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2,4-二氯苯酚(2,4-DCP)是一种广泛用于有机合成的酚类化合物,在农业、工业和医药等方面的应用日益广泛[1]. 由于储存和使用不善,2,4-DCP会通过农药、废水、泄漏等途径排放到环境中[2]. 据报道,在我国很多流域中均有2,4-DCP的检出,最高可达1 mg∙L−1[3 − 4]. 2,4-DCP能使蛋白质变性,对人体内分泌、神经和免疫系统造成永久性伤害,具有“三致”效应[5]. 已有较多文献报道了2,4-DCP对水生动物的毒性效应:Tsukazawa等[6]研究表明,斑马鱼幼鱼暴露于2,4-DCP浓度为2.5 mg∙L−1的水体中5 d时,可观察到脂质积累和ROS诱导;Hu等[7]发现,2,4-DCP可通过干扰性激素合成来诱导鱼类雌性化. 然而,目前对2,4-DCP的毒性评估在土壤生物方面研究尚少. 因此,开展2,4-DCP对土壤生物毒性研究,有助于更全面的评估其生态风险.
蚯蚓和微生物群落常用于毒理学研究,它们的生物量占据土壤生物的绝大部分,与土壤功能息息相关[8]. 蚯蚓经常作为评价土壤环境的指示生物,其表现出的毒性效应对土壤污染物的早期预警和生态危险评估具有重要意义[9]. 而微生物作为维持土壤生产力的重要组分,其多样性变化可在预测环境质量变化、评价土壤生态功能中发挥巨大作用[10]. 此外,蚯蚓作为土壤中最大的无脊椎动物,与微生物之间的关系作用密切,其通过活动以改变微生物群落结构和数量[11]. 已有研究表明蚯蚓可参与污染物的降解与转化,且其活动、分泌粘液和排泄等行为所产生的微生物在此过程中至关重要[12]. 因此,蚯蚓活动以及土壤微生物群落的改变也可能成为土壤中污染物降解的重要因素.
基于以上背景,本研究以赤子爱胜蚓(Eisenia fetida)作为受试生物,通过分析其致死浓度、抗氧化系统、病理学和行为特征变化来阐明2,4-DCP对蚯蚓的毒性;基于土壤中2,4-DCP的降解规律,采用高通量测序分析2,4-DCP、蚯蚓活动和蚓粪因素下土壤微生物群落变化. 为准确评估2,4-DCP对土壤环境的生态风险提供科学依据.
蚯蚓对2,4-DCP污染土壤的毒性响应及微生物群落影响
Toxic response of earthworms (Eisenia fetida) to 2,4-DCP contaminated soil and the impact of microbial community
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摘要: 2.4-二氯苯酚(2.4-DCP)作为合成农药、医药的中间体,在我国广泛生产和使用,其生态危害性已引起广泛关注. 然而,关于土壤动物、微生物对2.4-DCP的毒性响应知之尚少. 基于此,本研究以赤子爱胜蚓(Eisenia fetida)为受试生物,从致死浓度、抗氧化系统、病理学和行为特征角度评价2.4-DCP对蚯蚓的毒性,通过高通量测序分析土壤微生物群落变化. 此外,还探讨了蚯蚓对土壤中2.4-DCP的降解动力学和微生物多样性影响. 结果表明,2.4-DCP对蚯蚓7 d半致死浓度(LC50)值为29.55 mg∙kg−1,14 d为28.76 mg∙kg−1;蚯蚓体内超氧化物歧化酶(SOD)随暴露时间、浓度增加总体呈下降趋势(P<0.01),而过氧化氢酶(CAT)和丙二醛(MDA)呈缓慢上升趋势(P<0.01);当2.4-DCP暴露浓度为15 mg∙kg−1时,14 d可使蚯蚓表皮细胞发生凹陷并破裂,浓度达到25 mg∙kg−1时,蚯蚓表皮细胞丧失规则细胞排列结构,肠上皮细胞发生细胞间隙扩大和破裂现象;2.4-DCP浓度越高,对蚯蚓的行为特征影响越明显;2.4-DCP在添加蚯蚓和未添加蚯蚓土壤中的降解动力学均符合一级动力学模型,在添加蚯蚓土壤中的降解速率(0.20 d−1)明显高于未添加(0.08 d−1);2.4-DCP可抑制土壤微生物群落多样性,但蚯蚓活动、蚓粪的产生可分别增加土壤中一些具有降解有机物功能的好氧、厌氧菌属丰度. 本研究旨在揭示2.4-DCP与土壤动物及微生物的相互作用关系,为综合评价2.4-DCP的毒性和风险提供理论参考.Abstract: 2,4-dichlorophenol(2,4-DCP), as an intermediate in the synthesis of pesticides and pharmaceuticals, is widely produced and used in China, and its ecological hazard has aroused widespread concern. However, little is known about the toxic response of soil animals and microorganisms to 2,4-DCP. Based on this, this study took Eisenia fetida as the test organism, evaluate the toxicity of 2,4-DCP to earthworms from the perspective of lethal concentration, antioxidant system, pathology and behavioral characteristics, and analyze the changes of soil microbial community through high-throughput sequencing. In addition, the degradation kinetics and microbial diversity of 2,4-DCP by earthworms were also discussed. The results showed that the LC50 value of 2,4-DCP to earthworms was 29.55 mg∙kg−1 for 7 days, and 28.76 mg∙kg−1 for 14 days; The superoxide dismutase (SOD) in earthworms decreased with the increase of exposure time and concentration (P<0.01), while catalase (CAT) and malondialdehyde (MDA) increased slowly (P<0.01); When the exposure concentration of 2,4-DCP is 15 mg∙kg−1, the earthworm epidermal cells can be dented and ruptured for 14 days, when the concentration reaches 25 mg∙kg−1, the earthworm epidermal cells lose the regular cell arrangement structure, and the intestinal epithelial cells have the phenomenon of cell gap expansion and rupture; The higher the concentration of 2,4-DCP, the more obvious the effect on the behavioral characteristics of earthworms; The degradation kinetics of 2,4-DCP in the soil with and without earthworms conforms to the first-order kinetic model, and the degradation rate in the soil with earthworms (0.20 d−1) is significantly higher than that in the soil without earthworms (0.08 d−1); 2,4-DCP can inhibit the diversity of soil microbial community, but the activity of earthworms and the production of earthworm manure can respectively increase the abundance of aerobic and anaerobic bacteria in the soil that have the function of degrading organic matter. The purpose of this study is to reveal the interaction between 2,4-DCP and soil animals and microorganisms, and provide theoretical reference for comprehensive evaluation of 2,4-DCP toxicity and risk.
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Key words:
- 2,4-DCP /
- Eisenia fetida /
- toxic effects /
- microbial diversity.
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表 1 自然土壤法测得各浓度2,4-DCP梯度下蚯蚓死亡率(%)
Table 1. Mortality rate of earthworm under the gradient of 2,4-DCP measured by natural soil method(%)
时间/d
Time2,4-DCP浓度/(mg∙kg−1)
Concentration20 22.5 25 27.5 30 32.5 35 37.5 7 0 3.33±5.77 13.33±5.77 26.67±11.55 53.33±5.77 66.67±15.28 93.33±5.77 100 14 0 6.67±5.77 16.67±5.77 33.33±15.28 53.33±5.77 80±10 96.67±5.77 100 注:空白对照组中无蚯蚓死亡. Note: No earthworm died in the blank control group. 表 2 自然土壤法测得2,4-DCP对蚯蚓的LC50
Table 2. LC50 of 2,4-DCP to earthworm measured by natural soil method
时间/d
time毒性回归方程
Toxicity regression equations卡方值
Chi−square显著性
SigLC50(95%置信限)/(mg∙kg−1)
LC50(95% CL)7 y=17.447x-25.656 1.269 0.973 29.546(27.992—31.142) 14 y=17.313x-25.257 1.332 0.970 28.763(27.226—30.317) 表 3 各样本多样性指数结果
Table 3. The diversity index results of each sample
样品
Sample聚类
OTUs香农指数
ShannonChao1指数
ChaoACE指数
Ace辛普森指数
Simpson覆盖率
Coverage空白对照组土壤
Soil of blank control group857±16 4.60±0.09 862.00±14.74 860.02±13.92 0.09±0.02 1.00 2,4-DCP污染土壤
2,4-DCP contaminated soil700±33** 4.51±0.12 704.44±42.10** 703.67±41.83** 0.03±0.01** 1.00 污染后添加蚯蚓土壤
Pollution soil with earthworm685±41** 4.65±0.15 792.65±62.64 792.68±61.66 0.02±0.01** 1.00 蚯蚓粪便
Earthworm excrement225±15** 2.60±0.08** 309.91±33.41** 287.75±23.53** 0.17±0.03** 1.00 注:*表示P<0.05,**表示P<0.01.
Note:* represents P<0.05, ** represents P<0.01. -
[1] DUBEY M, KUMAR R, SRIVASTAVA S K, et al. ZnO/α-MnO2 hybrid 1D nanostructure-based sensor for point-of-care monitoring of chlorinated phenol in drinking water[J]. Materials Today Chemistry, 2022, 26: 101098. doi: 10.1016/j.mtchem.2022.101098 [2] FERNANDEZ M E, del ROSARIO MOREL M, CLEBOT A C, et al. Effectiveness of a simple biomixture for the adsorption and elimination of 2, 4-dichlorophenoxyacetic acid (2, 4-D) herbicide and its metabolite, 2, 4-dichlorophenol (2, 4-DCP), for a biobed system[J]. Journal of Environmental Chemical Engineering, 2022, 10(1): 106877. doi: 10.1016/j.jece.2021.106877 [3] 王钱森, 付先炜, 丁念琛, 等. 磁性氧化石墨烯负载酞菁铜的制备及去除2, 4-二氯苯酚研究[J]. 山东化工, 2022, 51(14): 4-7. WANG Q S, FU X W, DING N C, et al. Preparation of copper phthalocyanine supported on magnetic graphene oxide for removal 2, 4-dichlorophenol from aqueous solution[J]. Shandong Chemical Industry, 2022, 51(14): 4-7(in Chinese).
[4] FAN B, WANG X N, XIE Z Y, et al. Aquatic life criteria & human health ambient water quality criteria derivations and probabilistic risk assessments of 7 benzenes in China[J]. Chemosphere, 2021, 274: 129784. doi: 10.1016/j.chemosphere.2021.129784 [5] 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 Q. Preparation of Fe-Pd bimetallic catalyst supported on modified chitosan and its degradation performance of p-dichlorophenol[D]. Guangzhou: South China University of Technology, 2021 (in Chinese).
[6] TSUKAZAWA K S, LI L, TSE W K F. 2, 4-dichlorophenol exposure induces lipid accumulation and reactive oxygen species formation in zebrafish embryos[J]. Ecotoxicology and Environmental Safety, 2021, 230: 113133. [7] HU Y, LI D, MA X, et al. Effects of 2, 4-dichlorophenol exposure on zebrafish: Implications for the sex hormone synthesis[J]. Aquatic Toxicology, 2021, 236: 105868. doi: 10.1016/j.aquatox.2021.105868 [8] de BERNARDI A, MARINI E, CASUCCI C, et al. Ecotoxicological effects of a synthetic and a natural insecticide on earthworms and soil bacterial community[J]. Environmental Advances, 2022, 8: 100225. doi: 10.1016/j.envadv.2022.100225 [9] HOU K X, YANG Y, ZHU L, et al. Toxicity evaluation of chlorpyrifos and its main metabolite 3, 5, 6-trichloro-2-pyridinol (TCP) to Eisenia fetida in different soils[J]. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2022, 259: 109394. [10] 张芳, 郜红建, 葛高飞. 苯并[a]芘累积污染对土壤微生物群落功能多样性的影响[J]. 环境化学, 2017, 36(8): 1849-1857. doi: 10.7524/j.issn.0254-6108.2016123002 ZHANG F, GAO H J, GE G F. Effects of cumulative benzo(a)pyrene pollution on functional diversity of microbial community in soil[J]. Environmental Chemistry, 2017, 36(8): 1849-1857 (in Chinese). doi: 10.7524/j.issn.0254-6108.2016123002
[11] REN C L, TENG Y R, CHEN X Y, et al. Impacts of earthworm introduction and cadmium on microbial communities composition and function in soil[J]. Environmental Toxicology and Pharmacology, 2021, 83: 103606. doi: 10.1016/j.etap.2021.103606 [12] 潘政, 郝月崎, 赵丽霞, 等. 蚯蚓在有机污染土壤生物修复中的作用机理与应用[J]. 生态学杂志, 2020, 39(9): 3108-3117. PAN Z, HAO Y Q, ZHAO L X, et al. Mechanism and application of earthworm in bioremediation of soil contaminated with organic pollutants: A review[J]. Chinese Journal of Ecology, 2020, 39(9): 3108-3117 (in Chinese).
[13] 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. [14] International Organization for Standardization. Draft: Soil Quality-Avoidance Test for Determining the Quality of Soils and Effects of Chemicals on Behaviour-Part 1: Test with Earthworms (Eisenia fetida/andrei)[S]. Geneva: International Organization for Standardization, 2008. [15] AMORIM M J B, RÖMBKE J, SOARES A M V M. Avoidance behaviour of Enchytraeus albidus: Effects of Benomyl, Carbendazim, phenmedipham and different soil types[J]. Chemosphere, 2005, 59(4): 501-510. doi: 10.1016/j.chemosphere.2005.01.057 [16] 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: HJ 998—2018[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: HJ 998—2018[S]. Beijing: China Environmental Science Press, 2018(in Chinese).
[17] 乔文鹏, 乔玉辉, 孙振钧. 氯化镉、马拉硫磷和乙草胺对赤子爱胜蚓的单一急性毒性[J]. 农业环境科学学报, 2007, 26(B10): 514-518. QIAO W P, QIAO Y H, SUN Z. Acute toxicity of cadmium chloride, malathion, acetochlor on earthworm (Eisenia fetida)[J]. Journal of Agro-Environment Science, 2007, 26(B10): 514-518 (in Chinese).
[18] CHENG Y L, ZHU L S, SONG W H, et al. Combined effects of mulch film-derived microplastics and atrazine on oxidative stress and gene expression in earthworm (Eisenia fetida)[J]. Science of the Total Environment, 2020, 746: 141280. doi: 10.1016/j.scitotenv.2020.141280 [19] MARKAD V L, KODAM K M, GHOLE V S. Effect of fly ash on biochemical responses and DNA damage in earthworm, Dichogaster curgensis[J]. Journal of Hazardous Materials, 2012, 215/216: 191-198. doi: 10.1016/j.jhazmat.2012.02.053 [20] 王轶, 刁晓平, 张先勇. 莫能菌素对蚯蚓的生态毒理效应[J]. 农业环境科学学报, 2010, 29(6): 1091-1097. WANG Y, DIAO X P, ZHANG X Y. Ecotoxicological effects of monensin pollution on earthworm (Eisenia fetida)[J]. Journal of Agro-Environment Science, 2010, 29(6): 1091-1097(in Chinese).
[21] LI M Y, MA X X, WANG Y R, et al. Ecotoxicity of herbicide carfentrazone-ethyl towards earthworm (Eisenia fetida) in soil[J]. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2022, 253: 109250. [22] SOARES C, de SOUSA A, PINTO A, et al. Effect of 24-epibrassinolide on ROS content, antioxidant system, lipid peroxidation and Ni uptake in Solanum nigrum L. under Ni stress[J]. Environmental and Experimental Botany, 2016, 122: 115-125. doi: 10.1016/j.envexpbot.2015.09.010 [23] QIAO Z H, LI P Y, TAN J Q, et al. Oxidative stress and detoxification mechanisms of earthworms (Eisenia fetida) after exposure to flupyradifurone in a soil-earthworm system[J]. Journal of Environmental Management, 2022, 322: 115989. doi: 10.1016/j.jenvman.2022.115989 [24] HASCHEK W M, ROUSSEAUX C G, WALLIG M A. Haschek and Rousseaux's Handbook of Toxicologic Pathology (Fourth Edition)[M]. Academic Press. 2022: 1-12. [25] HE F L, LI X X, HUO C Q, et al. Evaluation of fluorene-caused ecotoxicological responses and the mechanism underlying its toxicity in Eisenia fetida: Multi-level analysis of biological organization[J]. Journal of Hazardous Materials, 2022, 437: 129342. doi: 10.1016/j.jhazmat.2022.129342 [26] 郭佳葳, 周世萍, 刘守庆, 等. 蚯蚓生物标志物在土壤生态系统监测中的应用研究进展[J]. 生态毒理学报, 2020, 15(5): 69-81 GUO J W, ZHOU S P, LIU S Q, et al. Advances in application of earthworm biomarkers in monitoring soil ecosystem[J]. Asian Journal of Ecotoxicology, 2020, 15(5): 69-81(in Chinese).
[27] 李芬, 林雪儿, 黄慧雯, 等. 探究蚯蚓对食用油污染土壤的回避行为[J]. 中学生物教学, 2020, 23: 67-69. LI F, LIN X E, HUANG H W, et al. Explore the avoidance behavior of earthworms to edible oil contaminated soil[J]. Middle School Biology Teaching, 2020, 23: 67-69 (in Chinese).
[28] 黄盼盼, 周启星. 石油污染土壤对蚯蚓的致死效应及回避行为的影响[J]. 生态毒理学报, 2012, 7(3): 312-316 HUANG P P, ZHOU Q X. Effects of petroleum-contaminated soil on lethality and avoidance behavior of the earthworm (Eisenia fetida)[J]. Asian Journal of Ecotoxicolog, 2012, 7(3): 312-316(in Chinese).
[29] 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 Y L, LI M Y, ZHANG X S, 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(in Chinese).
[30] 刘文凯, 熊海谦, 包细明, 等. 棚下牛粪养殖蚯蚓技术[J]. 湖北畜牧兽医, 2021, 42(7): 25-27 LIU W K, XIONG H Q, BAO X M, et al. Technology of cultivating earthworm with cow dung under shed[J]. Hubei Journal of Animal and Veterinary Sciences, 2021, 42(7): 25-27(in Chinese).
[31] WILSON W J, FERRARA N C, BLAKER A L, et al. Escape and avoidance learning in the earthworm Eisenia hortensis[J]. PeerJ, 2014, 2: e250. doi: 10.7717/peerj.250 [32] CUI G Y, AHMAD BHAT S, LI W J, et al. Gut digestion of earthworms significantly attenuates cell-free and-associated antibiotic resistance genes in excess activated sludge by affecting bacterial profiles[J]. Science of the Total Environment, 2019, 691: 644-653. doi: 10.1016/j.scitotenv.2019.07.177 [33] CHEN G W, YU H Q, LIU H X, et al. Response of activated sludge to the presence of 2, 4-dichlorophenol in a batch culture system[J]. Process Biochemistry, 2006, 41(8): 1758-1763. doi: 10.1016/j.procbio.2006.03.022 [34] 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 J, WANG C, HUANG Y, et al. Effects of earthworm on soil microbes and biological fertility: A review[J]. Chinese Journal of Applied Ecology, 2015, 26(5): 1579-1586(in Chinese).
[35] 张鑫爱. 低强度超声波强化含氮废水生物脱氮研究[D]. 太原: 太原理工大学, 2019. ZHANG X A. Study on enhanced biological denitrification of nitrogen-containing wastewater by low-intensity ultrasound[D]. Taiyuan: Taiyuan University of Technology, 2019(in Chinese).
[36] ZHENG M S, ZHOU N, LIU S F, et al. N2O and NO emission from a biological aerated filter treating coking wastewater: Main source and microbial community[J]. Journal of Cleaner Production, 2019, 213: 365-374. doi: 10.1016/j.jclepro.2018.12.182 [37] EL-BASSI L, ZIADI I, BELGACEM S, et al. Investigations on biofilm forming bacteria involved in biocorrosion of carbon steel immerged in real wastewaters[J]. International Biodeterioration & Biodegradation, 2020, 150: 104960. [38] ZHENG M Q, ZHU H, HAN Y X, et al. Comparative investigation on carbon-based moving bed biofilm reactor (MBBR) for synchronous removal of phenols and ammonia in treating coal pyrolysis wastewater at pilot-scale[J]. Bioresource Technology, 2019, 288: 121590. doi: 10.1016/j.biortech.2019.121590 [39] NORAMBUENA J, HANSON T E, BARKAY T, et al. Superoxide dismutase and pseudocatalase increase tolerance to Hg(Ⅱ) in Thermus thermophilus HB27 by maintaining the reduced bacillithiol pool[J]. mBio, 2019, 10(2): e00183-e00119. [40] 母显杰, 丁舒心, 许继飞, 等. 耐盐苯酚降解菌Staphylococcus sp. 的分离及降解特性[J]. 环境化学, 2020, 39(7): 1985-1995. doi: 10.7524/j.issn.0254-6108.2019050904 MU X J, DING S X, XU J F, et al. Isolation and degradation characteristics of a salt tolerant phenol degrading bacterium Staphylococcus sp. [J]. Environmental Chemistry, 2020, 39(7): 1985-1995 (in Chinese). doi: 10.7524/j.issn.0254-6108.2019050904
[41] JUNG H M, JUNG M Y, OH M K. Metabolic engineering of Klebsiella pneumoniae for the production of cis, cis-muconic acid[J]. Applied Microbiology and Biotechnology, 2015, 99(12): 5217-5225. doi: 10.1007/s00253-015-6442-3 [42] XING W, WANG Y, HAO T Y, et al. pH control and microbial community analysis with HCl or CO2 addition in H2-based autotrophic denitrification[J]. Water Research, 2020, 168: 115200. doi: 10.1016/j.watres.2019.115200 [43] DRAKE H L, HORN M A. As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[J]. Annual Review of Microbiology, 2007, 61: 169-189. doi: 10.1146/annurev.micro.61.080706.093139 [44] 蔡建林, TENG Hui Henry, 王钺博, 等. 方解石和钾长石在模拟蚯蚓肠液中的初始溶解动力学机理及意义[J]. 岩石矿物学杂志, 2022, 41(4): 818-834. CAI J L, HENRY T H, WANG Y B, et al. Kinetics and mechanistic implications of calcite and K-feldspar initial dissolution in simulated earthworm intestine fluid[J]. Acta Petrologica et Mineralogica, 2022, 41(4): 818-834(in Chinese).
[45] PASS D A, MORGAN A J, READ D S, et al. The effect of anthropogenic arsenic contamination on the earthworm microbiome[J]. Environmental Microbiology, 2015, 17(6): 1884-1896. doi: 10.1111/1462-2920.12712 [46] LIU P, YANG Y, LI M. Responses of soil and earthworm gut bacterial communities to heavy metal contamination[J]. Environmental Pollution, 2020, 265: 114921. doi: 10.1016/j.envpol.2020.114921 [47] GAO C Y, WANG A J, WU W M, et al. Enrichment of anodic biofilm inoculated with anaerobic or aerobic sludge in single chambered air-cathode microbial fuel cells[J]. Bioresource Technology, 2014, 167: 124-132. doi: 10.1016/j.biortech.2014.05.120 [48] CHEN M X, WANG W C, FENG Y, et al. Impact resistance of different factors on ammonia removal by heterotrophic nitrification–aerobic denitrification bacterium Aeromonas sp. HN-02[J]. Bioresource Technology, 2014, 167: 456-461. doi: 10.1016/j.biortech.2014.06.001