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Gelation Behavior by the Lanthanoid Adsorption of the Cyanobacterial Extracellular Polysaccharide Maiko K. Okajima,† Toshimitsu Higashi,† Ryuya Asakawa,† Tetsu Mitsumata,‡ Daisaku Kaneko,† Tatsuo Kaneko,*,† Tetsuya Ogawa,§ Hiroki Kurata,§ and Seiji Isoda§,| School of Materials Science, Japan Advanced Institute of Science and Technology, Asahidai, Nomi, Ishikawa 923-1292, Japan, Department of Polymer Science and Engineering, Graduate School of Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, 992-8510, Japan, and Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan Received August 28, 2010; Revised Manuscript Received September 9, 2010
The self-organization behavior of an extracellular polysaccharide (sacran) extracted from the cyanobacterium Aphanothece sacrum in response to lanthanoid ion adsorption was investigated. Consequently, cryogenic TEM images revealed that sacran could be cross-linked by Nd3+ trivalent ions and formed a fibrous nanostructural network containing water. Furthermore, sacran adsorbed trivalent metal ions at a 3:1 ratio, which was the theoretical ionic adsorption and showed more efficient adsorption than alginate based on electric conductivity titration. The critical gelation concentrations, Cg, where sacran formed tough gels upon metal ion binding were estimated. The Cg for trivalent metal ions was lower than that for divalent ions, and the Cg for lanthanoid ions was particularly low at 10-3 to 10-4 M, changing every four elemental numbers. The extracellular matrix of Aphanothece sacrum, sacran, may adsorb metal ions to create fibrous nanostructures that reinforce the jelly matrix.
1. Introduction Cyanobacteria, which live in the rivers and sea where industrial waste is discharged, produce and secrete polysaccharides with functional groups such as carboxylic acid, sulfates, phosphates, and amines that are responsible for ionic adsorption.1-8 These extracellular polysaccharides adsorb heavy metal ions in the water to prevent toxicity and protect the cells themselves. In addition, it has been reported that polysaccharides secreted outside of the cells are cross-linked with metal ions to flocculate and precipitate them.9,10 Therefore, there is a strong relationship between the extracellular polysaccharides produced by cyanobacteria and metal ions adsorption. The structure and function of the extracellular polysaccharide from the terrestrial jellylike structure formed by cyanobacteria representing Nostoc genus have been previously reported.11-13 However, there have been no reports on the role and formational mechanism of the extracellular polysaccharide produced by the jellylike structure formed by cyanobacteria in fresh water. Aphanothece sacrum is mass cultured in fresh water only in Japan and can be grown in groundwater containing various metal ions. Therefore, it is speculated that the extracellular polysaccharide made by this organism adsorbs these ions to create a jellylike material (gels) that protects the cells and may be useful as a scaffold for cell proliferation. In a previous study, we evaluated the material and structural properties of the novel polysaccharide “sacran” extracted from Aphanothece sacrum14,15 and reported that the sacran was a supergiant molecule (molecular weight: 1.6 × 107 g/mol) that had the ability to adsorb metal ions.16 However, it remains unknown whether the extracellular polysaccharide * To whom correspondence should be addressed. Tel: +81-761-51-1631. Fax: +81-761-51-1635. E-mail:
[email protected]. † Japan Advanced Institute of Science and Technology. ‡ Yamagata University. § Kyoto University. | Present affiliation: Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, 606-8501, Japan.
sacran actually adsorbs metal ions adsorption in intact Aphanothece sacrum and the formation mechanism of the jelly material (gels). Therefore, in this present study, a quantitative determination of the metal ions content adsorbed onto the sacran in Aphanothece sacrum was performed, and we investigated the gel formation behavior by estimating the critical gelation concentration of the sacran against various metal ions. Furthermore, the structure of the sacran-metal ions complex was examined.
2. Experimental Section Extraction. The A. sacrum samples were freeze-thawed and washed in pure water, followed by lyophilization. The samples were washed three times using a large amount of isopropanol with agitation and then collected by filtration using gauze. The isopropanol-washed samples were put into 0.1 M NaOH aq at 100 °C and agitated at constant temperature for 4 h to yield the transparent solution. The solution was neutralized with HCl until the pH value decreased to 8.0 to 9.0 and was then filtrated. Then, the filtrate was concentrated by rotary evaporator to create a highly viscous solution. The viscous solution was slowly poured in 100% isopropanol (1000 mL) to precipitate white fibrous material. The fibers were dissolved in hot water again, concentrated, and reprecipitated, and these operations were repeated three times in total. The fibrous precipitates in isopropanol were collected and dried using vacuum oven. Polarization Microscopy. The sacran solution of 0.5% was dropped in neodymium (Nd) chloride solution at a concentration of 10-4 M and, fibrous materials were obtained. The materials were observed under the cross-nicol using a first-order retardation plate (λ ) 530 nm) inserted into the light path. Electric Conductivity Measurements. The electric conductivity, σs, of the sacran aqueous solutions was measured by an AC two-terminal method using an LCZ meter (HIOKI 3532-50). The frequency range was 42 Hz to 5 MHz, and the applied voltage was 0.1 V. The sample cell used in the present study was a coaxial-cylindrical condenser with stainless-steel electrodes. The dielectric measurement was carried out at room temperature (∼20.0 ( 0.5 °C).
10.1021/bm101012u 2010 American Chemical Society Published on Web 10/05/2010
Self-Assembled Gels of Sacran-Lantanoids Observation of Cryo-TEM. Cryogenic transmission electron microscopic (cryo-TEM) images were obtained with a JEOL JEM2100F(G5) operated at an acceleration voltage of 200 kV at a magnification of 10 000. For the specimen preparation, a thin layer of sample solution was rapidly frozen to achieve cooling sufficiently fast, not to rearrange the water molecules into a crystalline form. After a little solution was placed on an electron microscopy microgrid, the excess solution on the grid was drained off with a filter paper, and the grid was immediately plunged in liquid propane maintained at ∼100 K in an immersion cryofixation apparatus (Leica, Reichert KF 80 plunger). Then, it was placed in a compartment of specially designed cryotransfer system attached to the cryo-TEM system. A liquid-helium stage was incorporated in the microscope to keep specimens around 4.2 K so that structures formed in the solution could be observed in vitreous water as they were in solution. Atomic Force Microscopy (AFM). All AFM experiments were performed using a commercial AFM unit (SPA-400, Seiko Instruments, Japan) equipped with a calibrated 20 µm xy-scan and 10 µm z-scan range PZT-scanner. For the imaging of the sacran by AFM, a stiff cantilever (SI-DF20, Seiko Instruments, force constant is 13 N/m in typical value, typical resonant frequency is 130 kHz, pyramidal tip shape, tip curvature radius is 10 nm) was used, and imaging was taken in the dynamic force modulation (DFM) mode at optimal force. The scan speed was 2 µm/sec. Measurement of the Critical Concentration of Gel Formation. Sacran solutions of 0.5% were dropped in various metallic chloride solutions, and the gels thus created were put in the metal ion solution for 10 min. The solutions containing the gels were then vigorously shaken. Next, we visually inspected whether the gels were broken by the shaking. The lowest concentration of metal ion solution at which the gels were not broken, even if the concentration of the metal ion solution was changed, was defined as the critical concentration of gel formation, Cg. Mechanical Moduli of the Gels. Water over the gel beads was thoroughly but carefully removed by the edge of a paper wiper. These wiped gel beads were put onto a glass substrate that was washed with pure water several times. The gel beads made a circle-shaped contact area between the glass substrate because of their own weight. We then measured the contact radius, A, under the glass substrates. After measuring the contact radius, the elastic modulus of the gel beads was estimated JKR theory19,20 using their contact area and weight.
3. Results and Discussion Metal Binding Behavior. Sacran had ∼11 mol % carboxylic acid and 12 mol % sulfate content and adsorbed metal ions at very low concentrations, and thus, gels made from sacran could enrich metal ions into them.16,17 Therefore, the sacran capability for metal ion adsorption was investigated. For example, the concentrations of Ca ions and Fe ions in the river where Aphanothece sacrum are found were 21 ppm and 86 ppb, respectively, and the concentrations of Ca ions and Fe ions in Aphanothece sacrum biomaterials were 270-400 ppm and 17-25 ppm, respectively. From this result, it was clear that the enrichment of the metal ions from the river water had occurred (12- to 19-fold for Ca ions and 200- to 290-fold for Fe ions). This suggests that the extracellular polysaccharide sacran functioned within a living organism for metal ions condensation. Consequently, the role of the sacran in the formation of the jelly material (gels) was examined by qualitatively evaluating the gel formation capability. In our previous study, we reported that when a sacran solution of 0.5 wt % was dropped in solutions of trivalent metal ion such as Fe3+ and Ln3+ with low concentrations (10-3 M), tough gels were formed, and this phenomenon did not occur in the case of divalent metal ions such as Ca2+, which formed a viscous slime state.16 These
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Figure 1. Sacran solution of 0.5% was dropped into a neodymium (Nd) chloride solution at a concentration of 10-4 M, and fibrous materials were obtained. These materials were observed under a cross-nicol microscope using a first-order retardation plate (λ ) 530 nm) inserted in the light path.
findings supported the above-mentioned result of higher condensation ratio of Aphanothece sacrum biomaterials for Fe ion than for Ca one. Furthermore, the same experiments were replicated on alginate, which was one of the widely studied metal-binding polysaccharides, but no alginate gels were formed in the presence of trivalent metal ions at concentrations of 10-3 M. Therefore, we speculated that sacran might have vigorous adsorption characteristics to trivalent metal ions at low concentrations and investigated the adsorption characteristics to the trivalent metal ions. We tried to investigate the Fe3+ binding to sacran chains, but the quantitative evaluation was quite difficult because of the unexpected formation of brown particles on the surface of gels even under nitrogen atmosphere. Then, we selected neodymium (Nd) ion for two reasons: (1) because we have already found the ion adsorbed very efficiently to sacran chains16 and (2) because Nd alloys are gaining attention in highperformance magnet of motors18 and Nd are required to recover from industrial waste liquids. A sacran solution of 0.5 wt % was dropped in an NdCl3 solution at a concentration of 10-3 M, and tough gels were also obtained, whereas Nd concentration was reduced to 10-4 M, and then not the gels but fibrous materials were obtained. The fibrous materials were sandwiched between glass coverslips and were observed under a polarizing microscope. It was confirmed that the fibers were oriented and had a negative birefringence, as evidenced by subtractive birefringence (blue color) in the fiber lying from upper left to lower right (Figure 1). In other words, the sacran chains were oriented in the direction of the fiber axis. In any other Ln3+ ions, the fibrous materials with the orientation similar to that of Nd3+ were also obtained. It seemed that the sacran molecular chains were cross-linked by metal ions to agglutinate by forming bundles. Electric Conductivity Titration. Positive charges of Nd ions adsorbing to the sacran chains erased their ionic function by forming a salt with the sacran anions and thus did not show any conductivity, whereas nonadsorbed Nd ions kept their charges and showed conductivity (σ). We then evaluated the number of Nd ions adsorbed to the sacran chains by measuring the σ value of the supernatant of the solution, where the fibrous sacran/Nd complexes were precipitated by vigorous agitation just after mixing (sacran concentration: 0.1 wt %). Prior to the measurement of supernatant, the σ value of the NdCl3 aqueous solution was measured, and it increased linearly with an increase in the concentration, as shown with the solid line in Figure 2a. From the Figure, the molar conductivity of NdCl3 was calculated from the inclination of the solid line as 345 S cm2/mol. The molar conductivity of Nd3+ was then estimated to be 166 S
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Figure 2. (a) Solid line shows electrical conductivity σ of the NdCl3 aqueous solution. Dotted line shows the conductivity difference σ′ between sacran-NdCl3 mixed aqueous solution and sacran solution. (b) Conductivity difference (∆σ) in NdCl3 between σ and σ′ (concentration of polysaccharides: 3 mM). ∆σ values in alginate were estimated by the analogous procedure with sacran. The inserted values are adsorption constants estimated using Langmuir theory.
cm2/mol by using the contribution of Nd3+ to the molar conductivity (∼48%). Mixing the sacran solution with NdCl3 also showed a linear increase in σ with a concentration increase (>0.5 mM), but the linearity was lost in the lowest range of concentrations ( 0.98) and was 6.5 × 104 M-1 for adsorption to the sacran chains versus 3.3 × 104 M-1 for adsorption to the alginate chains. This result indicated that the Nd3+ adsorption efficiency to the sacran anions was about twice as high as that to alginate. It can be considered that this reason is related to the fact that the anionic groups are concentrated on super giant sacran chains to form a deep well of negative potential. By this method, we investigated the complexation behavior of Ca2+/alginate and found a 1:2 (Ca2+: anions) stoichiometry and the adsorption constant of 2.0 × 103 M-1. However no ∆σ saturation of sacran was shown over the experimental concentration range (1 µm. However, one can observe fibrous substances of the same width but with lengths
