Preparation of nanofibers containing the microalga Spirulina (Arthrospira)

m o ty o Mar ran Electrospinning ich duc sta 00 issu ibers and for PEO concentrations in the spinning solution well below 4 wt.%. These results suggest to us fers a u d rang rtified More important for our purposes, clinical studies suggest that compounds in the microalgae have therapeutic functions: polysac- charides with anti-inflammatory effects (Matsui et al., 2003), fatty acids with antibacterial and anti-fungal properties (Borowitzka, 1995). Furthermore Calcium Spirulan, is believed to inhibit pul- monary metastasis in humans and to prevent the adhesion and proliferation of tumor cells (Mishima et al., 1998). Other character- istics which have been attributed to Spirulina biomass, are blood it allows to mimic the architecture of the extra cellular matrix enclosing the cells in tissues and also mechanically supporting them as well as its mechanical properties closely. Nanofibers scaf- folds are generally prepared via electrospinning and they tend to be composed of synthetic biocompatible polymers, of natural ones or blends of synthetic and natural polymers. The scaffold, i.e. in particular the material from which it is com- posed has to facilitate anchorage, migration and proliferation of the cells, to provide the three dimensional structure model of the tissue and it has to fulfill a diverse range of requirements concern- ing biocompatibility, biodegradability, architecture, sterilizability, * Corresponding author. Tel.: +49 (0) 6421 28 25964; fax: +49 (0) 6421 28 28916. Bioresource Technology 101 (2010) 2872–2876 Contents lists availab T els E-mail address: [email protected] (J. Wendorff). Drug Administration) as GRAS (Generally Recognized As Safe). It can be used as a nutritive or pharmaceutical additive with no risk to health. Spirulina has been produced on a large scale to be used as a nutritive, as a source of energy and for pharmaceutical, biochem- ical and fertilizer products. Because they are nutritionally rich in proteins, essential fatty acids, vitamins and minerals, microalgae have been considered in the USA, Japan, India and France for the production of foodstuffs to combat malnutrition (Henrikson, 1994). In the south of Brazil, on the edge of the Mangueira Lagoon, a pilot plant produced 50 kg per month of Spirulina to enrich food- stuffs, which were distributed as snacks for children in the region. The use of scaffolds for tissue engineering appears to be a good strategy in the treatment of tissue and organ lesions, such as spinal cord injury, skin burns, other lesions and also bone regeneration. The broad set of biological functions introduced above suggests that one might use Spirulina biomass with great success also in this particular rapidly growing area of Life Science. Tissue engineering aims to use cells cultivated in scaffolds to replace corresponding tissues destroyed by accident or illness in the human body. One ap- proach in the field of tissue engineering is the use of scaffolds onto which cells or human body cells respectively can be settled. Scaf- folds based on nanofiber nonwovens are highly promising because Microalgae Nanofibers Polymer Spirulina 1. Introduction Spirulina is a microalga which of functions highly favorable for a broa Science. It has, for instance, been ce 0960-8524/$ - see front matter � 2009 Elsevier Ltd. A doi:10.1016/j.biortech.2009.11.059 the use of the biomass containing nanofibers as extracellular matrices for stem cell culture and future treatment of spinal chord injury. � 2009 Elsevier Ltd. All rights reserved. nique set of biological e of applications in Life by the FDA (Food and cholesterol reduction, partial inactivation of HIV-1 replication in human cells (Hirahashi et al., 2002), stimulation of the immuno- logical system and intestinal lactobacillus, reduction of hyperlipid- emia, obesity, and negative effects of radiation, drugs and heavy metals (Costa et al., 2004; Jiménez et al., 2003). Keywords: system PEO/biomass is behaved surprisingly well in electrospinning. Very thin bead-free nanofibers with diameters of about 110 nm can be produced for different biomass contents of up to 67 wt.% of the nanof- Preparation of nanofibers containing the Michele Greque de Morais a, Christopher Stillings b, R Jorge Alberto Vieira Costa a, Joachim Wendorff b,* a Laboratory of Biochemical Engineering, School of Chemistry and Food, Federal Universi bDepartment of Chemistry, Philipps-Universität Marburg, Hans Meerwein Str., D-35032 cHematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio G a r t i c l e i n f o Article history: Received 28 September 2009 Received in revised form 10 November 2009 Accepted 13 November 2009 Available online 28 December 2009 a b s t r a c t Spirulina is a microalga wh porous scaffolds can be pro tribution was therefore to e diameters down to about 1 for subsequent studies in t Bioresource journal homepage: www. ll rights reserved. icroalga Spirulina (Arthrospira) land Dersch b, Markus Rudisile b, Patrícia Pranke c, f Rio Grande, P.O. Box 474, 96201-900, Rio Grande, RS, Brazil burg, Germany de do Sul, 90610-000, Porto Alegre, RS, Brazil offers biological functions highly favorable for tissue engineering. Highly ed by electrospinning containing biomass of Spirulina. The goal of this con- blish spinning conditions allowing to produce well defined nanofibers with nm and to produce nanofibers with various concentration of the biomass e engineering applications. The experimental results reveal that the blend le at ScienceDirect echnology evier .com/locate /bior tech porosity, incorporation and release of drugs, mechanical properties etc. Requirements to be met by the nanofiber scaffold are thus highly demanding, are highly multifunctional. They cannot really be met by synthetic polymers but possibly by a multifunctional material such as Spirulina biomass. A problem is that Spirulina bio- mass by itself cannot be electrospun to well defined nanofibers. So the concept to be evaluated in this paper is to blend it with a water soluble synthetic polymer i.e. polyethylene oxide (O–CH2–CH2)n known to yield good nanofibers in electrospinning. PEO is a low cost, biodegradable and biocompatible polymer, which has already been used in biomedical applications, such as the development of wound dressings. The goal of this contribution is to establish spin- ning conditions allowing to produce well defined nanofibers with diameters down to about 100 nm and to produce nanofibers with various concentration of the biomass for subsequent studies in tis- sue engineering applications. 2. Methods The morphology of nanofibers was investigated with a digital optical microscope (Keyence VH-Z500, Japan) and a scanning elec- Table 1 Concentration of polyethylene oxide (XP, w/v), concentration of Spirulina LEB 18 (XS, w/v), final concentration of solution (XF, w/v), concentration of Spirulina LEB 18 as a function of total concentration of solution (XST) and conductivity responses (C) and viscosity (g) of the solutions used to produce nanofibers. Sample XP (%) XS (%) XF (%) XST (%) C (mS cm�1) g (Pa s) SP1 4.0 1.3 5.3 24.5 0.0012 0.44 SP2 4.0 2.0 6.0 33.3 0.0016 0.43 SP3 4.0 4.0 8.0 50.0 2.82 0.48 SP4 4.0 8.0 12.0 66.6 4.71 0.60 SP5 4.0 12.0 16.0 75.0 6.26 0.92 SP6 3.8 0.2 4.0 5.0 0.0002 0.40 SP7 3.6 0.4 4.0 10.0 0.0004 0.40 SP8 3.4 0.6 4.0 15.0 0.0005 0.40 SP9 3.0 1.0 4.0 25.0 0.0009 0.38 SP10 2.6 1.4 4.0 35.0 0.0012 0.37 SP11 2.2 1.8 4.0 45.0 0.0016 0.33 SP12 1.8 2.2 4.0 55.0 0.0019 0.20 Standard 4.0 – 4.0 - 0.00007 0.40 M.G. de Morais et al. / Bioresource Technology 101 (2010) 2872–2876 2873 2.1. The microorganism and biomass pre-treatment The microalga used in this study was of the Spirulina LEB 18 (Arthrospira LEB 18) (Fig. 1) type isolated from the Mangueira La- goon in the south of Brazil, located between the Atlantic Ocean and Mirim Lagoon (between the latitudes 32�3200500S and 33�3105700S) (Morais et al., 2008). This microalga was cultivated in a pilot plant located on the edges of the Mangueira Lagoon in three raceway type bioreactors each with a 10,000 L capacity. Microalga biomass was collected with a 200 lm filter, concen- trated in a hydraulic press and subsequently extruded. After extru- sion, it was dried at 50 �C for 4 h in a tray dryer, ground in a ball grinder, vacuum packed and stored. The final biomass has the fol- lowing composition: 6.7% ash, 0.19% lipids, 5.3% moisture and 86% protein. 2.2. Preparation of the solutions for electrospinning Solutions of Spirulina LEB 18 biomass and polyethylene oxide (PEO) (900,000 g mol�1) were prepared at different concentrations and relative ratios (see Table 1) using water as a solvent. The sam- ples were homogenized in a magnetic agitator overnight at 21 �C. The PEO concentration of samples SP1 to SP5 was kept constant at 4.0 wt.% and the biomass concentration was increased. In sam- ples SP6 to SP12, the PEO concentration in the solutions was Fig. 1. Microalga Spirulina LEB 18. substituted by the biomass at constant overall concentration. The conductivities of the spinning solutions as well as the viscosities both being key parameters in electrospinning, are given in Table 1. It is apparent that the conductivities vary by several orders of mag- nitude whereas the viscosities stay within the same order of mag- nitude. Electrospinning was also carried for solutions containing just PEO to be used as a standard. 2.3. Electrospinning process In the electrospinning process, the solutions were pumped through needle shaped dies with an inner diameter of 0.45 up to 0.70 mm. The needle tip was selective as the positive electrode with the aluminum collector (Fig. 2) acting as the counter elec- trode. The distances between the needle tip and the collector were varied between 14.0 and 20.0 cm and the electrical potential from 6.3 to 31.3 kV. The flow rate of solution controlled by a pump at- tached to the needle varied between 0.7 and 3.5 lL min�1. The best conditions in terms of reproducibility and homogeneity of fiber formation turned out from our experiments to be a voltage of 24.3 kV, 2.5 lL min�1 flow rate, 15 cm distance from the needle tip to the collector and needle diameter of 0.45 mm. 2.4. Characterization of the nanofibers Fig. 2. Experimental apparatus used in electrospinning process. of biomass leads to a strong increase of the conductivity. This is obvious from the results for samples SP1 to SP5, in which the good approximation controlled just by the biomass concentration. Secondly the conductivity increases very strongly with the biomass So the general result is that the viscosities of all composite solu- tions to be electrospun are close to each other, i.e. within the same order of magnitude. 3.3. Fiber formation as function of the composition of the spinning solution Electrospinning of PEO solutions containing 4 wt.% of the poly- mer gives rise to well defined nanofibers with an average diameter of about 110 nm (108 ± 17 nm) as demonstrated in the literature. The topic to be discussed in the following is to what extent bio- mass can be introduced into the solution at constant or varying PEO concentration without disrupting nanofibers formation, with- out the formation of beaded fibers particularly in view of the fact that the both the viscosity and more strongly the conductivity of the solutions vary with composition. The general result to be discussed below inmore detail is that all compositions displayed in Table 1 with the exception of sample SP5 behave well in electrospinning yielding nanofibers. These appear green to the naked eye indicative of the incorporation of the bio- mass. Fig. 4 displays the optical images for electrospun fibers from the solutions SP2 and SP7 as examples, similar resultswere obtained Tec concentration at lower concentrations yet levels off at higher con- centration. Two reasons for this behavior come to the mind. First of all, one may assume that the solubility of the biomass is limited yet a close inspection of the solutions do not give any indication of phase separation. Secondly the biomass may have characteristics of a weak electrolyte where the degree of dissociation is strongly reduced as its concentration increases. In fact the highly complex composition of the biomass (see above) makes a more detailed analysis more than difficult. In any case the results discussed so PEO concentration was kept at 4.0% whereas the one of the bio- mass to Spirulina LEB 18 was increased from 1.3% to 12.0%. The conductivity ranged from 0.0012 mS cm�1 in the sample contain- ing 1.3% microalgal biomass up to 6.26 mS cm�1 in the solution with 12.0% biomass. A corresponding behavior was observed in samples SP6 to SP12, where the polymer was gradually replaced by the microalgal bio- mass and the total concentration of the solution was kept at 4.0%. The lowest conductivity was 0.0002 mS cm�1 in the 3.8 wt.% PEO solution (0.2 wt.% microalgal biomass); and the larg- est one amounted to 0.0019 mS cm�1 in the 1.8 wt.% PEO solution (2.2 wt.% biomass). Fig. 3 demonstrates the dependence of the conductivity on the concentration of the biomass for various concentrations of PEO in the solutions. Fig. 3 clearly reveals two facts. First of all the conductivity is to a tron microscope (SEM) (Jeol JSM-7500F, Germany). The mean diameter of the nanofibers was determined by measuring 30 different points, using SEM images. The diameter of the nanofibers was assessed by analysis of variance (ANOVA) and Tukey’s test to compare the means of the diameter at a significance level of 95%. The apparent viscosity and conductivity of solutions were mea- sured with a digital viscometer (Haake PK100, Germany) and dig- ital conductivity meter (Inolab, Germany), respectively. 3. Results and discussion Fiber formation by electrospinning is known to depend on a broad range of internal (solution properties) and external (electri- cal set-up) parameters the electric conductivity of the spinning solution and the viscosity being a major internal parameter. The conductivity will control, for instance, the presence or absence of bead formation. The dependence of both the viscosity and the con- ductivity on the composition of the spinning solutions will there- fore be discussed first. The expectation is that the conductivity will depend strongly on the composition since the biomass will to a certain extent yet contain salts of the cultivation medium. The medium used for cultivation of Spirulina contains: NaHCO3: 16.8 g L�1; NaNO3: 2.5 g L�1; K2HPO4: 0.5 g L�1; K2SO4: 1.0 g L�1; NaCl: 1.0 g L�1; MgSO4�7H2O: 0.2 g L�1; CaCl2: 0.04 g L�1; FeS- O4�7H2O: 0.01 g L�1; EDTA: 0.08 g L�1. 3.1. Dependence of conductivity on the composition of the spinning solutions The solution containing just 4 wt.% of PEO is characterized by a conductivity of 7 � 10�5 mS cm�1. Table 1 reveals that the addition 2874 M.G. de Morais et al. / Bioresource far show that the conductivities of the solution to be electrospun differ by up to five orders of magnitude which might influence the electrospinning process significantly. 3.2. Dependence of the viscosity on the composition of the spinning solutions Solutions containing 4 wt.% PEO display a viscosity of 0.40 Pa s. The viscosities in solutions with just 1.0, 10.0 and 20.0 wt.% bio- mass on the other hand amounted to 0.38, 0.44 and 0.57 Pa s, respectively. These results and the ones displayed in Table 1 on the dependence of the viscosity on the composition of the blends reveal first of all that the viscosities of all solutions differ not more than by about a factor of two. It furthermore is obvious that the PEO concentration enhances the viscosity more strongly than the biomass concentration at similar concentrations. A reduction of the PEO concentration while simultaneously increasing the bio- mass concentration causes the viscosity to decrease (samples SP6 to SP11) slightly. On the other hand one observes that the addition of biomass to a 4 wt.% PEO solution causes a weak approximately linear increase of the viscosity from 0.40 to 0.92 Pa s as the bio- mass concentration grows strongly from 1.3 wt.% up to 12 wt.%. 0.0 2.0 4.0 6.0 8.0 10.0 12.0 0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 Co nd uc tiv ity (m S/ cm ) Biomass Concentration (%) Fig. 3. Dependence of the conductivity on the concentration of the biomass for various concentrations of PEO in the solutions. hnology 101 (2010) 2872–2876 for instance for samples SP6, SP8. The concentration of biomass relative to PEO amounts to 33.3 wt.% for SP2 (PEO concentration in solution 4 wt.%) and to 10 wt.% for SP7 (PEO concentration in The results reported here resemble in many instances those ob- tained for nanofibers composed of sodium alginate. Safi et al. entr e Tec solution 3.6 wt.%). The fibers are homogeneous in diameter, no bead formation is found and the fiber diameters are definitely well below 0.5 lm. A more exact determination of the diameter cannot be achieved by optical microscopy but rather by SEM analysis. The samples SP3 and SP4, which have high microalgal biomass concentrations of 50 and 66.6 wt.%, and therefore strongly in- creased conductivities (2.82 and 4.71 mS cm�1, respectively) at slightly enhanced viscosities (0.48 and 0.60 Pa s). It is obvious that fibers free of beads with only slight undulations are produced with the nanofibers diameters amounting to 107 ± 13 and 110 ± 10 nm diameters. So the values are very close to the ones obtained for pure PEO solutions of approximately the same polymer concentration. Similar results were obtained for the samples SP9 and SP10, in which PEO was substituted by 25.0% and 35.0% biomass (Table 1). Nanofibers resulted which had diameters of 105 ± 18 nm and 107 ± 12 nm. The samples SP11 (1.8% PEO and 1.8% microalgal bio- mass) and SP12 (1.8% PEO and 2.2% microalgal biomass) having lower viscosities (0.20 and 0.33 Pa s, respectively) could be spun to fibers yet these showed beads. An interesting finding is that sample SP5 could not be electrospun to fibers since it was impos- Fig. 4. Optical images of fibers electrospun from SP2 (33.3 wt.% biomass, PEO conc solution 3.6 wt.%) (b). M.G. de Morais et al. / Bioresourc sible to pump the solution through the die. SP5 has indeed the highest viscosity as obvious from Table 1. Yet polymer solutions displaying viscosities well above this magnitude have been electro- spun, polyamide solution in formic acid being a good example. The viscosity was controlled in such cases by highly flexible polymer chains whereas in the case considered here the viscosity is con- trolled by a complex bio-based system with unknown internal de- grees of freedom and thus not easy to model. The aim of the investigations reported in this contribution was to find out whether nanofibers can be prepared by electrospinning containing a high concentration of the biomass of the microalgae Spirulina in view of its unique biological functions. The concept was to blend the biomass for this purpose with a synthetic poly- mer, with PEO in this case and to establish spinning solution parameters allowing to produce nanofibers free of beads. Very thin bead-free nanofibers with diameters of about 110 nm can be pro- duced for different biomass contents of up to 67 wt.% of the nanofi- bers and for PEO concentrations in the spinning solution well below 4 wt.%. The fiber diameter and morphology which result seem to be insensitive to the strong variation of the electric con- ductivity of the spinning solution varying by a factor of 105. It seems feasible that the biomass content in the fibers can even be larger than 67%, however the spinning die becomes jammed for biomass concentrations in the spinning solution surpassing about (2006) reported, for instance, those solutions with sodium alginate and PEO at ratios of 50:50 and 30:70, formed uniform fibers, with- out beads with diameters of 99.1 and 109.0 nm, respectively. Lu et al. (2006), obtained fibers with diameters of 117 and 228 nm by electrospinning with solutions of sodium alginate and PEO in a 2:1 and 1:1 ratio, respectively, (which corresponds to the sam- ples SP3 and SP4 in this study). 4. Conclusions Our studies discussed above and the ones reported in the liter- ature suggest that electrospinning is a very favorable approach to- wards the production of nanofibers scaffolds composed of very thin 10 wt.%. The high porosity of the nonwovens composed of such nanofibers which are obvious from Fig. 4 suggests that the compos- ite nanofibers system may offer vary favorable properties as scaf- fold in tissue engineering. Future studies will consider just this topic. ation in solution 4 wt.%) (a) and from SP7 (biomass 10 wt.%, PEO concentration in hnology 101 (2010) 2872–2876 2875 fibers containing high concentrations of bioactive biomass. The experimental results reveal that the blend system PEO/biomass is surprisingly well behaved in electrospinning. Acknowledgements The authors thank the CAPES/Probral (Coordenação de Aper- feiçoamento de Nível superior/Programa Brasil-Alemanha) for financially supporting this work. References Borowitzka, M.A., 1995. Microalgae as source of pharmaceuticals and other biologically active compounds. J. Appl. Physiol. 7, 3–15. Costa, J.A.V., Colla, L.M., Filho, P.F.D., 2004. Improving Spirulina platensis biomass yield using a fed-batch process. Bioresour. Technol. 92, 237–241. Henrikson, R., 1994. Barcelona: Ediciones S.A. Urano, Microalga Spirulina: Superalimento del futuro, ISBN: 84-7953-047-2. Hirahashi, T., Matsumoto, M., Hazeki, K., Saeki, Y., Ui, M., Seya, T., 2002. Activation of the human innate immune system by Spirulina: augmentation of interferon and NK cytotoxicity by oral administration of hot water extract of Spirulina platensis. Int. Immunopharmacol. 2, 423–434. Jiménez, C., Cossío, B.R., Niell, F.X., 2003. The feasibility of industrial production of Spirulina (Arthrospira) in Southern Spain. Aquaculture 221, 331–345. Lu, W.J., Zhu, Y.L., Guo, Z.X., Hu, P., Yu, J., 2006. Electrospinning of sodium alginate with poly(ethylene oxide). Polymer 47, 8026–8031. Matsui, M.S., Muizzuddin, N., Arad, S., Marenus, K., 2003. Sulfated polysaccharides from red microalgae have anti-inflammatory properties in vitro and in vivo. Appl. Biochem. Biotechnol. 104, 13–22. Mishima, T., Murata, J., Toyoshima, M., Fuji, H., Nakajima, M., Hayashi, T., Kato, T., Saiki, I., 1998. Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue-green alga Spirulina platensis. Clin. Exp. Metastas. 16, 541–550. Morais, M.G., Reichert, C.C., Dalcanton, F., Durante, A.J., Marins, L.F., Costa, J.A.V., 2008. Isolation and characterization of a new Arthrospira strain. Z. Naturforsch. 63c, 144–150. Safi, S., Morshed, M., Ravandi, S.A.H., Ghiaci, M.J., 2006. Study of electrospinning of sodium alginate, blended solutions of sodium alginate/ poly(vinyl alcohol) and sodium alginate/poly(ethylene oxide). Appl. Polym. Sci. 104, 3245–3255. 2876 M.G. de Morais et al. / Bioresource Technology 101 (2010) 2872–2876 Preparation of nanofibers containing the microalga Spirulina (Arthrospira) Introduction Methods The microorganism and biomass pre-treatment Preparation of the solutions for electrospinning Electrospinning process Characterization of the nanofibers Results and discussion Dependence of conductivity on the composition of the spinning solutions Dependence of the viscosity on the composition of the spinning solutions Fiber formation as function of the composition of the spinning solution Conclusions Acknowledgements References