Direct cell extraction from fresh and stored soil samples: Impact on microbial viability and community compositions

Highlights

• We optimized the procedure of soil cell extraction with two rounds of extraction.

• Blending + Tween 20 + 80% Nycodenz had the highest cell viability and yield.

• Repeated extraction increased the cell yield but reduced the cell viability.

• 4 °C short-term storage is suitable for efficient soil viable cell extraction.

Abstract

The direct extraction of viable microbes from soil samples is critical for the application of many single-cell related technologies. However, there are many aspects of extraction technologies that can impact the viability and diversity of extractable cells from fresh or stored soil samples. In this study, physical dispersion method, chemical dispersion method, and Nycodenz density gradient medium concentration were optimized with two sequential rounds of cell extraction from four soil samples having diverse physicochemical properties. Cell viability was quantified with fluorescence staining and flow cytometry. The viable microbial community compositions in soil extractable cells and soil samples were assessed after selective removal of DNA from dead cells. Among the four different extraction and purification methods, a protocol that included physical blending, Tween 20 treatment, and centrifugation with 80% Nycodenz, had the highest cell viability and yield. Repeated extraction increased the yield but reduced the cell viability. The over- or under-represented taxa in extractable cells might contribute to the bias of the extractable microbial communities. Using the optimized cell extraction procedure, the effect of soil storage conditions (4 °C, −80 °C, and air-drying) on yield, viability, and community composition of soil extractable cells were assessed. Cell viability decreased in all stored soil samples, but significant decreases in cell yield was only observed in air-dried soil samples. Microbial community compositions changed significantly in all stored soil samples, with the least changes were observed in soil stored at 4 °C, confirming that 4 °C short-term storage is suitable for highly efficient viable cell extraction. Taken together, the developed method offers great potential for advancing our analyses and understanding of soil microbial ecology and the role of individual microbes.

Introduction

Despite the abundant and diverse microorganisms in soil samples, the majority of soil bacteria and archaea remain uncultured (Steen et al., 2019), which hampers our understanding of the functions of these prokaryotes from soil microbial communities. Recent development of various single-cell techniques using directly extracted microbial cells provides valuable phenotypic and genomic information of these uncultured microbes (Eichorst et al., 2015). Also, such directly extracted soil microbial cells have been used for high-throughput culturing (Wang et al., 2014), direct quantification of bacterial abundance (Bressan et al., 2015; Frossard et al., 2016; Khalili et al., 2019), and extraction of high-molecular-weight DNA (Robe et al., 2003; Williamson et al., 2011). However, the relative low yield and low viability of typical soil cell extraction procedures remain a major challenge, and hence, how well the directly extractable soil cells represent the original soil samples remains largely unknown.

Intensive efforts have been invested to improve the direct microbial cell extraction efficiency from soil samples by focusing on the following aspects: i) separation of microbial cells from soil organic matter and soil particles. Physical dispersion (e.g. blending and sonication) and chemical dispersion (e.g., ionic or non-ionic buffers) are used alone or together to detach cells from soil particle surfaces (Bakken, 1985; Courtois et al., 2001; Lindahl and Bakken, 1995; Williamson et al., 2011). Previous studies have shown that physical and chemical dispersions largely determine cell extraction efficiencies (Courtois et al., 2001; Khalili et al., 2019; Williamson et al., 2011). But the cell extraction efficiency is highly variable depending on soil textures (Amalfitano and Fazi, 2008). ii) purification of the dispersed microbial cells. Several density gradient media have been used to purify microbial cells from soil matrices, including Nycodenz (Lindahl and Bakken, 1995), Histodenz (Frossard et al., 2016), sucrose (Liu et al., 2010), and sodium bromide (Laflamme et al., 2005). Nycodenz density gradient centrifugation is one of the most commonly used purification methods. Higher Nycodenz concentration has been reported to be beneficial for improving the overall soil cell extraction efficiency (Eichorst et al., 2015; Holmsgaard et al., 2011). iii) increasing the number of extraction/purification procedures. For example, three sequential rounds of extraction recover more cells than a single-pass extraction (Williamson et al., 2011). Despite these various methods developed, with currently reported soil cell extraction procedures, both dead and live cells are recovered after Nycodenz density centrifugation (Burkert et al., 2019; Whiteley et al., 2003). Also, cell extraction efficiencies reported in literature are largely based on the total number of cells extracted that includes both live and dead cells. Therefore, to truly assess the extraction efficiency meaningful for downstream microbial phenotypic characterization, it is important to focus on the efficiency of viable cell extraction along with examining the viable microbial community compositions of cells extracted from soils.

Another aspect to consider is how soil sample storage conditions affect the efficiency of viable cell extraction from soil samples. Soil samples are often stored before conducting physiological or molecular biological experiments on them. Many studies have examined the effect of soil storage conditions on microbial communities, but the results of these studies are inconsistent. Some studies have found that temperature and duration of storage have no effect on the overall microbial community composition (Dolfing, 2004; Lauber et al., 2010; Rubin et al., 2013). However, other studies have demonstrated that storage conditions significantly change soil microbial community composition (Černohlávková et al., 2009; Cui et al., 2014; Piao et al., 2010; Tzeneva et al., 2009). In these studies, the microbial community diversities were measured at the DNA level; however, it is unknown whether the various soil storage conditions significantly impact the viability and recovery of soil-extractable cells and soil-viable microbial community compositions.

Lastly, to evaluate cell viability, cells are often fluorescently labeled using live/dead staining reagents and quantified using microscopy or flow cytometry (Emerson et al., 2017). However, this technique does not provide any information on the viable microbial community composition. Viability PCR has been used in cell cultures or environmental samples (Carini et al., 2016; Emerson et al., 2017; Nocker et al., 2007). Viability dyes, such as propidium monoazide or ethidium monoazide, bind to DNA from dead or compromised cells and the dye-bound DNA is degraded when exposed to certain wavelength of light, enabling the analysis of DNA originated from viable cells only. In this study, both live/dead staining and viability PCR were used to evaluate the impact of extraction procedures and soil storage conditions on the viability and microbial community composition of soil-extractable cells using soil samples having diverse ranges of physicochemical properties.

In this study, we aimed to improve the yield and viability of soil extractable cells with two sequential rounds of cell extraction procedure. By comparing different physical and chemical dispersion methods, and Nycodenz density gradient medium concentrations in soil samples having diverse physicochemical properties, we expected to identify the combination offering the highest cell viability and yield. In addition, using the optimized cell extraction procedure, we sought to assess the effect of soil storage conditions (4 °C, −80 °C, and air-drying) on yield, viability, and community composition of soil extractable cells. We hypothesized that storage at 4 °C would be suitable for maintaining viable microbial cells in soil samples because storage at −80 °C and air-drying may exert physiological stresses on soil microbes.