atw International Journal for Nuclear Power | 04.2020

atw Vol. 65 (2020) | Issue 4 ı April
RESEARCH AND INNOVATION 232
| Fig. 4.
Luminescence spectra for the initial medium R red with 200 µM U(VI)
as well as cells and supernatants after 24 h exposure to 200 µM U(VI).
Spectra recorded after a delay of 0.1 µs.
medium as well as plant cells and
supernatants after exposure to U(VI).
These samples were examined by
means of TRLFS. Complexation with
inorganic and organic ligands present
in solution may change the speciation,
i.e. the physico-chemical form in
which the uranium is present in
solution. These changes in speciation
can be detected by TRLFS. Each U(VI)
species has a spectrum with characteristic
band positions and a typical
luminescence lifetime, which can
be determined by time-dependent
measurements. These data can be
used to determine how many and
which species occur in the system.
Reference substances can be used to
identify them. For biological samples,
cryo-TRLFS measurements are carried
out in particular, in which the samples
are cooled to -120 °C. This step is
necessary because numerous quenching
effects occur in the system due to
medium and cell components that
suppress the luminescence of U(VI).
At low temperatures, quenching
effects are suppressed as far as
possible and spectra can be obtained.
Figure 4 gives an overview of the
luminescence spectra for the initial
nutrient medium with 200 µM U(VI)
and cells and supernatants after 24 h
exposure to 200 µM U(VI).
It can be seen that the cell species
differs from the species in the medium
or the supernatant. According to the
literature [3] it can be assumed that
this cell species is formed by the
binding of UO 2
2+
to cell membranes
or cell walls. Especially phosphate
groups are of importance for the
UO 2
2+
sorption processes. A closer
look at the spectrum from the initial
medium and from the supernatant
after 24 h cell contact shows that
speciation in the medium changes
over time due to cell contact. From the
experiments it can be concluded that
the ((UO 2 ) 3 (OH) 5 ) + complex dominates
in the initial medium according
to the speciation calculations performed
(not shown) and the band
positions [2] of the luminescence
spectrum. However, the species that
dominates the luminescence in the
supernatant is different. The measurements
of the supernatants at different
exposure times showed that various
species occur in the supernatants over
time (not shown). Starting from the
medium species, the ((UO 2 ) 3 (OH) 5 ) +
complex [2], two more species appear
with increasing exposure time. Thus,
the sub-process of biocomplexation
could be detected spectroscopically
for B. napus cells. Both species represent
potential U(VI) complexes
with plant cell metabolites. To identify
these species it is necessary to identify
released metabolites and to investigate
their complexation behavior
with U(VI).
Plant cells release metabolites
in response to exposure
to uranium and europium
For the experiments on the enrichment
and identification of plant cell
metabolites only very high concentrations
of U(VI) and Eu(III) were
used and the exposure time was fixed
at 1 week in order to accumulate as
many metabolites as possible in the
nutrient medium. For the identification
of metabolites in the supernatants
after cell contact, sequentially
solid phase extraction, HPLC (highperformance
liquid chromatography)
and MS (mass spectrometry) measurements
were performed. For solid
phase extraction columns were used
that were suitable for enrichment of
phenolic compounds and flavonoids.
This procedure made it possible to
obtain chromatograms of the enriched
eluates from the extraction
(Figure 5).
| Fig. 5.
Chromatograms of solid phase extraction eluates of cell culture media after
plant cell exposure to 200 µM U(VI) or Eu(III) in comparison to those of
control samples. Peaks that are new or exhibit higher intensities compared
to the control sample are marked.
Of particular interest are peaks in
the chromatograms, which either
show increased intensities or are new,
compared to the control sample. A
first peak assignment is possible by
measuring reference compounds by
HPLC and comparing their retention
times with those found for the eluates
from the solid phase extraction. In the
literature, phenolic compounds and
flavonoids in particular are mentioned
as typical plant metabolites that are
exuded in response to heavy metal
stress. [21,26] In addition, however,
smaller molecules such as organic
acids or amino acids, peptides and
amines are also mentioned. [21] A
number of representatives from these
mentioned substance classes have
therefore been measured by HPLC.
Peaks, which matched in their retention
times for reference and solid
phase extraction eluates were examined
more closely. The cor responding
peaks were fractionated and two metabolites
could be identified using MS:
p-coumaric acid and fumaric acid.
However, the investi gation of the
complexation behavior of these
compounds with U(VI) showed that
these substances are not involved in
the formation of the two species
identified in the TRLFS measurements
of the supernatants. Therefore,
further investigations on metabolite
release are still necessary. However,
a complexation constant could be
obtained from the complexation
experiments with fumaric acid, which
will be used for calculations.
Summary
The investigation of the timedependent
bioassociation behavior as
well as the spectroscopic, spectrometric
and chromatographic investigation
of the formed cell metabolites
and metal species led to molecular
understanding of the interaction of
Research and Innovation
Studies on the Interaction of Plant Cells with U(VI) and Eu(III) and on Stress- induced Metabolite Release ı Jenny Jessat, Susanne Sachs, Robin Steudtner and Thorsten Stumpf