嗜吡啶红球菌Rp3生长及降解粪臭素特性研究

doi:10.13304/j.nykjdb.2021.0139

摘要/Abstract

摘要：

为获得适宜嗜吡啶红球菌（Rhodococcus pyridinivorans）Rp3生长的碳氮源并明确不同因素对Rp3菌株降解粪臭素的影响，采用单因素和正交试验优化Rp3菌株的发酵培养基，并采用高效液相色谱法测定培养液中粪臭素含量，分析最适碳氮源、金属离子、温度和传代对Rp3菌株生长和降解粪臭素效率的影响。结果表明，Rp3菌株能够以粪臭素为唯一碳源生长。碳氮源对Rp3菌株生长的影响排序依次为酵母浸粉>牛肉膏>蔗糖，适宜碳氮源用量为蔗糖16 g·L^-1、牛肉膏14 g·L^-1、酵母浸粉6 g·L^-1。培养液中不添加Mg²⁺时，粪臭素不能被降解，Mg²⁺被Fe²⁺、Mn²⁺、Zn²⁺替代后，Rp3菌株对粪臭素的降解率显著下降。Rp3菌株对粪臭素的降解率随温度的升高而增加，28~40 ℃，培养24 h，降解效果可达到82.9%~95.5%。碳氮源和传代不影响Rp3菌株对粪臭素的降解。研究结果表明，嗜吡啶红球菌Rp3是易培养、不易退化的粪臭素高效降解菌株，具有一定的开发潜力。

关键词: 嗜吡啶红球菌, 粪臭素, 生长, 降解

Abstract:

To obtain an optimal medium for the growth of Rhodococcuspyridinivorans strain Rp3 and clarify the influence of different factors on its ability to degrade skatole， the fermentation medium was optimized by single factor and orthogonal experiments， and the skatole content in the medium was analyzed by high performance liquid chromatography to determine the effects of optimal carbon and nitrogen， metal ions， temperature， and continuous subculture on the growth of strain Rp3 and its ability to degrade skatole. The results showed that strain Rp3 could utilize skatole as the sole carbon for its growth. The growth of strain Rp3 promoted by organic carbon and nitrogen was as following： yeast extract powder>beef extract>sucrose. The optimal carbon and nitrogen were sucrose 16 g·L^-1， beef extract 14 g·L^-1 and yeast extract 6 g·L^-1. Skatole couldn’t be degraded in the medium without Mg²⁺， and its degradation significantly reduced when Mg²⁺ was replaced by Fe²⁺， Mn²⁺ and Zn²⁺. The skatole degrading ability of strain Rp3 increased with the raised temperature. The degrading efficacy could reach 82.9%~95.5% when cultured at 28~40 ℃ for 24 hours. The carbon and nitrogen source and continuous subculture didn’t affect the skatole degradation by strain Rp3. The above results indicated that Rhodococcuspyridinivorans Rp3 was a potential strain to be developed further due to its convenient and stable culturing characteristics.

Key words: Rhodococcus pyridinivorans, skatole, growth, degradation

中图分类号:

S182

吴玉洪, 郭荣君, 马桂珍, 李世东. 嗜吡啶红球菌Rp3生长及降解粪臭素特性研究[J]. 中国农业科技导报, 2022, 24(6): 82-89.

Yuhong WU, Rongjun GUO, Guizhen MA, Shidong LI. Characteristics of the Growth of Rhodococcuspyridinivorans Rp3 and Ability to Degrade Skatole[J]. Journal of Agricultural Science and Technology, 2022, 24(6): 82-89.

图/表 6

图1 Rp3菌株在粪臭素为唯一碳源培养液中的生长量注：不同小写字母表示不同处理间差异在P<0.05水平显著。

Fig.1 Growth of strain Rp3 cultured in broth with skatole as sole carbonNote：The different lowercase letters indicate significant difference between different treatments at P<0.05 level.

表1 培养基各组分配比优化的正交试验统计结果

Table 1 Statistical results of orthogonal test for the optimization of the medium components

实验编号 Experiment number	A：蔗糖 Sucrose	B：牛肉膏 Beef extract	C：酵母浸粉 Yeast extract powder	活菌数对数值 Logarithm of cultivable bacteria/ （lg CFU·mL^-1）
1	A3	B2	C1	9.55±0.02 d
2	A2	B2	C3	9.79±0.01 a
3	A1	B2	C2	9.69±0.01 b
4	A2	B1	C2	9.65±0.02 bc
5	A2	B3	C1	9.62±0.02 c
6	A3	B1	C3	9.81±0.02 a
7	A3	B3	C2	9.77±0.02 a
8	A1	B3	C3	9.81±0.02 a
9	A1	B1	C1	9.50±0.04 d
K₁	29.00	28.96	28.67	—
K₂	29.06	29.03	29.11	—
K₃	29.13	29.20	29.41	—
k₁	9.67	9.65	9.56	—
k₂	9.69	9.68	9.70	—
k₃	9.71	9.73	9.80	—
极差R_i	0.04	0.08	0.24	—
主次顺序 Primary and secondary order	C>B>A
最优组合 Excellent combination	A3B3C3

图2 适宜Rp3菌株生长及粪臭素降解的碳氮源注：L2?N和L2分别为加入和不加入碳氮源的无机盐培养液，L2?N碳氮源为蔗糖16.0 g·L-1、牛肉膏14.0 g·L-1、酵母浸粉6.0 g·L-1；L2?N和L2无机盐为KH2PO4 0.5 g·L-1、K2HPO4 1.5 g·L-1、MgSO4 0.5 g·L-1、（NH4）2SO4 1.5 g·L-1。不同小写字母表示不同处理间差异在P<0.05水平显著。Note： L2?N and L2 indicate salt medium with and without carbon and nitrogen sources， respectively. The carbon and nitrogen sources of L2?N are sucrose 16.0 g·L-1， beef extract 14.0 g·L-1， yeast extract powder 6.0 g·L-1； Inorganic salts of L2?N和L2 are KH2PO4 0.5 g·L-1， K2HPO4 1.5 g·L-1， MgSO4 0.5 g·L-1 and （NH4）2SO4 1.5 g·L-1. Different lowercase letters indicate significant difference among different treatments at P<0.05 level.

Fig.2 Carbon and nitrogen sources for strain Rp3 growth and skatole degradation

图3 适宜Rp3菌株生长及粪臭素降解的金属离子注：不同小写字母表示不同处理间差异在P<0.05水平显著。

Fig.3 Metal ions for strain Rp3 growth and skatole degradationNote： Different lowercase letters indicate significant difference among different treatments at P<0.05 level.

图4 适宜Rp3菌株生长及粪臭素降解的温度注：不同小写字母表示不同处理间差异在P<0.05水平显著。

Fig.4 Optical temperature for strain Rp3 growth and skatole degradationNote： Different lowercase letters indicate significant difference among different treatments at P<0.05 level.

表2 Rp3 初始菌株与传代菌株生长和降解粪臭素能力比较

Table 2 Comparison of the growth and skatole degrading ability between the initial and subculture strain Rp3

培养时间 Incubation time/h	处理 Treatment	Rp3菌株生长量 Growth of strain Rp3（OD₆₀₀）	粪臭素含量 Skatole content/（mg·L^-1）	降解率 Degradation rate/%
24	Rp3-0	0.37±0.015	14.8±1.0	82.9±0.3 a
	CK-0	—	86.3±7.1	—
	Rp3-5	0.37±0.014	13.4±0.5	84.0±0.6 a
	CK-5	—	83.7±0.9	—
	Rp3-10	0.36±0.014	13.7±0.4	83.2±1.3 a
	CK-10	—	82.1±7.1	—
48	Rp3-0	0.78±0.017	0	100.0 a
	CK-0	—	78.0±2.3	—
	Rp3-5	0.80±0.012	0	100.0 a
	CK-5	—	78.7±0.92	—
	Rp3-10	0.80±0.018	0	100.0 a
	CK-10	—	78.7±0.5	—

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