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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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Functionality recovery after cryopreservation.Error bars represent error-propagated standard errors A) the MOB community on NMS and dNMS cultivation medium. The activity recovery was the percentage of specific MOR (mmol CH4 g−1 VS d−1) from the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3) B) The different functional members in the OLAND community. The activity recovery was the percentage of specific activity (mg N g−1 VSS d−1) for either aerobic or anaerobic ammonium oxidation (AOB and AnAOB) or nitrite oxidation (NOB), from the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3) C) short chain fatty acid production by the fecal microbiome. The activity recovery was the percentage of SCFA produced in the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3). Total SCFA, acetate, propionate and butyrate were measured.
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pone-0099517-g002: Functionality recovery after cryopreservation.Error bars represent error-propagated standard errors A) the MOB community on NMS and dNMS cultivation medium. The activity recovery was the percentage of specific MOR (mmol CH4 g−1 VS d−1) from the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3) B) The different functional members in the OLAND community. The activity recovery was the percentage of specific activity (mg N g−1 VSS d−1) for either aerobic or anaerobic ammonium oxidation (AOB and AnAOB) or nitrite oxidation (NOB), from the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3) C) short chain fatty acid production by the fecal microbiome. The activity recovery was the percentage of SCFA produced in the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3). Total SCFA, acetate, propionate and butyrate were measured.

Mentions: Two cultivation media (NMS and dNMS) were evaluated with the MOB mixed culture. The key specific activity (MOR) was comparable to the original activity, when a CPA was added prior to cryopreservation (Figure 2A), on both media. When no CPA was added, the average specific activity over 48 h was significantly lower than the original activity on both media (p<0.0001). The largest activity recovery was obtained when only DMSO was added as a CPA both with NMS (147.2±2.6%) and dNMS (156.1±10.1%), which exceeded the original activity. When DMSO+TT was used as a CPA, the original specific activity was obtained on NMS (96.4±10.7%) and dNMS (116.5±20%). Both on NMS and dNMS with DMSO+TT as a CPA the specific activity was not significantly different from the initial activity (p = 1).


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)

Functionality recovery after cryopreservation.Error bars represent error-propagated standard errors A) the MOB community on NMS and dNMS cultivation medium. The activity recovery was the percentage of specific MOR (mmol CH4 g−1 VS d−1) from the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3) B) The different functional members in the OLAND community. The activity recovery was the percentage of specific activity (mg N g−1 VSS d−1) for either aerobic or anaerobic ammonium oxidation (AOB and AnAOB) or nitrite oxidation (NOB), from the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3) C) short chain fatty acid production by the fecal microbiome. The activity recovery was the percentage of SCFA produced in the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3). Total SCFA, acetate, propionate and butyrate were measured.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0099517-g002: Functionality recovery after cryopreservation.Error bars represent error-propagated standard errors A) the MOB community on NMS and dNMS cultivation medium. The activity recovery was the percentage of specific MOR (mmol CH4 g−1 VS d−1) from the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3) B) The different functional members in the OLAND community. The activity recovery was the percentage of specific activity (mg N g−1 VSS d−1) for either aerobic or anaerobic ammonium oxidation (AOB and AnAOB) or nitrite oxidation (NOB), from the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3) C) short chain fatty acid production by the fecal microbiome. The activity recovery was the percentage of SCFA produced in the pre-freezing activity test (t0 to t1) that was obtained in each experimental condition in the post-freezing activity test (t2 to t3). Total SCFA, acetate, propionate and butyrate were measured.
Mentions: Two cultivation media (NMS and dNMS) were evaluated with the MOB mixed culture. The key specific activity (MOR) was comparable to the original activity, when a CPA was added prior to cryopreservation (Figure 2A), on both media. When no CPA was added, the average specific activity over 48 h was significantly lower than the original activity on both media (p<0.0001). The largest activity recovery was obtained when only DMSO was added as a CPA both with NMS (147.2±2.6%) and dNMS (156.1±10.1%), which exceeded the original activity. When DMSO+TT was used as a CPA, the original specific activity was obtained on NMS (96.4±10.7%) and dNMS (116.5±20%). Both on NMS and dNMS with DMSO+TT as a CPA the specific activity was not significantly different from the initial activity (p = 1).

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