Arthrobacter sp.W1共代谢杂环芳烃及其强化处理焦化废水的研究
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摘要
咔唑(CA)、二苯并呋喃(DBF)和二苯并噻吩(DBT)是典型的杂环芳烃，广泛存在于焦化废水中，具有“三致”作用，一旦释放到自然界便会对人体及环境造成较大的危害。相对于其它技术而言，生物法被认为是最经济、最彻底的治理此类杂环芳烃污染的方法。本研究目的在于从环境微生物学角度探究Arthrobacter sp. W1以苯酚为基质共代谢CA、DBF和DBT的特性及途径机理，结合磁性Fe3O4-结冷胶固定化技术提高其实用性，并以其作为强化菌剂，建立活化沸石曝气生物滤池处理实际焦化废水。为实际的杂环芳烃污染治理提供理论基础和技术支持。
研究以苯酚为基质对CA、DBF和DBT进行共代谢降解，并考察了菌株W1生长及休眠细胞共代谢CA、DBF和DBT的基本特性。Andrews抑制动力学分析表明，菌株W1生长细胞代谢苯酚、CA、DBF和DBT的qmax值分别为0.0471、0.0192、0.0184和0.0160h-1。菌株W1休眠细胞对不同浓度CA、DBF和DBT的代谢符合Michaelis-Menten方程，Vmax值分别为3.435，3.131和3.102mmol g cell-1h-1。在300min内，菌株W1休眠细胞耦合活化沸石可以将2mM苯酚、0.5mM萘、0.25mM CA、0.25mM DBF和0.25mM DBT均代谢至未检出，其出水IR值为20%，说明经处理后的焦化废水出水毒性显著降低。
利用二维线性离子阱-静电场轨道阱傅里叶变换组合式高分辨质谱仪(LC/MS/MS)和核磁共振(NMR)等化学分析手段鉴定出苯酚羟化酶基因工程菌(PHIND)和菌株W1共代谢CA的一系列新的代谢中间产物如1,2-dihydroxycarbazole、3,4-dihydroxycarbazole、2-oxo-4-(3-oxoindolin-2-yl)but-3-enoic acid、3-(1h-indol-2-yl)acrylic acid、2-(1h-indol-2-yl)acetaldehyde、2-吲哚乙酸、靛红和2-氨基苯乙醛酸，完善了CA共代谢途径。同时，鉴定出工程菌PHIND和菌株W1共代谢DBF和DBT的一系列中间产物，发现DBF和DBT共代谢途径与经典的DBF和DBT共代谢途径一致。为CA、DBF和DBT的生物修复及高价值中间产物的绿色生物合成奠定基础。
固定化细胞代谢CA、DBF和DBT的特性表明结冷胶最佳天然有机载体。磁性Fe3O4纳米载体的加入可以解决固定化细胞质子传递的限制，提高结冷胶固定化细胞对CA、DBF和DBT的代谢效率，且磁性Fe3O4最适浓度为80mg/L。磁性固定化细胞对不同浓度CA、DBF和DBT的代谢符合Michaelis-Menten方程，Vmax值分别为3.534，3.287和3.209mmol g cell-1h-1。相对于非磁性固定化细胞而言，磁性固定化细胞重复使用10次时，仍保持较高的苯酚、萘、CA、DBF和DBT代谢效率。在第10次使用时，磁性固定化细胞可以在240min内将焦化废水中2mM苯酚、0.5mM萘、0.25mM CA、0.25mM DBF和0.25mM DBT均代谢至未检出，且其出水IR值低于30%，说明经处理后的焦化废水毒性显著降低。
将菌株W1游离细胞及磁固定化细胞分别投加到活化沸石曝气生物滤池中进行生物强化处理含高浓度苯酚、萘、CA、DBF、DBT和NH3-N的实际焦化废水，研究结果表明，磁固定化细胞强化体系(G3反应体系)对焦化废水中所有污染物的代谢效果最好。反应体系连续运行试验表明，相对于非强化体系(G2反应体系)及游离细胞强化体系(G1反应体系)，G3反应体系耐冲击负荷能力最强，处理效果更稳定，反应体系运行后期，可以将600mg/L苯酚、200mg/L萘、60mg/L CA、90mg/L DBF、90mg/L DBT和30mg/L NH3-N均代谢至未检出。且经G3反应体系处理后的出水IR值为20%，说明经处理后焦化废水的毒性显著降低。同时，利用llumina MiSeq2000对各反应体系中微生物群落结构进行动态监测，进而揭示系统微观结构与宏观功能之间内在的联系。经生物强化的活性污泥体系，其微生物丰富度和多样性均有所增加，尤其是G3反应体系。G3反应体系中投加的Arthrobacter属为反应体系内的优势菌群，并且促进了反应体系中微生物群落的演替，推测出焦化废水中苯酚、萘、CA、DBF、DBT、COD和NH3-N等污染物高效去除取决于磁固定化细胞强化与土著微生物资源的共同作用。
Carbazole (CA), dibenzofuran (DBF) and dibenzothiophene (DBT) are recognized as the predominant nitrogen-, oxygen-, and sulfur-heterocyclic compound and have often been found in coking wastewater, which are known to possess toxic and mutagenic activities and can cause great harm to human health and the environment. Biological treatment is regarded as a feasible and cost-effective method for the removal of these refractory pollutants. The purposess of this study are to invesitigate the cometabolic degradation pathways of CA, DBF, and DBT by Arthrobacter sp. W1using phenol as the primary substrate, and the bioremediation of coking wastewater by bioaugmented zeolite-biological aerated filters (Z-BAFs) with mcrobial community dynamics.
The phenol-utilizing bacterium, Arthrobacter sp. W1, was used to cometabolically degrade CA, DBF, and DBT for the first time. Both the growing and washed cells of strain W1were capable of degrading CA, DBF, and DBT. Andrews confirmed that the presence of CA, DBF and DBT in the growing system would inhibit the cells growth and biodegradability of strain W1, and the qmax of CA, DBF and DBT were0.0471,0.0192,0.0184and0.0160h-1, respectively. The relationship between specific degradation rate and CA, DBF and DBT concentration by phenol-grown W1could be described well by Michaelis-Menten kinetics and the Vmax were3.435,3.131and3.102mmol g cell-1h-1, respectively. Real coking wastewater containing high concentration of2mM phenol,0.5mM naphthalene,0.25mM CA,0.25mM DBF,0.25mM DBT and30mg/L NH3-N could be completely degraded by phenol-grown W1coupling with activation zeolite within300min. Toxicity assessment indicated the effluent showed much lower toxicity than the untreated wastewater.
A series of newly intermediate metabolites such as1,2-dihydroxycarbazole,3,4-dihydroxycarbazole,2-oxo-4-(3-oxoindolin-2-yl)but-3-enoic acid,3-(1h-indol-2-yl)acrylic acid,2-(1h-indol-2-yl)acetaldehyde, indole-2-acetic acid, isatin, and2-aminophenyl glyoxylic acid derived from CA cometabolic degradation were identified by LC/MS/MS, LC/MS TOF accurate mass and NMR. The structures of these metabolites indicated that a novel cometabolic degradation pathway of CA was possessed by strain W1. Meanwhile, examination of metabolites by LC/MS TOF accurate mass suggested that strain W1was capable of degrading DBF and DBT via a successive hydroxylation and meta-cleavage pathway. These findings provide new insights into the cometabolic degradation process of CA, DBF and DBT and have potential applications in biotechnology and bioremediation.
The cometabolic degradation of CA, DBF and DBT by immobilized strain W1was also tested. Gellan gum and magnetic Fe3O4nanoparticle were selected as the optimal immobilization support and most suitable nanoparticle for immobilization, respcectively. The optimal concentration of magnetic Fe3O4nanoparticle was80mg/L. The relationship between specific degradation rate and the initial concentration of CA, DBF and DBT was described well by Michaelis-Menten kinetics and the Vmax were3.534，3.287and3.209mmol g cell-1h-1. The recycling experiments demonstrated that the magnetically immobilized cells coupling with activation zeolite showed highly bioremediation activity on the coking wastewater containing high concentration of phenol, naphthalene, CA, DBF and DBT during ten recycles. In the tenth cycle, magnetically immobilized cells coupling with activation zeolite could completely degrade coking wastewater after240min. Toxicity assessment indicated the treatment of the coking wastewater by magnetically immobilized cells with activation zeolite led to less toxicity than untreated wastewater.
Finally, coking wastewater was treated using Z-BAFs bioaugmented by adding free or magnetically immobilized Arthrobacter sp. W1. All treatments were carried out for a period of100days and the data indicated that G3(bioaugmented Z-BAF with magnetically immobilizedstrain W1) was the most efficient for treating coking wastewaters. Coking wastewater containing600mg/L phenol,200mg/L naphthalene,60mg/L CA,90mg/L DBF,90mg/L DBT and30mg/L NH3-N could be completely degraded G3. Illumina sequencing data revealed that the bacterial richness and diversity was recovered by both bioaugmented Z-BAF treatments and that the biodiversity was especially high after phenol, naphthalene, CA, DBF, DBT, and COD loading shocks. Notably, treatment with magnetically immobilized cells showed the highest biodiversity. Furthermore, both bioaugmentation treatments accelerated the shift of the bacterial community structure such that there was a more distinct difference from the structure of the starting bacterial community. Magnetically immobilized cells of strain W1was14%of the total bacterial population in G3on100thday. Thus, the data indicate that both the introduced strain W1and the indigenous degrading bacteria played a significant role in the treatments. These findings were also consistent with the treatment data of G3.
研究以苯酚为基质对CA、DBF和DBT进行共代谢降解，并考察了菌株W1生长及休眠细胞共代谢CA、DBF和DBT的基本特性。Andrews抑制动力学分析表明，菌株W1生长细胞代谢苯酚、CA、DBF和DBT的qmax值分别为0.0471、0.0192、0.0184和0.0160h-1。菌株W1休眠细胞对不同浓度CA、DBF和DBT的代谢符合Michaelis-Menten方程，Vmax值分别为3.435，3.131和3.102mmol g cell-1h-1。在300min内，菌株W1休眠细胞耦合活化沸石可以将2mM苯酚、0.5mM萘、0.25mM CA、0.25mM DBF和0.25mM DBT均代谢至未检出，其出水IR值为20%，说明经处理后的焦化废水出水毒性显著降低。
利用二维线性离子阱-静电场轨道阱傅里叶变换组合式高分辨质谱仪(LC/MS/MS)和核磁共振(NMR)等化学分析手段鉴定出苯酚羟化酶基因工程菌(PHIND)和菌株W1共代谢CA的一系列新的代谢中间产物如1,2-dihydroxycarbazole、3,4-dihydroxycarbazole、2-oxo-4-(3-oxoindolin-2-yl)but-3-enoic acid、3-(1h-indol-2-yl)acrylic acid、2-(1h-indol-2-yl)acetaldehyde、2-吲哚乙酸、靛红和2-氨基苯乙醛酸，完善了CA共代谢途径。同时，鉴定出工程菌PHIND和菌株W1共代谢DBF和DBT的一系列中间产物，发现DBF和DBT共代谢途径与经典的DBF和DBT共代谢途径一致。为CA、DBF和DBT的生物修复及高价值中间产物的绿色生物合成奠定基础。
固定化细胞代谢CA、DBF和DBT的特性表明结冷胶最佳天然有机载体。磁性Fe3O4纳米载体的加入可以解决固定化细胞质子传递的限制，提高结冷胶固定化细胞对CA、DBF和DBT的代谢效率，且磁性Fe3O4最适浓度为80mg/L。磁性固定化细胞对不同浓度CA、DBF和DBT的代谢符合Michaelis-Menten方程，Vmax值分别为3.534，3.287和3.209mmol g cell-1h-1。相对于非磁性固定化细胞而言，磁性固定化细胞重复使用10次时，仍保持较高的苯酚、萘、CA、DBF和DBT代谢效率。在第10次使用时，磁性固定化细胞可以在240min内将焦化废水中2mM苯酚、0.5mM萘、0.25mM CA、0.25mM DBF和0.25mM DBT均代谢至未检出，且其出水IR值低于30%，说明经处理后的焦化废水毒性显著降低。
将菌株W1游离细胞及磁固定化细胞分别投加到活化沸石曝气生物滤池中进行生物强化处理含高浓度苯酚、萘、CA、DBF、DBT和NH3-N的实际焦化废水，研究结果表明，磁固定化细胞强化体系(G3反应体系)对焦化废水中所有污染物的代谢效果最好。反应体系连续运行试验表明，相对于非强化体系(G2反应体系)及游离细胞强化体系(G1反应体系)，G3反应体系耐冲击负荷能力最强，处理效果更稳定，反应体系运行后期，可以将600mg/L苯酚、200mg/L萘、60mg/L CA、90mg/L DBF、90mg/L DBT和30mg/L NH3-N均代谢至未检出。且经G3反应体系处理后的出水IR值为20%，说明经处理后焦化废水的毒性显著降低。同时，利用llumina MiSeq2000对各反应体系中微生物群落结构进行动态监测，进而揭示系统微观结构与宏观功能之间内在的联系。经生物强化的活性污泥体系，其微生物丰富度和多样性均有所增加，尤其是G3反应体系。G3反应体系中投加的Arthrobacter属为反应体系内的优势菌群，并且促进了反应体系中微生物群落的演替，推测出焦化废水中苯酚、萘、CA、DBF、DBT、COD和NH3-N等污染物高效去除取决于磁固定化细胞强化与土著微生物资源的共同作用。
Carbazole (CA), dibenzofuran (DBF) and dibenzothiophene (DBT) are recognized as the predominant nitrogen-, oxygen-, and sulfur-heterocyclic compound and have often been found in coking wastewater, which are known to possess toxic and mutagenic activities and can cause great harm to human health and the environment. Biological treatment is regarded as a feasible and cost-effective method for the removal of these refractory pollutants. The purposess of this study are to invesitigate the cometabolic degradation pathways of CA, DBF, and DBT by Arthrobacter sp. W1using phenol as the primary substrate, and the bioremediation of coking wastewater by bioaugmented zeolite-biological aerated filters (Z-BAFs) with mcrobial community dynamics.
The phenol-utilizing bacterium, Arthrobacter sp. W1, was used to cometabolically degrade CA, DBF, and DBT for the first time. Both the growing and washed cells of strain W1were capable of degrading CA, DBF, and DBT. Andrews confirmed that the presence of CA, DBF and DBT in the growing system would inhibit the cells growth and biodegradability of strain W1, and the qmax of CA, DBF and DBT were0.0471,0.0192,0.0184and0.0160h-1, respectively. The relationship between specific degradation rate and CA, DBF and DBT concentration by phenol-grown W1could be described well by Michaelis-Menten kinetics and the Vmax were3.435,3.131and3.102mmol g cell-1h-1, respectively. Real coking wastewater containing high concentration of2mM phenol,0.5mM naphthalene,0.25mM CA,0.25mM DBF,0.25mM DBT and30mg/L NH3-N could be completely degraded by phenol-grown W1coupling with activation zeolite within300min. Toxicity assessment indicated the effluent showed much lower toxicity than the untreated wastewater.
A series of newly intermediate metabolites such as1,2-dihydroxycarbazole,3,4-dihydroxycarbazole,2-oxo-4-(3-oxoindolin-2-yl)but-3-enoic acid,3-(1h-indol-2-yl)acrylic acid,2-(1h-indol-2-yl)acetaldehyde, indole-2-acetic acid, isatin, and2-aminophenyl glyoxylic acid derived from CA cometabolic degradation were identified by LC/MS/MS, LC/MS TOF accurate mass and NMR. The structures of these metabolites indicated that a novel cometabolic degradation pathway of CA was possessed by strain W1. Meanwhile, examination of metabolites by LC/MS TOF accurate mass suggested that strain W1was capable of degrading DBF and DBT via a successive hydroxylation and meta-cleavage pathway. These findings provide new insights into the cometabolic degradation process of CA, DBF and DBT and have potential applications in biotechnology and bioremediation.
The cometabolic degradation of CA, DBF and DBT by immobilized strain W1was also tested. Gellan gum and magnetic Fe3O4nanoparticle were selected as the optimal immobilization support and most suitable nanoparticle for immobilization, respcectively. The optimal concentration of magnetic Fe3O4nanoparticle was80mg/L. The relationship between specific degradation rate and the initial concentration of CA, DBF and DBT was described well by Michaelis-Menten kinetics and the Vmax were3.534，3.287and3.209mmol g cell-1h-1. The recycling experiments demonstrated that the magnetically immobilized cells coupling with activation zeolite showed highly bioremediation activity on the coking wastewater containing high concentration of phenol, naphthalene, CA, DBF and DBT during ten recycles. In the tenth cycle, magnetically immobilized cells coupling with activation zeolite could completely degrade coking wastewater after240min. Toxicity assessment indicated the treatment of the coking wastewater by magnetically immobilized cells with activation zeolite led to less toxicity than untreated wastewater.
Finally, coking wastewater was treated using Z-BAFs bioaugmented by adding free or magnetically immobilized Arthrobacter sp. W1. All treatments were carried out for a period of100days and the data indicated that G3(bioaugmented Z-BAF with magnetically immobilizedstrain W1) was the most efficient for treating coking wastewaters. Coking wastewater containing600mg/L phenol,200mg/L naphthalene,60mg/L CA,90mg/L DBF,90mg/L DBT and30mg/L NH3-N could be completely degraded G3. Illumina sequencing data revealed that the bacterial richness and diversity was recovered by both bioaugmented Z-BAF treatments and that the biodiversity was especially high after phenol, naphthalene, CA, DBF, DBT, and COD loading shocks. Notably, treatment with magnetically immobilized cells showed the highest biodiversity. Furthermore, both bioaugmentation treatments accelerated the shift of the bacterial community structure such that there was a more distinct difference from the structure of the starting bacterial community. Magnetically immobilized cells of strain W1was14%of the total bacterial population in G3on100thday. Thus, the data indicate that both the introduced strain W1and the indigenous degrading bacteria played a significant role in the treatments. These findings were also consistent with the treatment data of G3.
引文
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