Degree Name

Master of Science (MS)

Semester of Degree Completion

2014

Thesis Director

Kai Hung

Abstract

Ferroplasma acidarmanus strain fer1 (fer1) is an archaeal microaerophilic acidophile (pH 0 to pH 3) with a chemolithotrophic metabolism. As an efficient consumer of sulfate, fer1 plays an important role in the biogeochemical process of pyrite dissolution at acidic site. Confirming the genomic prediction of metabolic gene functions in this organism requires functional assays, which cannot be carried out until a defined medium is obtained. The objective of the current study was to replace the 0.1% (w/v) yeast extract component in the growth medium, mfer, with defined components for carbon, nitrogen, and vitamin needs. In order to replace the 0.1% (w/v) yeast extract in mfer, possible sources for carbon (glucose, short-chain carbon compounds, organic acid mixture), nitrogen (alanine, ammonia, nitrate, Casamino Acids), and vitamins (10 vitamins) were tested in different combinations for their ability to support growth. Various growth conditions were also tested. Serial passages (1/100 dilution) were carried out to eliminate carry-over effects. Growth was monitored by optical density (590 nm), visual inspection, and most probable number estimation, as appropriate. Multiple independent trials with at least 2 repeats of each trial were conducted for statistical analyses. Results showed that optical density reading is a relatively reliable method to quantify the growth of fer1 in comparison with most probable number estimate (R2=0.904). A solid mfer medium was developed and shown to be able to support the growth of fer1, although no individual colony was observed. 48 well-plate was unsuitable for growth quantification but able to identify positive growth. Yeast extract was unable to support growth at either high (0.5% w/v) or low (0.01% w/v) concentrations. Glucose had no effect on fer1's growth with concentration of 0.1% w/v or 0.01% w/v. Increasing FeSO4 concentration from 20 g/L to 100 g/L inhibited growth of fer1. Base medium concentration significantly (p=0.046) increased the growth of fer1 in a medium with lower concentration of yeast extract and excessive accessible energy. No defined medium that support visible growth was found, mainly due to the unique carbon usage of fer1. Elucidation of a defined medium to support growth of fer1 continues. Discovering a defined medium will enable research into the metabolic capacities of fer1, which will enhance our understanding of how extremophiles exist at the boundaries of life and raise the accuracy of bioinformatic predictions.

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