Cryopreservation and Resuscitation of Natural Aquatic Prokaryotic Communities

Abstract

Experimental reproducibility in aquatic microbial ecology is critical to predict the dynamics of microbial communities. However, controlling the initial composition of naturally occurring microbial communities that will be used as the inoculum in experimental setups is challenging, because a proper method for the preservation of those communities is lacking. To provide a feasible method for preservation and resuscitation of natural aquatic prokaryote assemblages, we developed a cryopreservation procedure applied to natural aquatic prokaryotic communities. We studied the impact of inoculum size, processing time, and storage time on the success of resuscitation. We further assessed the effect of different growth media supplemented with dissolved organic matter (DOM) prepared from naturally occurring microorganisms on the recovery of the initially cryopreserved communities obtained from two sites that have contrasting trophic status and environmental heterogeneity. Our results demonstrated that the variability of the resuscitation process among replicates decreased with increasing inoculum size. The degree of similarity between initial and resuscitated communities was influenced by both the growth medium and origin of the community. We further demonstrated that depending on the inoculum source, 45-72% of the abundant species in the initially natural microbial communities could be detected as viable cells after cryopreservation. Processing time and long-term storage up to 12 months did not significantly influence the community composition after resuscitation. However, based on our results, we recommend keeping handling time to a minimum and ensure identical incubation conditions for repeated resuscitations from cryo-preserved aliquots at different time points. Given our results, we recommend cryopreservation as a promising tool to advance experimental research in the field of microbial ecology.

Keywords: aquatic environments; community composition; complex microbial communities; cryobiology; cryopreservation; culture; experimental microbiology; microbial ecology.

FIGURE 1

Schema displaying the cryopreservation procedure (created with BioRender.com).

FIGURE 2

Sampling sites and representative environmental conditions. (A) Sampling location of the SOLA coastal station and the Canet Lagoon. (B) Salinity (PSU) and (C) chlorophyll-a (μg L^–1) for the Canet lagoon and SOLA station. Salinity and chlorophyll data for SOLA were provided by the SOMLIT program (http://somlit-db.epoc.u-bordeaux1.fr/bdd.php; 03/05/2005-30/10/2018, n = 595). Salinity and chlorophyll data for the Canet lagoon were extracted from the IFREMER Surval service (https://wwz.ifremer.fr/surval/, 26/06/2001-07/09/2006, n = 87).

FIGURE 3

Growth and variability of resuscitated communities from experiment 1a (filter concentrated inocula; upper panels) and 1b (water inocula; lower panels). (A,B) Growth curves. Empty circles represent the individual replicates, while filled circles represent the replicates mean (n = 3). (C,D) Variance and (E,F) coefficient of variation for bacterial cells detected after 4, 5, and 6 incubation days in dependence on the inoculum size in triplicate incubations.

FIGURE 4

Growth of resuscitated communities incubated in different media from experiment 2. Growth curves of resuscitated communities originating from (A) SOLA and (B) the Canet lagoon. Error bars represent standard deviation among replicates (n = 3). Empty circles illustrate the individual replicates, while filled circles represent the mean of triplicate incubations.

FIGURE 5

Initial and resuscitated microbial assemblies from experiment 2. (A) PCoA biplot displaying the community similarities (based on ASVs) among the initial and resuscitated communities in different media. (B) Relative contribution of the 10 most abundant orders in the initial communities as well as in the resuscitated communities after 5 days incubation in different media. (C) Shannon diversity indices. (B) and (C) share the same y-axis labels.

FIGURE 6

Log2-fold-changes (L2FC) of absolute abundances of ASVs (>0.5% relative abundance in the initial inoculum) between the initial inoculum and the final incubation day. L2FC values of ASVs against their relative abundances in the initial sample from (A) SOLA and (B) Canet, respectively. Ranked L2FC values of ASVs and the assigned taxonomy at the order level if available or otherwise at the class level for (C) SOLA and (D) Canet, respectively. For each ASV, only the L2FC in one medium is displayed: we selected for each ASV among the media with positive L2FC the one exhibiting the highest statistical support for growth (lowest P-value). For ASVs that exhibited in all media exclusively negative L2FC values, we displayed the one with the lowest p-value. Negative L2FC values or positive L2FC values with Padj > 0.1 are displayed in empty circles. Positive L2FC values with 0.05 ≤ Padj < 0.1 are displayed in light green and positive L2FC values with P < 0.05 are displayed in dark green.

FIGURE 7

Growth of resuscitated communities and ARISA-based dendrogram displaying community composition similarity from experiment 3 and 4. (A) Growth curves and (B) dendrogram illustrating the influence of handling time (0 and 7.5 h) during the preparation of inocula for cryopreservation on community composition 5 days after resuscitation. (C) Growth curves, and (D) dendrogram illustrating the influence of storage time of cryopreserved inocula at –80°C (1.5, 6, and 12 months) on community composition 5 days after resuscitation. Empty circles in (A) and (C) indicate the individual replicates, while filled circles represent the mean value. Vertical lines represent the standard deviation.