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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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Average abundance-based Jaccard (Ružička) distances between experimental stages and conditions.The distances are displayed for MOB biomass cultivated on NMS (A), dNMS (C), OLAND biomass (B) and fecal biomass (D). The reference represents the distance between t0 and t1 whilst the other bars represent the distance between t0 and the several conditions at t3. Error bars (for MOB and fecal samples) represent standard deviations (n = 4 in A, n = 6 for reference in C and 4 for the other means, n = 4 in D). No reference is available for the OLAND biomass because time point t1 was not assessed using Illumina.
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pone-0099517-g006: Average abundance-based Jaccard (Ružička) distances between experimental stages and conditions.The distances are displayed for MOB biomass cultivated on NMS (A), dNMS (C), OLAND biomass (B) and fecal biomass (D). The reference represents the distance between t0 and t1 whilst the other bars represent the distance between t0 and the several conditions at t3. Error bars (for MOB and fecal samples) represent standard deviations (n = 4 in A, n = 6 for reference in C and 4 for the other means, n = 4 in D). No reference is available for the OLAND biomass because time point t1 was not assessed using Illumina.

Mentions: Cryopreservation was associated with greater community dissimilarities on NMS, regardless of the addition of a CPA (Figure 6A). Nonetheless, these dissimilarities (t0–t3) were not significantly different from the community dissimilarity of the first activity test (t0–t1, p = 0.12). With dNMS, the differences were significant (p = 0.01). More specifically, the dissimilarity with the inoculum was lower when a CPA was added. This dissimilarity was within the range of the dissimilarity of the pre-freezing activity test (t0–t1) when DMSO was used as a CPA (Figure 6C). The community dissimilarities in the reference (t0–t1), DMSO (t1–t3) and DMSO+TT (t1–t3) samples were not significantly different (pref-DMSO = 0.99, pref-DMSO+TT = 0.50, pDMSO-DMSO+TT = 0.57) while the dissimilarities between the reference and the samples with no CPA did significantly differ (p<0.0001).


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)

Average abundance-based Jaccard (Ružička) distances between experimental stages and conditions.The distances are displayed for MOB biomass cultivated on NMS (A), dNMS (C), OLAND biomass (B) and fecal biomass (D). The reference represents the distance between t0 and t1 whilst the other bars represent the distance between t0 and the several conditions at t3. Error bars (for MOB and fecal samples) represent standard deviations (n = 4 in A, n = 6 for reference in C and 4 for the other means, n = 4 in D). No reference is available for the OLAND biomass because time point t1 was not assessed using Illumina.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099517-g006: Average abundance-based Jaccard (Ružička) distances between experimental stages and conditions.The distances are displayed for MOB biomass cultivated on NMS (A), dNMS (C), OLAND biomass (B) and fecal biomass (D). The reference represents the distance between t0 and t1 whilst the other bars represent the distance between t0 and the several conditions at t3. Error bars (for MOB and fecal samples) represent standard deviations (n = 4 in A, n = 6 for reference in C and 4 for the other means, n = 4 in D). No reference is available for the OLAND biomass because time point t1 was not assessed using Illumina.
Mentions: Cryopreservation was associated with greater community dissimilarities on NMS, regardless of the addition of a CPA (Figure 6A). Nonetheless, these dissimilarities (t0–t3) were not significantly different from the community dissimilarity of the first activity test (t0–t1, p = 0.12). With dNMS, the differences were significant (p = 0.01). More specifically, the dissimilarity with the inoculum was lower when a CPA was added. This dissimilarity was within the range of the dissimilarity of the pre-freezing activity test (t0–t1) when DMSO was used as a CPA (Figure 6C). The community dissimilarities in the reference (t0–t1), DMSO (t1–t3) and DMSO+TT (t1–t3) samples were not significantly different (pref-DMSO = 0.99, pref-DMSO+TT = 0.50, pDMSO-DMSO+TT = 0.57) while the dissimilarities between the reference and the samples with no CPA did significantly differ (p<0.0001).

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
Related in: MedlinePlus