Question

Hi everyone! I am working on some anaerobic cultures, and I am looking for a good method to calculate the depletion rate of the added Nitrate in the culture medium. Which method do you recommend as a sensitive one for concentration of micro-molar? Would you please share your valuable experience and knowledge on the material and methods? Thanks in advance.
Roya

Answer 1

See the attached paper, they can quantify uM amounts of Nitrates in water using cyanobacterial hole-cell luminescent bio-reporters and the second one methods that use ultraviolet absorption spectrometry

Construction and characterization of a cyanobacterial bioreporter
capable of assessing nitrate assimilatory capacity in freshwaters
Natalia V. Ivanikova, R. Michael L. McKay, and George S. Bullerjahn*
Department of Biological Sciences, Life Sciences Building, Bowling Green State University, Bowling Green, OH 43403
Abstract
The use of cyanobacterial whole-cell luminescent bioreporters has enhanced our ability to monitor nutrient availability
in aquatic ecosystems. We have constructed a Synechocystis sp. strain PCC6803 bioluminescent reporter for the
assessment of nitrate bioavailability. Specifically, a 380–base pair DNA fragment containing the NtcA/B-dependent
nitrate/nitrite-activated nirA promoter (regulating expression of genes encoding nitrite reductase) was fused to the
bacterial luciferase genes, luxAB, and introduced into Synechocystis by genetic transformation. Characterization of this
strain, designated AND100, yielded dose-dependent increased bioluminescence coincident with increased nitrate
added to the growth medium from 1 to 100 µM. Bioluminescence in response to nitrate addition was light dependent
up to 50 µmol quanta m–2 s–1. Assessing environmental samples collected from oligotrophic Lake Superior, we
demonstrated that the onset of luminescence coincided with the drawdown of nitrate by simultaneously monitoring
nitrate depletion from reaction vessels. Nitrate in the Lake Superior samples was consistently underestimated by the
bioreporter. Only by following amendment of these samples with phosphate and iron was total nitrate accurately
reflected by the cyanobacterial bioreporter. Thus, strain AND100 can be used to elucidate factors that constrain use
of nitrate in freshwaters. This is pertinent to a system such as Lake Superior where the concentration of nitrate has
increased 6-fold in the last century. Indeed, pilot experiments with the bioreporter suggest that nutrient co-limitation
(P and Fe), as well as low light, may reduce the capacity for nitrate assimilation in field samples from Lake Superior.