茚虫威降解菌海藻酸钠微球制剂研制及应用效果

doi:10.3864/j.issn.0578-1752.2022.05.007

摘要/Abstract

摘要：

【目的】以海藻酸钠为载体制备降解菌斯氏假单胞菌（Pseudomonas stutzeri）ACCC 02521菌株微球制剂,确定该制剂降解农田土壤茚虫威的应用条件。【方法】通过微滴包埋成球法,将湿菌体重悬后加入海藻酸钠溶液,混匀后逐滴滴至CaCl₂溶液造粒,低温固定后,以0.9% NaCl溶液洗涤,测定降解菌微球的传质性能和机械强度,确定海藻酸钠最佳浓度。通过单因素优化获得最佳制剂配方,在3.0%海藻酸钠溶液中分别滴入1.0%—5.0% CaCl₂溶液、40—200 g·L^-1菌量或20—100 g·L^-1制剂,根据茚虫威降解率,分别确定降解菌微球制剂CaCl₂浓度、包埋菌量和用量。通过扫描电镜观察固菌前后微球形态、降解菌细胞形态和菌群分布情况。在不同类型土壤悬液、温度、pH或不同时间条件下投入定量降解菌微球制剂,通过计算释放菌量（CFU/mL）或茚虫威降解率,评价环境因素对制剂释放能力、降解效果和稳定性的影响。喷雾施用后2 d撒施降解菌微球制剂,采集表层土壤检测茚虫威残留量,确定田间施用剂量,明确制剂田间应用条件。茚虫威残留浓度变化均以HPLC法检测追踪。【结果】制剂由3.0%海藻酸钠、2.0% CaCl₂及80 g·L^-1降解菌组成,粒径约3.0 mm,降解菌微球粒径大小均匀,机械强度适中,传质性能、降解活性和贮藏稳定性良好。扫描电镜观测表明,降解菌在海藻酸钠微球中分布均匀,菌体形态正常。在10—30℃、pH为6.0—8.0的土壤中,降解菌稳定释放,对茚虫威降解率达到85%以上,释放性能不受土壤类型影响,稳定性良好,受环境条件影响小。田间喷施150 g·L^-1茚虫威乳油（EC）有效成分20 mg·L^-1后2 d撒施降解菌微球制剂90—900 kg·hm^-2时,茚虫威残留半衰期(T_1/2)缩短至2.49—3.32 d（空白对照区T_1/2为7.53 d）;有效成分5、20、50 mg·L^-1喷施后2 d,均匀撒施降解菌微球制剂量450 kg·hm^-2,土壤茚虫威残留T_1/2由6.03—7.45 d缩短至2.34—3.59 d。【结论】以海藻酸钠为载体制备降解菌斯氏假单胞菌微球制剂性能稳定,可显著降解农田土壤茚虫威残留,缩短残留半衰期,为土壤农药残留污染生物修复提供了技术和产品支撑,具有进一步优化和应用的潜力。

关键词: 茚虫威残留, 生物修复, 海藻酸钠, 斯氏假单胞菌, 降解菌制剂

Abstract:

【Objective】The objective of this study is to prepare microsphere of degrading bacterium Pseudomonas stutzeri (ACCC 02521) with sodium alginate as carrier, and to establish the application conditions for degrading indoxacarb in farmland.【Method】Through the micro drop embedding ball forming method, bacteria were suspended and added into sodium alginate solution. After mixing, it was dropped into CaCl₂ for granulation. It was washed with 0.9% NaCl after fixing at low temperature. The mass transfer performance and mechanical strength of degrading bacteria microspheres were measured to determine the optimal concentration of sodium alginate. The best formulation was obtained by single factor optimization. 1.0%-5.0% CaCl₂, 40-200 g·L ^-1 bacteria or 20-100 g·L^-1preparation were dropped into 3.0% sodium alginate, respectively. The CaCl₂ concentration, embedded bacteria and preparation dosage in the degrading bacteria microspheres were determined according to the degradation rate of indoxacarb. The morphology of microspheres, cell morphology and distribution of degrading bacteria were observed by scanning electron microscope. Quantitative degrading bacteria microspheres were put into different types of soil suspension, temperature, pH or treatment time. The effects of environmental factors on the release capacity, degradation effect and stability of degrading bacteria microspheres were evaluated by calculating the amount of released bacteria (CFU/mL) or the degradation rate of indoxacarb. Two days after the routine application of pesticides, the degrading bacteria microsphere preparation was applied. Topsoil was collected to detect the residue of indoxacarb, the field application of dose was determined, and the field application conditions of the preparation were determined. The residual concentrations of indoxacarb were detected and tracked by HPLC.【Result】The preparation was composed of 3.0% sodium alginate, 2.0% CaCl₂ and 80 g·L^-1 degrading bacteria. The particle size was about 3.0 mm. The degrading bacteria microspheres had uniform particle size, moderate mechanical strength, good mass transfer performance, degradation activity and storage stability. Scanning electron microscopy showed that the degrading bacteria were evenly distributed in sodium alginate microspheres and their morphology was normal. In the soil with 10-30℃ and pH of 6.0-8.0, the degrading bacteria were released stably, and the degradation rate of indoxacarb was more than 85%. The release performance was not affected by soil type, the stability was good, and was less affected by environmental conditions. When 90-900 kg·hm^-2 of degrading bacteria microspheres were applied 2 d after field spraying of 150 g·L^-1 of EC active ingredient 20 mg·L^-1, the residual half-life (T_1/2) of indoxacarb in soil was shortened to 2.49-3.32 d (7.53 d for blank control area); furthermore when 450 kg·hm^-2 of degrading bacteria microspheres were applied 2 d after spraying indoxacarb with 5, 20 and 50 mg·L^-1, the values of T_1/2 was shortened from 6.03-7.45 d to 2.34-3.59 d.【Conclusion】The preparation of degrading bacteria P. stutzeri microspheres with sodium alginate as carrier has stable performance, can significantly degrade indoxacarb residues in farmland and shorten T_1/2time, provides technical and product support for bioremediation of soil pesticide residue pollution, and has the potential for further optimization and application.

Key words: indoxacarb residue, bioremediation, sodium alginate, Pseudomonas stutzeri, degrading bacteria preparation

汪育泰,许智帆,刘婕,钟国华. 茚虫威降解菌海藻酸钠微球制剂研制及应用效果[J]. 中国农业科学, 2022, 55(5): 920-931.

WANG YuTai,XU ZhiFan,LIU Jie,ZHONG GuoHua. Preparation and Application of Indoxacarb Degrading Bacteria Immobilized Sodium Alginate Microspheres[J]. Scientia Agricultura Sinica, 2022, 55(5): 920-931.

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海藻酸钠用量 Sodium alginate dosage (%)	造粒难易程度 Granulation difficulty	形变程度 Deformation degree (mm)	RSD (%)	传质性能 Transfer performance (RawIntDen)
1.0	未成球No balling	—	—	—
1.5	成球较易Balling easily	2.34a	2.1	55045
2.0	成球易Balling more easily	2.03a	1.2	50970
2.5	成球易Balling more easily	1.84ab	1.3	48845
3.0	成球易Balling more easily	1.51b	1.8	45796
3.5	黏度较大,成球较难High viscosity, balling relatively difficult	1.18c	3.7	37073
4.0	黏度大,成球难Higher viscosity, balling difficult	0.83c	4.2	30928

菌液或微球及用量 Bacteria suspension or microspheres and their dosages	动力学方程 Kinetic equation (y=)	速率常数 Rate constant	半衰期T_1/2 (d)	相关系数 Correlation coefficient (R², %)
CK	1.0534e^-0.092^x	0.092	7.53	0.971
微球Microsphere CK	0.9397e^-0.110^x	0.110	6.30	0.899
菌液Bacteria suspension	1.0095e^-0.165^x	0.165	4.20	0.967
微球Microsphere 90 kg·hm^-2	0.8243e^-0.209^x	0.209	3.32	0.945
微球Microsphere 450 kg·hm^-2	0.9273e^-0.245^x	0.245	2.83	0.992
微球Microsphere 900 kg·hm^-2	0.9988e^-0.278^x	0.278	2.49	0.962

处理 Treatment	动力学方程 Kinetic equation (y=)	速率常数 Rate constant	半衰期 T_1/2 (d)	相关系数 Correlation coefficient (R², %)
5 mg·L^-1 CK	0.9739e^-0.115^x	0.115	6.03	0.975
20 mg·L^-1CK	1.3636e^-0.103^x	0.103	6.73	0.987
50 mg·L^-1CK	2.064e^-0.093^x	0.093	7.45	0.962
5 mg·L^-1+450 kg·hm^-2	0.8701e^-0.296^x	0.296	2.34	0.862
20 mg·L^-1+450 kg·hm^-2	1.0536e^-0.242^x	0.242	2.86	0.969
50 mg·L^-1+450 kg·hm^-2	1.6731e^-0.193^x	0.193	3.59	0.957