EVALUATING COMMUNITY INTERACTIONS AND CARBON FLOW IN METHANE-RICH ENVIRONMENTS USING METHANOTROPHIC-HETEROTROPHIC BACTERIAL CO-CULTURES

Moon-Escamilla, Rosa

EVALUATING COMMUNITY INTERACTIONS AND CARBON FLOW IN METHANE-RICH ENVIRONMENTS USING METHANOTROPHIC-HETEROTROPHIC BACTERIAL CO-CULTURES

dc.contributor.advisor	Krumholz, Lee
dc.contributor.author	Moon-Escamilla, Rosa
dc.contributor.committeeMember	Stevenson, Bradley
dc.contributor.committeeMember	Tanner, Ralph
dc.date.accessioned	2020-07-31T15:12:56Z
dc.date.available	2020-07-31T15:12:56Z
dc.date.issued	2020-07
dc.date.manuscript	2020-07-30
dc.description.abstract	Methane oxidizing bacteria play a significant role in global methane and carbon cycling and are present in many different global environments. Isolation and enrichment for novel methanotrophic species is an important way to learn more about the diversity and behaviors of these organisms. In this study, natural sediments from a creek and a landfill in Oklahoma were sampled and enriched for methane oxidizing bacteria. One novel alphaproteobacterial methane oxidizing species (Methylocystis sp. NLS7) was isolated from a landfill in Norman, OK. In addition, three heterotrophic species (Pseudomonas chlororaphis, Cupriavidus sp. HC, and Flavobacterium sp. HC) were isolated from Honey Creek, in Davis, OK, even though methane was the only provided carbon source in the enrichments. Species identity for these isolates was determined using 16S rRNA gene sequencing and whole genome sequencing. In addition, each isolate was imaged with transmission electron microscopy to visualize internal structures, as methanotrophs have unique intracytoplasmic membranes that are useful for characterization. TEM images showed concentric membranes within Methylocystis sp. NLS7, which are characteristic of the genus. TEM of Cupriavidus sp. HC revealed bright inclusion bodies which are likely polyhydroxyalkanoates (PHAs) used for carbon storage. Pseudomonas sp. HC and Flavobacterium sp. HC TEM images were comparable to published images of their respective genera. The discovery of heterotrophic species within methane enrichments led to further investigation of methanotrophic-heterotrophic interactions in co-culture, as previous literature indicates that the addition of heterotrophic species may present certain benefits to methanotrophic species. Co-cultures were constructed using Methylocystis sp. NLS7 and each of the three heterotrophic species. Co-cultures were provided methane as a sole carbon source, and then monitored for growth and methane oxidation to evaluate any potential stimulatory effects of co-culturing, compared to a pure culture methanotroph control. Growth and methane oxidation over time for Methylocystis sp. NLS7 were unaffected by the addition of heterotrophic species. However, the heterotrophs did grow in the co-cultures, given that the concentration of heterotroph cells at the end of the log phase was significantly higher than starting cell amounts for each co-culture. The heterotrophic species in each co-culture are not capable of oxidizing methane on their own, so it is likely they were utilizing by-products of methane metabolism which might include organic molecules, amino acids, or polysaccharides. Proposed future work includes transcriptome analysis to identify a carbon source for the heterotrophs within methane-fed co-cultures and to investigate carbon and metabolite flow within these co-cultures. Methanotrophic-heterotrophic community interactions represent a significant knowledge gap in microbial ecology. Enrichment for novel species and co-culture studies like these can provide insight into the diversity, behavior, and ecology of these organisms and how they influence carbon flow in natural environments.	en_US
dc.identifier.uri	https://hdl.handle.net/11244/325341
dc.language	en_US	en_US
dc.subject	methanotroph	en_US
dc.subject	co-culture	en_US
dc.subject	heterotroph interactions	en_US
dc.subject	TEM	en_US
dc.thesis.degree	Master of Science	en_US
dc.title	EVALUATING COMMUNITY INTERACTIONS AND CARBON FLOW IN METHANE-RICH ENVIRONMENTS USING METHANOTROPHIC-HETEROTROPHIC BACTERIAL CO-CULTURES	en_US
ou.group	College of Arts and Sciences::Department of Microbiology and Plant Biology	en_US