Tian, Wei, The biological
metabolism of nitrate and nitrite in Pseudomonas fluorescens K27 amended
with tellurium. Master of Science (Chemistry), December 2004, Sam Houston
State University, Huntsville, Texas, 82 pp. (pdf version
of this thesis)
The purpose of this research was to determine the initial steps in the nitrate and nitrite metabolism of Pseudomonas fluorescens K27, investigate tellurite influence on this reduction and whether K27 has a nitrate assimilatory system and determine if K27 has separate nitrate and nitrite reductase systems.
Experiments were carried out by inoculating Pseudomonas fluorescens K27 in growth medium with or without tellurite amendment and growing anaerobically. Samples were analyzed in regular time intervals for cell growth using optical density, for extracellular ammonium ion concentration using an ammonia-selective electrode, and for extracellular nitrate and nitrite content using colorimetric or UV-VIS spectrometry.
Nitrate is a preferred terminal electron acceptor for the anaerobic growth of K27 in tryptic soy broth medium. Nitrite acts as both a terminal electron acceptor and toxic reagent: low level nitrite supports the anaerobic growth of K27; high level nitrite inhibits the anaerobic growth of K27.
Nitrate and nitrite were reduced concomitantly in the anaerobic conditions studied; nitrate reduction rate was faster than nitrite reduction rate as measured by the disappearance of these anions in growing cultures. Part of the newly-formed nitrite bacterially produced was further reduced; the other unreduced nitrite accumulated to its highest level at the point in time at which nitrate was used up.
Ammonium ion was produced by K27 in the minimal medium used in other experiments with NO3- as the only N source. At the time point that the nitrate was used up, both nitrite and ammonium concentrations attained their highest level. After that, nitrite and ammonium concentrations gradually decreased.
In tryptic soy broth with added nitrate amended with tellurite, the specific growth rate of K27 was inhibited; nitrate uptake per bacterium also was decreased. In tryptic soy broth with added nitrate and with 0.1 mmol/L tellurite amendment, nitrate was used up in 5 days but nitrite was at high levels after 7 days. In this same medium with 0.2 mmol/L tellurite added, both nitrate and nitrite were not used up after 7 days.
In tellurite-amended conditions, before the point of change at which nitrite gained its highest concentration in solution, tellurite inhibited a little bit of the nitrate reduction and decreased nitrite reduction a lot. Therefore newly-formed nitrite concentration was increased faster than that in cultures without tellurite amendment. K27 apparently has separate nitrate and nitrite reductase systems because the reduction of these anions is carried on concomitantly no matter which anion is higher in concentration. Nitrate is used up first even in growth media of high levels of nitrite. On the other hand, nitrite reduction is not stopped even if the nitrate concentration is high.