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FEMS Microbiology Ecology 52 (2005) 129–137
Formation of insoluble magnesium phosphates during growthof the archaea Halorubrum distributum and Halobacterium salinarium
and the bacterium Brevibacterium antiquum
Aleksey Smirnov a, Natalia Suzina a, Natalia Chudinova b,Tatiana Kulakovskaya a,*, Igor Kulaev a
a Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russiab Institute of General and Inorganic Chemistry, Russian Academy of Sciences, 119191 Moscow, Russia
Received 23 June 2004; received in revised form 10 October 2004; accepted 28 October 2004
First published online 25 November 2004
Abstract
Stationary phase cells of the halophilic archaea Halobacterium salinarium and Halorubrum distributum, growing at 3–4 M NaCl,
and of the halotolerant bacterium Brevibacterium antiquum, growing with and without 2.6 NaCl, took up �90% of the phosphate
from the culture media containing 2.3 and 11.5 mM phosphate. The uptake was blocked by the uncoupler FCCP. In B. antiquum,
EDTA inhibited the phosphate uptake. The content of polyphosphates in the cells was significantly lower than the content of ortho-
phosphate. At a high phosphate concentration, up to 80% of the phosphate taken up from the culture medium was accumulated as
Mg2PO4OH Æ 4H2O in H. salinarium and H. distributum and as NH4MgPO4 Æ 6H2O in B. antiquum. Consolidation of the cytoplasm
and enlargement of the nucleoid zone were observed in the cells during phosphate accumulation. At phosphate surplus, part of the
H. salinarium and H. distributum cell population was lysed. The cells of B. antiquum were not lysed and phosphate crystals were
observed in the cytoplasm.
� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Halobacterium salinarium; Halorubrum distributum; Brevibacterium antiquum; Magnesium phosphate; Polyphosphate; Phosphate uptake
1. Introduction
Microorganisms play a significant role in the phos-
phorus turnover in nature. Using extracellular enzymes,
microorganisms convert organic phosphorus com-pounds into soluble forms, available for other genera-
tions of microorganisms and plants [1]. In addition,
they are able to dissolve natural phosphates (calcium,
aluminum or iron salts) [1,2] and, moreover, microbial
cells can take up phosphate (Pi) via specific transport
systems [3,4]. The ability to grow at both high and low
0168-6496/$22.00 � 2004 Federation of European Microbiological Societies
doi:10.1016/j.femsec.2004.10.012
* Corresponding author. Tel.: +7 95 9257448; fax: +7 95 9233602.
E-mail address: [email protected] (T. Kulakovskaya).
Pi concentrations is one of the adaptation mechanisms
to changing environmental conditions.
Microorganisms are able tomaintain a rather constant
intracellular Pi level, independent of its concentration in
the medium [3–5]. This is allowed by regulation of the Pi
uptake [6,7] and by accumulation of reserve phosphorus
compounds in cells [5]. The main phosphorus reserve in
most microorganisms comprises inorganic polyphos-
phates, linear polymers of orthophosphate [5,8–11].How-
ever, other reserve phosphorus compounds are known as
well. Some fungi possess polymeric orthophosphates of
metals [5], and cyanobacteria accumulate Pi in the cell
envelope [12]. A significant amount of pyrophosphatewas found in cells of some bacteria, algae and protozoa
. Published by Elsevier B.V. All rights reserved.
130 A. Smirnov et al. / FEMS Microbiology Ecology 52 (2005) 129–137
[5,13–15]. The cell wall teichoic acids of bacteria also can
act as a phosphate reserve [16].
The ability for phosphate accumulation is quite differ-
ent in various microorganisms. For example, Escherichia
coli takes up a minor amount of Pi from the culture med-
ium and has a low intracellular level of polyphosphates[4]. On the contrary, the bacteria involved in the process
of ‘‘enhanced biological phosphate removal’’ (EBPR) are
able to take up large quantities of Pi from waste [17–19].
The cells of such bacteria contain considerable amounts
of polyphosphates [17–19]. Previously, we have shown
that the halophilic archaeon Halobacterium salinarium
is very effective in accumulation of Pi from the culture
medium [20–22]. Moreover, insoluble magnesium phos-phate is formed during this accumulation [21,22]. It is
still unknown if other archaea or bacteria possess such
an unusual form of phosphorus reserve.
The aim of this work was to study the accumulation
of Pi and to identify the reserve phosphorus compounds
in the halophilic archaeon Halorubrum distributum and
halotolerant bacterium Brevibacterium antiquum in com-
parison with H. salinarium.
2. Materials and methods
2.1. Microorganisms
Two halophilic archaea were used in the work: H.
salinarium ET 1001, provided by Kalebina T.S. (Mos-cow State University), and H. distributum VKM-1739,
provided by All-Russian Collection of Microorganisms,
Russian Academy of Sciences (VKM RAS). Escherichia
coli K-12 and the halotolerant bacterium B. antiquum
VKM Ac-2118 (isolated from permafrost soils of the
Kolyma lowlands, Russia) were also provided by
VKM RAS [23].
2.2. Culture conditions
The cultures were maintained on solid slant agar media
at 4 �C for no longer than 1 month. The solid media con-
tained the same compounds as the liquid media, with the
addition of bactoagar (Difco, USA) (20 g/l).
The cultures of H. salinarium, H. distributum and E.
coli were grown in a liquid medium in flasks (mediumvolume of 200 ml) on a shaker (200 rpm) at 37 �C. Brev-ibacterium antiquum was grown under the same condi-
tions at 28 �C.The culture medium for H. salinarium contained per
liter: NaCl, 250 g (4.3 M); KCl, 2 g; sodium citrate, 3
g; MgSO4 Æ 7H2O, 20 g; peptone (Diacon, Russia), 7 g.
The culture medium for H. distributum contained per
liter: NaCl, 200 g (3.4 M); KCl, 2 g; sodium citrate, 3 g;sodium glutamate, 1 g; MgSO4 Æ 7H2O, 20 g; yeast ex-
tract (Serva, Germany), 5 g; FeSO4 Æ 7H2O – 36 mg;
MnCl2 Æ 4H2O, 0.36 mg. The pH was adjusted to 7.0
by adding NaOH.
The culture medium for B. antiquum contained per
liter: MgSO4 Æ 7H2O, 20 g; glucose, 5 g; yeast extract
(Serva, Germany), 3 g; peptone (Diacon, Russia), 5 g.
The effect of salinity was studied by adding 150 g/l NaCl(2.6 M) to the medium. Various concentrations of
MgSO4 Æ 7H2O (0, 5, 10, 20 g/l) were added to the cul-
ture medium to study the effects of Mg2+ ions.
The culture medium for E. coli contained per liter:
NaCl, 5 g; KCl, 1.5 g; NH4Cl, 1 g; CaCl2, 0.5 g;
MgSO4 Æ 7H2O, 20 g; C4H11NO3, 6 g; glucose, 10 g;
yeast extract (Serva, Germany), 3 g; peptone, 5 g. The
pH was adjusted to 7.0 by adding NaOH.Sterile K2HPO4 solution was added to the concentra-
tions of 0.8, 2.3, 7.8 and 11.5 mM as indicated in the leg-
ends to tables and figures.
2.3. Preparation of biomass for analyses
After growth in liquid medium, the biomass of H.
salinarium and H. distributum was harvested at 5000gfor 40 min and washed twice with a medium containing
per liter: NaCl, 250 g; KCl, 2 g; sodium citrate, 3 g;
MgSO4 Æ 7H2O, 20 g.
The biomass of B. antiquum and E. coli was harvested
at 5000g for 40 min and washed twice with distilled
water at 14,000g for 40 min. Wet biomass value was
used for calculations.
2.4. Extraction of phosphorus compounds from biomass
Fresh biomass samples were used for extraction of
phosphorus-containing compounds. First, the biomass
was extracted three times at 0 �C for 15 min with a mix-
ture of methanol:chloroform (3:1, v/v) during stirring to
obtain a phospholipid-containing fraction. After centri-
fugation at 5000g for 20 min, supernatants were com-bined. The amount of phosphorus in this fraction was
determined as Pi after the treatment with 3.5 M HClO4
at 150 �C.Residual biomass was used for extraction of Pi and
polyphosphates as described by Kulaev [5] and Wanner
[7]. The biomass was extracted by 0.5 N HClO4 at 0 �Cfor 30 min by stirring and then centrifuged under the
same conditions. The supernatant contained acid-solu-ble polyphosphates. The precipitate was extracted by
0.05 N NaOH at 0 �C for 30 min and centrifuged to
obtain an alkali-soluble polyphosphate fraction. The
content of Pi and polyphosphates as labile phosphorus
[21] was determined in the acid-soluble and alkali-solu-
ble fractions. The remaining precipitate was treated with
0.5 N HClO4 for 30 min at 90 �C. The amount of acid-
insoluble polyphosphates was estimated by the contentof Pi in hot perchlorate extract [21,24]. Pi was assayed
accordingly [25].
0
3
6
9
12
1 2 3 4 5 6 7
Time (d)
Pi i
n th
e m
ediu
m (
mM
)
0
3
6
9
12
Wet
bio
mas
s (g
/litr
e)
Fig. 1. Growth of H. distributum at 2.3 mM Pi (m) and 7.8 mM Pi (d).
Phosphate uptake by H. distributum during growth in the media with
2.3 mM Pi (n) and 7.8 mM Pi (s).
0
3
6
9
Pi i
n th
e m
ediu
m (
mM
)
9
12
15
18
ass
(g/li
tre)
(a)
(b)
A. Smirnov et al. / FEMS Microbiology Ecology 52 (2005) 129–137 131
2.5. Obtaining water-insoluble phosphates from biomass
The biomass of H. salinarium and H. distributum was
lysed by adding distilled water at 4 �C for 5–10 min in a
glass homogenizer with a teflon pestle. The biomass of
B. antiquum was frozen at �70 �C and extruded usinga French press (IBPM, Russia). The obtained homoge-
nate was suspended in distilled water.
In both cases, centrifugation was carried out at 5000g
for 20 min. White water-insoluble precipitate was
washed three times with distilled water in the same cen-
trifugation mode and analyzed.
2.6. Analysis of water-insoluble precipitate compositions
The compounds in the water-insoluble precipitates
were identified by elemental analysis using atomic emis-
sion spectroscopy with inductively coupled plasma
(ICP-AE8) (ISA Jobin Yvon, France). The content of
Pi in the precipitates was also determined after their
complete dissolution in 6 N HCl [21].
The compounds were identified by roentgen-phase as-say using the Gynie-de-Wolf chamber (Cu Ka irradia-
tion) (Enraf-Nonius, Holland) and the JCPDS database
(International Centre for Diffraction Data, 1999). The
infrared spectroscopy and the differential thermogravi-
metric analysis by derivatograph Q-1500D (MOM, Hun-
gary Optical factory, Budapest) were carried out as well.
2.7. Ultrathin sections
Biomass samples were fixed in 1.5% glutaraldehyde
solution in buffer A (0.05 M cacodylate, 0.08 M MgSO4,
pH 7.2) at 4�C for 1 h. Buffer A contained 4.3 M NaCl
in case of the halophilic archaea. The biomass was
washed three times in buffer A and additionally fixed
in 1% OsO4 solution in the same buffer for 3 h at 20
�C. After dehydration in a series of alcohols, the mate-rial was embedded in Epon 812 epoxyresin. Ultrathin
sections were mounted on supporting grids, contrasted
for 30 min in 3% uranylacetate solution in 70% alcohol,
and additionally contrasted by lead citrate according to
Reinolds [26]. Ultrathin sections were observed in an
electron microscope JEM-100B (JEOL, Japan) at an
accelerating voltage of 80 kV.
0
3
6
0 30 60 90 120 150 180
Time (h)
Wet
bio
m
Fig. 2. Growth of (a) and phosphate uptake from the medium (b) by
B. antiquum on the media with different initial Pi concentrations in the
absence and in the presence of 2.6 NaCl. (s) 2.3 mM Pi; (d) 11.5 mM
Pi; (n) 2.3 mM Pi + 2.6 NaCl; (m) 11.5 mM Pi + 2.6 M NaCl. The
medium contained 80 mM MgSO4.
3. Results
3.1. Pi uptake by H. distributum and B. antiquum during
growth
The Pi uptake from the culture medium was observed
during growth of the halophilic archaeon H. distributum
(Fig. 1). The cells of this archaeon took up �95% of the
medium Pi, at initial Pi concentrations of 0.8 (not
shown), 2.3 and 7.8 mM (Fig. 1). Similar results were
obtained earlier for another halophilic archaeon H. sali-
narium [21,22].
The Pi uptake was also observed during growth of the
halotolerant bacterium B. antiquum (Fig. 2). During sta-
tionary growth, nearly 90% of the Pi is taken up from
the medium, both at 2.3 and 11.5 mM Pi, when the cellsare grown without NaCl (Fig. 2(b)). The level of Pi up-
take by B. antiquum was lower in the medium containing
2.6 M NaCl, probably due to growth suppression (Fig.
2(b)). Thus, the ability to accumulate Pi from the culture
medium was similar in H. salinarium, H. distributum and
B. antiquum.
132 A. Smirnov et al. / FEMS Microbiology Ecology 52 (2005) 129–137
As a control, E. coli, with the well-studied Pi trans-
port [3,4], was grown in the medium containing the same
concentrations of Pi (2.3 and 11.5 mM) and MgSO4 (80
mM). During the stationary growth phase, only 20%
and 6% Pi was removed from the medium at 2.3 and
11.5 mM Pi, respectively (not shown). These amountscorresponded to the common ability of E. coli for Pi up-
take [4]. So, the microorganisms under study accumu-
lated Pi more effectively than E. coli.
3.2. Effect of the uncoupler FCCP on Pi uptake
We have investigated the effect of the uncoupler
FCCP (carbonyl cyanide p-(trifluoromethoxy) phen-ylhydrazone) on the growth of and the Pi uptake by
the cultures under study. Both the growth of H. distrib-
utum and the Pi uptake were suppressed by 0.005 mM of
FCCP (not shown). Similar effects were observed for H.
salinarium [22] and for B. antiquum (Table 1). It was not
unlikely that its uptake could occur via the DlH+-
dependent transport system, as known for many pro-
karyotes [27].So, the observed decrease of the Pi concentration in
the culture media depended on the growth as such of
the above microorganisms. It should be noted that pH
values in the media were 6.0–7.0 during cultivation
and therefore, the decrease of the Pi concentration in
the culture medium cannot be due to alkalization of
the medium in the course of cultivation or to chemical
precipitation of Pi.
3.3. Effect of the Mg2+ concentration on Pi accumulation
by B. antiquum
In some microorganisms, the Pi uptake depends on
the presence of bivalent cations such as Mg2+, Ca2+,
Co2+ and Mn2+ [28]. In particular, Acinetobacter has a
transport system carrying Mg2+ and Pi nearly in equi-molar amounts [29]. In the experiments described in Sec-
tions 3.1 and 3.2 the culture media contained 80 mM of
MgSO4. H. salinarium and H. distributum did not even
grow at 60 mM of MgSO4 whereas the growth of B.
antiquum was the same at various concentrations of this
compound (Table 2).
Table 1
The effects of FCCP (0.005 mM) and EDTA (4 mM) on Pi uptake by
B. antiquum
Wet biomass
after 70 h of
growth, g/l
Pi in the medium
after 70 h of
growth, mM
Control 14.8 1.3
FCCP added to 10-h culture 7.0 5.0
FCCP added to 20-h culture 7.5 4.3
EDTA added to 10-h culture 4.5 8.0
EDTA added to 20-h culture 9.0 5.7
The cells were grown with 11.5 mM Pi.
Therefore, the effect of Mg2+ on the Pi uptake was
studied for B. antiquum. The levels of Pi uptake were
the same at 20–80 mM MgSO4 (Table 2). It should be
noted that the culture medium without MgSO4 con-
tained some Mg2+ derived from yeast extract and pep-
tone. This amount was sufficient for growth and forcomplete removal of Pi from the culture medium with
2.3 mM Pi (Table 2). However, if MgSO4 was not added
into the culture medium with 11.5 mM Pi, its uptake de-
creased (Table 2). EDTA suppressed the growth of and
Pi uptake by B. antiquum (Table 1). Thus, Mg2+ ions are
essential for Pi uptake by B. antiquum.
3.4. Pi and polyphosphates in the biomass of H. distribu-tum and B. antiquum
In H. distributum cells, inorganic polyphosphates
were found in three fractions: in the acid-soluble, the al-
kali-soluble and the fraction of hot perchlorate extract
(Table 3). The main part of polyphosphates was ob-
served in the acid-soluble fraction (Table 3). The content
of these compounds increased at increasing Pi concen-tration in the culture medium, but the major part of
the phosphorus taken-up was revealed as Pi in H. dis-
tributum cells. The amount of Pi in the biomass in-
creased 7.5-fold at increasing Pi concentration in the
medium from 2.3 to 7.8 mM (Table 3). Similar results
were obtained earlier for H. salinarium [22].
In the cells of B. antiquum, the levels of Pi and
polyphosphates were low at 2.3 mM Pi and in the ab-sence of NaCl (Table 3). Apparently, this Pi concentra-
tion was not surplus and consumed Pi was used for the
biosynthesis of phosphorus-containing organic com-
pounds. In the presence of NaCl, the growth of B. antiq-
uum was inhibited (Fig. 2(a)), and even 2.3 mM Pi was a
surplus concentration. The total polyphosphate content
increased twice and the Pi content increased 15-fold as
compared with the cells grown without NaCl. At thesame time, 11.5 mM Pi was a surplus concentration in
both cases. About 70–80% of the Pi consumed by the
culture was revealed as orthophosphate. The level of
phosphorus in the phospholipid fractions of all microor-
ganisms did not depend on the Pi concentration in the
medium (Table 3).
Thus, the major part of th Pi accumulated in the bio-
mass of H. distributum, H. salinarium and B. antiquum
under Pi surplus was present as orthophosphate,
whereas most microorganisms accumulate phosphorus
mainly as polyphosphates [5,8,24].
3.5. Identification of inorganic phosphoric salts in the bio-
mass of H. salinarium, H. distributum and B. antiquum
During stationary growth, water-insoluble Pi-con-taining precipitates could be obtained from the biomass
Table 3
The content of some phosphorus compounds in the biomass of H. distributum and B. antiquum (mmol P/g of wet biomass)
The initial Pi concentration in the medium, mM
H. distributum B. antiquum
(�NaCl) (+NaCl)
2.3 7.8 2.3 11.5 2.3 11.5
Pi consumed from the medium 140 860 130 500 260 290
Pi in the biomass 100 760 6.2 340 92 240
Phospholipid fraction 4.0 4.4 8.7 8.0 6.4 6.2
Acid-soluble polyphosphates 28 87 2.6 49 16 15
Alkali-soluble polyphosphates 5.3 7.8 0.3 0.5 0.5 1.5
Polyphosphates of hot perchlorate extract 0.7 1.2 8.7 158 12.3 6.5
Cells were grown to the stationary growth phase: H. distributum for 5 days, B. antiquum for 60 h without NaCl and for 120 h with NaCl.
Table 2
The effects of MgSO4 added to the culture medium on the growth and Pi uptake (60 h of growth) by B. antiquum (without NaCl)
MgSO4, mM The initial concentration of Pi in the medium, mM
2.3 11.5
Wet biomass, g/l Pi in the medium, mM Wet biomass, g/l Pi in the medium, mM
0 12.0 0.4 12.0 6.0
20 14.2 0.3 13.2 1.1
40 13.7 0.5 13.8 0.9
80 14.0 0.4 14.5 0.8
A. Smirnov et al. / FEMS Microbiology Ecology 52 (2005) 129–137 133
of H. salinarium and H. distributum grown at 2.3 and 7.8
mM Pi in the culture medium, whereas in the case of B.antiquum in the same growth phase, such precipitates
could be obtained only in the medium with 11.5 mM
Pi. The precipitates completely dissolved in 6 N HCl at
room temperature. The levels of Pi per 1 g of wet bio-
mass in the precipitates were similar to that in Table 3.
The contents of phosphorus and metal cations in the
precipitates were determined by atomic emission spec-
troscopy with inductively coupled plasma (ICP-AE8).This method showed that the main components of the
precipitates were magnesium and phosphate (Table 4).
Table 4
Element composition of Pi-containing precipitates obtained from the bioma
Element, % Sample
H. salinariuma
Phosphorus 13
Magnesium 9.7
Calcium <0.05
Iron 0.025
Manganese 0.0022
Strontium
Sodium 0.3
Potassium <0.03
Lithium
The method of analysis was atomic emission spectroscopy with inductively ca Cells grown on a medium with 11.5 mM Pi to the stationary growth phb Cells grown on a medium with 7.8 mM Pi to the stationary growth phac Cells grown on a salt-free medium with 11.5 mM Pi to the stationary g
The roentgen-phase analysis demonstrated that the
major component of the above precipitates for H. sali-
narium and H. distributum was Mg2PO4OH Æ 4H2O
(International Centre for Diffraction Data, 1999, N
44-0774). For B. antiquum, the major compound of
phosphate-containing precipitates was NH4MgPO4 Æ6H2O (International Centre for Diffraction Data,
1999, N 15-0762). The presence of NHþ4 was confirmed
by infrared spectroscopy. The characteristic absorption
maximum at 1435–1470 cm�1 was observed [30] andthe spectrum was identical to that of NH4MgPO4 Æ6H2O in [31].
ss of H. salinarium, H. distributum and B. antiquum
H. distributumb B. antiquumc
12 12
8.1 10.8
0.008 0.54
0.016 0.44
0.0007 0.031
<0.00006 0.024
<0.003 0.3
0.94 <0.06
<0.002 <0.002
oupled plasma (ICP-AE8).
ase.
se.
rowth phase.
134 A. Smirnov et al. / FEMS Microbiology Ecology 52 (2005) 129–137
The content of H2O and NH4, determined by thermo-
gravimetry, corresponded to the stoichiometry of
NH4MgPO4 Æ 6H2O for the precipitate from B. antiq-
uum. The content of H2O and OH groups, determined
by same method, corresponded to the stoichiometry of
MgPO4OH Æ 4H2O for the precipitate of both archaea.Probably, the accumulation of insoluble magnesium
salts retains phosphorus in an osmotic-inert form. The
chemical composition of the precipitates explains the
need of magnesium ions for Pi accumulation. For
B. antiquum, the high content of NH4 suggests the possi-
bility of using such salt as a nitrogen reserve for the cells.
3.6. Ultrathin sections of H. distributum and B. antiquumgrown under phosphate surplus
Fig. 3(a) shows an electron microphotograph of a H.
distributum cell in a medium with low Pi (0.8 mM), in the
stationary growth phase. In the same growth phase, in
the medium with 7.8 mM Pi, the cytoplasm becomes
more dense and homogeneous and the nucleoid zone in-
creases (Fig. 3(b)). Moreover, the electron microphoto-graph also shows magnesium phosphate crystals (Fig.
3(c)). Light microscopy confirmed that some parts of
the cells were broken and free magnesium phosphate
Fig. 3. Electron microscopy of ultrathin sections of H. distributum cells. (a) C
Cells grown to the stationary phase in the medium with 7.8 mM Pi. (c) Ma
cytoplasm. The bar is 0.3 lm.
crystals appeared (not shown). We suggested that mag-
nesium phosphate was first accumulated in the cells and,
after cell lysis, occurred in the biomass as crystals. Sim-
ilar changes in the cell state were observed for H. salina-
rium [22].
Electron microscopy revealed the changes in the cellstructure of B. antiquum under massive Pi accumulation.
Fig. 4(a) shows the typical thin section of B. antiquum
cells grown at 2.3 mM Pi, when the Pi content in the cells
was low (Table 3). The cells have a rod-shaped structure
with the intracellular contents and cell wall states typical
of bacteria. However, under Pi surplus (11.5 mM) elec-
tron-dense areas are observed in the cell cytoplasm
and the nucleoid zone alters (Fig. 4(b)). Moreover, crys-tals that are probably water-insoluble phosphates were
observed inside some cells (Fig. 4(c)). In contrast to
the archaea under study, neither lysed cells nor free crys-
tals were revealed by electron and light microscopy at a
high level of Pi accumulation (not shown). Probably, the
resistance of B. antiquum to high contents of insoluble Pi
salts is due to its strong cell wall containing a consider-
able amount of peptidoglycan [32].Some cyanobacteria demonstrate the phenomenon of
posthumous mineralization of trichoms [13]. At 2.5 mM
Pi in the medium, Pi-containing mineral jackets were
ells grown to the stationary phase in the medium with 0.8 mM Pi. (b)
gnesium phosphate crystals. Arrows point to: 1 – nucleoid zone; 2 –
Fig. 4. Electron microscopy of ultrathin sections of B. antiquum cells. (a) Cells grown to the stationary phase (60 h) in the medium with 2.3 mM Pi
and 80 mMMgSO4. (b) Cells grown to the stationary phase (60 h) in the medium with 11.5 mM Pi and 80 mMMgSO4 and (c) Cells grown to the late
stationary phase (90 h) in the medium with 11.5 mM Pi and 80 mM MgSO4. Arrows point to: 1 – nucleoid zone; 2 – cytoplasm; 3 – crystal inclusions
in the cytoplasm. The bar is 0.3 lm.
A. Smirnov et al. / FEMS Microbiology Ecology 52 (2005) 129–137 135
formed on the cell surface [13]. However, in our case
microscopy revealed no such phenomenon. It should
be noted that only 5–9% of the total Pi was convertedinto a soluble form after washing the biomass of B.
antiquum with 10 mM EDTA solution (not shown). This
confirms the intracellular localization of accumulated Pi
in B. antiquum.
4. Discussion
The cells of H. salinarium, H. distributum and B.
antiquum took up Pi from the culture medium and accu-
mulated it in the biomass to the levels of 0.9, 0.5 and
0.65 mmol Pi g�1 of wet biomass, respectively. The
above levels are similar to those of the eubacteria from
activated sludge, which are characterized by the highest
Pi uptake. For example, Acinetobacter johnsonii [33],
Microlunatus phosphovorus [34] and Rhodocyclus sp.
[35] accumulated 0.8, 1.0, and 0.6 mmol Pi g�1 of wet
biomass, respectively. The recombinant strains of E.
coli, with mutations enhancing the activities of Pi trans-port systems, accumulated 0.4 mmol Pi g
�1 of wet bio-
mass [36]. The high ability of the microorganisms in
this study to remove Pi from the medium is of interest
for developing biotechnological phosphate removal
processes.
Up to 80% of phosphate taken up from the culture
medium was stored as Mg2PO4OH Æ 4H2O in H. salina-
rium andH. distributum and as NH4MgPO4 Æ 6H2O in B.
antiquum. Deposition of water-insoluble phosphate in a
cell maintains the concentration of free Pi ions in the
cytoplasm at a constant level, as in case of polyphos-
phates accumulation.
It should be noted that energy (�10 kcal/mol) is re-
quired for the synthesis of one phosphoanhydride bond
in the polyphosphate molecule [5]. The Pi uptake is also
an energy-consuming process. In case of polyphosphate
136 A. Smirnov et al. / FEMS Microbiology Ecology 52 (2005) 129–137
accumulation, a cell spends energy for Pi uptake and
polyphosphate synthesis. On the other hand, during
accumulation of magnesium phosphate, the energy is
spent on Pi uptake only. The accumulation of magne-
sium phosphate by a cell requires much less energy than
the accumulation of polyP, and might be preferable un-der energy limitation. Probably, such reservation is of
ancient origin.
The cells of H. salinarium [22], H. distributum and B.
antiquum were able to use the accumulated magnesium
phosphate during growth on Pi-limited media. Despite
the death of part of the cell populations of H. salinarium
and H. distributum, the formation of insoluble magne-
sium phosphate might be a useful factor for further sur-vival of the population as a whole on a Pi-limited
medium. Microorganisms, which are able to convert
phosphate into insoluble forms and to use it again as
phosphorus source for their own growth under changed
conditions, could be important participants of the phos-
phorus circulation in the environment.
The question arises if microorganisms of other sys-
tematic groups, which are not halophilic or halotolerant,might accumulate such compounds. For example, the
bacteria of activated sludge accumulate phosphorus
mainly as Pi or as polyphosphates, depending on the
carbon source type and availability and on the presence
of bivalent cations in wastewater [29,37–39]. Possibly,
the inclusions observed in the cells of B. antiquum are
similar to membrane-enclosed intracellular structures
containing polyphosphates and Pi, which were foundin the cells of Agrobacterium tumifaciens [15].
This work has revealed the ability of microorganisms
for massive Pi uptake, formation of insoluble phospho-
rus compounds and utilization of this reserve under Pi
limitation for biosynthetic processes, which demon-
strates an important role of some prokaryotes in the
phosphorus circulation in the environment.
Acknowledgements
This work was supported by Grant 02-04-48544 from
the Russian Foundation for Basic Research and Grant
1382.2003.4, supporting the leading scientific schools
of Russian Federation. We are thankful to Dr. A. Sereda
and Dr. E.V. Murashova for their help in the chemicalanalysis of phosphate-containing precipitates and to
Mrs. Elena Makeeva for assistance in the preparation
of the manuscript.
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