 mg·kg−1、40.38 mg·kg−1、60.57 mg·kg−1和80.76 mg·kg−1。Yield: 产量; NUPE: 氮素吸收率; NUE: 氮素利用率; NR: 硝酸还原酶; NiR: 亚硝酸还原酶; GDH: 谷氨酸脱氢酶; GOGAT: 谷氨酸合成酶; GS: 谷氨酰胺合成酶。CK is the control without nitrogen ferilizer. T1, T2, T3, T4 and T5 are applicaiton of alkaline thermal hydrolysis liquid of 0 mg·kg−1, 20.19 mg·kg−1, 40.38 mg·kg−1, 60.57 mg·kg−1 and 80.76 mg·kg−1 based on nitrogen application, respectively. NUPE: nitrogen absorption efficiency; NUE: nitrogen use efficiency; NR: nitrate reductase; NiR: nitrite reductase; GDH: glutamate dehydrogenase; GOGAT: glutamate synthase; GS: glutamine synthase.
Figure 5. Redundancy analysis (RDA) of relationship at different periods (A: the second week; B: the fourth week; C: the sixth week) between nitrogen-related enzymes activies to yield, nitrogen uptake, and utilization
图 6 不同时期氮素相关酶与品质、氮素累积量的相关性分析
SP: 可溶性蛋白; SS: 可溶性糖; VC: 维生素C; TN: 氮素累积量; Yield: 产量; NR: 硝酸还原酶; NiR: 亚硝酸还原酶; GDH: 谷氨酸脱氢酶; GOGAT: 谷氨酸合成酶; GS: 谷氨酰胺合成酶。SP: soluble protein; SS: soluble sugar; VC: vitamin C; TN: nitrogen accumulation; NR: nitrate reductase; NiR: nitrite reductase; GDH: glutamate dehydrogenase; GOGAT: glutamate synthase; GS: glutamine synthase. *: P≤0.05; **: P≤0.01.
Figure 6. Correlation analysis of nitrogen-related enzymes and quality, nitrogen accumulation at different periods
表 1 热碱液的理化性质
Table 1. Physical and chemical properties of alkaline thermal hydrolysis liquid
性质 Property 含量 Content 性质 Property 含量 Content 有机碳 Organic carbon (g∙L−1) 153.49±9.37 Mg (mg∙L−1) 25.08±1.19 腐殖酸 Humus (g∙L−1) 8.11±0.08 Fe (mg∙L−1) 54.41±5.0 蛋白质 Protein (g∙L−1) 63.6±1.9 Mn (mg∙L−1) 0.27±0.03 多肽 Polypeptide (g∙L−1) 116.7±12.1 Cu (mg∙L−1) 0.12±0.02 游离氨基酸 Free amino acids (g∙L−1) 54.67±5.21 Zn (mg∙L−1) 1.64±0.13 NH4+-N (g∙L−1) 1.28±0.13 Hg (μg∙L−1) 66.23±5.88 NO3−-N (g∙L−1) 0.19±0.04 As (mg∙L−1) 1.78±0.09 N (g∙L−1) 44.08±0.32 Cd (μg∙L−1) 12.29±3.84 P (g∙L−1) 316.0±18.9 Pb (mg∙L−1) 3.02±0.12 K (g∙L−1) 8.75±0.05 Cr (mg∙L−1) 1.81±0.08 Ca (g∙L−1) 55.3±3.7 pH 10.05±0.08 表 2 不同热碱液施用量处理对小青菜氮素吸收利用的影响
Table 2. Effects of different alkaline thermal hydrolysis liquid treatments on nitrogen absorption, utilization of Brassica chinensis
处理
TreatmentSPAD 产量
Yield (g∙plant−1)地上部氮素累积
Aboveground nitrogen accumulation (mg∙plant−1)氮素吸收率
Nitrogen uptake efficiency (g∙g−1)氮素利用率
Nitrogen use efficiency (%)CK 22.37±1.13b 40.53±2.59d 83.59±2.81d — — T1 40.30±0.53a 51.23±1.39c 118.46±2.40c 31.59±0.02b 16.67±0.06a T2 40.07±0.87a 63.38±2.44b 147.15±5.95ab 39.24±0.02a 16.87±0.02a T3 40.83±0.49a 69.20±6.49a 157.23±3.20a 41.93±0.02a 19.55±0.02a T4 39.37±0.24a 68.41±3.92a 156.12±1.02ab 41.63±0.02a 19.26±0.02a T5 40.40±0.23a 63.12±2.18b 138.83±9.68b 37.02±0.03ab 14.65±0.03a 同列不同字母表示不同处理间在P<0.05水平差异显著。CK为不施氮肥对照, T1、T2、T3、T4和T5分别为施氮肥基础上施用热碱液0 mg·kg−1、20.19 mg·kg−1、40.38 mg·kg−1、60.57 mg·kg−1和80.76 mg·kg−1。Different letters in the same column indicate significant differences among treatments at P<0.05 level. CK is the control without nitrogen ferilizer. T1, T2, T3, T4 and T5 are applicaiton of alkaline thermal hydrolysis liquid of 0 mg·kg−1, 20.19 mg·kg−1, 40.38 mg·kg−1, 60.57 mg·kg−1 and 80.76 mg·kg−1 based on nitrogen application, respectively. 表 3 不同热碱液施用量处理对小青菜品质的影响
Table 3. Effects of different alkaline thermal hydrolysis liquid treatments on quality of Brassica chinensis
处理
Treatment可溶性糖
Soluble sugar content (mg·g−1)可溶性蛋白
Soluble protein content (mg·g−1)维生素C
Vitamin C content (mg·kg−1)硝酸盐
Nitrate content (mg·kg−1)CK 4.64±0.50c 6.48±0.51c 33.18±1.56c 74.00±5.03d T1 6.70±0.13ab 7.99±0.61b 36.04±2.43c 962.00±46.35a T2 7.42±0.32a 9.18±0.18ab 36.82±1.27bc 822.00±47.03b T3 8.08±0.26a 10.48±0.38a 45.90±0.66a 668.00±28.75c T4 7.11±0.75a 10.19±0.57a 45.61±4.83a 696.25±67.19c T5 5.20±0.61bc 9.02±0.51ab 43.56±1.44ab 976.75±20.48a 同列不同字母表示不同处理间在P<0.05水平差异显著。CK为不施氮肥对照, T1、T2、T3、T4和T5分别为施氮肥基础上施用热碱液0 mg·kg−1、20.19 mg·kg−1、40.38 mg·kg−1、60.57 mg·kg−1和80.76 mg·kg−1。Different letters in the same column indicate significant differences among treatments at P<0.05 level. CK is the control without nitrogen ferilizer. T1, T2, T3, T4 and T5 are applicaiton of alkaline thermal hydrolysis liquid of 0 mg·kg−1, 20.19 mg·kg−1, 40.38 mg·kg−1, 60.57 mg·kg−1 and 80.76 mg·kg−1 based on nitrogen application, respectively. 表 4 不同时期小青菜氮同化相关酶活性与产量、氮素吸收利用的相关性
Table 4. Pearson’s correlation analysis of nitrogen-related enzymes activities and yield, nitrogen absorption and utilization at different periods
氮素同化相关酶
Nitrogen assimilation-related enzyme第2周 The second week 第4周 The fourth week 第6周 The sixth week 产量
YieldNUPE NUE 产量
YieldNUPE NUE 产量
YieldNUPE NUE NR 0.331 0.482* −0.514* 0.007 −0.107 −0.241 0.632** 0.746** −0.240 NiR 0.068 0.276 −0.214 0.013 0.142 −0.118 0.340 0.304 −0.051 GDH −0.258 −0.307 0.007 −0.297 −0.202 −0.124 −0.609** −0.674** 0.192 GOGAT 0.542* 0.401 −0.181 0.227 0.493* −0.367 0.354 0.288 −0.161 GS 0.169 0.337 −0.400 −0.354 −0.259 −0.105 −0.463 −0.541* 0.232 *表示显著性为P<0.05, **表示显著性为P<0.01; NR: 硝酸还原酶; NiR: 亚硝酸还原酶; GDH: 谷氨酸脱氢酶; GOGAT: 谷氨酸合成酶; GS: 谷氨酰胺合成酶; NUPE: 氮素吸收效率; NUE: 氮素利用率。* and ** indicate significant correlations at P<0.05 and P<0.01 levels, respectively. NR: nitrate reductase; NiR: nitrite reductase; GDH: glutamate dehydrogenase; GOGAT: glutamate synthase; GS: glutamine synthase; NUPE: nitrogen absorption efficiency; NUE: nitrogen use efficiency. 表 5 不同时期氮同化相关的酶活性冗余分析
Table 5. Redundancy analysis of enzymes activities related to nitrogen assimilation at different periods
氮素同化相关酶
Nitrogen assimilation related enzyme第2周 The second week 第4周 The fourth week 第6周 The sixth week Explains (%) F P Explain (%) F P Explain (%) F P NR 1.7 0.6 0.466 13.8 3.6 0.064 38.8 10.2 0.01 NiR 2.6 0.9 0.366 2.2 0.6 0.454 <0.1 <0.1 0.94 GDH 28.4 9.9 0.014 8.8 1.9 0.162 17.8 6.2 0.022 GOGAT 28.5 6.4 0.014 15.6 3.2 0.092 32.5 41.7 0.002 GS 5.1 1.9 0.198 12.1 2.2 0.16 0.4 0.5 0.604 NR: 硝酸还原酶; NiR: 亚硝酸还原酶; GDH: 谷氨酸脱氢酶; GOGAT: 谷氨酸合成酶; GS: 谷氨酰胺合成酶。NR: nitrate reductase; NiR: nitrite reductase; GDH: glutamate dehydrogenase; GOGAT: glutamate synthase; GS: glutamine synthase. -
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