Journal of Biotechnology 61 (1998) 135141

Characterization of exopolysaccharides produced by 19

halophilic strains of the species Halomonas eurihalina
Victoria Bejar
a,
*, Inmaculada Llamas
a
, Concepcio n Calvo
b
, Emilia Quesada
a
a
Exopolysaccharide Research Group, Department of Microbiology, Faculty of Pharmacy, Uni6ersity of Granada,
18071 Granada, Spain
b
Water Institute, Uni6ersity of Granada, Granada, Spain
Received 23 September 1997; received in revised form 19 January 1998; accepted 23 January 1998
Abstract
The formation, chemical composition and rheological properties of the exopolysaccharides (EPS) produced by 19
strains belonging to Halomonas eurihalina have been compared in two different culture media. Our aim was to screen
several strains isolated from saline soils to select those producing maximum EPS yield and good rheological
properties. We found that MY medium was best for the production of EPS in all the strains studied. Maximum EPS
production was 1.6 g l
1
with strain H212 grown in this medium. The pattern of the chemical composition of the
polysaccharides was affected by the strain in question and by the culture medium. All EPS studied had an unusually
high sulphate content. Furthermore, the exopolymer from strain H96 contained signicant amounts of uronic acid.
EPS from strain H96, cultivated in dened NH medium, behaved in an interesting way rheologically; when the pH
of the polymer solution was decreased to 3.0 a gel with a viscosity of 30 000 cP formed. 1998 Elsevier Science B.V.
All rights reserved.
Keywords: Halomonas eurihalina; Exopolysaccharides; Halophilic bacteria
1. Introduction
Bacterial growth is often accompanied by the
production of exopolysaccharides (EPS), which
have important ecological and physiological func-
tions. Increasing interest is being generated in the
study of these molecules because of their wide
applications in food, pharmaceutical, petroleum
and other industries (Dawes, 1990; Sutherland,
1990). Nevertheless, the strains used for the indus-
trial production of EPS belong to a small number
of taxa, such as Xanthomonas campestris (Evans et
al., 1979), Pseudomonas (Jarman, 1979), Azoto-
bacter (Jarman et al., 1978), Sphingomonas (Lobas
et al., 1992), Alcaligenes (Sutherland, 1990), etc.
There are still good prospects, however, for devel- * Corresponding author. E-mail: vbejar@platon.ugr.es
0168-1656/98/$19.00 1998 Elsevier Science B.V. All rights reserved.
PII S0168-1656(98)00024-8
V. Bejar et al. / Journal of Biotechnology 61 (1998) 135141 136
oping new polysaccharides with better properties
than those of the existing polymers because of the
wide diversity offered by microorganisms.
Volcaniella eurihalina is a moderately halophilic
bacteria described by us in 1990 (Quesada et al.,
1990). Recently, Mellado et al. (1995) reclassied
this microorganism on the basis of its 16S rRNA
sequence and proposed that Volcaniella eurihalina
should be transferred to the genus Halomonas.
Moderately halophilic bacteria are dened as be-
ing those which grow best in media containing
0.52.5 M salts and they constitute the most
important eubacteria group which lives in hyper-
saline habitats (Kushner and Kamekura, 1988).
In previous reports we have studied the produc-
tion, chemical composition and rheological prop-
erties of the exopolysaccharide produced by strain
F2-7 of Halomonas eurihalina (Quesada et al.,
1993, 1994; Bejar et al., 1996). The most notable
characteristic of this EPS was its capacity to
increase the viscosity of solutions at low pH val-
ues (Calvo et al., 1995). This property would
make it valuable for use in the food industry as an
additive in salad sauces or citric desserts, where
the pH is usually acidic.
In the course of our studies in saline soils we
have isolated a group of H. eurihalina strains. All
these strains show mucoid growth in solid media.
Thus, we decided to evaluate the quantity of EPS
produced by each strain in two different media.
We also determined their chemical composition
and rheological properties. The main object of
this work is focused: (i) to determine whether EPS
production is common to all strains of H. euri -
halina, and (ii) to select the best strains of this
species for the production of polysaccharides with
adequate rheological properties and any other
interesting features for industrial application.
2. Materials and methods
2.1. Bacterial strains
A total of 18 strains of H. eurihalina isolated
from saline soil in Alicante (Southern Spain) were
tested for their ability to produce exopolysaccha-
ride (EPS) in two different media. Strain F2-7
(producer of EPS V2-7) was also used as reference
strain.
2.2. Growth media
For the isolation of EPS, strains of H. euri -
halina were grown for 8 days at 32C in a complex
medium (MY) (Moraine and Rogovin, 1966) or in
a minimal medium (NH) with glucose as the sole
carbon and energy source (Ng and Hu, 1989).
Both media were supplemented with a sea-salt
solution (Rodr guez-Valera et al., 1981) to reach
7.5% (w/v) salt concentration.
2.3. Isolation and purication of EPS
The cultures were centrifuged at 36000g in a
Sorvall RC-5B refrigerated centrifuge for 60 min
and the supernatants were then tangentially
ltered with 100000 Da ultralters (Minitan Sys-
tem, Millipore) and precipitated with 3 vols of
cold ethanol. This solution was kept at 4C
overnight before being centrifuged. The EPS was
resuspended in distilled water and puried by
ultracentrifugation at 226000g for 60 min in a
Beckman L8-M ultracentrifuge. The pellet was
dissolved in water, dialyzed against distilled water
for 24 h, lyophilized and then weighed.
The EPS was puried in an anion-exchange
column (Hitrap Q, Pharmacia) equilibrated with a
0.02 M tris-amino-methane hydrochloric acid
buffer (pH 7.5). Polysaccharides were applied to
the column and washed with the same buffer. The
column was eluted with a linear gradient of NaCl
from 0 to 0.4 M at a ow rate of 0.5 ml min
1
.
The fractions were tested qualitatively for carbo-
hydrate (Dubois et al., 1956) and protein contents
(Lowry et al., 1951; Bradford, 1976).
2.4. Analytical procedures
We made the following colorimetric analyses:
total carbohydrates (Dubois et al., 1956), proteins
(Lowry et al., 1951; Bradford, 1976, in both meth-
ods we used albumin from Sigma as standard),
hexosamines (Johnson, 1971), uronic acids (Blu-
menkrantz and Asboe-Hansen, 1973) and acetyl
residues (McComb and McCready, 1957).
V. Bejar et al. / Journal of Biotechnology 61 (1998) 135141 137
Table 1
EPS yields of Halomonas eurihalina strains
MY medium NH medium
%
b
g l
1
g l
1a
%
Range of 0.81.6 12.427.7 0.20. 8.220.7
values
c
19.3 0.3 Mean values
c
15.3 1.2
26.2 0.3 1.4 17.3 Strain F2-7
18.3 0.5 Strain H28 18.1 1.2
27.7 0.2 1.6 20.1 Strain H212
0.9 Strain H214 27.4 0.5 16.7
14.9 0.4 0.9 13.1 Strain H217
1.3 Strain H96 22.5 0.6 20.7
a
Grams of EPS per liter of culture medium. Each value is a
mean of three determinations.
b
Grams of EPS per gram of dry cell weight. Each value is a
mean of three determinations.
c
Data corresponding to the 19 strains of Halomonas euri -
halina.
Cationic (Na
+
, K
+
, Ca
2+
, Mg
2+
) and sul-
phate contents were determined with a Dionex
DX-300 gradient chromatography system with
chemical suppression of the eluent conductivity.
For cations we used an 18 mmol l
1
hy-
drochloric acid solution as eluent with deion-
ized water with a specic resistance of 18 MV
and the regenerant was a 10 mmol l
1
tetra-
butylammonium hydroxide solution. To deter-
mine sulphates, the eluent was a Na
2
CO
3
/
NaHCO
3
mixture made by diluting 10 ml
0.18 mol l
1
Na
2
CO
3
/0.17 mol l
1
NaHCO
3
of
concentrated eluent to a nal volume of 1 l in
18 MV of deionized water. Finally, we used a
17.5 mmol l
1
H
2
SO
4
solution as regenerant.
2.5. Thin-layer chromatography (TLC)
The neutral sugar composition of the EPS
was analysed by TLC in cellulose as described
before (Quesada et al., 1993; Bejar et al., 1996).
2.6. Rheological studies
Lyophilized samples obtained under the con-
ditions described above were dissolved in dis-
tilled water to give 1% (w/v) solutions. The
viscosity of the solutions was measured with a
Brookeld viscosimeter RTV tted with a small
sample adapter (Brookeld Engineering Labora-
tories, MA). Determinations were made at 25C
and at different shear rates (1.5, 3 and 6 rpm).
The inuence of pH on the viscosity was stud-
ied by lowering the pH of the EPS solutions
with 1 N HCl to 3.0.
3. Results
3.1. Producti6ity
The MY medium encouraged stronger growth
of all the strains of H. eurihalina than the NH
medium. Since EPS production was always
linked to biomass the highest yields were also
obtained in MY medium. The production of
EPS varied between strains of H. eurihalina.
Ten strains produced more than 1 g of EPS
per l of culture medium; strain H212 reached
maximum productivity in the MY medium (1.6
g l
1
). In the NH medium, productivity was
lower for all strains, at about 0.20.6 g l
1
(Table 1).
3.2. EPS purication
All the EPS were obtained by centrifugation,
tangential ltration, ethanol precipitation and
ultracentrifugation. EPS from six representative
strains was also puried by ionic chromatogra-
phy. The strains F2-7, H28, H214, H217 and
H96 synthesized the polymers with the best
rheological properties, whilst strain H212 pro-
duced the maximum EPS yield. Purication by
ionic chromatography showed that most of
the polymer was eluted with 0.1 M NaCl,
showing that only one type of EPS existed
and that this polysaccharide was anionic. Ana-
lytical determinations of the fractions de-
monstrated that the EPS produced contains
a small percentage of proteins, which cannot
be eliminated by the purication processes
used.
V. Bejar et al. / Journal of Biotechnology 61 (1998) 135141 138
Table 2
Chemical composition of EPS produced by Halomonas eurihalina strains
Range of Mean value
a
Strain F2-7 Strain H28 Strain H212 Strain H214 Strain H217 Strain H96
values
a
Carbohy-
drates
3144.1 37 37 44.3 MY 43.7 34.7 42.1 31.6
medium
1357 36.3 28.6 NH 20.7 37.8 25.8 42.2 55.4
medium
Proteins
Lowry
method
917.1 12.5 13.9 15.5 MY 15.1 14.3 17.1 10.4
medium
NH 7.414.3 10.2 9 13.5 10.6 10.4 10 10.4
medium
Bradford
method
5.98.9 6.7 7.5 7.1 MY 6.2 6.3 7.1 8.5
medium
NH 1.64.7 2.9 3.2 1.6 4.7 2.4 1.7 4
medium
Uronic
acids
0.68.1 1.5 1.3 1.7 MY 2 0.8 1.2 8.1
medium
NH 0.611.1 1.2 0.8 1 1.8 1.1 1.6 11.1
medium
Hexosamines
1.52.9 2.4 2.4 2.9 MY 2.8 2.6 2.8 2.2
medium
1.42.9 2.2 2.3 NH 2.7 2.7 2.9 1.4 1.4
medium
Acetyls
0.10.7 MY 0.3 0.5 0.4 0.7 0.4 0.2 0.1
medium
0.13.4 1.5 1.3 1.1 NH 3.4 1.3 3.3 0.1
medium
Sulfates
N.D N.D 11.2 24.7 22.2 18.1 18.7 MY 12
medium
N.D N.D 2.5 3.2 3.2 NH 6.3 5.4 1.3
medium
Data are expressed as percentages of total dry weight of EPS. Each value is a mean of at least three determinations.
a
Data corresponding to 19 Halomonas eurihalina strains.
N.D., not determined.
3.3. Chemical composition
The gross chemical composition of the EPS is
set out in Table 2. The culture medium modied
the chemical composition of the EPS. Acetyls had
the highest values in the EPS extracted from the
NH medium. Carbohydrates, proteins and sul-
phates were higher in the EPS obtained from the
V. Bejar et al. / Journal of Biotechnology 61 (1998) 135141 139
MY medium (with the exception of the carbohy-
drate content in the EPS from strain H96). The
protein content varied according to the analytical
method applied. Thus, the protein values were
higher when Lowrys method was used (Lowry et
al., 1951). Uronic acids and hexosamines were
also present in all the EPS from H. eurihalina.
EPS from the H96 strain had the highest percent-
ages of uronic acids in both the MY and NH
media [8.1 and 11.1% (w/w), respectively]. Sul-
phate content determination by ionic chromato-
graphy showed concentrations ranging between
24.7% (w/w) in strain H28 cultivated in MY and
1.3% (w/w) in strain H96 cultivated in NH.
On the other hand, the cation content of the
EPS produced by the H. eurihalina strains culti-
vated in MY ranged from 18.5% (w/w) (strain
F2-7) to 22% (w/w) (strain H96). In the EPS
obtained from the NH medium, the cation con-
tent was lower, showing values ranging from
11.2% (w/w) (strain H96) to 19.8% (w/w) (strain
H217). The specic cation content followed the
order: Na
+
\Ca
2+
\Mg
2+
\K
+
.
Neutral sugar composition tests made by TLC
showed similar results between the EPS from H.
eurihalina strains. They all contained glucose,
mannose and rhamnose as principal neutral
sugars.
3.4. Rheological properties
We made a viscosimetric analysis of EPS pro-
duced by H. eurihalina strains and studied the
inuence of pH on this behaviour (Table 3).
All polysaccharides from strains of H. euri -
halina dissolved well in distilled water. At pH 7.0,
the viscosity of the EPS solutions was not particu-
larly high, with values ranging from 15 to 32.1 cP,
and 26.1 to 100 cP in MY and NH media, respec-
tively. Nevertheless, ve strains showed an inter-
esting rheological behaviour when the pH was
decreased, the viscosity of the solution increasing
concomitantly. Maximum viscosity was reached
by the EPS from strain H96, acidic solutions from
this EPS showing viscosities of 16600 cP (MY
medium) and 30000 cP (NH medium).
All EPS from H. eurihalina had pseudoplastic
rheological properties, that is, the viscosity of the
Table 3
Viscosity of Halomonas eurihalina EPS solutions
MY medium NH medium
pH 3 pH 7 pH 3 pH 7
26.1-100 10 54 Range of 1532.1
value
a

30 000 16 600
800 30 441 60.1 Strain F2-7
3600 30.1 78.2 30.1 Strain H28
10 84.2 Strain 32.1 118
H212
34.1 33.2 842 Strain 44.1
H214
100 Strain 1360 24 22
H217
16 600 30 000 Strain H96 48.1 28.1
Viscosity of EPS solutions at a shear rate of 1.5 rpm. Each
value is a mean of three determinations.
a
Data corresponding to the 19 Halomonas eurihalina strains.
solution decreased as the shear rate increased. To
evaluate this behaviour we measured the change
in Brookeld viscosity with increasing shear rate.
4. Discussion
Bacterial strains isolated from saline soils and
belonging to H. eurihalina have shown their abil-
ity to produce extracellular polymers in two dif-
ferent culture media: MY and NH.
In a previous work (Bejar et al., 1996), we
selected a complex medium (MY) and a dened
medium (NH) (glucose as sole carbon and energy
source) as the most appropriate media for study-
ing the EPS of H. eurihalina F2-7. With this strain
EPS production reached its maximum during the
late stationary phase (Quesada et al., 1993) and its
chemical composition depended upon the culture
medium used (Bejar et al., 1996). In this study, all
the H. eurihalina strains screened produced EPS
under the same conditions as strain F2-7 de-
scribed previously. This is an important character-
istic from the taxonomic point of view and may
serve as useful trait for sorting out halophilic
V. Bejar et al. / Journal of Biotechnology 61 (1998) 135141 140
microorganism in the species H. eurihalina. The
genus Halomonas (Mellado et al., 1995) currently
includes several species of moderately halophilic
bacteria, validly published, but up to date, none
of them are characterized by EPS production,
with the exception of H. eurihalina.
EPS of H. eurihalina strains studied have the
same types of neutral sugars but different compo-
sition in uronic acid, hesoxamines, acetyl and
sulphate content.
By ion exchange chromatography we deter-
mined that H. eurihalina excreted only one class
of exopolysaccharide. The samples maintained
protein impurities even after this purication pro-
cess. We think that these results could be due to
the existence of some amino acids linked to the
polymer as the same occurred with biodispersan
produced by Acinetobacter calcoaceticus (Elkeles
et al., 1994).
The exopolysaccharides from H. eurihalina
strains contained signicant quantities of sul-
phates. The percentage found in strain F2-7 culti-
vated in MY medium was 11.5% (w/v), this value
was higher than 2.7% (w/v) determined in a previ-
ous work (Bejar et al., 1996) using the method of
Dodgson and Price (1962). Therefore, ion ex-
change chromatography seems to be a more accu-
rate method for determining the sulphate content.
Although sulphates are unusual components of
microbial EPS, they have been described in those
polymers deriving from microorganisms isolated
from hypersaline habitats (Anto n et al., 1988;
Matsuda et al., 1992; Guezennec et al., 1994).
Sulphate polysaccharides provide interesting ap-
plications for the pharmaceutical industry as an-
tiviral (Okutani, 1992), antitumoral (Inoue et al.,
1988) and anticoagulant (Nishino et al., 1989).
Thus, sulphated EPS from H. eurihalina strains
may be applied to these elds.
The large amount of uronic acid present in the
EPS from strain H96 could be useful in biodetox-
ication and water treatment as it happens with
other microbial EPS (Geddie and Sutherland,
1993). Bacterial exopolysaccharides of industrial
interest do not contain large amounts of uronic
acid, with the exception of gelan and the bacterial
alginates (Guezennec et al., 1994), which have gel
forming properties (Roller and Dea, 1992).
Moreover, the EPS produced by strains of H.
eurihalina have another interesting property,
which is their capacity to gelify at acidic pH. We
have previously described that solutions of exo-
polysaccharide V2-7 extracted from MY medium
increased in viscosity up to 800 cP as the pH
decreased (Calvo et al., 1995). We have now
demonstrated that other strains of H. eurihalina
behave in the same way. EPS from strain H96 has
the best rheological behaviour, reaching a viscos-
ity value of 30000 cP at pH 3.0. This gelicant
capacity may be due to its high uronic acid con-
tent. In conclusion, this exopolymer is exception-
ally attractive for industrial application, bearing
in mind not only its unique chemical composition
but also the high viscosity of its solution at acidic
pH.
Acknowledgements
Financial support was provided by grants from
Spanish Ministerio de Educacio n y Cultura
(BIO95-0497) and the Junta de Andaluc a. We
also thank our colleague Dr J. Trout for revising
the English text.
References
Anto n, J., Meseguer, I., Rodriguez-Valera, F., 1988. Produc-
tion of an extracellular polysaccharide by Haloferax med-
iterranei. Appl. Environ. Microbiol. 54, 23812386.
Bejar, V., Calvo, C., Moliz, J., D az-Mart nez, F., Quesada,
E., 1996. Effect of growth conditions on the rheological
properties and chemical composition of Volcaniella euri -
halina exopolysaccharide. Appl. Biochem. Biotechnol. 59,
7786.
Blumenkrantz, N., Asboe-Hansen, G., 1973. New method for
quantitative determination of uronic acids. Anal. Biochem.
54, 484489.
Bradford, M.M., 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal. Biochem. 72,
248254.
Calvo, C., Ferrer, M.R., Mart nez-Checa, F., Bejar, V., Que-
sada, E., 1995. Some rheological properties of the extracel-
lular polysaccharide produced by Volcaniella eurihalina
F2-7. Appl. Biochem. Biotechnol. 55, 4554.
Dawes, E.A., 1990. Novel Biodegradable Microbial Polymers,
Kluver Academic Publisher, Netherlands.
V. Bejar et al. / Journal of Biotechnology 61 (1998) 135141 141
Dodgson, K.S., Price, R.G., 1962. A note on the determina-
tion of the ester sulphate content of sulphated polysaccha-
rides. Biochem. J. 84, 106110.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A.,
Smith, F., 1956. Colorimetric method for determination of
sugars and related substances. Anal. Chem. 28, 350356.
Elkeles, A., Rosenberg, E., Ron, E.Z., 1994. Production and
secretion of polysaccharide biodispersan of Acinetobacter
calcoaceticus A2 in protein secretion mutants. Appl. Envi-
ron. Microbiol. 60, 46424645.
Evans, C.G.T., Yeo, R.G., Ellwood, D.C., 1979. Continuous
culture studies on the production of extracellular polysac-
charides. In: Berkely, R.C.W., Gooday, G.W., Ellewood,
D.C. (Eds.), Microbial Polysaccharides and Polysaccha-
rases, Academic Press, London, pp. 5164.
Geddie, J.L., Sutherland, I.W., 1993. Uptake of metals by
bacterial polysaccharides. J. Appl. Bacteriol. 74, 467472.
Guezennec, J.G., Pignet, P., Raguenes, G., Deslandes, E.,
Lijour, Y., Gentric, E., 1994. Preliminary chemical charac-
terization of unusual eubacterial exopolysaccharides of
deep-sea origin. Carbohydr. Polym. 24, 287294.
Inoue, K., Korenaga, H., Tanaka, N.G., Sakamoto, N.,
Kadoya, S., 1988. The sulfated polysaccharidepeptidogly-
can complex potently inhibits embryonic angiogenesis and
tumor growth in the presence of cortisone acetate. Carbo-
hydr. Res. 81, 135142.
Jarman, T.R., Deavin, L., Slocombe, S., Righelato, R.C.,
1978. Investigation on the effect of environmental condi-
tions on the rate of exopolysaccharide synthesis in Azoto-
bacter 6inelandii. J. Gen. Microbiol. 107, 5964.
Jarman, T.R., 1979. Bacterial alginate synthesis. In: Berkeley,
R.C.W., Gooday, G.W., Ellwood, D.D. (Eds.), Microbial
Polysaccharides and Polysaccharases, Academic Press,
London, pp. 3545.
Johnson, A.R., 1971. Improved method of hexosamine deter-
mination. Anal. Biochem. 44, 628635.
Kushner, D.J., Kamekura, M., 1988. Physiology of halophilic
eubacteria. In: Rodriguez-Valera, F. (Eds.), Halophilic
Bacteria, vol. 1, CRC Press, Boca Raton, FL, pp. 109
140.
Lobas, D., Schumpe, S., Deckwer, W.D., 1992. The produc-
tion of gellan exopolysaccharide with Sphingomonas
paucimobilis E2 (DSM-6314). Appl. Microbiol. Biotechnol.
37, 411415.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.,
1951. Protein measurement with the Folin phenol reagent.
J. Biol. Chem. 193, 265275.
Matsuda, M., Worawattanamateekul, W., Okutani, K., 1992.
Simultaneous production of muco and sulfated polysaccha-
rides by marine Pseudomonas. Nippon Susian Gakkaishi
58, 17351741.
McComb, E.A., McCready, R.M., 1957. Determination of
acetyl in pectin and in acetylated carbohydrate polymers.
Anal. Chem. 29, 819821.
Mellado, E., Moore, E.R.B., Nieto, J.J., Ventosa, A., 1995.
Phylogenetic interferences and taxonomic consequences of
16S ribosomal DNA sequence comparison of Chromo-
halobacter marismortui, Volcaniella eurihalina and Deleya
halophila, and reclassication of V. eurihalina as
Halomonas eurihalina comb. nov. Int. J. Syst. Bacteriol. 45,
712716.
Moraine, R.A., Rogovin, P., 1966. Kinetics of polysaccharide
B-1459 fermentation. Biotechnol. Bioeng. 8, 511524.
Ng, T.K., Hu, W.S., 1989. Adherence of emulsan producing
Acinetobacter calcoaceticus to hydrophobic liquids. Appl.
Microbiol. Biotechnol. 31, 480485.
Nishino, T., Yokoyama, G., Dobashi, K., Fujihara, M.,
Nagumo, T., 1989. Isolation, purication and characteriza-
tion of fucose containing sulfated polysaccharides from the
brown seaweed Ecklonia kurome and their blood-anticoag-
ulant activities. Carbohydr. Res. 186, 119129.
Okutani, K., 1992. Antiviral activities of sulfated derivatives of
a fucosamine containing polysaccharide of marine bacterial
origin. Nippon Suisan Gakkaishi 58, 927930.
Quesada, E., Bejar, V., Calvo, C., 1993. Exopolysaccharide
production by Volcaniella eurihalina. Experientia 49, 1037
1041.
Quesada, E., del Moral, A., Bejar, V., 1994. Comparative
methods for isolation of Volcaniella eurihalina exopolysac-
charide. Biotechnol. Technol. 8, 701706.
Quesada, E., Valderrama, M.J., Bejar, V., Ventosa, A.,
Gutierrez, M.C., Ruiz-Berraquero, F., Ramos-Cormen-
zana, A., 1990. Volcaniella eurihalina gen. nov., sp. nov., a
moderately halophilic nonmotile gram-negative rod. Int. J.
Syst. Bacteriol. 40 (3), 261267.
Rodr guez-Valera, F., Ruiz-Berraquero, F., Ramos-Cormen-
zana, A., 1981. Characteristics of heterotrophic bacterial
populations in hypersaline environments of different salt
concentrations. Microbial Ecol. 7, 235243.
Roller, S., Dea, I.C.M., 1992. Biotechnology in the production
and modication of biopolymers for foods. Crit. Rev.
Biotechnol. 12, 261277.
Sutherland, I.W., 1990. Biotechnology of Microbial Exo-
polysaccharides, Cambridge Press, Cambridge.
.
.