Simple, Rapid, Chemical-Free Concentration of Bacteriophages from Anaerobic Digestion Sludge and the Impact of Cold Storage on Viral Community Composition

Bacteriophages, the most abundant biological entities on Earth, remain vastly understudied and are often referred to as microbial “dark matter” due to the challenges in isolating and characterizing them. This is especially true in complex matrices like anaerobic digestion sludge. The first part of the study presents a simple and cost-effective protocol for phage concentration from anaerobic digester samples, optimized for subsequent extraction of nucleic acids enabling virome analysis. The entire workflow requires less than one hour to process ten samples under optimal conditions (excluding further downstream processes), using as little as 1 mL of sludge per sample. Phages were concentrated using an ultrafiltration-based approach with protein concentrators (100K MWCO), eliminating the need for expensive instrumentation or chemical reagents. The second part compares the impact of cold storage conditions by refrigeration (4 °C) for one week and a single freeze–thaw cycle at −20 °C on viral community composition.

PCoA visualization and statistical analysis using PERMANOVA revealed no significant difference between ultrafiltered samples and unfiltered negative controls (p = 0.66), suggesting that ultrafiltration does not introduce bias in community profiling. On the other hand, refrigeration demonstrated minimal impact (p = 0.14), while a single freeze–thaw cycle significantly altered the community structure (p = 0.01).

Together, these findings demonstrate that the proposed protocol enables reliable phage recovery and emphasize the importance of storage conditions in preserving community integrity. The method’s scalability and minimal input requirements make it well-suited for broader application in environmental virology and wastewater research.