Optimized Cryopreservation of Mixed Microbial Communities for Conserved Functionality and Diversity

Abstract

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at −80°C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

Cryopreservation of artificial gut microbiota produced with in vitro fermentation technology

“…However, low concentrations of DMSO might result in cellular toxicity (Galvao et al ., 2014), which limits its use for FMT unless a removal step is used before transplantation. Additionally, DMSO alone showed no significant improvement in SCFA production by faecal microbiota after frozen storage and reactivation compared to the control without cryoprotectant (Kerckhof et al ., 2014). Glycerol prevents hydrogen bonding between water molecules and thus prevents formation of intracellular ice crystals during freezing (Koh, 2013).…”

Preservation of Microbial Pure Cultures and Mixed Communities

“…However, for mixed communities or samples, not only specific activities need to recover after preservation, but also the composition of the revived community, both species richness and evenness, should be identical to the original. Still, maintained functionality and community structure after cryopreservation have already been successfully demonstrated for denitrifying biomass of a fluidized, methanol-fed denitrification reactor (10), a methanotrophic coculture, an oxygen-limited autotrophic nitrification/denitrification biofilm, and fecal samples from a human donor (13). These examples should encourage further preservation attempts of mixed communities and environmental samples to provide backup biological material for future research.…”

“…For these cultures, it should be tested if CPA addition results in better, equal, or worse recovery rate after preservation. For enrichments of Candidatus Kuenenia stuttgartiensis (12) and Candidatus Methylomirabilis oxyfera (Heylen, Ettwig, Jetten, unpublished), a methanotrophic coculture and an OLAND biofilm (13), the use of DMSO improved the specific activity recovery rate. The activity recovery of cryopreserved fecal material from human donor was not influenced by DMSO addition (13).…”

“…Research on storage of these kinds of samples is in its infancy. Still, the limited reports available demonstrate that their stable cryopreservation is possible using similar simple protocols as for pure cultures (9)(10)(11)(12)(13). Embedding of cells in a matrix such as exopolysaccharides in aggregates or biofilm-covered particles in soils or sediments provides extra protection to the cells during preservation and is a major advantage compared to planktonic pure cultures.…”

“…We refer to reference works such as The Prokaryotes and Bergey's Manual of Systematic Bacteriology, which, next to detailed information on isolation and growth, also include sections on maintenance and preservation for functionally or phylogenetically related groups. Despite the well-known organism-dependent nature of preservation, the wide applicability of a very simple cryopreservation protocol was recently demonstrated for a broad range of fastidious microorganisms and mixed communities (12,13,(15)(16)(17). Nevertheless, a preservation procedure should always be verified before long-term storage.…”