Limits...
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.

Show MeSH

Related in: MedlinePlus

Phylogenetic tree of OTU consensus sequences in the fecal material 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 treatment-wise means (n = 2 except for fecal inoculum n = 1) of the 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. Classification was done based upon the Greengenes taxonomy (adapted to mothur from [53]) with the naïve Bayesian classifier implemented in mothur (Wang algorithm). Black arrows indicate OTUs with differential abundance among the experimental conditions.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4061060&req=5

pone-0099517-g005: Phylogenetic tree of OTU consensus sequences in the fecal material 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 treatment-wise means (n = 2 except for fecal inoculum n = 1) of the 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. Classification was done based upon the Greengenes taxonomy (adapted to mothur from [53]) with the naïve Bayesian classifier implemented in mothur (Wang algorithm). Black arrows indicate OTUs with differential abundance among the experimental conditions.

Mentions: The most abundant microorganisms in the fecal microbiome were representatives of the Lachnospiraceae, Bacteroidaceae, Ruminococcacae, Enterococcaceae, Enterobacteriaceae, Verrucomicrobiaceae, Bifidobacteriaceae and Clostridiales Family XI. Incertae sedis families (Figure S3). A total of 18 different families, with documented associations with the fermentative metabolism in the gut, were investigated for their relative abundance before and after cryopreservation (Figure S6). Variable results were obtained in the different taxonomic groups (Table S2). Similar to the MOB and OLAND mixed community, most OTUs from the fecal microbiome remained present in all experimental stages and conditions, irrespective of CPA addition (Figure 5). Of all observed OTUs, 29% (representing 8.5% of total sequences) were not detected in at least one of the experimental conditions (pre-freezing, post-freezing with or without CPA). This percentage excludes the OTU presence in the fecal inoculum due to the drastic change of the community upon first cultivation. Addition of CPA during cryopreservation of the fecal biomass was necessary to maintain comparable community dissimilarities to the reference activity test (Figure 6D), but differences in community dissimilarity between the conditions with and without CPA were not significant (p = 0.24).


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 fecal material 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 treatment-wise means (n = 2 except for fecal inoculum n = 1) of the 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. Classification was done based upon the Greengenes taxonomy (adapted to mothur from [53]) with the naïve Bayesian classifier implemented in mothur (Wang algorithm). Black arrows indicate OTUs with differential abundance among the experimental conditions.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099517-g005: Phylogenetic tree of OTU consensus sequences in the fecal material 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 treatment-wise means (n = 2 except for fecal inoculum n = 1) of the 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. Classification was done based upon the Greengenes taxonomy (adapted to mothur from [53]) with the naïve Bayesian classifier implemented in mothur (Wang algorithm). Black arrows indicate OTUs with differential abundance among the experimental conditions.
Mentions: The most abundant microorganisms in the fecal microbiome were representatives of the Lachnospiraceae, Bacteroidaceae, Ruminococcacae, Enterococcaceae, Enterobacteriaceae, Verrucomicrobiaceae, Bifidobacteriaceae and Clostridiales Family XI. Incertae sedis families (Figure S3). A total of 18 different families, with documented associations with the fermentative metabolism in the gut, were investigated for their relative abundance before and after cryopreservation (Figure S6). Variable results were obtained in the different taxonomic groups (Table S2). Similar to the MOB and OLAND mixed community, most OTUs from the fecal microbiome remained present in all experimental stages and conditions, irrespective of CPA addition (Figure 5). Of all observed OTUs, 29% (representing 8.5% of total sequences) were not detected in at least one of the experimental conditions (pre-freezing, post-freezing with or without CPA). This percentage excludes the OTU presence in the fecal inoculum due to the drastic change of the community upon first cultivation. Addition of CPA during cryopreservation of the fecal biomass was necessary to maintain comparable community dissimilarities to the reference activity test (Figure 6D), but differences in community dissimilarity between the conditions with and without CPA were not significant (p = 0.24).

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