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Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity.

Kerckhof FM, Courtens EN, Geirnaert A, Hoefman S, Ho A, Vilchez-Vargas R, Pieper DH, Jauregui R, Vlaeminck SE, Van de Wiele T, Vandamme P, Heylen K, Boon N - PLoS ONE (2014)

Bottom Line: 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.This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.

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.

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Timeline and sampling strategy of the cryopreservation setup.The time is shown in days. In red the activity tests are shown. The freezing and thawing are shown in dark grey. DNA sampling was executed at t0, t1 and t3.
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pone-0099517-g001: Timeline and sampling strategy of the cryopreservation setup.The time is shown in days. In red the activity tests are shown. The freezing and thawing are shown in dark grey. DNA sampling was executed at t0, t1 and t3.

Mentions: The experimental design, over a three month period of cryopreservation, is outlined in Figure 1. Each source of biomass at t0 was divided in three parts: one part was subjected to cryopreservation, with or without addition of CPA, another part was subjected to the reference activity test, and a final part was sampled for DNA extraction and biomass quantification. At the end of the reference activity test, biomass was again sampled for DNA extraction and biomass quantification (t1). After 106 days, biomass was resuscitated (t2) and used as inoculum for the post-freezing activity test. At the end of the post-freezing activity test biomass was sampled again for quantification and DNA extraction (t3). DNA sampling at this point allows to investigate the active community after resuscitation and a standard batch activity test.


Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity.

Kerckhof FM, Courtens EN, Geirnaert A, Hoefman S, Ho A, Vilchez-Vargas R, Pieper DH, Jauregui R, Vlaeminck SE, Van de Wiele T, Vandamme P, Heylen K, Boon N - PLoS ONE (2014)

Timeline and sampling strategy of the cryopreservation setup.The time is shown in days. In red the activity tests are shown. The freezing and thawing are shown in dark grey. DNA sampling was executed at t0, t1 and t3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061060&req=5

pone-0099517-g001: Timeline and sampling strategy of the cryopreservation setup.The time is shown in days. In red the activity tests are shown. The freezing and thawing are shown in dark grey. DNA sampling was executed at t0, t1 and t3.
Mentions: The experimental design, over a three month period of cryopreservation, is outlined in Figure 1. Each source of biomass at t0 was divided in three parts: one part was subjected to cryopreservation, with or without addition of CPA, another part was subjected to the reference activity test, and a final part was sampled for DNA extraction and biomass quantification. At the end of the reference activity test, biomass was again sampled for DNA extraction and biomass quantification (t1). After 106 days, biomass was resuscitated (t2) and used as inoculum for the post-freezing activity test. At the end of the post-freezing activity test biomass was sampled again for quantification and DNA extraction (t3). DNA sampling at this point allows to investigate the active community after resuscitation and a standard batch activity test.

Bottom Line: 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.This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Gent, Belgium.

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.

Show MeSH