Beyond pollutant removal - understanding the biochemical mechanism of sulfonamide degradation in wastewater and the contribution of ipso-substitution.

超越污染物去除 - 了解废水中磺酰胺降解的生化机制以及自身替代的贡献。

• 批准号: 273126418
• 负责人: Professor Dr. Harald Horn, since 6/2016
• 金额: --
• 依托单位: Lehrstuhl für Wasserchemie und Wassertechnologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/273126418?language=en
• 关键词: Beyond; pollutant; removal; understanding; biochemical

Sulfonamide antibiotics are are only insufficiently removed in wastewater treatment plants, and can therefore be readily detected in the aquatic environment. Constant exposure of microorganisms even to non-inhibitory concentrations has been shown to induce the propagation of antibiotic resistances, which is a cause of concern with regard to the rising number of reports on multi-resistant pathogens. Microbacterium sp. strain BR1 has been isolated from an enrichment culture from activated sludge and is able to degrade sulfonamide antibiotics as a source of carbon and energy. In this strain, enzymes and their encoding genes have been identified which are responsible sulfonamide degradation. An FMN-dependent monooxygenase, encoded by the sadA gene, was shown to catalyze ipso-substitution of sulfonamides, initiating their fragmentation to 4-benzoquinone-imine, sulfite and the moiety previously attached to the sulfo group. While 4-benzoquinone-imine serves as the source of carbon and energy, the mostly heterocyclic moiety was shown to remain a dead-end metabolite in culture supernatants.In the proposed project, we will evaluate whether the identified dead-end metabolites can also be linked to biological sulfonamide degradation. At the same time it will be investigated if the presence of homolog bacterial genes detected in selected wastewater treatment plants can be attributed to sulfonamide degrading activity. We will obtain genome sequences from further isolated sulfonamide degrading strains to investigate the presence of sadA homologs. From homolog sequences, a consensus sequence will be calculated to serve as a base for PCR primers to specifically amplify and quantify sadA homologs in activated sludge. Sequences flanking these homologs will be analyzed for the presence of gene clusters resembling that in Microbacterium sp. strain BR1 Sulfonamide removal rate of the sludges will be determined by quantification of sulfonamides and dead-end metabolites in influent and effluent of the wastewater treatment plants. Additionally, the ability of the sludge to degrade artificially added sulfamethoxazole will be assayed.For pure strains, sulfonamide degradation at various nutrient conditions will be assayed to establish thresholds for up- or down-regulation of genes associated with ipso-substitution of sulfonamides.Our overall aim is to transfer findings from the preceding project funded by the SNF and others to provide a better understanding of sulfonamide degradation in wastewater treatment plants, compared to the current practice, which is a mere balance of influent and effluent concentrations of the parent compound, or at best acetylated derivatives of that. Bringing all ends together, it will be possible to assess how much sulfonamides are really degraded, and which contribution to that stems from homologs of the FMN-dependent monooxygenase isolated in the preceding project.

磺酰胺类抗生素在废水处理厂中仅被充分去除，因此在水生环境中很容易被检测到。已显示，即使在非抑制浓度下持续接触微生物也会诱导抗生素耐药性的传播，这是关于多重耐药病原体报告数量不断增加的一个令人担忧的原因。微杆菌属菌株BR 1已从活性污泥的富集培养物中分离出来，并且能够降解作为碳源和能源的磺胺类抗生素。在该菌株中，已经鉴定了负责磺胺降解的酶及其编码基因。由sadA基因编码的FMN依赖性单加氧酶被证明催化磺胺类药物的ipso取代，引发它们断裂为4-苯醌亚胺、亚硫酸盐和先前连接到磺基的部分。虽然4-苯醌亚胺作为碳和能量的来源，但大多数杂环部分被证明在培养上清液中仍然是死端代谢物。在拟议的项目中，我们将评估所鉴定的死端代谢物是否也与磺胺的生物降解有关。同时，将调查在选定的污水处理厂中检测到的同源细菌基因的存在是否可归因于磺胺降解活性。我们将从进一步分离的磺胺降解菌株中获得基因组序列，以研究sadA同源物的存在。从同源物序列中，将计算共有序列以用作PCR引物的碱基，以特异性扩增和定量活性污泥中的sadA同源物。将分析这些同源物侧翼的序列，以确定是否存在类似于微杆菌属菌株BR 1中的基因簇。将通过定量污水处理厂的流入物和流出物中的磺胺和死端代谢物来确定污泥的磺胺去除率。此外，将测定污泥降解人工添加的磺胺甲恶唑的能力。对于纯菌株，将测定在各种营养条件下的磺胺降解，以建立与ipso相关的基因的上调或下调的阈值，我们的总体目标是转移由SNF和其他人资助的先前项目的发现，以更好地了解磺胺类药物的降解，与目前的实践相比，目前的实践仅仅是母体化合物或其最好的乙酰化衍生物的流入物和流出物浓度的平衡。将所有目的结合在一起，将有可能评估有多少磺胺类药物真正被降解，以及在前一个项目中分离的FMN依赖性单加氧酶的同源物对此的贡献。