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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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Phylogenetic tree of OTU consensus sequences in the OLAND samples.Sequences were aligned using the mothur implementation of the NAST algorithm with the Silva v102 reference alignment. RAxML was used to construct an extended majority rule bootstrap consensus tree with the GTR + GAMMA substitution model and 1000 bootstrap iterations. This bootstrap consensus tree was visualized using iTol. The colored bars represent log transformed absolute abundances with the log transformation as suggested by Anderson and colleagues [74] with base 10. Before transformation the samples were rarefied to the lowest sequence count. Black arrows point out OTUs that have a differential presence among experimental conditions. Classification was done based upon the Greengenes taxonomy (adapted to mothur from [53]) with the naïve Bayesian classifier implemented in mothur (Wang algorithm).
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pone-0099517-g004: Phylogenetic tree of OTU consensus sequences in the OLAND samples.Sequences were aligned using the mothur implementation of the NAST algorithm with the Silva v102 reference alignment. RAxML was used to construct an extended majority rule bootstrap consensus tree with the GTR + GAMMA substitution model and 1000 bootstrap iterations. This bootstrap consensus tree was visualized using iTol. The colored bars represent log transformed absolute abundances with the log transformation as suggested by Anderson and colleagues [74] with base 10. Before transformation the samples were rarefied to the lowest sequence count. Black arrows point out OTUs that have a differential presence among experimental conditions. Classification was done based upon the Greengenes taxonomy (adapted to mothur from [53]) with the naïve Bayesian classifier implemented in mothur (Wang algorithm).

Mentions: The main constituents of the OLAND mixed community were representatives of the Comamonadaceae, Flavobacteriaceae, Nitrosomonadaceae, Rhodocylaceae and OD1 incertae sedis families as well as the Bacteroidetes order (Figure S2). Overall, most OTUs occurred in each condition irrespective of CPA addition, and only 15% of OTUs (representing 5.3% of total sequences) were not detected in at least one of the experimental conditions (Figure 4). Most of the OTUs that did not occur in every condition required the addition of a CPA to persist after cryopreservation. In the samples where a CPA was added, global community dissimilarity to the inoculum was lower, regardless of the type of CPA used (Figure 6B).


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)

Phylogenetic tree of OTU consensus sequences in the OLAND samples.Sequences were aligned using the mothur implementation of the NAST algorithm with the Silva v102 reference alignment. RAxML was used to construct an extended majority rule bootstrap consensus tree with the GTR + GAMMA substitution model and 1000 bootstrap iterations. This bootstrap consensus tree was visualized using iTol. The colored bars represent log transformed absolute abundances with the log transformation as suggested by Anderson and colleagues [74] with base 10. Before transformation the samples were rarefied to the lowest sequence count. Black arrows point out OTUs that have a differential presence among experimental conditions. Classification was done based upon the Greengenes taxonomy (adapted to mothur from [53]) with the naïve Bayesian classifier implemented in mothur (Wang algorithm).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099517-g004: Phylogenetic tree of OTU consensus sequences in the OLAND samples.Sequences were aligned using the mothur implementation of the NAST algorithm with the Silva v102 reference alignment. RAxML was used to construct an extended majority rule bootstrap consensus tree with the GTR + GAMMA substitution model and 1000 bootstrap iterations. This bootstrap consensus tree was visualized using iTol. The colored bars represent log transformed absolute abundances with the log transformation as suggested by Anderson and colleagues [74] with base 10. Before transformation the samples were rarefied to the lowest sequence count. Black arrows point out OTUs that have a differential presence among experimental conditions. Classification was done based upon the Greengenes taxonomy (adapted to mothur from [53]) with the naïve Bayesian classifier implemented in mothur (Wang algorithm).
Mentions: The main constituents of the OLAND mixed community were representatives of the Comamonadaceae, Flavobacteriaceae, Nitrosomonadaceae, Rhodocylaceae and OD1 incertae sedis families as well as the Bacteroidetes order (Figure S2). Overall, most OTUs occurred in each condition irrespective of CPA addition, and only 15% of OTUs (representing 5.3% of total sequences) were not detected in at least one of the experimental conditions (Figure 4). Most of the OTUs that did not occur in every condition required the addition of a CPA to persist after cryopreservation. In the samples where a CPA was added, global community dissimilarity to the inoculum was lower, regardless of the type of CPA used (Figure 6B).

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