FEMS Microbiology Letters 35 (1986) 43-47
Published by Elsevier
43
FEM02438
Selective synthesis of polysaccharides by Rhizobium trifolii,
strain TA-1
(Rhizobium trifolii; EPS; CPS; fl-l,2-glucans)
L.P.T.M. Z e v e n h u i z e n
Laboratory of Microbiology, Agricultural University. Itesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands
Received 13 January 1986
Accepted after revision 19 February 1986
1. S U M M A R Y
Rhizobium trifolii strain TA-1, produces one of
each of the exocellular polysaccharides EPS, CPS
and fl-l,2-glucans as a major product during
cultivation in glutamic acid-mannitoi-salts (GMS)
medium at 25 °. In batch culture, the major exocellular polysaccharide product was acidic exopolysaccharide (EPS) under conditions of air
saturation; capsular polysaccharide (CPS) under
conditions of N-limitation and moderate oxygen
supply; and cyclic fl-l,2-glucans at high cell density and severe oxygen limitation.
cultivation in 0.1% glutamic acid - 1% mannitol salts medium (50 ml) in 100-ml Erlenmeyer flasks
on a shaker at 25 °. Most of the strains were found
to produce mainly EPS and only little CPS, but R.
trifolii strain TA-1, was exceptional in that it
produced mostly CPS and only small amounts of
EPS under these conditions [1,2]. When R. trifolii
TA-1 was cultivated in a fermenter under forced
aeration (100% oxygen concentration) a highly
viscous culture was obtained, which contained EPS
as the major product, and less CPS. These current
experiments describe further the influence of cultural conditions on the ratios of E P S / C P S / g l u c a n s
synthesised by R. trifolii TA-I.
2. I N T R O D U C T I O N
3. M A T E R I A L S A N D M E T H O D S
Fast-growing rhizobia produce 3 different types
of exopolysaccharides. These are, firstly, highmolecular-weight EPS, which are excreted into the
medium and cause the cultures to become highly
viscous; secondly, neutral insoluble CPS, which
are deposited as voluminous masses around the
cells; and thirdly cyclic fl-l,2-glucans, which are
accumulated in the cells and excreted into the
medium.
Rhizobium leguminosarum and R. trifolii strains
have been tested for polysaccharide production by
3.1. Cultivation of organisms
GMS-medium was composed of (g/l) glutamic
acid, 1; mannitol, 10; K 2 H P O 4, 1; MgSO 4 • 7H20,
0.2; CaCl 2, 0.04; trace elements (mg/l): FeC! 3.
6H20, 2.5; H3BO3, 0.01; Z n S O 4 . 7 H 2 0 , 0.01;
CoCl 2 • 6H20, 0.01; CuSO 4 • 5H20, 0.01; MnC12,
1.0; N a E M o O 4 . 2 H 2 0 , 0.01; biotin, 10 ~tg/l;
thiamine, 100 # g / l ; p H 7.0. 4 × G M S medium
was composed as follows (g/l): glutamic acid, 4;
mannitol, 20; KEHPO4, 2; M g S O 4 . 7 H 2 0 , 0.2;
0378-1097/86/$03.50 '~ 1986 Federation of European Microbiological Societies
44
CaCI 2, 0.05; biotin, 20 t~g/l; thiamine, 200/.tg/1;
trace elements as above. For routine experiments
50 ml of medium in 100 ml Erlenmeyer flasks
were inoculated with 1 ml of preculture and incubated on a gyratory shaker at 25 °. Whole cultures were removed from the shaker after different
periods of incubation and analysed as follows.
3.2. A nalysis of cultures
Samples of 10 ml were centrifuged at high
speed to separate cells and supernatant liquid.
Supernatant liquid (0.1 ml of samples) was
analysed for hexose content with the anthronesulphuric acid reaction and expressed as glucose
equivalents, and for uronic acid content with mhydroxydiphenyl-sulphuric acid and expressed as
glucuronic acid equivalents. EPS concentrations
were based on the uronic acid content of the
supernatant and calculated using the known ratios
of hexose : uronic acid (3 : 1) of EPS and expressed
as the sum of the glucose and glucuronic acid
equivalents. Glucan concentrations were deduced
from the hexose (glucose) portion, which was not
accounted for in the calculation of EPS concentration. CPS was extracted from the cell pellet with 1
N NaOH and measured with anthrone-suiphuric
acid and expressed as galactose equivalents.
3.3. Isolation and purification of polysaccharides
EPS was isolated by precipitation with 3 vols.
of ethanol added to the cell-free supernatant under stirring, dissolving the gelatinous precipitate in
water, reprecipitating with ethanol and freeze-drying.
fl-l,2-Glucans were extracted as follows. The
alcoholic supernatant, after EPS isolation, was
concentrated to 1/20th of the original culture
volume, and a further 3 vols. of ethanol were
added. Additional precipitated EPS was removed
by centrifugation, and more alcohol was added to
about 10 vols. On standing, a fine white precipitate formed which was collected by centrifugation,
dissolved in water and freeze-dried. This material
still contained uronic acid-containing substances
(oligosaccharides) which could be removed by anion-exchange chromatography over a DEAE-Trisacryl column which passed the neutral glucan
fraction but retained the acidic substances. The
neutral glucan fraction was further purified over
Ultragel AcA202 and freeze-dried.
CPS was extracted from the cell pellet by stirring in 1 N NaOH at room temperature, followed
by centrifugation of the cells and precipitation of
CPS by the addition of 1 vol. of ethanol. After
centrifugation of CPS it was washed several times
with ethanol/water (1 : 1) and water until neutral,
and then freeze-dried.
3.4. Chemical methods
Colorimetric determinations of hexoses with
anthrone-sulphuric acid and of hexuronic acids
with m-hydroxydiphenyl-sulphuric acid on unhydrolysed material, pyruvate and mannitol determinations, were done as described earlier [3], as
were gas-chromatographic separations of component sugars of hydrolysates (2-N-trifluoroacetic
acid. 100°C, 6 h) and of partially methylated
sugars resulting from methylation analysis. Viscosity measurements were carried out with a Ubbelohde viscometer at 25°C. Separation of cyclic
B-1,2-glucans was achieved by HPTLC on silica
gel glass plates (Merck) in the system Nb u t a n o l / e t h a n o l / w a t e r (5 : 5:4, v / v / v ) in the
ascending way (3-4 times); spray: 5% sulphuric
acid in ethanol followed by heating at 100°C [10].
Protein of cells was measured by the method of
Lowry et al. [16].
4. RESULTS AND DISCUSSION
4.1. Identification of isolated polysaccharide fractions from R. trifolii, strain TA-I
EPS of R. trifolii TA-I has the compositions
o-glucose/D-galactose/D-glucuronic acid/pyruvic
acid (5 : 1 : 2 : 2) (approx. molar ratios) [3]. Methylation analysis of EPS has afforded a pattern of
partially methylated sugars [4] which was common
to the methylated sugar patterns of EPSs from a
number of R. leguminosarum, R. trifolii and
Rhizobium phaseoli strains [5]. The complete structure of this type of EPS has been elucidated [6-8],
leading to the repeating unit I of Fig. 2.
Isolated glucans from R. trifolii TA-1 were
completely built up from glucose, and on methylation analysis yielded exclusively 3,4,6-tri-O-
45
RHIZOBIUM TRIFOLII TA-1
monnitol
--~ 4 -G Ic A --~ 4-G ICA --*/. -G Ic~4-GIc--~
~-mg/ml
Produc, yields
EPS: 9,3 %
CPS 20 %
E
O
fial
Pyr
5.
Is
addlh0n
aa
"--b3- H an --~ 3 fJal--W.,-Olc--~ 1111
lC
Gat
T
moJmt
85 mg/h/g protein
.3.2
pyr
Yield(PlS):32%
~.------- E PS
~'3
O
Prod r a t e
?6rng/h/g
u
£L
ILl
protein
2
/
£PS
I
o
•
o
U,
h/g
¢:
X
/
profeln
3,-.
•
2
~
8
10
12
J
•
08
lt,.
s ° ~ C P S
/ i
2
°
6
I
Yield : 10"/.
20 i
4
|
6
i
8
i
10
!
days
Fig. 2. Polysaccharide synthesis by R. trifolii TA-1 durin~
batch cultivation in a 1-1 Kluyver flask containing 0.5 1 GMS
medium under forced aeration (600 ml air/min) at 25°C. Cell
protein, 600 txg/ml; EPS, acidic exopolysaccharide in glucose
and glucuronic acid equivalents.
16 "
days
Fig. 1. Polysaccharide synthesis by R. trifolii TA-I during
mannitol-fed batch culture. Cultivation took place in 50 ml of
medium in 100 ml Erlenmeyer flasks on a shaker at 25°C. Cells
were grown in 0.1% glutamic acid/0.5% mannitol/salts medium
for 6 days. and then extra mannitol (1%) was added to the
nitrogen-limited stationary culture. Cell protein, 600 #g/ml of
culture: EPS, total of excreted polysaccharides in glucose
equivalents; CPS, capsular polysaccharides in galactose equivalents. Specific rates of mannitol consumption and of polysaccharide synthesis are indicated by the numbers, pH values
remained between 6-8.
methyl-D-glucose. H P T L C of the cyclic glucans on
silica gel showed 3 m a i n c o m p o n e n t s . By c o m p a r i son with a p a r t i a l h y d r o l y s a t e of the glucans,
which showed
glucose and
a series of
o l i g o s o p h o r a o s e s with D P s of 2 - 1 8 , it was estim a t e d that the native glucan m i x t u r e of R. trifolii
TA-1 had ring sizes of 16, 17 a n d 18 glucose
residues (structure III, Fig. 3) [9,10].
C P S of R. trifolii TA-1 had a c o m p o s i t i o n of
D - g l u c o s e / D - g a l a c t o s e / o - m a n n o s e (1 : 4 : 1). Its
p r i m a r y structure was d e t e r m i n e d by a c o m b i n a tion of m e t h y l a t i o n analysis a n d p e r i o d a t e o x i d a tion leading to structure II of Fig. 1 [11].
~2- OIc~ 2-OIC~'-2-GIC)
~
(Ill)
?
m /ml
lo
"oroZ
~s
o.
12rnglhlgproteli
E
/:
~-Z---
._
/
,~,~
2
,~.
4
6
,
8
.
tO
~--~--
E PS
i
.
.
12
,
14
.'\
16
days
Fig. 3. Polysaccharide synthesis by R. trifolii TA-1 during
batch cultivation in 4 x GMS medium. Cultivation took place
in 100 ml of medium in 300-ml Erlenmeyer flasks at 25°C on a
shaker. Protein of cells, 1700 #g/ml of culture.
46
4.2. Influence of cultural conditions on pol),saccharide production by R. trifolii TA-1
On routine cultivation in GMS medium in
Erlenmeyer flasks with shaking, R. trifolii TA-I
excreted soluble polysaccharides (EPS) during the
growth phase and during the N-limited stationary
phase, but gradually EPS excretion stopped and
was taken over by production of insoluble CPS in
excess of carbon of the medium [2]. When the
process was carried out in 2 steps, cell growth took
place during the first stage in the complete medium
with 0.5% mannitol, with production of EPS and
lesser amounts of CPS. In the second stage, when
extra mannitol (1%) was added, only small
amounts of soluble polysaccharides (mainly
glucans) were excreted, but CPS production continued at a constant rate of 17 m g / h / g protein,
up to a concentration of 3 g / l and product yield
of 20% (Fig. 1).
In a second experiment, R. trifolii TA-1 was
growth in GMS medium under forced aeration in
a Kiuyver flask. Acidic EPS without any glucan
was produced at a high rate of 76 m g / h / g protein
in a overall yield of 32%, and CPS in a much
lower yield of 10%. calculated from the mannitol
consumed (Fig. 2). A highly viscous culture solution (r/: 164 cP) was obtained.
Attempts were made to increase the concentration of CPS by using a 4-fold concentration of
glutamic acid in the 4 x GMS medium, but only
low amounts of EPS and of CPS were produced,
and instead up to 4 g / l of low-molecular weight
/3-1,2-glucans were excreted. Glucans appeared
only late in the process, with a production rate of
12 m g / h / g protein and an overall yield of 20%
(Fig. 3).
Depending on the mode of cultivation of R.
trifolii TA-1, it is possible to direct polysaccharide
synthesis towards one product or another: EPS
under conditions of air saturation, CPS under
conditions of N-limitation and moderate oxygen
supply in shaking cultures, and /3-1,2-glucans in a
medium with 4-fold N-source concentration,
thereby creating a dense cell population and consequently severe oxygen limitation. From these
experiments it follows that oxygen supply has a
pronounced effect on the nature of the principal
product.
Rhizobiaceae are notable for their complex patterns of polysaccharide products, in the form of
viscous EPS, insoluble CPS. cyclic /3-1,2-glucans
and octasaccharide repeating units of EPS. Agrobacterium usually produces cyclic /3-1,2-glucans
(1 2 g/l) together with large amounts of succinoglycan a n d / o r curdlan (up to 14 g/l) and
octasaccharide repeating units [12]. Spontaneous
mutants have been obtained which exclusively
p r o d u c e either curdlan or succinoglycan.
Rhizobium cultures were found to contain small
amounts of /3-1,2-glucans (100-400 mg/l), often
in combination with large amounts of viscous EPS
a n d / o r octasaccharide repeating units of EPS [13].
EPS-negative mutant strains of R. phaseoli have
been prepared which only excrete cyclic /3-1,2glucan (approx. 1 g/l) without the formation of
acidic poly- and oligosaccharides [14]. These experiments were carried out in high carbohydrate
(2-4%). N-limited media by cultivating the
bacteria in Erlenmeyer flasks at 25 30°C for 5--6
days with shaking, but without special attention to
variation in cultural conditions and to incubation
times. Since certain polysaccharide mutant strains
have proved to be unstable or less productive,
especially during continuous cultivation [15], it is
suggested that investigations are carried out with
highly productive wild-type strains and with adjustment of specific cultural conditions and growth
phases to the optimal production of desired polysaccharides. These may then be obtained from a
single bacterial strain in amounts of g / l of culture, according to separate procedures in (fed)
batch processes.
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