牛骨炭载体菌剂（HD）协同蚯蚓生物降解土壤中的2,4-DCP及对微生物群落的影响

doi:10.16085/j.issn.1000-6613.2023-2148

摘要/Abstract

摘要：

以典型氯酚类化合物2,4-二氯苯酚（2,4-DCP）为目标污染物，牛骨炭作为载体负载HD菌株制备固体工程菌剂，对污染土壤中的2,4-DCP进行降解实验，利用高通量测序揭示2,4-DCP胁迫下的土壤与蚯蚓肠道微生物群落演替、变化。结果表明：牛骨炭粒径160~250μm、热解4h时，牛骨炭负载的活菌数最多，达2.42×10⁹cfu/g；当土壤中2,4-DCP浓度为15mg/kg时，协同修复组效果显著高于空白对照组和蚯蚓修复组；扫描电镜图表明，HD菌株可稳定负载于牛骨炭的表面空隙中，且固体工程菌剂表面呈多孔蜂窝状结构；微生物测序结果表明蚯蚓、固体工程菌剂的投入均可增加土壤微生物总数和多样性。本研究旨在为有机污染土壤生物修复技术发展提供理论与应用参考。

关键词: 2,4-二氯苯酚, 蚯蚓, 动力学, 固定化, 生物技术

Abstract:

2,4-Dichlorophenol (2,4-DCP), a typical chlorophenol compound, was used as the target pollutant. Bovine bone char was used as a carrier to load the HD bacterial strain, thereby producing solid engineered bacterial agents to degrade 2,4-DCP in contaminated soils. The succession and changes of microbial communities in soil and earthworm guts under the stress of 2,4-DCP were revealed using high-throughput sequencing. The results showed that when the bovine bone char, with a particle size range of 160—250μm, was pyrolyzed for 4h, the viable bacteria count was the highest, reaching 2.42×10⁹cfu/g. When the concentration of 2,4-DCP in the soil was 15mg/kg, the effect of solid engineered bacterial agent combined with earthworm remediation group was significantly higher than that of the blank control group and the earthworm-only group. Scanning electron microscopy images showed that strain HD could be stably loaded into the surface voids of bovine bone char, and the surface of the solid engineered bacterial agent showed a porous honeycomb structure. The results of microbial sequencing showed that the input of earthworms and solid engineered bacterial agent could increase the total number and diversity of soil microorganisms. This study aims to provide theoretical and practical references for the development of bioremediation techniques for organic polluted soils.

Key words: 2,4-dichlorophenol, earthworm, kinetics, immobilization, biotechnology

中图分类号:

吴佳楠, 张华, 李哲, 徐珊, 尹勇, 张文艺. 牛骨炭载体菌剂（HD）协同蚯蚓生物降解土壤中的2,4-DCP及对微生物群落的影响[J]. 化工进展, 2024, 43(12): 6896-6904.

WU Jianan, ZHANG Hua, LI Zhe, XU Shan, YIN Yong, ZHANG Wenyi. Synergistic biodegradation of 2,4-DCP in soil by bovine bone char-based bacterial agent (HD) with earthworms and its impact on microbial communities[J]. Chemical Industry and Engineering Progress, 2024, 43(12): 6896-6904.

图/表 7

表1 牛骨炭粒径和热解时间优化

序号	粒径/μm	热解时间/h	热解温度/℃
1	250~550	4	350
2	160~250	4
3	120~160	4
4	106~120	4
5	<106	4
6	160~250	2
7	160~250	3
8	160~250	4
9	160~250	5
10	160~250	6

图1 不同牛骨炭粒径和热解时间对菌剂中活菌数的影响

图2 350℃热解下牛骨炭的原始形貌图(a)、(b)和负载菌株HD形貌图(c)、(d)

图3 土壤中2,4-DCP的降解动力学

图4 各样本多样性指数

图5 各样本间微生物群落的OTUs差异性分析和PcoA分析

图6 各样本的门和属水平Circos图

参考文献 34

1	MA Yanbo, HAN Jian, GUO Yongyong, et al. Disruption of endocrine function in in vitro H295R cell-based and in in vivo assay in zebrafish by 2,4-dichlorophenol[J]. Aquatic Toxicology, 2012, 106: 173-181.
2	何骞. 改性壳聚糖负载铁钯双金属催化剂的制备及对二氯苯酚降解性能研究[D]. 广州: 华南理工大学, 2021.
	HE Qian. Preparation of chitosan derivative supported Fe/Pd bimetallic catalyst and its degradation performance on 2,4-dichlorophenol[D]. Guangzhou: South China University of Technology, 2021.
3	HU Yan, LI Dong, MA Xuan, et al. Effects of 2,4-dichlorophenol exposure on zebrafish: Implications for the sex hormone synthesis[J]. Aquatic Toxicology, 2021, 236: 105868.
4	Aurang ZEB, LI Song, WU Jiani, et al. Insights into the mechanisms underlying the remediation potential of earthworms in contaminated soil: A critical review of research progress and prospects[J]. Science of the Total Environment, 2020, 740: 140145.
5	顾浩天, 袁永达, 张天澍, 等. 蚯蚓修复污染土壤的作用与机理研究进展[J]. 江苏农业科学, 2021, 49(20): 30-39.
	GU Haotian, YUAN Yongda, ZHANG Tianshu, et al. Research progress on effect and mechanism of earthworms in remediation of contaminated soil[J]. Jiangsu Agricultural Sciences, 2021, 49(20): 30-39.
6	WANG Yikun, WANG Zijing, HUANG Jung-Chen, et al. Bioremediation of selenium-contaminated soil using earthworm Eisenia fetida: Effects of gut bacteria in feces on the soil microbiome[J]. Chemosphere, 2022, 300: 134544.
7	曹佳. 蚯蚓和菌根真菌促进土霉素降解及其修复污染土壤的协同机制[D]. 北京: 中国农业大学, 2018.
	CAO Jia. Cooperation between earthworm and AM fungi on the degradation and remediation of oxytetracycline in soil[D]. Beijing: China Agricultural University, 2018.
8	LU Yanfei, LU Mang. Remediation of PAH-contaminated soil by the combination of tall fescue, arbuscular mycorrhizal fungus and epigeic earthworms[J]. Journal of Hazardous Materials, 2015, 285: 535-541.
9	周东兴, 王兵, 邓杰, 等. 蚯蚓与细菌协同作用对土壤中聚丙烯酰胺的降解效果[J]. 江苏农业科学, 2017, 45(6): 259-262.
	ZHOU Dongxing, WANG Bing, DENG Jie, et al. The synergistic effect of earthworms and bacteria on the degradation of polyacrylamide in soil[J]. Jiangsu Agricultural Sciences,2017,45(6): 259-262.
10	王新, 柳文睿, 侯佳文, 等. 菌剂制备及其修复农药污染土壤的研究进展[J]. 化工环保, 2022, 42(1): 10-15.
	WANG Xin, LIU Wenrui, HOU Jiawen, et al. Research progresses on preparation of microbial agent and remediation of pesticides-contaminated soil[J]. Environmental Protection of Chemical Industry, 2022, 42(1): 10-15.
11	朱晓丽, 张婵娟, 张星, 等. 生物炭固定化硫酸盐还原菌对镉污染土壤的钝化修复[J]. 环境科学学报, 2023, 43(5): 421-429.
	ZHU Xiaoli, ZHANG Chanjuan, ZHANG Xing, et al. Remediation of Cd²⁺ contaminated soil by biochar immobilized sulfate reducing bacteria[J]. Acta Scientiae Circumstantiae, 2023, 43(5): 421-429.
12	Organization for Economic Co-operation and Development. Guideline for Testing of Chemicals No 222, Earthworm Reproduction Test (Eisenia fetida/andrei) [S]. Paris: Organization for Economic Co-operation and Development, 2016.
13	生态环境部. 土壤和沉积物挥发酚的测定 4-氨基安替比林分光光度法: [S]. 北京: 中国环境出版社, 2018.
	Ministry of Ecological Environment. Soil and sediment Determination of volatile phenolic compounds 4-AAP spectrophotometric method: [S]. Beijing: China Environmental Science Press, 2018.
14	XU Xiaoyi, Chenpei LYU, YOU Xiaolu, et al. Nitrogen removal and microbial diversity of activated sludge entrapped in modified poly(vinyl alcohol)-sodium alginate gel[J]. International Biodeterioration & Biodegradation, 2017, 125: 243-250.
15	ZHANG Yansong, YU Zehai, HU Yunshuang, et al. Immobilization of nitrifying bacteria in magnetic PVA-SA-diatomite carrier for efficient removal of NH₄ ⁺-N from effluents[J]. Environmental Technology & Innovation, 2021, 22: 101407.
16	郭印丽, 李梦耀, 张晓松, 等. 2, 4-二氯苯酚在黄土性土壤中的吸附与解吸[J]. 应用化工, 2014, 43(9): 1640-1643.
	GUO Yinli, LI Mengyao, ZHANG Xiaosong, et al. Study on the adsorption and desorption of 2,4-diehlorophenol in the loess soil[J]. Applied Chemical Industry, 2014, 43(9): 1640-1643.
17	曾华冲, 杨利芝, 徐宏勇, 等. Fenton试剂氧化法修复2,4-二氯酚污染土壤的研究[J]. 生态环境, 2008, 17(1): 221-226.
	ZENG Huachong, YANG Lizhi, XU Hongyong, et al. Remediation of 2,4-dichlorophenol-contaminated soils by Fenton’s reagent oxidation[J]. Ecology and Environment Sciences, 2008, 17(1): 221-226.
18	吴炜龙, 陈艺杰, 卫婷, 等. 蚯蚓驱动的滨海盐碱农田土壤中多环芳烃生物降解的机制研究[J]. 生态环境学报, 2023, 32(11): 1996-2006.
	WU Weilong, CHEN Yijie, WEI Ting, et al. Mechanisms of earthworm-driven biodegradation of polycyclic aromatic hydrocarbons in coastal saline agricultural soils[J]. Ecology and Environmental Sciences, 2023, 32(11): 1996-2006.
19	黄枫城, 吴伟健, 张伟健, 等. 不同温度生物炭促进五氯酚生物降解的机制[J]. 环境科学与技术, 2022, 45(9): 32-42.
	HUANG Fengcheng, WU Weijian, ZHANG Weijian, et al. Mechanism of biochar promoting pentachlorophenol biodegradation at different temperatures[J]. Environmental Science & Technology, 2022, 45(9): 32-42.
20	吕纯剑, 高红杰, 张羽. 内分泌干扰物2,4-二氯苯酚和双酚A对潜流人工湿地系统中微生物群落结构的影响[C]//中国环境科学学会学术年会论文集, 2016: 1015-1020.
	Chunjian LYU, GAO Hongjie, ZHANG Yu. Effects of endocrine disruptors 2,4-dichlorophenol and bisphenol A on microbial community structure in subsurface flow constructed wetland system[C]//Proceedings of the Annual Conference of Chinese Society of Environmental Sciences, 2016: 1015-1020.
21	曹佳, 王冲, 皇彦, 等. 蚯蚓对土壤微生物及生物肥力的影响研究进展[J]. 应用生态学报, 2015, 26(5): 1579-1586.
	CAO Jia, WANG Chong, HUANG Yan, et al. Effects of earthworm on soil microbes and biological fertility: A review[J]. Chinese Journal of Applied Ecology, 2015, 26(5): 1579-1586.
22	岑福, 殷根深, 蒋明星, 等. 邻苯二甲酸酯(PAEs)对不同植物根际微生物群落的影响[J]. 贵州农业科学, 2024, 52(1): 66-73.
	CEN Fu, YIN Genshen, JIANG Mingxing, et al. Effects of phthalic acid esters (PAEs) on rhizosphere microbial communities of different plants[J]. Guizhou Agricultural Sciences, 2024, 52(1): 66-73.
23	黄雪颖, 陈威, 王旭, 等. 焦化废水对菌藻颗粒污泥的微生物群落及代谢基因的影响[J]. 河南科技大学学报(自然科学版), 2022, 43(5): 100-104, 10.
	HUANG Xueying, CHEN Wei, WANG Xu, et al. Effect of coking wastewater on microbial communities and metabolic genes in microalgae bacteria granular sludge[J]. Journal of Henan University of Science and Technology (Natural Science), 2022, 43(5): 100-104, 10.
24	黄湘云, 钟文军, 刘训杰, 等. 基于环境DNA的复合污染土壤生物评价和胁迫诊断[J]. 环境科学, 2023, 44(7): 4130-4141.
	HUANG Xiangyun, ZHONG Wenjun, LIU Xunjie, et al. Biological evaluation and key stress factor diagnosis of compound contaminated soil based on environmental DNA[J]. Environmental Science, 2023, 44(7): 4130-4141.
25	王彬浩, 关晓彤, 颜庆云, 等. 含氟有机废水处理过程活性污泥微生物群落研究进展[J]. 微生物学通报, 2019, 46(8): 2020-2037.
	WANG Binhao, GUAN Xiaotong, YAN Qingyun, et al. Research progresses of activated sludge microbial communities in fluorine-containing organic wastewater treatment processes[J]. Microbiology China, 2019, 46(8): 2020-2037.
26	杨庆, 王铸, 许欣宇, 等. 基于MMI技术对印染废水中NP降解关键功能菌群的识别、构建与评估[J]. 工业水处理, 2023, 43(11): 15-26.
	YANG Qing, WANG Zhu, XU Xinyu, et al. Identification, construction and evaluation of key functional flora for NP degradation in printing and dyeing wastewater based on MMI technology[J]. Industrial Water Treatment, 2023, 43(11): 15-26.
27	杨思德, 常兴平, 潘政, 等. 蚯蚓对金霉素污染土壤酶活性和微生物群落的影响[J]. 农业环境科学学报, 2021, 40(6): 1268-1280.
	YANG Side, CHANG Xingping, PAN Zheng, et al. Effects of earthworms on soil enzyme activities and microbial communities of chlortetracycline-contaminated soils[J]. Journal of Agro-Environment Science, 2021, 40(6): 1268-1280.
28	陆玉兰. 污泥好氧堆肥堆体空气阻流区分布变化及氧传递规律[D]. 桂林: 桂林理工大学, 2022.
	LU Yulan. Distribution change and oxygen transfer law of obstructed gas region during sludge aerobic compost[D]. Guilin: Guilin University of Technology, 2022.
29	翟亚萍, 王绍明, 刘鸯, 等. 不同种植地苜蓿根际土壤细菌群落结构多样性差异分析[J]. 新疆农业科学, 2021, 58(5): 955-964.
	ZHAI Yaping, WANG Shaoming, LIU Yang, et al. Study on structural diversity of bacterial community in rhizosphere soil of alfalfa in parts of northern foot of Tianshan Mountains[J]. Xinjiang Agricultural Sciences, 2021, 58(5): 955-964.
30	MA Weiwei, HAN Yuxing, XU Chunyan, et al. Enhanced degradation of phenolic compounds in coal gasification wastewater by a novel integration of micro-electrolysis with biological reactor (MEBR) under the micro-oxygen condition[J]. Bioresource Technology, 2018, 251: 303-310.
31	HU Shunli, XU Chuangchuang, XIE Yanghe, et al. Metagenomic insights into the diversity of 2,4-dichlorophenol degraders and the cooperation patterns in a bacterial consortium[J]. Science of the Total Environment, 2024, 912: 168723.
32	张婉莹, 朱清禾, 杨洁. 强化氧化后污染源浅层地下水中氯苯类污染物的自然衰减与微生物群落演化[J]. 环境工程学报, 2023, 17(8): 2674-2683.
	ZHANG Wanying, ZHU Qinghe, YANG Jie. Natural attenuation and microbial community evolution of chlorobenzene pollutants in shallow groundwater after enhanced oxidation[J]. Chinese Journal of Environmental Engineering, 2023, 17(8): 2674-2683.
33	刘雯静, 张克峰, 丁万德. 淀粉/聚乙烯醇固体碳源对污水厂尾水的脱氮效果[J]. 中国给水排水, 2024, 40(5): 75-81.
	LIU Wenjing, ZHANG Kefeng, DING Wande. Effect of starch/polyvinyl alcohol solid carbon sources on nitrogen removal of tail water from municipal wastewater treatment plant[J]. China Water & Wastewater, 2024, 40(5): 75-81.
34	SONG Biao, GONG Jilai, TANG Wangwang, et al. Influence of multi-walled carbon nanotubes on the microbial biomass, enzyme activity, and bacterial community structure in 2,4-dichlorophenol-contaminated sediment[J]. Science of the Total Environment, 2020, 713: 136645.

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