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Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Microbiology

Year Degree Awarded

2018

Month Degree Awarded

May

First Advisor

James F. Holden

Second Advisor

Kristen DeAngelis

Third Advisor

Klaus Nusslein

Fourth Advisor

Steven Petsch

Subject Categories

Environmental Microbiology and Microbial Ecology | Microbial Physiology

Abstract

Approximately 1 giga ton (Gt, 1015 g) of CH4 is formed globally per year from H2, CO2, and acetate through methanogenesis, largely by methanogens growing in syntrophic association with anaerobic microbes that hydrolyze and ferment biopolymers. However, our understanding of methanogenesis in hydrothermal regions of the subseafloor and potential syntrophic methanogenesis at thermophilic temperatures is nascent. This dissertation shows that thermophilic H2 syntrophy can support methanogenesis within natural microbial assemblages at hydrothermal vents and that it can be an important alternative energy source for thermophilic autotrophs in marine geothermal environments. This dissertation also elucidates H2 stress survival strategies of the H2 producing heterotrophs and the H2 consuming methanogens as well as their cooperation with one another for survival.

The growth of natural assemblages of thermophilic methanogens from Axial Seamount was primarily limited by H2 availability. Heterotrophs supported thermophilic methanogenesis by H2 syntrophy in microcosm incubations of hydrothermal fluids at 80°C supplemented with tryptone only. Based on 16S rRNA gene sequencing, H2 producing heterotrophs, Thermococcus, and H2-consuming methanogens, Methanocaldococcus were abundant in 80°C tryptone microcosms from an Axial Seamount hydrothermal vent. In order to model the impact of H2 syntrophy at hyperthemophilic temperatures, a coculture was established consisting of a H2-producing hyperthermophilic heterotroph and a H2-consuming hyperthermophilic methanogen.

The model organisms, hyperthermophilic heterotroph Thermococcus paralvinellae and the hydrogenotrophic methanogen Methanocaldococcus jannaschii, were examined in monocultures and cocultures for their H2 stress and syntrophy strategies. In monocultures, H2 inhibition changed the growth kinetics and the transcriptome of T. paralvinellae. A significant decrease in batch phase growth rates and steady state cell concentrations was observed with high H2 background. Metabolite production measurements, RNA-seq analyses of differentially expressed genes, and in silico experiments performed with a constraint-based metabolic network model showed that T. paralvinellae produces formate by a formate hydrogenlyase to survive H2 inhibition. H2 limitation on M. jannaschii caused a significant decrease in batch phase growth rates and CH4 production rates but also caused a significant increase in cell yields. In cocultures, H2 syntrophy relieved H2 stress for T. paralvinellae but not for M. jannaschii. T. paralvinellae only produced formate when grown in monoculture while no formate was detected during growth in coculture. While M. jannaschii was capable of growth and methanogenesis solely on the H2 produced by T. paralvinellae, the growth and CH4 production rates of M. jannaschii decreased in coculture compared to when M. jannaschii was grown in monoculture.

Available for download on Sunday, November 11, 2018

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