Title

Cultivation Optimization of Methylosinus Trichosporium Ob3B for Methanobactin Recovery: Structural Effects of Dissolved Oxygen and Cell Density on the Molecular Stability of Methanobactin

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological and Environmental Sciences

Date of Award

Summer 2015

Abstract

Methanobactin (mbs) are Cu-binding chromopeptide secreted by methanotrophs, as a part of methanotrophic copper acquisition. The methanobactin (mb) produced from Methylosinus trichosporium (mb-OB3b; C45H58N10O16S5; Mw 1154.26) has become the archetype molecule due to the extensive molecular and functional characterization. However, our preliminary results indicate potential purity issues in previous characterization attempts. The mb-OB3b prepared following the standard purification resulted in heavily contaminated mb samples with a minimum of 40% methanotrophic siderophores. In our attempts to improve mb-OB3b sample quality, we found another contamination source, the decomposed mb-OB3b molecules. Further studies have demonstrated that mb-OB3b production as well as quality may be related to the oxygen saturation level. Methylosinus trichosporium OB3b was cultivated in Bioflo110 bioreactor, where agitation speed, temperature, gas flow (ambient air and CH4), and copper availability were controlled in semi-continuous batch mode. Dissolved oxygen, pH, and cell density were monitored as potential indicators of in situ mb-OB3b productivity levels. The Cu and dissolve oxygen (DO) levels were changed accordingly. Secreted mb-OB3b was isolated by tangential flow filtration followed by elution using an HP20 Diaion column. The information obtained from recorded DO levels, Cell Density measurements, and data provided by UV-Vis spectrophotometer were analyzed. Mass spectrometry analysis agrees with the spectrophotometric data showing a decrease in methanobactin ion counts as DO decrease during the cultivation for mb productions. Findings suggest that increased oxygen tension is an important requirement for the quality production of mb.

Advisor

Dongwon Choi

Subject Categories

Biology | Life Sciences

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