Eriksen, Jr., Lynn Reduction and methylation of various selenium anions by Pseudomonas fluorescens K27 in bioreactor-based batch cultures. Master of Science (Chemistry), August, 1999, Sam Houston State University, Huntsville, Texas, 75 pp. (pdf version of this thesis)


The purpose of the experiments in this work was to track the changes in cellular growth, pH and concentrations of specific volatile headspace compounds produced by batch cultures of Pseudomonas fluorescens K27 amended with various selenium salts using a commercial bioreactor. Research of this nature has not been carried out before.
Batch cultures inoculated with Pseudomonas fluorescens K27 were grown in a New Brunswick BioFlow III Batch/Continuous Fermentor with a 10% tryptic soy broth, 3% potassium nitrate growth media. Changes in cellular growth were followed by extracting liquid cultures from the fermentor during the experiment and using a spectrometer to analyze for optical density at 526 nm. Changes in pH were monitored by pH probe measurements of the same liquid cultures used to take optical density readings.

Bioreactor headspace concentrations were measured by extracting headspace via gas tight syringe from the fermentor. Separation and detection of headspace compounds were carried out by gas chromatography equipped with sulfur chemiluminescence detection. Also, an effort was made to trap gas purged headspace compounds with a 30% hydrogen peroxide solution for analysis of selenium by atomic absorption spectroscopy detection in order to determine the selenium mass balance.

Batch cultures amended with single or mixed concentrations of selenate and/or selenite produced repeatable results similar to prior established work. In general, control cultures exhibited a specific growth rate near previously reported work for unamended batch cultures: 0.30 h -1 . Ten millimolar selenate cultures showed trends that suggest that selenate is less toxic than selenite. Also, results from 2:1 and 1:2 selenate to selenite mixed cultures suggest that a higher concentration of one type of selenium oxyanion in the mix causes a change in specific growth rate based upon the toxicity of the higher concentration selenium oxyanion as compared to 1:1 selenate to selenite amended cultures. Again, selenite appears to be more toxic than selenate to this organism.

Changes in pH over the experimental time course showed the most variability between cultures amended with 1:0, 2:1, and 1:2 selenate to selenite. In general, the 2:1 amendments exhibited a drop in pH while the 1:0, 0:1 and 1:1 selenate to selenite cultures displayed an increase in pH.

The headspace trapping experiments showed that only very small amounts of volatile organoselenium species are purged at the flow rates examined. Less than 0.05% of the added Se was trapped after 10 hours of approximately 2 mL/min continuous nitrogen gas flow.

Thomas G. Chasteen
Thesis Director

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