title: 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.
authors.affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.
authors.forename: Frederiek-Maarten
authors.initials: FM
authors.lastname: Kerckhof
authors.affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.
authors.forename: Emilie N P
authors.initials: EN
authors.lastname: Courtens
authors.affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.
authors.forename: Annelies
authors.initials: A
authors.lastname: Geirnaert
authors.affiliation: Laboratory of Microbiology, Faculty of Sciences, Ghent University, Gent, Belgium.
authors.forename: Sven
authors.initials: S
authors.lastname: Hoefman
authors.affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.
authors.forename: Adrian
authors.initials: A
authors.lastname: Ho
authors.affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.
authors.forename: Ramiro
authors.initials: R
authors.lastname: Vilchez-Vargas
authors.affiliation: Microbial Interactions and Processes Research Group, Department of Medical Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Dietmar H
authors.initials: DH
authors.lastname: Pieper
authors.affiliation: Microbial Interactions and Processes Research Group, Department of Medical Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Ruy
authors.initials: R
authors.lastname: Jauregui
authors.affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.
authors.forename: Siegfried E
authors.initials: SE
authors.lastname: Vlaeminck
authors.affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Van de Wiele
authors.affiliation: Laboratory of Microbiology, Faculty of Sciences, Ghent University, Gent, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Vandamme
authors.affiliation: Laboratory of Microbiology, Faculty of Sciences, Ghent University, Gent, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Heylen
authors.affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.
authors.forename: Nico
authors.initials: N
authors.lastname: Boon
mesh_terms: D052818:Autotrophic Processes; D018441:Biofilms; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D019605:Euryarchaeota / Q000235:genetics / Q000254:growth & development; D005243:Feces / Q000382:microbiology; D006801:Humans; D050296:Microbial Viability; D064307:Microbiota; D010802:Phylogeny; D019936:Probiotics
chemical_list: D003451:Cryoprotective Agents