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Purification and identification of secondary metabolites from marine derived Streptomyces rochei 6CM016 and their antimicrobial activities
New Biotechnology · Volume 29S · September 2012
Poster 1.3.47
Poster 1.3.48
Polyhydroxyalkanoates production and recovery with Ralstonia eutropha from plant and animal oils
Biodiversity and bioactivity of some marine sediment derived actinomycetes from Turkey
S.L. Riedel 1,2,∗ , C. Brigham 1 , J. Bader 2 , S. Koenig 2 , C.K. Rha 3 , U. Stahl 2 , A.J. Sinskey 1,4,5
¸ etinel Aksoy 1 , Orc¸un Kalkan 1 , Atac¸ Kadriye Özcan 1 , Semiha C 1,∗ Uzel , E. Esin Hames-Kocabas 2 , M. Baki Yokes¸ 3 , Erdal Bedir 2
1
1
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA 2 Department of Applied and Molecular Microbiology, Technische Universität Berlin, Germany 3 Biomaterials Science and Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA 4 Engineering Systems Division, Massachusetts Institute of Technology, Cambridge, MA, USA 5 Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Ralstonia eutropha is the model organism for production of polyhydroxyalkanoates (PHA), biodegradable and biocompatible alternatives to petroleum-based plastics. There is great interest in developing scalable, low cost polymer production processes on high carbon, commodity plant and animal oil feedstocks. We researched wild type and recombinant R. eutropha metabolizing various feedstocks (palm oils, waste frying oil, and tallow) for production and recovery of polyhydroxybutyrate (PHB) or poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)). P(HB-co-HHx) containing short chain HB and medium chain HHx monomers enhances several processing properties (e.g. melting temperature, crystallinity and flexibility) of the PHA polymer. We demonstrated recombinant R. eutropha in fed batch fermentations on palm oil producing over 140 g/L cell dry weight (CDW) of biomass with more than 70% PHA per CDW with ∼20 mol% HHx. Our data suggest the scalable fermentation process with a space time yield better than 1 g PHA/L/h. We further showed the potential use of waste fats as carbon feedstocks for PHA production in batch or extended batch fermentations. Initial promising tallow and waste frying oil carbon source results suggest an adapted fed batch process. We have also demonstrated P(HB-co-HHx) recovery from both dry and wet cells. Ethyl acetate showed both high recovery levels and high purities (up to 99%) from dry cells. Simpler recovery from wet cells, however, potentially saves time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) proved the most favourable solvent for PHA recovery with purities of up to 99% and total recovery yields of up to 84%. http://dx.doi.org/10.1016/j.nbt.2012.08.192
Ege University, Faculty of Science, Department of Biology, Basic and Industrial Microbiology Section, 35100 Bornova, Izmir, Turkey 2 Ege University, Faculty of Engineering, Department of Bioengineering, 35100 Bornova, Izmir, Turkey 3 Halic Univ, Fac Arts & Sci, Dept Mol Biol & Genet, TR-34381 Istanbul, Turkey In recent years marine environments are proved as a rich source of microorganisms including actinomycetes. These marine derived actinomycetes are prolific sources of many bioactive compounds including antibiotics. In this study, a total of 33 samples were collected from coastal sediments of Turkey from 10 different locations and 1 sample was from Van (Soda) Lake. Four different selective isolation media were used for actinomycetes isolation including; modified actinomycetes isolation agar (MAIA), M1 agar, M6 agar and modified R2A agar. MAIA was found best medium for recovery of actinomycetes but M6 was found superior than others for the isolation of antibiotic producing actinomycetes strains. In total, 269 actinomycetes strain were isolated and 70 (23.64/%) of the strains were found active against at least one test microorganism. Consequently, coastal sediments of Turkey were found rich in actinomycetes diversity and this diversity provides an untouched source for the isolation of new and potent antimicrobial compounds. Keywords: Biodiversity; Antimicrobial activity; Marine sediment; Actinobacteria Acknowledgements: This study was financed mainly by The Scientific and Technological Research Council of Turkey (TUBITAK, Project No: 109S361) and partly by “The Research Fund of Ege University 2009 Fen 061” project. Kadriye Özcan was supported by TUBITAK 2211 doctorate scholarship programme. http://dx.doi.org/10.1016/j.nbt.2012.08.193 Poster 1.3.49 Purification and identification of secondary metabolites from marine derived Streptomyces rochei 6CM016 and their antimicrobial activities Semiha Cetinel Aksoy 1 , Atac¸ Uzel 1,∗ , E. Esin Hames-Kocabas 2 , Ikhlas A. Khan 3 , Erdal Bedir 2 1
Ege University, Faculty of Science, Department of Biology, BornovaIzmir, Turkey 2 Ege University, Faculty of Engineering, Department of Bioengineering, Bornova-Izmir, Turkey 3 University of Mississippi, National Center for Natural Products Research, University, MS 38677, USA Aim of this study was purification and identification of antimicrobial secondary metabolites from marine derived Streptomyces rochei 6CM016. Strain 6CM016 was isolated from a sediment sample collected from Kas¸-Antalya. Initial fermentation was performed www.elsevier.com/locate/nbt S69
New Biotechnology · Volume 29S · September 2012
in 250 ml flasks containing 50 ml of M1 medium. Inoculated medium was incubated at 28◦ C for 10 days at 150 rpm. Fermentation broth was extracted with EtOAc and antimicrobial activity was tested using disc diffusion method. The isolate was found to be active against enteropathogenic Escherichia coli 0157:H7 (RSKK 234), methicillin resistant Staphylococcus aureus ATCC 43300 and Candida albicans DSMZ 5817. Then, 56 l of fermentation broth was obtained from a scale-up fermentation study, and was extracted with EtOAc after removal of the cells via filtration. The extract was concentrated in vacuo at 35◦ C yielding 1.92 g of brown residue. Bioassay-guided fractionations by using high performance flash chromatography (Biotage) and preparative thin layer chromatography on normal-phase/reverse-phase silica gel and Sephadex LH20 led to the isolation of five compounds [06CM016-01 (plicacetin), 06CM016-02, 06CM016-03 (norplicacetin), 06CM016-05 and 06CM016-08]. Structures of the compounds were elucidated as nucleoside-type secondary metabolites, three of which were new molecules for nature, by spectral methods (1D-, 2D-NMR and HR-ESI-MS). All of the compounds showed strong antimicrobial activity versus test organisms with MIC values ranging from 4 to16 g/ml. Keywords: Marine derived actinomycetes; Streptomyces rochei; Secondary metabolites; Nucleoside analogs Acknowledgements: This study was financed mainly by Scientific and Technological Research Council of Turkey (TUBITAK, Project No: 109S361) and partly by “The Research Fund of Ege University 2010 Fen 012” project.
whereas, the optimal conditions were sucrose as a carbon source, amount of sucrose 50 g/l, diammonium hydrogen citrate as a nitrogen source, amount of nitrogen 2 g/l, temperature 37 ◦ C, amount of inoculums 3 × 108 CFU/ml and incubation time 72 hours for isolate 73%. For 28%, 27 and 45.3 g/l for lactic acid amount were found, respectively before and after optimization. For 73%, 15 and 51.7 g/l for lactic acid amount were found, respectively before and after optimization. 28% and 73% isolates optimized lactic acid production abilities were identified as Lactobacillus brevis and Streptococcus sp. according to API CHL 50 and biochemical test results. From the results obtained in this study, we can conclude that two isolates are excellent microbial producers of lactic acid from sucrose. Due to its high potentiality in conversion of sucrose to lactic acid, these selected isolates can be exploited industrially for developing a novel technology. Keywords: Oral lactic acid bacteria; Mouth microbiota; Medium optimization; Lactic acid http://dx.doi.org/10.1016/j.nbt.2012.08.195 Poster 1.3.51 Study of the influential environmental variables in the production of DHA through the fermentation of Crypthecodinium cohnii niga 1,2,∗ , J.C. Gentina 1 César Ruminot 1 , M.E. Zu˜ 1 2
http://dx.doi.org/10.1016/j.nbt.2012.08.194 Poster 1.3.50 Investigation of lactic acid production abilities of bacteria isolated from oral microbiota ¸ abuk 3 Ya˘ gmur Toptas¸ 1,∗ , Gülc¸in Akca 2 , Ahmet C 1
Graduate School of Natural and Applied Sciences, Eskis¸ehir Osmangazi University, Eskis¸ehir, Turkey 2 Department of Microbiology, Faculty of Dentistry, Gazi University, Ankara, Turkey 3 Department of Biology, Faculty of Arts and Sciences, Eskis¸ehir Osmangazi University, Eskis¸ehir, Turkey Lactic acid is a commercially viable product. Today, the production of lactic acid through fermentation process is possible using the members of the genus Lactobacillus. Mouth is suitable environment for many microorganisms such as lactic acid bacteria. In this study, lactic acid production abilities of some lactic acid bacteria isolated from mouths of patients were investigated. After biochemical characterization of isolates, it is chosen two isolates having lactic acid production potential for further studies. Using these strains, the effects of carbon source, amount of carbon, nitrogen source, and amount of nitrogen, temperature, amount of inoculums and incubation time on lactic acid production were systematically tested. Optimal conditions for lactic acid production have been found to be: sucrose as a carbon source, amount of sucrose 50 g/l, diammonium hydrogen citrate as a nitrogen source, amount of nitrogen 8 g/l, temperature 42◦ C, amount of inoculums 4.5 × 108 CFU/ml and incubation time 72 hours for isolate 28#; S70
www.elsevier.com/locate/nbt
Pontificia Universidad Catolica de Valparaiso, Valparaiso, Chile Creas Conicyt-Regional Gore Valparaíso R06I1004, Chile
Crypthecodinium cohnii is a heterotrophic marine microorganism characterized by accumulate lipids (∼40% dry weight) and have a profile rich in DHA (30–50%). The DHA is a fatty acid omega 3, essential due its synthesis and functionality. The focus of this work is to study the culture environmental conditions that maximize cell growth and lipid accumulation. It shows the effect of illuminance, of glucose and ethanol concentrations both as carbon source, of the nitrogen source as limiting nutrient, besides the pH effect and the temperature through an experimental central design compound 22 using responses such as volumetric productivity (QDHA ) and specific growth rate (μ). Specific grow rate of C. cohnii culture decreased when illuminance was 5000 lx. C. cohnii, grew faster in glucose in comparison with ethanol as carbon source, being 20 g/l of glucose the optimal concentration under the studied conditions. When C. cohnii was grew using nitrogen as limiting nutrient, it was observed a significant increment of lipid content for both carbon source, glucose (from 9.7% to 24.2%) and ethanol (from 7.9% to 29.6%). The results showed significant effect of temperature on the μ for both sources of carbon. For the QDHA was found significant effects of temperature for the case of glucose and of pH and the temperature using ethanol. These studies serve as the basis for future fermentations where is integrated the obtained information. http://dx.doi.org/10.1016/j.nbt.2012.08.196
Poster 1.3.47
Poster 1.3.48
Polyhydroxyalkanoates production and recovery with Ralstonia eutropha from plant and animal oils
Biodiversity and bioactivity of some marine sediment derived actinomycetes from Turkey
S.L. Riedel 1,2,∗ , C. Brigham 1 , J. Bader 2 , S. Koenig 2 , C.K. Rha 3 , U. Stahl 2 , A.J. Sinskey 1,4,5
¸ etinel Aksoy 1 , Orc¸un Kalkan 1 , Atac¸ Kadriye Özcan 1 , Semiha C 1,∗ Uzel , E. Esin Hames-Kocabas 2 , M. Baki Yokes¸ 3 , Erdal Bedir 2
1
1
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA 2 Department of Applied and Molecular Microbiology, Technische Universität Berlin, Germany 3 Biomaterials Science and Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA 4 Engineering Systems Division, Massachusetts Institute of Technology, Cambridge, MA, USA 5 Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA Ralstonia eutropha is the model organism for production of polyhydroxyalkanoates (PHA), biodegradable and biocompatible alternatives to petroleum-based plastics. There is great interest in developing scalable, low cost polymer production processes on high carbon, commodity plant and animal oil feedstocks. We researched wild type and recombinant R. eutropha metabolizing various feedstocks (palm oils, waste frying oil, and tallow) for production and recovery of polyhydroxybutyrate (PHB) or poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)). P(HB-co-HHx) containing short chain HB and medium chain HHx monomers enhances several processing properties (e.g. melting temperature, crystallinity and flexibility) of the PHA polymer. We demonstrated recombinant R. eutropha in fed batch fermentations on palm oil producing over 140 g/L cell dry weight (CDW) of biomass with more than 70% PHA per CDW with ∼20 mol% HHx. Our data suggest the scalable fermentation process with a space time yield better than 1 g PHA/L/h. We further showed the potential use of waste fats as carbon feedstocks for PHA production in batch or extended batch fermentations. Initial promising tallow and waste frying oil carbon source results suggest an adapted fed batch process. We have also demonstrated P(HB-co-HHx) recovery from both dry and wet cells. Ethyl acetate showed both high recovery levels and high purities (up to 99%) from dry cells. Simpler recovery from wet cells, however, potentially saves time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) proved the most favourable solvent for PHA recovery with purities of up to 99% and total recovery yields of up to 84%. http://dx.doi.org/10.1016/j.nbt.2012.08.192
Ege University, Faculty of Science, Department of Biology, Basic and Industrial Microbiology Section, 35100 Bornova, Izmir, Turkey 2 Ege University, Faculty of Engineering, Department of Bioengineering, 35100 Bornova, Izmir, Turkey 3 Halic Univ, Fac Arts & Sci, Dept Mol Biol & Genet, TR-34381 Istanbul, Turkey In recent years marine environments are proved as a rich source of microorganisms including actinomycetes. These marine derived actinomycetes are prolific sources of many bioactive compounds including antibiotics. In this study, a total of 33 samples were collected from coastal sediments of Turkey from 10 different locations and 1 sample was from Van (Soda) Lake. Four different selective isolation media were used for actinomycetes isolation including; modified actinomycetes isolation agar (MAIA), M1 agar, M6 agar and modified R2A agar. MAIA was found best medium for recovery of actinomycetes but M6 was found superior than others for the isolation of antibiotic producing actinomycetes strains. In total, 269 actinomycetes strain were isolated and 70 (23.64/%) of the strains were found active against at least one test microorganism. Consequently, coastal sediments of Turkey were found rich in actinomycetes diversity and this diversity provides an untouched source for the isolation of new and potent antimicrobial compounds. Keywords: Biodiversity; Antimicrobial activity; Marine sediment; Actinobacteria Acknowledgements: This study was financed mainly by The Scientific and Technological Research Council of Turkey (TUBITAK, Project No: 109S361) and partly by “The Research Fund of Ege University 2009 Fen 061” project. Kadriye Özcan was supported by TUBITAK 2211 doctorate scholarship programme. http://dx.doi.org/10.1016/j.nbt.2012.08.193 Poster 1.3.49 Purification and identification of secondary metabolites from marine derived Streptomyces rochei 6CM016 and their antimicrobial activities Semiha Cetinel Aksoy 1 , Atac¸ Uzel 1,∗ , E. Esin Hames-Kocabas 2 , Ikhlas A. Khan 3 , Erdal Bedir 2 1
Ege University, Faculty of Science, Department of Biology, BornovaIzmir, Turkey 2 Ege University, Faculty of Engineering, Department of Bioengineering, Bornova-Izmir, Turkey 3 University of Mississippi, National Center for Natural Products Research, University, MS 38677, USA Aim of this study was purification and identification of antimicrobial secondary metabolites from marine derived Streptomyces rochei 6CM016. Strain 6CM016 was isolated from a sediment sample collected from Kas¸-Antalya. Initial fermentation was performed www.elsevier.com/locate/nbt S69
New Biotechnology · Volume 29S · September 2012
in 250 ml flasks containing 50 ml of M1 medium. Inoculated medium was incubated at 28◦ C for 10 days at 150 rpm. Fermentation broth was extracted with EtOAc and antimicrobial activity was tested using disc diffusion method. The isolate was found to be active against enteropathogenic Escherichia coli 0157:H7 (RSKK 234), methicillin resistant Staphylococcus aureus ATCC 43300 and Candida albicans DSMZ 5817. Then, 56 l of fermentation broth was obtained from a scale-up fermentation study, and was extracted with EtOAc after removal of the cells via filtration. The extract was concentrated in vacuo at 35◦ C yielding 1.92 g of brown residue. Bioassay-guided fractionations by using high performance flash chromatography (Biotage) and preparative thin layer chromatography on normal-phase/reverse-phase silica gel and Sephadex LH20 led to the isolation of five compounds [06CM016-01 (plicacetin), 06CM016-02, 06CM016-03 (norplicacetin), 06CM016-05 and 06CM016-08]. Structures of the compounds were elucidated as nucleoside-type secondary metabolites, three of which were new molecules for nature, by spectral methods (1D-, 2D-NMR and HR-ESI-MS). All of the compounds showed strong antimicrobial activity versus test organisms with MIC values ranging from 4 to16 g/ml. Keywords: Marine derived actinomycetes; Streptomyces rochei; Secondary metabolites; Nucleoside analogs Acknowledgements: This study was financed mainly by Scientific and Technological Research Council of Turkey (TUBITAK, Project No: 109S361) and partly by “The Research Fund of Ege University 2010 Fen 012” project.
whereas, the optimal conditions were sucrose as a carbon source, amount of sucrose 50 g/l, diammonium hydrogen citrate as a nitrogen source, amount of nitrogen 2 g/l, temperature 37 ◦ C, amount of inoculums 3 × 108 CFU/ml and incubation time 72 hours for isolate 73%. For 28%, 27 and 45.3 g/l for lactic acid amount were found, respectively before and after optimization. For 73%, 15 and 51.7 g/l for lactic acid amount were found, respectively before and after optimization. 28% and 73% isolates optimized lactic acid production abilities were identified as Lactobacillus brevis and Streptococcus sp. according to API CHL 50 and biochemical test results. From the results obtained in this study, we can conclude that two isolates are excellent microbial producers of lactic acid from sucrose. Due to its high potentiality in conversion of sucrose to lactic acid, these selected isolates can be exploited industrially for developing a novel technology. Keywords: Oral lactic acid bacteria; Mouth microbiota; Medium optimization; Lactic acid http://dx.doi.org/10.1016/j.nbt.2012.08.195 Poster 1.3.51 Study of the influential environmental variables in the production of DHA through the fermentation of Crypthecodinium cohnii niga 1,2,∗ , J.C. Gentina 1 César Ruminot 1 , M.E. Zu˜ 1 2
http://dx.doi.org/10.1016/j.nbt.2012.08.194 Poster 1.3.50 Investigation of lactic acid production abilities of bacteria isolated from oral microbiota ¸ abuk 3 Ya˘ gmur Toptas¸ 1,∗ , Gülc¸in Akca 2 , Ahmet C 1
Graduate School of Natural and Applied Sciences, Eskis¸ehir Osmangazi University, Eskis¸ehir, Turkey 2 Department of Microbiology, Faculty of Dentistry, Gazi University, Ankara, Turkey 3 Department of Biology, Faculty of Arts and Sciences, Eskis¸ehir Osmangazi University, Eskis¸ehir, Turkey Lactic acid is a commercially viable product. Today, the production of lactic acid through fermentation process is possible using the members of the genus Lactobacillus. Mouth is suitable environment for many microorganisms such as lactic acid bacteria. In this study, lactic acid production abilities of some lactic acid bacteria isolated from mouths of patients were investigated. After biochemical characterization of isolates, it is chosen two isolates having lactic acid production potential for further studies. Using these strains, the effects of carbon source, amount of carbon, nitrogen source, and amount of nitrogen, temperature, amount of inoculums and incubation time on lactic acid production were systematically tested. Optimal conditions for lactic acid production have been found to be: sucrose as a carbon source, amount of sucrose 50 g/l, diammonium hydrogen citrate as a nitrogen source, amount of nitrogen 8 g/l, temperature 42◦ C, amount of inoculums 4.5 × 108 CFU/ml and incubation time 72 hours for isolate 28#; S70
www.elsevier.com/locate/nbt
Pontificia Universidad Catolica de Valparaiso, Valparaiso, Chile Creas Conicyt-Regional Gore Valparaíso R06I1004, Chile
Crypthecodinium cohnii is a heterotrophic marine microorganism characterized by accumulate lipids (∼40% dry weight) and have a profile rich in DHA (30–50%). The DHA is a fatty acid omega 3, essential due its synthesis and functionality. The focus of this work is to study the culture environmental conditions that maximize cell growth and lipid accumulation. It shows the effect of illuminance, of glucose and ethanol concentrations both as carbon source, of the nitrogen source as limiting nutrient, besides the pH effect and the temperature through an experimental central design compound 22 using responses such as volumetric productivity (QDHA ) and specific growth rate (μ). Specific grow rate of C. cohnii culture decreased when illuminance was 5000 lx. C. cohnii, grew faster in glucose in comparison with ethanol as carbon source, being 20 g/l of glucose the optimal concentration under the studied conditions. When C. cohnii was grew using nitrogen as limiting nutrient, it was observed a significant increment of lipid content for both carbon source, glucose (from 9.7% to 24.2%) and ethanol (from 7.9% to 29.6%). The results showed significant effect of temperature on the μ for both sources of carbon. For the QDHA was found significant effects of temperature for the case of glucose and of pH and the temperature using ethanol. These studies serve as the basis for future fermentations where is integrated the obtained information. http://dx.doi.org/10.1016/j.nbt.2012.08.196