- Email: [email protected]
Poly-β-hydroxybutyrate (PHB) Production by Cyanobacteria
Abstracts / New Biotechnology 33S (2016) S1–S213
plex (Ag-Ab) (with no corresponding binding to free antibodies); SC are atypical protein ligands because they bind outside of the protein’s active site (related with conformational changes resulting from function-related destabilization of the protein) 3) in vivo interactions between SC and Ag-Ab complexes (local inflammatory conditions), followed by their progressive elimination from the organism. Research methodology include spectrophotometry, microscopic and dynamic light scattering technique and molecular modelling of interactions between carbon nanotubes, SC and drugs. Results show the ability of SC to disperse hydrophobic CNT in aqueous solution and form capacious drug carriers. Results show pH-dependent release of drugs and accumulate on the cell surface. http://dx.doi.org/10.1016/j.nbt.2016.06.791
O4-10 Using flow cytometry to monitor cell physiology of magnetotactic bacteria in fermentation processes Alfred Fernández-Castané 1,∗ , Hong Li 1 , Matthias Franzreb 2 , Owen R.T. Thomas 1 , Tim W. Overton 1 1 2
University of Birmingham, United Kingdom Karlsruhe Institute of Technology, Germany
Magnetotactic bacteria (MTB) are a group of microorganisms that naturally synthesize single-domain ferrimagnetic nanoparticles of magnetite contained within subcellular membrane bound organelles called ‘magnetosomes’ [1]. The unique characteristics of MTB dictate the need for delicate manipulation and carefully – controlled conditions for growth. Moreover, growing MTB to relatively high cell densities whilst simultaneously preserving healthy magnetosome production requires accurate control of nutrient supply and process parameters [2]. Here in this study we detail a simple fermentation strategy employing a pH-stat approach and minimal control of process parameters to cultivate Magnetospirillum gryphiswaldense MSR-1 in 5L bioreactors. We have used a magnetic spectrophotometry measurement system to track (quasi-online) and compare magnetosome production during growth. Flow cytometry analysis was used to monitor physiological parameters during fermentation experiments, enabling determination of cell numbers and the fraction of healthy/damaged cells by intracellular staining. Our results represent a benchmark for the establishment of a platform for the production of functional magnetic nanoparticles synthesized via biological routes. We anticipate the scalability of this process for industrial purposes aiming to efficiently develop innovative capture techniques of target products using magnetic separation. References [1] Yan, et al. Microbiol Res 2012;167(9):507–19. [2] Sun, et al. Appl Microbiol Biotechnol 2008;79(3):389–97.
http://dx.doi.org/10.1016/j.nbt.2016.06.792
S19
O4-11 Production of poly(3-hydroxyalkanoate) biopolymers from syngas using Rhodospirillum rubrum: Turning waste into treasure Stéphanie Follonier ∗ , Stephanie Karmann, Marco Romanino, Manfred Zinn HES-SO Valais-Wallis, Switzerland Poly(3-hydroxyalkanoates) (PHA) are bio-based and biodegradable alternatives to conventional polymers derived from fossil fuels. In this work we assessed a novel type of production process that relies on fermentations using syngas (CO, CO2 , H2 and N2 ) as main substrate and Rhodospirillum rubrum as CO-metabolizing and PHA-producing strain. Syngas can be obtained from the pyrolysis of organic wastes and thus represents an inexpensive, non food-competitive carbon source. A cutting-edge process analytical technology platform including measurements of dissolved oxygen and redox potential, gas concentrations by mass spectrometry as well as cell concentration and PHA content by flow cytometry was set up to monitor the bioprocess and cell physiology. Preliminary experiments revealed the difficulty of culturing R. rubrum with syngas as sole carbon source. In particular, yeast extract and acetate addition were found to be essential for cell growth and PHA production, respectively. Moreover, cell growth was shown to be limited by CO mass transfer. In order to enhance the overall productivity, we designed a semi-continuous process with a heterotrophic, aerobic growth phase on fructose prior to the PHA production from syngas and acetate. This strategy proved to be feasible but only if an anaerobic adaptation phase on fructose was included in-between. Fructose exhibited better cell growth than succinate and malate when considering both the aerobic and anaerobic phases and was therefore chosen as carbon source for the heterotrophic growth phase. We are currently further investigating the effect of the different growth conditions (aerobic, anaerobic, syngas) on the cell physiology and PHA production. http://dx.doi.org/10.1016/j.nbt.2016.06.793
O4-12 Poly--hydroxybutyrate (PHB) Production by Cyanobacteria Roberta Carpine 1,∗ , Giuseppe Olivieri 1 , Klaas Hellingwerf 2 , Antonino Pollio 1 , Gabriele Pinto 1 , Antonio Marzocchella 1 1 2
Università degli Studi di Napoli Federico II, Italy University Van Amsterdam, Italy
Polyhydroxyalkanoates (PHAs) are a group of building block of biodegradable plastics. They are produced by a wide variety of microorganisms, mainly as reservoir for the energy and carbon in excesses with respect to the physiologic requirement. Polyhydroxybutyrate (PHB) is a short-chain-length PHA characterized by interesting chemical and physical properties. A potential sustainable route to produce the PHB is via biotechnological production by means of cyanobacteria. These microorganisms may synthesize PHB under photoautotrophic conditions. However, the systematic characterization of the PHB production via cyanobacteria fermentation is still missing. Present contribution reports the characterization of the PHB production by cyanobacteria. Tests on Synechocystis PCC6803, a strain known to produce PHB, have been carried out to assess the optimial media formulation to maximize the PHB production. The
S20
Abstracts / New Biotechnology 33S (2016) S1–S213
best assessed performance was characterized by cyanobacteria PHB content of 7.7% wt and PGB productivity 7 g/m3 day. Five cyanobacterium strains have been investigated using the optimal assessed medium to find the best PHB producer: S. PCC6803, S. aquatilis, S. fuscopigmentosa, Synechococcus nidulans, and Chloregloeopsis fritshii. S. PCC6803 and S. aquatilis have been selected as best strains for PHB production: composition about 8%wt, productivity about 7 g/m3 day. The main difference between the two strains regards the growth phase coupled with the PHB production: the S. aquatilis produces PHB since the beginning of the growth, the S. PCC6803 starts to produce PHB after the nitrogen starvation. http://dx.doi.org/10.1016/j.nbt.2016.06.794
O4-13 Microencapsulation of bioactive phenolics from wine wastes in polymers and kinetics of their release
develop a more effective insulin delivery system for the treatment of diabetes, two well-characterized insulin-loaded nanogels with opposite zeta potential (−15.94 ± 0.449 mV for insulin:CMCS/CSNGs(−) and +17.15 ± 0.492 mV for insulin:CMCS/CS-NGs(+)) were obtained. The adhesion of insulin:CMCS/CS-NGs(−) were 3 folds and 1.3 folds compared with insulin:CMCS/CS-NGs(+) in rat jejunum and ileum and it was 1.8 folds for the permeation through ex vivo jejunum. Although positive and negative insulin:CMCS/CSNGs have almost the same capacity to effect the TEER of Caco-2 cell monolayers. Insulin:CMCS/CS-NGs(−) had a higher permeation through Caco-2 cell monolayers than insulin:CMCS/CS-NGs(+) when the temperature was 4 ◦ C and at 37 ◦ C there was a opposite result. The study demonstrated that insulin:CMCS/CS-NGs(−) was probably a potential insulin delivery for the treatment of diabetes and also could effectively prevent the occurrence of complications. The transport mechanisms of insulin:CMCS/CS-NGs(−) and insulin:CMCS/CS-NGs(+) in different intestinal segments needed further study. http://dx.doi.org/10.1016/j.nbt.2016.06.796
Maria Liakopoulou-Kyriakides ∗ , Alexandra Moschona Aristotle University of Thessaloniki, Greece
O4-15
Wine wastes (white local variety “Malagouzia” and red variety “Syrah”) are a rich source of various valuable secondary byproducts such as phenolics among others. Phenolic compounds can be considered as high added-value by-products and the use of industrial wastes could greatly reduce their production cost and increase the margin profit of the products. We have previously reported the isolation of specific phenolic fractions, from red and white wine wastes, using solvent extractions and sorption/desorption techniques, possessing 92–95% of the estimated activities (antioxidant, antiplatelet, anti-inflammatory etc.) of the initial crude extract. Specifically, the antioxidant activity was determined by measuring the inhibition of DPPH and found to be 82%, the anti-inflammatory activity was determined by measuring the inhibition of COX1 (99%) and COX2 (78%), and antiplatelet activity in vitro, with 98% inhibition of platelet aggregation induced by collagen. An attempt to encapsulate these phenolics in order to retain chemical stability and biological activities is presented here. Several polymers, such as alginate and chitosan and mixtures of them, were examined. A mixture of 2%, w/v, sodium alginate and 2% w/v chitosan was found to perform better, shown encapsulation efficiency up to 70% of the initial phenolic content. Release experiments of phenolic compounds with EtOH (70%), 0.1 M HCl and sodium citrate 0.2 M showed that in the first two cases, a higher than 50% of the total phenolic content was released in the first 6 hours. These data suggest that encapsulated phenolics may have several applications in food and pharmaceutical industries.
Polyvinyl alcohol (PVA)-degrading enzyme: Structure analysis, heterologous overexpression, and applications
http://dx.doi.org/10.1016/j.nbt.2016.06.795
O4-16
O4-14
Novel mutants of Gluconacetobacter with different cellulose synthesis ability
Safety and pathways of intestinal absorption for chitosan nanocarriers Xi Guang Chen Ocean University of China, China Nanocarriers are highly efficient vehicles to deliver drugs and bioactive ingredients in disease therapy. We have developed tens of chitosan derivatives, fabricated into nano-sized entities and studied their potential and bioactivities as drug delivery carriers. To
Guocheng Du Jiangnan University, China Polyvinyl alcohol (PVA), with its many desirable physical and chemical characteristics, is widely used in textile sizing, adhesives, fiber coating, and et al. However, this extensive usage, particularly in the textile industry, has resulted in large amounts of PVA being discharged in the wastewater, causing environmental pollution due to the difficulty in degrading PVA. In contrast, PVA degradation using microbial enzymes is an environmentally friendly approach that can save energy and reduce the problems caused by the treatment of sewage from the textile desizing stage. Microbial degradation of PVA entails a two-step metabolism. In the first step two neighboring alcohols are oxidized to form a diketone structure in the polymer by PVA oxidase or PVA dehydrogenase. In the second step oxidized PVA (OPA) is hydrolyzed by OPA hydrolase (OPH, also known as ß-diketone hydrolase, BDH). In this report, we first introduce the structural analysis of OPA, and then address the expression and production of OPA and OPH, and finally introduce the application of OPA and OPH in the PVA degradation. http://dx.doi.org/10.1016/j.nbt.2016.06.797
∗ , Anna Cielecka, Małgorzata ˛ Marzena Jedrzejczak-Krzepkowska Ryngajłło, Katarzyna Kubiak, Stanisław Bielecki
Lodz University of Technology, Poland Bacterial NanoCellulose (BNC) is an exopolysaccharide synthesized by various species of bacteria, such as the genus Gluconacetobacter, Agrobacterium, Rhizobium and Sarcina. The Gluconacetobacter species are known to be one of the most efficient BNC producers. This polymer protects the cells from harmful environmental factors (UV light, desiccation). BNC has unique prop-
plex (Ag-Ab) (with no corresponding binding to free antibodies); SC are atypical protein ligands because they bind outside of the protein’s active site (related with conformational changes resulting from function-related destabilization of the protein) 3) in vivo interactions between SC and Ag-Ab complexes (local inflammatory conditions), followed by their progressive elimination from the organism. Research methodology include spectrophotometry, microscopic and dynamic light scattering technique and molecular modelling of interactions between carbon nanotubes, SC and drugs. Results show the ability of SC to disperse hydrophobic CNT in aqueous solution and form capacious drug carriers. Results show pH-dependent release of drugs and accumulate on the cell surface. http://dx.doi.org/10.1016/j.nbt.2016.06.791
O4-10 Using flow cytometry to monitor cell physiology of magnetotactic bacteria in fermentation processes Alfred Fernández-Castané 1,∗ , Hong Li 1 , Matthias Franzreb 2 , Owen R.T. Thomas 1 , Tim W. Overton 1 1 2
University of Birmingham, United Kingdom Karlsruhe Institute of Technology, Germany
Magnetotactic bacteria (MTB) are a group of microorganisms that naturally synthesize single-domain ferrimagnetic nanoparticles of magnetite contained within subcellular membrane bound organelles called ‘magnetosomes’ [1]. The unique characteristics of MTB dictate the need for delicate manipulation and carefully – controlled conditions for growth. Moreover, growing MTB to relatively high cell densities whilst simultaneously preserving healthy magnetosome production requires accurate control of nutrient supply and process parameters [2]. Here in this study we detail a simple fermentation strategy employing a pH-stat approach and minimal control of process parameters to cultivate Magnetospirillum gryphiswaldense MSR-1 in 5L bioreactors. We have used a magnetic spectrophotometry measurement system to track (quasi-online) and compare magnetosome production during growth. Flow cytometry analysis was used to monitor physiological parameters during fermentation experiments, enabling determination of cell numbers and the fraction of healthy/damaged cells by intracellular staining. Our results represent a benchmark for the establishment of a platform for the production of functional magnetic nanoparticles synthesized via biological routes. We anticipate the scalability of this process for industrial purposes aiming to efficiently develop innovative capture techniques of target products using magnetic separation. References [1] Yan, et al. Microbiol Res 2012;167(9):507–19. [2] Sun, et al. Appl Microbiol Biotechnol 2008;79(3):389–97.
http://dx.doi.org/10.1016/j.nbt.2016.06.792
S19
O4-11 Production of poly(3-hydroxyalkanoate) biopolymers from syngas using Rhodospirillum rubrum: Turning waste into treasure Stéphanie Follonier ∗ , Stephanie Karmann, Marco Romanino, Manfred Zinn HES-SO Valais-Wallis, Switzerland Poly(3-hydroxyalkanoates) (PHA) are bio-based and biodegradable alternatives to conventional polymers derived from fossil fuels. In this work we assessed a novel type of production process that relies on fermentations using syngas (CO, CO2 , H2 and N2 ) as main substrate and Rhodospirillum rubrum as CO-metabolizing and PHA-producing strain. Syngas can be obtained from the pyrolysis of organic wastes and thus represents an inexpensive, non food-competitive carbon source. A cutting-edge process analytical technology platform including measurements of dissolved oxygen and redox potential, gas concentrations by mass spectrometry as well as cell concentration and PHA content by flow cytometry was set up to monitor the bioprocess and cell physiology. Preliminary experiments revealed the difficulty of culturing R. rubrum with syngas as sole carbon source. In particular, yeast extract and acetate addition were found to be essential for cell growth and PHA production, respectively. Moreover, cell growth was shown to be limited by CO mass transfer. In order to enhance the overall productivity, we designed a semi-continuous process with a heterotrophic, aerobic growth phase on fructose prior to the PHA production from syngas and acetate. This strategy proved to be feasible but only if an anaerobic adaptation phase on fructose was included in-between. Fructose exhibited better cell growth than succinate and malate when considering both the aerobic and anaerobic phases and was therefore chosen as carbon source for the heterotrophic growth phase. We are currently further investigating the effect of the different growth conditions (aerobic, anaerobic, syngas) on the cell physiology and PHA production. http://dx.doi.org/10.1016/j.nbt.2016.06.793
O4-12 Poly--hydroxybutyrate (PHB) Production by Cyanobacteria Roberta Carpine 1,∗ , Giuseppe Olivieri 1 , Klaas Hellingwerf 2 , Antonino Pollio 1 , Gabriele Pinto 1 , Antonio Marzocchella 1 1 2
Università degli Studi di Napoli Federico II, Italy University Van Amsterdam, Italy
Polyhydroxyalkanoates (PHAs) are a group of building block of biodegradable plastics. They are produced by a wide variety of microorganisms, mainly as reservoir for the energy and carbon in excesses with respect to the physiologic requirement. Polyhydroxybutyrate (PHB) is a short-chain-length PHA characterized by interesting chemical and physical properties. A potential sustainable route to produce the PHB is via biotechnological production by means of cyanobacteria. These microorganisms may synthesize PHB under photoautotrophic conditions. However, the systematic characterization of the PHB production via cyanobacteria fermentation is still missing. Present contribution reports the characterization of the PHB production by cyanobacteria. Tests on Synechocystis PCC6803, a strain known to produce PHB, have been carried out to assess the optimial media formulation to maximize the PHB production. The
S20
Abstracts / New Biotechnology 33S (2016) S1–S213
best assessed performance was characterized by cyanobacteria PHB content of 7.7% wt and PGB productivity 7 g/m3 day. Five cyanobacterium strains have been investigated using the optimal assessed medium to find the best PHB producer: S. PCC6803, S. aquatilis, S. fuscopigmentosa, Synechococcus nidulans, and Chloregloeopsis fritshii. S. PCC6803 and S. aquatilis have been selected as best strains for PHB production: composition about 8%wt, productivity about 7 g/m3 day. The main difference between the two strains regards the growth phase coupled with the PHB production: the S. aquatilis produces PHB since the beginning of the growth, the S. PCC6803 starts to produce PHB after the nitrogen starvation. http://dx.doi.org/10.1016/j.nbt.2016.06.794
O4-13 Microencapsulation of bioactive phenolics from wine wastes in polymers and kinetics of their release
develop a more effective insulin delivery system for the treatment of diabetes, two well-characterized insulin-loaded nanogels with opposite zeta potential (−15.94 ± 0.449 mV for insulin:CMCS/CSNGs(−) and +17.15 ± 0.492 mV for insulin:CMCS/CS-NGs(+)) were obtained. The adhesion of insulin:CMCS/CS-NGs(−) were 3 folds and 1.3 folds compared with insulin:CMCS/CS-NGs(+) in rat jejunum and ileum and it was 1.8 folds for the permeation through ex vivo jejunum. Although positive and negative insulin:CMCS/CSNGs have almost the same capacity to effect the TEER of Caco-2 cell monolayers. Insulin:CMCS/CS-NGs(−) had a higher permeation through Caco-2 cell monolayers than insulin:CMCS/CS-NGs(+) when the temperature was 4 ◦ C and at 37 ◦ C there was a opposite result. The study demonstrated that insulin:CMCS/CS-NGs(−) was probably a potential insulin delivery for the treatment of diabetes and also could effectively prevent the occurrence of complications. The transport mechanisms of insulin:CMCS/CS-NGs(−) and insulin:CMCS/CS-NGs(+) in different intestinal segments needed further study. http://dx.doi.org/10.1016/j.nbt.2016.06.796
Maria Liakopoulou-Kyriakides ∗ , Alexandra Moschona Aristotle University of Thessaloniki, Greece
O4-15
Wine wastes (white local variety “Malagouzia” and red variety “Syrah”) are a rich source of various valuable secondary byproducts such as phenolics among others. Phenolic compounds can be considered as high added-value by-products and the use of industrial wastes could greatly reduce their production cost and increase the margin profit of the products. We have previously reported the isolation of specific phenolic fractions, from red and white wine wastes, using solvent extractions and sorption/desorption techniques, possessing 92–95% of the estimated activities (antioxidant, antiplatelet, anti-inflammatory etc.) of the initial crude extract. Specifically, the antioxidant activity was determined by measuring the inhibition of DPPH and found to be 82%, the anti-inflammatory activity was determined by measuring the inhibition of COX1 (99%) and COX2 (78%), and antiplatelet activity in vitro, with 98% inhibition of platelet aggregation induced by collagen. An attempt to encapsulate these phenolics in order to retain chemical stability and biological activities is presented here. Several polymers, such as alginate and chitosan and mixtures of them, were examined. A mixture of 2%, w/v, sodium alginate and 2% w/v chitosan was found to perform better, shown encapsulation efficiency up to 70% of the initial phenolic content. Release experiments of phenolic compounds with EtOH (70%), 0.1 M HCl and sodium citrate 0.2 M showed that in the first two cases, a higher than 50% of the total phenolic content was released in the first 6 hours. These data suggest that encapsulated phenolics may have several applications in food and pharmaceutical industries.
Polyvinyl alcohol (PVA)-degrading enzyme: Structure analysis, heterologous overexpression, and applications
http://dx.doi.org/10.1016/j.nbt.2016.06.795
O4-16
O4-14
Novel mutants of Gluconacetobacter with different cellulose synthesis ability
Safety and pathways of intestinal absorption for chitosan nanocarriers Xi Guang Chen Ocean University of China, China Nanocarriers are highly efficient vehicles to deliver drugs and bioactive ingredients in disease therapy. We have developed tens of chitosan derivatives, fabricated into nano-sized entities and studied their potential and bioactivities as drug delivery carriers. To
Guocheng Du Jiangnan University, China Polyvinyl alcohol (PVA), with its many desirable physical and chemical characteristics, is widely used in textile sizing, adhesives, fiber coating, and et al. However, this extensive usage, particularly in the textile industry, has resulted in large amounts of PVA being discharged in the wastewater, causing environmental pollution due to the difficulty in degrading PVA. In contrast, PVA degradation using microbial enzymes is an environmentally friendly approach that can save energy and reduce the problems caused by the treatment of sewage from the textile desizing stage. Microbial degradation of PVA entails a two-step metabolism. In the first step two neighboring alcohols are oxidized to form a diketone structure in the polymer by PVA oxidase or PVA dehydrogenase. In the second step oxidized PVA (OPA) is hydrolyzed by OPA hydrolase (OPH, also known as ß-diketone hydrolase, BDH). In this report, we first introduce the structural analysis of OPA, and then address the expression and production of OPA and OPH, and finally introduce the application of OPA and OPH in the PVA degradation. http://dx.doi.org/10.1016/j.nbt.2016.06.797
∗ , Anna Cielecka, Małgorzata ˛ Marzena Jedrzejczak-Krzepkowska Ryngajłło, Katarzyna Kubiak, Stanisław Bielecki
Lodz University of Technology, Poland Bacterial NanoCellulose (BNC) is an exopolysaccharide synthesized by various species of bacteria, such as the genus Gluconacetobacter, Agrobacterium, Rhizobium and Sarcina. The Gluconacetobacter species are known to be one of the most efficient BNC producers. This polymer protects the cells from harmful environmental factors (UV light, desiccation). BNC has unique prop-