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. 

Methane (CH4) is a prominent greenhouse gas that contributes to the negative impacts of global warming and climate change, whose emissions have more than doubled since the Industrial Revolution primarily due to anthropogenic sources. The main pathways in which methane moves through the environment are methanogenesis and methanotrophy. Methane is primarily generated by acetoclastic methanogenesis in wetlands while it can be oxidized both aerobically and anaerobically. Wetlands are important methane emission sources at 177 - 284 Tg CH4 year-1. The Tres Rios Wetland (TRW) is a constructed facility to complete nutrient removal of treated municipal wastewater, and has shown low emissions of methane. Whether such low emissions could be achieved through active anaerobic oxidation of methane (AOM) is not known, and the main objective of this work is to evaluate the rates of AOM in TRW. In this study an isotopic method and a mass balance method were utilized to determine the rate of AOM from top sediments found at Tres Rios at various locations and in two sets of sampling. The results showed that evidence of AOM occurred in the sediments of both sampling events conducted. The first sampling set showed evidence of AOM at all locations along a transect, showing that oxidation of methane is indeed occurring in Tres Rios sediments. Evidence from both methodologies suggested that high methanogenesis rates occurred at the outside location closest to the water. The second sampling set showed that the highest rate of AOM occurred at the outlet location, with the lowest rate occurring in the middle location. DNA extractions and PCR images resulted in a poor DNA yield, and inability to extract DNA. It was determined that the isotopic approach was less accurate than the mass balance approach due to unexpected delta CH4 values. It was determined that dilutions of CH4 ppm lead to less accurate isotopic measurements needed to estimate AOM rates using a 13C pulse technique. Literature review suggests that factors including water presence, temperature, redox potential, and plant presence can be influential in the oxidation of methane. This AOM assay can be beneficial in better understanding how methane cycles at Tres Rios, and can provide opportunities for future research in determining which factors influence the oxidation of methane in different locations throughout wetlands.

Tropical peatlands play a critical role in global carbon storage and greenhouse gas flux, yet the role of microbial communities in these ecosystems remains poorly understood. Methane-oxidizing bacteria (MOB) are considered an efficient biological filter for methane and can mitigate its release into the atmosphere, facilitating an ecosystem’s capacity to become a net sink. Prokaryotic gene amplicon surveys targeting a unique biomarker instead of a universal one (i.e., 16S rRNA) can reveal a more comprehensive analysis of microbial communities with ecological functions (i.e., methanotrophy). The alpha subunit of particulate methane monooxygenase (pmoA) is commonly targeted as a phylogenetic biomarker for both aerobic and anaerobic MOB. Here, we tested three different primer sets and investigated their ability to assess methanotrophic diversity across three biogeochemically distinct tropical peatland sites in the Pastaza-Marañón foreland basin (PMFB) in western Amazonia. The results showed that sequencing using 16S rRNA and pmoA genes revealed differences in MOB taxonomic identification in 21 tropical peat soils. Beta diversity analysis of pmoA genes suggests that site location is not the main driver of differences in MOB community makeup. This work offers insight into the strengths and weaknesses of targeted gene amplicon surveys using 16S and pmoA from tropical peat soils as a case study.