Optimization of viral resuspension methods for carbon-rich soils along a permafrost thaw gradient

Abstract

Permafrost stores approximately 50% of global soil carbon (C) in a frozen form; it is thawing rapidly under climate change, and little is known about viral communities in these soils or their roles in C cycling. In permafrost soils, microorganisms contribute significantly to C cycling, and characterizing them has recently been shown to improve prediction of ecosystem function. In other ecosystems, viruses have broad ecosystem and community impacts ranging from host cell mortality and organic matter cycling to horizontal gene transfer and reprogramming of core microbial metabolisms. Here we developed an optimized protocol to extract viruses from three types of high organic-matter peatland soils across a permafrost thaw gradient (palsa, moss-dominated bog, and sedge-dominated fen). Three separate experiments were used to evaluate the impact of chemical buffers, physical dispersion, storage conditions, and concentration and purification methods on viral yields. The most successful protocol, amended potassium citrate buffer with bead-beating or vortexing and BSA, yielded on average as much as 2-fold more virus-like particles (VLPs) g−1 of soil than other methods tested. All method combinations yielded VLPs g−1 of soil on the 108 order of magnitude across all three soil types. The different storage and concentration methods did not yield significantly more VLPs g−1 of soil among the soil types. This research provides much-needed guidelines for resuspending viruses from soils, specifically carbon-rich soils, paving the way for incorporating viruses into soil ecology studies.

Uncovering a hidden diversity: optimized protocols for the extraction of bacteriophages from soil

“…We compared the most promising elution buffers found in literature to maximize bacteriophage suspension from agricultural soil samples (Figure 1). For this, the most commonly used (or elsewhere optimized) elution buffers such as SM buffer [9, 10], amended potassium citrate (AKC) [1], 10 % beef extract [3, 18] or PBS amended with beef extract [13] were assessed and compared in viral yield using plaque assay. When soil samples were suspended using SM or AKC buffer, as little as 0.5 to 5 % of all spiked phages could be recovered (Figure 2a and 2b).…”

“…Soil samples were spiked with an artificial viral community (ΦA511, Φ2638AΔLCR, ΦT7 at 10 6 PFU/g soil) and bacteriophage yield was assessed using plaque assay. ( a ) SM buffer [9, 10]; 10 % beef extract buffer [3, 18]; PBS supplemented with beef extract [32]; AKC [1]. ( b ) Elution amelioration measured by plaque assay (Mean ± SD) when using optimized PPBS instead of literature proposed elution buffer.…”

“…Soil bacteriophages are a vital part of soil bacterial ecology and a major reservoir of genetic material that contributes to biological evolution and diversity. They also influence the flow of nutrients as a potential cause of microbial distribution and mortality [1, 2]. Soil is known to harbour a vast abundance of phages (10 8 −10 9 pfu·g −1 ), with their numbers exceeding those of co-occurring bacteria by 10 – 1000 fold [3–5].…”

“…Given the physicochemical diversity of soils, the soil matrix complexity and its high microbial diversity [2], it is not surprising that no universal phage extraction protocol or standardization towards viral elution, concentration and DNA extraction have yet been proposed. Only few phage extraction protocols from soil samples have been suggested in literature, which frequently suffer from a low recovery rate of phages (< 5 %), and downstream metagenome analyses therefore require DNA amplification [1, 3, 7–17]. Moreover, the most often applied rather harsh methods may render phage isolation impossible, or imply the use of equipment that is not available in standard laboratories.…”

“…Among those studies, soil phage extraction has included a wide range of elution media, such as deionized water [7, 8], SM buffer [9–11], potassium citrate buffer [4], 10 % beef extract [3, 18], modified potassium citrate buffer [1], Na/K buffer [12], or phosphate buffered saline (PBS) supplemented with beef extract [13]. Those elution media are commonly combined with mechanical approaches to disrupt phage soil interactions, such as homogenization [13], sonication [3, 4, 14], vortexing [1, 3, 11], shaking [8, 10, 15], magnetic stirring [18] or bead-beating [16, 17]. Despite these diverse approaches, the elution and recovery of soil phages remains the major bottleneck in the extraction, since more than 90 % of viruses tend to absorb to soil particles [2, 19].…”