一株高效溶磷产红青霉培养条件优化及其溶磷特性

doi:10.13560/j.cnki.biotech.bull.1985.2023-0268

摘要/Abstract

摘要：

为了减少农业生产中化学肥料的使用，本研究利用无机磷培养基对富磷的茶树（Camellia sinensis L.）根际微生物进行筛选，获得一株对磷酸三钙具有高效降解能力的真菌菌株JL-1，经鉴定为产红青霉（Penicillium rubens）。通过检测菌株JL-1在溶磷过程中发酵液的pH值、磷含量和有机酸含量变化发现，该菌株在无机磷培养基中，发酵液pH值与葡萄糖酸含量、pH值与磷含量以及葡萄糖酸与磷含量分别呈显著负相关、显著负相关和显著正相关，且在电子显微镜下观察到磷酸三钙颗粒表面存在被侵蚀痕迹，深入分析发现菌株JL-1通过分泌葡糖糖酸实现侵蚀磷酸三钙与溶磷的目的。通过单因素试验、Plackett-Burman设计试验、最陡爬坡试验和中心组合试验等一系列试验对培养条件进行优化发现，菌株JL-1溶解磷酸三钙的最佳碳源和氮源分别是葡萄糖和硫酸铵。葡萄糖含量、磷酸三钙含量和温度是影响菌株JL-1溶磷能力的主要因素，在葡萄糖29.8 g/L，磷酸三钙7.1 g/L，温度31.9℃的条件下，菌株JL-1在无机磷培养基中的溶磷能力达到最佳，溶磷量达到1 194.15 mg/L，是初始值的近3倍。在缺磷土壤中，该菌株能显著促进小麦（Triticum aestivum L.）生长，其根长、株高和总鲜重分别提高57.9%、36.4%和42.9%，进一步说明产红青霉JL-1具有良好的解磷、促生功能。菌株JL-1优良的溶磷特性与溶磷能力为其在生物肥料方向的开发和应用提供了参考。

关键词: 产红青霉, 茶树根际, 溶磷作用, 磷酸三钙, 葡萄糖酸, 响应面优化, 促生

Abstract:

In order to reduce the use of chemical fertilizers in agricultural production, a fungal strain JL-1 with high efficiency in degrading tricalcium phosphate was isolated from phosphorus-rich tea（Camellia sinensis L.）rhizosphere soil by using inorganic phosphorus screening medium, and identified as Penicillium rubens. The changes of pH value, phosphorus content and organic acid content of fermentation broth of strain JL-1 in the process of phosphorus solubilization showed that there was a significant negative correlation between pH value and gluconic acid content, a significant negative correlation between pH value and phosphorus content, and a significant positive correlation between gluconic acid and phosphorus content in the fermentation broth of strain JL-1. And the surface erosion marks of tricalcium phosphate particles were observed under the electron microscope. Further analysis uncovered that strain JL-1 eroded tricalcium phosphate and dissolved phosphorus by secreting gluconic acid. The culture conditions were optimized through a series of experiments including single factor experiment, Plackett-Burman design experiemnt, steepest climb experiment and central combination experiment. The results showed that glucose and ammonium sulfate were the optimal carbon and nitrogen sources for dissovling tricalcium phosphate by strain JL-1. Glucose content, tricalcium phosphate content and temperature were significant factors affecting the phosphorus solubility of strain JL-1. Under the conditions of 29.8 g/L glucose, 7.1 g/L tricalcium phosphate and 31.9℃, the phosphorus solubility of strain JL-1 in inorganic phosphorus medium, the solubilized phosphorus of the strain reached 1194.15 mg/L, which was nearly three times of the initial value. In phosphorus-deficient soil, this strain significantly promoted the growth of T. aestivum L., with its root length, plant height and total fresh weight increased by 57.9%, 36.4% and 42.9% respectively, indicating that the strain JL-1 had good functions of phosphate-dissolving and growth promotion. With the excellent phosphorus-dissolving characteristics and phosphate-dissolving ability, the strain JL-1 will provide a reference for the development and application in the field of biological fertilizer.

Key words: Penicillium rubens, rhizosphere of Camellia sinensis L., phosphorus-dissolving, tricalcium phosphate, gluconic acid, response surface optimization, growth promotion

赵光绪, 杨合同, 邵晓波, 崔志豪, 刘红光, 张杰. 一株高效溶磷产红青霉培养条件优化及其溶磷特性[J]. 生物技术通报, 2023, 39(9): 71-83.

ZHAO Guang-xu, YANG He-tong, SHAO Xiao-bo, CUI Zhi-hao, LIU Hong-guang, ZHANG Jie. Phosphate-solubilizing Properties and Optimization of Cultivation Conditions of Penicillium rubens: A Highly Efficient Phosphate Solubilizer[J]. Biotechnology Bulletin, 2023, 39(9): 71-83.

图/表 16

参考文献 42

[1]	陈雯彬, 欧阳泽怡, 欧阳硕龙, 等. 低磷胁迫对植物生长及生理特性影响的研究进展[J]. 湖南林业科技, 2020, 47(3): 132-136, 146
	Chen WB, Ouyang ZY, Ouyang SL, et al. Research progress on effects of low phosphorus stress on plant growth and physiological characteristics[J]. Hunan For Sci Technol, 2020, 47(3)132-136, 146
[2]	鲁如坤. 我国土壤氮、磷、钾的基本状况[J]. 土壤学报, 1989, 26(3): 280-286.
	Lu RK. General status of nutrients(N, N, K)in soils of China[J]. Acta Pedol Sin, 1989, 26(3): 280-286.
[3]	Sharma SB, Sayyed RZ, Trivedi MH, et al. Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils[J]. SpringerPlus, 2013, 2(1): 1-14. doi: 10.1186/2193-1801-2-1
[4]	李廷亮, 谢英荷, 洪坚平, 等. 施磷水平对晋南旱地冬小麦产量及磷素利用的影响[J]. 中国生态农业学报, 2013, 21(6): 658-665.
	Li TL, Xie YH, Hong JP, et al. Effects of phosphorus application rates on winter wheat yield and phosphorus use efficiency in drylands of South Shanxi Province[J]. Chin J Eco Agric, 2013, 21(6): 658-665. doi: 10.3724/SP.J.1011.2013.00658 URL
[5]	何迪, 耿丽平, 郭佳, 等. 草酸青霉菌HB1溶磷能力及作用机制[J]. 农业工程学报, 2020, 36(2): 255-265.
	He D, Geng LP, Guo J, et al. Ability and mechanism of Penicillium oxalicum HB1 solubilizing phosphates[J]. Trans Chin Soc Agric Eng, 2020, 36(2): 255-265.
[6]	池景良, 郝敏, 王志学, 等. 解磷微生物研究及应用进展[J]. 微生物学杂志, 2021, 41(1): 1-7.
	Chi JL, Hao M, Wang ZX, et al. Advances in research and application of phosphorus-solubilizing microorganism[J]. J Microbiol, 2021, 41(1): 1-7.
[7]	唐哲, 杨洪一, 李丽丽. 解磷真菌的研究进展与应用前景[J]. 安徽农业科学, 2014, 42(32): 11287-11288, 11296.
	Tang Z, Yang HY, Li LL. Advance and application prospect of phosphate-solubilizing fungi[J]. J Anhui Agric Sci, 2014, 42(32): 11287-11288, 11296.
[8]	孟建宇, 李蘅, 杨鸿儒, 等. 内蒙古荒漠灌木根际解磷菌多样性及其解磷和产铁载体能力[J]. 环境科学研究, 2021, 34(11): 2714-2721.
	Meng JY, Li H, Yang HR, et al. Diversity of phosphorus-solubilizing bacteria in rhizosphere of desert shrubs in Inner Mongolia and their phosphorus-solubilizing and siderophore-producing capabilities[J]. Res Environ Sci, 2021, 34(11): 2714-2721.
[9]	Illmer P, Schinner F. Solubilization of inorganic calcium phosphates-solubilization mechanisms[J]. Soil Biol Biochem, 1995, 27(3): 257-263. doi: 10.1016/0038-0717(94)00190-C URL
[10]	郭艺鹏, 李静, 杨越, 等. 枣根际解磷细菌解磷特性及影响因素分析[J]. 河南农业大学学报, 2018, 52(1)96-103
	Guo YP, Li J, Yang Y, et al. Characteristics and influencing factors of phosphate solubilizing bacteria in jujube rhizosphere[J]. J Henan Agric Univ, 2018, 52(1)96-103
[11]	Bakri MM. Tri-calcium and zinc phosphates solubilization by Aspergillus niger and its relation to organic acids production[J]. BioNanoSci, 2019, 9(2): 238-244. doi: 10.1007/s12668-019-0604-1
[12]	唐岷宸, 李文静, 宋天顺, 等. 一株高效解磷菌的筛选及其解磷效果验证[J]. 生物技术通报, 2020, 36(6): 102-109. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0969
	Tang MC, Li WJ, Song TS, et al. Screening of a highly efficient phosphate-solubilizing bacterium and validation of its phosphate-solubilizing effect[J]. Biotechnol Bull, 2020, 36(6): 102-109.
[13]	王莉晶, 高晓蓉, 吕军, 等. 解磷真菌C2'的分离鉴定及其在土壤中实际解磷效果的研究[J]. 土壤通报, 2009, 40(4):771-775.
	Wang LJ, Gao XR, Lyu J, et al. Phosphate-solubilizing mechanism of C2’ and its actual phosphate-solubilizing effect in soil[J]. Chin J Soil Sci, 2009, 40(4): 771-775.
[14]	乔欢, 吴小芹, 王早. 一株嗜松青霉JP-NJ4的解磷特性[J]. 微生物学通报, 2014, 41(9): 1741-1748.
	Qiao H, Wu XQ, Wang Z. Phosphate-solubilizing characteristic of a Penicillium pinophilum strain JP-NJ4[J]. Microbiol China, 2014, 41(9): 1741-1748.
[15]	王聪, 凌娟, 张燕英, 等. 海洋固氮菌和解磷菌的分离鉴定及发酵条件优化[J]. 微生物学报, 2018, 58(5): 817-829.
	Wang C, Ling J, Zhang YY, et al. Isolation, characterization and culture optimization of nitrogen-fixing and phosphate-solubilizing bacteria from rhizosphere sediments of Halophila ovalis[J]. Acta Microbiol Sin, 2018, 58(5): 817-829.
[16]	黄达明, 李倩, 管国强, 等. 一株解磷细菌的筛选、鉴定及其溶磷培养条件的优化[J]. 生物技术通报, 2015, 31(2): 173-178. doi: 10.13560/j.cnki.biotech.bull.1985.2015.02.026
	Huang DM, Li Q, Guan GQ, et al. Selection, identification and medium optimization of a phosphate-solubilizing bacterium[J]. Biotechnol Bull, 2015, 31(2): 173-178.
[17]	胡山, 牛世全, 龙洋, 等. 河西走廊盐碱土壤中一株高效溶磷菌的鉴定及条件优化[J]. 微生物学通报, 2017, 44(2): 358-365.
	Hu S, Niu SQ, Long Y, et al. Identification of an efficient phosphorus solubilizing bacteria from saline-alkali soil in the Hexi Corridor and optimization of its condition[J]. Microbiol China, 2017, 44(2): 358-365.
[18]	魏景超. 真菌鉴定手册[M]. 上海: 上海科学技术出版社, 1979.
	Wei JC. Handbook of fungal identification[M]. Shanghai: Shanghai Scientific & Technical Publishers, 1979.
[19]	徐丹丹, 赵邯郸, 王秀然, 等. 两种测定植酸酶酶活的方法比较及发酵条件研究[J]. 湖北农业科学, 2019, 58(12): 134-137.
	Xu DD, Zhao HD, Wang XR, et al. Comparison of two methods for the determination of phytase activity and fermentation conditions[J]. Hubei Agric Sci, 2019, 58(12): 134-137.
[20]	刘胜亮, 朱舒亮, 祁先慧, 等. 四株解磷菌分泌有机酸与溶解磷酸三钙能力的研究[J]. 新疆农业科学, 2017, 54(6): 1114-1121.
	Liu SL, Zhu SL, Qi XH, et al. Study on organic acid secreted from 4 strains phosphorus-solubilizing bacteria and calcium phosphate dissolving ability[J]. Xinjiang Agric Sci, 2017, 54(6): 1114-1121.
[21]	Safari Motlagh MR, Farokhzad M, Kaviani B, et al. Endophytic fungi as potential biocontrol agents against Sclerotium rolfsii sacc. The causal agent of peanut white stem rot disease[J]. Cells, 2022, 11(17): 2643. doi: 10.3390/cells11172643 URL
[22]	García-Calvo L, Rodríguez-Castro R, Ullán RV, et al. Penicillium chrysogenum as a fungal factory for feruloyl esterases[J]. Appl Microbiol Biotechnol, 2023, 107(2/3): 691-717. doi: 10.1007/s00253-022-12335-w
[23]	Larena I, Sabuquillo P, Melgarejo P, et al. Biocontrol of Fusarium and Verticillium wilt of tomato by Penicillium oxalicum under greenhouse and field conditions[J]. J Phytopathol, 2003, 151(9): 507-512. doi: 10.1046/j.1439-0434.2003.00762.x URL
[24]	Martinez-Beringola ML, Salto T, Vázquez G, et al. Penicillium oxalicum reduces the number of cysts and juveniles of potato cyst nematodes[J]. J Appl Microbiol, 2013, 115(1): 199-206. doi: 10.1111/jam.12213 pmid: 23560806
[25]	罗加儿. 利用微生物对木质素、染料及天然产物进行转化的研究[D]. 长春: 吉林大学, 2022.
	Luo JE. Study on the transformation of lignin, dyes and natural products by microorganisms[D]. Changchun: Jilin University, 2022.
[26]	Yasser MM, Mousa ASM, Marzouk Marym A, et al. Molecular identification, extracellular enzyme production and antimicrobial activity of endophytic fungi isolated from Solanum tuberosum L. in Egypt[J]. Biosci, Biotech Res Asia, 2019, 16(1): 135-142. doi: 10.13005/bbra/ URL
[27]	孔德兴, 徐永清, 杨燕, 等. 向日葵列当毛蕊花糖苷的提取工艺优化及其抗氧化活性[J]. 西北农林科技大学学报: 自然科学版, 2022, 50(8): 103-113.
	Kong DX, Xu YQ, Yang Y, et al. Extraction optimization and antioxidant activities of acteoside from Orobanche cumana[J]. J Northwest A&F Univ Nat Sci Ed, 2022, 50(8): 103-113.
[28]	吴海燕, 金荣德, 范作伟, 等. 解磷巨大芽孢杆菌(Bacillus megaterium)的溶磷机理探讨[J]. 吉林农业大学学报, 2014, 36(2): 171-175.
	Wu HY, Jin RD, Fan ZW, et al. Mechanism of solubilizing phosphate by Bacillus megaterium[J]. J Jilin Agric Univ, 2014, 36(2): 171-175.
[29]	Buch A, Archana G, Naresh Kumar G. Metabolic channeling of glucose towards gluconate in phosphate-solubilizing Pseudomonas aeruginosa P4 under phosphorus deficiency[J]. Res Microbiol, 2008, 159(9/10): 635-642. doi: 10.1016/j.resmic.2008.09.012 URL
[30]	韩杨. 耐盐解磷菌的筛选、鉴定及其在盐胁迫下对甘草的促生作用[D]. 天津: 天津科技大学, 2013.
	Han Y. Screening and identification of salt-tolerant phosphate-solubilizing bacteria and its growth-promoting effect on Glycyrrhiza uralensis fisch under salt stress[D]. Tianjin:Tianjin University of Science & Technology, 2013.
[31]	刘春菊, 王彩霞, 张玉芹, 等. 一株高效解磷青霉菌株的筛选与鉴定[J]. 山东农业科学, 2017, 49(12): 50-53.
	Liu CJ, Wang CX, Zhang YQ, et al. Screening and identification of one efficient phosphate-solubilizing strain from Penicillium[J]. Shandong Agric Sci, 2017, 49(12): 50-53.
[32]	张建峰, 苗天瑶, 张嘉旭, 等. 1株溶磷真菌的分离鉴定及溶磷特性分析[J]. 西北农林科技大学学报: 自然科学版, 2017, 45(12): 121-128.
	Zhang JF, Miao TY, Zhang JX, et al. Screening, identification and phosphate solubilizing properties of a fungi strain isolated from saline-alkali land[J]. J Northwest A&F Univ Nat Sci Ed, 2017, 45(12): 121-128.
[33]	谭海霞, 李丽艳, 王连龙, 等. 耐盐溶磷菌YJC19的鉴定及不同培养条件对其溶磷效果的影响[J]. 江苏农业科学, 2022, 50(9): 235-239.
	Tan HX, Li LY, Wang LL, et al. Identification of salt-tolerant phosphate-solubilizing bacteria YJC19 and effects of different culture conditions on its phosphate-solubilizing effect[J]. Jiangsu Agric Sci, 2022, 50(9): 235-239.
[34]	张洁婧, 王虹, 宋科稷, 等. 一株高效溶磷真菌耐盐能力及其对羊草生长的影响[J]. 吉林农业大学学报, 2021, 43(4): 433-438.
	Zhang JJ, Wang H, Song KJ, et al. Salt tolerance of an efficient phosphorus-soluble fungus and its effect on growth of Chinese wildrye[J]. J Jilin Agric Univ, 2021, 43(4): 433-438.
[35]	李小军, 吉俐, 张景宁, 等. 解磷真菌JL-7的筛选及其作用于低品位磷矿的肥效研究[J]. 福建农业学报, 2022, 37(9): 1237-1244.
	Li XJ, Ji L, Zhang JN, et al. Efficiency improvement of low-grade phosphorus fertilizer by phosphate-solubilizing fungus JL-7[J]. Fujian J Agric Sci, 2022, 37(9): 1237-1244.
[36]	林钰栅, 刘景春, 卢豪良, 等. 红树根际解磷真菌筛选及其对桐花树生长的影响[J]. 农业环境科学学报, 2022, 41(5): 950-958.
	Lin YS, Liu JC, Lu HL, et al. Screening phosphate-solubilizing fungi from the mangrove rhizosphere and their effect on Aegiceras corniculatum seedling growth[J]. J Agro Environ Sci, 2022, 41(5): 950-958.
[37]	孙冉, 张素, 吴臣林, 等. 黑曲霉解磷能力的影响因素及培养条件优化[J]. 应用生态学报, 2020, 31(6): 1963-1970. doi: 10.13287/j.1001-9332.202006.033
	Sun R, Zhang S, Wu CL, et al. Influencing factors of phosphate solubilizing capacity of Aspergillus niger and optimization of its culture condition[J]. Chin J Appl Ecol, 2020, 31(6): 1963-1970.
[38]	尚晓静, 侯瑞, 徐芳玲, 等. 2株蓝莓溶磷内生真菌的筛选、鉴定及溶磷效果评价[J]. 江苏农业科学, 2022, 50(20): 246-252.
	Shang XJ, Hou R, Xu FL, et al. Screening, identification and evaluation of phosphorus-solubilizing effect of two endophytic fungi from blueberry[J]. Jiangsu Agric Sci, 2022, 50(20): 246-252.
[39]	于淼, 吴红艳, 冯健, 等. 解磷菌623-3的鉴定、培养条件优化及应用[J]. 四川农业大学学报, 2020, 38(5): 572-579.
	Yu M, Wu HY, Feng J, et al. Identification, optimization of culture conditions and application of dephosphorizing bacteria 623-3[J]. J Sichuan Agric Univ, 2020, 38(5): 572-579.
[40]	张云霞, 雷鹏, 许宗奇, 等. 一株高效解磷菌Bacillus subtilis JT-1的筛选及其对土壤微生态和小麦生长的影响[J]. 江苏农业学报, 2016, 32(5): 1073-1080.
	Zhang YX, Lei P, Xu ZQ, et al. Screening of a high-efficiency phosphate solubilizing bacterium Bacillus subtilis JT-1 and its effects on soil microecology and wheat growth[J]. Jiangsu J Agric Sci, 2016, 32(5): 1073-1080.
[41]	龙赛波, 张晓东, 王志远, 等. 三七重茬土壤中高效解磷菌的筛选和鉴定[J]. 安徽农业科学, 2016, 44(12): 82-83.
	Long SB, Zhang XD, Wang ZY, et al. Isolation and identification of phosphate-solubilizing bacteria from continuous cropping soil of Panax notoginseng[J]. J Anhui Agric Sci, 2016, 44(12): 82-83.
[42]	李静, 艾加敏, 余天飞, 等. 一株溶磷真菌的鉴定及其促生特性研究[J]. 福建农业学报, 2021, 36(10): 1224-1230.
	Li J, Ai JM, Yu TF, et al. Identification and growth-promoting effect of a phosphate-solubilizing fungus on wheat seedlings[J]. Fujian J Agric Sci, 2021, 36(10): 1224-1230.

序号 No.	葡萄糖 Glucose/（g·L^-1）	硫酸铵 Ammonium sulfate/（g·L^-1）	无机盐 Inorganic salt/（g·L^-1）	磷酸三钙 Tricalcium phosphate/（g·L^-1）	温度 Temperature/℃	转速 Rotate speed/（r·min^-1）	初始 pH Initial pH	接种量 Inoculated amount/%
1	15	0.75	1.44	5.0	26	160	8	1
2	15	0.75	0.96	5.0	26	200	6	3
3	10	0.75	1.44	7.5	26	160	6	3
4	10	0.75	1.44	5.0	30	200	8	1
5	10	0.50	1.44	5.0	30	200	6	3
6	10	0.50	0.96	5.0	26	160	6	1
7	10	0.50	0.96	7.5	26	200	8	1
8	15	0.50	1.44	7.5	26	200	8	3
9	15	0.75	0.96	7.5	30	200	6	1
10	10	0.75	0.96	7.5	30	160	8	3
11	15	0.50	1.44	7.5	30	160	6	1
12	15	0.50	0.96	5.0	30	160	8	3

序号 No.	葡萄糖 Glucose/（g·L^-1）	硫酸铵 Ammonium sulfate/（g·L^-1）	无机盐 Inorganic salt/（g·L^-1）	磷酸三钙 Tricalcium phosphate/（g·L^-1）	温度 Temperature/℃	转速 Rotate speed/（r·min^-1）	初始 pH Initial pH	接种量 Inoculated amount/%
1	15	0.75	1.44	5.0	26	160	8	1
2	15	0.75	0.96	5.0	26	200	6	3
3	10	0.75	1.44	7.5	26	160	6	3
4	10	0.75	1.44	5.0	30	200	8	1
5	10	0.50	1.44	5.0	30	200	6	3
6	10	0.50	0.96	5.0	26	160	6	1
7	10	0.50	0.96	7.5	26	200	8	1
8	15	0.50	1.44	7.5	26	200	8	3
9	15	0.75	0.96	7.5	30	200	6	1
10	10	0.75	0.96	7.5	30	160	8	3
11	15	0.50	1.44	7.5	30	160	6	1
12	15	0.50	0.96	5.0	30	160	8	3

方差来源Source	平方和Sum of squares	自由度df	均方Mean square	F 值F value	P值P value
Model	1.603E+05	8	20 033.75	46.95	0.004 6
A-Glucose	1.369E+05	1	1.369E+05	320.83	0.000 4
B-Ammonium sulfate	3.17	1	3.17	0.007 4	0.936 7
C-Inorganic salt	2.23	1	2.23	0.005 2	0.946 9
D-Tricalcium phosphate	12 973.79	1	12 973.79	30.41	0.011 7
E-Temperature	7 137.98	1	7 137.98	16.73	0.026 4
F-Rotational speed	1 879.75	1	1 879.75	4.41	0.126 7
G-Initial pH	1 119.21	1	1 119.21	2.62	0.203 8
H-Inoculated quantity	264.05	1	264.05	0.618 9	0.488 9
Residual	1 280.01	3	426.67
Cor Total	1.616E+05	11

方差来源Source	平方和Sum of squares	自由度df	均方Mean square	F 值F value	P值P value
Model	1.603E+05	8	20 033.75	46.95	0.004 6
A-Glucose	1.369E+05	1	1.369E+05	320.83	0.000 4
B-Ammonium sulfate	3.17	1	3.17	0.007 4	0.936 7
C-Inorganic salt	2.23	1	2.23	0.005 2	0.946 9
D-Tricalcium phosphate	12 973.79	1	12 973.79	30.41	0.011 7
E-Temperature	7 137.98	1	7 137.98	16.73	0.026 4
F-Rotational speed	1 879.75	1	1 879.75	4.41	0.126 7
G-Initial pH	1 119.21	1	1 119.21	2.62	0.203 8
H-Inoculated quantity	264.05	1	264.05	0.618 9	0.488 9
Residual	1 280.01	3	426.67
Cor Total	1.616E+05	11

显著影响因素 Significant influencing factor	水平 Level	溶磷量Amount of dissolved phosphorus/（mg·L^-1）
A-Glucose/（g·L^-1）	25	809.63
	30	822.28
	35	805.27
B-Tricalcium phosphate/（g·L^-1）	6	419.92
	7	437.79
	8	426.46
C-Temperature/℃	30	456.92
	32	521.49
	34	486.18