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Related Experiment Video

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A Small Volume Procedure for Viral Concentration from Water
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Method evaluation for viruses in activated sludge: Concentration, sequencing, and identification.

Yulin Zhang1, Xiawan Zheng1, Weifu Yan1

  • 1Environmental Microbiome Engineering and Biotechnology Lab, Department of Civil Engineering, The University of Hong Kong, Pokfulam, Road, Hong Kong, China.

The Science of the Total Environment
|October 17, 2024
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Summary

This study optimizes methods for analyzing viruses in activated sludge (AS), recommending flocculation, filtration, and resuspension (FFR) and tangential flow filtration for concentration. geNomad and long-read sequencing are best for viral identification in AS.

Keywords:
Activated sludgeFlocculation filtration and resuspensionPhagesTangential flow filtrationVirusWastewater treatment plants

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Area of Science:

  • Environmental microbiology
  • Virology
  • Wastewater treatment

Background:

  • Activated sludge (AS) is a crucial microbial ecosystem in wastewater treatment, hosting abundant viruses.
  • Viral communities in AS are understudied due to challenges in concentration, extraction, and sequencing.
  • Understanding AS viruses is vital for optimizing wastewater treatment processes.

Purpose of the Study:

  • To evaluate and compare different methods for viral analysis in activated sludge.
  • To identify optimal concentration techniques, sequencing approaches, and bioinformatic tools for AS virome studies.
  • To provide insights into the viral composition and host interactions within AS ecosystems.

Main Methods:

  • Comparison of four viral concentration methods: flocculation, filtration, resuspension (FFR), and ultracentrifugation.
  • Evaluation of two sequencing approaches: short-read and long-read sequencing.
  • Assessment of four viral identification bioinformatic tools, with a focus on geNomad.

Main Results:

  • FFR yielded the longest viral DNA, while ultracentrifugation provided the highest DNA yield.
  • FFR and tangential flow filtration (100 kDa) are recommended for concentrating viruses from large AS volumes.
  • geNomad and long-read sequencing demonstrated superior performance for viral identification and assembly statistics.
  • Multiple concentration methods are necessary for a comprehensive viral catalog.
  • The AS virome is dominated by phages (95.1%), primarily Caudovirales, with virulent phages targeting Pseudomonadota.

Conclusions:

  • This study provides a methodological framework for effective viral research in activated sludge.
  • Recommended methods include FFR or tangential flow filtration for concentration and geNomad with long-read sequencing for identification.
  • The findings highlight the dominance of virulent phages in AS and their primary hosts.