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Fungal biotransformation of terpenes
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
death, and by means of fluorescence activated cell sorting to distinguish between necrotic and apoptotic cell death. Results show that Chinese Hamster Ovaries (CHO) cells will enter the apoptotic pathway when subjected to low levels of hydrodynamic stress (around 2.0 Pa) in oscillating, extensional flow. In contrast, necrotic death prevails when the cells are exposed to hydrodynamic stresses around 1.0 Pa in simple shear flow or around 500 Pa in extensional flow. The obtained threshold values in small-scale model system were subsequently used to predict the impact of the hydrodynamic stress on cell damage during cell cultivation in two lab-scale units: a stirred tank bioreactor (STR) and a novel lobed Taylor—Couette (LTC) bioreactor. After a detailed characterization of all used system by Computational Fluid Dynamics (CFD) and the description of the occurring hydrodynamic stresses within the small-scale model system, a CHO cell line was used to verify the predictive power of the small-scale model systems and the CFD analysis for cell damage in stirred bioreactors. It was found that both units allow cell cultivation once the maximal hydrodynamic stress encountered by the suspended cells is below 1 Pa. Moreover, the LTC bioreactor featuring a narrow distribution of the hydrodynamic stress compared to the STR enables higher cell densities, faster growth and a higher volumetric productivity compared to the STR. Furthermore, cell cultivation without the supplementation of cell protective agents, like Pluronic F-68, is feasible.
0.01 g L−1 of Fe2 SO4 ). Biotransformation was started by adding 1% (v/v) of terpene, and the flasks were incubated in rotary shaker at 30 ◦ C and 150 rpm. At 0, 24, 48, 72 and 96 h after the addition of the terpene, samples were extracted with the same volume of ethil acetate. A Gas Chromatograph (GC/FID—–Agilent 6890) was used to analyse the volatiles compounds formed during the biotransformation. The working conditions were: injector 250 ◦ C, detector-FID 250 ◦ C (make-up gas He 1 mL min−1 ) and a DB-5 capillary column (30 m × 0.32 mm × 0.25 m). The oven temperature was programmed from 80 ◦ C (5 min) at 20 ◦ C/min until 200 ◦ C (5 min), with a final holding time of 5 min. The results of this work indicated that the strains are capable to biotransform some terpenes, however the yields were small to a direct industrial application. Volatiles products identification were made by a GC/MS (Shimadzu QP5000) in the same conditions. Using limonene as substrate, one Penicillium sp. strain was able to produce carvone, cis- and trans-carveol by an hydroxylation at the 6-position of the carbon. Moreover it was found that this strain also produced traces of an interesting aroma compound labelled as p-mentha-1,8-dien3-one, with a sweet and fruity aroma.
doi:10.1016/j.nbt.2009.06.111
Fermentation broth rheological modelling during production of Pantoea agglomerans PBC-1, a biocontrol agent of post-harvest fruit diseases
doi:10.1016/j.nbt.2009.06.112
2.6.007
Poster 2.6.006 M.E. Lima-Costa Fungal biotransformation of terpenes A.P. Dionísio ∗ , G. Molina, J. Lemos Bicas, S. Dias, G.M. Pastore UNICAMP - University of Campinas, Campinas, Brazil
Nowadays, with more than 22,000 individual identified compounds, terpenes represent the largest group of natural products. They are secondary metabolites of plants that are produced, in part, as a defense against microorganisms and insects, in addition to their pollinator-attractive properties. Biotransformation of terpenes represents a very attractive alternative to production of aromas, because takes place under mild conditions, does not generate toxic wastes and it allows to produce “natural” aromas that can be used as fragrances and flavors in the industry. In this work the fungal biotransformation of terpenes was investigated. For this, nine fungal strains (Aspergillus spp., Penicillium spp. and Paecilomyces spp.) were isolated from Brazilian fruits and tested with five terpenes ((1S)-(−)-␣-pinene, (R)-(+)-limonene, ␥-terpinene, farnesene and citronellol). For each strain, 1 cm2 of three-day-old precultures on a Petri dish were homogenized with an ultraturrax, contained 50 mL yeast malt medium (glucose 10 g L−1 , peptone 5 g L−1 , malt extract 3 g L−1 , yeast extract 3 g L−1 ). The flasks were incubated in rotary shaker at 30 ◦ C and 150 rpm for 72 h. The fungal culture was filtered through Whatman no. 1 filter. The concentrated biomass was placed in a 250 mL conical flask containing 50 mL of mineral medium (0.5 g L−1 of MgSO4; 3.0 g L−1 of NaNO3 ; 1.0 g L−1 of K2 HPO4 ; 0.5 g L−1 of KCl;
University of Algarve - CDCTPV, Faro, Portugal
Biomass production of biocontrol agent is a great technological challenge for its commercial success. An important feature of a fermentation process is the change in rheological behaviour during fermentation due to changing of substrate composition, morphology of the microorganism and biomass concentration which creates heterogeneity in the broth medium, resulting in compartmentalization of dissolved oxygen concentration, pH and substrate availability, with consequences in terms of biomass viability, yield and biomass productivity. Pantoea agglomerans PBC-1 cultures were grown either in Erlenmeyer flasks or in a 3-L mechanically stirred bioreactor, in medium with yeast extract as nitrogen source and glucose, sucrose or alternatively carob extracts as carbon sources. The higher microbial viable population with good antagonist efficiency was achieved for carob extracts and for sucrose, respectively, 3—4 × 109 cfu ml−1 and 2—3 × 109 cfu ml−1 , after 20 h growth, when the available sugar was fully depleted. Rheological analyses were carried out on fresh samples collected at regular intervals, using a UL adapter mounted on a LVDV-II + Pro Brookfield viscometer. Different mathematical models Oswaldde Waele (power law), Bingham equation, Casson equation and Herschel—Bulkley equation, were adjusted to describe the rheological characteristics of the broth suspension culture. It was verified that P. agglomerans PBC-1 culture fits with Oswald-de Waele model (power law), as expected. Nevertheless the higher confidence of fit
www.elsevier.com/locate/nbt S191
ABSTRACTS
death, and by means of fluorescence activated cell sorting to distinguish between necrotic and apoptotic cell death. Results show that Chinese Hamster Ovaries (CHO) cells will enter the apoptotic pathway when subjected to low levels of hydrodynamic stress (around 2.0 Pa) in oscillating, extensional flow. In contrast, necrotic death prevails when the cells are exposed to hydrodynamic stresses around 1.0 Pa in simple shear flow or around 500 Pa in extensional flow. The obtained threshold values in small-scale model system were subsequently used to predict the impact of the hydrodynamic stress on cell damage during cell cultivation in two lab-scale units: a stirred tank bioreactor (STR) and a novel lobed Taylor—Couette (LTC) bioreactor. After a detailed characterization of all used system by Computational Fluid Dynamics (CFD) and the description of the occurring hydrodynamic stresses within the small-scale model system, a CHO cell line was used to verify the predictive power of the small-scale model systems and the CFD analysis for cell damage in stirred bioreactors. It was found that both units allow cell cultivation once the maximal hydrodynamic stress encountered by the suspended cells is below 1 Pa. Moreover, the LTC bioreactor featuring a narrow distribution of the hydrodynamic stress compared to the STR enables higher cell densities, faster growth and a higher volumetric productivity compared to the STR. Furthermore, cell cultivation without the supplementation of cell protective agents, like Pluronic F-68, is feasible.
0.01 g L−1 of Fe2 SO4 ). Biotransformation was started by adding 1% (v/v) of terpene, and the flasks were incubated in rotary shaker at 30 ◦ C and 150 rpm. At 0, 24, 48, 72 and 96 h after the addition of the terpene, samples were extracted with the same volume of ethil acetate. A Gas Chromatograph (GC/FID—–Agilent 6890) was used to analyse the volatiles compounds formed during the biotransformation. The working conditions were: injector 250 ◦ C, detector-FID 250 ◦ C (make-up gas He 1 mL min−1 ) and a DB-5 capillary column (30 m × 0.32 mm × 0.25 m). The oven temperature was programmed from 80 ◦ C (5 min) at 20 ◦ C/min until 200 ◦ C (5 min), with a final holding time of 5 min. The results of this work indicated that the strains are capable to biotransform some terpenes, however the yields were small to a direct industrial application. Volatiles products identification were made by a GC/MS (Shimadzu QP5000) in the same conditions. Using limonene as substrate, one Penicillium sp. strain was able to produce carvone, cis- and trans-carveol by an hydroxylation at the 6-position of the carbon. Moreover it was found that this strain also produced traces of an interesting aroma compound labelled as p-mentha-1,8-dien3-one, with a sweet and fruity aroma.
doi:10.1016/j.nbt.2009.06.111
Fermentation broth rheological modelling during production of Pantoea agglomerans PBC-1, a biocontrol agent of post-harvest fruit diseases
doi:10.1016/j.nbt.2009.06.112
2.6.007
Poster 2.6.006 M.E. Lima-Costa Fungal biotransformation of terpenes A.P. Dionísio ∗ , G. Molina, J. Lemos Bicas, S. Dias, G.M. Pastore UNICAMP - University of Campinas, Campinas, Brazil
Nowadays, with more than 22,000 individual identified compounds, terpenes represent the largest group of natural products. They are secondary metabolites of plants that are produced, in part, as a defense against microorganisms and insects, in addition to their pollinator-attractive properties. Biotransformation of terpenes represents a very attractive alternative to production of aromas, because takes place under mild conditions, does not generate toxic wastes and it allows to produce “natural” aromas that can be used as fragrances and flavors in the industry. In this work the fungal biotransformation of terpenes was investigated. For this, nine fungal strains (Aspergillus spp., Penicillium spp. and Paecilomyces spp.) were isolated from Brazilian fruits and tested with five terpenes ((1S)-(−)-␣-pinene, (R)-(+)-limonene, ␥-terpinene, farnesene and citronellol). For each strain, 1 cm2 of three-day-old precultures on a Petri dish were homogenized with an ultraturrax, contained 50 mL yeast malt medium (glucose 10 g L−1 , peptone 5 g L−1 , malt extract 3 g L−1 , yeast extract 3 g L−1 ). The flasks were incubated in rotary shaker at 30 ◦ C and 150 rpm for 72 h. The fungal culture was filtered through Whatman no. 1 filter. The concentrated biomass was placed in a 250 mL conical flask containing 50 mL of mineral medium (0.5 g L−1 of MgSO4; 3.0 g L−1 of NaNO3 ; 1.0 g L−1 of K2 HPO4 ; 0.5 g L−1 of KCl;
University of Algarve - CDCTPV, Faro, Portugal
Biomass production of biocontrol agent is a great technological challenge for its commercial success. An important feature of a fermentation process is the change in rheological behaviour during fermentation due to changing of substrate composition, morphology of the microorganism and biomass concentration which creates heterogeneity in the broth medium, resulting in compartmentalization of dissolved oxygen concentration, pH and substrate availability, with consequences in terms of biomass viability, yield and biomass productivity. Pantoea agglomerans PBC-1 cultures were grown either in Erlenmeyer flasks or in a 3-L mechanically stirred bioreactor, in medium with yeast extract as nitrogen source and glucose, sucrose or alternatively carob extracts as carbon sources. The higher microbial viable population with good antagonist efficiency was achieved for carob extracts and for sucrose, respectively, 3—4 × 109 cfu ml−1 and 2—3 × 109 cfu ml−1 , after 20 h growth, when the available sugar was fully depleted. Rheological analyses were carried out on fresh samples collected at regular intervals, using a UL adapter mounted on a LVDV-II + Pro Brookfield viscometer. Different mathematical models Oswaldde Waele (power law), Bingham equation, Casson equation and Herschel—Bulkley equation, were adjusted to describe the rheological characteristics of the broth suspension culture. It was verified that P. agglomerans PBC-1 culture fits with Oswald-de Waele model (power law), as expected. Nevertheless the higher confidence of fit
www.elsevier.com/locate/nbt S191