Cultivation and Detection of Novel Archaea from Great Boiling Springs

RM 218

Candidate phyla currently constitute a significant amount of the overall phylogenetic diversity of microbes. Because they are not closely related to cultivated microbes, their potential physiology and ecology are much more difficult to infer than for uncultivated microbes that are within a known genus or family. This project aims to cultivate a member of the archaeal candidate phylum Aigarchaeota, Group 1 Aigarchaeota (Aig G1) in the laboratory. Based on cultivation-independent genomic data available from several Aig G1 members in Great Boiling Spring, 22 different types of growth media were designed. Variables that were tested in these media included different combinations of organic and inorganic growth substrates, amounts of oxygen, and incubation temperatures. These media were inoculated in the field with freshly collected sediment, incubated in the laboratory, and transferred (1/100 volume) after 3-4 weeks of growth. At various time points, DNA was extracted from culture samples and the abundance of Aig G1 and total Bacteria and Archaea was assessed by quantitative polymerase chain reaction (qPCR). From the 22 growth conditions, two cultures yielded growth of Aig G1. These growth conditions included hot spring mat extract with 2% oxygen and casamino acids with 10% oxygen, both incubated at 80°C. Aig G1 Abundance was 1-20% of the microorganisms grown in culture where the densities reached up to 107 16S rRNA gene copies/ mL, and were stably maintained after several transfers. These are the first reported stable laboratory cultures of Aig G1 and will serve as a basis for understanding the physiology of this lineage.