1. UNESP—Univ Estadual Paulista FACULDADE DE CIÊNCIAS AGRÁRIAS E VETERINÁRIAS CÂMPUS DE JABOTICABAL DEPARTAMENTO DE TECNOLOGIA aboratório de Bioquímica de Microrganismos e Plantas Laboratório de Bioquímica de Microrganismos e Plantas * Email: teluque@yahoo.com.brteluque@yahoo.com.br Tereza Cristina Luque Castellane *, Érica Mendes Lopes, João Carlos Campanharo, & Eliana Gertrudes de Macedo Lemos a* “PRODUCTION OF EXOPOLYSACCHARIDE FROM RHIZOBIA WITH POTENTIAL BIOEMULSIFYING APPLICATIONS”

2. INTRODUCTION Rhizobia are typical Gram- negative bacteria with a cytoplasmic and an outer membrane separated by a periplasmic space.  Like many other bacteria, are among the most well-known exopolysaccharides (denote EPS) producers;  Excrete large amounts of these polysaccharides in the rhizosphere. Source: http://bacmap.wishartlab.com/organisms/763

3. INTRODUCTION Source: Rodríguez-Navarro et al. (2007) 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. Characterized as EXOPOLYSACCHARIDES, to distinguish them from any polysaccharides that might be found within the cell.

4. Fig. 1. Samples of Exopolysaccharides driedFig. 2. EPS Solution Campanharo, J.C. Thesis. (2006). UNESP, Univ Estadual Paulista, Brasil. 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.

5. AIM 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. 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.

6. Table 1. Bacterial strains included in the study Strain a Description SEMIA 4080Rhizobium tropici (identical to PRF 81 ; Soil Biology & Biochemistry 39, 867- 876, 2007) MUTZC3 * This mutant strain was obtained by transposon Tn5 mutagenesis of R. tropici SEMIA4080 (Castellane, T.C.L. Thesis, 2011. UNESP, Univ Estadual Paulista, Brasil. ) METHODOLOGY a 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

7. METHODOLOGY 200200 400400 600600 800800 200 400 600 800 3:1 (v/v) ethanol:supernatant ratio to precipitate the EPS (J Gen Appl Microbiol. 1990, 136, 2511-2519) 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 24 h OD 600 nm = 1.5 Centrifuged (12000 × g, 4 ºC, 40 min) to separate the cell and supernatant EPS dried samples - EPS isolation EPS Production; Composition HPLC diesel oil 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 Figure 2. Schematic steps involved in the studies of Rhizobial EPS

8. METHODOLOGY EPS dried and dyalisis samples EPS Solution (5 g/L; 5%, w/v ) 24 h to ensure their full hydration Rheological evaluation the maximum shear rate value was 100 s -1 at 25 °C in triplicate Hydrocarbon / Oil EPS Solution Vortex (2 min) Emulsification index (E 24 %)

9. 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). INITIAL SCREENING WITH DIFFERENT CONCENTRATION OF DIESEL OIL IN THE GROWTH MEDIUM

10. 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). MUTZC3 14.25% 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. 20.47% Mean values (±standard deviation) within the same column not sharing a common superscript differ significantly (P < 0.05). EVALUATION OF EXOPOLYSACCHARIDE PRODUCTION

11. SAMPLES Monomers Compositions (%) GlucoseGalactose EPS from SEMIA 4080 (Medium with 3% Sucrose) 74.4322.54 EPS from SEMIA 4080 (Medium with 3% Sucrose, 0.1% Diesel) 71.1325.54 EPS from MUTZC3 (Medium with 3% Sucrose) 72.8724.16 EPS from MUTZC3 (Medium with 3% Sucrose, 0.1% Diesel) 74.3721.92 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

12. (…) in previous studies, Rheological Properties of EPS Source: Castellane et al. Carbohydr. Polym. (2014) 111:191-7

13. Rheological Properties of EPS 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) SEMIA 4080 Castellane et al. Carbohydr. Polym. (2014) 111:191-7 Differences in rheoligal properties of EPS wt and EPS mut when grown on medium PSYL with and without diesel oil Viscosity values - higher than those of the EPSs from strains in previous studies

14. Rheological Properties of EPS 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) MUTZC03 Castellane et al. Carbohydr. Polym. (2014) 111:191-7

15. SAMPLESHYDROCARBON/OIL E 24 % Culture mediumCell-free mediumEPS (5 %, wt/v) SEMIA 4080 (3% Sucrose)Liquid paraffin oil54.4223.1958.06 Hexane49.6331.9643.51 SEMIA 4080 (3% Sucrose, 0.1% Diesel)Liquid paraffin oil74.3436.9338.89 Hexane83.0956.8934.67 MUTZC3 (3% Sucrose)Liquid paraffin oil26.4549.841.85 Hexane51.6134.7459.83 MUTZC3 (3% Sucrose, 0.1% Diesel)Liquid paraffin oil60.9338.6260.42 Hexane89.5941.3942.58 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 a Results are expressed as percentages of the total height occupied by the emulsion; values are means of at least three determinations Emulsification index (E 24 %)

16. These results indicate that the heteropolysaccharide could be used as an emulsifying and emulsion-stabilizing agent; “green” alternative These properties make the biopolymer an attractive, “green” alternative to synthetics, for use as a stabilizing or rheology modifying agent. CONCLUSIONS

17. 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 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.

18. Tereza Cristina Luque Castellane; Érica Mendes Lopes; João Carlos Campanharo; Eliana G. de Macedo Lemos egerle@fcav.unesp.br Acknowledgment