Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (15): 3250-3263.doi: 10.3864/j.issn.0578-1752.2021.15.010


Identification of Functional Substances from Rice Straw Obtained by Pyrolysis and Enzymolysis and Its Effect

TANG SiYu(),LIU QiuMei,MENG XiaoHui,MA Lei,LIU DongYang(),HUANG QiWei,SHEN QiRong   

  1. Jiangsu Key Laboratory for Organic Solid Waste Utilization, Nanjing Agricultural University/Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing 210095
  • Received:2020-09-20 Accepted:2020-12-17 Online:2021-08-01 Published:2021-08-10
  • Contact: DongYang LIU;


【Objective】 To explore the effect of agricultural wastes (rice straw) on plant growth, the identification and analyzation of the small molecules that may exist in the high-temperature extracts and enzymolysis solution of rice straw were performed, and the promoting effect of these substances on cucumber growth was also evaluated.【Method】 The high-temperature extract solution of rice straw was prepared by high temperature water extraction at 115℃ for 30 min. The condensation of the extracellular proteins from Trichoderma guizhouense NJAU 4742 under the induction of rice straw was obtained by ammonium sulfate precipitation method, which was used to hydrolyze the rice straw to prepare rice straw enzymatic hydrolysate. The high-temperature extract and enzymolysis solution of rice straw were diluted into different times, and the growth-promoting effects were verified by cucumber hydroponic experiment. The high-temperature extract and enzymatic hydrolysis of rice straw were identified and compared by UHPLC-QE-MS non-target metabolomics detection technology. Under the control of the acquisition software (Xcalibur 4.0.27, Thermo), the QE mass spectrometer collects primary and secondary mass spectrometry data in the information-related acquisition mode for the high-temperature extract and enzymatic hydrolysate of rice straw. Through the self-written R program package (the kernel was XCMS), the original data was processed for peak identification, peak extraction, peak alignment and integration, and then it was matched with the BiotreeDB (V2.1) self-built MS database for substance annotation. Finally, the growth-promoting effects of some identified substances were verified by the cucumber hydroponic experiment.【Result】 The results showed that both high-temperature extract and enzymatic hydrolysate liquid of rice straw could promote the growth of cucumber seedlings at appropriate concentrations, and the enzymatic hydrolysate of rice straw showed significant growth promoting effect on cucumber when diluted for 100 times. Compared with the CK, the dry weight of overground part, underground part and plant height of cucumber plants treated with 100 times dilution increased by 52.64%, 55.05% and 21.43%, respectively, and the number of plant root tips increased by 31.95%. The high-temperature extract of rice straw owned the best growth promotion effect when it was diluted 50 times, in which the dry weight of overground part, the underground part and the plant height of cucumber plants increased by 44.16%, 63.38% and 55.56%, respectively, and the number of plant root tips increased by 64.44%, compared with CK. The UHPLC-QE-MS non-target detection technology results showed that 714 different substances were identified in the high-temperature extract of rice straw and 638 different substances were identified in the enzymatic hydrolysis solution, among which acetylcholine, L-carnitine and myo-inositol were screened out. Meanwhile, the standard products of these three substances were added to the cucumber root system, and the results showed that they all had considerable promotion effect on cucumber growth. Acetylcholine at the concentration of 1 μmol·L-1, 10 μmol·L-1 and 100 μmol·L-1 could all promote the growth of cucumber. Compared with CK, the exogenous acetylcholine at 1 μmol·L-1 concentration increases the dry weight of cucumber shoots by 54.69% and the dry weight of roots by 73.67%, the number of root tips increased by 130.5%; Exogenous L-carnitine was beneficial to the growth of cucumber plants at the concentration of 0.1 mmol·L-1 and 1 mmol·L-1. Compared with CK, the dry weight of cucumber shoots increased by 33.79% and 30.19%, and the dry weight of roots increased by 44.97% and 48.82%, the number of cucumber root tips increased by 41.8% and 49.9%, respectively; Exogenous myo-inositol at the concentration of 0.05 mmol·L-1 and 0.1 mmol·L-1 could promote the growth of cucumber. Compared with CK, the dry weight of cucumber shoots increased by 36.66% and 30.15%, roots dry weight increased by 69.82% and 51.78%, and the number of cucumber root tips increased by 149.0% and 96.7%, respectively.【Conclusion】 In brief, both the high-temperature extract and the enzymatic hydrolysate of rice straw could significantly promote the growth of cucumber, and the acetylcholine, L-carnitine and myo-inositol were detected by LCMS in the pyrolysis and enzymatic hydrolysate of rice straw, which were considered as the functional substances in rice straw.

Key words: rice straw, high-temperature extraction, enzymatic hydrolysis, LCMS, growth promotion

Fig. 1

SDS-PAGE electrophoresis analysis results A was SDS-PAGE analysis results, and B was the protein concentration; the T1: supernatant of Trichoderma guizhouense NJAU 4742 fermented for 5 days under the induction of rice straw; T2: crude enzyme solution obtained by ammonium sulfate precipitation on T1"

Fig. 2

The effect of rice straw enzymatic hydrolysate on cucumber seedlings growth"

Table 1

Effects of rice straw enzymatic hydrolysate on some growth indexes of cucumber seedlings"

Stem diameter (mm)
Plant height (cm)
根系指标Root index
Total roots length (cm)
Root surface area (cm2)
Root tips
CK 4.20±0.20d 11.2±0.5c 1046.8±69.4b 162.71±5.34c 1111±114b
25 4.47±0.17c 11.6±0.4c 774.1±117.9c 128.97±8.78a 1124±239b
50 4.88±0.13b 13.0±0.6ab 1153.1±87.6b 194.44±11.99ab 1483±160a
100 5.14±0.06a 13.6±0.8a 1391.9±121.7a 204.39±15.17a 1466±82a
200 4.29±0.06cd 12.6±0.3b 1060.4±60.0b 177.11±10.74bc 1115±76b

Fig. 3

Effects of high-temperature extract of rice straw on cucumber seedlings growth"

Table 2

Effects of high-temperature extract of rice straw on some growth indexes of cucumber seedlings"

Stem diameter (mm)
Plant height (cm)
根系指标Root index
Total roots length (cm)
Root surface area (cm2)
Root tips
CK 3.88±0.05b 10.8±0.3c 909.4±20.8bc 132.07±5.25c 1094±14e
200 3.91±0.10b 13.2±0.6b 869.9±18.4c 135.40±10.94c 1207±53d
100 3.94±0.10b 13.5±0.4b 938.1±30.1b 154.23±6.95b 1463±29c
50 4.41±0.09a 16.8±0.4a 1225.4±48.0a 190.00±9.39a 1799±51a
25 0.11±0.11a 16.8±0.3a 1172.3±43.0a 187.81±3.26a 1670±56b

Fig. 4

Total ion current diagram of rice straw high-temperature extract and enzymatic hydrolysate A: Rice straw high-temperature extract; B: Rice straw enzymatic hydrolysate"

Table 3

Screening results of LC-MS/MS"

Substance name
MS2 score
time (s)
mass ratio
绝对峰面积平均值 Average absolute peak area
Enzymatic hydrolysate
High temperature extract
乙酰胆碱 Acetylcholine 0.82 351.71 146.12 0.00 1232294.50 1304286316.78
左旋肉碱 L-Carnitine 0.99 348.51 162.11 0.00 1057738.90 974337335.47
肌醇 myo-Inositol 0.92 387.48 203.05 2533514.18 438705294.96 35929119.59

Fig. 5

Analysis result of the screened substance by LC-MS"

Fig. 6

Effects of different concentrations of acetylcholine chloride, L-carnitine and myo-Inositol on growth of cucumber seedlings"

Table 4

The effects of different concentrations of acetylcholine chloride, L-carnitine and myo-Inositol on growth indexes of cucumber seedlings"

Substance name
Leaf area
SPAD value
Stem diameter (mm)
根系指标Root index
Total roots length (cm)
Root surface area (cm2)
Root tips
Acetylcholine chloride
CK 17.90±1.20b 35.9±1.1c 3.28±0.04c 584.3±24.8b 98.65±4.91b 727±70c
1 μmol·L-1 36.5±0.60a 46.5±0.8a 3.82±0.02ab 901.7±25.6a 133.54±3.32a 1676±29a
10 μmol·L-1 36.41±2.26a 44.9±0.3b 3.89±0.05a 869.9±90.5a 125.78±10.33a 1643±107a
100 μmol·L-1 34.98±3.30a 44.1±0.3b 3.74±0.06b 882.4±81.6a 127.03±7.55a 1313±126b
CK 17.90±1.20b 35.9±1.1c 3.28±0.04c 584.3±24.8b 98.65±4.91b 727±70c
0.1 mmol·L-1 25.10±1.47a 39.2±1.0b 3.70±0.06a 686.5±63.5a 118.38±3.93a 1031±45a
1 mmol·L-1 22.17±3.34a 41.2±0.7a 3.68±0.01a 744.±45.8a 109.87±3.78a 1090±21a
5 mmol·L-1 22.11±1.95a 42.6±0.5a 3.53±0.03b 520.6±41.5b 86.77±7.34c 896±67b
CK 17.90±1.20b 35.9±1.1c 3.28±0.04b 584.3±24.8c 98.65±4.91c 727±70c
0.05 mmol·L-1 29.79±3.09a 44.0±0.4a 3.95±0.01a 1026.8±74.1a 138.27±5.83a 1810±186a
0.1 mmol·L-1 27.13±3.10a 44.5±0.2a 3.82±0.11a 808.4±18.3b 112.23±9.55b 1430±125b
1 mmol·L-1 - - 2.86±0.15c 102.8±18.3d 18.96±6.23d 477±93d
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