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近年来,许多有机污染物在河流、地下水和湖泊中被发现,包括抗生素、农药和微塑料,并且含量维持在痕量浓度[1]。其中,抗生素和农药被广泛用于医疗和农业领域,这些污染物在痕量浓度下长期存在于水体中,会对饮用水造成严重危害[2]。长期过度使用抗生素导致细菌产生耐药性,从而降低抗生素对人类疾病的治疗效果[3]。因此,减少诱发抗性基因(antibiotic resistance genes, ARGs)的风险尤为重要。有研究[4]表明,氮循环细菌暴露在β-内酰胺类抗生素和寡营养环境下,成为主要抗性基因宿主。非抗生素化学物质,例如季铵化合物、微塑料可以诱导微生物应激反应,促进ARGs水平转移[5-6]。KURENBACH等[7]研究发现,除草剂可将抗生素的最小抑菌浓度提高6倍,促进微生物抗性基因产生。
活性炭具有发达的孔隙结构和不错的经济效益,被普遍用作饮用水高级处理的吸附剂。同时,活性炭可以与水体中微生物结合,利用微生物去除污染物,因此,活性炭通常被称为生物活性炭[8-9]。大量微生物附着于生物活性炭表面,ARGs的水平转移在微生物群落之间增强,从而引起耐药性细菌的增殖[10-11]。此外,有研究[12-13]表明,生物膜促进消毒过程中消毒副产物的产生,尤其是含氮类消毒副产物(nitrogenous disinfection byproducts formation potential, N-DBPs),究其原因是胞外聚合物(extracellular polymeric substance, EPS)、可溶性微生物产物和死细胞可以作为消毒副产物前驱体参与了反应。LI等[14]研究表明,环境胁迫促使微生物过度分泌EPS确保生存。蛋白质、多糖和核酸是EPS的主要成分,此外,蛋白质比多糖更容易促进N-DBPs的产生,因为其含有高含量的有机氮[15-16]。
LYU等[17-18]研究发现,吸附在双反应中心(dual-reaction-center, DRC)贫电子区的有机物能被高效降解,同时吸附在富电子区的H2O2被还原成羟基自由基或活性氧。有研究表明,微生物可以通过生物膜吸收不溶性电子供体提供的电子,例如,硫氧化硫化杆菌(Sulfobacillus thermosulfidooxidans)通过外膜蛋白从石墨表面电子供体吸收电子[19]。因此,电子从吸附在催化剂贫电子区的有机物到富电子区的微生物。这种滤料-微生物协同作用避免微生物从污染物中获得能量,削弱污染物对微生物的胁迫效应。
本研究通过浸渍和表面络合法制备了DRC催化剂(HCLL-S8-M),将其用于饮用水深度处理工艺。使用溶解性有机碳(dissolved organic carbon, DOC)的质量浓度、SUVA (specific ultraviolet absorbance)、高效凝胶排阻色谱(high-performance size exclusion chromatography, HPSEC)和三维荧光(excitation-emission matrix fluorescence, EEM)参数反映HCLL-S8-M滤柱对水质处理效果。通过实时荧光定量PCR (polymerase chain reaction)检测了微生物ARGs的相对丰度,使用气相色谱分析水体中N-DBPs生成势(N-DBPs formation potential, N-DBPs FP)。通过宏基因组学分析结合EPS的理化性质探讨微生物群落结构的变化,阐明催化剂、微生物之间的相互作用。探讨了在饮用水处理过程中,微生物协同HCLL-S8-M削弱复合微污染物对微生物胁迫效应的机制,为降低N-DBPs和ARGs风险提供理论依据。
催化剂填料表面微生物对微污染物胁迫的响应
Response of catalyst microorganisms to the stress effects of micropollutants
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摘要: 目前复合微污染水源条件下,饮用水中复合微污染物对微生物带来的胁迫效应会引发含氮类消毒副产物生成势(nitrogenous disinfection byproducts formation potential, N-DBPs FP)和抗性基因(antibiotic resistance genes, ARGs)水质风险。本研究基于催化剂(HCLL-S8-M)作为新型生物滤料建立反应器处理砂滤后水,考察生物滤池出水N-DBPs FP和ARGs丰度,对滤料胞外聚合物(extracellular polymeric substance, EPS)进行分析,通过宏基因组学分析水体中微生物群落结构和胁迫效应基因表达水平。结果表明,在相同复合微污染物条件下(磺胺嘧啶和2,4-二氯苯氧乙酸各1 µg·L−1),HCLL-S8-M滤柱出水中卤代乙腈和卤代硝基甲烷的总生成势为522.30 ng·L−1,比椰壳活性炭滤柱出水低171.70 ng·L−1,ARGs相对丰度也比椰壳活性炭滤柱出水低约4倍。这得益于HCLL-S8-M滤柱可以高效去除包括微污染物在内的溶解性有机碳、SUVA和高分子质量的有机物。HCLL-S8-M滤柱和出水中微生物分泌更少的EPS,从而减少的N-DBPs前驱体和削弱ARGs的水平转移。宏基因组学分析结果表明,微生物在HCLL-S8-M滤柱出水中的氧化应激反应功能基因表达比椰壳活性炭滤柱下降。HCLL-S8-M滤柱减弱微污染物对微生物的胁迫效应,降低N-DBPs FP和ARGs,有助于控制微生物应激反应造成间接饮用水水质风险。Abstract: Under the current conditions of composite micro-polluted water sources, the stress effects of composite micropollutants in drinking water for microbes can cause water quality risks of nitrogenous disinfection byproducts formation potential (N-DBPs FP) and antibiotic resistance genes (ARGs). In this study, the catalyst HCLL-S8-M was used as a new biofilter to build the reactor treating the effluent from sand filtration. The abundances of N-DBPs FP and ARGs in the biofilter effluent were studied, the filter media and extracellular polymeric substance (EPS) was analyzed, and metagenome sequencing was used to analyze the microbial community structure and gene expression levels of stress effect in water bodies. The results showed that under the exposure of the same complex micropollutants conditions (1 µg·L−1 of sulfadiazine and 1 µg·L−1 of 2,4-dichlorophenoxyacetic acid), the total formation potential of haloacetonitriles and halonitromethanes was 522.30 ng·L−1, which was 171.70 ng·L−1 lower than that of coconut shell activated carbon, the relative abundance of ARGs was around 4 times lower than that of coconut shell activated carbon effluent. This was due to HCLL-S8-M could efficiently remove dissolved organic carbon (including micropollutants), SUVA and organics with high molecular weight. Microorganisms in the HCLL-S8-M filter and effluent secreted few EPS, which resulted in reduced precursors of N-DBPs and horizontal transfer of ARGs. Metagenome analysis revealed that oxidative stress functional gene expression of microorganisms decreased on HCLL-S8-M compared to coconut shell activated carbon. HCLL-S8-M attenuated the stress effects of micropollutants on microorganisms, and decreased N-DBPs FP and ARGs, which will contribute to control the indirect drinking water quality risks of micropollutants from the microbial stress response during biofiltration.
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图 8 微生物属水平的PCA分析、前20种细菌的相对丰度、典型相关分析、共现网络分析和抗氧化相关酶基因绝对丰度
Figure 8. PCA analysis of microbes at genus level, relative abundances of the top 20 bacteria, canonical correlation analysis, co-occurrence network analysis and expression profiles for related enzyme genes of antioxidant with absolute abundance
表 1 滤料表面EPS蛋白质二级结构占比情况
Table 1. Percentage of protein secondary structures of EPS on filters surface
蛋白质二级结构 波数/cm−1 蛋白质二级结构所占比例% 椰壳活性炭 HCll-S8-M 聚合链 1 625~1 610 29.30 18.79 β-折叠 1 640~1 630 12.07 19.13 无规则卷曲 1 645~1 640 7.44 22.90 α-螺旋 1 657~1 648 19.99 18.10 三向螺旋 1 672~1 659 19.80 14.36 反平行/β-折叠 1 695~1 680 11.42 6.71 表 2 图8(d)中OTU对应的门、属和绝对丰度
Table 2. Phylum, Genus and absolute abundance corresponding to OTU in Fig.8(d)
OTU 门 属 进水 椰壳活性炭滤柱出水 HCLL-S8-M滤柱出水 OTU1 Actinobacteria Mycolicibacterium 11256 836 813 710 48 896 OTU2 Proteobacteria Reyranella 1293 900 2435 894 1453 014 OTU3 Proteobacteria Sphingopyxis 1910 720 2058 776 266 966 OTU4 Proteobacteria Limnobacter 811 708 207 092 2695 466 OTU5 Proteobacteria Bradyrhizobium 1039 726 901 014 1369 452 OTU6 Proteobacteria Phenylobacterium 622 486 1255 288 1228 110 OTU7 Proteobacteria Hyphomicrobium 396 548 2410 284 273 686 OTU8 Bacteroidota Sediminibacterium 253 564 70 144 2271 874 OTU9 Proteobacteria Novosphingobium 66 532 213 954 2314 156 OTU10 Proteobacteria Sphingomonas 1183 450 1111 608 259 714 OTU11 Actinobacteria Mycobacterium 1410 282 989 004 32 716 OTU12 Actinobacteria Nocardioides 420 912 1880 760 5 356 OTU13 Proteobacteria Methylobacterium 787 802 1274 470 170 738 OTU14 Proteobacteria Bosea 824 698 1122 304 97 046 OTU15 Proteobacteria Methylotenera 29 230 1726 124 59 582 OTU16 Proteobacteria Falsiroseomonas 1738 764 12 182 21 782 OTU17 Proteobacteria Rhodobacter 860 160 340 022 446 816 OTU18 Proteobacteria Roseomonas 1223 196 74 974 31 470 OTU19 Proteobacteria Brevundimonas 928 140 178 374 84 584 OTU20 Proteobacteria Caulobacter 69 884 113 844 797 672 OTU21 Proteobacteria Methylophilus 114 028 524 408 333 398 OTU22 Proteobacteria Tabrizicola 504 872 201 308 263 828 OTU23 Proteobacteria Piscinibacter 77 592 61 154 826 228 OTU24 Proteobacteria Aquisediminimonas 1 130 948 778 3 852 OTU25 Proteobacteria Sphingobium 187 780 628 680 137 014 OTU26 Proteobacteria Curvibacter 12 124 695 130 112 822 OTU27 Proteobacteria Afipia 77 162 326 084 409 316 OTU28 Proteobacteria Aestuariivirga 291 710 435 756 81 166 OTU29 Proteobacteria Solimonas 619 750 87 994 17 044 OTU30 Proteobacteria Aquabacterium 15 084 98 008 529 220 OTU31 Proteobacteria Methyloversatilis 3 124 477 578 42 298 OTU32 Proteobacteria Mesorhizobium 156 102 248 982 41 414 OTU33 Bacteroidota Haliscomenobacter 380 072 53 280 1 696 OTU34 Nitrospirae Nitrospira 61 004 298 250 27 688 OTU35 Actinobacteria Pimelobacter 279 370 83 954 550 OTU36 Proteobacteria Methylibium 11 040 83 316 268 244 OTU37 Actinobacteria Miltoncostaea 21 526 331 566 4 632 OTU38 Proteobacteria Rubrivivax 139 984 113 738 96 944 OTU39 Proteobacteria Rhodoplanes 11 736 251 922 9 530 OTU40 Proteobacteria Pseudomonas 80 188 101 058 74 944 OTU41 Actinobacteria Mycobacteroides 128 118 106 338 20 612 OTU42 Proteobacteria Pseudorhodoplanes 45 956 160 012 44 112 OTU43 Proteobacteria Legionella 19 490 194 794 29 928 OTU44 Proteobacteria Rhodoferax 17 382 165 722 48 922 OTU45 Proteobacteria Hydrogenophaga 22 826 70 754 76 062 OTU46 Proteobacteria Methylorubrum 6 972 154 324 8 248 OTU47 Proteobacteria Acidovorax 84 840 38 906 34 702 OTU48 Proteobacteria Ideonella 19 214 26 610 52 772 OTU49 Proteobacteria Aquariibacter 856 2 218 9 060 -
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