Accepted Manuscript Title: Bacterial polyhydroxybutyrate for electrospun fiber production Authors: Francisca Acevedo, Pamela Villegas, Viviana Urtuvia, Jeyson Hermosilla, Rodrigo Navia, Michael Seeger PII: DOI: Reference:
S0141-8130(17)31411-3 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.08.066 BIOMAC 8058
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
20-4-2017 18-7-2017 10-8-2017
Please cite this article as: Francisca Acevedo, Pamela Villegas, Viviana Urtuvia, Jeyson Hermosilla, Rodrigo Navia, Michael Seeger, Bacterial polyhydroxybutyrate for electrospun fiber production, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bacterial polyhydroxybutyrate for electrospun fiber production Francisca Acevedo a,b#, Pamela Villegasc, Viviana Urtuviac, Jeyson Hermosillaa, Rodrigo Naviaa,d, Michael Seegerc a
Scientific and Technological Bioresource Nucleus, BIOREN, Universidad de La Frontera, Casilla 54-D, Temuco, Chile
b
Department of Basic Sciences, Faculty of Medicine, Universidad de La Frontera, Casilla 54-D, Temuco, Chile c
Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química & Centro de Biotecnología (CBDAL), Universidad Técnica Federico Santa María, Valparaíso, Chile
d
Department of Chemical Engineering, Faculty of Engineering and Sciences & Centre for Biotechnology and Bioengineering (CeBiB), Universidad de La Frontera, Casilla 54-D, Temuco, Chile
#Corresponding author: F. Acevedo. Tel. +56 45 2596711 Fax: +56 45 2732402. E-mail: [email protected]
Abstract Nano- and microfibers obtained by electrospinning have attracted great attention due to its versatility
and
potential
for
applications
in
diverse
technological
fields.
Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by microorganisms such as the bacterium Burkholderia xenovorans LB400. In particular, LB400 cells are capable to synthesize poly(3-hydroxybutyrate) (PHB) from glucose.
The aim of this study was to produce and characterize electrospun fibers obtained from bacterial PHBs. Bacterial strain LB400 was grown in M9 minimal medium using xylose and mannitol (10 g L-1) as the sole carbon sources and NH4Cl (1 g L-1) as the sole nitrogen source. Biopolymer-based films obtained were used to produce fibers by electrospinning. Diameter and morphology of the microfibers were analyzed by scanning electron microscopy (SEM) and their thermogravimetric properties were investigated. Bead-free fibers using both PHBs were obtained with diameters of less than 3 µm. The surface morphology of the microfibers based on PHBs obtained from both carbon sources was different, even though their thermogravimetric properties are similar. The results indicate that the carbon source may determine the fiber structure and properties. Further studies should be performed to analyze the physicochemical and mechanical properties of these PHB-based microfibers, which may open up novel applications.
Keywords: Polyhydroxyalkanoate; electrospinning; electrospun fiber
1. Introduction Electrospinning has garnered attention in recent years due to its potential for applications in various fields. Electrospun fibers have shown great applicability for novel materials in tissue engineering, wound healing, bioactive molecules delivery as well as sensor, filtration, composite reinforcement and nanoelectronic applications [1-4]. Electrospinning consists of a syringe through which a polymer solution is pumped, a high voltage source and a collector. The pendant drop of polymer solution held by surface tension forces at the tip of the syringe is electrified upon application of high voltage. As a result, an electrostatic repulsion is established between like charges within the polymer solution,
resulting in Coulomb forces due to the external field [2]. When the strength of the electric field exceeds a threshold value that overcomes the surface tension of the polymer solution, a jet is produced from the pendant drop [2]. The jet undergoes stretching and whipping while traveling toward the collector. The solvent evaporates during this process and then a solid non-woven fibrous matrix is deposited on the collector [2]. A number of diverse polymeric materials have been electrospun, including synthetic and natural polymers. Among natural polymers, polyhydroxyalkanaotes (PHAs) have useful properties such as biodegradability, thermoplasticity, biocompatibility and non-toxicity [5]. PHAs are promising materials in biomedical and agricultural applications, among others. Li et al. (2008) [6] have described PHA nanofiber matrices as combining the advantages of biodegradation, improved mechanical strengths and the nanostructure of a natural extracellular matrix, leading to better compatibility. PHAs are polyesters synthesized by microorganisms under carbon excess and a limited concentration of essential nutrients such as nitrogen or phosphate [7]. The structural composition of PHAs depends on the carbon compound supplied as the growth substrate and the bacterial strain [5,7,8]. Burkholderia xenovorans LB400 is a model bacterium to study the metabolism of aromatic compounds and the synthesis of biotechnological products [9,10]. Recently, we reported that LB400 cells grown on glucose are able to synthesize the bioplastic poly(3-hydroxybutyrate) (PHB) [11]. Staining of LB400 cells grown on mannitol with Sudan Black B suggests the synthesis of PHAs [11]. Strain LB400 possesses the key enzyme for polyhydroxyalkanote synthesis: PhaC polymerase class I. Strain LB400 can grow on xylose and mannitol as the sole carbon sources. Under these conditions, the synthesis of PHAs by strain LB400 is expected. Therefore, the aim of this study was to produce and characterize electrospun fibers based on bacterial PHBs synthesized by strain LB400 from xylose and mannitol.
2. Materials and Methods
2.1. Bacterial growth and PHB extraction
Burkholderia xenovorans strain LB400 was grown in M9 minimal medium [9,10] using xylose and mannitol (10 g L-1) as the sole carbon sources and 1 g L-1 NH4Cl as the sole nitrogen source at 30°C. LB400 cells grown until stationary phase were harvested, centrifuged and freeze-dried. PHB was extracted with chloroform at 30°C. The solvent was evaporated to a small volume with a rotary evaporator. Finally, the solution was transferred to a Petri dish and dried at room temperature to obtain a PHB film.
2.2. PHB fiber production by electrospinning
2.2.1. PHB solutions
The dissolving solutions of the PHB film were prepared by dissolving 0.5 g PHB in 10 mL chloroform using an ultrasound bath at 50°C for 24 h.
2.2.2. Electrospinning procedure
The electrospinning apparatus (NEU-BM, China) was equipped with a high-voltage power supply with a maximum voltage of 50 kV. The flow rate of the biopolymer solution was controlled by a precision pump to maintain a steady flow from the capillary outlet. The
electrospinning experiments were carried out under ambient conditions (25°C and 50% relative humidity). Preliminary assays were performed to find the conditions for obtaining bead-free fibers. For each sample the negative and positive applied voltage was adjusted at 2 kV and 25 kV, respectively. The flow rate of the biopolymer solution was fixed at 0.5 mL/h and the rotor speed was 25 rpm. The fibers were collected on aluminum foil at a distance of 25 cm.
2.3. Electrospun fiber characterization
2.3.1. Morphology
Fiber diameter (Yd) and morphology index (Ym) were determined by micrographs obtained by SEM (scanning electron microscopy). A variable pressure scanning electron STEM SU3500 microscope (Hitachi, Tokyo, Japan) was used; hence, metal coating on the samples was not required. Three images were used for each fiber sample and at least 50 different segments were randomly measured to obtain an average diameter. For the quantification of the response morphology (Y m), a qualitative valuation from the fiber characteristics was performed as previously described by Rodrigo et al. (2012) [12]. This valuation made it possible to determine a quantitative value for this response. Thus, some qualitative features related to the defective fibers were established and measured to obtain an index. The first step is to assign a value to the presence of beads, agglomeration and sphere in the fibers as a negative feature. Micrographs were taken by SEM from each experiment and divided into 20 portions. If at least one particle in a portion displayed one of the above-mentioned defects, one point (1) was assigned; otherwise, zero points (0) were
assigned. To calculate the morphological index (Ym), Eq. 1 was used. With this protocol, when the defects are not very prominent the index is closer to a unit.
𝑌𝑚 = 1 −
𝑁𝑏𝑒𝑎𝑑𝑠 + 𝑁𝑎𝑔𝑔𝑙𝑜𝑚𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠 + 𝑁𝑠𝑝ℎ𝑒𝑟𝑒𝑠 𝑁𝑓𝑒𝑎𝑡𝑢𝑟𝑒𝑠 × 𝑁𝑝𝑜𝑟𝑡𝑖𝑜𝑛𝑠
(Eq. 1)
where Ym represents the morphology index, Nbeads is the number of portions with the beads feature, Nagglomerations is the number of portions on the micrograph with presence of agglomeration and Nspheres correspond to the numbers of portions with spheres. N features is equal to 3 and Nportions is 20.
2.3.2. Thermal analyses of PHB fibers
Thermal properties of PHB fibers were determined using differential scanning calorimetry (DSC, STA 6000 Perkin Elmer, USA) in a nitrogen atmosphere (50 mL/min) at 20°C. Thermogravimetric analyses (TGA) were recorded from 20 to 600°C at a heating rate of 15°C/min. The measurements were carried out in triplicate. Poly[(R)-3-hydroxybutyric acid] from Sigma Aldrich was used as the control (molecular weight ~260 g/mol).
3. Results and Discussion
In this study, bead-free fibers based on both PHBs produced by strain LB400 from xylose and mannitol were obtained (Fig. 1a and b). The morphologies of the electrospun fibers are loopy, indicating that there is a limited electrostatic repulsion force acting along the fiber
during the spinning process. This is because in this case the electrospinning process parameters were not optimized and will be studied in future works. The fiber diameter (Yd) and morphology index (Ym) using PHB from xylose were around 2.6 µm and 0.9625, respectively. Using PHB from mannitol, the Yd was lower, i.e., 2.2 µm, and Ym was 0.9875. Most of the diameter frequencies were in the range between 2 and 4 µm for both PHB types (Fig. 2a and b). As shown in Fig.1, the surface morphology of the two microfibers is different even though they were produced from a similar polymer, suggesting that the different carbon sources may determine the microfiber properties and structure. However, other parameters should also be studied. In fact, previous studies have report that PHB percentage used in the preparation of the solution subjected to electrospinning results in different microfiber morphology [13]. It has also been reported that different polymer crystallinity produces different microfibers [13]. PHB crystallinity depends on various factors, including how the polymers are dried. Therefore, different microfiber structures may be obtained starting from the same polymer composition obtained from different cultures due to changing drying parameters.
TGA profiles and first-derivative curves (differential thermogravimetric analyses, DTG) for PHB fibers produced using xylose and mannitol as the carbon source, respectively, were compared with commercial PHB (> 98% purity), as shown in Fig. 3a. Two main degradation steps were observed with PHB fibers from xylose. A lower weight loss, corresponding to the moisture present in the sample, was approximately 2.9% from 72°C to 100°C. Then, a second decomposition step was produced showing a high mass loss between 284 and 566°C, and the remaining mass was 1.1%. The point at which 50% of the material was decomposed (TD50%)
was about 297°C. PHB fibers produced from mannitol also showed two decomposition steps. A lower weight loss, attributed to the moisture in the sample, was approximately 6.5% from 70°C to 99°C. The second decomposition step with an important mass weight loss occurred between 92°C and 551°C, and the remaining mass was 0.3%. TD50% was approximately 298°C. Commercial PHB showed a high mass loss between 286°C and 321°C, and the remaining mass was 0%. TD50% was in this case approximately 293°C. In all cases, the second decomposition was attributed to the degradation of the abundant carbon groups of the PHB. Differential scanning calorimetric (DSC) curves of PHB fibers from xylose and mannitol and commercial PHB are shown in Fig. 3b. PHB fibers from xylose showed an endothermic peak at 172°C between 147 and 198°C and a ΔH = 29.111 J/g. This transition temperature corresponding to Tm is similar to the value reported by Sigma Aldrich for PHB. An endothermic peak of greater intensity was noted at 298ºC with a temperature range between 281 to 338ºC and a ΔH = 390.835 J/g. PHB fibers from mannitol showed two endothermic peaks of medium intensity, the first at 61°C, from 26 to 149°C, was attributed to the degradation of residues, impurities and solvent in the sample. The second endothermic peak was observed at 172°C between 159 and 194°C and a ΔH = 22.882 J/g. This transition temperature was similar to the value reported by Sigma Aldrich (172°C) for the pure PHB compound. A high intensity endothermic peak was observed at 300°C between 284 and 336°C and a ΔH = 360.291 J/g was obtained. For commercial PHB, a small endothermic peak was observed at 177°C ranging between 160°C and 217 °C with a ΔH = 62.273 J/g. Compared with the produced fibers, commercial PHB showed a higher intensity for the endothermic peak at 297°C from 287 to 341°C and a ΔH = 616.480 J/g was observed. This peak may correspond to the condensation of the polymer present in the three samples.
4. Conclusions
Electrospun microfibers were produced using biobased PHB synthesized by strain LB400 from the substrates xylose and mannitol. Microfiber physicochemical properties are dependent on the applied voltage and the input solution viscosity. In addition, PHB fiber properties, such as diameter and morphology index, seem to be dependent on the carbon substrate used for the PHB synthesis. Future studies will focus on the application of PHB microfibers in several technological fields.
Acknowledgments The authors acknowledge financial support from CONICYT through FONDECYT (11140127 & 1151174), USM (131342 & 131562), Red RIABIN and CNBS. The funders had no role in the study design, data collection or analyses, decision to publish or preparation of the manuscript.
References [1] W.G. Ciu, Y. Zhou, J. Chang, Electrospun nanofibrous materials for tissue engineering and drug delivery, Sci. Tech. Adv. Mater. 11 (2010) 1-11. [2] A.J. Meinel, O. Germershaus, T. Luhmann, H.P. Merkle, L. Meinel, Electrospun matrices for localized drug delivery: Current technologies. and selected biomedical applications, Eur. J. Pharm. Biopharm. 81 (2012) 1–13. [3] D.-G.Yu, L.-M. Zhu, K. White, C. Branford-White, Electrospun nanofiber-based drug delivery systems, Health. 1 (2009) 67-75.
[4] R. Rošic, J. Pelipenko, J. Kristl, P. Kocbek, S. Baumgartner, Properties, engineering and applications of polymeric nanofibers: Current research and future advances, Chem. Biochem. Eng. Q. 26 (2012) 417–425. [5] S. Ciesielski, J. Możejko, R. Kiewisz, Bacterial polyhydroxyalkanoates: Still fabulous?, Microbiol. Res. 192 (2016) 271–282. [6] X.-T. Li, Y. Zhang, G.-Q. Chen, Nanofibrous polyhydroxyalkanoate matrices as cell growth supporting materials. Biomaterials. 29 (2008) 3720–3728. [7] S. Ciesielski, J. Możejko, G. Przybylek, The influence of nitrogen limitation on mclPHA synthesis by two newly isolated strains of Pseudomonas sp., J. Microbiol. Biotechnol. 37 (2010) 511–520. [8] G.G.-Q. Chen, Natural Functions and Applications. In Plastics from Bacteria. In: Chen, G.G.-Q. (Ed), Springer-Verlag Berlin Heidelberg, Springer, Berlin, 2010.Vol. 11, pp. 450. [9] M.J. Vargas-Straube, B. Cámara, M. Tello, F. Montero-Silva, F. Cárdenas, M. Seeger, Genetic and functional analysis of the biosynthesis of a non-ribosomal peptide siderophore in Burkholderia xenovorans LB400, PloS ONE, 11(2016), e0151273. [10] M.J. Romero-Silva, V. Mendez, L. Agullo, M. Seeger, Genomic and functional analyses of the gentisate and protocatechuate ring-cleavage pathways and related 3-hydroxybenzoate and 4-hydroxybenzoate peripheral pathways in Burkholderia xenovorans LB400, PloS ONE. 8(2013), e56038. [11] V. Urtuvia, P. Villegas, M. González, M. Seeger, Bacterial production of the biodegradable plastics polyhydroxyalkanoates, Int. J. Biol. Macromol. 70 (2014) 208-213. [12] R. Rodrigo, C.A. Toro, J. Cuellar, Influence of the geometric factors of the experimental device used in suspension polymerization on the properties of poly(styrene-codivinylbenzene) microparticles, J. Appl. Polym. Sci. 124 (2012) 1431-1446.
[13] B. Mahaling, D.S. Katti, Fabrication of micro-structures of poly [(R)-3-hydroxybutyric acid] by electro-spraying/-spinning: understanding the influence of polymer concentration and solvent type, J. Mater. Sci. 49(2014) 4246-4260.
Figure captions a)
Fig. 1. SEM micrographs of electrospun microfibers produced from bacterial PHBs. a) PHB produced by strain LB400 from xylose and b) PHB produced by strain LB400 from mannitol.
Fiber Diameter (µm)
40
30
20
10
0
9-10
8-9
7-8
6-7
5-6
4-5
3-4
2-3
1-2
0-1
>11
50
>11
b) 10-11
Fiber Diameter (µm)
10-11
9-10
8-9
7-8
6-7
5-6
4-5
3-4
2-3
1-2
0-1
Frequency (%) Frequency (%)
a)
50
40
30
20
10
0
Fig. 2. Frequency (%) of the diameter range of electrospun microfibers based on bacterial PHBs produced from xylose and mannitol. a) PHB produced from xylose; b) PHB produced from mannitol.
b)
Fig. 3. TGA thermograms, derivative curves of TGA (DTG) and DSC curves of microfibers of PHB produced from xylose and mannitol. a) TGA thermograms, DTG (derivative curves of TGA) and b) DSC curves of nanofibers using xylose (green line), mannitol (blue line) and commercial PHB (red line).
S0141-8130(17)31411-3 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.08.066 BIOMAC 8058
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
20-4-2017 18-7-2017 10-8-2017
Please cite this article as: Francisca Acevedo, Pamela Villegas, Viviana Urtuvia, Jeyson Hermosilla, Rodrigo Navia, Michael Seeger, Bacterial polyhydroxybutyrate for electrospun fiber production, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bacterial polyhydroxybutyrate for electrospun fiber production Francisca Acevedo a,b#, Pamela Villegasc, Viviana Urtuviac, Jeyson Hermosillaa, Rodrigo Naviaa,d, Michael Seegerc a
Scientific and Technological Bioresource Nucleus, BIOREN, Universidad de La Frontera, Casilla 54-D, Temuco, Chile
b
Department of Basic Sciences, Faculty of Medicine, Universidad de La Frontera, Casilla 54-D, Temuco, Chile c
Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química & Centro de Biotecnología (CBDAL), Universidad Técnica Federico Santa María, Valparaíso, Chile
d
Department of Chemical Engineering, Faculty of Engineering and Sciences & Centre for Biotechnology and Bioengineering (CeBiB), Universidad de La Frontera, Casilla 54-D, Temuco, Chile
#Corresponding author: F. Acevedo. Tel. +56 45 2596711 Fax: +56 45 2732402. E-mail: [email protected]
Abstract Nano- and microfibers obtained by electrospinning have attracted great attention due to its versatility
and
potential
for
applications
in
diverse
technological
fields.
Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by microorganisms such as the bacterium Burkholderia xenovorans LB400. In particular, LB400 cells are capable to synthesize poly(3-hydroxybutyrate) (PHB) from glucose.
The aim of this study was to produce and characterize electrospun fibers obtained from bacterial PHBs. Bacterial strain LB400 was grown in M9 minimal medium using xylose and mannitol (10 g L-1) as the sole carbon sources and NH4Cl (1 g L-1) as the sole nitrogen source. Biopolymer-based films obtained were used to produce fibers by electrospinning. Diameter and morphology of the microfibers were analyzed by scanning electron microscopy (SEM) and their thermogravimetric properties were investigated. Bead-free fibers using both PHBs were obtained with diameters of less than 3 µm. The surface morphology of the microfibers based on PHBs obtained from both carbon sources was different, even though their thermogravimetric properties are similar. The results indicate that the carbon source may determine the fiber structure and properties. Further studies should be performed to analyze the physicochemical and mechanical properties of these PHB-based microfibers, which may open up novel applications.
Keywords: Polyhydroxyalkanoate; electrospinning; electrospun fiber
1. Introduction Electrospinning has garnered attention in recent years due to its potential for applications in various fields. Electrospun fibers have shown great applicability for novel materials in tissue engineering, wound healing, bioactive molecules delivery as well as sensor, filtration, composite reinforcement and nanoelectronic applications [1-4]. Electrospinning consists of a syringe through which a polymer solution is pumped, a high voltage source and a collector. The pendant drop of polymer solution held by surface tension forces at the tip of the syringe is electrified upon application of high voltage. As a result, an electrostatic repulsion is established between like charges within the polymer solution,
resulting in Coulomb forces due to the external field [2]. When the strength of the electric field exceeds a threshold value that overcomes the surface tension of the polymer solution, a jet is produced from the pendant drop [2]. The jet undergoes stretching and whipping while traveling toward the collector. The solvent evaporates during this process and then a solid non-woven fibrous matrix is deposited on the collector [2]. A number of diverse polymeric materials have been electrospun, including synthetic and natural polymers. Among natural polymers, polyhydroxyalkanaotes (PHAs) have useful properties such as biodegradability, thermoplasticity, biocompatibility and non-toxicity [5]. PHAs are promising materials in biomedical and agricultural applications, among others. Li et al. (2008) [6] have described PHA nanofiber matrices as combining the advantages of biodegradation, improved mechanical strengths and the nanostructure of a natural extracellular matrix, leading to better compatibility. PHAs are polyesters synthesized by microorganisms under carbon excess and a limited concentration of essential nutrients such as nitrogen or phosphate [7]. The structural composition of PHAs depends on the carbon compound supplied as the growth substrate and the bacterial strain [5,7,8]. Burkholderia xenovorans LB400 is a model bacterium to study the metabolism of aromatic compounds and the synthesis of biotechnological products [9,10]. Recently, we reported that LB400 cells grown on glucose are able to synthesize the bioplastic poly(3-hydroxybutyrate) (PHB) [11]. Staining of LB400 cells grown on mannitol with Sudan Black B suggests the synthesis of PHAs [11]. Strain LB400 possesses the key enzyme for polyhydroxyalkanote synthesis: PhaC polymerase class I. Strain LB400 can grow on xylose and mannitol as the sole carbon sources. Under these conditions, the synthesis of PHAs by strain LB400 is expected. Therefore, the aim of this study was to produce and characterize electrospun fibers based on bacterial PHBs synthesized by strain LB400 from xylose and mannitol.
2. Materials and Methods
2.1. Bacterial growth and PHB extraction
Burkholderia xenovorans strain LB400 was grown in M9 minimal medium [9,10] using xylose and mannitol (10 g L-1) as the sole carbon sources and 1 g L-1 NH4Cl as the sole nitrogen source at 30°C. LB400 cells grown until stationary phase were harvested, centrifuged and freeze-dried. PHB was extracted with chloroform at 30°C. The solvent was evaporated to a small volume with a rotary evaporator. Finally, the solution was transferred to a Petri dish and dried at room temperature to obtain a PHB film.
2.2. PHB fiber production by electrospinning
2.2.1. PHB solutions
The dissolving solutions of the PHB film were prepared by dissolving 0.5 g PHB in 10 mL chloroform using an ultrasound bath at 50°C for 24 h.
2.2.2. Electrospinning procedure
The electrospinning apparatus (NEU-BM, China) was equipped with a high-voltage power supply with a maximum voltage of 50 kV. The flow rate of the biopolymer solution was controlled by a precision pump to maintain a steady flow from the capillary outlet. The
electrospinning experiments were carried out under ambient conditions (25°C and 50% relative humidity). Preliminary assays were performed to find the conditions for obtaining bead-free fibers. For each sample the negative and positive applied voltage was adjusted at 2 kV and 25 kV, respectively. The flow rate of the biopolymer solution was fixed at 0.5 mL/h and the rotor speed was 25 rpm. The fibers were collected on aluminum foil at a distance of 25 cm.
2.3. Electrospun fiber characterization
2.3.1. Morphology
Fiber diameter (Yd) and morphology index (Ym) were determined by micrographs obtained by SEM (scanning electron microscopy). A variable pressure scanning electron STEM SU3500 microscope (Hitachi, Tokyo, Japan) was used; hence, metal coating on the samples was not required. Three images were used for each fiber sample and at least 50 different segments were randomly measured to obtain an average diameter. For the quantification of the response morphology (Y m), a qualitative valuation from the fiber characteristics was performed as previously described by Rodrigo et al. (2012) [12]. This valuation made it possible to determine a quantitative value for this response. Thus, some qualitative features related to the defective fibers were established and measured to obtain an index. The first step is to assign a value to the presence of beads, agglomeration and sphere in the fibers as a negative feature. Micrographs were taken by SEM from each experiment and divided into 20 portions. If at least one particle in a portion displayed one of the above-mentioned defects, one point (1) was assigned; otherwise, zero points (0) were
assigned. To calculate the morphological index (Ym), Eq. 1 was used. With this protocol, when the defects are not very prominent the index is closer to a unit.
𝑌𝑚 = 1 −
𝑁𝑏𝑒𝑎𝑑𝑠 + 𝑁𝑎𝑔𝑔𝑙𝑜𝑚𝑒𝑟𝑎𝑡𝑖𝑜𝑛𝑠 + 𝑁𝑠𝑝ℎ𝑒𝑟𝑒𝑠 𝑁𝑓𝑒𝑎𝑡𝑢𝑟𝑒𝑠 × 𝑁𝑝𝑜𝑟𝑡𝑖𝑜𝑛𝑠
(Eq. 1)
where Ym represents the morphology index, Nbeads is the number of portions with the beads feature, Nagglomerations is the number of portions on the micrograph with presence of agglomeration and Nspheres correspond to the numbers of portions with spheres. N features is equal to 3 and Nportions is 20.
2.3.2. Thermal analyses of PHB fibers
Thermal properties of PHB fibers were determined using differential scanning calorimetry (DSC, STA 6000 Perkin Elmer, USA) in a nitrogen atmosphere (50 mL/min) at 20°C. Thermogravimetric analyses (TGA) were recorded from 20 to 600°C at a heating rate of 15°C/min. The measurements were carried out in triplicate. Poly[(R)-3-hydroxybutyric acid] from Sigma Aldrich was used as the control (molecular weight ~260 g/mol).
3. Results and Discussion
In this study, bead-free fibers based on both PHBs produced by strain LB400 from xylose and mannitol were obtained (Fig. 1a and b). The morphologies of the electrospun fibers are loopy, indicating that there is a limited electrostatic repulsion force acting along the fiber
during the spinning process. This is because in this case the electrospinning process parameters were not optimized and will be studied in future works. The fiber diameter (Yd) and morphology index (Ym) using PHB from xylose were around 2.6 µm and 0.9625, respectively. Using PHB from mannitol, the Yd was lower, i.e., 2.2 µm, and Ym was 0.9875. Most of the diameter frequencies were in the range between 2 and 4 µm for both PHB types (Fig. 2a and b). As shown in Fig.1, the surface morphology of the two microfibers is different even though they were produced from a similar polymer, suggesting that the different carbon sources may determine the microfiber properties and structure. However, other parameters should also be studied. In fact, previous studies have report that PHB percentage used in the preparation of the solution subjected to electrospinning results in different microfiber morphology [13]. It has also been reported that different polymer crystallinity produces different microfibers [13]. PHB crystallinity depends on various factors, including how the polymers are dried. Therefore, different microfiber structures may be obtained starting from the same polymer composition obtained from different cultures due to changing drying parameters.
TGA profiles and first-derivative curves (differential thermogravimetric analyses, DTG) for PHB fibers produced using xylose and mannitol as the carbon source, respectively, were compared with commercial PHB (> 98% purity), as shown in Fig. 3a. Two main degradation steps were observed with PHB fibers from xylose. A lower weight loss, corresponding to the moisture present in the sample, was approximately 2.9% from 72°C to 100°C. Then, a second decomposition step was produced showing a high mass loss between 284 and 566°C, and the remaining mass was 1.1%. The point at which 50% of the material was decomposed (TD50%)
was about 297°C. PHB fibers produced from mannitol also showed two decomposition steps. A lower weight loss, attributed to the moisture in the sample, was approximately 6.5% from 70°C to 99°C. The second decomposition step with an important mass weight loss occurred between 92°C and 551°C, and the remaining mass was 0.3%. TD50% was approximately 298°C. Commercial PHB showed a high mass loss between 286°C and 321°C, and the remaining mass was 0%. TD50% was in this case approximately 293°C. In all cases, the second decomposition was attributed to the degradation of the abundant carbon groups of the PHB. Differential scanning calorimetric (DSC) curves of PHB fibers from xylose and mannitol and commercial PHB are shown in Fig. 3b. PHB fibers from xylose showed an endothermic peak at 172°C between 147 and 198°C and a ΔH = 29.111 J/g. This transition temperature corresponding to Tm is similar to the value reported by Sigma Aldrich for PHB. An endothermic peak of greater intensity was noted at 298ºC with a temperature range between 281 to 338ºC and a ΔH = 390.835 J/g. PHB fibers from mannitol showed two endothermic peaks of medium intensity, the first at 61°C, from 26 to 149°C, was attributed to the degradation of residues, impurities and solvent in the sample. The second endothermic peak was observed at 172°C between 159 and 194°C and a ΔH = 22.882 J/g. This transition temperature was similar to the value reported by Sigma Aldrich (172°C) for the pure PHB compound. A high intensity endothermic peak was observed at 300°C between 284 and 336°C and a ΔH = 360.291 J/g was obtained. For commercial PHB, a small endothermic peak was observed at 177°C ranging between 160°C and 217 °C with a ΔH = 62.273 J/g. Compared with the produced fibers, commercial PHB showed a higher intensity for the endothermic peak at 297°C from 287 to 341°C and a ΔH = 616.480 J/g was observed. This peak may correspond to the condensation of the polymer present in the three samples.
4. Conclusions
Electrospun microfibers were produced using biobased PHB synthesized by strain LB400 from the substrates xylose and mannitol. Microfiber physicochemical properties are dependent on the applied voltage and the input solution viscosity. In addition, PHB fiber properties, such as diameter and morphology index, seem to be dependent on the carbon substrate used for the PHB synthesis. Future studies will focus on the application of PHB microfibers in several technological fields.
Acknowledgments The authors acknowledge financial support from CONICYT through FONDECYT (11140127 & 1151174), USM (131342 & 131562), Red RIABIN and CNBS. The funders had no role in the study design, data collection or analyses, decision to publish or preparation of the manuscript.
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Figure captions a)
Fig. 1. SEM micrographs of electrospun microfibers produced from bacterial PHBs. a) PHB produced by strain LB400 from xylose and b) PHB produced by strain LB400 from mannitol.
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Fig. 2. Frequency (%) of the diameter range of electrospun microfibers based on bacterial PHBs produced from xylose and mannitol. a) PHB produced from xylose; b) PHB produced from mannitol.
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Fig. 3. TGA thermograms, derivative curves of TGA (DTG) and DSC curves of microfibers of PHB produced from xylose and mannitol. a) TGA thermograms, DTG (derivative curves of TGA) and b) DSC curves of nanofibers using xylose (green line), mannitol (blue line) and commercial PHB (red line).
