响应面法改良牟氏角毛藻培养基

doi:10.13304/j.nykjdb.2022.0430

摘要/Abstract

摘要：

为优化牟氏角毛藻的培养基配方，提高牟氏角毛藻生长速度及细胞密度，采用响应面法筛选f/2培养基中的NaNO₃、NaH₂PO₄·H₂O、Na₂SiO₃和C₆H₈FeNO₇质量浓度并进行验证。结果表明，根据单因素试验筛选出牟氏角毛藻相对适宜的营养盐质量浓度范围为：NaNO₃ 10～30 mg·L^-1，NaH₂PO₄·H₂O 1.0～3.0 mg·L^-1，Na₂SiO₃ 20～80 mg·L^-1，C₆H₈FeNO₇ 0.1～0.5 mg·L^-1；通过响应面优化，4种营养盐对牟氏角毛藻生长的影响强弱依次为NaNO₃>Na₂SiO₃>NaH₂PO₄·H₂O>C₆H₈FeNO₇，最佳质量浓度分别为NaNO₃ 21.8 mg·L^-1、NaH₂PO₄·H₂O 2.93 mg·L^-1、Na₂SiO₃ 54.2 mg·L^-1、C₆H₈FeNO₇ 0.158 mg·L^-1。使用优化后的培养基培养该藻，密度提高27.95%，显著高于f/2培养基组的细胞密度（P<0.05），第4天后试验组牟氏角毛藻光合系统PSⅡ最大光能转换效率显著高于对照组（P<0.05）。综上所述，该方法筛选得出的营养盐质量浓度可以显著提高牟氏角毛藻的生长速度及细胞密度，结果为牟氏角毛藻的高密度培养提供数据参考。

关键词: 牟氏角毛藻, 响应面法, 营养盐, 细胞密度

Abstract:

In order to optimize the medium formula of Chaetoceros muelleri and improve its growth rate and cell density，response surface methodology was used to screen and verify the mass concentration of NaNO₃， NaH₂PO₄·H₂O， Na₂SiO₃ and C₆H₈FeNO₇ in f/2 medium. The results showed that， in accordance with single factor， the nutrient concentrations suitable for C. muelleri were screened， and the mass concentrations of NaNO₃， NaH₂PO₄·H₂O， Na₂SiO₃ and C₆H₈FeNO₇ were 10～30， 1.0～3.0， 20～80， 0.1～0.5 mg·L^-1， respectively. After being optimized by responding surface， the effects of four nutrient salts on the growth of C. muelleri were as follows： NaNO₃>Na₂SiO₃>NaH₂PO₄·H₂O>C₆H₈FeNO₇， and the optimal mass concentration of them were： NaNO₃ 21.8 mg·L^-1， NaH₂PO₄·H₂O 2.93 mg·L^-1， Na₂SiO₃ 54.2 mg·L^-1 and C₆H₈FeNO₇ 0.158 mg·L^-1. Cell density of C. muelleri in optimized culture medium group enhanced 27.95%， significantly higher than that of f/2 medium group （P<0.05）. At 4^th days， the maximum light conversion efficiency of photosynthetic system PSII in experimental group was significantly higher than that in control group （P<0.05）. To sum up， the selected nutrient mass concentration could significantly improve the growth rate and cell density of C. muelleri， which could provide data reference for high-density culture of C. muelleri.

Key words: Chaetoceros muelleri, response surface, nutrient, cell density

中图分类号:

S963.21⁺3

李琦, 张树林, 张达娟, 贾滢暄, 王泽斌. 响应面法改良牟氏角毛藻培养基[J]. 中国农业科技导报, 2023, 25(8): 225-233.

Qi LI, Shulin ZHANG, Dajuan ZHANG, Yingxuan JIA, Zebin WANG. Optimization of Culture Medium of Chaetoceros muelleri by Response Surface Methodology[J]. Journal of Agricultural Science and Technology, 2023, 25(8): 225-233.

图/表 11

参考文献 21

1	李志军,薛长湖,林洪.微藻中的活性物质及其保健食品的研究与开发[J].山东商业职业技术学院学报,2003,3(2):75-76.
2	梁英,黄徐林,田传远,等.海洋药源微藻研究进展[J].中国海洋大学学报,2016,46(11):32-43.
	LIANG Y, HUANG X L, TIAN C Y, et al.. Advances in studies on marine medicinal microalgae [J]. J. Ocean Univ. China,2016,46(11):32-43.
3	赵子续,曲木,张宝龙,等.饲料中添加多种微藻对锦鲤生长、体色及抗氧化指标的影响[J].养殖与饲料,2021,20(5):9-16.
4	MOHSENI A, FAN L H, RODDICK F A. Impact of microalgae species and solution salinity on algal treatment of wastewater reverse osmosis concentrate [J/OL]. Chemosphere,2021,285:131487[2022-04-22]. .
5	胡一丞,吴悦,陆丰逸,等.牟氏角毛藻与微拟球藻、青岛大扁藻混合培养技术的初步研究[J].当代水产,2016,41(6):77-78.
6	朱昔恩,黎大勇,熊建华,等.接种密度、氮、磷、碳、硅、维生素对牟氏角毛藻生长的影响[J].普洱学院学报,2018,34(6):9-14.
	ZHU X E, LI D Y, XIONG J H, et al.. Effects of salinity, temperature and light on the growth of Chaetoceros muelleri from the coast of Guangxi [J]. J. Puer Univ., 2018,34(6):9-14.
7	林权卓,吴悦,陆丰逸,等.牟氏角毛藻规模化培育过程中营养因子的筛选[J].科学养鱼,2016(10):50-51.
8	张贵杰,孟二力.不同氮源对牟氏角毛藻增殖的影响[J].河北渔业,2006(2):25-26.
9	于瑾,蒋霞敏,梁洪,等.氮、磷、铁对牟氏角毛藻生长速率的影响[J].水产科学,2006(3):121-124.
	YU J, JIANG X M, LIANG H, et al.. Influence of nitrogen, phosphorus and iron nutrition on the growth rate of Chaetoceros muelleri [J]. Fisheries Sci., 2006(3):121-124.
10	褚以文.微生物培养基优化方法及其OPTI优化软件[J].国外医药(抗生素分册),1999,20(2):58-60, 66.
11	郝学财,余晓斌,刘志钰,等.响应面方法在优化微生物培养基中的应用[J].食品研究与开发,2006,27(1):38-41.
	HAO X C, YU X B, LIU Z Y, et al.. The application of response surface methodology in optimization of microbial media [J]. Food Res. Dev., 2006,27(1):38-41.
12	王立原.响应面法在结构优化应用上的研究[D]. 上海:上海海洋大学,2018.
	WANG L Y. The response surface method in structural optimization study on the application [D]. Shanghai : Shanghai Ocean University,2018.
13	孙晓富,董超,李文静,等.响应面法优化纳豆芽孢杆菌发酵制备多肽螯合钙工艺[J].中国酿造,2021,40(6):119-123.
	SUN X F, DONG C, LI W J, et al.. Optimization of preparation process of peptides chelated calcium from Bacillus natto fermentation by response surface methodology [J]. China Brewing,2021,40(6):119-123.
14	宋立立,李志国.响应面法优化枯草芽孢杆菌产蛋白酶的发酵条件[J].饲料研究,2020,43(7):81-85.
	SONG L L, LI Z G. Optimization of fermentation conditions for protease production by Bacillus subtilis using response surface methodology [J]. Feed Res., 2020,43(7):81-85.
15	马若欣,王学魁,曹春晖.氮浓度和光照强度对小新月菱形藻生长和总脂含量的影响[J].天津科技大学学报,2009,24(3):31-34.
	MA R X, WANG X K, CAO C H. Effects of nitrate concentrations and light intensity on the growth and total lipid contents of Nitzschia Closterium f.minutissima [J]. J. Tianjin Univ. Sci. Technol., 2009,24(3):31-34.
16	张力.底栖硅藻的规模化培养及其在刺参育苗中的应用[D]. 扬州: 扬州大学,2013.
	ZHANG L. Large-scale culture of benthic diatoms and its application to the breeding of the sea cucumber Apostichopus japonicus [D]. Yangzhou :Yangzhou University,2013.
17	张国庆,邓湘云,李建保,等.氮、磷、铁、硅营养盐对牟氏角毛藻生长的影响[J].河北渔业,2013(3):8-11.
	ZHANG G Q, DENG X Y, LI J B, et al.. Effects of nitrogen, phosphorus, iron and silicon on the growth of Chaetoceros muelleri [J]. Hebei Fisheries,2013(3):8-11.
18	SHIFRIN N S, CHISHOLM S W. Phytoplankton lipids: inter specific differences and effects of nitrate, silicate and light-dark cycles [J]. J. Phycol.,1981,17(4):374-384.
19	如扎·哈布都拉,王萍,王阳,等.Box-Behnken响应面法优化阿里红总多糖水提醇沉工艺[J].化学与生物工程,2020,37(3):29-36.
	Ruzha·Habudula WANG P, WANG Y, et al.. Optimization of water extraction and alcohol precipitation process of fomes of ficinalis ames total polysaccharide with Box-Behnken response surface methodology [J]. Chem. Bioeng.,2020,37(3):29-36.
20	许天阳,董坤园,宋凤媛,等.Box-Behnken响应面法优化炒赤芍炮制工艺[J].中国药房,2019,30(20):2845-2850.
	XU T Y, DONG K Y, SONG F Y, et al.. Optimization of processing technology of fried radix paeoniae by Box-Behnken response surface method [J]. China Pharmacy, 2019,30(20):2845-2850.
21	李莉,张赛,何强,等.响应面法在试验设计与优化中的应用[J].试验室研究与探索,2015,34(8):41-45.
	LI L, ZHANG S, HE Q, et al.. Application of response surface methodology in experiment design and optimization [J]. Res. Exploration Laboratory,2015,34(8):41-45.

方差来源 Variance sources	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean squares	F值 F value	P值 P value	显著性 Significance
模型Model	44 720.85	14	3 194.35	29.89	<0.000 1	**
A-A	11 797.01	1	11 797.01	110.39	<0.000 1	**
B-B	575.47	1	575.47	5.38	0.035 9	*
C-C	878.77	1	878.77	8.22	0.012 4	*
D-D	322.61	1	322.61	3.02	0.104 2
AB	258.41	1	258.41	2.42	0.142 3
AC	704.90	1	704.90	6.60	0.022 3	*
AD	12.74	1	12.74	0.119 3	0.735 0
BC	730.62	1	730.62	6.84	0.020 4	*
BD	1 021.76	1	1 021.76	9.56	0.008 0	**
CD	1 962.93	1	1 962.93	18.37	0.000 8	**
A²	22 529.99	1	22 529.99	210.81	<0.000 1	**
B²	1 679.08	1	1 679.08	15.71	0.001 4	**
C²	6 714.11	1	6 714.11	62.82	<0.000 1	**
D²	4 466.73	1	4 466.73	41.80	<0.000 1	**
残差Residual	1 496.19	14	106.87
失拟项Lack of fit	1 280.10	10	128.01	2.37	0.210 5
纯误差Pure error	216.09	4	54.02
总和 Cor total	46 217.04	28

方差来源 Variance sources	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean squares	F值 F value	P值 P value	显著性 Significance
模型Model	44 720.85	14	3 194.35	29.89	<0.000 1	**
A-A	11 797.01	1	11 797.01	110.39	<0.000 1	**
B-B	575.47	1	575.47	5.38	0.035 9	*
C-C	878.77	1	878.77	8.22	0.012 4	*
D-D	322.61	1	322.61	3.02	0.104 2
AB	258.41	1	258.41	2.42	0.142 3
AC	704.90	1	704.90	6.60	0.022 3	*
AD	12.74	1	12.74	0.119 3	0.735 0
BC	730.62	1	730.62	6.84	0.020 4	*
BD	1 021.76	1	1 021.76	9.56	0.008 0	**
CD	1 962.93	1	1 962.93	18.37	0.000 8	**
A²	22 529.99	1	22 529.99	210.81	<0.000 1	**
B²	1 679.08	1	1 679.08	15.71	0.001 4	**
C²	6 714.11	1	6 714.11	62.82	<0.000 1	**
D²	4 466.73	1	4 466.73	41.80	<0.000 1	**
残差Residual	1 496.19	14	106.87
失拟项Lack of fit	1 280.10	10	128.01	2.37	0.210 5
纯误差Pure error	216.09	4	54.02
总和 Cor total	46 217.04	28

[1]	赵秀英, 黄晴雯, 曹浩杰, 王杰, 李瑞姣, 聂冬霞, 韩铮, 赵志辉. 响应面法优化禾谷镰刀菌产脱氧雪腐镰刀菌烯醇及其衍生物的液体培养条件[J]. 中国农业科技导报, 2023, 25(7): 222-233.
[2]	李晓东, 苏丽, 李晓捷, 李杰, 徐英江, 常丽荣, 于仁成, 杨德周, 逄少军. 2021—2022年山东荣成海带产区大规模溃烂灾害综合调查分析[J]. 中国农业科技导报, 2023, 25(1): 206-222.
[3]	王凯强, 杨雪, 李常风, 段晓, 彭晴, 乔宇, 石波. 响应面法优化低聚木糖诱导大豆抗毒素合成条件[J]. 中国农业科技导报, 2022, 24(10): 208-217.
[4]	戴阳军1,胡舰1,2,周莹2,左波1,石怡雪1. Plackett-Burman设计及响应面法优化无花果干复合酶解工艺[J]. 中国农业科技导报, 2018, 20(7): 146-153.
[5]	陈金娥,刘慧,赵志刚,张海容*. 响应面法优化超声波提取三七根多糖工艺研究[J]. 中国农业科技导报, 2018, 20(4): 138-146.
[6]	徐澜1,张鹏飞1,雷丽霞1,张海容2,王创云3. 响应面法优化微波辅助提取吴茱萸多糖工艺[J]. 中国农业科技导报, 2016, 18(4): 174-180.
[7]	熊夏宇,曾新安*,王满生,张智宏. 响应面法优化脉冲电场辅助提取油菜花粉中黄酮类物质工艺研究[J]. 中国农业科技导报, 2015, 17(5): 88-93.
[8]	辛崇博,孜力汗,刘晨光,白凤武*. 响应面法优化玉米芯同步糖化发酵预处理条件[J]. , 2013, 15(5): 173-180.
[9]	王金玲1,刘晓平2,高玮华1,赵凤艳3,吕长山3. 解磷巨大芽胞杆菌液体发酵的培养基优化[J]. , 2013, 15(2): 185-192.
[10]	张宁,刘爽,刘冬莲*. 超声波协同固体酸水解花生壳制备乙酰丙酸的研究[J]. , 2013, 15(1): 164-169.