Expanding Membrane Based Isolation of Terrestrial Bacteria: Effects of Microaerophilic and Vitamin Proficient Conditions on Amazonian Peat

Due to complex requirements and relationships found in terrestrial soil environments, less than 2% of bacteria has been cultured using traditional cultivation methods. The soil substrate membrane system (SSMS) is a method designed to overcome these limitations by incorporating the environmental soil as substrate. This work examines the improvements achievable through SSMS in combination with two variables known to affect microbial growth: microaerophilic conditions and vitamin availability, on Peruvian peatland soils of varying nutrient levels; poor (San Jorge), intermediate (Quistococha), and rich (Buena Vista). First, a preliminary study was performed to enhance the knowledge of SSMS applications. Following, soil samples were pre-incubated according to their treatments and inoculated onto membranes for 3 weeks. New membranes were inoculated from the first membrane's enrichment and incubated for 2 weeks. Verified microcolonies were transferred onto dilute media (dR2G 1:5 or RAVAN) through direct streaking and spreading of dilutions (10-3, 10-5, 10-7). Colony appearance was monitored with colonies being isolated and purified. Buena Vista produced the largest, most diverse microcolonies as well as the most isolates. Quistococha produced the fewest microcolonies and isolates and was the only Peatland with increased success rates in the control group. Nearly a 4:1 recovery of isolates was observed for Buena Vista's and San Jorge's treatment groups compared to their control groups. With nearly 300 isolates in isolation and sequencing, it can be concluded that SSMS improves the recovery of terrestrial bacteria, and ongoing work aims to identify the recovered isolates.