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Spectroscopic study of the purple sulfur bacteria

Chromatium sp. cultured in an aqueous medium

ARTICLE in MOSCOW UNIVERSITY PHYSICS BULLETIN · JUNE 2007

Impact Factor: 0.25 · DOI: 10.3103/S0027134907030101

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Svetlana Patsaeva
Lomonosov Moscow State University
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ISSN 0027-1349, Moscow University Physics Bulletin, 2007, Vol. 62, No. 3, pp. 170–173. © Allerton Press, Inc., 2007.
Original Russian Text © A.S. Milyukov, S.V. Patsaeva, V.I. Yuzhakov, E.L. Rostovtseva, 2007, published in Vestnik Moskovskogo Universiteta. Fizika, 2007, No. 3, pp. 40–43.

Spectroscopic Study of the Purple Sulfur Bacteria

Chromatium sp. Cultured in an Aqueous Medium
A. S. Milyukova, S. V. Patsaevaa, V. I. Yuzhakova, and E. L. Rostovtsevab
a
Department of General Physics, Faculty of Physics, Moscow State University,
Leninskie gory, Moscow, 119992 Russia
e-mail: spatsaeva@mail.ru
b Faculty of Biology, Moscow State University, Leninskie gory, Moscow, 119992 Russia

Received March 1, 2006

Abstract—A spectroscopic study of the photosynthetic purple sulfur bacteria of the Chromatium genus is per-
formed at various growth stages and under different culture conditions. Absorption and luminescence spectra
of the bacterial cells are found to be useful in estimating their populations within certain concentration ranges.
The relative proportion between the porphyrin and blue-band luminescence intensities can be used as an indi-
cator of the physiological state of the culture.
DOI: 10.3103/S0027134907030101

INTRODUCTION erations with enormously high numbers of cells. Their

The littoral zones of seas are particularly vulnerable
to the growing anthropogenic influence. Therefore, bio-
logical monitoring of these zones, considered as a part
of a more general monitoring of the entire biosphere, is
especially important for the assessment of the state of a
local biota in intensely polluted areas [1].
Ecological monitoring can be effective only with a
sufficiently developed network of observatories regu-
larly collecting primary information. The list of the abi-
otic parameters to be monitored is rather common: tem-
perature, salinity, concentrations of main elements, etc.
The situation with biotic parameters (especially for
populations of particular species) is much more com-
plicated. Identification of some species is impossible
without the involvement of highly qualified specialists.
A hope to get around this difficulty is shown by the con-
cept of indicator species, whose number is used to
judge changes in the entire community. In this context,
the role of express tests for assessing the state of a spe-
cific biological community becomes increasingly high.
Spectral analysis is successfully used in studying
phytoplankton [2, 3] and testing organic substances [4–
6] dissolved in natural waters. We made an attempt to
apply spectral analysis to count the population and to
assess the state of purple sulfur bacteria, which are an
important component of the littoral biota.
We experimented with the purple sulfur bacteria of
the genus Chromatium. The photosynthetic purple bac-
teria are widely spread in nature. They can be found in
almost every water basin and in the soil. Their predom-
inant habitats are fresh and sea waters containing
hydrogen sulfide. In stagnant basins enriched with
organic matter, the purple bacteria form mass agglom-
photosynthetic range lies from 800 to 900 nm [7].
Representatives of the Chromatium genus are the
most prevalent microorganisms in natural waters. They
grow in the bulk of the water and accumulate in the
benthos, easily adapting to variations of salinity, con-
tent of dissolved oxygen, concentration of organic sub-
stances, temperature, and pH. These microorganisms
are an important component of the sulfur cycle in water
basins. Their ability to oxidize hydrogen sulfide is help-
ful in removing this toxic substance [8].
In the future, spectroscopy of the physiological state
of purple sulfur bacteria may provide an instrument for
biological monitoring of water basins by express
detecting the response of the phototrophic bacterial
community to changes in the ambient physicochemical
conditions.
This work was designed to study the spectral prop-
erties of purple sulfur bacteria of the Chromatium
genus at various stages of their growth under different
conditions. The absorption spectra, as well as the spec-
tra of luminescence excitation and emission, were
recorded at different stages of the culture growth (expo-
nential growth, stationary phase, etc.) and for the cul-
tures grown under different light conditions. The absorp-
tion spectra were recorded within a 200- to 900-nm
range using a Specord M40 spectrophotometer. The
luminescence spectra were excited at wavelengths of
270 and 390 nm and detected using the Jobin Yvon 3CS
and Perkin Elmer LS55 instruments over a range of up
to 800 nm. All the measurements were performed in
standard quartz cells with a 1-cm-long optical path.

170
 SPECTROSCOPIC STUDY OF THE PURPLE SULFUR BACTERIA Chromatium sp.
D D
1.5 for cultures grown
in luminostat
porphyrins at daylight
in darkness
1.0
bacteriochlorophyll800
0.5
0
400 500 600 700 800
Wavelength, nm
Fig. 1. Absorption spectra of Chromatium sp. cultured
under different conditions.
ABSORPTION SPECTRA
OF THE Chromatium sp. CULTURES
The absorption spectra of bacterial culture Chroma-
tium sp. (Fig. 1) display strongly pronounced absorp-
tion peaks of protoporphyrin (at λ = 390 nm) and a cer-
tain form of bacteriochlorophyll (at λ = 800 nm). The
sulfur bacteria owe their purple color to carotinoids,
which intensely absorb in the blue-green spectral range.
However, the peaks specific for individual carotinoid
compounds are indiscernible against a broad back-
ground due to the absorption of other organic com-
pounds and scattering in the suspensions of bacterial
cells, which is especially strong in the in-vivo cultures
(in contrast to that in the extracts made with organic
solvents). The contribution of scattering increases at
shorter wavelengths and in the UV region prevails over
the absorption caused by the cellular pigments.
When the culture with a known cell number (deter-
mined by a direct counting [9]) was diluted with the
nutrient medium, the optical density monotonically
decreased over the entire spectral range studied. The
gradual dilution of the culture with the nutrient medium
allowed us to determine the concentration range where
the absorption spectra are applicable to count the bac-
terial populations. Optical densities in the regions of
absorption of the purple bacteria pigments (protopor-
phyrins (390 nm), carotinoids (600 nm), and bacterio-
chlorophyll (800 nm)) are linear with the bacteria con-
centration in water up to 6 million cells per ml (Fig. 2).
At higher concentrations, no reliable result was
obtained because of the sedimentation of cells during
the measurements.
A comparison between the optical densities of the
7-day-old bacterial cultures grown in darkness, under
daylight, and in a luminostat (Fig. 1) shows that a
MOSCOW UNIVERSITY PHYSICS BULLETIN Vol. 62
171
1.5
390 nm
600 nm
800 nm
1.0
0.5
0 1 2 3 4 5 6 7
Concentration, 106 cell/ml
Fig. 2. Optical density vs. the bacteria concentration.
higher illumination intensity results in a higher growth
rate at the exponential stage.
LUMINESCENCE SPECTRA
OF A Chromatium sp. CULTURE
Three main bands are observed in the UV and visi-
ble parts of the luminescence spectra of the Cromatium
sp. bacterial culture:
(i) a UV band of the tryptophan residues of protein
complexes with a maximum at λ = 340–350 nm was
excited by wavelengths below 270 nm;
(ii) a broad blue-band luminescence with a maxi-
mum at λ = 440–450 nm;
(iii) narrow bands of porphyrins with the emission
maxima at 616 and 680 nm and the excitation maxi-
mum at 390 nm.
Figure 3 shows the concentration dependences of
the intensity of different luminescence bands in the
spectrum obtained on the excitation at 390 nm.
To determine the applicability of the luminescence
method for estimating the bacteria concentration, the
bacterial culture was successively diluted with the
nutrient medium to a known extent. It is found that the
intensities of all three fluorescence bands initially grow
in proportion to the cell count and, then, tend to saturate
(i.e., deviate from the linear increase and, sometimes,
even decrease). The saturation is observed at a cell con-
centration of about 2 million cells per ml and may be
caused by a too high absorption of the excitation light
and the reabsorption of the emitted fluorescence light in
a highly concentrated solution.
Thus, the intrinsic fluorescence of the purple sulfur
bacteria in an aqueous medium can be used for quanti-
tative estimation of their concentration in the range of a
linear growth of the fluorescence intensity (up to 2 mil-
lion cells per ml).
No. 3 2007
172 MILYUKOV et al.

I, rel. units Excitation at 390 nm

600 I, rel. units
fl335
500 fl440 For cultures grown
fl616 60 at daylight
400
40 7-day-old
300 28-day-old
200 20

100 0

0 1 2 3 4 5 60
In luminostat
Concentration, 106 cell/ml
40 7-day-old
Fig. 3. Intensity of different luminescence bands vs. the 28-day-old
bacteria concentration during the exponential growth phase. 20

0
The luminescence intensity depends not only on the
cell number, but also on the conditions of their growth.
Figure 4 shows the luminescence spectra obtained for 60
In darkness
the excitation at 390 nm in the bacterial cultures grown
40 7-day-old
under different conditions (in darkness, under natural
28-day-old
light conditions, and in a luminostat). For comparison,
20
each plot shows spectra for the 7- and 28-day-old cul-
tures (exponential and stationary phases, respectively). 0
For the 7-day-old cultures, the intensities of the blue- 400 450 500 550 600 650 700
band luminescence and fluorescence of porphyrins are Wavelength, nm
maximal for the cultures grown in the luminostat and
minimal for the cultures grown in darkness. These Fig. 4. Fluorescence spectra of the purple bacteria grown
results agree very well with the corresponding absorp- under different conditions and in different physiological
tion spectra. During the following hold of the samples states.
for three weeks at natural illumination, the number of
cells in the cultures grown under different conditions the corresponding changes in the relative proportions
tends to equalize, which is evident from comparison of of the porphyrin and blue-band luminescence intensi-
the intensities of blue-band luminescence of different ties [10, 11].
samples. As time elapses, the fluorescence band of the
porphyrin pigments fades and almost vanishes in the Special experiments performed with the cultures of
stationary phase. different age have shown that ageing results in an
almost twofold increase in the ratio between the UV
Therefore, the porphyrin fluorescence intensity in and blue-band intensities. In contrast, the ratio between
the Chromatium sp. culture depends not only on the the porphyrin and blue-band luminescence intensities
illumination level, but also on the growth phase. The decreases by a factor of 2.5 (see table). These ratios can
“ageing” of the culture is accompanied by a pro- be used as indicators of the physiological state of the
nounced decrease in the porphyrin fluorescence and cell culture.

Relative intensities of the porphyrin (Ip) and blue-band (If) CONCLUSIONS

fluorescence maxima
(1) Absorption and luminescence spectra of bacte-
Concen- Young culture Mature culture rial cells can be used for their in vivo quantitation in
tration, I , rel. I , rel. Ip, rel. If, rel. aqueous media within a certain range of the cell con-
p f Ip/If Ip/If
% units units units units centrations. Optical density measured in the absorption
bands of the purple bacteria pigments—protoporphy-
10 14 26 0.54 8 35 0.22 rins (390 nm) and bacteriochlorophyll (800 nm)—
15 24 40 0.60 13 56 0.23 grows linearly with the bacteria concentration up to
22 25 45 0.55 10 44 0.23 6 million cells per ml. The fluorescence of the purple
bacteria due to the tryptophan residues (350 nm)
33 18 48 0.38 14 87 0.16 excited at 270 nm, as well as the blue-band lumines-
50 33 58 0.57 11 49 0.22 cence (450 nm) of cells excited at 390 nm, can be used

MOSCOW UNIVERSITY PHYSICS BULLETIN Vol. 62 No. 3 2007

 SPECTROSCOPIC STUDY OF THE PURPLE SULFUR BACTERIA Chromatium sp.
to estimate the bacteria concentrations up to 2 million
cells per ml.
(2) The luminescence spectra taken of the bacteria
cultures grown under different light conditions showed
that, during the exponential growth, the highest and
lowest bacterial counts were obtained for the cultures
grown in a luminostat and darkness, respectively. Dur-
ing the following 3-week-long stationary phase under
natural illumination, this difference in the cell counts
diminishes; at the same time, the ratio of the cellular
pigments changes, thus, leading to a change in the rel-
ative intensities of the luminescence bands.
(3) The experiments with the cultures in different
growth phases revealed that the ratio of intensities of
the porphyrin and blue luminescence bands can be used
as an indicator of the physiological state of purple sul-
fur bacteria.
The results obtained can be used to elaborate an
express method for ecological monitoring.
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