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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2023, Vol. 43 Issue (5): 45-54    DOI: 10.13523/j.cb.2211020
    
Effects of Magnetic Nano-metals on Growth Characteristics and Intracellular Ectoine Accumulation of Halomonas sp. XH26
WANG Ming-xiang1,GUO Min1,GAO Xiang1,LI Yong-zhen1,HAN Rui2,ZHU De-rui1,SHEN Guo-ping1,**()
1 Research Center of Basic Medical Science, Medical College, Qinghai University, Xining 810016, China
2 Qinghai Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Sciences, Qinghai University, Xining 810016, China
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Abstract  

Objective: Ectoine, a compatible solute, is widely distributed in halophilic bacteria to resist extreme environmental conditions. The aim of this study is to increase the accumulation of ectoine in wild-type Halomonas campaniensis sp. XH26. Methods: Magnetic nanoparticles (Fe3O4 NPs) were used, and single factor analysis, Plackett-Burman design and the response surface method were performed to evaluate the intracellular accumulation of ectoine and bacterial growth. The feasibility of nano-metal application in Halomonas fermentation was further discussed. Results: According to single factor analysis, Fe3O4 NPs can promote the strain growth and accumulation of ectoine. The optimal period for adding Fe3O4 NPs was the logarithmic growth stage of the strain. Plackett-burman and the response surface results revealed that ectoine accumulation of the strain reached 640.28 mg/L in shaker fermentation, under the optimized conditions(Fe3O4 NPs: 0.05 g/L; NaCl: 1.53 mol/L; MSG: 0.03 mol/L), which was 63.61% higher than that of the wild strain (391.35 mg/L). Transmission electron microscopy indicated that Fe3O4 NPs accumulated on the surface of bacterial cell membrane, which may play a catalytic role through surface adsorption. Conclusions: In summary, Fe3O4 NPs can effectively promote the accumulation of ectoine, and the combination of Plackett-Burman and the response surface method can better optimize the fermentation conditions of the strain. This study provides a new technical idea and reference for the subsequent application of magnetic nano-metal particles in the industrial production of ectoine by fermentation.

Key words： Halomonas sp.      Ectoine      Fe3O4 NPs      High-performance liquid chromatography      Response surface method     
Received: 10 November 2022      Published: 01 June 2023
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WANG Ming-xiang
GUO Min
GAO Xiang
LI Yong-zhen
HAN Rui
ZHU De-rui
SHEN Guo-ping
Cite this article:

WANG Ming-xiang, GUO Min, GAO Xiang, LI Yong-zhen, HAN Rui, ZHU De-rui, SHEN Guo-ping. Effects of Magnetic Nano-metals on Growth Characteristics and Intracellular Ectoine Accumulation of Halomonas sp. XH26. China Biotechnology, 2023, 43(5): 45-54.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2211020     OR     https://manu60.magtech.com.cn/biotech/Y2023/V43/I5/45

实验 因素 变量 水平
-1 0 +1
Plackett-Burman Fe3O4 NPs/(g/L) X1 0.01 - 0. 1
pH X2 7.2 - 8.4
L-谷氨酸钠(MSG,mol/L) X3 0.02 - 0.05
天冬氨酸(L-Asp,mmol/L) X4 0 - 2
NaCl/(mol/L) X5 0 - 2
温度 (T,℃) X6 25 - 40
虚拟因素 X7~X9 - - -
Box-Behnken Fe3O4 NPs/(g/L) A 0.02 0.045 0.07
NaCl/(mol/L) B 0.02 0.03 0.04
L-谷氨酸钠(MSG,mol/L) C 1 1.5 2
Table 1 Factor levels involved in the tests
Fig.1 Effects of Fe3O4 NPs on XH26 strain bacterial growth and intracellular ectoine accumulation under different conditions (a)Strain growth curve after adding Fe3O4 NPs at the initial growth stage (b) Strain growth curve with Fe3O4 NPs added in logarithmic phase (c)Ectoine accumulation of strains in different periods; a:Control group; b:Adding simultaneously group ; c:Logarithmic phase added group (d)Accumulation of ectoine in 0.1 g/L Fe3O4 NPs-treated strains at different pH; c:Control group (e) Ectoine accumulation of 0.1 g/L Fe3O4 NPs-treated strain under different NaCl concentrations (f) Ectoine accumulation of 0.1 g/L Fe3O4 NPs treated strains under different MSG concentrations; c:Control group * P<0.05, ** P<0.001
Fig.2 Growth curve of XH26 strain and Ectoine accumulation under different concentration of Fe3O4 NPs (a),(b) Growth curves of the strain under different concentrations of Fe3O4 NPs (c),(d) Intracellular Ectoine accumulation under different concentrations of Fe3O4 NPs in strains;c: Control group * P<0.05, ** P<0.001
方差来源 平方和 自由度 均方 F P 显著性
模型 153 600 6 25 595.07 8.33 0.017 2 *
X1 90 158.70 1 90 158.70 29.33 0.002 9 *
X2 8 838.72 1 8 838.72 2.88 0.150 7
X3 21 632.21 1 21 632.21 7.04 0.045 3 *
X4 967.11 1 967.11 0.3146 0.599 1
X5 24 946.95 1 24 946.95 8.12 0.035 9 *
X6 7 026.74 1 7 026.74 2.29 0.191 0
残差 15 369.51 5 3 073.90
总离差 168 900 11
Table 2 Results of Plackett-Buman square difference analysis
实验编号 因素 Ectoine/(mg/L)
Fe3O4 NPs /(g/L) 氯化钠/(mol/L) L-谷氨酸钠/(mol/L)
1 0.02 1 0.03 471.04
2 0.07 1 0.03 543.40
3 0.02 2 0.03 372.04
4 0.07 2 0.03 581.10
5 0.02 1.5 0.02 509.55
6 0.07 1.5 0.02 476.49
7 0.02 1.5 0.04 382.82
8 0.07 1.5 0.04 521.09
9 0.045 1 0.02 520.90
10 0.045 2 0.02 471.84
11 0.045 1 0.04 423.37
12 0.045 2 0.04 497.98
13 0.045 1.5 0.03 601.87
14 0.045 1.5 0.03 648.04
15 0.045 1.5 0.03 624.96
16 0.045 1.5 0.03 635.35
17 0.045 1.5 0.03 628.80
Table 3 Box-Behnken design and response
方差来源 平方和 自由度 均方 F P 显著性
模型 114 500 9 12 723.42 14.88 0.000 9 *
A 18 685.34 1 18 685.34 21.85 0.002 3 *
B 159.76 1 159.76 0.186 8 0.678 6
C 2 946.05 1 2 946.05 3.44 0.105 8
AB 4 671.72 1 4 671.72 5.46 0.052 1
AC 7 338.49 1 7 338.49 8.58 0.022 0 *
BC 3 823.57 1 3 823.57 4.47 0.072 3
A2 21 208.53 1 21 208.53 24.80 0.001 6 *
B2 17 755.01 1 17 755.01 20.76 0.002 6 *
C2 29 953.66 1 29 953.66 35.02 0.000 6 *
残差 5 986.62 7 855.23
失拟项 4 838.53 3 1 612.84 5.62 0.064 4
纯误项 1 148.09 4 287.02
总离项 120 500 16
Table 4 Variance analysis for the response surface model
Fig.3 Response surface of pairwise interactions between Fe3O4 NPs, NaCl, and MSG
Fig. 4 Transmission electron microscopic view of strain treated with Fe3O4 NPs. (a) Control group (b)-(d) were all strains supplemented with Fe3O4 NPs
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