FRASE SULLA CARATTERIZZAZIONE FILOGENETICA. The results obtained by the optimization phase, showed that diesel oil resulted the better carbon source ...
Graziano, M., Rizzo, C., Michaud, L., Porporato, E.M.D., De Domenico, E., and Lo Giudice, A. Department of Animal Biology and Marine Ecology, University of Messina – Viale F. Stagno d’Alcontres, 98166 Messina (Italy)
ISOLATION of potential biosurfactant producers from the sea-pen Pteroeides spinosum (Ellis, 1794). INTRODUCTION
Biosurfactants (BSs) are amphipatic compounds produced by microorganisms, which act at interface between substances of different polarity, such as water/oil, water/air. Microorganisms use different organic compounds as carbon source and energy for th eir growth. When such source is represented by insoluble substrates such as hydrocarbons, the cell uptake is facilitated by the production of specific substances, the biosurfactants. The aim of this study was to monitor the biosurfactants production of bacteria isolated from Cnidaria Anthozoa (Octocorallia), by considering that biological matrices have been poorly considered until now for this purpose.
MATERIAL and METODS
Adult specimens of Pteroeides spinosum were aseptically collected at Giardini Naxos (Messina), and homogenized. Enrichment cultures were performed by inoculating homogenates in mineral soil medium ONR7a supplemented with crude oil (1%) (PBE). Part of the samples were used to set up crops from natural samples (PBN). Serial dilutions of homogenates were prepared by using filter-sterilized water, and used to inoculate solidified mineral medium ONR7a supplemented with different carbon source (IPA-OILPCB). A first screening of isolates was performed by using DCPIP assay (2,6dichlorophenol indophenol). BS production screening was performed on strains resulted positive to the DCPIP test, by using standard tests with specific interv als of 48h: optical density, emulsification, E24 index detection, blood agar assay, C-TAB assay, surfac e tension measurement. Positive strains were taxonomically identified by 16S rDNA sequencing. A second screening was performed on most interesting strains, by analyzing the effect of different carbon source (crude oil, diesel oil, tetradecane) and temperature (15 °C, 25 °C, 30 °C). An extrac tion of biosurfactant was performed on most promising strain, and preliminarly characterized by using colorimetric assays.
Campione Arricchimenti
Campione Arricchimenti
Campione BP1
B6P
Campione Arricchimenti B1L
B6L
RESULTS
Among 120 strains, 90 resulted positive to the of DCPIP, by showing a different level of hydrocarbon degradation. During the first sc reening procedure, 5 strains produced stable emulsions and were choose for further experiments. FRASE SULLA CARATTERIZZAZIONE FILOGENETICA. The results obtained by the optimization phase, showed that diesel oil resulted the better carbon source for all tested strains, with maximum values o f stable emulsion shown by the strain PBN 295 (XX). All temperature v alues resulted promising for emulsification activity of strains All strains gave better results during incubation at 25°C and 30°C, and among them the isolate PBN 298 produced stable emulsion during incubation at all temperature values. The colorimetric assays were carried out on biosurfactant extracted from the strain Vibrio sp. PBN295. The total carbohydrate content was determined according with phenol-sulfuric acid method (Dubois et al., 1956), while the quantization of the proteins was performed according to Bradford method (1976)
Emulsioni (%) **
E2 4 (%) ***
ST(mN/m) †
Micr. Assay
PBE119 ONR 7a IPA 20 PBE120 ONR 7a IPA 60 PBE121 ONR 7a IPA 26.7 PBE122 ONR 7a IPA 53.3 PBE123 ONR 7a IPA 30 PBE124 ONR 7a IPA 56.7 PBE127 ONR 7a IPA 26.7 PBE128 ONR 7a IPA 37.7 PBE129 ONR 7a IPA 33.3 PBE132 ONR 7a IPA 66.7 PBE133 ONR 7a IPA 20 PBE134 ONR 7a IPA 12.5 PBE135 ONR 7a IPA 26.7 PBE137 ONR 7a IPA 40 PBE138 ONR 7a IPA 20 PBE139 ONR 7a IPA 24.3 PBE140 ONR 7a IPA 100 PBE143 ONR 7a IPA 26.7 PBE145 ONR 7a IPA 86.7 PBE146 ONR 7a IPA 33.3 PBE147 ONR 7a IPA 35 Terreno di Emulsioni Ceppo (%) PBE150 provenienza* ONR 7a IPA 53.3** PBE161 ONR7a BP 46.7 PBE119 ONR 20 PBE164 ONR7a7aIPA BP 33.3 PBE173 ONR7a7aIPA BP 100 PBE120 ONR 60 PBE121 ONR 26.7 PBE175 ONR7a7aIPA BP 13.3 PBE122 ONR 53.3 PBE177 ONR7a7aIPA BP 100 PBE123 ONR 30 PBE178 ONR7a7aIPA BP 100 PBE124 ONR 56.7 PBE180 ONR7a7aIPA BP 100 PBE127 ONR 26.7 PBE181 ONR7a7aIPA BP 100 PBE128 ONR IPA 37.7 PBE182 ONR 6.7 7a 7a BP Terreno di Emulsioni Ceppo PBE129 ONR 33.3 PBE183 ONR7a7IPA 60** aC provenienza* (%) PBE132 ONR 66.7 PBE184 ONR7a7IPA 100 aC PBE133 ONR IPA 20 7a PBE185 ONR C 73.3 PBN 215 ONR7a7PET 55 a PBE134 ONR 12.5 7a7IPA PBE190 ONR 100 PBN 217 ONR PET 0 aC 7a PBE135 ONR 26.7 7a7IPA PBE191 ONR 100 PBN 219 ONR PET 40 aC 7a PBE137 ONR 40 7a7IPA PBE192 ONR C 100 PBN 221 ONR PET 100 7a a PBE138 IPA 20 PBN 301 ONR ONR 100 PBE201 ONR7a C 100 7a7 aIPA PBE139 ONR 24.3 PBE207 ONR7a7IPA 100 aC PBE140 ONR 100 7a7IPA PBN 231 ONR PET 10 PBE208 ONR C 73.3 a 7a PBE143 ONR 26.7 7a IPA PBN 232 ONR 40 PBE209 ONR 80 7a7PET aC PBE145 ONR IPA 86.7 PBN 233 ONR7a7a PET 50 PBE146 ONR7a7a IPA 33.3 PBN 234 ONR PET 10 PBE147 ONR7a7a IPA 35 PBN 235 ONR PET 45 PBE150 ONR7a7a IPA 53.3 PBN 236 ONR PET 20 PBE161 ONR7a7aPET BP 46.7 Terreno di Emulsioni PBN 237 ONR 3 Ceppo (%) PBE164 ONR7a7aPET BP 33.3 PBN 238 provenienza* ONR 65 ** PBE173 ONR 7a BP 100 PBE119 ONR 20 PBE175 ONR7a7aIPA BP 13.3 75 PBN 295 ONR 7a IPA PBE177 ONR7a7aIPA BP 100 PBE120 ONR 60 PBN 296 ONR 100 7a IPA PBE178 ONR7a7aIPA BP 100 PBE121 ONR 26.7 PBN 297 ONR IPA 100 7a PBE180 ONR7a7aIPA BP 100 PBE122 ONR 53.3 PBN 443 ONR 60 7a IPA PBE181 ONR7a7aIPA BP 100 PBE123 ONR 30 PBE182 ONR7a7aPCB BP 6.7 PBE124 ONR IPA 56.7 PBN 391 ONR 75 7a PBE183 ONR7a7PCB 60 aC PBE127 ONR IPA 26.7 PBN 393 ONR 100 7a PBE184 ONR C 100 a PBE128 ONR7a7a7PCB IPA 37.7 PBN 394 ONR 85 PBE185 ONR7a7PCB 73.3 aC PBE129 ONR IPA 33.3 PBN 395 ONR 33 7a PBE190 ONR7a7PCB 100 aC PBE132 ONR 66.7 PBN 396 ONR 50 7a IPA PBE191 ONR C 100 7 a PBE133 ONR7a IPA 20 PBE192 ONR7a7IPA 100 aC PBE134 ONR 12.5 PBE201 ONR7a7IPA 100 aC PBE135 ONR 26.7 PBE207 ONR7IPA C 100 PBE137 ONR 40 7a a PBE208 ONR7IPA 73.3 aC PBE138 ONR 20 7a PBE209 ONR7IPA 80 aC PBE139 ONR 24.3 7a PBE140 ONR7a IPA 100 PBE143 ONR7a IPA 26.7 PBE145 ONR7a IPA 86.7 PBE146 ONR7a IPA 33.3 PBE147 ONR7a IPA 35 PBE150 ONR7a IPA 53.3 PBE161 ONR7a BP 46.7 PBE164 ONR7a BP 33.3 PBE173 ONR7a BP 100 PBE175 ONR7a BP 13.3 PBE177 ONR7a BP 100 PBE178 ONR7a BP 100 PBE180 ONR7a BP 100 PBE181 ONR7a BP 100 PBE182 ONR7a BP 6.7 PBE183 ONR7 a C 60 PBE184 ONR7 a C 100 PBE185 ONR7 a C 73.3 PBE190 ONR7 a C 100 PBE191 ONR7 a C 100 PBE192 ONR7 a C 100 PBE201 ONR7 a C 100 PBE207 ONR7 a C 100 PBE208 ONR7 a C 73.3 PBE209 ONR7 a C 80
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 E2 40(%) *** 0 0 0 0 0 0 50 0 0 25.00 0 E24 (%) 00 *** 0 -00 28.6 -0 -00 -0 -00 00 -00 -0 23.5 -0 -0 -0 -0 E2 4-0(%) *** -0 0 340 -0 -50 -0 25.00 0 -00 -0 -00 -0 28.6 -0 00 00 00 00 00 23.5 0 0 0 0 0 0 0 0 0 0 0 0 5 0 25.00 0 0 0 0 28.6 0 0 0 0 0 23.5
24.4±0.62 25.4±1.78 23.1±0.75 23.8±2.72 26.2±1.33 26.4±3.06 28.5±1.15 27.3±1.21 29.3±3.98 29.5±1.50 28.4±5.14 27.8±1.79 29.4±3.87 28.9±2.31 26.5±3.29 31.4±2.94 28.7±1.62 25.7±2.25 26.3±0.12 27.3±0.81 27.2±3.06 ST(mN/m) † 24.1±1.69 27.8±2.03 24.4±0.62 25.7±2.03 26.0±0.00 25.4±1.78 23.1±0.75 26.3±1.44 23.8±2.72 29.3±1.91 26.2±1.33 27.2±2.26 26.4±3.06 25.4±1.34 28.5±1.15 26.2±1.20 27.3±1.21 24.5±2.72 ST(mN/m) 29.3±3.98† 27.1±3.42 29.5±1.50 28.2±2.05 28.4±5.14 31.0±5.57 25.0±0.46 27.8±1.79 32.0±2.40 24.9±3.61 29.4±3.87 31.7±2.47 22.5±1.41 28.9±2.31 27.3±0.78 22.8±1.62 26.5±3.29 28.5±1.41 29.5±1.91 31.4±2.94 27.3±1.70 28.7±1.62 22.9±.028 27.8±0.71 25.7±2.25 22.1±1.11 25.8±0.64 26.3±0.12 24.6±0.07 27.3±0.81 24.0±0.42 27.2±3.06 23.0±.1.03 24.1±1.69 23.6±0.42 27.8±2.03 ST(mN/m) 23.3±1.60 † 25.7±2.03 27.8±0.39 26.0±0.00 24.4±0.62 26.3±1.44 29.0±2.11 29.3±1.91 25.4±1.78 29.9±0.47 27.2±2.26 23.1±0.75 25.1±2.44 25.4±1.34 23.8±2.72 36.8±4.27 26.2±1.20 26.2±1.33 24.5±2.72 26.4±3.06 25.6±2.85 27.1±3.42 28.5±1.15 23.2±3.53 28.2±2.05 27.3±1.21 29.2±5.35 31.0±5.57 29.3±3.98 35.6±6.70 32.0±2.40 29.5±1.50 21.3±2.91 31.7±2.47 28.4±5.14 27.3±0.78 27.8±1.79 29.5±1.91 29.4±3.87 27.3±1.70 28.9±2.31 27.8±0.71 26.5±3.29 25.8±0.64 31.4±2.94 28.7±1.62 25.7±2.25 26.3±0.12 27.3±0.81 27.2±3.06 24.1±1.69 27.8±2.03 25.7±2.03 26.0±0.00 26.3±1.44 29.3±1.91 27.2±2.26 25.4±1.34 26.2±1.20 24.5±2.72 27.1±3.42 28.2±2.05 31.0±5.57 32.0±2.40 31.7±2.47 27.3±0.78 29.5±1.91 27.3±1.70 27.8±0.71 25.8±0.64
+ + + + + + + + + + + + + + + + Micr. Assay + + + +++++Micr. + + Assay + + + + ++ ++++ -++ + ++++-+ -+ -+ -+ Micr. -+ Assay ++ +++++++++ -+ -+++++ + ++ + + + + ++ + -+-
Ceppo
≈ 5 ,6 mg/ml
≈ 1 1 5 mg/ml
Terreno di provenienza*
PBN 295 – Vibr io s p.
+ + + + + + + + + + + + + + + + + -
!
! !
Effect Of Carbon Source
!
PBE 178 – Br evibacter ium s p.
!
PBE 209 – Br evibacter ium s p.
!
!
PBE 181 – Br evibacter ium s p.
!
PBE 190 – Br evibacter ium s p.
!
!
!
Effect Of Temperature
PBN 295 – Vibr io s p.
PBE 178 – Br evibacter ium s p.
PBE 209 – Br evibacter ium s p.
! !
!
PBE 181 – Br evibacter ium s p.
PBE 190 – Br evibacter ium s p.
≈ 5 2 mg/ml ≈ 1 ,8 mg/ml
!
!
P ro tei n co n ten t o f b i osu rfa cta nt p ro d u ced b y Vi b ri o sp . PB N2 95 .
!
Gl u ci d co n ten t o f b o i su rfacta n t p ro d u ced b y Vi b ri o sp . PB N2 95 .
!
Isolates deriving from enrichment cultures resulted affiliated to Brevibacterium genus, while strain PBN295 resulted belonging to Vibrio genus.
CONCLUSION
Phylum'o'classe' Actinobacteria ! ! ! ! ! Gammaproteobacteria! !
Ceppo' !
Allineamento,'AN**' !
PBE178 PBE181! PBE190! PBE209!
GQ274005.1, Brevibacterium sp. BS01 GQ274005.1, Brevibacterium sp. BS01! GQ274005.1, Brevibacterium sp. BS01! GQ274005.1, Brevibacterium sp. BS01!
PBN295
FN687911.1, Vibrio sp. BFLP-10
Origine*' Sim†'(%)' ! ! PBE! 99! PBE! 99! PBE! 99! PBE! 99! ! PBN 98!
The choice of biological matrices was promising for isolating biosurfactant produced bacteria. The screening tests was used to select the potential producer quickly, and revealed the potentiality of each strains. This allowed to focalized the improver experiment on the most promising strains. The strains, however, have shown greater ability in emulsion production, by suggesting a probable BS with high molecular weight. The optimization study determined the optimal conditions for each strain, and showed good performances both at different incubation temperatures and different carbon sources. The genus Brevibacterium has been reported only recently in regard to the BS production, while there are no references about the genus Vibrio in this context.
!
