No time to die: Comparative study on preservation protocols for anaerobic fungi

• DOI: 10.3389/fmicb.2022.978028
• Publication Date: 2022-09-26T08:50:19Z
• Authors: Julia Vinzelj; Akshay Joshi; Diana Young; Ljubica Begovic; Nico Peer; Lona Mosberger; Katharina Cécile ...; Heribert Insam; Veronika Flad; Magdalena Nagler; Sabine Marie Podm...
• Keywords: Cryopreservation; Long-term storage; 0303 health sciences; Neocallimastigomycota; long-term storage; Anaerobic fungus; short-term storage; Resting stage; 660.6: Biotechnologie; cryopreservation; Microbiology; Short-term storage; QR1-502; 03 medical and health sciences; Environmental biotechnology; 579: Mikrobiologie; Preservation technique; anaerobic fungi; preservation techniques; FOS: Environmental biotechnology; Culture preservation

Anaerobic fungi (AF, phylum Neocallimastigomycota) are best known for their ability to anaerobically degrade recalcitrant lignocellulosic biomass through mechanic and enzymatic means. While biotechnological potential is well-recognized, applied research on AF still hampered by the time-consuming cost-intensive laboratory routines required isolate, maintain, preserve cultures. Reliable long-term preservation of specific strains would aid basic as well research, but commonly used protocols can show erratic survival rates usually exhibit only moderate resuscitation success up one or two years after preservation. To address both, variability, issues, we have set a cross-laboratory, year-long study. We tested five different AF. The experiments were performed at three laboratories (Austria, Germany, Switzerland) with same morphologically distinct isolates ( Anaeromyces mucronatus, Caeocmyces sp. , Neocallimastix cameroonii ) living in stable co-culture naturally occurring, syntrophic methanogens. could that handling greatly contributes variability results, especially mucronatus . Cryopreservation (mature) liquid nitrogen had highest overall (85–100%, depending strain laboratory). Additionally, agar 39°C surprisingly high 9 months, if pieces containing mature thalli resuscitated. This low-cost, low-effort method replace consecutive batch cultivation periods 6 while done cryopreservation nitrogen. Regardless method, however, preserving several replicates (>three) highly advisable.