Wu Qinglong, Liu Wen-Tso
Division of Environmental Sciences and Engineering, Faculty of Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore.
Water Res. 2009 Mar;43(4):1101-9. doi: 10.1016/j.watres.2008.11.039. Epub 2008 Dec 6.
Procedures to extract and count sludge viruses-like particles from municipal sewage treatment plant were optimized by epifluorescence microscopy using SYBR Green I as a stain. The highest indigenous virus yields from the bulk of the anaerobic digestion sludge and influent (solid) were obtained by utilizing 10 mM sodium pyrophosphate as eluant solution with vortex and 1 min of sonication. The use of 1x phosphate buffered saline as eluant with vortex and 1 min of sonication yields highest indigenous virus from activated sludge. The efficiency of extracting indigenous viruses by sodium pyrophosphate-ultrasound treatment was about 62% of the extractable virus particles from activated sludge and 87% for anaerobic digestion sludge, respectively. Samples treated with DNase had decreased, but not significant, virus counts, suggesting a minor effect of extracellular DNA on virus count. Following the optimized procedure, we investigated the abundance and diversity of virus particles in the wastewater stream of a municipal treatment plant. The concentrations of virus particles ranged from 0.28 x 10(9) ml(-1) to 27.04 x 10(9) ml(-1). Transmission electron microscopy (TEM) showed a high variety of virus morphotypes in sludge. Pulsed-field gel electrophoresis (PFGE) revealed a diverse and dynamic viral community in different stages of the system with genome sizes ranging from 33 kb to >350 kb with most of the viral DNA in the 30-80 kb and 200-350 kb size ranges. Collectively, our study suggested that indigenous viruses are abundant and dynamic in the municipal wastewater treatment system and may play an important role in functioning of the system.
通过使用SYBR Green I作为染色剂的落射荧光显微镜法，优化了从城市污水处理厂提取和计数污泥类病毒颗粒的程序。通过使用10 mM焦磷酸钠作为洗脱液，涡旋并超声处理1分钟，可从大部分厌氧消化污泥和进水（固体）中获得最高的本地病毒产量。使用1x磷酸盐缓冲盐水作为洗脱液，涡旋并超声处理1分钟，可从活性污泥中获得最高的本地病毒产量。焦磷酸钠-超声处理提取本地病毒的效率分别约为活性污泥中可提取病毒颗粒的62%和厌氧消化污泥的87%。用DNase处理的样品病毒计数有所下降，但不显著，表明细胞外DNA对病毒计数的影响较小。按照优化程序，我们研究了城市污水处理厂废水流中病毒颗粒的丰度和多样性。病毒颗粒浓度范围为0.28×10⁹ ml⁻¹至27.04×10⁹ ml⁻¹。透射电子显微镜（TEM）显示污泥中有多种病毒形态类型。脉冲场凝胶电泳（PFGE）揭示了系统不同阶段中一个多样且动态的病毒群落，基因组大小范围从33 kb到>350 kb，大多数病毒DNA在30 - 80 kb和200 - 350 kb大小范围内。总体而言，我们的研究表明本地病毒在城市污水处理系统中丰富且动态变化，可能在该系统的功能发挥中起重要作用。
