Rygaard Anita Mac, Thøgersen Mariane Schmidt, Nielsen Kristian Fog, Gram Lone, Bentzon-Tilia Mikkel
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark
Appl Environ Microbiol. 2017 Apr 17;83(9). doi: 10.1128/AEM.00243-17. Print 2017 May 1.
Only 1% of marine bacteria are currently culturable using standard laboratory procedures, and this is a major obstacle for our understanding of the biology of marine microorganisms and for the discovery of novel microbial natural products. Therefore, the purpose of this study was to investigate if improved cultivation conditions, including the use of an alternative gelling agent and supplementation with signaling molecules, improve the culturability of bacteria from seawater. Replacing agar with gellan gum improved viable counts 3- to 40-fold, depending on medium composition and incubation conditions, with a maximum of 6.6% culturability relative to direct cell counts. Through V4 amplicon sequencing we found that culturable diversity was also affected by a change in gelling agent, facilitating the growth of orders not culturable on agar-based substrates. Community analyses showed that communities grown on gellan gum substrates were significantly different from communities grown on agar and that they covered a larger fraction of the seawater community. Other factors, such as incubation temperature and time, had less obvious effects on viable counts and culturable diversity. Supplementation with acylated homoserine lactones (AHLs) did not have a positive effect on total viable counts or a strong effect on culturable diversity. However, low concentrations of AHLs increased the relative abundance of sphingobacteria. Hence, with alternative growth substrates, it is possible to significantly increase the number and diversity of cultured marine bacteria. Serious challenges to human health, such as the occurrence and spread of antibiotic resistance and an aging human population in need of bioactive pharmaceuticals, have revitalized the search for natural microbial products. The marine environment, representing the largest ecosystem in the biosphere, harbors an immense and virtually untapped microbial diversity producing unique bioactive compounds. However, we are currently able to cultivate only a minute fraction of this diversity. The lack of cultivated microbes is hampering not only bioprospecting efforts but also our general understanding of marine microbes. In this study, we present a means to increase the number and diversity of cultured bacteria from seawater, showing that relatively simple changes to medium components may facilitate the isolation and growth of hitherto unknown bacterial orders.
目前，使用标准实验室程序只能培养出1%的海洋细菌，这对我们了解海洋微生物生物学以及发现新型微生物天然产物构成了重大障碍。因此，本研究的目的是调查改进的培养条件，包括使用替代胶凝剂和添加信号分子，是否能提高海水中细菌的可培养性。用结冷胶替代琼脂可使活菌数增加3至40倍，这取决于培养基组成和培养条件，相对于直接细胞计数，可培养性最高可达6.6%。通过V4扩增子测序，我们发现可培养多样性也受到胶凝剂变化的影响，有利于在基于琼脂的底物上不可培养的菌目的生长。群落分析表明，在结冷胶底物上生长的群落与在琼脂上生长的群落显著不同，并且它们覆盖了更大比例的海水群落。其他因素，如培养温度和时间，对活菌数和可培养多样性的影响不太明显。添加酰化高丝氨酸内酯（AHLs）对总活菌数没有积极影响，对可培养多样性也没有强烈影响。然而，低浓度的AHLs增加了鞘脂菌的相对丰度。因此，使用替代生长底物有可能显著增加培养的海洋细菌的数量和多样性。对人类健康的严峻挑战，如抗生素耐药性的出现和传播以及需要生物活性药物的老龄化人口，重新激发了对天然微生物产品的探索。海洋环境是生物圈中最大的生态系统，蕴藏着巨大且几乎未被开发的微生物多样性，能产生独特的生物活性化合物。然而，目前我们只能培养出这一多样性中的极小一部分。缺乏可培养的微生物不仅阻碍了生物勘探工作，也妨碍了我们对海洋微生物的总体认识。在本研究中，我们提出了一种增加海水中培养细菌数量和多样性的方法，表明对培养基成分进行相对简单的改变可能有助于分离和培养迄今未知的菌目。
