除磷菌CP-7的筛选及其降解特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-1271

摘要/Abstract

摘要：

为实现生活污水中磷的生物降解和有效处理,从兰州市某生活污水处理厂分离出一株除磷菌开展强化生物除磷的研究。（1）结合生理生化特性鉴定、16S rDNA测序分析和系统发育树构建,结果显示该菌株与产酸克雷伯氏菌株（Klebsiella oxytoca strain）具有很高的同源性。（2）按5%（V/V）的接种量将该菌接种到复筛培养基中,在30℃,150 r/min 的恒温振荡培养箱中进行培养,每隔12 h测定TP浓度,对其降解特性进行初步研究发现该菌在72 h内对TP的降解率为58.52%。（3）考察碳源、氮源和C/N最优条件,得出此菌株最适碳源为葡萄糖、最适氮源为硫酸铵、最佳C/N为5∶1。（4）考察接种量、pH、温度、初始NaCl浓度和初始磷酸盐浓度等因素适应性,得出当接种量8%（V/V）、pH 9、温度30℃、初始NaCl浓度2.5 g/L、初始磷酸盐浓度70 mg/L时该菌株对TP的降解率最佳,分别为75.94%、90.19%、65.48%、57.02%、52.26%;同时,当接种量2%-14%（V/V）、pH 4.0-9.0、温度15-40℃、初始NaCl 浓度0-15 g/L、初始磷酸盐浓度45-90 mg/L时该菌株对TP均具有不同程度降解能力,且相关性分析显示,菌株TP降解率与接种量、pH、初始NaCl浓度和初始磷酸盐浓度相关性显著。（5）考察该菌株与其他菌株相互作用,将该菌株与一株铜绿假单胞菌进行复配,发现两者能协同增强除磷,两者比例4∶1时,TP降解率能达到99.43%。本实验为除磷菌株下一阶段的实际应用奠定了基础,并为生物强化除磷技术提供了更多的菌种选择。

关键词: 污水处理, 除磷菌, 筛选分离, 降解特性

Abstract:

In order to achieve the biodegradation and effective treatment of phosphorus in domestic sewage,a strain of dephosphorization bacteria was isolated from a domestic sewage treatment plant in Lanzhou to carry out enhanced biological phosphorus removal research. 1）Combined with physiological and biochemical characterization,16S rDNA sequencing analysis and phylogenetic tree construction,the results showed that the strain had a high homology with the Klebsiella oxytoca strain. 2）The bacterium was inoculated into the double screening medium at the inoculation rate of 5%（V/V）and cultured in a constant temperature oscillation incubator at 30℃ and 150 r/min. The concentration of TP was measured every 12 h. The preliminary study on the degradation characteristics of the bacterium showed that the degradation rate of TP was 58.52% within 72 h. 3）The optimal conditions of carbon source,nitrogen source and C/N were investigated. The optimal carbon source was glucose,nitrogen source was ammonium sulfate,and the optimal C/N was 5∶1. 4）By investigating the adaptability of inoculation amount,pH,temperature,initial NaCl concentration and initial phosphate concentration,it was concluded that the degradation rate of TP was the best when the inoculation amount was 8%（V/V）,pH was 9,temperature was 30℃,initial NaCl concentration was 2.5 g/L and initial phosphate concentration was 70 mg/L. The degradation rates of TP were 75.94%,90.19%,65.48%,57.02% and 52.26% respectively. At the same time,when the inoculation amount was 2%-14%（V/V）,pH was 4.0-9.0,temperature was 15-40℃,initial NaCl concentration was 0-15 g/L,initial phosphate concentration was 45-90 mg/L,the strain demonstrated varied degrees of degradation ability to TP. Correlation analysis showed that the TP degradation rate of the strain was significantly correlated with inoculation amount,pH,initial NaCl concentration and initial phosphate concentration. 5）The interaction between the strain and other strains was investigated,and the strain was mixed with a Pseudomonas aeruginosa strain. It was found that the two strains synergistically enhanced phosphorus removal. When the ratio of the two strains was 4∶1,the TP degradation rate reached 99.43%. This experiment lays a foundation for the practical application of dephosphorization bacteria in the next stage,and provides more bacterial species selection for biological enhanced phosphorus removal technology.

Key words: wastewater treatment, dephosphorization bacteria, screening and isolation, degradation characteristics

王亚军, 司运美. 除磷菌CP-7的筛选及其降解特性研究[J]. 生物技术通报, 2022, 38(7): 258-268.

WANG Ya-jun, SI Yun-mei. Screening and Degradation Characteristics of a CP-7 Strain of Dephosphorization Bacteria[J]. Biotechnology Bulletin, 2022, 38(7): 258-268.

图/表 15

图1 CP-7菌株菌落特征 A：革兰氏染色电镜形态图;B：固体培养基形态特征（划线接种）;C：固体培养基形态特征（两点接种）

Fig.1 Colony characteristics of CP-7 strain A：Gram staining electron microscope morphology. B：Morphological characteristics of solid medium（streaked inoculation）. C：Morphological characteristics of solid medium（two-point inoculation）

表1 CP-7菌株的形态特征

Table 1 Morphological characteristics of CP-7 strain

形态特征 Morphological characteristics	CP-7
形状Shape	卵圆形Oval
菌落大小Size of the colony/mm	2-8
颜色Color	乳白色Ivory
边缘Edge	波状Undulating
光泽Luster	有Exist
质地Texture	蜡状;黏稠;湿润;易挑起Waxy,sticky,moist,easy to lift
高度Height	隆起Upheaval
透明度Pellucidity	不透明Opacity

表2 CP-7菌株的生理生化特征

Table 2 Physiological and biochemical characteristics of CP-7 strain

特征Characteristics	CP-7
利用葡萄糖Use of glucose	+
利用柠檬酸盐Use of citrate	+
产硫化氢Hydrogen sulfide	-
明胶液化Gelatin liquefaction	+
甲基红Methyl red	-
精氨酸双水解酶Arginine double hydrolase	-
赖氨酸脱羧酶Lysine double hydrolase	+
鸟氨酸脱羧酶Ornithine decarboxylase	-
脲酶Urease	+
V-P	+

表3 通过16S rDNA基因序列分析鉴定CP-7菌株

Table 3 Identification of CP-7 strain by 16S rDNA gene sequence analysis

菌株 Strain	物种 Species	NCBI 登录号 NCBI accession No.	相似度 Percent similarity
	Klebsiella oxytoca strain JCM 1665	NR_112010.1	99.785%
CP-7	Klebsiella grimontiistrain SB73	NR_159317.1	99.427%
	Klebsiella oxytoca strain ATCC 13182	NR_118853.1	99.213%

图2 CP-7菌株基于16S rDNA的系统发育树

Fig. 2 Phylogenetic tree of CP-7 strain based on 16S rDNA

图3 CP-7菌株生长曲线

Fig.3 Growth curve of CP-7 strain

图4 碳源对CP-7菌株总磷降解效果的影响 1：乙酸钠;2：葡萄糖;3：蔗糖;4：草酸钠;5：淀粉

Fig. 4 Effect of carbon source on the TP degradation of CP-7 strain 1：Sodium acetate. 2：Glucose. 3：Sucrose. 4：Sodium oxalate. 5：Starch

图5 氮源对CP-7菌株总磷降解效果的影响 1：亚硝酸钠;2：硫酸铵;3：氯化铵;4：硝酸钠;5：硝酸钾

Fig. 5 Effect of nitrogen source on TP degradation of CP-7 strain 1：Sodium nitrite. 2：Ammonium sulfate. 3：Ammonium chloride. 4：Sodium nitrate. 5：Potassium nitrate

图6 C/N对CP-7菌株总磷降解效果的影响

Fig. 6 Effect of C/N on the TP degradation of CP-7 strain

图7 接种量对CP-7菌株总磷降解效果的影响

Fig. 7 Effect of inoculation amount on the TP degradation of CP-7 strain

图8 pH对CP-7菌株总磷降解效果的影响

Fig. 8 Effect of pH on the TP degradation of CP-7 strain

图9 温度对CP-7菌株总磷降解效果的影响

Fig. 9 Effect of temperature on the TP degradation of CP-7 strain

图10 初始NaCl浓度对CP-7菌株总磷降解效果的影响

Fig. 10 Effect of initial NaCl concentration on the TP degradation of CP-7 strain

图11 初始磷酸盐浓度对CP-7菌株总磷降解效果的影响

Fig. 11 Effect of initial phosphate concentration on the TP degradation of CP-7 strain

图12 YZ-1菌株对CP-7菌株总磷降解效果的影响

Fig. 12 Effect of YZ-1 strain on the TP degradation of CP-7 strain

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