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.
Highlights
In a bio-based economy, the exploitation of microbial resources represents a valuable solution for many of the current sustainability issues [1,2,3,4,5,6]
No optimized approach to maintain a reproducible mixed community inoculum is available to date
Recent studies on preservation of fastidious pure cultures have shown the effectiveness of complex media for cryopreservation of methanotrophic bacteria (MOB) [18], aerobic and anaerobic ammonia-oxidizing bacteria (AerAOB and AnAOB) [19,20] and nitrite oxidizing bacteria [21]
Summary
In a bio-based economy, the exploitation of microbial resources represents a valuable solution for many of the current sustainability issues [1,2,3,4,5,6] Both single strains and consortia of different microorganisms with various interconnected functions (i.e. microbiomes [7]) have been employed. Recent studies on preservation of fastidious pure cultures have shown the effectiveness of complex media for cryopreservation of methanotrophic bacteria (MOB) [18], aerobic and anaerobic ammonia-oxidizing bacteria (AerAOB and AnAOB) [19,20] and nitrite oxidizing bacteria [21] These complex cryopreservation media exploit the concerted protective effects of a fast penetrating CPA (DMSO) and the innate cryoprotective effects of carbon-rich media (Trehalose and Tryptic Soy Broth, TT). Apart from the choice of CPA, which has been indicated to be one of the most determining factors for cryopreservation success [17], a rigorous protocol for freezing, thawing, resuscitation and storage with as less temperature variations as possible is essential for successful cryopreservation [16]
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