Rain-Franco Angel, de Moraes Guilherme Pavan, Beier Sara
UMR 7621 Laboratoire d'Océanographie Microbienne, Observatoire Océanologique de Banyuls-sur-Mer, Sorbonne Université, Banyuls-sur-Mer, France.
Graduate Program in Ecology and Natural Resources (PPGERN), Laboratory of Phycology, Department of Botany, Universidade Federal de São Carlos, São Carlos, Brazil.
Front Microbiol. 2021 Jan 28;11:597653. doi: 10.3389/fmicb.2020.597653. eCollection 2020.
Experimental reproducibility in aquatic microbial ecology is critical to predict the dynamics of microbial communities. However, controlling the initial composition of naturally occurring microbial communities that will be used as the inoculum in experimental setups is challenging, because a proper method for the preservation of those communities is lacking. To provide a feasible method for preservation and resuscitation of natural aquatic prokaryote assemblages, we developed a cryopreservation procedure applied to natural aquatic prokaryotic communities. We studied the impact of inoculum size, processing time, and storage time on the success of resuscitation. We further assessed the effect of different growth media supplemented with dissolved organic matter (DOM) prepared from naturally occurring microorganisms on the recovery of the initially cryopreserved communities obtained from two sites that have contrasting trophic status and environmental heterogeneity. Our results demonstrated that the variability of the resuscitation process among replicates decreased with increasing inoculum size. The degree of similarity between initial and resuscitated communities was influenced by both the growth medium and origin of the community. We further demonstrated that depending on the inoculum source, 45-72% of the abundant species in the initially natural microbial communities could be detected as viable cells after cryopreservation. Processing time and long-term storage up to 12 months did not significantly influence the community composition after resuscitation. However, based on our results, we recommend keeping handling time to a minimum and ensure identical incubation conditions for repeated resuscitations from cryo-preserved aliquots at different time points. Given our results, we recommend cryopreservation as a promising tool to advance experimental research in the field of microbial ecology.
水生微生物生态学中的实验可重复性对于预测微生物群落的动态变化至关重要。然而，在实验设置中控制用作接种物的天然微生物群落的初始组成具有挑战性，因为缺乏保存这些群落的合适方法。为了提供一种保存和复苏天然水生原核生物组合的可行方法，我们开发了一种应用于天然水生原核生物群落的冷冻保存程序。我们研究了接种物大小、处理时间和储存时间对复苏成功率的影响。我们进一步评估了添加了由天然微生物制备的溶解有机物（DOM）的不同生长培养基对从两个具有不同营养状态和环境异质性的地点获得的初始冷冻保存群落恢复的影响。我们的结果表明，随着接种物大小增加，重复实验中复苏过程的变异性降低。初始群落和复苏群落之间的相似程度受生长培养基和群落来源的影响。我们进一步证明，根据接种物来源，初始天然微生物群落中45%-72%的优势物种在冷冻保存后可被检测为活细胞。处理时间和长达12个月的长期储存对复苏后的群落组成没有显著影响。然而，根据我们的结果，我们建议将处理时间保持在最短，并确保在不同时间点从冷冻保存的等分试样中进行重复复苏时具有相同的培养条件。鉴于我们的结果，我们建议将冷冻保存作为推进微生物生态学领域实验研究的一种有前景的工具。
