蛭弧菌对污泥的生物裂解脱水减量并协同削减抗生素抗性基因作用与机理

The existing conventional municipal waste sludge dewatering processes generally are not efficient in disrupting sludge cells, which result in insufficient releases of sludge intracellular water and low sludge dewatering efficiencies. The required additions of conditioning chemical reagents cause high cost of sludge treatment, increased inorganic mass content in sludge, and potential secondary pollution. Bdellovibrio-and-like organisms (BALOs) are a group of small and ubiquitous predatory parasitic bacteria with unique ability to penetrate and lyse a wide variety of bacteria for proliferation. In this proposal, BALOs will be screened from municipal sludge and applied for the development of a new technology for sludge biolysis and dewaterability improvement. BALO has a distinct parasitic biphasic life cycle. Both the hindering effects of sludge extracellular polymeric substances (EPS) on the movement ability of BALOs in their free-attack phase towards hosts and their acceleration potential as the energy source will be investigated. Polyhydroxyalkanoates (PHA) commonly exist in bacteria as the energy storage substances. The ability of PHA to ensure and enhance the movement and the attack performance of BALOs in sludge will be addressed. At the same time, the reduction of antibiotic resistance genes (ARGs) in sludge by BALOs in their growth phase will be revealed, considering BALOs’ requirement to degrade and utilize host genetic materials for proliferation. In summary, the BALOs’ sludge biolysis effects with the possible mechanisms on the improvement of both sludge dewaterability and its environmental and hygienic safety will be explored. The reaction control parameters and regulation strategies will be determined. This study will provide an environmentally friendly and cost-effective ecological solution to alleviate the pressure of sludge treatment and disposal.

现有常规市政污泥脱水工艺对污泥细胞破壁裂解困难，胞内水难以释放，污泥脱水效率低；而化学调理剂的投加更导致污泥处理费用高、无机干质增加、易产生二次污染。蛭弧菌是一类具有宿主广谱性的小型寄生性细菌。本项目拟利用蛭弧菌的专性噬菌特性，自市政污泥中筛选出具有高效裂解污泥活性的土著蛭弧菌并用于污泥的脱水减量新技术研究。结合蛭弧菌的二态生活周期特点，本项目将重点阐释污泥胞外多聚物对进攻期蛭弧菌向宿主菌运动趋近的阻滞影响以及作为能源物质对蛭弧菌裂解污泥活性的促进潜能，掌握作为能源储备物质的聚羟基脂肪酸酯对保证与提高蛭弧菌运动与攻击水平的作用规律，探讨繁殖期蛭弧菌降解利用宿主菌遗传物质进行增殖并协同消减污泥中抗生素抗性基因的能力，以期系统掌握蛭弧菌裂解污泥以提高其脱水性能与环境卫生安全性的作用与机理，明确并优化反应关键控制参数与调控策略，为缓解污泥处理处置压力提供一条环境友好、经济高效的生态解决途径。

本项目重点探讨了借助好氧专性噬菌蛭弧菌的独特增殖特性以提高市政污泥脱水性能并协同消减抗生素抗性基因(ARG)的潜力与作用机理。当污泥浓度为15-25g/L、pH为中性、溶解氧浓度为3-4mg/L、反应温度为30℃时将筛选的高效裂解D15菌株与污泥混合反应16-24h,可获最好污泥裂解效率，污泥比阻(SRF)和毛细吸水时间(CST)分别可降低50%和20%以上。通过筛选出的与D15具有互补宿主裂解谱的高效污泥裂解蛭弧菌D18菌株与D15按1：1体积比复配，可最有效促进污泥脱水性能，CST下降率较单一D15菌剂作用提高(25±4.6)%。污泥中的胞外多聚物(EPS)经蛭弧菌生物裂解处理后，蛋白质和多糖含量显著降低，其中紧密型EPS(TB-EPS)中多糖、蛋白质含量下降率分别达(60.5±1.7)%和(78.6±2.6)%，TB-EPS中色氨酸类蛋白和络氨酸类蛋白的三维荧光强度同步降低，预示蛭弧菌生物裂解时可分解利用TB-EPS中营养物质，并对特定蛋白质具有能量趋向性。当污泥TB-EPS作为培养基质时，蛭弧菌捕食活性相对于对照组提高了(51±1.6)%。另鉴于受攻击宿主菌细胞的裂解释放了胞内物质，经蛭弧菌处理的污泥中聚羟基脂肪酸酯(PHA)浓度增加了(46.5±1.7)%。外源投加PHA可显著促进蛭弧菌裂解污泥效率。当PAH浓度为2.5g/L时，污泥CST经蛭弧菌作用8小时可达到最低值，并相对空白对照组和无外源PHA的实验组分别降低了(28±3.1)%和(17±3.4)%。针对污泥中9种典型ARG研究发现，蛭弧菌可有效裂解内嵌入不同ARGs重组质粒的大肠杆菌；是否携带ARG片段不影响细胞裂解效率。蛭弧菌可使污泥中9种ARGs的去除率达到(2.5±1.3)% -(87.3±1.0)%，其中tet Q的去除效率最高。高通量测序结果显示蛭弧菌降低了污泥中携带ARGs的宿主病原菌(分枝杆菌、蛇颈囊杆菌、伯克霍尔德菌和拟杆菌)的相对丰度污泥并同时减少了病原菌(不动杆菌、气单胞菌、梭状芽胞杆菌、克雷伯氏菌和假单胞菌等)的相对丰度，推测对ARGs潜在宿主的有效攻击以及对胞内ARGs的降解重构是蛭弧菌削减污泥中ARGs，协同提高污泥脱水性能和生态安全性的重要机制。本课题为形成以蛭弧菌应用为核心的、绿色安全与经济高效的污泥生物裂解脱水减量新技术体系提供了必要的理论支持和数据支撑。