摘要

       近些年来,抗生素抗性基因(Antibiotic resistance genes,ARGs)作为一种重要的新型环境污染物,其污染问题日益严峻并逐渐受到国内外研究学者的广泛关注。污水处理厂(Wastewater treatment plants,WWTPs)废水污泥被认为是ARGs的重要储存库,传统的污水处理工艺往往会使ARGs在生物处理单元从废水转移到固体污泥当中,导致污泥中的ARGs大量富集。因此,剩余污泥的处理处置方法不当可能会造成ARGs的二次污染,对环境安全和人类健康造成潜在威胁。无论采用何种方式对剩余污泥进行处理处置,都需要预先对污泥进行脱水处理以降低污泥含水率、减少污泥体积,其中,促进污泥脱水的生物预调理法具有价格低廉、处理方便和环境友好等优点。课题组采用一株从矿坑废水中分离纯化而得的嗜酸性氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans XJF8,NCBI Accession:KF378735)在实验室内构建了生物酸化铁氧化深度脱水工艺,前期研究表明该工艺能够促进污泥深度脱水(含水率60%),能够实现污泥的减量化。本文采用高通量定量PCR技术,探究了该工艺对市政污水污泥中8类典型ARGs(含移动基因元件)的40个基因亚型的去除效果。结果表明,该工艺对污泥中能够检出的16种目的ARGs亚型均有良好的去除效果(去除率为9.40%~68.18%),其中去除效果最好的为mphA-02基因。我们通过16S r RNA高通量测序技术对工艺处理过程中污泥样本的菌群结构进行了分析,发现处理后变形菌门、厚壁菌门和酸杆菌门的物种相对丰度显著提高,而酸性敏感菌和厌氧菌则明显减少。为了探究环境因子(pH和Fe~(2+))与污泥样本和污泥菌群之间的相互关系,我们采用R语言工具对三者进行了RDA分析,结果表明pH和Fe~(2+)对污泥样本和污泥菌群有显著影响(P0.05),并与变形菌门、厚壁菌门和互养菌门呈正相关关系。同时,定量PCR结果显示供试菌Acidithiobacillus ferrooxidans XJF8在工艺处理各阶段的生物量很低(低于0.05%),这可能是由于该菌作为自养菌的生长特性决定的,我们认为该菌的存在改变了污泥环境条件进而影响污泥菌群,从而实现目的ARGs的去除。为此,本文利用斯皮尔曼相关性检验(Spearman correlation)分析了污泥菌群与目的ARGs亚型之间的潜在关联,发现变形菌门和厚壁菌门的菌属对ARGs的去除贡献最大。最后,本文对供试菌株Acidithiobacillus ferrooxidans XJF8进行了全基因组扫描测序,将获得的基因序列与COG、GO、KEGG和CARD等数据库进行blast比对得到基因注释信息,使得从基因水平上对该菌的基本结构功能有了初步了解,为进一步探究Acidithiobacillus ferrooxidans XJF8在本工艺体系及其他污染治理应用中的作用机制奠定了基础。

       In recent years, Antibiotic resistance genes (ARGs), as an important new environmental pollutant, have become increasingly serious and have gradually attracted widespread attention from domestic and foreign researchers. Wastewater treatment plant (Wastewater treatment plants, WWTPs) wastewater sludge is considered to be an important storage reservoir for ARGs. Traditional wastewater treatment processes often cause ARGs to be transferred from wastewater to solid sludge in biological treatment units, resulting in ARGs in sludge. A large amount of enrichment. Therefore, improper treatment and disposal methods of surplus sludge may cause secondary pollution of ARGs, posing potential threats to environmental safety and human health. Regardless of the method used to treat and dispose of the remaining sludge, it is necessary to pre-dehydrate the sludge to reduce the water content of the sludge and reduce the volume of the sludge. Convenience and environmental friendliness. The research group used a strain of Acidithiobacillus ferrooxidans (Acidithiobacillus ferrooxidans XJF8, NCBI Accession: KF378735) isolated and purified from mine wastewater to construct a biological acidified iron oxidation deep dehydration process in the laboratory. It can promote the deep dewatering of sludge (60% moisture content) and realize the reduction of sludge. In this paper, high-throughput quantitative PCR technology was used to explore the effect of this process on the removal of 40 genotypes of 8 typical ARGs (including mobile genetic elements) in municipal sewage sludge. The results show that the process has a good removal effect on the 16 target ARGs subtypes that can be detected in the sludge (removal rate is 9.40%~68.18%), and the best removal effect is the mphA-02 gene. We used 16S r RNA high-throughput sequencing technology to analyze the flora structure of the sludge samples during the process, and found that the relative abundance of the species of the Proteobacteria, Firmicutes and Acidobacteria was significantly increased after treatment. Acid-sensitive bacteria and anaerobes were significantly reduced. In order to explore the relationship between environmental factors (pH and Fe~(2+)), sludge samples and sludge flora, we used R language tools to perform RDA analysis on the three, and the results showed that pH and Fe~(2 +) It has a significant impact on the sludge samples and the sludge flora (P0.05), and is positively correlated with the phylum Proteobacteria, Firmicutes and Cotrophic bacteria. At the same time, the results of quantitative PCR showed that the biomass of the tested bacteria Acidithiobacillus ferrooxidans XJF8 at each stage of the process was very low (less than 0.05%), which may be due to the growth characteristics of the bacteria as an autotrophic bacteria. Existence changes the environmental conditions of the sludge and then affects the sludge flora, so as to achieve the purpose of removing ARGs. To this end, this paper uses Spearman correlation to analyze the potential association between the sludge flora and the target ARGs subtype, and finds that the bacteria of the Proteobacteria and Firmicutes contribute the most to the removal of ARGs. . Finally, this article performed a whole-genome scan and sequencing of the tested strain Acidithiobacillus ferrooxidans XJF8, and blast compared the obtained gene sequence with databases such as COG, GO, KEGG and CARD to obtain gene annotation information. A preliminary understanding of the basic structure and functions has laid a foundation for further exploring the mechanism of Acidithiobacillus ferrooxidans XJF8 in this process system and other pollution control applications.

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