1,2-DCA降解菌Ancylobacter sp. PY9的分离鉴定及固定化应用

doi:10.13560/j.cnki.biotech.bull.1985.2026-0485

摘要/Abstract

摘要：

目的筛选高效降解1,2-二氯乙烷（1,2-DCA）的菌株并通过固定化技术进一步提升其性能，为后续生物修复的工程应用提供基础。方法对1,2-DCA污染土壤富集驯化，通过高通量测序技术考察富集液中微生物群落结构特征。从富集液中筛选分离1,2-DCA高效降解菌株，采用16S rRNA基因序列分析和生理生化特征分析对菌株进行鉴定，采用L₉（3⁴）正交实验优化聚乙烯醇（PVA）-海藻酸钠（SA）-秸秆生物炭复合载体的制备条件，对菌株进行固定化应用研究。结果在Ancylobacter sp.相对丰度较高的富集液中分离并初步鉴定了一株1,2-DCA高效降解菌株Ancylobacter sp. PY9，其最适生长条件为pH 6-7、30 ℃、NaCl 5 g/L。固定化最优条件为8% PVA、2% SA、1%秸秆生物炭和1% CaCl₂（w/v），固定化PY9在高盐（30、40 g/L）、极端酸碱（pH=5、13）以及高温（50 ℃）/低温（10-20 ℃）条件下的降解效率均优于游离菌，固定化颗粒在循环利用5次后仍保持降解率在89%，稳定性良好。菌株PY9降解1,2-DCA符合一级反应动力学，游离态PY9可在8 h内完全降解300 mg/L的1,2-DCA，固定化PY9在500 mg/L高浓度1,2-DCA下降解率仍可达98%。结论在不同富集液群落中获得一株1,2-DCA高效降解菌株PY9，经优化的PVA-SA-秸秆生物炭复合固定化体系可显著提升菌株环境耐受性且对高浓度1,2-DCA具备高效降解能力，固定化颗粒重复利用性强。

关键词: 1,2-二氯乙烷, 生物修复, Ancylobacter, 固定化颗粒, 降解动力学

Abstract:

Objective Screening strains that efficiently degrade 1,2-dichloroethane (1,2-DCA ) and further improving its performance through immobilization technology, thereby providing a basis for subsequent engineering applications of bioremediation. Method The enrichment and domestication of 1,2-DCA contaminated soil were performed, and the microbial community characteristics in the enrichment culture were investigated by high-throughput sequencing technology. The efficient 1,2‑DCA‑degrading strain was screened and isolated from the enrichment culture. The strain was identified based on 16S rRNA gene sequence analysis and physiological and biochemical characteristics. The preparation conditions of polyvinyl alcohol (PVA)-sodium alginate (SA)-straw biochar composite carrier were optimized by L₉(3⁴) orthogonal experiment, and the application of the immobilized beads of the strain was investigated. Result An efficient degrading strain Ancylobacter sp. PY9 was isolated and preliminarily identified from the enrichment culture with high relative abundance. The optimum growth conditions were pH 6-7, 30 ℃ and NaCl 5 g/L. The optimal immobilization conditions were 8% PVA, 2% SA, 1% straw biochar and 1% CaCl₂ (w/v). The immobilized PY9 exhibited higher 1,2‑DCA degradation efficiency than free cells under high salt (30, 40 g/L), extreme pH (5, 13), high temperature (50 ℃), and low temperature (10-20 ℃) conditions. Moreover, the immobilized beads maintained a degradation rate of 89% after five cycles of reuse, indicating good stability. The degradation process of 1,2-DCA by strain PY9 followed first-order reaction kinetics. Free PY9 could completely degrade 300 mg/L 1,2-DCA within 8 h, while the degradation rate of immobilized PY9 could still reach 98% at 500 mg/L high concentration of 1,2-DCA. Conclusion An efficient 1,2-DCA-degrading strain PY9 was obtained from different enrichment cultures. The optimized PVA-SA-straw biochar composite immobilization system significantly enhanced the environmental tolerance of the strain and improved its degradation efficiency of the high concentration of 1,2-DCA. Moreover, the immobilized beads exhibited strong reusability.

Key words: 1,2-DCA, bioremediation, Ancylobacter, immobilized particles, degradation kinetics

吴志国, 张雪杨, 徐金娜, 于春倩, 蔡文华, 杨宗政. 1,2-DCA降解菌Ancylobacter sp. PY9的分离鉴定及固定化应用[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0485.

WU Zhi-guo, ZHANG Xue-yang, XU Jin-na, YU Chun-qian, CAI Wen-hua, YANG Zong-zheng. Isolation, Identification and Immobilization Application of 1,2-DCA Degrading Bacterium Ancylobacter sp. PY9[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0485.

图/表 12

图1 1，2-DCA降解菌群的富集及降解特性A：四组富集液对1,2-DCA的降解效果；B：富集液物种丰度图

Fig. 1 Enrichment and degrading characteristics of 1,2-DCA degrading microbial consortiumA: Degradation efficiency of 1,2-DCA by four groups of enrichment cultures. B: Species abundance of the enrichment cultures

表1 富集液的α多样性指数

Table 1 α-diversity indices of enrichment cultures

样本 Sample	Chao1	Shannon	Simpson	Goods_coverage
F1	136.714	4.013	0.855	1.000
F2	138.000	3.478	0.861	1.000
F3	207.500	3.541	0.872	0.999
F4	136.789	3.364	0.814	1.000

图2 菌株PY9系统发育树

Fig. 2 Phylogenetic tree of strain PY9

图3 菌株PY9的生长特性A：菌株PY9的生长曲线；B：pH对菌株PY9生长的影响；C：温度对菌株PY9生长的影响；D：NaCl浓度对菌株PY9生长的影响

Fig. 3 Growth characteristics of strain PY9A: Growth curve of strain PY9. B: Effects of pH on the growth of strain PY9. C: Effects of temperature on the growth of strain PY9. D: Effects of NaCl concentration on the growth of strain PY9

图4 菌株PY9关键降解基因扩增及1,2-DCA降解中间产物分析A：dhlA、dhlB基因电泳图；B：1,2-DCA生物降解培养液的气相色谱图；C：15.2 min色谱峰对应的质谱碎片图

Fig. 4 Amplification of key degrading genes and analysis of 1,2-DCA degrading intermediates in strain PY9A: Electrophoretogram of dhlA and dhlB genes. B: GC-MS results of 1,2-DCA biodegradation medium. C: EI fragment spectrum of the 15.2 min chromatographic peak

表2 固定化颗粒基本特性

Table 2 Characteristics of the immobilized beads

颗粒编号 Bead No.	球形度 Sphericity	机械强度 Mechanical strength	传质性能 Mass transfer performance （%）	含水率 Water content （%）	降解率 Degradation rate （%）	溶胀率 Swelling ratio （%）
1	+	++	49.80	85.00	66.59	2.33
2	++	++	64.28	86.00	65.61	3.39
3	+++	++	64.45	86.50	66.52	3.19
4	+	++	58.04	88.50	63.15	4.48
5	+++	+++	69.62	83.00	62.50	1.38
6	+	+	57.58	90.50	64.50	3.53
7	++	++	51.33	85.50	62.04	1.45
8	++	++	45.49	86.00	59.03	2.71
9	+	+++	65.13	86.00	53.46	1.96

图5 固定化颗粒SEM图A：未加生物炭颗粒表面；B：添加生物炭颗粒表面；C、D：添加生物炭颗粒内部

Fig. 5 SEM image of immobilized particlesA: Surface without biochar particles. B: Surface with biochar particles. C, D: Interior with biochar particles

图6 固定化与游离态PY9对1，2-DCA的降解特性A：不同处理体系的降解效果；B：pH对菌株PY9降解的影响；C：温度对菌株PY9降解的影响；D：NaCl浓度对菌株PY9降解的影响

Fig. 6 Degradation characteristics of 1,2-DCA by immobilized and free PY9A: Degradation efficiency of different treatment systems. B: Effect of pH on degradation by strain PY9. C: Effect of temperature on degradation by strain PY9. D: Effect of NaCl concentration on degradation by strain PY9

图7 固定化颗粒循环利用性

Fig. 7 Reusability of immobilized beads

图8 固定化与游离态菌株PY9的1,2-DCA降解动力学A：固定化PY9对不同初始浓度1,2-DCA的降解曲线；B：游离态PY9对不同初始浓度1,2-DCA的降解曲线；C：固定化PY9的一级降解动力学；D：游离态PY9的一级降解动力学；E：固定化PY9的动力学拟合曲线；F：游离态PY9 动力学拟合曲线

Fig. 8 Degradation kinetics of 1,2-DCA by immobilized and free strain PY9A: Degradation curves of 1,2-DCA with different initial concentrations by immobilized PY9. B: Degradation curves of 1,2-DCA with different initial concentrations by free PY9. C: First-order degradation kinetics of immobilized PY9. D: First-order degradation kinetics of free PY9. E: Kinetic fitting curves of immobilized PY9. F: Kinetic fitting curves of free PY9

表3 固定化PY9和游离态PY9的降解拟合参数

Table 3 Degradation fitting parameters of immobilized and free PY9

Item	初始浓度 Initial concentration （mg/L）	R²	K （h^-1）	t_1/2 （h）
固定化 PY9 Immobilized PY9	100	0.991 2	0.613 7	1.181 4
	200	0.995 7	0.527 3	1.333 1
	300	0.989 3	0.373 7	1.677 5
	400	0.954 2	0.178 2	2.418 0
	500	0.968 1	0.088 6	3.116 8
游离态PY9 Free PY9	100	0.942 4	1.005 5	0.687 7
	200	0.980 9	0.851 2	0.854 3
	300	0.979 1	0.591 7	1.217 9
	400	0.975 6	0.232 7	2.151 2
	500	0.918 7	0.141 8	2.646 5

表4 不同1,2-DCA降解菌株的降解性能对比

Table 4 Degradation comparison of different 1,2-DCA degrading strains

菌株 Strain	浓度 Concentration （mg/L）	降解条件（pH/温度/摇床转速/接种量） Degradation conditions （pH/ temperature （℃）/ rotate speed （r/min）/ inoculum）	时间 Time （h）	降解率 Degradation rate （%）	文献 Reference
Ancylobacter sp. PY9	300	7/ 30/ 180/ 初始OD₆₀₀ 0.058	8	100	本研究
Ancylobacter sp. J3	300	7/ 30/ 180/ 菌体干重34.29 mg/L	24	100	［15］
Ancylobacter sp. BL0	120	7/ 30/ 180/ 初始OD₆₀₀ 0.01	6	100	［9］
Ancylobacter sp. BL1	300	7/ 30/ 180/ 初始OD₆₀₀ 0.044	36	100	［14］
Ancylobacter sp. YN5	200	9/ 30/ 180/ 初始OD₆₀₀ 0.05	16	100	［16］
Starkeya sp. T-2	200	7/ 30/ 160/ 初始OD₆₀₀ 0.035	19	100	［28］
Klebsiella VA 8391	99	7.5/ 30/ 220/ 未描述	5	25	［29］
Pseudomonas sp. DCA1	247	7/ 25/ 剧烈振荡/ 初始OD₆₀₀ 0.13	5.5	100	［30］

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