“PRODUCTION OF EXOPOLYSACCHARIDE FROM
RHIZOBIA WITH POTENTIAL BIOEMULSIFYING
APPLICATIONS”
Tereza Cristina Luque Castellane*, Érica Mendes Lopes, João Carlos
Campanharo, & Eliana Gertrudes de Macedo Lemosa*
UNESP—Univ Estadual Paulista
FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS
CÂMPUS DE JABOTICABAL
DEPARTAMENTO DE TECNOLOGIA
Laboratório de Bioquímica de Microrganismos e Plantas
*Email: teluque@yahoo.com.br
INTRODUCTION
many
other
Rhizobia are typical Gram-  Like
negative bacteria with a
bacteria, are among the
cytoplasmic and an outer
most
well-known
membrane separated by a
exopolysaccharides
periplasmic space.
(denote EPS) producers;
 Excrete large amounts
of
these
polysaccharides in the
rhizosphere.
Source: http://bacmap.wishartlab.com/organisms/763
INTRODUCTION
Characterized as
EXOPOLYSACCHARIDES, to
distinguish them from any
polysaccharides that might
be found within the cell.
Figure 1. Scheme of the rhizobial cell
surface showing the position of surface
polysaccharides that might be involved
in rhizobial attachment to legume
roots. OM, outer membrane; PS,
periplasmic space; PG, peptideglycan
layer; PM, cytoplasmic membrane; EPS,
Exopolysaccharide; CG, cyclic glucan;
PL, phospholipid; MP, membrane
protein; KPS, capsular polysaccharide
(K-antigens); LPS, lipopolysaccharide.
Source: Rodríguez-Navarro et al. (2007)
EPS are an important class of
biopolymers with great ecological and
biotechnological importance.
Viscosifiers and syneresis-lowering agents, for their
pseudoplastic rheological behavior and water-binding
capacity, bioflocculating and bioadsorption of heavy
metals from waste water and natural water .
Fig. 1. Samples of Exopolysaccharides dried
Fig. 2. EPS Solution
Campanharo, J.C. Thesis. (2006). UNESP, Univ Estadual Paulista, Brasil.
AIM
In the context that Rhizobium species are likely to be involved in
a broad range of functions in diverse ecosystems and the
commercial and ecological importance of the rhizobial EPS.
The
production,
the
rheological
properties and the emulsifying properties of
the exopolysaccharides from wild-type and
mutant strains of Rhizobium tropici SEMIA 4080
were investigated.
METHODOLOGY
Table 1. Bacterial strains included in the study
Straina
SEMIA 4080
MUTZC3*
a
Description
Rhizobium tropici (identical to PRF 81 ; Soil
Biology & Biochemistry 39, 867- 876, 2007)
This mutant strain was obtained by
transposon Tn5 mutagenesis of R. tropici
SEMIA4080 (Castellane, T.C.L. Thesis, 2011.
UNESP, Univ Estadual Paulista, Brasil. )
Numbers refer to the SEMIA Collection/FEPAGRO, also registered as a faithful
Brazilian depositary institute for Ministry of Environment (MMA) under no.
075/2013/SECEX/CGEN.
* an uncharacterized mutant strain
METHODOLOGY
24 h
OD 600 nm = 1.5
The frozen (-80 °C) cultures were activated by growing them
cultivated on solid PGYA medium (Campanharo, J. C., 2006)
containing glycerol (10 g L-1), as a carbon source
Liquid PSYL medium (Campanharo, J. C. 2006)
(with and without diesel oil) plus sucrose (3%
w/v) . Incubated for 96 h at 140 rpm and 29 ºC
10 % pre inoculum (v/v)
with
without
Centrifuged (12000 × g, 4 ºC, 40 min)
to separate the cell and supernatant
8
0
0
800
6
0
0
600
4
0
0
2
0
0
EPS isolation
400
3:1 (v/v) ethanol:supernatant ratio to
precipitate the EPS (J Gen Appl
Microbiol. 1990, 136, 2511-2519)
200
EPS dried
samples -
Figure 2. Schematic steps involved in the studies of Rhizobial EPS
EPS Production;
Composition HPLC
METHODOLOGY
EPS dried and
dyalisis samples
Emulsification index (E24 %)
EPS
Solution
(5 g/L; 5%, w/v )
24 h to ensure their full hydration
Hydrocarbon / Oil
Rheological evaluation
Vortex (2 min)
EPS Solution
•
•
the maximum shear rate value
was 100 s-1
at 25 °C in triplicate
Cell growth (OD600)
INITIAL SCREENING WITH DIFFERENT CONCENTRATION OF
DIESEL OIL IN THE GROWTH MEDIUM
SEMIA 4080
MUTZC3
Figure 3. Growth of Rhizobium tropici strains (SEMIA4080 and MUTZC3) on different concentrations of
diesel oil plus 3% sucrose, as carbon source. The values are the means ±SD of three different experiments
performed on different days, determined by measuring the optical density after 96 hour incubation (each
experiment was conducted in triplicate).
EVALUATION OF EXOPOLYSACCHARIDE PRODUCTION
Table 2. Evaluation of the differences in the exopolysaccharide (EPS) production and
cell dry weight (CDW) between Rhizobium tropici strains (SEMIA4080 and MUTZC3) on
two different medium PSYL (with and without diesel oil).
14.25%
MUTZC3
20.47%
Mean values (±standard deviation) within the same column not sharing a common superscript differ
significantly (P < 0.05).
 We observed that the maximum amounts of rhizobia biomass
(measured as cell dry weight) and EPS were obtained when
diesel oil was added to the medium.
 This correlates to significant increases of 14.25 and 20.47%
respectively for the control and the mutant strains.
EVALUATION OF MONOSACCHARIDE COMPOSITION OF EPS
Table 3 Comparative monosaccharide composition of EPS (%) produced by the wild-type
(SEMIA4080) and mutant (MUTZC3) strains of Rhizobium tropici
EPS from SEMIA 4080
Monomers Compositions (%)
Glucose
Galactose
74.43
22.54
(Medium with 3% Sucrose)
EPS from SEMIA 4080
71.13
25.54
(Medium with 3% Sucrose, 0.1% Diesel)
EPS from MUTZC3
72.87
24.16
(Medium with 3% Sucrose)
EPS from MUTZC3
74.37
21.92
SAMPLES
(Medium with 3% Sucrose, 0.1% Diesel)
(…) in previous studies, Rheological Properties of EPS
Source: Castellane et al. Carbohydr. Polym. (2014) 111:191-7
Rheological Properties of EPS
viscosidade
tensão
1
(Pa.s)
n(Pa.s)
100
100
10
10
1
1
(Pa)
100
0.01
0.01
0.1
1
y
10
100
(s-1)
Castellane et al. Carbohydr. Polym.
(2014) 111:191-7
0,1
0,1
SEMIA 4080
0,01
0,01
0,01
 Differences
in
rheoligal
properties of EPSwt and EPSmut
when grown on medium PSYL
with and without diesel oil
 Viscosity values - higher than
those of the EPSs from strains
in previous studies
0,1
1
(s-1)
10
100
Figure 5. Shear rate versus viscosity (Pa.s)
and shear rate versus shear stress (1/s), for
the EPS samples (10 g L-1) from wild-type
strain SEMIA 4080, with diesel oil plus
sucrose as carbon sources, at 25 °C.
(Not publish data)
Rheological Properties of EPS
n(Pa.s)
100
1
0.01
0.01
0.1
1
10
100
y (s-1)
Castellane et al. Carbohydr. Polym.
(2014) 111:191-7
MUTZC03
Figure 5. Shear rate versus viscosity (Pa.s)
and shear rate versus shear stress (1/s), for
the EPS samples (10 g L-1) from mutant strain
MUTZC03, with diesel oil plus sucrose as
carbon sources, at 25 °C.
(Not publish data)
Emulsification index (E24 %)
Table 3. Stability of emulsifying activity of culture medium, a substantially cell-free medium
and exopolysaccharide (EPS) from a bacterial cell culture in which wild-type SEMIA 4080 or
mutant (MUTZC3) strains were grown
SAMPLES
SEMIA 4080 (3% Sucrose)
HYDROCARBON/OIL
Culture medium
Cell-free medium
EPS (5 %, wt/v)
Liquid paraffin oil
54.42
23.19
58.06
Hexane
49.63
31.96
43.51
74.34
36.93
38.89
Hexane
83.09
56.89
34.67
Liquid paraffin oil
26.45
49.8
41.85
Hexane
51.61
34.74
59.83
Liquid paraffin oil
60.93
38.62
60.42
Hexane
89.59
41.39
42.58
SEMIA 4080 (3% Sucrose, 0.1% Diesel) Liquid paraffin oil
MUTZC3 (3% Sucrose)
MUTZC3 (3% Sucrose, 0.1% Diesel)
aResults
E24 %
are expressed as percentages of the total height occupied by the emulsion; values are means of at least three
determinations
CONCLUSIONS
 These
results
indicate
that
the
heteropolysaccharide could be used as an
emulsifying and emulsion-stabilizing agent;
 These properties make the biopolymer an
attractive, “green” alternative to synthetics,
for use as a stabilizing or rheology modifying
agent.
Our most recent publications
T.C.L. Castellane, M.V.F. Lemos, E.G.M. Lemos, Evaluation of the
biotechnological potential of Rhizobium tropici strains for exopolysaccharide
production. Carbohydr. Polym. (2014) 111:191-7
T.C.L. Castellane, M.R. Persona, J.C. Campanharo E.G.M. Lemos, Production of
Exopolysaccharide from Rhizobia with Potential Biotechnological and
Bioremediation Applications. International Journal of Biological
Macromolecules 74 (2015) 515–522.
C. Moretto, T.C.L. Castellane, et. al. Chemical and rheological properties of
exopolysaccharides produced by four isolates of rhizobia, International Journal
of
Biological
Macromolecules
(2015),
http://dx.doi.org/10.1016/j.ijbiomac.2015.07.056
T.C.L. Castellane, A.M. Otoboni, E.G.M. Lemos, Characterization of
exopolysaccharides produced by rhizobia species , Revista Brasileira Ciência do
Solo, (2015), in press.
Acknowledgment
Tereza Cristina Luque Castellane;
Érica Mendes Lopes;
João Carlos Campanharo;
Eliana G. de Macedo Lemos
egerle@fcav.unesp.br